Annals of Sri Lanka Department of Agriculture. 2006.8:1-10.

MOTHER PLANT SELECTION OF PROMISING MANGO LAND RACES (Mangifera indica L.) BY FRUIT CHARACTER EVALUATION

S.J. ARASAKESARY1, K. SIVATHEPAN1 and K. SRIBALASUNDARAM2 1Agricultural Research Station, Thirunelvely 2District Agriculture Office, Jaffna

ABSTRACT

The polyembryonic and monoembryonic nature of mango (Mangifera indica L.) has led to the evolution of innumerable cultivars and varieties. Every tree from a seed is potentially a new type. On introduction from various places and from selections locally, the cultivars or varieties received various names. Proper selection of mother plants and their planting materials is important to researchers, nurserymen and growers. Hence initially a study was conducted to evaluate and select promising trees of four mango varieties as land races and establish these in a germplasm collection. Thirteen Karuthai colomban (KC), five Vellai colomban (VC), four Willard (WD) and four Ambalavi (AM) tress were evaluated on their fruit characteristics. Fruit traits were scored on a scale developed locally based on the Indian Council of Agriculture Research guide. Three KC types were found to be generally differing in some external fruit characters while only meagre differences in shape of fruit and flesh color were found in VC, WD and AM entries. Two KC, two VC, two WD and two AM entries were selected based on the score values. One KC entry was classified as a different genotype. The parent trees of all selected trees were labelled. Two plants with similar fruit characteristics known locally as Kodima (KM), but of unknown origin, were also selected. True to type grafts of above selections have been established in a germplasm block at the District Agricultural Training Centre, Jaffna.

KEYWORDS: Evaluation, Mango land races, Mother plants, Scion.

INTRODUCTION

Polyembryony and monoembryony combined with the cross pollinated nature of Mango (Mangifera indica L.) led to the production of innumerable cultivars and varieties. Every tree from the seed is potentially a new variety (Gangolly et al., 1957). The vegetative propagation of mango started probably just 300 years ago and this helped the preservation of outstanding cultivars which originated as chance seedling (Gangolly et al., 1957). The cultivars on introduction from various places received different names in the region in particular. Identification of varieties only using vegetative characters at the time of purchasing of plants is not advisable since a long time is taken by the trees to come into bearing and before an accurate identity, based on fruit characters too is made.

About 47900 mango trees are available in Jaffna District (Anon., 2004). Information on location, fruit qualities and other related characteristics of these trees are scanty and very little work has been reported. 2 ARASAKESARY et al.

Nurserymen often produce grafted seedlings from their old trees and such plants seldom produce quality fruits. Proper selection of planting materials and mother plants is very important for nurserymen, fruit growers and researchers. Such work will help to improve this crop and fruit production. On the other hand, there are many outstanding trees which are underutilized from which quality scions could be taken for the production of plants. There is an urgent need to evaluate and locate such mango trees as many trees have been abandoned or destroyed by the armed conflict, Tsunami and the construction of buildings, roads etc.

This paper stems from a study conducted to evaluate mango trees by their fruit characters and locate most productive mango trees in Jaffna district so that true to type plant seedlings from such selected mother plants could be established at the District Agricultural Training Centre (DATC), Jaffna. It could serve as a collection of mango germplasm for further development in mango.

MATERIALS AND METHODS

Potential mango growing areas in Jaffna district were selected for this study in the year 2005. Information was gathered from agriculture extension staff, consumers and local traders on high quality fruit producing mango trees of four popular cultivars, namely Karuthai colomban (KC), Vellai colomban (VC), Willard (WD) and Ambalavi (AM). At the outset sixty trees were observed. Twenty eight trees were selected for recording data. These trees were labelled as indigenous cultivars (IC) with first letter of genus and species (MI). Abbreviations were used for the names of Agriculture Instructor ranges and local names of cultivars ending with the year of selection and with a serial number (Singh, 2000). Three fruits of uniform maturity from each tree were used for observing fruit characters such as form, stalk, base, apex, shoulder, cavity and beak using the Mango descriptor published by the Indian Council of Agricultural Research (ICAR). Fully mature fruits were hand picked and were allowed to ripen under ambient conditions. Well ripe fruits were randomly selected for the evaluation of internal characters. The most practical useful traits such as fruit weight, flesh weight percentage to the non-edible parts, fibre content, flesh colour, aroma and sweetness were considered for selection purposes. Aroma and sweetness were graded by a panel. These commercially important traits were scored according to an arbitrary scale developed locally (considering local preferences) with the help of ICAR (Table 1). Best preferred traits were scored with the highest values (Table 2) and the total scores and fruit character were used for selection of suitable mother plant (Appendices 1 and 2). Scions from selected trees were used to establish true to type grafts at DATC, Jaffna. MOTHER PLANT SELECTION OF MANGO 3

Table 1. Arbitrary scale developed for scoring fruit characters of mango germplasm in Jaffna district.

S Fruit Flesh Fibre content Flesh colour Aroma Sweetness N wt (g) wt(%) (by feeling) Weight S % S Fibre- S Colour S Range S Range S Range c range c ness c range c c c o o o o o o r r r r r r e e e e e e 1 150-250 1 <60 1 Fibre 1 Light 1 Acidic 1 None/ 1 yellow Acidic

2 251-350 2 60-70 2 Moderate 2 Yellow 2 Less 2 Less 2 fibre 3 351-450 3 70-75 3 Less 3 Deep 3 Medium 3 Medium 3 fibre yellow 4 451-550 4 75-80 4 Little 4 Light 4 Strong 4 Sweet 4 fibre orange 5 >551 5 >80 5 Almost 5 Orange 5 Very 5 More 5 nil strong sweet

RESULTS AND DISCUSSION

The cultivars, on introduction from various sources have been deliberately or otherwise given entire new names and this has caused a great deal of confusion in naming of these trees. Names used in this study have been in use traditionally in Sri Lanka.

Score values of thirteen KC, five VC, four WD and four AM fruits are given in Table 2. These values ranged from 12 to 19 in these cultivars. Some KC and AM scored higher values of 19; WD and VC scored maximum score of 18. These values confirmed traditional preference of these cultivars in the Jaffna region.

Karuthai colomban

This variety (commonly abbreviated as KC but is abbreviated as KKM in this paper) is the most preferred in the peninsula and fetches the highest commercial value. A large vigorous tree with stocky trunk produces fruit with medium thick skin, primuline yellow when ripe. Flesh of the fruit is juicy and tasty with mild pleasant flavour. Among fifteen trees, three distinct types were found which varied in fruit and canopy structure, fruit shoulder and cavity (Appendix 1). Fruit traits such as aroma and sweetness also differed slightly. 4 ARASAKESARY et al.

Table 2. Score values of fruit characters of twenty six mango trees of Karuthai colomban, Vellai colomban, Willard and Ambalavi and two trees of new type.

Name of the Tree Frui Flesh Fibrenes Flesh Aroma Sweetnes Total t wt ratio s colour s score value IC/MI/KEE/KKM/501 2 1 3 4 1 2 13 IC/MI/THO/KKM/502 1 2 2 4 4 1 14 IC/MI/THO/KKM/503 1 4 1 3 2 1 12 IC/MI/THO/KKM/504 3 3 3 3 1 1 14 IC/MI/THO/KKM/505 1 3 3 3 4 3 17 IC/MI/NAL/KKM/506 2 2 3 3 4 3 17 IC/MI/NAL/KKM/507 2 2 3 3 4 3 17 IC/MI/NAL/KKM/508 3 3 3 3 4 3 19 IC/MI/NAL/KKM/509 2 2 3 3 4 3 17 IC/MI/NAL/KKM/510 3 2 3 3 4 3 18 IC/MI/NAL/KKM/511 2 3 3 3 2 1 14 IC/MI/CHA/KKM/512 2 3 3 3 2 1 14 IC/MI/CHA/KKM/513 2 2 3 3 4 3 17

IC/MI/UDU/VLK/501 1 4 4 2 3 1 15 IC/MI/VEI/VLK/502 1 3 4 2 4 3 17 IC/MI/URU/VLK/503 2 3 4 2 4 3 18 IC/MI/CHA/VLK/504 1 1 4 2 3 1 12 IC/MI/CHA/VLK/505 1 2 4 2 3 3 15

IC/MI/KEE/WLD/501 1 2 4 3 4 3 17 IC/MI/NAL/WLD/502 1 2 4 3 4 3 17 IC/MI/CHA/WLD/503 1 4 4 3 4 3 19 IC/MI/NAL/WLD/504 1 3 4 3 4 3 18

IC/MI/URU/AMV/501 1 1 4 4 3 1 14 IC/MI/CHA/AMV/502 1 1 4 4 3 3 16 IC/MINAL/AMV/503 1 3 4 4 4 3 19 IC/MI/NAL/AMV/504 1 3 4 4 4 3 19

New type IC/MI/CHA/KM/501 3 4 5 5 4 3 24 IC/MI/CHA/KM/502 3 4 5 5 4 3 24

Some of these types are as follows: IC/MI/KEE/KKM/501: It had top round canopy. Fruits had round shoulder, ovate oblong form with tapering base. IC/MI/THO/THO/KKM/505 produced oblong forms, falling abruptly with rounded base fruit and IC/MI/NAL/KKM-508 produced oblong oblique forms ending in a moderate curve shoulder with rounded base fruits (Appendix 1). Single KKM fruit weighed an average of 350g (Appendix 2) with the range of 250-400g. As shown in Table 2, trees with the highest score (19) IC/MI/NAL/KKM/508 and IC/MI/THO/KKM/505(18) were selected as potential mother plants. The entry IC/MI/KEE/KKM/501 though obtaining lower value (13), was selected as germplasm (Appendix 2). MOTHER PLANT SELECTION OF MANGO 5

Vellai colomban

In this paper Vellai colomban is abbreviated as VLK. This variety is a vigorous, regular bearing spreading tree producing soft sparsely fibrous golden yellow flesh. Each fruit weighed an average of 210 g with the range of 170-260 g. IC/MI/VEICHA/VLK/502 and IC/MI/URU/VLK/503 were selected with score values of 18 and 17 respectively (Appendix 2).

Willard

This variety is abbreviated as WLD in this paper. Fruits of this cultivar are attractive giving tasty flesh. It has peculiar aroma and colour. This variety is found in almost every home garden in this region owing to its attractive small canopy. Single fruit weighed around 200 g with a range of 180-240 g. Fruit score values varied from 14 to 18 (Table 2). Higher scored two trees namely IC/MI/CHA/WLD/503 and IC/MI/NAL/WLD/504 were selected as mother plants.

Ambalavi

Abbreviated as AMV, it produces medium, oblong fruits with thin smooth skin at times tending to be warty. Flesh is sour to moderately sweet with moderate fibre. Single AMV fruit weighed around 220g. Score values ranged from 14 to 19 (Table 2). The entries IC/MI/NAL/AMV/503 and IC/MI/NAL/AMV/504 were selected as mother plants.

New type

During this study two similar trees of completely new type mango called - Kodima (Creeping mango) abbreviated as KM were found by chance. Origin of this cultivar could not be traced. These two trees appeared closeby and this may be a recent introduction. Trees had drooping canopy appearing like a creeper. Fruits were medium with an average weight ranging between 280-400 g and of attractive size and shape and had very little fibre. This type scored the highest value of 24 (Table 2). This locally called Kodima is an underutilized one.

CONCLUSIONS

Of the twenty-six mango trees of four popular varieties in this region, three Karuthai colomban, two Vellai colomban, two Willard and two Aambalavi had been selected as potential mother plants. These trees were labelled permanently. Location of these trees was easily identifiable. A new type mango, found by chance could be observed further and developed as a new variety. True to type grafts of selected land races are being established at 6 ARASAKESARY et al.

DATC, Jaffna. Scions are available from the selected mother plants for propagation. Bud wood could be made available to the departmental institutions and nurserymen once the established plants growing at Germplasm block at DATC, Jaffna bear fruits and the genotypic characters are confirmed.

ACKNOWLEDGEMENTS

Authors thankfully acknowledge Mr. S. Tharmarajah, DD (RARDC, Vavuniya and Mr. S. Selvarajah, Former ADA (Ext.), Jaffna for their valuable guidance in this study. District Secretary Mr. K. Ganesh is highly appreciated for providing funds to undertake this study. Agricultural Instructors of relevant ranges are also acknowledged. Authors wish to thank Mr. P. Atputhachandiran (AI) for his assistance during the preparation of the manuscript.

REFERENCES

Anon., 2004. Statistical handbook, District Secretariat, Jaffna. Gangolly, S.R., R. Singh, S. Katyal and D. Singh, 1957. The mango, Indian Council of Agricultural Research, New Delhi. Pp 1-239. Singh, B.D. 2000. Plant breeding principles and methods, Kalyani Publishers, New Delhi. Pp 17-47. MOTHER PLANT SELECTION OF MANGO 7

Appendix 1. Fruit characters of twenty six mango trees of Karuthai colomban, Vellai colomban, Willard, Ambalavi and two trees of new type.

Name of the tree Form Stalk Base Apex Shoulder Cavity Beak IC/MI/KEE/KKM/501 Ova.Obl Obq Tap Shl Ron Slt Apn IC/MI/THO/KKM/502 Obl Obq Tap Shl Fal.Avr Slt Apn IC/MI/THO/KKM/503 Obl.Obq Obq Obl.Rol Shl Fal.Avr Slt Apn IC/MI/THO/KKM/504 Obl Obq Obl.Rol Shl Fal.Avr Slt Apn IC/MI/THO/KKM/505 Obl Obq Rol Shl Fal.Avr Slt Apn IC/MI/NAL/KKM/506 Obl.Obq Obq Rol Shl End.Mcr Slt Apn IC/MI/NAL/KKM/507 Obl Obq Rol Shl End.Mcr Slt Apn IC/MI/NAL/KKM/508 Obl.Obq Obq Rol Shl End.Mcr Slt Apn IC/MI/NAL/KKM/509 Obl.Ovl Obq Rol Shl End.Mcr Slt Apn IC/MI/NAL/KKM/510 Obl.Ovl Obq Rol Shl End.Mcr Slt Apn IC/MI/NAL/KKM/511 Obl.Ovl Obq Rol Shl End.Mcr Slt Apn IC/MI/CHA/KKM/512 Obl.Ovl Obq Rol Shl End.Mcr Slt Apn IC/MI/CHA/KKM/513 Obl.Ovl Obq Rol Shl End.Mcr Slt Apn

IC/MI/UDU/VLK/501 Obl.Elp Squ Tap Ron Fal.Avr Abs Apn IC/MI/VEI/VLK/502 Obl.Elp Squ Tap Ron Fal.Avr Abs Apn IC/MI/URU/VLK/503 Obl.Elp Squ Tap Ron Fal.Avr Abs Apn IC/MI/CHA/VLK/504 Obl.Elp Squ Tap Ron Fal.Avr Abs Apn IC/MI/CHA/VLK/505 Obl.Elp Squ Tap Ron Fal.Avr Abs Apn 8 ARASAKESARY et al.

IC/MI/KEE/WLD/501 Ron Obq Flt Bro.Ron Ron Slt Apn

IC/MI/NAL/WLD/502 Ron Obq Flt Bro.Ron Ron Slt Apn IC/MI/CHA/WLD/503 Ron Obq Flt Bro.Ron Ron Slt Apn

IC/MI/NAL/WLD/504 Ron Obq Flt Bro.Ron Ron Slt Apn

IC/MI/URU/AMV/501 Obl Squ Tap Bro.Ron Fal.Avr Abs Slt.Prm

IC/MI/CHA/AMV/502 Obl Squ Tap Bro.Ron Fal.Avr Abs Slt.Prm

IC/MINAL/AMV/503 Obl Squ Tap Bro.Ron Fal.Avr Abs Slt.Prm

IC/MI/NAL/AMV/504 Obl Squ Tap Bro.Ron Fal.Avr Abs Slt.Prm

New type IC/MI/CHA/KM/501 Obl.Ovl Obl Ron Ron Fal.Avr Slt Apl IC/MI/CHA/KM/502 Obl.Ovl Obl Ron Ron Fal.Avr Slt Apl

Ova.Obl-Ovate Oblong Obl- Oblong Ron- Roundish Obq-Oblique Squ-Square Tap-Tapering Obq.ron-Obliquely rounded Flt.Flattned Slt-Slight Shl-Shallow Bro-Ron-broadly rounded Apn-Pointed beak Fal.Abr-Falling abruptly Abs-Absent Slt.prm-Slightly prominent End.Mcr- Ending in a moderate curve

AI ranges- KEE- Keerimalai THO- Tholpuram NAL- Nallur CHA- Chavakachcheri UDU- Uduvil URU- Urumpirai MOTHER PLANT SELECTION OF MANGO 9

Appendix 2. Average value of important fruit characters of twenty six mango trees of Karuthai colomban, Vellai colomban, Willard and Ambalavi and two trees of new type.

Name of the tree Fruit wt(g) Flesh Fibre content Flesh Aroma sweetness Remarks ratio (%) (feeling) colour IC/MI/KEE/KKM/501 260 57.7 Less De-yell Acidic Sweet Selected IC/MI/THO/KKM/502 250 64.0 Less De-yell Strong Less Sweet IC/MI/THO/KKM/503 250 76.0 Fibrous De-yell Light Less Sweet IC/MI/THO/KKM/504 360 78.0 Little De-yell Acidic Less Sweet IC/MI/THO/KKM/505 240 75.0 Little De-yell Strong More sweet Selected IC/MI/NAL/KKM/506 280 75.0 less De-yell Strong More sweet IC/MI/NAL/KKM/507 260 69.2 little De-yell Strong More sweet IC/MI/NAL/KKM/508 400 78.0 little De-yell Strong More sweet Selected IC/MI/NAL/KKM/509 320 68.7 little De-yell Strong More sweet IC/MI/NAL/KKM/510 400 70.0 little De-yell Strong More sweet IC/MI/NAL/KKM/511 300 73.0 little De-yell Less Less Sweet IC/MI/CHA/KKM/512 300 73.0 Less De-yell Less More sweet IC/MI/CHA/KKM/513 300 66.7 Less De-yell Strong Less Sweet

IC/MI/UDU/VLK/501 170 76.5 Less DA-yell Strong Less sweet IC/MI/VEI/VLK/502 200 75.0 Less DA-yell Strong More sweet Selected IC/MI/URU/VLK/503 260 73.0 Less DA-yell Medium More sweet Selected IC/MI/CHA/VLK/504 200 75.0 Less DA-yell Medium More sweet IC/MI/CHA/VLK/505 180 73.1 Less DA-yell Medium More sweet

IC/MI/KEE/WLD/501 210 61.0 Least Da-yell Strong More sweet IC/MI/NAL/WLD/502 230 70.0 Least Da-yell Strong More sweet 10 ARASAKESARY et al.

IC/MI/CHA/WLD/503 180 77.8 Least Da-yell Medium More sweet Selected IC/MI/NAL/WLD/504 240 70.1 Least Da-yell Medium More sweet Selected

IC/MI/URU/AMV/501 200 58.0 Less Li-yell Medium Less sweet IC/MI/CHA/AMV/502 200 62.0 Less Li-yell Medium More sweet IC/MINAL/AMV/503 220 70.0 Less Li-yell Strong More sweet Selected IC/MI/NAL/AMV/504 220 70.0 Less Li-yell Strong More sweet Selected

New type IC/MI/CHA/KM/501 380 78.9 Less Or-yel Strong More sweet IC/MI/CHA/KM/502 400 77.5 Less Or-yel Strong More sweet

Dee.yell-deep yellow Da.yell- Dark yellow Or.yell-Orange yellow Li.Yell-Light yellow KKM-Karutha colomban, VLK-Vella colomban, WLD-Willard, AMV-Ambalavi, KM- Kodima

AI ranges: KEE- Keerimalai THO- Tholpuram NAL- Nallur CHA- Chavakachcheri UDU- Uduvil URU- Urumpirai Annals of Sri Lanka Department of Agriculture. 2006.8:11-18.

DEVELOPMENT OF AN EFFECTIVE INTEGRATED PEST MANAGEMENT SYSTEM FOR MELON FLY, Bactrocera cucurbitae INFESTING COMMERCIAL CULTIVATION OF GHERKIN, Cucumis sativus

K.A.N.P. BANDARA1, C. KUDAGAMAGE1, D.P. SENADEERA 2 and G.S. PRATHAPASIGNHA3 1Department of Agriculture, Peradeniya 2HJS Condiments Ltd, Biyagama 3 Sun Frost Ltd, Katugoda

ABSTRACT

Pickling cucumbers (Gherkins) account for 20% of country’s total fruit and vegetable export valued about Rs. 625 million in 2005. However, Melon fly, Bactocera cucurbitae infestation jeopardizes production and export of this crop. Therefore, the industry urges implementation of an effective and farmer acceptable Integrated Pest Management (IPM) system against this infestation. The purpose of the present study was to develop farmer accepted IPM technologies. Different IPM compatible techniques were evaluated against the fly infestation in farmer fields in four major gherkin cultivating areas in Anuradhapura, Kurunegala Mahiyangana and Polonnaruwa districts during three cultivating seasons (2004-2005). Use of male lure traps (Cue-lure®), spot application of protein baits developed by Horticultural Crop Research and Development Institute (HORDI) and imported from Australia (Fruit fly lure®) along with other field sanitary practices were compared with blanket spraying of the recommended insecticide fenthion (Lebaycid®). A field survey was also carried out to explore potential natural enemies aiming to develop biological control methods to support IPM. Cue-lure traps placed at 120 cm above the ground level attracted the highest number of male flies compared to the traps placed at other heights. The two protein baits developed by HORDI and Fruit fly lure® equally attracted female flies in the laboratory tests. However, results obtained from field evaluations failed to reflect their effects due to high variability of the fly population in different locations which were used as replicates. Area-wide fly management using bait spray combined with high density of cue-lure traps in Anuradapura and Kurunegala recorded the lowest melon fly infestation (1.3% and 0.19% respectively) while untreated fields in the same locality recorded very high fruit infestation (20% and 8% respectively). Laboratory studies revealed that high soil moisture at early pupal development stages stimulated adult emergence explaining the increase of fly population in gherkin fields after rain followed by a drought. A pupal parasitoid, Tetrastichus sp. (Hymenoptera: Eulophidae) was recorded in Polpithigama (Kurunegala District) but its parasitism was low (0.2%). The findings suggest that implementation of area-wide IPM system with spot spraying of protein bait and placement of high density cue-lure traps with good field sanitary practices would be effective for controlling melon fly infestation. Further studies on parasitoids of melon fly are needed to develop effective biological control methods to improve IPM.

KEYWORDS: Bactrocera cucurbitae, Gherkins, Management, Melon fly. 12 BANDARA et al.

INTRODUCTION

Several species of economically important crops belonging to family cucurbitaceae including cucumbers, melon, pumpkin and gourds are grown in Sri Lanka and their extent is approximately 16,000 ha. Pickling cucumbers (Gherkins) which have an export potential are grown approximately in 500 ha annually by out-growers attached to main exportres of processed gherkins (HJS Condiments Limited and Sunfrost Limited) which account for 20% of country’s total fruit and vegetable export valued about Rs. 625 million in 2005. Melon fly, cucurbitae has become the major constraint for the production of all types of cucurbitaceous crops including gherkins. The fly deposit eggs beneath the skin of the fruits and the emerging larvae feed on the flesh destroying the fruit prematurely (Vijaysegaran, 1996). As the salad cucumbers are harvested prematurely, the developing larvae could be seen inside the fruits and its damage is visible even after their processing. As the overseas customers refuse products with damaged fruits, melon fly infestation may adversely affect the production and export of the crop. On the other hand, the use of chemical pesticides failed to give satisfactory control over the problem and their use in salad cucumbers is limited due to the risk of pesticide residues. As a result the industry urges effective and farmer acceptable integrated management system for melon fly in managing the infestation. The study intended to develop such a system incorporating methods suitable for local conditions.

Several IPM compatible techniques including use of male lure traps (Cue-lure®), spot application of three protein baits namely HORDI A and HORDI B developed by Horticultural Crop Research and Development Institute (HORDI) and Fruit fly lure® imported from Australia, along with other field sanitary practices were compared with blanket spraying of the Department of Agriculture (DOA) recommended insecticide, fenthion (Lebaycid®) and with the untreated check in farmer fields. A field survey was also carried out to explore potential natural enemies aiming to develop biological control methods to support IPM. Laboratory and field studies were carried out to investigate the effect of soil moisture for adult emergence and to study the optimum height for placement of Cue-lure® traps. The possibility of using these techniques in area-wide management of this pest was also studied.

MATERIALS AND METHODS

All field experiments were conducted in farmer fields in main gherkin growing areas in Anuradhapura, Kurunegala, Mahiyangana and Polonnaruwa districts during three cultivating seasons. Laboratory experiments were conducted at HORDI. The experiments were in IPM SYSTEM FOR MINIMIZING MELON FLY DAMAGE 13

Randomized Complete Block Design (RCBD) and data were analyzed by ANOVA and LSD procedure. Optimum height for placing cue-lure traps

The para-pheramone, cue-lure® traps were prepared by absorbing the material into plugs of cotton wool were placed in clear plastic bottles (1 l capacity) fixed with two, 1 cm diameter plastic tubes (4.0 cm in length) to the lid and bottom of the bottle. The traps were placed at different heights (30, 60, 90, 120 and 150 cm) from the ground level of gherkin fields in three cultivating areas (Mahiyangana, Polpithigama and Anuradhapura) and fly counts were taken at weekly intervals from June to November, 2004. Ten traps were placed at each location and the cue-lure plugs were replaced at monthly intervals.

Efficacy of protein baits to attract female flies

Three different protein bait formulations, HORDI Bait A, HORDI Bait B and Fruit fly lure® were evaluated for their attractiveness against 10-day-old female flies (9-10 days old) using screen cage method adopted by Ekanayake and Bandara (2003). The experiment was conducted in HORDI laboratory, Gannoruwa between 8.00am-9.00am. The number of flies settling on different bait formulations and on the untreated control (water) were recorded in two minute intervals and the recordings were started 10 min after the introduction of adults. After each recording the cages were rotated 90°.

Effect of soil moisture on adult emergence from soil inhabited pupae

Previous observations of trap counts have shown that rain after prolonged dry spell increases population of melon flies. Therefore, the experiment was designed to investigate the relationship between soil moisture level and adult emergence. Forty pupae (0-6 hour old) were placed at 1 cm depth in air-dried clay loam soil in 15x10x6 cm transparent plastic boxes. Water was added to each box at different time periods (On the same day, 2, 3 and 4 days) after pupal introduction up to 50% of the water holding capacity of the soil samples. Adult emergence in each box was recorded daily for 20 days. The experiment was replicated 4 times.

Field experiments

Comparison of spot spraying of protein bait with cover spray of fenthion

Field experiments were conducted in farmer fields at four locations representing main gherkin growing districts, Anuradhapura, Mahiyangana, Kurunegala and Polonnaruwa during three seasons (yala 2004, 14 BANDARA et al. intermediate season 2004 and maha 2004/05) with farmer participation. All field experiments were in RCBD with three replicates. Cluster of farmer fields approximately 1000 m2 each located closer to each other was considered as one block and different treatments were allocated to different farmers in each block.

Farmers were given gherkin, Cucurmis sataivam (cv. Vlasset B) seeds and advised to follow recommended cultivation practices. Farmers assigned for different melon fly control practices were instructed to initiate the control practices at 50% flowering stage of the crop and continue up to the end of the harvesting. Farmers were advised to collect infested fruits into black polythene bags (>600) and tie them up to avoid adult emergence as a common control practice. In yala 2004 and intermediate season, three treatments, spot application of protein bait developed by HORDI, spot application of Protein bait (Fruit fly lure) and cover spray of Fenthion (Lebaycid) were tested. In maha 2004/5, in addition to these three treatments, a plot without any melon fly control practices (untreated control) was also maintained. At harvesting, weight and number of total and melon fly damaged fruits were recorded.

Effectiveness of area-wide management on melon fly infestation

Melon fly adults have high flying capacity, high fecundity and life span. As a result area-wide management was shown to be more effective than practicing in small individual farms. Therefore, this field experiment was conducted in two isolated locations at Madiyawa in Kurunegala district and at Digampathana in Anuradhapura district with 2 and 4 acres of cucumber cultivations respectively, to demonstrate the importance of implementing the IPM package in a large area. The control practices used in this package include setting up of Cue-lure traps at equal distance surrounding the entire cultivation area (60 traps/ha), spot application of fruit fly lure in 5-7 day intervals with sanitary practices such as destruction of crop residues and collection of damaged fruits into black garbage bags to prevent adult emergence. Yield data were taken as in the previous field experiments.

Collection of melon fly parasitoids

Five hundred soil inhabiting melon fly pupae were collected from each field and kept in sand trays for adult emergence. Parasitoids emerged were collected, preserved and identified.

RESULTS AND DISCUSSION

Optimum height for placing cue-lure traps IPM SYSTEM FOR MINIMIZING MELON FLY DAMAGE 15

Lure traps placed at 120, 90 and 60 cm above the ground vegetation attracted significantly higher number of male flies when compared with the rest of the height levels (Table 1). Therefore, placement of lure traps at 60-120 cm above ground level is recommended.

Table 1. Male flies attracted to cue-lure® traps placed in different heights at different locations. Trap Number of male flies attracted/trap/30 days height (cm) Mahiyangana Polpithigama Anuradhapura Mean* 60 65 09 08 27abc 90 49 19 28 28ab 120 43 34 37 38a 150 14 07 17 13bc 180 10 03 05 06c CV% 57% *Means followed by the same letter are not significantly different at P=0.05.

Efficacy of protein baits to attract female flies

All tested protein baits attracted significantly higher number of female melon flies as compared to untreated control (water). In two tests, HORDI bait A showed significantly higher level of fly attraction over other two bait formulations. However, in eight tests there was no significant difference on attractiveness among different protein baits (Table 2).

Table 2. Melon fly attraction to different protein bait formulations in laboratory cage tests.

Treatment Mean number of flies attracted*/10 flies Test Test Test Test Test Tes Tes Tes Test Tes 1 2 3 4 5 t 6 t 7 t 8 9 t 10 HORDI bait 0.3ab 4.2a 4.4a 0.9ab 0.9ab 1.7a 4.9a 0.0b 3.5a 0.8b A HORDI bait 2.3a 1.6ab 1.1bc 2.2a 2.2a 0.8a 2.3b 4.7a 1.2ab 1.4b B Fruit fly 2.3a 0.9b 2.7ab 1.1ab 1.1ab 0.8a 0.0b 0.0b 0.3b 3.4a lure® Control 0.0b 0.0b 0.0c 0.3b 0.3b 0.1a 0.0b 0.0b 0.0b 0.0b *Means in a column followed by the same letter are not significantly different at P=0.05.

Effect of soil moisture on adult emergence from soil inhabited pupae

It was indicated that increase of soil moisture level at the early pupal stages induce adult emergence (Table 3). Studies on pupal survival in several soil textures, irrigated at different times of pupal development using mediteranean fruit fly, Ceratitis capitata also showed similar effect (Del Pino et al., 1997). According to their results, in sandy soil the highest emergence of 16 BANDARA et al. adults occurred when the soil was irrigated during early stage of pupal development. They also reported that when irrigated the sandy is soil at the end of the pupal period, most of the adults emerged from the puparium, but only a few were able to leave out the soil. However, in clay loam soil the highest adult emergence was observed, when it was irrigated at the end of the pupal development. This information could be useful to explain the increase of melon fly population in major gherkin growing areas after rain followed by a prolonged drought period. Therefore, farmers are advised to strengthen their fly management programs under such situations.

Table 3. Effect of soil moisture on adult emergence of pupae at different maturity stages in sandy soil.

Age of the pupae when moisture added Number of adults emerged/40 pupae 1 36 2 13 3 8 4 7

Field experiments

The level of melon fly infestation was not significantly different among different control practices in yala 2004 (Table 4). As no cultivation was maintained without treatment, it was difficult to conclude whether this low infestation was due to the treatments or due to low melon fly population during the period.

Table 4. Effectiveness of three melon fly control practices on melon fly infestation of gherkin fruits (yala, 2004).

Treatment Infested fruits (%) Kurunegala Anuradhapura Mahiyangana HORDI Protein Bait A 0.33 0.01 0.01 Fruit fly lure® 0.16 0.01 0.00 Fenthion, Lebaycid® 0.19 0.01 0.01 CV% 49 82 109

In intermediate season, 2004 only Anuradhapura recorded a little higher level of infestation and HORDI protein bait-A recorded the lowest infestation (Table 5). However, there was no significant difference among treatments on their infestation levels. As in the previous season, no untreated control field was maintained in this season too. Hence, no conclusion could be made on the effect of different treatments on fly infestation. IPM SYSTEM FOR MINIMIZING MELON FLY DAMAGE 17

Table 5. Effectiveness of three melon fly control practices on melon fly infestation of gherkin fruits (Intermediate season, 2004).

Treatment Infested fruits (%) Kurunegala Anuradhapura Mahiyangana HORDI Protein Bait A 0.09 1.60 0.01 Fruit fly lure® 0.03 2.20 0.00 Fenthion, Lebaycid® 0.02 2.50 0.01 CV= 54.1 In maha 2004/5, comparatively high melon fly infestation was observed in Anuradhapura and in some areas of Kurunegala district. Though comparatively low fruit infestation was recorded in Fenthion treated and both bait sprayed plots, when compared to the untreated control, the pooled values failed to show significant difference among them (Table 6). This is mainly due to the high variability of fly population in different locations within the district, which were used as blocks in the analysis process.

Table 6. Effectiveness of three melon fly control practices on melon fly infestation of gherkin fruits (maha 2004/5).

Treatment Infested fruits (%) Kurunegala Anuradhapura Mahiyangana Giritale HORDI protein 0.58 9.55 0.03 0.0 bait A Fruit fly lure 8.21 5.19 0.01 0.0 Fenthion, 0.89 2.85 0.05 0.0 Lebaycid® Control 8.25 20.26 0.11 0.0 CV=117%

Area-wide management in suppressing melon fly infestation

Non-replicated, area-wide management program carried out in two locations in Anuradhapura (Digampathana) and Mahiyangana (Madiyawa) combining cue-lure traps, spot spraying of protein bait and sanitary practices recorded zero fruit infestation where as 20% and 8% infestations, respectively were recorded in untreated plots in the same areas. This study highlighted the importance of implementing area-wide management programs for highly mobile pests such as fruit flies.

Parasitoids of melon fly

The pupal parasitism of melon flies was low and only a few parasitoids emerged from Polpithigama (Kurunegala District). The parasitoid was identified as a Tetrastichus species (Hymenoptera: Eulophidae). Several species of Tetrastichus have been recorded from fruit fly pupae in various parts of the world and attempts have also been made to use them in bio-control 18 BANDARA et al. programs (Wharton, 1989). Therefore, it is very important to carry out a complete survey on parasitoids of the pest and develop technology to use them in IPM programs.

CONCLUSIONS

Melon fly infestation can be minimized implementing area- wide Integrated Management package consisting of crop sanitation, spot application of protein bait and placing of high density cue-lure traps. Protein baits developed by HORDI (HORDI A and HORDI B) and Fruit fly lure®, a bait formulation imported from Australia, were equally effective.

ACKNOWLEDGMENTS

The authors wish to thank Sri Lanka Council for Agriculture Research Policy (CARP) for the financial assistance provided for this program. REFERENCES

Del Pino, A. A., M.T.G Putruele and A. Garrido. 1997. Effect of soil moisture and texture on pupal survival of the Mediteranean fruit fly, Ceratitis capitata. OILB Srop. 20(8): 38-45. Ekanayake, H.M.R.K and K.A.N.P Bandara. 2003. Protein baits to manage fruit flies of economic importance. Annals of the Sri Lanka Department of Agriculture 5: 85-92. Vijaysegaran, S. 1996. Fruit fly research and development in tropical Asia. In Management of Fruit Flies in the Pacific., ACIAR Proceedings No. 76. Eds. A.J.Allwood and R.A.I. Drew. Australion Center for International Agriculture Research, Canbera. Pp 21-29. Wharton, R.A. 1989. Classical biological control of fruit-infesting Tephritidae In Fruit flies: Their Biology, Natural Enemies and Control, Eds. A.S Robinson and G. Hooper. Elsevier Science Publishers, Amsterdam. Pp 303-313. Annals of Sri Lanka Department of Agriculture. 2006.8:19-28.

CONTRIBUTION OF MICRONUTRIENTS IN ORGANIC MANURES FOR GROWTH AND GRAIN YIELD OF RICE GROWN IN BATALAGODA SOIL SERIES OF LOW COUNTRY INTERMEDIATE ZONE

W.M.J. BANDARA, S.B.A.WEERAWARNA and V.R.B. UDAGALADENIYA MENIKE Rice Research and Development Institute, Batalagoda, Ibbagamuwa

ABSTRACT

In addition to N, P and K fertilizer applications, it has been found that the use of few micronutrients increased the growth and grain yield of rice grown in Low Humic Gley soils (Batalagoda soil series) of Low Country Intermediate Zone (LCIZ). Organic manures contain small amounts of micronutrients. Therefore, a field experiment was conducted for 2 seasons using 11 treatments viz. no fertilizer (T 1), recommended level of NPK fertilizer (T2), NPK+ all micronutrients (T3), NPK +Zn (T4), NPK+ rice straw (5 t/ha) (T5), NPK + cattle manure (5 t/ha) (T6), NPK + poultry manure (2 t/ha) (T7), NPK + green manure (10 t/ha Gliricidia sepium) (T8), NPK +(Zn +Cu) each at 2.5 kg/ha (T9), NPK + Mg +S (T10), NPK+Cu (T11) to determine the contribution of micronutrients from different organic manure sources to increase the grain yield of rice grown in LHG soils of LCIZ. Results showed that application of Zn significantly increased growth and grain yield of rice in this soil. Cattle and poultry manure at given rates can be used as supplementary sources of Zn for this soil, but rice straw and green manure (Gliricidia sepium) cannot supply the required quantity of Zn to rice.

KEYWORDS: LHG soil, Micronutrients, Organic manures, Rice yield.

INTRODUCTION

Annual extent of rice cultivation in Sri Lanka is 0.89 m ha (Abeysiriwardana and Sandanayaka, 2000). Ninety eight percent of this area is cultivated with new improved varieties, which require high levels fertilizer application to obtain high yields. Continuous cultivation for a long period and mismanagement of soils have resulted in depletion of fertility of rice soils (Sillanpaa, 1972). Rice soils display a great deal of heterogeneity with respect to total and available micronutrients. Increased cropping intensity, introduction of high yielding varieties and high analysis fertilizers at enhanced rate have resulted in micronutrient deficiencies in rice soils (Ponnamperuma, 1977). Ponnamperuma reported that submergence results in an increase in concentration of water soluble iron, manganese and molybdenum, decrease in concentration of copper and zinc and no appreciable changes in boron concentration. Much research has been done the world over on the availability and behavior of micronutrients in rice soils and on the alleviation of micronutrient deficiencies and toxicities, (Deb, 1992; Bandara and Silva, 2000a). The micronutrient status of LHG soils in the Low Country Intermediate and Dry zones has been examined by many researchers and found that certain micronutrients are limiting in this soil series (Deb, 1992; 20 BANDARA et al.

Bandara and Silva, 2000a). Bandara and Silva (2000b) reported that Zn and Cu availability in LHG soils (Batalagoda series) is low and application of these nutrients as inorganic fertilizers increases grain yield. However, only few rice farmers use green manure, rice straw, animal manure and other organic manure though the beneficial effects of these materials have been emphasized by researchers (Nagarajah and Amarasiri, 1977). FAO (1985) reported that the quantities of animal manure produced annually in Sri Lanka could replace approximately 50% of the plant nutrients consumed as chemical fertilizers. The use of animal manures for rice cultivation is scarce in Sri Lanka and is limited mostly to upland crops (Jayasundara, 2001). Very limited research information is available on the use of animal waste in rice cultivation. The role of animal manure in highland crop cultivation has been reported by Wijewardana (1993 and 1995). Animal manures can supply significant quantities of major plant nutrients (Maraikar and Amarasiri, 1988; Wijewardana and Yapa, 1999) and micro nutrients (Kemppainen, 1986). Generally, the rice growing LHG soils of the LCIZ are inherently low in fertility, while the demand for nutrients by the presently used improved varieties is high (Wickramasinghe and Wijewardana, 2000)

Application of rice straw, green manure and animal manure are recommended to increase rice yield. However, the contribution of micronutrients from these manures has not been studied in detail. The objective of this study was to determine the contribution of micronutrients supplied through organic manures for the growth and grain yield of rice grown in Batalagoda soil series (LHG soils) of LCIZ.

MATERIALS AND METHODS

A Field experiment was conducted in 2001/02 maha in the imperfectly drained Batalagoda soil series (LHG, Plinthaaqults) which are the lower members of the drainage catena in association with Red Yellow Podzolic soils (Panabokke, 1996) in the IL1 region of the LCIZ.

In this study, 11 treatments (Table 1) were tested in a randomized complete block design with 3 replicates to determine the contribution of micronutrients available from rice straw (RS), cattle manure (CM), poultry manure (PM), and green manure (GM) for the growth and grain yield of rice grown in LHG soils of the LCIZ. Iron was not included in the experiment as there was sufficient Fe in this soil. Nitrogen, phosphorus and potassium were applied at rates and times of application recommended by the Department of Agriculture. Rates of Mg, S and micronutrients added in each treatment are given in Table 1. Sources of P, K, S, Mg, B, Cu, Mn, Mo and Zn were triple super phosphate, muriate of potash, elemental sulfur, magnesium chloride, sodium tetra borate, copper sulphate, manganese dioxide, ammonium molybdate and zinc sulphate, respectively. Eighteen-day old rice ORGANIC MANURE FOR RICE CULTIVATION 21 seedlings (Bg352) were transplanted, 3 plants per hill at 15x15 cm spacing on ploughed leveled 6 m x3 m lowland plots surrounded by 30 cm wide bunds.

Table 1. Rates of Mg, S and micronutrients applied in different treatments (Bandara and Silva, 2000b).

Treatment Nutrient rates (kg/ha) Mg S B Cu Mn Mo Zn T1 No fertilizer 0 0 0 0 0 0 0 T2 NPK 0 0 0 0 0 0 0 T3 NPK+all MN 10 10 1.0 2.5 5.0 1.0 2.5 T4 NPK+Zn 0 0 0 0 0 0 2.5 T5 NPK+ RS (5t/ha) 0 0 0 0 0 0 0 T6 NPK+CM (5t/ha) 0 0 0 0 0 0 0 T7 NPK+PM (2.5t/ha) 0 0 0 0 0 0 0 T8 NPK+GM (10t/ha) 0 0 0 0 0 0 0 T9 NPK+Zn+Cu 0 0 0 2.5 0 0 2.5 T10 NPK+Mg+S 10 10 0 0 0 0 0 T11 NPK+Cu 0 0 0 2.5 0 0 0 N- 120 kg/ha P – 22 kg/ha K- 42 kg/ha MN – micronutrients, RS – rice straw, CM – cattle manure, PM- poultry manure, GM- green manure (Gliricidia sepium)

Phosphorus, K, S, Mg and all micronutrients were broadcast and incorporated into soil by puddliing just before transplanting. Nitrogen as urea was split applied according to the Departmental of Agriculture. Manual weed control and chemical methods of pest control were adopted.

Yield components (number of panicles per meter square, number of filled grains per panicle, thousand grain weight) and yield data were recorded.

Soils were analyzed to determine fertility status of the experimental field. Soils were extracted with relevant extracting solutions and Cu, Fe, Mn and Zn concentrations were determined by atomic absorption spectrophotometry and Boron by colorimetry (PCARR, 1990).

Plant samples taken at maximum tillering stage were oven dried at 85°C for 8 h and ground to 1 mm size in a stainless steel mill. A sample of 1 gm was digested with diacid mixture (HNO3:HCl, 3:1) and Cu, Zn, Fe and Mn were determined by atomic absorption spectrophotometry. Boron was determined by colorimetry. The experiment was repeated with the same treatments tested in the same plots. Nutrient contents of each organic manure used were determined using standard procedures (PCARR, 1990). 22 BANDARA et al.

RESULTS AND DISCUSSION

Soil properties

The soil at the experimental site was sandy clay loam in texture, with near neutral pH, low organic matter, extractable P and exchangeable K contents. Cation exchange capacity was also low. Iron content was high while S and Mn were in the sufficiency range and Cu and B marginal. The soil was deficient in Zn (Table 2).

Nutrient contents in organic manures

Nutrient contents in organic manures are given in Table 3. Except K and Fe, other nutrients are low in RS when compared to CM, PM and gliricidia, a green manure (GM). PM has high level of all essential nutrients including B, Cu, Mn, Zn and Fe. CM also has high levels of micronutrients while GM has comparatively low levels. But green manure has the highest level of N compared to the other 3 sources. Therefore, CM and PM are good sources of micronutrients and GM has the 3rd place in the nutrient supply out of all tested materials. But, GM is the best source of N while RS is the best source of K.

Table 2. Chemical properties of Batalagoda soil series (LHG soils) of the experimental site at Batalagoda and critical levels for rice crop growth.

Property Analytical value Critical level* pH (1:5 soil: water) 5.9 6.1-7.2 Total N (%) 0.085 0.25 Organic matter (%) 1.2 3.0 Olsen P (mg/kg) 8.9 10 CEC *(c mol +/kg ) 6.2 15-20

Ca**(c mol +/kg) (1N NH4OAC at pH 7) 3.2 >1 Mg**(c mol +/kg) (1N NH4OAC at pH 7) 1.01 >1 K**(c mol +/kg )(1N NH4OAC at pH 7) 0.16 0.2 S+ mg/kg 12 10 B++ mg/kg Hot water extraction 1.1 0.8 Cu++++ mg/kg (AAAC-EDTA) 1.1 1.0 Fe+++ mg/kg (AAAC- at pH 4.8) 265 5 Mn++++ mg/kg (AAAC-EDTA) 24 10 Zn++++ mg/kg (AAAC-EDTA) 0.82 1.0 * Source: S.Yoshida, 1981 Fundamentals of rice crop science. International Rice Research Institute, Manila, Philippines **- 1N NH4OAc (pH= 7) extraction + - 0.01 M Ca(H2PO4)2 extraction ++ - Hot water extraction +++ - Acid Ammonium Acetate (AAAC) pH 4.8 extraction ++++ - AAAC- EDTA (Ethylene Diamine Tetra Acetic Acid) extraction ORGANIC MANURE FOR RICE CULTIVATION 23

Table 3. Nutrient content in different organic manures used in the experiment.

Organi N P K Mg S B Cu Mn Fe Zn c (% ) (%) (%) (%) (%) mg/k mg/k mg/k mg/k mg/k manure g g g g g RS 0.59 0.10 1.50 0.20 0.08 04 06 35 75 05

CM 1.4 0.77 0.87 0.6 0.5 08 31 225 28 204

PM 2.98 1.26 1.57 0.55 0.53 52 82 345 55 242

GM 4.8 0.18 1.8 0.25 0.12 06 08 23 18 22

Nutrient contents in soil at tillering stage of the crop

At tillering stage of the crop, soil N contents in all treatment plots except the no fertilizer plot were in the sufficient range (Table 4). Plots treated with CM, PM and GM had the highest levels of N. Where P content is concerned highest P was in plots treated with the 3 types of organic manures. The RS treated plot also had a sufficient amount of P. Rice straw and PM treated plots had the highest soil K content while Mg treated plot had the highest exchangeable magnesium content. Other plots had marginal Mg contents. Likewise S treated plot had the highest S level although all plots had sufficient levels of S at this growth stage.

Where micronutrients are concerned, B in soil of all plots at this stage were sufficient for crop growth and application of either B fertilizer or organic manures increased B levels in soil. Similar trend was observed for Cu. The Fe and Mn contents were also in sufficient range. However, in comparison to the no fertilizer plot, Fe contents in all other plots were low. This may be due to the interaction effect of Fe with other elements added to soil. Similar interaction effects have been observed for Zn and N (Bandara et al., 2003) and Zn and P (Bandara et al., 2004) in the LHG soils in the LCIZ. Except in the Zn fertilizer, CM and PM treatments, the soil Zn contents in all other treatments were below the critical level (Table 4). Rice straw and GM recorded lower Zn contents. 24 BANDARA et al.

Table 4. Nutrient contents in soil at 6 weeks after planting.

Treatment Nutrient content Nutrient content (mg/kg) N P K Mg S B Cu Mn Fe Zn (%) mg/ (c mol (c mol mg/ kg /kg) /kg) kg No fert 0.09 8.8 0.12 0.88 10 1.0 1.0 12 189 0.6 NPK 0.23 17 0.21 0.86 12 1.0 0.9 13 154 0.7 NPK+all other 0.22 18 0.22 0.81 11 1.3 1.4 18 123 1.5 nutrients NPK+ Zn 0.21 16 0.21 0.82 12 1.1 1.2 15 126 1.6 NPK+Straw 0.20 20 0.25 0.84 15 1.1 1.2 16 95 0.8 NPK +CM 0.24 23 0.23 0.91 16 1.6 1.3 14 102 1.2 NPK+PM 0.25 24 0.24 0.93 18 1.9 1.4 16 92 1.4 NPK+GM 0.26 22 0.20 0.88 15 1.3 1.2 13 122 0.97 NPK+Zn+Cu 0.22 13 0.2 0.81 12 1.1 1.5 12 98 1.4 NPK+Mg+S 0.20 12 0.19 1.02 30 1.2 1.1 12 96 0.82 NPK+Cu 0.21 11 0.22 0.77 13 1.2 1.2 13 141 0.65 Critical level* 0.25 10 0.2 >1 10 0.8 1.0 12 30- 1.0 300 * Source - S.Yoshida, 1981 Fundamentals of rice crop science. International Rice Research Institute, Manila, Philippines

Nutrient content in shoots of rice plants grown at tillering stage of the crop

Except the no fertilizer plot, plant tissues from all other treatments have sufficient N and application of GM gave the highest N level in plant tissue (Table 5). Similar results were observed for tissue P and K contents. Where tissue Mg and S contents are concerned, highest Mg and S contents were in plots treated with inorganic Mg and S fertilizers. However, tissues from other treatments also had sufficient levels of Mg and S. Soil available Cu and B were sufficient to supply more than the critical levels. However, application of inorganic B and Cu, CM or PM increased the tissue B and Cu contents (Table 5). Tissue Mn and Fe contents in all treatments except the no fertilizer treatment recorded sufficient levels. Rice straw which provides high amounts of K reduced the Fe level in tissues significantly. This interaction was earlier reported by Bandara and Gunatilaka (1994). Table 5 shows that inorganic fertilizer or organic manures significantly affected the tissue Zn content. Application of either inorganic Zn fertilizer, CM or PM increased tissue Zn above the critical level (Table 5). Therefore, it can be concluded that CM and PM can be used as supplementary sources of Zn for rice cultivation. ORGANIC MANURE FOR RICE CULTIVATION 25

Table 5. Nutrient content in shoots of Bg352 at 6 weeks after planting.

Treatment % mg/kg N P K Mg S B Cu Mn Fe Zn No fert 1.5 0.10 1.4 0.11 0.12 6 5 18 135 8 NPK 2.2 0.24 1.9 0.16 0.17 8 6 22 101 11 NPK+all other 2.9 0.30 2.3 0.25 0.23 12 12 39 118 24 nutrients NPK+ Zn 2.8 0.28 2.3 0.22 0.21 9 6 42 122 25 NPK+Straw 2.5 0.26 2.5 0.21 0.22 7 7 28 95 9 NPK +CM 2.9 0.28 2.6 0.23 0.24 12 10 33 102 16 NPK+PM 2.9 0.30 2.5 0.24 0.22 13 11 31 113 18 NPK+GM 3.0 0.27 2.4 0.22 0.23 9 8 28 133 14 NPK+Zn+Cu 2.7 0.28 2.4 0.21 0.22 8 11 28 120 20 NPK+Mg+S 2.6 0.22 1.9 0.24 0.22 9 9 29 101 10 NPK+Cu 2.6 0.22 2.0 0.20 0.18 7 12 29 112 9 Critical level* 2.5 0.10 1.5 0.12 0.15 5 5 20 70 15 * Source - S.Yoshida, 1981 Fundamentals of rice crop science. International Rice Research Institute, Manila, Philippines

Yield components

Addition of all nutrients (T3) gave the highest number of panicles/m2 (NPM), number of filled grains per panicles (NFGP) and thousand grain weight (TGW) (Table 6). Addition of the 2.5 kg Zn/ha gave similar values, which were not significantly different to that obtained in T3. This indicates that the main limiting nutrient in this soil is Zn (Bandara and Silva, 2001). Yield component values obtained in these two treatments were significantly higher than that in the NPK treatment (T2) confirming that the soil is Zn deficient. Addition of Cu, Mg and S has no significant effect on yield components (Table 6). However, addition of Cu significantly increased TGW. Rice straw (T5) significantly increased NFGP and TGW compared to NPK fertilizer treatment. This may be due to the high K release from RS which affects grain filling. Addition of CM, PM and GM increased the NPM of the crop indicating that all 3 organic manures can supply micronutrients especially Zn in adequate amounts (Table 6). Only CM (T6) or PM (T7) can increase the NFGP and TGW to the same extents as treatments T3 and T4. Therefore, CM and PM are the best sources of micronutrients. Rice straw and green manures could not supply micronutrients in required levels.

Grain yield

The no fertilizer treatment (T1) gave the lowest grain yield compared to other treatments. Application of NPK increased grain yield of rice during both seasons. 26 BANDARA et al.

Table 6. Effect of different treatments on yield components.

Treatment Yield components (Mean of two season) Number of Number of 1000 grain panicles/m2 filled grain/ weight (g) panicle

T1, No fert 266c 78c 23.2c T2, NPK 282c 105b 24.5b

T3, NPK + all MN 370a 129a 25.5a

T4, NPK + Zn (2.5 kg) 352a 127a 25.3a

T5, NPK+RS (5t) 330b 120ab 25.1a

T6, NPK+CM (5t) 361a 121ab 25.4a

T7, NPK+PM (2t) 365a 130a 25.5a

T8, NPK+GM (10t) 358a 116b 24.8b

T9, NPK+Cu+ Zn (2.5 kg each) 349a 128a 25.4a

T10, NPK+Mg+S 320b 114b 24.6b

T11, NPK+ Cu 332b 110b 25.0a CV % 4.6 6.7 5.8 Treatment * * * Treatment x Season ns ns ns * - Significant at 5% probability level Means followed by the same letter in each column are not significantly different at 5% probability level by DMRT

Table 7. Effect of different treatments on grain yield.

Treatment Grain yield t/ha 2001/02maha 2002yala T1 No Fert 3.29c 3.87c T2 NPK 4.83b 5.48b T3 NPK+ all MN 5.44a 6.79a T4 NPK + Zn (2.5 kg) 5.42a 6.51a T5 NPK+Straw (5t) 5.13ab 6.08b T6 NPK+CM (5t) 5.41a 6.39a T7 NPK+PM (2t) 5.53a 6.63a T8 NPK+GM (10t) 5.28ab 6.03b T9 NPK+Cu+ Zn (2.5 kg each) 5.40a 6.62a T10 NPK+Mg+S 5.02b 5.78b T11 NPK+ Cu 5.12b 5.82b CV % 6.3 7.2 Treament * * * significant at 5% probability level Means followed by the same letter in each column are not significantly different at 5% probability level by DMRT

Application of all nutrients (T3) to the crop significantly increased grain yield compared to T2 indicating that micronutrient application is essential to increase grain yield in this soil (Table 7). However, yields in treatments, T3 and T4 were similar, indicating most limiting micronutrients in ORGANIC MANURE FOR RICE CULTIVATION 27

these soils is Zn. Application of Mg, S and Cu (T10 and T11 treatments) increased grain yield but the increase was not significantly different from T2. Application of organic manures increased the grain yield during both seasons. Both CM and PM gave significant yield increases (Table 7) while RS and GM did not increase yields significantly.

CONCLUSIONS

The Batalagoda soil series (LHG soil) of the LCIZ have sufficient levels of Mn and Fe. Boron and Cu contents of these soils are marginal, while zinc level is deficient. Application of all micronutrients increased soil and plant nutrient contents to sufficient levels and increased yield components and grain yield. Significant yield increases were observed by Zn application. Application of all organic manures increased soil fertility levels and plant nutrient contents. They also increased yield components and grain yield. But, significant increases were obtained by CM and PM application showing the ability of CM and PM to supply Zn in required quantities for rice grown in this soil. In contrast, RS and GM could not supply the required amounts of Zn. Further studies should be carried out to confirm these results.

ACKNOWLEDGEMENTS

The authors thank staff of the Soil Science Division and laborers of the Rice Research and Development Institute, Batalagoda, Ibbagamuwa for their help in conducting laboratory analysis and field experiments.

REFERENCES

Abeysiriwardana, D.S.D.Z. and C. A. Sandanayake. 2000. Future rice research as directed by trends in cultivated extent and yields of rice during the recent past. Proceedings of the Annual Symposium of the Department of Agriculture Pp 372-380. Bandara, W.M.J. and G.A Gunatilaka. 1994. Effect of applied K and P on bronzing in rice grown in iron toxic mineral soils. J. Nat. Sci. Cou. Sri Lanka 22(3): 219- 230. Bandara,W.M.J. and L.C. Silva. 2000a. An assessment of micronutrient requirement for rice grown in LHG soils of low country dry zone. Proceedings of the Annual Symposium of the Department of Agriculture Pp. 35-46. Bandara,W.M.J. and L.C. Silva. 2000b. Effects of applied micronutrients on growth and grain yields of rice grown in LHG soils of low country intermediate zone. J. Soil Sci. Soc. of Sri Lanka 12: 40-50. Bandara,W.M.J. and L.C. Silva. 2001. Rice crop response to zinc application in LHG soils of low country intermediate zone. Annals of the Sri Lanka Department of Agriculture 3:9-18. 28 BANDARA et al.

Bandara, W.M.J., W.M.A.D.B. Wickramasinghe, D.N. Sirisena and L.C. Silva. 2003. Effect of applied Zn on N use efficiency, Growth and grain yield of rice grown in LHG soils of LCIZ. Annals of the Sri Lanka Department of Agriculture 5: 23-32. Bandara, W.M.J., D.B. Wickramasinghe and L.C. Silva. 2004. Effects of applied P on Zn availability, growth and grain yields of rice grown in LHG soils of low country intermediate zone. Annals of the Sri Lanka Department of Agriculture 6:39-48. Deb, D.L. 1992. Development of soil and plant analytical methods for micronutrient and sulfur in Sri Lanka. GCPF/SRL/047/NET Field Document No.11. FAO. 1985. Food and Agricultural Organization, Soil Bulletin 40:12-16. Jayasundara, J.M.P.B. 2001. Effect of integrated application of organic and chemical fertilizers on dry seeded rice (Oryza sativa L.) Annals of the Sri Lanka Department of Agriculture 2001 3:61-70. Kemppainen, E. 1986. Utilization of animal manure. In report on the FAO seminar and training cource on micronutrients and macronutrients in soils and agriculture. Jokioinen, Finland, June 2-14 :114-120. Mariakar, S. and S.L. Amarasiri. 1988. Plant nutrient contents in animal waste. Tropical Agriculturist 144: 79-87. Nagarajah, S. and S.L. Amarasiri. 1977. Use of organic materials as fertilizers for lowland rice in Sri Lanka. Soil organic matter studies, IAEA 1:97-104. Panabokke , C.R. 1996. Soils and Agro-Ecological Enviroment of Sri Lanka Natural Resources series No.2. Natural Resources Energy and Science Authority, Colombo, Sri Lanka. 39-40p. PCARR (Philippines Council For Agriculture & Resources Research) 1990. Standard methods of analysis for soil, plant tissue , water and fertilizer). Farm Resources and Systems Research Division, Philippines Council for Agriculture and Resources Research. Losbanos, Laguna, Philippines. Ponnamperuma, F.N. 1977. Behavior of minor elements in paddy soils. Research Paper Series 8, International Rice Research Institute. p10. Sillanpaa, M.1972. Trace elements in soils and agriculture. In FAO soils Bulletin, No.17. 1972. Food and Agricultural Organization of the United Nations, Rome and Swedish International Development Authority (SIDA). Wickramasinghe, W.M.A.D.B and J.D.H. Wijewardena. 2000. Soil and nutrient management for increasing soil fertility towards increased rice productivity in Sri Lanka. Proceedings of the Annual Symposium of the Department of Agriculture, Pp. 465-482. Wijewardana, J.D.H. 1993. Effect of organic and chemical fertilizers on vegetable cultivation in up country Intermediate zone. Tropical Agriculturist 149:1-11. Wijewardana, J.D.H. 1995. Effect of animal manure and chemical fertilizers on the growth and yield of tomato. Krushi 15(1): 7-10. Wijewardana, J.D.H. and U.W.S.P. Yapa. 1999. Effects of combined use of animal manures and chemical fertilizers on potato and vegetable cultivation in upcountry of Sri Lanka. Sri Lankan Journal of Agricultural Science 136: 68-82. Annals of Sri Lanka Department of Agriculture. 2006.8:29-34.

EFFECT OF ZINC AND BORON ON GROWTH, YIELD AND FRUIT QUALITY OF PAPAYA (Carica papaya) GROWN IN NON-CALCIC BROWN SOILS

A.G. CHANDRAPALA, S.H.S.A. DE SILVA, H.A.P. JAYALATH and K.L.D. KULARATHNA Regional Agricultural Research and Development Centre, Aralaganwila

ABSTRACT

Micronutrients are generally not included when applying chemical fertilizers. Increased yields through intensive cropping and use of high yielding varieties have contributed towards accelerated depletion of micronutrients in soils. An experiment was conducted at Aralaganwila Research Station in 2004 in non-calcic brown soils to quantify the effects of zinc and boron on yield and bumpy fruit formation in papaya, a common problem in papaya cultivation in Sri Lanka. Zinc and boron applied separately at three levels (5, 10, 15 kg/ha) and together (10 kg/ha), were tested on growth, yield and fruit quality of papaya (var. Red lady). The highest crop growth and yield without bumpy fruit formation was observed in the zinc + boron treatment. High amount of bumpy fruits with low sugar content was observed in plants without boron treatment. However, significant growth and yield differences were not observed within different zinc and boron levels. Although significant differences on fruit quality were not observed under zinc deficient condition, zinc and boron are necessary to get high yields without the formation of bumpy fruits.

KEYWORDS: Boron, Bumpy fruits, Papaya, Zinc.

INTRODUCTION

Market demand for fruits has increased in the recent past due to increased income of farmers, urbanization, export demand, health and nutritional awareness etc. The export potential of fruits is very high and export market maintains a high standard in the quality of fruits; special attention is needed to ensure continuous supply of quality fruits. Efficient nutrient management is essential for obtaining optimum productivity of fruit crops. Unfortunately, nutrient management is the most neglected aspect of fruit crop production in Sri Lanka. Therefore, yields of the fruit crops are neither their optimum levels nor consistent in both quality and quantity. Even the improved varieties provide low quality fruits due to insufficient nutrient management. Nutrient imbalance in soils causes low fertilizer use efficiency, low yields and low farmer profit. It also results in further depletion of the most deficient nutrients in soil. Once a nutrient reaches the critical level, yields decline even when large amounts of other nutrients are applied (Anon., 2000). Hence the importance of balanced fertilization must be realized in increasing crop yields. At present, only N, P and K fertilizers are applied to fruit crops. Hence, plants depend on soil reserves for a number of micronutrients. However, soil cannot be considered as a source of continuous 30 CHANDRAPALA et al. supply of plant nutrients forever. Furthermore, increased yields through intensive cropping and use of high yielding varieties, losses of micronutrients through leaching, application of decreasing proportion of farmyard manure compared with commercial fertilizers are the primary factors that contribute towards accelerated exhaustion of the supply of available micronutrients to soil.

It is apparent that micronutrient deficiencies are far more widespread than estimated. Micronutrient deficiencies which are only local, may become more serious in the future, occurring extensively over new areas and creating widespread and complicated production restrictions if they are not properly diagnosed and remediated in time and taken precautions to eliminate them.

In the recent past appearance of bumpy or lumpy fruit formation was commonly observed in many papaya cultivations, causing high economic loss to farmers. Bumpy fruit formation in papaya has been reported by several researchers (Munzos et al., 1966; Wang and Ko, 1975; Atkinson, 1991) and most workers have demonstrated that this disorder is associated with B deficiency occurring in many papaya growing countries such as Australia, Malaysia, Taiwan and Hawaii. The problem has been overcome by application of B. It has also been reported that B availability is dependant on soil factors, climatic conditions and cultural practices.

Zinc also functions as the metal component of a series of enzymes in many fruit crops. The most important enzymes activated by this element are carbonic anhydrase and number of dehydrogenases. Zinc deficiency is thought to restrict RNA synthesis, which in turn inhibits protein synthesis. Zinc deficient plants are thus poor in growth (Tisdale et al., 1985). Zinc is also involved in auxin production. Shoots and buds of Zn deficient plants contain very low auxin contents. This cause dwarfism and growth reduction. The net results are stunted plants and prolonged duration of growth. Zinc also plays a major role in yield and quality of fruits, especially flower initiation, fruit setting and fruit remaining. Zinc deficiency may depress plant yields by as much as 50 percent without producing any symptoms in fruit crops (Katyal and Randhawa, 1983). Therefore, the objective of this study was to quantify the effects of different levels of Zn and B on growth, yield and fruit quality of papaya in Non-Calcic Brown soils.

MATERIALS AND METHODS

The study was conducted from 2004 yala to end of 2005 yala season at the Regional Agriculture Research and Development Centre, Aralaganwila in the Low Country Dry Zone (DL2b) in Non-Calcic Brown soils which is the major soil group of the Mahaweli system B area. The study area EFFECTS OF ZINC AND BORON ON PAPAYA 31 has a distinctly uni-modal rainfall pattern with an average annual rainfall of 1100mm (Panabokke, 1996).

Papaya (variety: Red lady, F-1 hybrid) one month old plants were planted at 3 x 3 m spacing on raised beds in April 2004. The experimental design was a Randomized Complete Block with four replicates and three plants per treatment within a replicate. One meter deep drains were constructed around the field to facilitate the draining of excess water. Insect pest control, weed management, fertilizer (except micronutrient) application and other crop management practices were done according to the recommendations of the Department of Agriculture (Anon., 1991). Zinc and B were applied in four equal doses at three months interval. Ammonium sulphate was used to supply part of the nitrogen requirement in each treatment to balance the S applied as zinc sulphate. Zinc and B were applied as borax and zinc sulphate respectively, tested separately at three rates (5, 10, 15 kg/ha) and in combination (10 kg/ha).

Plant growth parameters were recorded as number of leaves per plant, stem diameter, crown diameter and plant height at 50% flowering stage of the crop. Number of fruits per plant, fruit weight and fruit quality characteristics in each plant such as sugar content (Refractometer method), number of bumpy and normal fruits, fruit length and diameter were measured in randomly selected fruits in each and every plant.

RESULTS AND DISCUSSION

Table 1. Effects of B and Zn application on plant growth.

Treatment Plant height No. of Crown Basal (cm) leaves diameter diameter (cm) difference (cm) NPK 35.96c 3.41b 28.3b 2.13e NPK + B-5 79.39b 8.45a 129.6a 3.19d NPK + B-10 81.12b 8.70a 130.3a 3.27d NPK + B-15 86.40ab 9.41a 139.3a 3.38cd NPK + Zn-5 88.32ab 9.50a 139.9a 3.57bc NPK + Zn-10 91.57ab 9.79a 147.0a 3.61bc NPK + Zn-15 101.70ab 10.58a 149.2a 3.62b NPK + B-10 + Zn-10 117.20a 10.63a 150.0a 4.36a

CV % 26.62 24.11 13.6 4.46 Means with same letters are not significantly different at 5% probability level

The mean plant growth parameters for different treatments are shown in Table 1. The highest plant growth was observed in the NPK + B - 10 + Zn - 10 followed by NPK + Zn - 15 treatments. The lowest plant growth 32 CHANDRAPALA et al. was recorded in NPK only (control) treatment (Table 1). Poor crop growth observed in the no Zn and no B treatments may be due to the retardation of growth functions, which may require sufficient supply of these nutrients. Cook (1964) reported the importance of boron in the development, growth and formation of new cells in the plant meristem.

Boron is also required for the development and differentiation of tissues. In its absence, abnormal formation and development of tissues were reported (Katyal and Randhawa, 1983). Tisdale et al. (1985) also reported the importance of Zn in the synthesis of tryptophane, a component of some protein and a compound needed for the production of growth hormone like indole acetic acid. Reduced growth hormone production in Zn deficient plants causes the shortening of internodes and small leaves (Tisdale et al., 1985).

Table 2. Effects of Zn and B application on fruit quality and yield.

Treatment Fruits / plant Fruit Sugar content Fruit yield (Brix value) length (cm) (kg/plant) NPK 5.41d 7.39c 5.6d 19.51d NPK + B-5 11.60c 18.18b 7.8c 21.40c NPK + B-10 16.30bc 18.70b 8.0c 21.74c NPK + B-15 16.40bc 18.74b 8.1c 22.76b NPK + Zn-5 18.00b 19.73b 8.1c 23.41b NPK + Zn-10 18.40b 19.94b 9.8b 25.37a NPK + Zn-15 20.23b 20.00b 10.3b 25.41a NPK + B-10 + Zn-10 29.50a 37.98a 13.3a 25.81a

CV % 14.13 14.32 6.1 2.70 Means with same letters are not significantly different at 5% probability level

The highest fruit yield of 37.98 kg/ha was obtained in the B + Zn -10 treatment and this was significantly higher than the yields obtained when B and Zn were applied separately. There was no significant increase in fruit yields with increasing rates of either B or Zn application. Zinc and B levels already contained in the soil also may be a reason for the lack of sensitivity of plants to the higher doses of Zn and B.

The results of the present experiment also suggest the importance of Zn for the yield and fruit quality of papaya. Yield parameters (fruits per plant and fruit weight) were higher under no Zn treatments than no B treatments. But, the differences were not significant (Table 2). The requirement of B for the proper pollination and fruit set may be the reason for the low yield under B deficient condition (Romheld and Marscher, 1991). Katyal and Randhawa (1983) also reported that the sterility and malformation of reproductive organs under no B conditions cause low fruit yield. EFFECTS OF ZINC AND BORON ON PAPAYA 33

The lowest crop growth and yield were observed in the control treatment where both Zn and B were not applied. Low Zn and B availability and high nutrient leaching in the light textured soils of the experimental site may be the reason for the lowest yield under control treatment. Tisdale et al. (1985) also reported lower availability of Zn and B in light textured soil.

Zinc and B availability is also highly pH dependent. It is higher in acid soils, whereas in alkaline soils the availability is very low. Severe Zn and B deficiencies are often noticed in high pH soils (Romheld, 1991).

The inability of increasing fruit yield may be the true reflection of Liebigs law of minimum factors. In many cases the significant increase of growth and yield parameters were observed only in the presence of both Zn and B. The lowest sugar content of fruits (brix value) was also observed in the B deficient condition (Table 2). Lack of translocation of sugars into the fruits from the leaves may be the reason for lower sugar content under the B deficient condition (Tisdale et al., 1985). Katyal and Randwa (1983) also observed that if B deficiency occurs, there is assimilation of product accumulation in the leaves causing the lack of sugar content in fruits.

Table 3. Effects of Zn and B application on bumpy fruit formation.

Treatment % Bumpy % Normal fruits fruits NPK 40 60 NPK + B-5 0 100 NPK + B-10 0 100 NPK + B-15 0 100 NPK + Zn-5 33 67 NPK + Zn-10 25 75 NPK + Zn-15 31 69 NPK + B-10 + Zn-10 0 100

The effects of Zn and B application on bumpy fruit formation are shown in Table 3. Formation of bumpy fruits was observed only under the B deficient condition (Table 3). Different levels of Zn application and treatment without Zn and B (control) showed the different levels of bumpy fruit formation. Thanthirige and Wijesundara (1999) also reported that the addition of 15 and 20g of borax per plant had significantly reduced the percentage of bumpy fruit formation under low country Wet Zone conditions. They also observed the reduction of bumpy fruit formation under application of B in the 20 g/plant than in the 10 g/plant. This finding contradicts the finding of the present experiment, where clear relationship with the different levels of B was not observed possibly due to the climatic differences between the two experimental sites. Clear relationship between the bumpiness and the rate of Zn application was also not observed possibly due to the insensitivity 34 CHANDRAPALA et al. of papaya bumpy fruit formation to the Zn fertilization. Percentage of bumpy fruit formation under B deficient condition varies from 80.2 to 89.0 in low country Wet Zone (Thanthirige and Wijesundara, 1999). However according to the findings of present experiment, formation of bumpy fruits was only 40% even at B deficient condition (Table 3). The high percent of bumpy fruit formation under wet zone than dry zone may be due to the difference in climatic conditions prevailing in two regions. High amount of rainfall and poor drainage induce the B deficiency condition (Katyal and Randhawa, 1983). Although the precise role of B in plants is unknown, it is involved in regulation of carbohydrate metabolism, synthesis of amino acids and proteins, translocation of sugars, proper pollination and new cell development in meristematic tissues (Tisdale et al., 1985).

CONCLUSIONS

The present study reveled that the lowest rate of Zn and B (5 kg/ha) is sufficient to increase the crop growth, yield and quality of Papaya to a satisfactory level. Although Zn had no effect on fruit quality, it had a significant effect on growth and yield of papaya. However, only the Zn and B combination increased the growth, yield and fruit quality of papaya.

REFERENCES

Anon., 1991. Crop recommendation technoguide. Department of Agriculture, Sri Lanka. Anon., 2000. Balanced fertilizer use for sustainable agriculture in India. PPIC program, Gurgon - 122016, Haryana. Pp 4-6 Atkinson, I. 1991. Papaw growing. Agfat, H6.1.19 NSW. Agriculture and Forestry. Australia. Cook, R.L. 1964. Hunger signs in crops. 3rd ed., David Mckay Co., New York. Pp 123-126. Katyal, J.C. and N.S. Randhawa (1983). Micronutrients. FAO fertilizer and plant nutrition bulletin 7: 11-16 Munzos, S.M., G.F. Kocher and P.A Villalobos. 1966. Cultivation of papaya. Agric. Tech., Satigo 26: 106-113. Panabokke, C. R. 1996. The great soil groups in Sri Lanka, their environmental setting, main characteristics and taxonomic placement. In Soils and Agro-ecological Environment in Sri Lanka, Natural Resources, Energy and Science Authority, Colombo, Sri Lanka. 30-40. Romheld, V. and H. Marscher. 1991. Functions in micronutrients in plants. In Micronutrients in Agriculture. No 4. Soil Science Society of America, Madison, Wisconsin. Thanthirige, M.K. and C. Wijesundara (1999). Bumpy fruit formation in papaya (Carica papaya L.) Proceeding of the Annual Symposium of the Department of Agriculture. Pp137-145. Tisdale, S.L., W.L. Nelson, J.D. Deaton and J.L. Havlin (1985). Soil fertility and fertilizers. 5th edition, Macmillan Publishing Company, New York. Pp 71-72. Wang, D.N. and W.H. Ko. 1975. Relationship between deformed fruit diseases of papaya and boron deficiency. Phytopathology 65: 445-447. Annals of Sri Lanka Department of Agriculture. 2006.8:35-48.

PEST RISK ANALYSIS: RISK OF Spongospora subterranea f.sp. subterranea J.A. Toml. PATHOGEN CAUSING POWDERY SCAB OF POTATO TO SRI LANKA

R.S.Y. DE SILVA, K.R.K. KARUNACHANDRA and W.D.K.N. FERNANDO National Plant Quarantine Service, Katunayake

ABSTRACT

Recent detection of symptoms similar to powdery scab of potato (PSP) caused by Spongospora subterranea f.sp. subterranea J.A. Toml. prompted the plant quarantine to take emergency phytosanitary measures. According to the Sanitary and Phytosanitary agreement under World Trade Organization (WTO/SPS Agreement), the implementation of new phytosanitary measures requires justification by way of risk analysis. Hence a pest risk analysis (PRA) was done for powdery scab of potato because of the necessity to review country’s policy on import of seed potato tubers. Even though several pathways could help introduction of the pest, the most troublesome pathways are seed tubers and rooted plantlets of potato. There is little chance for it to associate with true seeds and in vitro cultures of potato. Climate, topography, soil conditions, cultural practices, human and animal movement, and susceptibility of popularly grown potato varieties are conducive for the establishment, survival and spread of the pathogen in the endangered area i.e. Nuwara Eliya and Badulla districts. Production cost will be increased and marketability of potato will be severely affected which in turn reduce the income of the farmers. Thus, Spongospora subterranea f.sp. subterranea, pathogen causing PSP deserves to be treated as a quarantine pest. During the import of potato seed tubers and rooted plantlets, the appropriate level of protection (ALOP) against this pest for Sri Lanka is considered to be the importation of seed potato from “pest free place of production”, regular monitoring during growing season, phytosanitary certification, specific testing for PSP and inspection on arrival.

KEYWORDS: Pest risk analysis, Potato, Powdery scab, Quarantine pest, Spongospora subterranea f.sp. subterranea.

INTRODUCTION

Keeping vigilance on new pests appearing in the country is important in determining the necessary phytosanitary safeguards for their management (IPPC, 2003). Appearance of certain disease symptoms in experimental plots of the Seed Certification Service in Sita Eliya where imported potato seed tubers had been planted were diagnosed as powdery scab of potato (PSP). No detailed reports of the occurrence of this pest in Sri Lanka were so far published (CMI, 1987 and CABI, 2005). Causal agent of PSP is Spongospora subterranea f.sp. subterranea J.A. Toml. (Kunkel, 1915; Melhus et al., 1916; Harrison et al., 1997; Tsror (Lahkim) et al., 1999). Heeding to the various comments made by the plant pathologists, the phytosanitary requirements for the importation of seed tubers were updated listing this fungus as a quarantine pest. Area freedom of the pest was imposed as the condition to prevent its introduction. No other management options were considered because urgency warranted the imposition of this new 36 DE SILVA et al. phytosanitary action. Alleging this action as trade restrictive, the suppliers of seed potato requested clarifications from the Director General of Agriculture of Sri Lanka.

Imposition of new phytosanitary requirements during import of plant material must be based on scientific justification (Anon., 1998). This pest risk analysis (PRA) was done to show scientific and other technical justification manifesting transparency as required by our international obligations under the Agreement on Sanitary and Phytosanitary Measures of World Trade Organization (WTO/SPS). Another objective of reporting this PRA is to allow local agricultural scientists and other stakeholders to closely examine it and make comments before opening it for international community. To our knowledge, this is the second report on risk analysis done in Sri Lanka, the first being that of De Silva et al. (2005) on carnation nursery stocks.

METHODOLOGY

WTO/SPS agreement signed by the countries including Sri Lanka stipulate that any PRA for a plant pest should be done according to the international standards adopted by the International Plant Protection Convention (IPPC). Two International Standards on Phytosanitary Measures (ISPMs) No. 2 (IPPC, 1996a) and No. 11 (IPPC, 2003) describe the procedure for doing a PRA. We adopted the procedure given in these two ISPMs for this PRA.

PRA of powdery scab of potato (PSP)

Stage 1: Initiation

PRA initiated by the review or revision of a policy

The change in policy on PSP during import of seed potato tubers resulted in a dispute between Sri Lanka and the supplying countries. They requested scientific justification for the phytosanitary action taken. Subsequently, the Director General of Agriculture who is the authority responsible for the implementation of the Plant Protection Act of Sri Lanka directed the National Plant Quarantine Service to do this PRA. The authors were not aware of any PRA done earlier in Sri Lanka for PSP or its causal agent.

PRA area

The annual extent under potato cultivation in Sri Lanka is around 6000 to 7000 ha (Kelaniyangoda et al., 2004) and is concentrated in Nuwara Eliya (Up country wet zone) and Badulla districts (Up country PEST RISK ANALYSIS OF POWDERY SCAB OF POTATO 37 intermediate zone) and in Jaffna and Kalpitiya areas (Low country Dry zone). Hence, these areas will be considered as the PRA areas.

Information

Several sources were consulted for doing this PRA. These included, but not limited to, Crop Protection Compendium (CABI, 2005), the Internet, and several journal articles available in libraries within Sri Lanka.

Conclusion of initiation

The pest is Spongospora subterranea f.sp. subterranea J.A. Toml., a fungus causing PSP. Review or revision of the policy on import of seed potato tubers warranted this PRA. There is a possibility of this pest entering potato growing regions in Sri Lanka and hence it is a possible candidate for application of phytosanitary measures.

Stage 2: Pest risk assessment

Pest categorization: Identity of the pest

PSP is caused by Spongospora subterranea f.sp. subterranea J.A. Toml. Detailed description of the fungus can be obtained from Kunkel (1915), Melhus et al. (1916), Harrison et al. (1997), Miller (2001) and CABI (2005). The fungus is an obligate parasite. Synonyms (Index Fungorum, 2004) are Erysibe subterranea Wallr., Sorosporium scabies (Berk.) A.A. Fisch. Waldh., Spongospora scabies (Berk.) C. Massal., Spongospora subterranea (Wallr.) Lagerh., and Tuburcinia scabies Berk.

Primary hosts are Solanum tuberosum (Potato) and Lycopersicon esculentum (Tomato). Secondary hosts are Capsicum annuum (Bell pepper), Datura stramonium (Devils trumpet), Nicandra physalodes (Apple of Peru), Nicotiana rustica (Wild tobacco), Physalis peruviana (Cape gooseberry), Solanum marginatum (White-edged night shade), Solanum nigrum (Black nightshade), Tussilago farfara (Colt’s foot) and Ullucus tuberosus (Ulluco). Affected plant stages are pre-emergence, vegetative growing and flowering stages, and post-harvest storage period. Affected plant parts are whole plant, leaves, stems, roots and vegetative organs (CABI, 2005).

Presence or absence in PRA area

Now, PSP is practically found in potato producing areas in the world from latitudes 65’ N to 53’ S and at high altitudes in the Tropics where moist and cool climates are found (Tsror (Lahkim) et al., 1999; CABI, 2005). 38 DE SILVA et al.

Its occurrence in Sri Lanka is based on doubtful reports, which had never been confirmed by detailed studies. The incidences were reported only from cultivations of government regulatory agencies that planted imported seed tubers.

Regulatory status

Sri Lanka has taken emergency measures to regulate S. subterranea f.sp. subterranea causal agent of PSP.

Potential for establishment and spread in PRA area

The pathogen can be transmitted with planting of diseased tubers (Kunkel, 1915; Melhus et al., 1916; Harrison et al., 1997; De Nazareno and Boschetto, 2002). Most of the potato varieties grown in the PRA area are known to be susceptible to PSP (Harrison et al., 1997). Also, the cooler climates and soil factors in the Nuwara Eliya and Badulla districts where potato is extensively grown could be ideal for survival and spread of the pathogen. Other two areas, Jaffna and Puttalam may not be ideal for PSP due to prevailing high temperatures.

Potential for economic consequences in PRA area

Potato is a high-income crop in the country. Any problematic pest may definitely affect the net return to the farmers (Nugaliyadde et al., 2000), when compared with reports from PSP endemic areas (Falloon et al., 1995; Falloon et al., 1996; Harrison et al., 1997; de Nazareno and Boschetto, 2002; Iftikhar et al., 2002).

Conclusion of pest categorization

The evidence points out that Spongospora subterranea f.sp. subterranea has the potential to be considered as a quarantine pest and hence it is necessary to continue the PRA.

Assessment of the probability of introduction and spread

Identification of pathways for entry of PSP into PRA area

The pathogen causing PSP can enter the PRA area with several pathways because it has been associated with several host and non-host material (Table 1). After studying PSP in an area of improved pasture in New South Wales, Letham et al. (1988) suggested that the pathogen had entered the area with pasture seed. However, the probability of the pathogen associated with seeds (preferred means of importing alternate hosts) of tomato, tobacco PEST RISK ANALYSIS OF POWDERY SCAB OF POTATO 39 etc. appears to be low, if not negligible. The resting spores of the pathogen can pass through the digestive system of animals fed with infected tubers, and hence, fresh dropping from these animals could also be a pathway (Harrison et al., 1997; Gilat Research Center, 2001; Secor and Rivera-Varas, 2004).

Table 1. Association of PSP with pathways and its probability of entry into PRA area.

Pathway Likelihood of association Probability of entry Host (Solanum tuberosum): True seeds Negligible Negligible In vitro cultures Very low Very low Rooted plantlets High High Seed tubers High High Alternate hosts: Seeds Negligible Negligible In vitro cultures Negligible Negligible Rooted plantlets High High Non-host planting material: Seeds (grass) Moderate Moderate Seeds (other) Low Low Bulbs, tubers, corms, rhizomes High High In vitro cultures Negligible Negligible Non-host plant products: Planting media (Peat, compost, High High organic manure) Forest litter Unknown Unknown Non-plant material: Soil High High Rock, stones and gravels Very low Very low Conveyance/carrier: Passengers High Low Baggages Low Low Carriers Low Low Containers by airfreight Low Low Containers by sea freight Low Low Refrigerated containers by air Low Low Refrigerated containers by sea Low Low Parcels (mail) Low Low Animals (horses, cows, pets, birds) Moderate Low Animals (honeybees, insects) Negligible Negligible Other articles Uncertain Uncertain

Probability of pest being associated with the pathways at origin

Annually, Sri Lanka imports seed potato from countries such as Australia, France, Germany, India, the Netherlands, New Zealand, Poland, the United Kingdom, and the USA where PSP is prevalent (Harrison et al., 1997; CABI, 2005). If in the future, Sri Lanka envisages importing seed potato from any other PSP endemic country, the risk is the same as that of the present suppliers. Also, Sri Lanka exchanges other plant material with these countries. 40 DE SILVA et al.

Probability of survival during transport or storage

Without the host, S. subterranea f.sp. subterranea can survive for several years as resting spore balls known as cystosori (Harrison et al., 1997; Iftikhar et al., 2002). Thus, the pest can withstand any adverse conditions during transport. With sufficient water, the cystosori may produce infectious zoospores, which may be viable for 2-14 days depending on the availability of favourable conditions (Harrison et al., 1997).

Probability of pest surviving existing pest management options

Recent escalation of PSP in some countries due to banning of certain chemical control strategies (Harrison et al., 1997) indicates that the pathogen could survive the present day pest management programs. Hughes (1980), on the other hand, reported that in Queensland, PSP was becoming a less serious disease. Chemical treatments give only a partial control of PSP (Braithwaite et al., 1994; Falloon et al., 1996; Secor and Rivera-Varas, 2004). Phasing out of methyl bromide in few years may also pose additional problems for the control of the disease.

Probability of transfer to a suitable host

Potato is extensively grown in the PRA area. Hence, after entry, the pathogen could easily find a suitable host. If it comes with seed potato tubers, the host is already available. The other alternate hosts are also found in the PRA area (Senarathna, 2001). At present, with the facilities available with the Plant Quarantine Officers who inspect the imported seed potato at entry ports in Sri Lanka, the PSP pathogen could pass without detection. Thus, there is an overall high risk of PSP pathogen entering the PRA area, if associated with seed tubers and rooted plantlets of potato, contaminated soil, and any underground plant parts with soil.

Probability of establishment

Detection of certain symptoms similar to PSP indicates that the pathogen has already entered several times into the PRA area. Even though the conditions are favourable for its establishment, it is strange why the disease has not manifested in the fields of farmers who may have planted seed tubers from the same lots. Perhaps the inoculum was not large and, with time, the disease may manifest, if proper control measures are not taken. Also, the farmers could misidentify the disease as common scab. Melhus et al. (1916) found that some infested soils may not readily produce potato crops with PSP. Initial inoculum level e.g. number of cystosori in soil does not have any effect in subsequent disease development (Harrison et al., 1997). Hughes (1980) also observed that, under warm weather, potato crops grown in soils PEST RISK ANALYSIS OF POWDERY SCAB OF POTATO 41 contaminated with S. subterranea might not develop PSP symptoms. All these indicate that there is a huge information gap with regard to what factors trigger on PSP development in a potato crop.

Extensive cultivation of potato in Nuwara Eliya and Badulla districts will invariably offer enough hosts for the pathogen to complete its life cycle. The alternate hosts such as tomato, wild solanaceous plants are abundant in the area. Pathogen causing PSP survives best under cool and moist conditions (Harrison et al., 1997). Soils with high moisture holding capacity, free water in the soil matrix and alternating dry and wet periods increase the severity of PSP (Harrison et al., 1997). Optimum soil temperatures favouring development of PSP are 11-18°C (Gilat Research Center, 2001; Secor and Rivera-Varas, 2004) and frequently, the diurnal temperatures in Nuwara Eliya and Badulla districts fluctuate within this range (Nugaliyadde et al., 2000). On the other hand, temperature variations in Jaffna and Puttalam areas may exceed the upper limit of the optimal for PSP.

According to Harrison et al. (1997), there are conflicting reports on how cultural practices affect the development of PSP. The effect of liming, compost, organic manure and the season of planting may not be consistent. Chemical treatments of soil and seed tubers only partially control this disease (Braithwaite et al., 1994). Falloon et al. (1996) observed that low rates of mancozeb treatment increased the disease incidence whereas higher rates reduced the same. Some chemical treatments, which had the potential to control PSP, were found to be phytotoxic to potato plants and reduce sprouting (Braithwaite et al., 1994; Falloon et al., 1996). Furthermore, field application of some chemicals may not be practical (Harrison et al., 1997).

The occurrence of different pathotypes of S. subterranea was not demonstrated (Melhus et al., 1916; Harrison et al., 1997). This may be one of the reasons for slow manifestation of PSP. There is no information on the effects of present pest management options practised by our farmers for control of PSP. Reproductive and dispersal mechanisms adopted by the pathogen also favour its establishment (Melhus et al., 1916; Harrison et al., 1997). Growing potatoes in the same field with little or no break between successive crops also help the pathogen establishment under favourable environmental conditions (Melhus et al., 1916; Harrison et al., 1997). Thus, S. subterranea f.sp. subterranea has a good potential for establishment in the PRA area (Table 2) irrespective of the pathway of introduction. 42 DE SILVA et al.

Table 2. Probability of establishment (after infection) of PSP pathogen in the PRA area.

Evaluation factor Likelihood of establishment Availability, quantity and distribution of hosts High Environmental suitability High Potential adaptation of the pathogen Information gap Reproductive strategy of the pathogen High Method of pathogen survival High Cultural practices and control measures • Effect of existing control programmes High • Effect of eradication programs Information gap Overall risk High

Probability of spread after establishment

Factors that may help the spread of PSP pathogen are considered in Table 3. The terraced nature of the upcountry (Nuwara Eliya district) helps the spread of zoospores with running surface water. Cleaning and disinfecting all implements that were used in the contaminated fields may reduce the spread of the disease but the procedures are not very practical (Harrison et al., 1997).

Table 3. Probability of spread after establishment in the PRA area.

Evaluation factor Likelihood of spread Suitability of the natural environment for natural spread of the disease High Absence of natural barriers (desert/arid area) High Potential for dispersal with people, commodities or conveyances High Planting of seed potatoes harvested in the previous season High Potential natural enemies, biological control of pathogen Unknown Overall risk High

Conclusion on the probability of introduction and spread

PSP has a relatively high potential for introduction with seed tubers and rooted plantlets of potato, soil and other non-host material that have come in contact with contaminated fields. After introduction, spread of the pathogen will occur but may take some time. Therefore, it is necessary to continue this PRA.

Conclusion regarding endangered areas

The conditions in Nuwara Eliya and Badulla districts will promote the PSP. However, Jaffna and Puttalam areas are not conducive for PSP. PEST RISK ANALYSIS OF POWDERY SCAB OF POTATO 43

Assessment of potential economic consequences

Here, we present semi-quantitative impact of PSP on the economy in the PRA area. We envisage making a more detailed analysis in due course. However, here we would like to state that the perceived or extrapolated occurrence of PSP in the PRA area will significantly increase the cost of production of potato. Crop failures will also lead to import of consumption potato. The government will need to allocate more money for research and management of PSP. Therefore, occurrence of PSP in Nuwara Eliya and Badulla districts will invariably cause unacceptable economic consequences.

Degree of uncertainty

The failure of Sri Lankan Plant Quarantine officials to detect PSP during inspection at the entry ports is due to lack of proper identification tools and techniques. Sri Lanka has continuously imported seed potatoes from countries where PSP is found, but until recently, the authorities considered it as another form of deep-pitted scab. This risk assessment is based on the experience of other countries. Economic impact was deduced by extrapolating what occurred in PSP endemic areas.

Conclusion of the pest risk assessment stage

The seed tubers and rooted plantlets of potato, soil and underground parts of any plant from contaminated fields pose a high risk. The agro-ecological factors are conducive for the establishment and spread of the pathogen in Nuwara Eliya and Badulla districts. The difficulty in detecting the pest by untrained persons also adds to the risk. If the pathogen increases to epidemic proportions, which is possible under prevailing conditions, it will certainly be of concern to Sri Lanka. Thus, Spongospora subterranea f.sp. subterranea, pathogen causing PSP can be considered as a quarantine pest.

Stage 3: Pest risk management

Level of risk

Unattainable nature of zero risk has prompted the countries to agree on risk management strategies when implementing phytosanitary measures for regulated pests (IPPC, 1995). The country that faces the impending threat can determine the Appropriate Level of Protection (ALOP), which should be consistent for all pathways via which the threat appears (IPPC, 2003). The safeguards should create a minimal impediment to international movement of people, commodities and conveyances (IPPC, 1995). There are several applicable safeguards to achieve the ALOP and these 44 DE SILVA et al. could be taken at country of origin, pre-border, border and post-border. The options are intended to reduce to an acceptable level or to prevent the PSP infestation of the identified risky pathways. Since Sri Lanka is compelled to import seed potatoes, prohibition due to PSP is not a viable option.

Identification of risk management options

Area freedom

Biology of S. subterranea f.sp. subterranea indicates that delimiting pest free areas (IPPC, 1996b) in a bigger scale is more restrictive than what is required. When migration ability of the pathogen of PSP is considered, its effects on a considerable level in potato tubers to be used for planting in the next season will be manifested only after few years of initial infestation. However, there is little knowledge on this aspect. Under such circumstances, pest free places of production (IPPC, 1999) could be considered acceptable as the required phytosanitary measure. Regular inspection of the crop, perhaps at the beginning and at harvest should be done to confirm the pest freedom. Soil, plant parts esp. roots and, at harvest, the tubers should be inspected and then tested to determine the absence of PSP. There is no effective way of controlling PSP, but several integrated pathogen management strategies can be adopted to minimize the risk from the disease including cultivation of resistant varieties, applying chemical treatments, cultural practices and legislative measures (Braithwaite et al., 1994; Falloon et al., 1996; Harrison et al., 1997).

Specific testing for the pest

Several bioassay techniques have been devised to detect the pathogen causing PSP (Harrison et al., 1997; Bell et al., 1999; CABI, 2005; Iftikhar et al., 2002; Merz et al., 2004). These techniques are time consuming. PCR would be a rapid and reliable test (van de Graaf et al., 2003; Qu et al., 2005) but much work is needed to devise a suitable protocol for Sri Lanka. Hence, the country should either depend on tests done in the exporting country or on other bio-assay techniques.

Treatment

As already mentioned, chemical treatments do not result in the eradication of the pathogen from the seed tubers or other articles. Often, they resulted in phytotoxic effects (Falloon et al., 1995; Falloon et al., 1996). Fumigation with methyl bromide could be used but soon it has to be phased out. PEST RISK ANALYSIS OF POWDERY SCAB OF POTATO 45

Certification

The issue of phytosanitary certificates in compliance with ISPM No. 12 (IPPC, 2001) could be considered as another risk management option. An additional declaration must be provided indicating the PSP freedom in the place of production after appropriate monitoring and testing.

Inspection on arrival

Microscopic observations if done by trained officers, will reveal the spore balls or cystosori of the fungus. Appropriate sampling and laboratory testing will indicate the presence/absence of the PSP pathogen in the consignment. However, the symptoms could easily be misidentified as those of deep-pitted scab (Harrison et al., 1997). Need for further studies on devising a rapid and inexpensive detection technique for PSP is thus highlighted.

Post-entry quarantine

Seed potatoes are imported into Sri Lanka in commercial quantities. After obtaining plant quarantine clearance, the consignments are directly taken to planting sites. Demand for the seed potatoes cannot be completely met by local production due to several reasons including limitation of suitable land. Therefore, post-entry quarantine screening of the imported consignments before release is not a viable option. However, any material imported as germplasm could undergo post-entry quarantine screening and evaluation.

Other pathways

Sri Lanka prohibits import of soil, compost, forest litter, organic manure and some growing media that pose high risk due to a multitude of pest problems. As for the importation of other non-host planting material with underground parts, inspection and testing at an accredited laboratory before export and phytosanitary certification for freedom from PSP will meet the required ALOP.

Efficacy and impact

The risk management options outlined above are sufficient to keep PSP pathogen away from the PRA area. They will not have any adverse impact on trade between Sri Lanka and countries supplying seed potato. The producers will not incur any additional costs for monitoring and testing of PSP in their fields because it could be integrated to their present pest management programs. 46 DE SILVA et al.

Conclusion of pest risk management

Seed potato tubers and rooted potato plantlets should originate from PSP free places of production. Phytosanitary certificate must contain additional declarations to show that the place of production is free from S. subterranea f.sp. subterranea as verified by field monitoring and testing. Upon arrival, the consignment must be inspected and sampled followed by laboratory testing. Prohibition of import of soil and other plant-derived growth media must be continued. As for other non-host material, inspection and laboratory testing will give the necessary protection against risk of PSP.

CONCLUSIONS

The causal agent of powdery scab of potato Spongospora subterranea f.sp. subterranea J.A. Toml. must be considered as a quarantine pest for Sri Lanka since it fulfills all relevant criteria such as absence in the PRA area, high potential for introduction and spread, and unacceptable economic and environmental impact. The safeguards that could be taken to meet the ALOP in the order of preference are importation of the risky material from pest free places of production, regular monitoring during growing season, phytosanitary certification with relevant additional declaration, specific testing for the pathogen and inspection on arrival. Continued vigilance must be kept for detecting the fungus in imported consignments. Regulatory stand taken as an emergency phytosanitary measure must be continued. Also, there is urgent need to train our Plant Quarantine officers on the detection of PSP.

ACKNOWLEDGEMENTS

The authors wish to thank Dr. C Kudagamage, Director General of Agriculture of Sri Lanka for entrusting the principal author to do this PRA. They feel appreciative of the continuous encouragement given by Dr. (Mrs.) H.M.R.K. Ekanayake, Deputy Director (Plant Quarantine). Thanks are also extended to Dr. R.G.A.S. Rajapakse, Research Officer (Plant Pathology), Horticultural Crop Research and Development Institute for the friendly discussions on powdery scab of potato.

REFERENCES

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PRODUCT DEVELOPMENT OF DRUMSTICK (Moringa olifera) USING POD SCRAPINGS AND SEEDS

S. EKANAYAKE1, C.K. RANAWANA1 and P.G.R. HEMANTHI KUMARI2 1Food Research Unit, Horticultural Crop Research and Development Institute, Gannoruwa, Peradeniya 2Postgraduate Institute of Science, University of Peradeniya

ABSTRACT

Drumstick (Moringa oleifera) is a nutrient rich vegetable crop with a seasonal glut and the surplus is lost due to the lack of related products and preservation methods. Therefore, this study was undertaken to find a better preservation method as an attempt to make it available throughout the year and popularize the consumption. Variety ‘Jaffna’ was selected for the preservation of products considering its higher availability in the market. Blanched pod scrapings and seeds were used for the preparation of products. Salt free paste, salt added paste, pod scrapings and seeds in 2% brine, pod scrapings and seeds in a series of potassium metabisulfite (KMS) solutions (containing 500, 1000 and 1500 ppm of sulfur dioxide) were prepared. All products were bottled and stored at room temperature and tested for physicochemical, microbiological and sensory characteristics. The pH, and vitamin C content declined during storage while total soluble solid contents increased. A rapid colour change was observed in salt free paste. Retention of vitamin C was significantly high in salt added paste, during storage. No microbial colonies were present on samples preserved in both 1000ppm and 1500 ppm sulfur dioxide solutions whereas the other products showed a very low microbial density even after three months of storage. All presented products, pod scrapings in 2% brine, 2% salt added paste and pod scrapings in 500 ppm SO2 were highly accepted in the sensory evaluation. These treatments can be used to preserve drumstick products in bottles for more than three months.

KEYWORDS: Drumstick, Paste, Preservation, Products, Sensory evaluation.

INTRODUCTION

Drumstick (Moringa oleifera) is an economically important crop grown in dry zone, especially in Jaffna, Mannar and Puttlum (Jayaweera, 1982). Flowers and fruits appear twice a year and so there are two crops, in July to September and March to April. Some common varieties found in Sri Lanka are Jaffna, Chavacacheri, Makandura 1 (MK1), Makandura 2 (MK2), Kalpitiya Local, Miti Murunga, Lunu Murunga and Rana Murunga. Pods are harvested when they are still young and snap easily. These immature or slightly matured pods are used as a vegetable. They are cut into slices and used in culinary preparations; they are also pickled (Ramachandran et al., 1980). Almost all parts of the tree have been used for medicine and it is used in the treatment of ascites, rheumatism, venomous bites and as cardiac and circulatory stimulants (Siveragen et al., 1994). 50 EKANAYAKE et al.

Drumstick is a seasonal crop and the shelf life of drumstick does not exceed more than 16 days, even under favorable storage conditions (Damodran et al., 1999). Yet the application of various methods of vegetable preservation such as drying or dehydration, freezing, thermal processing and chemical preservation are minimal with regard to the preservation of drumstick. Therefore, considerable amount of this nutrient rich vegetable is lost yearly. Postharvest losses of drumstick could be reduced substantially, if suitable processing methods are available to local processors to undertake on- site processing of drumstick.

The objective of this study was to develop a value added product from drumstick and to evaluate the physicochemical properties and consumer acceptability of the developed product.

MATERIALS AND METHODS

Selection of variety

Different varieties of drumstick were harvested and pod morphology, percent moisture content (Askar and Treptow, 1993), flesh colour (RHS colour chart), flesh content, total soluble solid (TSS) content, vitamin C content and the pH (Kaushal and Bhat, 1999) were determined. Considering the availability of the crop, Jaffna variety was selected for product preparation.

Preparation of drumstick paste

Pods were cut open and flesh and seeds were scraped into a vessel. These scraped pieces and seeds were blanched for 3 min and blended with the addition of one-third of water from the weight of the drumstick sample and paste was prepared. Table salt 2% (w/w) was added to the portion of the prepared paste and was heated up to 80°C and immediately transferred to pre-sterilized glass bottles and was exhausted for 10 min and sealed immediately. Sealed bottles were sterilized for 15 min to destroy any microbes and inactivate the enzymes present. Then the bottles were allowed to cool and labeled. The same procedure was followed to prepare salt free paste.

Bottling of drumstick pod scrapings and seeds in 2% salt solution

Pod scrapings and seeds were blanched for 3 minutes and were filled in glass bottles with 2% salt solution. Bottles were exhausted for 10 min, sealed and retorted for 15 min. PRODUCT DEVELOPMENT OF DRUMSTICK 51

Bottling of drumstick pod scrapings and seeds in potassium metabisulfite (KMS) solution

Pod scrapings and seeds were blanched for 3 min and were filled in glass bottles containing 500, 1000, 1500 ppm of sulphurdioxide solutions respectively. These were exhausted for 10 min, sealed and retorted for 15 min. Then the bottles were allowed to cool and labeled.

Determination of physicochemical properties of prepared drumstick products

Physicochemical properties of the products were analyzed once a month for three consecutive months. Total soluble solids (TSS), color, pH, and vitamin C content were analyzed for all prepared drumstick products.

Microbial examination of the products

Total colony count of the prepared product was examined after three months of storage using colony count method (AOAC, 1984).

Sensory evaluation of drumstick products

Drumstick curries were prepared from three products after 3 months of stored samples (salt added paste, pod scrapings and seeds in brine solution, pod scrapings and seeds in 500 ppm KMS solution). Salt free paste was not subjected to sensory evaluation due to its unfavorable colour development during storage. Consumer preference of drumstick products was evaluated in terms of taste, aroma, consistency, nature of curry and overall acceptance using the ranking test. Twenty consumer panelists were used for the evaluation. For each sample, sensory ratings of different perceptions were subjected to analysis of variance (ANOVA), using the statistical analysis system (SAS) computer package and mean differences were determined by the Duncan Multiple Range Test (DMRT) at p= 0.05. Friedman test was carried out to determine the significant difference of the overall acceptability among products with the use of Minitab statistical package (Watts et al., 1989).

RESULTS AND DISCUSSION

Physicochemical properties of fresh pods

Pod morphology of drumstick varieties is shown in Table 1. Miti Murunga had higher percentage of edible portion when compared with other varieties. It also had higher vitamin C content and pH value (Table 2). 52 EKANAYAKE et al.

Table 1. Pod morphology of selected drumstick varieties.

Variety Features Jaffna Straight pods, both stem end and blossom end are blunt. Little amount of dark patches are present on green pod surface. Makandura 1 (MK1) Purplish stripes are present on green pod surface and stem end is reddish purple coloured. Both ends of the pod are blunt. Pod stem is straight and cylindrical. Makandura 2 (MK2) Blossom end of the pod is acute and purple colour is present on stem end. Transverse section is triangular in shape. Kalpitiya local Dark stripes are present on greenish surface. Both stem end and blossom end are acute. Pod stem is slightly curled. Miti murunga Pod surface is rough and dull green in colour. Both ends of the pods are blunt. Transverse section is triangular in shape.

Table 2. Physicochemical properties of fresh pods.

Variety Edible Average Moistur pH TSS (0Brix) Vitamin portion pod length e (%) C (%) (cm) (mg/100g ) Jaffna 71.8 52.7 85.9 5.6 8.5 116.6 MK1 71.7 40.2 87.8 5.6 8.2 117.0 MK2 72.3 45.0 89.8 5.8 8.0 117.1 Kalpitiya local 72.6 61.3 96.3 5.8 8.6 116.0 Miti murunga 80.5 34.2 86.2 6.1 5.0 117.6

Physicochemical properties of drumstick products

Values of pH among prepared products are not significantly different in the 2nd month, while it is significant initially, 1st month and 3rd month at 5% level. As a whole, pH values of all products have been decreased during storage (Table 3).

Table 3. Product pH values during storage.

Storage period (Months) Product Initial 1st month 2nd month 3rd month Salt free paste 5.3700b 5.3033d 5.2367a 5.1967ab Salt added paste 5.9233b 5.9233a 5.920a 4.6800b Pod scraping in brine 7.7267a 5.8367a 5.1100a 4.9500ab

Pod scraping in 500ppm SO2 5.7333b 5.7067b 5.5100a 5.4067a Pod scraping in 1000ppm SO2 5.6167b 5.4667c 5.0567a 4.7867b Pod scraping in 1500ppm SO2 5.4933b 5.4100c 5.9767a 4.9167ab Pooled standard deviation 0.4932 0.0494 0.3757 0.2277 P-value 0.0010 0.0000 0.408 0.020 Means in a column having a common letter are not significantly different by 5% DMRT PRODUCT DEVELOPMENT OF DRUMSTICK 53

Total soluble solid (TSS) contents of all products differ significantly from each other at initial and during storage at 5% level and the highest values are present in salt added paste (Table 4). It is observed that TSS content is increased with time during storage except salt free paste stored in the third month. Increase of TSS is possible with the decrease of pH or increase of acidity as acids are non-sugar soluble solids (Askar and Treptow, 1993).

Table 4. Total soluble solids (Brix) in products during storage.

Storage period (Months) Product Initial 1st month 2nd month 3rd month Salt free paste 2.0000c 3.0000b 3.9000c 2.9000c Salt added paste 3.4333a 4.8000a 5.0667a 5.6000a Pod scraping in brine 2.3333b 2.9333c 4.6670b 4.6433b

Pod scraping in 500ppm SO2 1.2667d 1.3333d 1.8000d 2.0333e

Pod scraping in 1000ppm SO2 1.2167d 1.1800d 1.3.167e 1.8167e Pod scraping in 1500ppm SO2 1.1833d 1.2367d 1.4400de 2.4400d

Pooled standard deviation 0.1093 0.15920 0.2280 0.1350 P-value 0.0000 0.0000 0.408 0.020 Means in a column having a common letter are not significantly different by 5% DMRT

Vitamin C content of all products is significantly different from each other at initial level and during storage (Table 5). Even though initially the lowest vitamin C content was observed in salt added paste, it reached high value after 2nd month at 5% significant level. Vitamin C can be lost initially, with the cutting of fresh pods before blanching, due to the ascorbic acid oxidase present in pressed pod juice (Anon, 1962). Vitamin C can also be destroyed by oxidation especially in high temperature, processing and storage (Dauthy, 1995). The presence of sulfur dioxide and ascorbic acid may easily be susceptible to oxidation. This may be another reason for the lower vitamin C content of drumstick preserved in sulfur dioxide solution.

Table 5. Vitamin C content (mg/100g) of the products during storage.

Storage period (Months) Product Initial 1st month 2nd month 3rd month Salt free paste 43.273ab 33.946a 23.730b 19.621b Salt added paste 34.847b 32.085a 28.899a 28.699a Pod scraping in brine 52.997a 23.537b 19.350c 12.723c

Pod scraping in 500ppm SO2 52.977a 6.990c 5.534e 4.880d

Pod scraping in 1000ppm SO2 51.653ab 7.831c 6.313de 5.550d Pod scraping in 1500ppm SO2 53.420a 8.415c 7.333d 5.569d

Pooled standard deviation 7.149 0.1.275 0.825 0.171 P-value 0.039 0.0000 0.000 0.000 Means in a column having a common letter are not significantly different by 5% DMRT 54 EKANAYAKE et al.

Products prepared from drumstick showed yellowish colour upon processing. Not much colour change was observed in all products during storage except salt free paste (Table 6).

Table 6. Colour changes of product during storage based on Royal Horticultural Society (RHC) colour chart.

Storage period (Months) Product Initial 1st month 2nd month 3rd month Salt free paste Y G 154D Y 8C GY 160B GY 160B Salt added paste Y G 154D GY 160A GY 160A GY 160A Pod scraping in brine Y G 154D Y 4D Y 2D Y 2D

Pod scraping in 500ppm SO2 Y G 154D Y 4D Y 2D Y 2D

Pod scraping in 1000ppm SO2 Y G 154D Y 4D Y 2D Y 2D Pod scraping in 1500ppm SO2 Y G 154D Y 4D Y 2D Y 2D YG= Yellow green group, Y= Yellow, GY= Grayed yellow group

Microbial examination of the products

As shown in Table 7, the average numbers of colonies per gram of some products vary from 4 to 20.

Table 7. Total colony count of the product after three months of storage.

Product Average number of colonies/g of sample Salt free paste 20 ± 2 Salt added paste 10 ± 1 Pod scraping in brine 8 ± 2

Pod scraping in 500ppm SO2 4 ± 1 Pod scraping in 1000ppm SO2 0 Pod scraping in 1500ppm SO2 0

Sensory evaluation

The statistical analysis revealed that there were no significant differences among four prepared curries using three drumstick products and fresh scrapings in terms of taste, colour, nature of curry and consistency, except aroma at 5% significant level (Table 8). Summary of overall acceptability results showed that there was no significant difference among all four curries prepared from each product (Table 9). It is evident that curries prepared from preserved products are almost close to the fresh curry in overall acceptance. PRODUCT DEVELOPMENT OF DRUMSTICK 55

However, the comments given by the panelists revealed that curries prepared from salt added paste were the best product among the three products tested. Table 8. Summary of analysis of sensory evaluation data.

Treatment Sensory property

Taste Aroma Colour Nature of Consistency curry EM SR EM SR EM SR EM SR EM SR Pod scrapings in 2% brine 78.125 56.0 1.687 59.5 72.188 49.5 62.687 55.5 66.00 48.5

Freshly prepared 69.875 45.0 69.437 51.0 73.437 53.5 58.687 42.5 66.250 46.5 paste Pod scrapings in 500ppm 72.125 46.5 63.312 36.5 72.188 50.5 64.937 56.5 66.500 46.5 SO2 Salt added 76.875 52.5 68.813 53.0 70.937 46.5 58.938 45.5 71.250 58.5 paste P-value 0.478 0.037 0.786 0.152 0.34 EM= Estimated median, SR= Sum of ranks

Table 9. Summary of overall acceptability.

Treatment EM SR Freshly prepared paste 70.750 45.0 Pod scrapings in 2% brine 73.125 53.5 Pod scrapings in 500ppm SO2 70.625 48.0 Salt added paste 73.000 53.0 P-value 0.629 EM= Estimated median, SR= Sum of ranks

CONCLUSIONS

Most of the physicochemical properties were almost same in most of the prepared products. Salt added paste had lower vitamin C reduction rate during storage compared to the other products. Products of salt added paste, pod scrapings and seeds in 2% brine and pod scrapings and seeds in 500ppm sulferdioxide solution were highly accepted by consumers in terms of sensory properties. All these three treatments can be used to preserve drumstick products in bottles for more than three months.

REFERENCES 56 EKANAYAKE et al.

Anon., 1962. Wealth of India Council Sci., Ind. Res. Val VI, 425-428. AOAC, 1984. Official method of analysis. Association of Official Analytical Chemists. USA. 579-580. Askar, A. and H. Treptow. 1993. Quality assurance in tropical fruit processing. Springer- Verlae, Berlin, Heidelberg, New York. 10-114. Damodran, T., S. Anubu, R.S. Azhakiamanuvalan and P. Vennila. 1999. Packaging methods to prolong the shelf life of moringa (Moringa olifera) cv PKMI during transit. South Indian Horticulture 47: 1-6. Dauthy, M.E. 1995. Fruit and vegetable processing, Agriculture Bulletin, FAO, Rome. Jayaweera, D.M. 1982. Medicinal plants used in Sri Lanka. NARESA, Vol.4, 100. Kaushal, B.B.I. and A. Bhat. 1999. Studies on physicochemical properties of fruit leather blended with sprouted soy slurry. Indian Food Packer 53(5): 18-21. Ramachandran, C., K. Peter, and P.K. Gopal Krishnan. 1980. Drumstick (Moringa olifera), Multipurpose Indian vegetable. Economic Botany 34(3): 276-203. Siveragen, V.V. and I. Balachandran. 1994. Ayurvedic drugs and their plant sources. Oxford and IBH publishing Co. Pot Ltd. 445-447. Watts, B.M., G.L.Ylimaki, L.E. Jeffery, and L.G. Elias. 1989. Basic sensory method for food evaluation. International Development Research Center. Canada. Annals of Sri Lanka Department of Agriculture. 2006.8:57-67.

EFFECT OF IN-SITU DECOMPOSITION OF COIR-DUST AND POULTRY LITTER IN PLANTING HOLES ON YIELD AND FERTILIZER USE EFFICIENCY OF BANANA

J.M.P.B. JAYASUNDARA1 and G.A.A.S.L. GUNATHILAKA2 1Horticultural Crop Research and Development Institute, Gannoruwa, Peradeniya 2Regional Agricultural Research and Development Centre, Makandura

ABSTRACT

An efficient nutrient management technique is needed in order to ensure continuous supply of quality banana fruits to meet export demands. The repeated use of only chemical fertilizers at four-months interval may not be efficient and economically viable under rain-fed conditions prevailing in Sri Lanka. Therefore, the effects of decomposition of coir-dust (CD), poultry litter (PM) and a combination of these in the planting hole along with chemical fertilizers applied at four and six-month intervals on performance of banana were studied during six crop cycles. The changes in C/N ratio of composting mixtures exhibited high correlation with major nutrient contents in plant tissues. Leaf analysis indicated that gradual decomposition of CD and PM mixture was able to regulate a continuous supply of N and K from applied fertilizers to the crop for a longer period, resulting in consistent yields and superior fruit quality performances, when compared to the current recommendation. In-situ decomposition of CD and PM in 1:1 ratio was an economically feasible substrate, which enhanced applied fertilizer use efficiency by slow release of plant nutrients and increased sustainable production. In-situ decomposition of even only coir-dust also showed a significant contribution in improving applied fertilizer use efficiency, especially, when fertilizer was applied at six-month intervals.

KEYWORDS: Chemical fertilizer, Coir-dust, In-Situ decomposition, Poultry litter.

INTRODUCTION

In the recent past, Sri Lanka’s local and export markets for banana has increased considerably. Therefore, it is necessary to ensure the continuous supply of quality fruits. Increased and sustainable yields could be achieved through an efficient nutrient management technique, especially, using an Integrated Nutrient Management (INM) system, which plays a key role in achieving optimum crop yields in a sustainable manner (Chundawat, 2001).

Market oriented growers use larger quantities of chemical fertilizers, which account for nearly one third of the cost of cultivation without further yield increase. The integrated use of organic manure and chemical fertilizers under local conditions has been reported to result in growth and yield increases (Pinto, 1986). On the other hand, studies conducted with slow or controlled release fertilizers have shown significant effect on growth, yield and quality of different perennial fruit crops (Ram et al., 1999). Organic 58 JAYASUNDARA AND GUNATHILAKA materials applied into the planting holes before planting by allowing them to decompose in-situ were found to result in slow nutrient release of applied chemical fertilizers (Mc Connel et al., 1993). On the other hand, CD absorbs and retains moisture for longer periods (Jayasundara, 1997), and this could enhance the efficiency of applied chemical fertilizers. It was also found that problems caused by high concentrations of minerals in organic manures like PM, which are in exchangeable form, could be solved by composting with organic substances having high C/N ratios (Fujiwara, 1998). The C/N ratio and particle size of the material used to mix with PM play a significant role in retaining NPK in the soil (Hansen et al., 1989).

Banana is grown as a mono or mixed crop under coconut in many parts of Makandura region in the low country intermediate zone (IL-1) of Sri Lanka. The rainfall prevailing in this region is not sufficient to retain adequate soil moisture throughout the year (Jayasundara, 1997). Therefore, depletion of soil moisture at the critical stages of the banana crop leads to poor fertilizer use efficiency, which often leads to low yields. In this region, the predominant soils are Red Yellow Podzolic soils with soft and hard laterites (Panabokke, 1967). Low water holding capacity is considered to be the main limiting factor for better fertilizer use efficiency (Jayasundara, 1997). Therefore, fertilizer application at 4 months interval as currently recommended by the Department of Agriculture needs re-assessment under these conditions. Improving soil moisture retention using locally available organic manure may overcome these problems.

An investigation was therefore carried out to study the composting effect of CD and PM (deep litter) in planting holes on the efficiency of applied chemical fertilizers in relation to growth, yield and fruit quality of six crop cycles of banana under rain-fed conditions.

MATERIALS AND METHODS

A field experiment was conducted with kolikuttu banana over six crop cycles at the Regional Agricultural Research Centre, Makandura. The following treatments were tested in a Randomized Complete Block Design (RCBD) with four replicates.

Treatment Application interval (months)

1. Recommended chemical fertilizers (Rec-4) (4) control 2. Rec. (4 )+ CD (4) 3. Rec. (6) + CD (6) 4. Rec. (4) + PM (4) 5. Rec. (6) + PM (6) 6. Rec. (4) + PM + CD (4) 7. Rec. (6) + PM + CD (6) IN-SITU DECOMPOSITION OF ORGANIC MATERIALS ON BANANA 59

Planting holes of 60 cm x 60 cm x 60 cm dimension were prepared at 3 m x 3 m spacing, 5 kg each of CD and PM were applied at the beginning of the experiment according to the treatment schedule and mixed with soil and planted with one banana sucker after cutting down the pseudo- stem 60 cm above the corm in order to obtain a uniform crop at the beginning. The experiment was conducted under rainfed conditions for six crop cycles. Two months after planting, chemical fertilizers were applied to all the treatments according to DOA recommended rates (Urea – 120 g, TSP – 80 g and MOP – 255 g per plant) and mixed with composting mixture. This was repeated at four and six months interval for relevant treatments. Application of PM and CD was repeated every six months into 30 cm deep furrows prepared with the radius of 60 cm away from the base of the plant and thoroughly mixed with topsoil for relevant treatments.

A composite soil sample was collected from the root zone (20 - 30 cm depth) before planting for site characterization. Composite samples of the composting mixture in the planting hole of each treatment were collected after each harvest and analyzed for organic C and total N contents using Walkley and Black (1965) method and Kjeldhahl method respectively and C/N was calculated. The central part of the fifth leaf including lamina and midrib was collected at flowering stage (Ray, 1988) from each treatment and analyzed for N by Kjeldhal method and K by flame photometric method.

At harvest, yield parameters such as number of fruits, hands and fruit weight per bunch were recorded. Fruit quality parameters such as fruit length and diameter were also recorded and flesh: peel ratio calculated at a similar ripening stage.

RESULTS AND DISCUSSION

Chemical characteristics of the soil

The soil at the experimental site was Red Yellow Podzolic with soft and hard laterites and slightly acidic with low total N, available P and organic matter contents (Table 1).

Table 1. Some important chemical characteristics of the soil at the experimental site.

Soil property Value

pH (1:1) water 5.10 Total N (%) 0.11 Olsen P (mg/kg) 1.60 Exch. K (mg/kg) 28.20 Organic matter (%) 1.13 60 JAYASUNDARA AND GUNATHILAKA

Concentrations of N and K contents in the leaves of six consecutive banana suckers

Effects of the treatments on of N and K contents in the leaves of six consecutive suckers are presented in Figures 1 and 2, respectively. 4

3 . 5

3

2 . 5

2

1 . 5

1

0 . 5 1 2 3 4 5 6 Sucker Number

Rec(4) Rec(4)+CD Rec(4)+PM Rec(4)+CD+PM

Chemical fertilizers applied at 4 months interval

4

3 . 5

3

% 2 . 5 N

f a

e 2 L

1 . 5

1

0 . 5 1 2 3 4 5 6

Sucker Number Rec(6)+CD Rec(6)+PM Rec(6)+CD+PM

Recommended fertilizers applied at 6 month intervals

Figure 1. Treatment effects on leaf N of six consecutive banana suckers.

Leaf N status

The leaf N in only recommended fertilizer added plants was drastically increased in the second sucker with application of fertilizers at four months interval and drastically reduced in the third and fourth suckers and with application of second dose of fertilizers increased in the fifth sucker. A similar trend was observed when only PM was integrated with recommended fertilizers. In contrast, leaf N content was consistent throughout the study period in CD treated plants. Incorporation of PM with CD showed further IN-SITU DECOMPOSITION OF ORGANIC MATERIALS ON BANANA 61 enhancement of leaf N ranging from 3.02 to 3.65 %. When chemical fertilizer was applied even at six months interval, leaf N declined drastically in only chemical fertilizer added plants whereas in CD and PM treated plants showed a sustained and higher N concentration in leaf tissues (Fig. 1).

Bhargava and Chadha (1993) observed the range of concentration of optimum leaf N for higher banana yields as 3.05% -3.35% and maintanance of this range was achieved satisfactorily by CD and PM mixture when fertilizer was applied at either four or six months interval.

4

3.5

3 )

% 2.5 (

K

f a

e 2 L

1.5

1

0.5 1 2 3 4 5 6

Sucker Number

Rec(4) Rec(4)+CD Rec(4)+PM Rec(4)+CD+PM

Recommended fertilizers applied at 4 month intervals

4

3.5

3

% 2.5 K

f a

e 2 L

1.5

1

0.5 1 2 3 4 5 6 Sucker Number Rec(6)+CD Rec(6)+PM Rec(6)+CD+PM

Recommended fertilizers applied at 6 month intervals Figure 2. Treatment effects on leaf K of six consecutive banana suckers. 62 JAYASUNDARA AND GUNATHILAKA

Leaf K status

Leaf K contents in consecutive six suckers also exhibited a similar trend as leaf N. Maximum leaf K was maintained consistently and reached an optimum level of 3.11% in the CD + PM treatment. Similar observations have been reported by Bhargava and Chandha (1993). Integrated use of chemical fertilizers with only CD showed a similar trend but leaf K levels were lower. In contrast, the chemical fertilizer only and in combination with PM treatments gave the lowest K levels (Fig. 2). Thus it is evident that leaf N and K maintenance was not possible when chemical fertilizer was integrated with either CD or PM at four or six months interval (Figs. 1 and 2).

Relationship between C/N ratio of composting mixtures and leaf N and K contents

The relationship between C/N ratio of the composting mixtures in different treatments and leaf N and K contents showed that there was a poor correlation in most treatments. However, a significant negative linear relationship was observed between leaf N content and C/N ratio when CD and PM were used (Fig. 3). Addition of fertilizer N and readily available N from PM promotes decomposition of the composting mixture resulting in the release of mineral N for quick uptake by the plant. As a result, with decrease of C/N ratio of the composting mixture, leaf N concentration increased (Fig.3). The pattern of increase was found consistent throughout the study period (Fig.1) and therefore, regular supply of mineral N was assured. On the other hand, a curvilinear relationship was observed between C/N ratio of the above mixture and the leaf K concentration. The most important factor here is to maintain the soil fertility status for a longer period of time in order to ensure higher and consistent banana yields. This could be achieved when organic materials, which have wide C/N ratio (100 - 200), are subject to slow decomposition. It has been reported that the gradual reduction of C/N ratio of composting mixture has a greater impact on N and K retention in the soil (Hansen et al., 1989). On the other hand, humic substances produced during decomposition of highly lignin containing materials such as CD, sow-dust are capable of retaining N, P and K in the soil for longer periods than fertilizer alone (Prasad and Singhania, 1989). Further, it was evident that, application of already composted organic material into the planting hole is less beneficial than materials, which have to be decomposed under the planting hole conditions (Neilson et al., 1994). This information are in agreement with the results of this study.

Overall results revealed that in-situ decomposition of CD and PM has a higher ability to retain N and K for longer period and their application may enhance the efficient use of applied chemical fertilizer. In addition, higher moisture retention capacity of CD promotes regular nutrient IN-SITU DECOMPOSITION OF ORGANIC MATERIALS ON BANANA 63 uptake by plants. On the other hand, application of PM increases exchangeable K and available N content in the soil. As a result, combined application of these two materials may provide an excellent composting material that behaves as a controlled or slow-release N and K supplying amendment for perennial fruit crops.

3.8

3.6 y = -0.0169x + 5.7964

) 2 % R = 0.8556

3.4 (

K K

) / / ) 3.2

%

( 3

N N

af af 2.8

Le 2.6 y = -0.0003x2 + 0.0897x - 3.674

R2 = 0.8109 2.4

2.2

2 120 140 160 180 200 C/N Ratio

% N Linear (% N) % K Poly. (% K)

Figure 3. Relationship between C/N ratio of substrate Rec(4)+CD+PM and concentrations of N and K in the leaf.

Crop yield

Yield parameters collected at the harvest of six consecutive banana crops are illustrated in Figure 4 and Table 2. 64 JAYASUNDARA AND GUNATHILAKA

800

700

600

500 s t i u r f

f 400 o . o

N 300

200

100

0

) 4 D D M M M M c( C C P P P P e )+ )+ )+ )+ + + R (4 (6 4 6 D D c c c( c( C C e e e e )+ )+ R R R R (4 (6 ec ec R R Treatment

Crop-1 Crop-2 Crop-3 Crop-4 Crop-5 Crop-6

Figure 4. Effect of treatments on total number of fruits in six consecutive bunches.

Table 2. Effect of treatments on fruit weight (kg)/bunch of six consecutive crop cycles.

Treatment Crop Total yield (kg) 1 st 2 nd 3rd 4 th 5 th 6 th

Rec(4) 8.6 cd 9.8 c 6.3 d 7.0 d 6.2 d 7.1 c 45.0 d Rec(4)+CD 9.4 c 10.8 b 9.8 b 10.6 b 9.7 b 9.9 b 60.2 b Rec(6)+CD 8.4 cd 9.2 c 9.0 bc 9.8 c 8.9 bc 9.1 bc 54.4 c Rec(4)+PM 9.5 c 10.3 b 9.1 bc 10.0 b 8.9 bc 9.1 bc 56.9 bc Rec(6)+PM 9.1 c 10.0 bc 8.5 c 8.2 bc 7.9 d 7.5 c 51.2 c Rec(4)+CD+PM 12.6 a 12.8 a 11.9 a 12.1a 11.8 a 11.6 a 72.8 a Rec(6)+CD+PM 10.9 b 12.2 a 11.9 a 12.0 a 11.1 a 11.0 a 69.1 a

CV (%) 18.5 16.2 21.5 17.3 15.5 19.2 15.4 Means followed by the same letter are not significantly different at the 0.05 probability level in the same column

Fruit weight per bunch obtained with the combined application of CD and PM with chemical fertilizers was superior to the other treatments and produced higher and consistent yields for six consecutive crops (Table 2). No significant yield reduction was observed when chemical fertilizers were applied either at 4 or 6 months interval together with CD and PM. On the other hand, yield was not consistent when only PM was applied with chemical fertilizers (Table 2). However, composting of only CD under in-situ conditions also promoted consistent yields but they were lower when IN-SITU DECOMPOSITION OF ORGANIC MATERIALS ON BANANA 65 compared to CD and PM combination. The chemical fertilizer only, control treatment, produced the lowest yields and was not consistent during the six crop cycles. The yield increase in CD and PM treated plants was found to be due to increase in number of fruits/bunch. (Fig. 4). Pinto (1986) reported that the yield increase in banana due to application of organic manure was mainly due to increase in number of fruits/bunch and fruit weight.

Cumulative fruit yield

Total number of fruits harvested during six crop cycles increased from 490 to 710 when chemical fertilizer was integrated with CD and PM. Addition of only CD with chemical fertilizer increased the number of fruits from 490 to 630 (Fig. 4). Total fruit weight in the six crops increased from 44 kg to 74 kg with the use of CD and PM compared to chemical fertilizer alone. Fruit yield increased by 16 kg when CD was incorporated with recommended chemical fertilizers. However, no significant yield difference was observed when chemical fertilizer was applied either at 4 or 6 months interval with CD and PM (Table 2).

Fruit quality parameters

The average fruit quality parameters for the six crop cycles are presented in Table 3.

Table 3. Fruit quality parameters of the first ratoon crop of banana with respect to treatments.

Treatment Fruit length (cm) Fruit girth (cm) Flesh:Peel ratio

Rec (4) 7.20 bc 9.90 c 4.60 b Rec (4) + CD 7.35 b 10.10 b 4.80 ab Rec (6) + CD 7.30 b 9.92 c 4.60 b Rec (4) + PM 7.65ab 11.21 ab 5.11 a Rec (6) + PM 7.32b 10.24 b 5.23 a Rec (4) + CD+PM 8.40a 11.61 a 5.28 a Rec (6) + CD+PM 8.36a 11.40 a 5.19 a CV (%) 28.6 23.3 20.6 Means of each column followed by the same letter are not significantly different at the 0.05 level.

Fruit length and girth increased significantly with application of CD and PM compared to the recommended fertilizer application. However, no differences were observed in fruit length and fruit girth when chemical fertilizer was integrated with CD and PM and applied either at 4 or 6 months interval. It appears that increase in fruit length and fruit girth is another factor that has contributed to yield increase. 66 JAYASUNDARA AND GUNATHILAKA

Cost effectiveness

The cost effectiveness of the different treatments for six crop cycles is illustrated in Figure 5.

Figure 5. Cost effectiveness of respective treatments.

The maximum and comparable net profit was observed when CD and PM were incorporated with recommended chemical fertilizer applied either at 4 or 6 months interval. The lowest was observed when only PM was incorporated with recommended chemical fertilizer applied at 6-months interval. However, application of CD with fertilizer gave an acceptable rate of return. The net benefit was increased from 1 million to 1.5 million/ha/six crop cycles when both CD and PM were incorporated with recommended chemical fertilizer.

CONCLUSIONS

The information generated by this study indicated that in-situ decomposition of 1:1 ratio of CD and PM along with recommended chemical fertilizers in planting holes provides an excellent medium in order to ensure regular and controlled supply of N and K for banana crop. Application of CD alone also regulates the N and K supply of added fertilizer. Higher and consistent yields with good quality characteristics are achievable if CD and IN-SITU DECOMPOSITION OF ORGANIC MATERIALS ON BANANA 67

PM are applied to planting holes together with recommended chemical fertilizers either at 4 or 6 months interval with higher economic returns.

ACKNOWLEDGEMENTS

Author wishes to acknowledge the assistance provide by Dr. R. Seneviratne (Chemist) and laboratory staff at Horticultural Crop Research and Development Institute and Coconut Research Institute by analyzing soil and leaf samples.

REFERENCES

Bhargava, B.S and K.L.Chadha. 1993. Leaf nutrient guide for fruit crops. Advance in Horticulture (New Delhi) 2: 973-1029. Chundawat, B.S. 2001. Integrated nutrient management in tropical and sub tropical fruits. Indian Journal of Horticulture 58(1): 59-69. Fujiwara, S.1998. Decomposition of poultry manure compost mixed with sow dust and its effect of application. Bulletin of the Kanagawan Horticulture Experiment Station 63(36): 1-100. Hansen, R.C, H.M. Keener and H.A.J. Hoitink. 1989. Poultry manure composting: an exploratory study. Transactions of the ASAE 32(6): 2151-2158. Jayasundara, J.M.P.B.1997. Use of coir-dust for agriculture. AGTEC in brief. Dept.of Agriculture, Sri Lanka. McConnell, D.B., A. Shiralipour and W.H. Smith. 1993. Compost application improves soil properties. Bio Cycle 34(4): 61-63. Neilson, G.H., B. Benlah, E.J. Hogue and R. Utkhede. 1994. Planting hole amendments modify growth and fruiting of apples on replanted sites. Horticulture Science 29(2): 82-85. Panabokke, C.R. 1967. The soils of Ceylon and use of fertilizer. Metro Ltd. Colombo, Sri Lanka.151p. Pinto, M.E.R. 1986. Response of “Embul” banana to manuring in increasing growth and yields. Proceeding. Annual Research Conference, Department of Agriculture, Sri Lanka. 24. Prasad, R.A. and R.A. Singhania.1989. Effect of different types of enriched manures and time of incubation on soil properties. Journal of the Indian Society of Soil Science. 37(2): 319-322. Ram, R.A, M.S.Rajput and S.R.Bhriguvanshi. 1999. Effect of controlled- released fertilizers on growth, yield and fruit quality of guava. Indian Journal of Horticulture 56(2): 104-111. Ray, D.P. 1988. Nutrient correlation in leaf tissues with different nutrient levels in banana. Indian Agriculturist 32(4): 249-256. Walky P. and Black C.A. Ed. 1965. Methods of soil analysis, Part 2, American Society of Agronomy. 223-254. Annals of Sri Lanka Department of Agriculture. 2006.8:69-77.

A COMPARATIVE ECONOMIC ANALYSIS OF HYBRID RICE AND SEED PRODUCTION IN SRI LANKA

S.N.JAYAWARDENA1, S.W.ABEYSEKERA1, K.D.S.KIRIWATHTHUDUWAGE1*, M.M.P.MUTHUNAYAKE1, S.BANDARA2 and C.SENENAYAKE3 1Rice Research and Development Institute, Batalagoda, Ibbagamuwa 2Agriculture Research Station, Girandurukotte, 3CIC, Seed Farm, Hingurakgoda

ABSTRACT

Rice Research and Development Institute (RRDI) has identified hybrid rice as one of the possible options to meet future rice demand in Sri Lanka. RRDI released its 1st rice hybrid variety, Bg407H, in 2005 for commercial cultivation. However, yield and economic advantage of hybrid rice cultivation as well as seed crop has not been studied. This study was undertaken to compare the yield, costs and returns of hybrid rice cultivation with those of inbred rice varieties and to estimate the achievable hybrid seed (F1) yield and cost of cultivation under large-scale seed production programs. Analysis was performed using the data reported in the National Co-ordinated Rice Varietal Testing (NCRVT) program and the reports submitted to varietal release committee (VRC) in 2005. Grain yield and cost of cultivation data were collected from a sample of 10 farmers at Girandurukotte, Ampara and Batticaloa who cultivated hybrid rice Bg407H as well as inbred varieties. F1 seed yields and cost of cultivation were estimated using the data collected from the large scale seed production plots established at Batalagoda, Girandurukotte and CIC farm at Hingurakgoda in maha 2005/2006 season. Hybrid rice has shown around 1.2 t/ha (21.6%) yield advantage over the inbred varieties. The cost of cultivation of hybrid rice (Rs. 65900/ha) is higher than the transplanted inbred rice (Rs. 62962/ha), but still profitable to go for hybrid rice cultivation. Girandurukotte was the best location for hybrid rice seed production. F1 seed yield was around 0.65 t/ha and the cost of cultivation was Rs. 95230/ha which is 1.7 times higher than that of inbred rice cultivation. The current cost of production of F 1 seed was Rs.147/kg and this could be substantially brought down by increasing the F1 seed yield through better synchronization of flowering of parental lines and by adopting labour saving techniques in crop establishment to lower the cost of production.

KEYWORDS: Cost of production, Hybrid rice, Seed production.

INTRODUCTION

Rice plays a major role in Sri Lankan agriculture as it is the staple food of nearly 20 million people in the country. It is a source of livelihood for approximately 800,000 farm families and more than 30% of the total labour force is directly or indirectly involved in the rice sector (Rajapaksa et al., 2000). Significant gains have been achieved in national rice production during the last two decades mainly through development and adoption of high- yielding varieties. However, in the recent past few years, national rice productivity has not increased substantially and it has come to a stagnation trend (Handawala 1994; Abeysekera and Abeysiriwardena, 2000). Present

* Present Address: Haleys Agro (Pvt) Ltd, Colombo. 70 JAYAWARDENA et al. national rice yield of 3.9 t/ha (Dept. of Census and Statistics, 2005) should be increased up to 4.8 t/ha to meet the demand by 2010 (Emitiyagoda and Weerasinghe, 2000). However, achieving the productivity targets in future is a challenge as resource base, especially land and water, is limiting.

Hybrid rice technology exploits the biological phenomenon of hybrid vigor (Hetrosis) to increase the yield potential of rice varieties through farmer’s use of F1 hybrid seeds. This technology has been successfully developed and commercialized in China since 1976. At present China cultivates about 50% of its total rice area with rice hybrids, which out-yielded inbred high-yielding varieties by a margin of 1.5 t/ha (IRRI, 2002). Vietnam and India also have succeeded in producing and cultivating rice hybrids (Dat Van Tran, 2002). Considering the future challenges of producing more rice with less land, less water and chemicals, the Rice Research and Development Institute (RRDI) identified hybrid rice as one of the options to increase rice yield beyond that of semi-dwarf inbred, especially under the irrigated ecosystem.

Sri Lanka started its hybrid rice research and development program in 1994 at RRDI, Batalagoda and has been able to identify several rice hybrids with 1.0-1.5 t/ha yield advantage over the best inbred grown under similar environment conditions (Abeysekera and Abeysiriwardena, 2000). After extensive efforts, RRDI released its first hybrid rice variety Bg407H in 2005 and seed production program has been started. High cost of F1 seeds has been one of the biggest constraints in the popularization of hybrid rice production in many countries (Dat Van Tran, 2002). Low seed yield due to poor seed setting in Cytoplasmic Male Sterile (CMS) lines and high cost of cultivation due to their demand for intensive management are two major reasons for high cost of F1 seeds.

This study was undertaken to compare the yield, costs and returns of hybrid rice cultivation with those of inbred rice varieties and to estimate the achievable F1 seed yield and cost of cultivation under large-scale seed production programs.

METHODOLOGY

Grain yield of hybrid rice variety, Bg407H and the test inbred rice variety, Bg387 was collected from the data reports of National Coordinated Rice Varietal Testing (NCRVT) program of RRDI and the reports submitted to VRC committee in 2005. NCRVT trials have been conducted at eight locations (Table 1) in different agro-ecological zones (dry, intermediate and wet zone) in yala 2004. However, grain yield data were collected only from the dry and intermediate zones used for this study as wet zone did not have very much potential for hybrid rice. Grain yield and cost of cultivation PROSPECTS FOR HYBRID RICE 71 data were collected from a sample of 10 farmers at Girandurukotte (4), Ampara (3) and Batticaloa (3) who cultivated hybrid rice Bg407H as well as inbred varieties. A structured questionnaire was used to collect the farm-level data on yield and cost of cultivation. A purposive sampling was used to select the respondents. The data on F1 seed yields and cost of cultivation were estimated from the large scale seed production plots established at Batalagoda (1 ha), Agriculture Research Station (ARS), Gindurukotte (5 ha), and Chemical Industries Colombo (CIC) seed production farm at Hingurakgoda (1.1 ha) during 2004-2006. T-test was performed to compare the yield between hybrid and inbreds in different locations.

RESULTS AND DISCUSSION

Yields of hybrid and inbred rice

Grain yields of hybrid and inbred varieties tested in NCRVT trials is given in Table 1. Out of eight NCRVT locations, except two locations at Girandurukotte and Ambalantota, in the other locations hybrid variety, Bg407H had given higher yields over the best inbred variety Bg357. The average yield of hybrid Bg407H tested in eight locations in NCRVT trials during yala 2004 was 6.3 t/ha and inbred rice yielded 5.6 t/ha in the best inbred test variety Bg357. The average yield advantage was 0.7 t/ha (12.5%) over the inbred rice variety (Table 1).

Table 1. Yields of Bg407H and Bg358 NCRVT trials conducted in the dry and intermediate zones of Sri Lanka during yala 2004.

Location Bg407H Bg357 Yield advantage Yield (t/ha) (t/ha) (t/ha) advantage (%) Aralaganwila 5.7 5.3 0.4 ns 7.5 Ambalantota 6.7 7.1 -0.4 ns -5.6 Batalagoda 5.5 4.0 1.5 ** 37.5 Girandurukotte 4.9 6.4 -1.5 ** -23.4 Mahailluppallama 5.0 4.9 0.1 ns 2.0 Paranthan 7.9 6.0 1.9 ** 31.6 Samanthurai 4.9 4.1 0.8 ** 19.5 Vavunia 9.7 7.1 2.6 ** 36.6 Mean 6.3 5.6 0.7 * 12.5 Source: Results of the NCRVT, 2004/05, Annual report of RRDI, 2005. *, ** are 5% and 1% probability levels of significance respectively. ns- Not significant.

Hybrid variety, Bg407H recorded higher yield over the inbred in all farmers’ fields. The average yields of hybrid and inbreds were 6.8 t/ha and 5.2 t/ha respectively (Table 2). Yield advantage of hybrid was 1.2 t/ha (30.7%). The overall yield advantage of hybrid was calculated as a mean of yield of NCRVT and farmer’s field yield. Therefore, the overall advantage of hybrid was 1.2 t/ha (21.6%). These results clearly demonstrated the higher 72 JAYAWARDENA et al. yield potentials of hybrid variety, although considerable variation in grain yield occurred across the locations.

Table 2. Yield comparison of hybrid and inbred in farmers’ fields in yala 2005.

No of Plot size Yield Yield of inbred Yield advantage Location farmers of the (t/ha (t/ha) (t/ha) (%) hybrid ) rice (ha) System C Hobariyawa 2 0.5 6.4 5.2 (Ld 356) 1.1 ** 7.5 Swasakthipura 2 0.5 8.4 4.2 (Bg 358) 4.2 ** 52.3 Batcoaloa AkkaraiPatthu 2 0.5 7.1 6.5 (Bg 450) 0.6 * 9.2 Nindavur 1 1 8.1 7.2 (Bg 403) 0.9 ** 12.5 Ampara Rajagama 1 0.3 5.2 4.3 (Bg 379/2) 0.9 ** 20.9 Ruhunugama 1 0.5 6.9 4.8 (Bg 357 ) 2.1 ** 43.7 Tissapura 1 0.5 6.2 4.7 (Bg 357) 1.5 ** 31.9 Mean 6.8 5.2 1.6 ** 30.7 Source: Report submitted to VRC committee on Bg407H, Annual report of RRDI, 2005. *, ** are 5% and 1% probability levels of significance respectively. ns- Not significant. Farmer’s varieties are in parentheses

Cost of production of hybrid and inbred rice

Costs of cultivation of inbred rice under direct seeding, transplanting and hybrid under transplanting were Rs. 54,552, 62,962 and 65,038/ha respectively (Table 3). The profit derived from the same was Rs. 24,198, 23,288 and 30,407/ha respectively.

Table 3. Yield (t/ha), cost of cultivation and profit for inbred, hybrid rice and F1 seed production-during maha 2004/05.

Description Direct Transplanting Transplanting F1 seed seeding (inbred rice) (hybrid rice) production (inbred rice) Labour cost* (Rs/ha) 28,023 39,360 39,360 56,960 Machinery cost (Rs/ha) 11,232 11,232 11,232 11,590 Input cost (Rs/ha) 15,217 12,370 14,645 26,680 Total cost of cultivation 54,552 62,962 65,038 95,230 (Rs/ha) Labour units (Man 88 123 123 178 days/ha) Yield** (t/ha) 52,50 5750 6363* 0.65 Gross return*** (Rs/ha) 78,750 86,250 95,445 14,9790 Profit (Rs/ha) 24,198 23,288 30,407 54,560 Cost of production 10.39 11.45 10.22 146.50 (Rs/kg) PROSPECTS FOR HYBRID RICE 73

*Wage rate at Rs.320/day ** Calculated hybrid rice yield is 21.6 % higher than the inbred rice (average yield of NCRVT & Farmer’s field) *** paddy Rs.15/kg This shows that direct seeding of inbred rice is more profitable than that of transplanting. If a farmer wants to adopt transplanting he needs to invest additional Rs.8490/ha. Almost all the additional cost accounts for extra labour required for nursery management and field establishment. However, yield advantage due to transplanting was 500 kg/ha (Rs. 7,500/ha) which is not adequate to cover the extra cost. Therefore, finding alternative methods for manual transplanting is essential to cut down the labour cost. Seedling broadcasting is one of the possible options which require comparatively less amount of labour for nursery management and establishment (Jayawardena et al., 2004). Total cost of cultivation of hybrid rice is around Rs.2500 higher than that of inbred. The additional cost in hybrid rice accounts for hybrid seed paddy. But, still generate higher profit than the inbred rice due to its ability to produce higher yield.

Returns of hybrid rice cultivation

Adoption of a new hybrid/inbred variety by the farmers would be determined by the relative profitability over the inbred varieties. Three basic factors determine the relative profitability of a new hybrid variety over the currently cultivated inbred varieties: grain yield, additional input cost, and market price. The estimated yield advantage of hybrid rice was taken as 21.6 % (1.1 t/ha). This yield advantage of hybrids is comparable with the yield advantages (20-25%) recorded from countries such as Bangladesh, India, Philippines and Vietnam (IRRI, 2002). The grain quality of hybrid rice was comparable with popular inbred rice varieties. Therefore, hybrid rice is also priced as inbred rice. Assuming an average yield advantage of 1.1 t/ha, the additional income derived from hybrid rice cultivation is Rs. 16500/ha. This is about Rs. 7119/ha extra income from hybrid rice cultivation over the inbred rice cultivation under transplant conditions (Table 3). The increase in income is about 31 % which is adequate for the farmers to shift from inbred to hybrid rice cultivation.

F1 seed yield

Estimated F1 seed yields at Batalagoda, Girandurukotte and Hingurakgoda were 0.64, 0.72 and 0.59 t/ha respectively. The overall mean yield was 0.65 t/ha which is lower than the previously recorded yield of 2.06 t/ha under experimental conditions (Kiriwaththuduwage et al., 2004). Low yield was mainly due to the poor synchronization of CMS and pollen parent (R) lines, where, CMS lines flowered earlier than R lines. If proper synchronization took place, higher yield would have been achieved due to the availability of higher pollen load from R lines as the plot sizes were larger. Low seed yield was observed in all the locations due to the same reason. In an 74 JAYAWARDENA et al. earlier study (Kiriwaththuduwage et al., 2002) it was found that Girandurukotte was the best location for hybrid rice seed production. This could be due to its favorable environmental conditions for higher seed setting.

Other than the F1 seed yield, additional paddy yields of 1.4, 1.9 and 1.6 t/ha were harvested from R lines respectively from Batalagoda, Girandurukotte and Hingurakgoda locations. The overall average yield received from the R line was 1.6 t/ha. The paddy grain quality of the R line is comparable with recommended inbred varieties and, therefore, it can be considered as an additional benefit from the hybrid rice seed production plots. As in F1 seed production, the highest R line yield was recorded from Girandurukotte.

Cost of cultivation of hybrid rice seed (F1) production

Cost of cultivation of hybrid seed production was estimated based on F1 seed yield of 0.65 t/ha (averaged over the three locations, Batalagoda, Girandurukotte and Hingurakgoda). The computed cost of cultivation of hybrid rice seed production was Rs. 95,230/ha (Table 4) which is Rs. 40,678 (73.2%) higher than that of transplanted inbred rice. Among all components, labour alone accounted for about 60 % of the total input cost because of additional labour requirement in seed production. The cost of Gibberellic acid (GA3) is 13%. GA3 is sprayed at flowering to enhance elongation of cells and thereby increases stem elongation in CMS rice resulting in complete exertion of panicle out of the flag leaf sheath and thus increasing outcrossing. This suggests that finding alternatives to cut down the labour, such as seedling broadcasting and GA3 would help reduce the cost of cultivation.

Based on the above total cost of production and seed yield, cost per kg of hybrid rice seeds was estimated at Rs. 147. This is about five times the cost of inbred rice seed production.

Grain quality of R line was as good as other inbred varieties and could be valued as a normal inbred variety and, therefore, an additional income generated from R line was Rs. 24,000/ha. Thus, including additional income generated from the R line, a net profit of Rs. 71,000/ha could be obtained from hybrid rice seed production. The profit generated from hybrid rice seed production is Rs. 54,560 or 2.25 times higher than that of inbred rice cultivation. Therefore, production of hybrid rice seed would be more attractive to the producers if there is an increasing and steady demand. PROSPECTS FOR HYBRID RICE 75

Table 4. Cost of cultivation per hectare for hybrid rice (F1) seed production.

Operation Labour Labour Mechiner Material Total (man days) cost(Rs) y cost cost cost Nursery preparation 10 3200 3200 Land preparation 8590 8590 Clearing & plastering 13 4160 4160 Leveling & lay out 8 2560 2500 5060 Transplanting R line 9 2800 2800 Transplanting CMS 20 6400 6400 Weed control 17 5440 2750 8190 Fertilizer application 3 960 7790 8750 GA3 application 2.5 800 12000 12800 Pollination 2.5 800 800 Roughing 15 4800 4800 Pest & disease control 3 960 1640 2600 Water management 14 4480 4480 Harvesting R line 8 2560 2560 Harvesting CMS line 30 9600 9600 Threshing, Winnowing 23 7360 3000 10360 & transport Total 178 56960 11590 26680 95230

It is hoped that the present F1 seed yield could be increased with the advancement of seed production technologies as adopted in other countries (Mao et al., 1998). Breakeven prices for different levels of seed yields were estimated (Table 5). These results revealed that there is a possibility of bringing down the present cost of production of hybrid rice seeds considerably through the advancement of F1 seed production technologies for which seed production research component of the hybrid rice research program should be strengthened.

Table 5. Unit cost of hybrid rice seed at different seed yield levels.

Yield level (kg/ha) Cost of production (Rs./kg) 500 190 750 127 1000 95 1250 76 1500 63 2000 48 2250 42 76 JAYAWARDENA et al.

CONCLUSIONS

Hybrid rice produced nearly 1.0 t/ha (22 %) higher yield and generated around 30% more higher profit than that of presently cultivated best inbred rice varieties under transplanted conditions. Profitability could be increased further by adopting labour saving techniques such as seedling broadcasting. Among the tested locations, Girandurukotte was found to be the best for F1 seed production. At present, around 0.7 t/ha hybrid rice seed F1 yield could be obtained under large-scale seed production programs. However, this F1 yield could be further increased through better synchronization of parental lines. The cost of production of F1 seed is Rs.95230/ha which is 80 % higher than that of inbred rice cultivation under transplanted conditions. The current cost of production of F1 seed is Rs.147/kg which could be brought down substantially by increasing F1 seed yield as well as by adopting labour saving techniques and substituting for high cost GA3. These results suggest that the cultivation of hybrid rice and production of hybrid rice seed is more profitable than the inbred rice cultivation. There exists a possibility to lower the F1 seed cost.

REFERENCES

Abeysekera, S.W. and D.Z.De. Abeysiriwardena. 2000. Recent development in hybrid rice research in Sri Lanka. Proceedings of the Annual Symposium of the Department of Agriculture, Pp 9-17. Abeysekera, S.W., D.Z.De. Abeysiriwardena and D.B. Weeratunga. 2002. Status, progress, policies and expectation from the hybrid rice research and development in Sri Lanka, Proc. of the workshop on policy support for rapid adoption of hybrid rice on large-scale production in Asia, Hanoi, Vietnam. 22-23 May 2001. Pp 107-121. Dat Van Tran. 2002. Hybrid rice for food security: Recent progress and large-scale production issues. Proceeding of the workshop on policy support for rapid adoption of hybrid rice on large-scale production in Asia. Hanoi, Viet Nam, 22-23 May 2001.Pp 17-35. Emitiyagoda, G.A.M. S. and S. Weerasinghe, 2000. Rice productivity enhancement in Sri lanka: Yaya (Tract) program. Proceedings of the Annual Symposium of the Department of Agriculture, Pp 395-408. Handawala, J. 1994. Soil quality: Assessment of degradation and restoration. Pro. of the workshop on soil quality of Sri Lanka. Sri Lanka Foundation Institute, Colombo, 23-25, March 1994, p 35-38. International Rice Research Institute. 2002. Terminal report of IRRI/ADB project. Development and use of hybrid rice in Asia. 10p. Jayawardena, S.N., S.W. Abeysekera., K.D.S. Kiriwaththuduwage, H.M.J.K. Herath, C.S. Silva,; S. Herath and P. Dissanayke. 2004. Seedling-broadcasting as an alternative method to manual transplanting in hybrid rice cultivation. Annals of the Sri Lanka Department of Agriculture 6:277-281. PROSPECTS FOR HYBRID RICE 77

Kiriwaththuduwage, K.D.S., S.W. Abeysekera and D.Z.De. Abeysiriwardena 2002. Hybrid rice seed production as influenced by location and season in Sri Lanka. Annals of the Sri Lanka Department of Agriculture 6:283-286. Kiriwaththuduwage, K.D.S., S.W. Abeysekera , D.Z.De. Abeysiriwardena, S.N.Jayawardena and M.U.N. Pushpakumara. 2004. Influence of Gibberellic acid (GA3) application on hybrid rice seed production under experimental conditions. Annals of the Sri Lanka Department of Agriculture 6:283-286. Mao, C.X., S.S. Virmani and I. Kumar. 1998. Technological innovations to lower the cost of hybrid seed production. In S.S. Virmani, E.A. Siddiq and K. Muralidharan, eds. Advances in hybrid rice technology. IRRI, Phillippines. Pp 111-128. Rajapaksa, R.M.T, C.A. Sandanayake and B.D. Pathinayake. 2000. Foot prints in rice variety improvement and its impact on rice production in Sri Lanka. Proceedings of the Annual Symposium of the Department of Agriculture, Pp 423-433. RRDI, 2005, Annual report. Batalagoda, Sri Lanka. Virmani, S. 1994. Heterosis and hybrid rice breeding. Spinger-Verlag, New York. Pp 94-106. Annals of Sri Lanka Department of Agriculture. 2006.8:79-90.

WATERSHED BASED SOIL CONSERVATION PLANNING USING GIS AND REMOTE SENSING

H.K. KADUPITIYA1 and P. MADANA2 1Horticultural Crop Research and Development Institute, Gannoruwa, Peradeniya 2Extension and Training Centre, Peradeniya

ABSTRACT

Human induced land degradation and its consequences are an increasing threat to sustainable land utilization in Sri Lanka. Therefore, arresting land degradation through land resource management is the immediate challenge, which necessitates proper planning. Although watershed based conservation approaches are the best, in many instances they have not been practiced. Conservation planning aiming at sustainable development of slopy lands requires assessment of the present status of land and mapping priority areas based on their susceptibility to land degradation. This research was a case study carried out in 185 km2 at Doragala-Kothmale area in the central highlands with the view of generating required information in the form of a GIS map for watershed based soil conservation planning, using GIS and remote sensing. Watersheds and sub-watersheds within the study area were delineated based on terrain and its runoff generating characteristics. Erosion prone areas were identified using a cumulative erosion index which was computed from the ratings given to three main soil erosion causative factors ie. land use/cover, slope and soil erodibility. Rainfall erosivity was not considered since the study area was not extensive to observe any substantial variation of the same. Multi-spectral IRS LISS III satellite image acquired in March 1998 was used to run a supervised classification for identifying land use/cover types. The land cover of the study area was classified into five land use/cover classes: Agriculture, Forest, Pinus, Grass and Tea. Moreover, the management levels of both forest and tea land use classes were differentiated using Normalized Vegetation Index (NDVI). Further, an index for land use was developed considering the susceptibility of land use/cover classes to erosion. Soil erodibility index was assigned for each soil type according to the erodibility values. An index of slope was derived based on slope steepness, calculated from Digital Elevation Model. The information on slop, erodibility index, land use, land cover and management were combined and a GIS map was developed. The sub watersheds in the study area were prioritised for soil conservation planning based on their relative susceptibility to erosion. The study confirmed that application of GIS and RS techniques to generate geographic information required for watershed based land management and conservation planning is possible particularly under the situations where financial resources are limited.

KEYWORDS: Conservation, GIS, Remote sensing, Soil erosion, Watershed.

INTRODUCTION

Land degradation and its consequences have been identified as an increasing threat to sustainable agriculture in Sri Lanka (Nayakekorala, 1998). Sri Lanka is not endowed with unlimited land resources and hence it should be utilised carefully while maintaining the productive capacity. The watershed based planning is the best recommended approach to harmonize the 80 KADUPITIYA AND MADANA use of soil, water and vegetation in a way that conserves these resources and maximizes their productivity (Bharat and Scott, 2005). Although the watershed is the appropriate hydrological unit for conservation planning, man made land boundaries have been used for many soil conservation programs. That may be associated with resource limitation. Under the situations where resources are limited, they should be allocated for implementation of conservation programs assigning priority based on soil erosion vulnerability assessments. Hence, watershed based conservation planning in diversified slopy areas requires assessing the present situation of land and mapping priority areas based on their susceptibility to land degradation and delineation of watersheds and sub watersheds.

Generation of information and subsequent characterisation of watersheds are prerequisites for effective watershed based conservation planning. Geographic information systems (GIS) and remote sensing can effectively be used for conservation planning as they provide facilities for rapid assessment of the more accurate and real-time information on land and water resources (Lillesand and Kiefer, 1994).

This research is a case study carried-out at Doragala-Kothmale area located in Mid country of Sri Lanka with the overall objective of generating and mapping information on erosion vulnerability of the land and prioritise watersheds for conservation according to the erosion severity.

The study area covers an extent of 18500 ha and lies between latitude 7°02´ to 7°10´ N and longitude 80°34´ to 80°42´ E in Kandy district. The area spreads into the Southeastern side from Gampola and it includes the surrounding area and north side of the Kothmale reservoir. Larger part of the study area falls into WM2 agro-ecological zone and the rest falls into WU2 agro-ecological zone (Agro-ecological map, 1976). Forest, grasslands, upcountry vegetables and tea are the prominent land use/cover types in the area. The great soil groups found in the study area are Immature Brown Loams, Mountain Regosols, Red Yellow Podsolic and Reddish Brown Latosolic (Panabokke, 1996).

MATERIALS AND METHODS

Indian Remote Sensing (23.3 m grid sized IRS LISS III) satellite image acquired in March 1998, topographical map at the scale of 1:50,000 of Survey Department and the general soil map (Panabokke, 1996) were used to extract necessary information for watershed based conservation planning.

Land cover is one of the major factors which influence the soil erosion status of the land. Erosion levels under different land uses vary from REMOTE SENSING AND GIS FOR SOIL CONSERVATION PLANNING 81 negligible to high (Table 1) in accordance with their characteristics, for instance, ground cover and canopy cover.

Since the land use has been changed drastically over the past few years, instead of using existing land use data in topographical maps of the Survey Department, recently acquired satellite image was used for assessing land use and land cover characteristics. Supervised classification algorithm was run in raster-based ILWIS environment for multi-spectral IRS satellite image based on training data that were collected through fieldwork to prepare land use/cover map of the study area. Five land use and cover classes: Seasonal Agriculture, Forest, Pinus, Grass, and Tea were considered for land use classification. Moreover, the management levels of both forest and tea land use classes were differentiated into two classes using Normalized Vegetation Index (NDVI) which represents the healthy vegetation cover (Dhruba el al., 2001). Forest cover was reclassified into dense forest and open forest. Tea cover was reclassified into well-covered tea and poorly covered tea.

Table 1. Erosion status, index and area percentage under land use/cover.

Land use/cover Area % Erosion status* Index Agriculture 12.8 High 5 Dense forest 8.1 Negligible 1 Grass 38 Low- medium 3 Open forest 11.3 Low 2 Pinus 4.1 Medium – high 4 Poorly covered tea 17.7 Medium – high 4 Water logged area 0.9 Negligible 1 Well covered tea 7.1 Negligible 1 *Source: Nayakekorala (1998)

Determination of slope classes

Elevation contours at 20 m intervals were extracted from scanned topographical maps through screen digitising. Digital Elevation Model (DEM) was developed using contour interpolation techniques and the pixel size of the DEM which is 25 m was adjusted to be almost the same as the resolution of IRS data. Slope map of the area was generated considering the elevation difference of neighbouring pixels and ground pixel size of DEM.

Delineation of watersheds

Drainage lines have been extracted in grid based Arcinfo 8.5 software, using flow direction of each grid cell. Using the drainage map and flow direction map, watersheds were delineated considering the flow contributing cells to the lowest point of each drainage line. 82 KADUPITIYA AND MADANA

Data analysis

Index for land use/cover was assigned with regard to the erosion status of each land use/cover type. The slope map was reclassified into seven slope (percent) classes 0-10, 10-20, 20-30, 30-40, 40-50, 50-60 and >60. Each percentage slope class was given a slope index value on a rating of 1 to 5. Soil erodibility index was defined based on erodibility of each soil type. Index values have been adjusted to get a maximum of 5.

Identifying erosion prone areas

Only three causative factors namely slope, soil erodibility and land cover/use were considered for identification of erosion prone areas. Since the study area was not extensive to observe any considerable variation of rainfall, erosivity factor was not considered. Susceptibility of land to erosion was estimated based on cumulative index of these three main causative factors. Cumulative index was calculated spatially by adding above- mentioned three indices. The study area has been divided into five equal areas considering the erosion status (negligible, low, moderate, high and very high). Noise reduction or the smoothening was done by applying a 3 by 3 majority filter on the erosion status map.

Prioritisation of sub watersheds

Sub-watersheds of the study area for conservation planning were prioritized considering the extent subjected to high and very high erosion. They were classified into four priority levels.

Extraction of watershed characteristics for conservation planning

Following information of watersheds was extracted using the analytical capabilities of ILWIS: a) Extent and the distribution of different land use/cover types b) Spatial distribution of slope and extents under each slope class c) Erosion potential d) Distribution and lengths of drainage lines

RESULTS AND DISCUSSION

Field verification indicated that the overall accuracy of land use/land cover classification of IRS image was more than 80%. Classification accuracy of pines is higher than 95%. Distinguishing grasslands from croplands was difficult, thus the classification accuracies were low. Poor separation of these two classes was associated with their ground cover REMOTE SENSING AND GIS FOR SOIL CONSERVATION PLANNING 83 conditions that prevailed in March. Agricultural lands were mostly covered by dry shrubs, grasses or occasionally consisted of patches with bare soil after the harvesting. However, the classification accuracies of the grasslands and croplands can be improved by incorporating an additional image acquired in late December or early January, as it is the period in which the agricultural lands are in full use in the area of study. Classification results show that larger extent (38%) of the area is covered by grass, and the Plate could be an overestimation due to the confusion with harvested agricultural lands.

The extent of poorly covered tea is nearly 18% of the total land area. However, this category includes both poorly managed tea lands and also pruned tea lands at the time of image acquisition. However, past records of respective tea lands revealed that there were no pruned tea lands within the study area at the time of image acquisition, thus the poor cover is totally due to poor management. Therefore, this information provides a valuable clue for a planner to identify and prioritise the areas when launching crop productivity enhancement programs. Besides, the erosion status of these tea lands ranged between medium to high, hence it needs attention. The details of land use/cover of the study area are presented in Plate 1 and Table 1.

Plate 1. Land cover map derived from IRS LISS III satellite image.

As shown in Plate 2, the elevation of the study area ranged between 400 - 1900 m from the mean sea level, with the highest elevation to the east and the lowest to the northwest. The study area was sub-divided into 186 sub-watersheds of which the extents ranged from 13 to 286 ha (Plate 3). 84 KADUPITIYA AND MADANA

Although in this particular study the area was divided into 186 sub-catchments, GIS allows the user to go for any detailed level of micro- watershed, depending on the feasibility of implementation.

The map indicating percentage slope classes is presented in Plate 4. Percentage slope classes, area and slope index are given in Table 2. Field accuracy check for slope percentages showed that the slope map derived from 20 m-interval elevation contours gives reliable results when considering the average slope percentage of each pixel. The results revealed that, more than a half of the area (52%) falls into the 10 - 30% slope category while the area with the slope of 30 % or higher is nearly 37 % of the total area.

Table 2. Slope class, area and the slope index.

Slope class (%) Area (%) Rating Slope Index < 10 10.2 1 0.71 10 – 20 24.4 2 1.43 20 – 30 27.7 3 2.14 30 – 40 14.3 4 2.86 40 – 50 9.4 5 3.57 50 – 60 4.7 6 4.29 > 60 9.3 7 5

Plate 4. Slope classes (slope percentages) of the area. REMOTE SENSING AND GIS FOR SOIL CONSERVATION PLANNING 85

Plate 2. Digital elevation mode (Elevation in meters).

Plate 3. Sub-watersheds and drainage network of the study area. 86 KADUPITIYA AND MADANA REMOTE SENSING AND GIS FOR SOIL CONSERVATION PLANNING 87

Plate 6. Priority watersheds for conservation planning.

Plate 7. Extracted characteristics for a selected micro-watershed. 88 KADUPITIYA AND MADANA REMOTE SENSING AND GIS FOR SOIL CONSERVATION PLANNING 89

As shown in Table 3, the variability of the soil erodibility within the study area is minimal due to the dominant coverage (95%) of the same soil group, which is Red Yellow Podzolic (RYP). Area under other soil groups is very small and belongs to the lower slope class ranges as well.

Table 3. Erodibility, index and area percentage of each soil type.

Soil type Area % Erodibility* Index Immature Brown Loams 2 0.27 5 RYP (with Mountain Regosols) 22 0.22 4.1 RYP 73 0.22 4.1 Reddish Brown Latosolic 3 0.17 3.1 * Munasinghe, 2002

Total area was divided into five categories of erosion status, which are namely negligible, low, moderate, high and very high as shown in Plate 5. Prioritised micro-watersheds based on the extents prone to high and very high erosion are presented in Plate 6. As shown in Table 4, 34 micro watersheds, which totally form an extent of 22% of the total study area belong to first priority category and draws the foremost attention when planning and implementing conservation programs. The second priority group covers 30% of the extent while 22% and 26% of the total extent fall under third and forth priority group, respectively.

Plate 5. Classification of the area according to the susceptibility to erosion.

Extracted information including extents and distribution of different land use/cover, spatial distribution of slope, erosion potential and, locations and lengths of drainage lines of a selected micro-watershed for conservation planning is shown in Plate 7. Drainage network was finalized after the adjustments based on field accuracy checks. 90 KADUPITIYA AND MADANA

Table 4. Priority areas and sub-watersheds for conservation planning.

Number of Area (ha) Area % Priority level sub-watersheds 1 34 3794 22 2 49 5278 30 3 45 3904 22 4 59 4590 26

CONCLUSIONS

Information extraction for watershed-based soil conservation is convenient in a GIS environment. Remotely sensed data provide considerably accurate information on land use and land cover types and their spatial distribution. The land use and land cover data extracted from IRS satellite images by unsupervised classification can effectively be used for index-based erosion modelling. Digital elevation model created by interpolating elevation contours at 20m intervals can be used to develop slope class map of sloping highlands with reasonable accuracy. Automated drainage line estimation followed by limited field verification provides realistic base for off-farm conservation especially for riverbank and gully conservation planning. Algorithm for watershed delineation can be performed automatically within a GIS, applying the principle of flow contributing cells to any predefined flow outlet located along the drainage network. Simple index-based erosion modelling approach using soil erosion causative factors covering slope and soil erodibility appears to be a practical method for identification of erosion- prone areas in sloping highlands. The delineation of micro-watersheds and presentation of results on micro-watershed basis provide the necessary framework for implementation of development programs on watershed basis.

REFERENCES

Department of Agriculture. Agro-meteorological map of Sri Lanka. 1976. Land Use Division. Sri Lanka. Bharat, R.S. and C. A. Scott. 2005. Watershed Management Challenges: Introduction and Overview In Watershed Management Challenges Improving Productivity, Resources and Livelihoods Eds. B.R. Sharma et al. IWMI. Sri Lanka 1-21. Dhurba, P.S. and J. A. Zinck. 2001. Land use classification in mountainous areas: Integration of image processing, digital elevation data and field knowledge (application to Nepal). International Journal of Applied Earth Observation and Geo- information 3(1):78-85. Lillesand, T.M. and R.W. Kiefer. 1994. Remote sensing and image interpretation. John Willey & Sons. New York. 749 p. Munasinghe, M.A.K. and S.M.V. Puspakumara. 2002. Identification of erosion prone areas for delineation of conservation areas in Sri Lanka (unpublished). Panabokke, C.R. 1996. Soils and agro-ecological environment of Sri Lanka. Natural Resources Series No 2. NARESA. Sri Lanka. 207 p. Annals of Sri Lanka Department of Agriculture. 2006.8:107-114.

SUCCESSFUL DEVELOPMENT OF INBRED LINES AND HYBRIDS OF MAIZE (Zea mays L.) AT MAHA ILLUPPALLAMA

K.M. KARUNARATNE Field Crop Research and Development Institute, Maha Illuppallama

ABSTRACT

Field Crop Research and Development Institute, Maha Illuppallama, initiated a Maize hybrid development program in 1998 using inbred lines from International Maize and Wheat Improvement Centre (CIMMYT), Mexico, to meet the rising hybrid seed demand, which is currently being met by importing hybrid seed by the private sector. Initially 165 single crosses were developed using CIMMYT inbred lines and one promising hybrid (KH 86) was released for farmer cultivation in 2004. This work was further strengthened by a local inbred line development program, initiated subsequently. Twelve source materials with diverse origin were used for development of 45 lines by self-pollination for seven generations. Hybrids were developed between non-sister lines and evaluated in order to find better genetic combinations. Test-crosses too were developed using a promising Mexican inbred line as the pollen parent to determine differences in genetic potential between the lines (combining ability). Selected hybrids were compared with parents and controls to estimate standard heterosis, heterobeltiosis and commercial heterosis. Based on five seasons of testing, 35 promising hybrids, which were found out-performing or in par with local hybrid and exotic commercial hybrids were selected for further testing for multi-location adaptation.

KEYWORDS: Heterosis, Inbred lines, Single crosses, Test crosses.

INTRODUCTION

Maize occupies the largest area (30,000 - 50,000 ha per annum) among the crop group Other Field Crops in Sri Lanka. It is the second most important cultivated cereal and the foremost feed grain in Sri Lanka. The current annual requirement of maize seed for animal feed is about 200,000 t, with imports of about 150,000 t, worth of over rupees 2500 million. To achieve self-sufficiency in maize, country has two options, namely extensive approach and intensive approach, where improved technology, including cultivars bear a high significance.

To ensure a healthy grain yield, operations such as, selection of suitable cultivars, early planting, drainage, tillage practices, balanced fertility and timely cultivation and harvest were found important. Rising income in much of the developing world and the consequent enhanced demand for meat and poultry products have resulted in a rapid increase in the demand for maize as a livestock feed. As a result, the world maize consumption has changed during the period 1982-84 to 1994-1996, from 91 to 98 kg/yr/head. Malawi 108 KARUNARATNE has the highest per capita consumption of 137 kg/year of maize in the world, followed by Mexico with 127 kg/year (Michael and Pandey, 1998). In the past 40 years, total maize production in many countries in the world, including USA and China, has increased by several fold due to cultivation of hybrids while land area devoted to the crop has virtually decreased. Shull (1908) was the first author to report increased yields from F1 crosses between inbred lines, which is a result of strength of stalks and of roots, and resistance to specific diseases and insect pests etc. The cumulative interaction of many favourable dominant or partially dominant genes is the cause of hybrid vigour in hybrids.

A program of inbred line development was initiated at Field Crop Research and Development Institute, Maha Illuppallama to develop more adaptable inbred lines and hybrids with higher yield potential over exotic hybrids to substitute imports of hybrid seed upon which currently heavy expenditure is being made. The high heterosis displayed by the hybrids, in relation to mid- parent (heterosis), better parent (hetero-beltiosis) and over commercial hybrid (commercial heterosis) justify further evaluations of the lines and hybrids, from which the most superior ones are expected to be nominated for national release in the near future.

MATERIALS AND METHODS

Development of maize inbred lines initiated during maha 1998/99, for which 12 different source materials of diverse origin were selected. Promising CIMMYT and local germplasm were used as source materials (FCRDI, 1999-2000). The standard method was adopted for line development in which selections were carried out in inter and intra row progenies. During maha 2000/01, 18 single cross hybrids were developed as early generation hybrids among selected S3 lines. Ruwan and Pacific 11 were included as local checks in an experiment conducted under Randomized Complete Block Design (RCBD) with 4 replicates, designed for comparison of performances of the hybrids. Two rows of 5.0 m length were planted for each treatment at recommended spacing of 60cm between rows and 30cm within the row. To compare heterosis, seven selected non-related single cross hybrids were compared with their parents and Pacific 11, the commercial check, during maha 2001/02, as a further step. Data were collected on days to 50% flowering, plant and ear height, and individual plot yields. During maha 2002/03, 120 single crosses developed with more advanced lines were tested in 3 experiments. During maha 2003/04, 60 selected hybrids were tested in 3 experiments. Test-crosses were developed during yala 2003 and were tested in replicated trials conducted at Maha Illuppallama for 3 consecutive seasons, for studying the combining ability of the lines. Further evaluations are underway by developing new hybrids and test-crosses to obtain consistency of favorable performance of the inbred lines and their combinations. Thirty-five such combinations are seed multiplied during maha 2005/06 for INBRED LINE DEVELOPMENT 109 multi-location testing and in Variety Adaptability Testing in the seasons ahead. Performances of individual experiments are tabulated below.

RESULTS AND DISCUSSION

Grain yields of the early generation hybrids during maha 2000/01, were highly encouraging. The grain yields were in the range of 3.50 - 7.25 t/ha, eleven hybrids performing better than the local open pollinated check variety, Ruwan and nine hybrids out-performing the commercial exotic hybrid Pacific 11. Flowering of the lines varied between 57 to 62 days and the maturity reached in 105-110 days after seeding (Table 1). Plant height ranged between 210 to 260 cm, the tallest contributing the highest grain yield. In general, the longer the time from planting to flowering, the greater the plant height and the greater the grain weight/plant (CIMMYT, 1985). Ear placement was satisfactory in all hybrids except in one, which had a higher placement of ears of over 50% on the stem. The performance of hybrids was highly satisfactory and hence advancement of line development and selection was justifiable and substantial.

Table 1. Performance of early generation hybrids developed with S3 lines in RCBD trial conducted at FCRDI, Maha Illuppallama during maha 2000/01.

Hybrid Yield Days to Palnt height Ear height (t/ha) flowering (cm) (cm)

P36-5-2 X Ki 31-4 7.27a 58 fg 260 a 107 bcde P31-2 X B1 6.37 ab 57 g 230 defg 89 gh C2-6 X S5-2 6.21 ab 57 g 214 gh 82 hi P36-2-2 X Ki 32-6 6.18 abc 62 ab 234 bcdef 116 ab AC22 X Ki 32-8 6.06 abc 61 bcde 249 abc 113 bc Ac 145-4 X KI 42-3 6.03 abc 59 cdefg 232 bcdef 99 cdefg C2-5 X S5-7 5.95 abc 58 fg 214 gh 67 I AC145-10 X Ki42-4 5.85 abcd 62 ab 250 ab 129 a Pacific 11 5.62 bcde 58 fg 217 fgh 91 fgh P31-3 X T 20 5.38 bcde 61 bcde 211 h 84 h P36-4 X T 2 5.27 bcde 59 defg 237 bcde 82 hi P36--2-1 X C2-2 5.25 bcde 59 efg 221 efgh 93 efgh C2-9 X Ki 31-7 5.03 bcde 61 abc 216 fgh 90 defgh Ruwan 4.71 cdef 59 efg 231 cdefg 92 efgh T 18 X Ki 32-5 4.45 def 60 cdef 214 gh 99 cdefg P 36-3 X T 16 4.40 def 62 ab 240 bcd 105 bcdef T 16 X B 6 4.32 ef 61 abcd 240 bcd 109 bcd Ac 145-1 X Ki 32-9 3.51 fg 63 a 226 defgh 105 bcdef P 31-14 X T 7 3.43 fg 61 acbd 231 cdefg 88 gh Ki 32-2 X S5-11 2.84 g 61 abc 234 bcdef 96 defgh Grand Mean 5.18 62 230 97 LSD 0.64 0.91 8.09 6.59 CV(%) 19.43 2.39 5.56 10.73 110 KARUNARATNE

Similar performances were displayed when seven unrelated hybrids were compared with 14 parents during maha 2001/02 (Table 2). Grain yields of the hybrids ranged between 3.4 t and 5.00 t/ha, while yields of parent lines ranged from 0.32 - 2.60 t/ha. Of the seven hybrids tested, 4 hybrids performed better than the commercial check, which produced a grain yield of about 4.11 t/ha. Heterosis, of the hybrids in comparison to mid parent, better parent (heterobeltiosis), and commercial check (commercial heterosis) were calculated for major characters and presented in Table 3. The highest commercial heterosis for grain yield was produced by the hybrid C2 - 5 X S 5-7, which is about 25%. Three hybrids produced positive commercial heterosis values and three produced negative heterosis values for grain yield (Table 3).

Table 2. Performance of local inbred lines and their single crosses in RCBD trial conducted at FCRDI, Maha Illuppallama during maha 2001/02.

Hybrid Yield Days to Plant height Ear height (t/ha) flowering (cm) (cm) P36-5-2 X Ki 31-4 3.75 b 54 hi 202 a 93 abc P31-2 X B1 4.48 ab 53 ij 187 abc 98 ab C2-6 X S5-2 3.44 bc 52 j 167 cde 73 bcdef P36-2-2 X Ki 32-6 4.34 ab 58 de 205 a 72 bcdef AC22 X Ki 32-8 3.41 bc 56 fg 190 ab 105 a Ac 145-4 X KI 42-3 4.34 ab 54 hi 187 abc 100 a C2-5 X S5-7 4.99 a 52 j 172 bcd 85 abcd Ki 41-4 0.20 I 59 cd 200 a 90 abc P 36-5-2 0.82 fghi 56 fg 125 hi 55 fg P 31-2 1.51 def 55 gh 123 hi 57 efg B 1 2.34 cde 62 a 173 bcd 97 ab S 5-2 1.48 efg 61 ab 122 hi 42 g C 2-6 1.38 efgh 59 cd 128 ghi 47 fg Ki 32-6 0.93 fghi 57 ef 122 hi 63 defg P 36-2-2 0.32 hi 61 ab 127 ghi 53 fg Ac 22 0.39 ghi 59 cd 118 I 45 g Ki 32-8 1.60 def 60 bc 147 efg 82 abcde Ac 145-4 0.33 hi 62 a 118 I 67 cdefg Ki 42-3 2.60 cd 58 de 152 def 72 bcdef C 2-5 1.07 fhgi 56 fg 127 ghi 57 efg S 5-7 1.24 efghi 57 ef 143 fgh 58 efg Pacific 11 3.99 ab 55 gh 165 de 85 abcd Grand Mean 2.23 57 154 72 LSD 0.41 0.58 8.03 9.62 CV(%) 30.24 1.67 8.52 21.81

Hybrids that out-yielded the check varieties tested during maha 2003/04 were selected for further studies to achieve consistency in performance (Table 4). Five hybrids out-performed the local check Sampath in the first experiment and five hybrids were outstanding compared to the exotic hybrid (Pacific 984) and 11 hybrids out-yielded the local check Sampath, in the 2nd experiment. In the 3rd experiment 16 hybrids performed better than the local hybrid. Mean yield of Pacific 684 was high due to less number of evaluations. INBRED LINE DEVELOPMENT 111

Table 3. Heterosis, heterobeltiosis and commercial heterosis of early generation hybrids calculated yield data of field experiment conducted at FCRDI, Mahailluppallama during maha 2001/02.

HYbrid Days to Plant Ear height Grain yield flower (%) height (%) (%) (%) P36-5-2 X Ki 31-4 Heterosis -5.75 24.06 28.6 435.7 Heterobeltiosis -7.84 0.8 3.33 357.3 Commercial heterosis -2.3 22.1 9.7 -6.0 P31-2 X B1 Heterosis -8.88 25.8 28.3 130.7 Heterobeltiosis -13.47 7.6 1.75 88.6 Commercial heterosis -3.6 13.0 15.6 12.0 C2-6 X S5-2 Heterosis -13.7 33.3 64.7 142.0 Heterobeltiosis -15.25 2.9 57.2 134.0 Commercial heterosis -6.69 0.96 -13.76 -13.7 P36-2-2 X Ki 32-6 Heterosis -1.52 65.1 74.2 600.0 Heterobeltiosis -4.29 61.9 60.5 366.6 Commercial heterosis 4.88 24.2 18.3 8.77 AC22 X Ki 32-8 Heterosis -5.88 43.5 65.8 211.0 Heterobeltiosis -7.13 29.6 28.6 112.5 Commercial heterosis 1.26 15.15 23.5 -14.78 Ac 145-4 X KI 42-3 Heterosis -10.66 38.6 44.3 196.5 Heterobeltiosis -13.96 23.8 39.6 66.5 Commercial heterosis -3.07 13.0 17.6 8.52 C2-5 X S5-7 Heterosis -9.15 27.6 48.0 333.9 Heterobeltiosis -9.74 19.4 45.7 305.4 Commercial heterosis -6.69 4.0 0.0 25.0

Finally test-cross performance was considered for identifying lines with the best performance (Table 5) and 3 season data indicated that the lines Ki 42-5 (5.18 t/ha), Ki 32-6 (4.96 t/ha), T -2 (4.88 t/ha), T-7 (4.80 t/ha), Ki 42-6 (4.74 t/ha) and P 36-5-1 (4.33 t/ha) were outstanding and would be further studied in the future evaluations. Thirty-five hybrids identified through test-cross and single- cross performance were seed multiplied for multi-location testing in the seasons ahead. 112 KARUNARATNE

Table 4. Performance of the single crosses in RCBD experiment conducted at FCRDI, Maha Illuppallama during maha 2003/04.

Hybrid Yield Days to Hybrid Yield Days to Hybrid Yield Days to (t/ ha) flowering (t/ ha) flowering (t/ ha) flowering

KH 364 5.40 abc 52 KH 348 4.88 abcd 53 KH 179 4.94 bcdef 53 KH 183 5.02 abcd 53 KH 349 3.46 f 57 KH 177 4.58 cdef 53 KH 365 4.69 cde 58 KH350 4.40 cdef 53 KH185 4.87 bcdef 53 KH 366 4.32 de 54 KH351 5.64 ab 55 KH188 5.14 bcde 57 KH 367 4.83 cde 56 KH190 4.70 bcde 53 KH190 5.21 bcde 55 KH 368 3.35 f 58 KH352 5.48 ab 53 KH184 4.18 ef 57 KH 369 3.96 ef 60 KH353 4.81 abcd 53 KH186 4.95 bcdef 59 KH 370 5.09 abcd 61 KH354 5.41 ab 53 KH181 4.45 def 55 KH 371 5.23 abcd 53 KH355 5.67 a 55 KH194 5.92 ab 59 KH 372 5.90 ab 54 KH356 5.17 abc 58 KH191 4.92 bcdef 57 KH 373 5.34 abc 56 KH357 5.22 abc 58 KH200 4.88 bcdef 60 KH 374 5.92 ab 53 KH358 3.79 ef 53 KH211 4.55 cdef 59 KH 375 4.57 cde 53 KH359 4.11 def 53 KH304 4.43 def 59 KH 376 4.92 cde 58 KH360 5.22 abc 55 KH206 5.65 abc 59 KH 278 4.91 cde 56 KH361 4.12 def 53 KH214 5.43 bcd 59 KH 377 5.09 abcd 58 KH362 5.46 ab 55 KH215 5.26 bcde 59 KH 378 5.11 abcd 53 KH215 5.24 abc 58 KH212 5.69 abc 55 KH 379 5.93 a 60 KH363 4.39 cdef 58 KH218 6.68 a 59 KH 380 4.30 def 58 Sampath 4.79 abcd 60 KH288 3.85 f 60 KH 214 4.96 bcd 56 Pacific 984 5.31 abc 60 KH196 4.83 bcdef 59 KH 381 4.36 de 58 KH235 4.67 cdef 62 Sampath 5.26 abcd 63 KH222 5.19 bcde 57 Sampath 4.63 cdef

LSD = 0.41 0.37 0.44 CV(%)= 13.76 11.55 14.81 INBRED LINE DEVELOPMENT 113

Table 5. Performance of the lines in test crosses at FCRDI, Maha Illuppallama during maha 2003/04, yala 2004 and maha 2004/05.

Entry Maha 2003/04 Yala 2004 Maha 04/05 Mean yield (t/ha) yield (t/ha) yield (t/ha) yield(t/ha) CML 20 X T-2 4.28 5.97 4.38 4.88 CML 20 X T -16 3.82 4.11 2.81 3.58

CML 20 X C2 -2 3.85 5.22 3.74 4.27 CML 20 X C2 -3 4.38 4.74 4.18 4.43 CML 20 X C2 -6 4.42 5.17 2.86 4.15 CML 20 X C2 -9 3.89 4.81 3.05 3.88 CML 20 X S 5 - 8 4.24 5.26 2.98 4.16 CML 20 X S 5 - 11 4.97 4.59 3.51 4.39 CML 20 X B - 6 3.78 4.79 3.27 3.95 CML 20 X B - 12 4.66 5.07 3.06 4.26 CML 20 X P 31-7-2 4.32 4.29 4.45 4.35 CML 20 X P 31-3 4.62 - - - CML 20 X Ki 42-4 4.53 5.48 3.57 4.53 CML 20 X Ki 42-5 5.80 5.97 3.76 5.18 CML 20 X Ki 42-6 4.95 5.73 3.55 4.74 CML 20 X P 36-4 4.47 5.12 3.55 4.38 CML 20 X Ki 31-4 4.76 5.33 4.50 4.86 CML 20 X Ki 32-3 4.07 5.97 4.00 4.68 CML 20 X Ki 32-5 4.62 5.71 3.13 4.49 Sampath 5.42 5.30 4.91 3.90 4.88 Pacific 984 6.86 5.64 - - 6.25 CML 20 X Ki 32-6 5.35 5.67 3.85 4.96 CML 20 X Ki 32-7 5.21 - - - CML 20 X Ki 32-8 5.93 - - - CML 20 X Ki 32-9 4.81 - - - CML 20 X Ki 32-10 5.34 - - - CML 20 X T -1 4.61 4.97 - - CML 20 X T -7 5.53 5.51 3.37 4.80 CML 20 X T -10 6.56 - - - CML 20 X S 5 - 3 5.18 - - - CML 20 X P 31-7-1 4.74 4.59 3.15 4.16 CML 20 X P 31-12 5.46 4.75 3.31 4.51 CML 20 X P 31-14 6.03 - - - CML 20 X Ki 42-1 5.69 - - - CML 20 X Ki 42-3 5.71 - - - CML 20 X P 36-2-2 4.92 - - - CML 20 X P 36-3 4.83 - - - CML 20 X P 36-5-1 4.79 4.02 4.18 4.33 CML 20 X AC 145-1 4.41 - - - CML 20 X AC 145-3 4.72 - - - LSD(0.05) NS 0.46 NS NS CV(%) 17.22 16.32 31.74 24.89 114 KARUNARATNE

CONCLUSIONS

Performance of some crosses was highly satisfactory, out-yielding local hybrid and commercial hybrids included as controls in number of experiments conducted. Thirty-five selected promising hybrids are being evaluated for including the most promising 8-10 hybrids in National Coordinated Variety Trials for identifying the best hybrids for nominating for national release.

REFERENCES

CIMMYT, 1985. Institutional Innovations in National Agricultural Research, 25 Hallauer, 1988. Corn and Corn Improvement. ASA. Field Crop Research and Development Institute, (1999- 2000). Research Reports, Maha Illuppallama. Michael, M.M. and S. Pandey, 1998. Maize Seed Industries. CIMMYT Publication, 32-76, 335- 337. Shull, G.H. 1908. The composition of a field of maize. American Breeders' Association Report 5: 51-59. Annals of Sri Lanka Department of Agriculture. 2006.8:115-124.

PROPAGATION OF BAEL FRUITS (BELI) [Aegle marmelos (L.) corr.] BY GRAFTING

K.W. KETIPEARACHCHI, C. KUDAGAMAGE, J. DE ZOYSA, D.H.N.W. HETTIGE, J.P.K.L. GAMLATH, M.B. WEERASURIYA and D. BANDARA Horticultural Crop Research Unit, Horticultural Crop Research and Development Institute, Gannoruwa, Peradeniya

ABSTRACT

Beli (Aegle marmelos (L.) corr.) is a naturally propagated plant through seeds and roots. The characteristics of seedlings show morphological variations and use of root cutting is destructive and not suitable for large scale propagation. These have restricted the commercial cultivation and popularization of beli in Sri Lanka. Present study was conducted to identify a suitable grafting method in large scale propagation of beli plants. Seedlings with 5 mm stem girth were selected as root stocks and scion woods with 4 nodes were taken from a promising beli cultivar. The wedge, splice and side vineer grafting methods were tested. Percentage success, days taken to initial bud take on, days to emergence of first leaf, shoot length and number of leaves emerged were recorded. Percentage success of wedge grafting showed the highest results (86.7%) and for side vineer and splice the success rates were 80% and 66.7%, respectively. Mean shoot length of successful grafts in wedge grafts was 22.4 cm after 12 weeks and it was 18.6 cm for splice and 7.8 cm for side vineer. Average number of leaves in the successful grafts was 15.5 in the wedge grafted plants while it was 8.3 and 4.0 respectively, for splice and side vineer grafts. Thus, wedge grafting is a promising propagation technique for rapid multiplication of beli.

KEYWORDS: Beli fruit, Grafting, Propagation.

INTRODUCTION

Bale fruit (Beli in Sinhala) is a multi-purpose tree. The roots, bark, wood, unripe fruits, ripe fruit pulp, fruit shell, flowers and leaves etc., are extensively used for various needs such as food and medicine. Ripened beli fruit is highly nutritious with high levels of carbohydrate, protein, fat, carotene, vitamins and minerals.

The origin of beli is in India (Srivastava and Singh, 1999) and has been introduced to Sri Lanka in ancient times. It is widely distributed in the dry, wet and intermediate zones of Sri Lanka and a naturalized tree to the local conditions resembling an indigenous plant. Beli is more prized for its medicinal virtues than its edible qualities. In Sri Lanka beli is widely used in traditional medicines. Fresh fruits are used for making herbal drinks and many other value added products such as juice, cordial, jam, beli latex and dried pulp in confectioneries. Thus, beli is a potential crop with many industrial applications. Therefore, there is a great potential for this fruit both commercially and environmentally. 116 KETIPEARACHCHI et al.

Although beli has been in use from time immemorial, hardly any effort has been made for the improvement of the crop or to cultivate it in an organized manner. Socio-economic studies have shown that beli is a popular home garden crop with versatile uses. However, most of the existing trees are naturally regenerated and there are no selected or improved varieties in cultivation. Therefore, introduction of good varieties or cultivars of beli fruit and cultivation of these in large scale mainly for the development of industrial uses have become necessary at present. For increasing the cultivation, a large quantity of planting materials of elite cultivars or varieties is required.

Beli is usually propagated by seeds (Wickremasinghe, 2002; Misra, 1999; Morton, 1987). However, it is a heterozygous crop and therefore seedlings show much variation in form, size, texture of rind, quality and quantity of pulp, flavour, number of seeds etc. Also, seedling plants take about 6-7 years to reach the bearing stage (http://www.worldagroforestry.org). Thus to overcome these defects and for rapid multiplication of superior types, vegetative propagation is essential. Beli could be vegetatively propagated by root cuttings, grafting, air layering and budding (Wickremasinghe, 2002). However, scientific information on vegetative propagation of beli fruit is very scarce and further there are no such records in Sri Lanka. Therefore, present study was conducted at the Horticultural Research Unit 2, Horticultural Crop Research and Development Institute, Gannoruwa with the objective of identifying a suitable grafting method for beli fruit propagation.

MATERIALS AND METHODS

Beli seedlings were produced from healthy seeds collected from a single plant from outstanding germplasm found from Polonnaruwa under plant house conditions. Root stock seedlings were raised in 8’’ x 12’’ polythene pots filled with normal potting mixture (sand: top soil: compost = 1:1:1). Seedlings those reached 5 mm stem girth were selected as root stocks and the bud woods having 4 nodes and similar size to rootstock were taken as scions from a characterized promising mother tree. The grafting methods of wedge, splice and side vineer were practised for beli at the same time under equal conditions with the objective of selecting the best performing grafting method.

For the wedge grafting (Plate 1) root stocks were cut down at about 5cm above the soil surface to a depth of 1.5-2 cm. Then edge of the scion was cut to a “V” shaped wedge with long even sloping sides and the scion and root stock were fitted together. The bud union was bound with a polythene strip and kept those grafts under propagator condition. PROPAGATION OF BELI BY GRAFTING 117

Preparation of the root stock Insertion of the scion into Tying with polythene strips the stock

Plate 1. Main steps of wedge grafting

For splice grafting (Plate 2), after removing the top of the root stock, a clear horizontal cut was made 5 cm above the soil level. Then a downward cut was made at an angle of 45° about 2.5 cm above the soil level. Then a downward cut was made starting from top to bottom until the second cut met the first cut. Then the scion with 4 nodes was taken and made a cut similar to the cut in root stock. Scion and root stock were fitted together and tied with a strip of polythene and kept under propagator condition.

Preparation of the root stock and Fitting the scion and root stock scion wood

Plate 2. Main steps of splice grafting.

For the first step for the side vineer (Plate 3), short downward cut about 5-6mm in length was made at 45° angle. Then the stem was carved by a downward cut to meet the first and it was about 2.5 cm above the first cut. Then a part of the silver wood was removed. The scions with 4 nodes were taken and a piece of wood having same length as the wound in root stock was stripped off from its base. Scion was put into the wound made in stock plant and tied it well by polythene strips. The top of the root stock was left intact, until the union had taken place. 118 KETIPEARACHCHI et al.

Preparation of the root Insertion of the scion Kept under single stock into the stock propagator

Plate 3. Main steps of side vineer grafting.

All the grafts were kept under single propagator condition until the bud took on and then at the time of shoot emergence, hardening process was started. Misting was done regularly to keep the grafts moist.

The experiment was laid out with Complete Randomized Design with 3 replicates. The plot size was 5 plants per replicate. Percentage success or percentage bud take on (total no. of successful grafts/total no. of grafts/100), the days taken to initial bud take on, number of buds in a successful graft, average shoot length, leaves per shoot, average number of leaves in the successful grafts were recorded at weekly intervals. The data were statistically analyzed using SAS package.

RESULTS AND DISCUSSION

Percentage success (Percentage bud take on)

Present study was undertaken to identify a suitable method for grafting beli. The method which gave successful results in the shortest duration would be the best out of all the methods. In spite of that, vegetative growth after the bud union had taken place was also measured.

The percentage of initial bud take on which indicates the success of grafting was recorded weekly and indicated in Figure 1. Percentage success was higher in wedge grafting compared to other two grafting methods tested. Mean percentage success was 86.67% for wedge grafting and it was 80 % and 66.67% for splice and side vineer grafts, respectively. Interaction effect also indicates higher percentage of success in wedge grafting over the other grafting methods. However, there was no difference (P= 0.05) between the 3 methods tested on percentage success (Table1). PROPAGATION OF BELI BY GRAFTING 119

Average days taken for success in grafting was different (P= 0.05) between wedge and side vineer methods (Table 1). Side vineer grafting recorded the highest time period of 46.2 days compared to the wedge grafting (31.6 days)

100

90 n

o 80

e k a

t 70

d u

b 60

e g a

t 50 n e c

r 40 e p

e

g 30 a r e

v 20 A

10

0 1 2 3 4 5 6 7 8 9 10 Weeks after the treatment Wedge Splice Side vineer

Figure 1. Effect of different grafting methods on percentage success.

Table 1. Mean effect of the method of grafting on percentage success and average days taken to success (alpha =0.05).

Treatment Mean % success Mean average days taken to success Wedge 86.67 a 31.617 b Splice 80.00 a 39.290 ab Side Vineer 66.67 a 46.240 a LSD (P=0.05) 29.783 9.1131 Means with same letter are not significantly different at P=0.05.

The average days taken to union under 3 grafting methods is illustrated in Figure 2. It shows the average data of 3 replicates. For wedge grafting it took 21 days for initial bud take on and percentage success observed at that time was 20%. It took 28 days for splice and 35 days for side vineer. The initial percentage success was 20% and 40%, respectively for splice and side vineer.

According to the results, percentage success and days taken for success were comparatively better with wedge grafting. There were variations 120 KETIPEARACHCHI et al. within the same treatment with time and it may be due to the physiological variations among the scion wood used (Fig. 2).

70

60 n o

50 e k a t

d u b 40 o t

n e k a t

s 30 y a d

e g a

r 20 e v A

10

0 1 2 3 4 5 Plant No Wedge Splice Side Vineer

Figure 2. Effect of different grafting methods on days taken to bud take on.

Vegetative growth after the bud union was measured weekly in terms of number of buds, average shoot length, number of leaves per shoot and number of leaves per graft. A vigorous vegetative growth is important in large scale planting material production. Therefore, special attention was given to the growth parameters to select the best grafting method out of the tested techniques.

Number of buds

Buds are the starting points of leaves. Number of active buds in the scion wood in different grafting methods is shown in Figure 3. Though there were 4 nodes in almost all the scion woods taken, the highest number of bud emergence was observed in the wedge grafted plants while the lowest was in side vineer grafts (Figure 3). The time taken to emergence of initial bud was 3 weeks in wedge grafted plants and it was 5 weeks for side vineer grafts. Number of buds in the successful grafts of splice grafts was almost similar to wedge grafts and the reason for the lower number of buds in side vineer grafts may be due to the remaining rootstock until the union had taken place. Statistical analysis indicates that the response of grafting method affected the number of buds activated (P=0.05). Among the grafting methods tested, number of buds in scion wood was higher in the wedge grafts while it was significantly lower in side vineer grafts (Table 2). PROPAGATION OF BELI BY GRAFTING 121

6

5 e t a

c 4 i l p e r

/

s 3 d u b

f o

o 2 N

1

0 3 4 5 6 7 8 9 10 11 No of weeks after grafting Wedge Splice Side Vineer Figure 3. Number of buds in the wedge, splice and side vineer grafts.

Average shoot length

Average shoot length in the wedge and splice grafts was almost similar throughout the experimental period and the values were significantly higher than in the side vineer grafts (Fig. 4 and Table 2).

250

200 ) m m (

h 150 t g n e l

t o o h s

e 100 g a r e v A

50

0 3 4 5 6 7 8 9 10 11 Weeks after grafting

Wedge Splice Side Vineer Figure 4. Average shoot length for the wedge, splice and side vineer grafts with time.

Leaves per shoot 122 KETIPEARACHCHI et al.

Leaves are the sources of photosynthesis and the number of leaves produced from the scion may enhance growth and increase the assimilates. Figure 5 shows the number of leaves per shoot in the different grafts. It is evident that the wedge grafts produced leaves 3 weeks after grafting, while the side vineer grafts took 5 weeks. The leaves per shoot in side vineer grafts fluctuated drastically and the reason may be the continued growth of root stock. Wedge and splice grafts behaved in a similar manner. Differences in number of leaves per shoot between wedge and side vineer and that between splice and side vineer, were significant. Shoot growth showed no significant difference in the wedge and splice grafts (Table 2).

Number of leaves per graft

The effect of grafting method on number of leaves was recorded weekly (Fig. 6). Average number of leaves in the wedge and splice grafts were similar and significantly higher than in the side vineer grafts (Table 2). The low leaf production observed in the side vineer grafts may be due to the antagonistic effect of the growing root stock.

4

3.5

3 t o

o 2.5 h s

r e p

s 2 e v a e l

f o

1.5 o N

1

0.5

0 3 4 5 6 7 8 9 10 11 Weeks after grafting W edge Splice Side Vineer

Figure 5. Leaves per shoot for the wedge, splice and side vineer grafts with time. PROPAGATION OF BELI BY GRAFTING 123

4

3.5

3 t f

a 2.5 r G

r e p

2 s e v a e 1.5 L

1

0.5

0 3 4 5 6 7 8 9 10 11 Weeks after grafting

Wedge Splice Side Vineer Figure 6. Leaves per shoot for the wedge, splice and side vineer grafts with time.

Table 2: Effect of the method of grafting on vegetative growth of beli.

Treatment No. of buds Average shoot Leaves per Leaves per length shoot graft Wedge 3.5804 a 118.79 a 2.4852 a 9.9348 a Splice 2.9922 b 105.40 a 2.3893 a 8.3863 a Side 1.4863 c 30.19 b 1.4437 b 2.8667 b Vineer LSD 0.5801 20.499 0.3605 1.5537 Means with the same letter are not significantly different at P=0.05.

Thus it may be generalized, that the vegetative growth parameters of the wedge and splice grafts, were similar, but significantly different to that of the side vineer grafts.

Hence for vegetative multiplication, wedge and splice grafting seem better methods than side vineer grafting. Comparing wedge and splice grafting, the former seemed a better method when considering percentage of successful grafts, growth performances and number of days taken to produce buds. Success of grafting depends on the compatibility between the root stock and the scion wood. The better performances of wedge grafting may be due to the close intact of the scion wood with the rootstock in wedge shape cut. The earliest bud activation in wedge grafting may be also due to the fast callus development and the quick cambial growth on close intact and the shape of the wedge grafting. This may enhance the quicker absorption and translocation of nutrients and water to the scion wood. This could result in quick bud initialization and vigorous vegetative growth. Splice grafting also makes a 124 KETIPEARACHCHI et al. close intact between the root stock and scion wood but this method is practically difficult owing to the shape of the cutting edge. In the side vineer method, growth of the scion wood is affected due to the active growth of the root stock.

CONCLUSIONS

Beli can be successfully propagated by wedge, splice or side vineer grafting methods. However, wedge grafting seems better with minimum days taken for initial bud take on and producing 86.7% successful grafts with vigorous vegetative growth.

REFERENCES http:/www.worldagroforestry.org/SEA/products/afdbases/AF/asp/speciesinfo.asp Misra, K.K. 1999. Bael. Department of Horticulture, G.B. Pant University of Agriculture and Technology, Pantnagar-263 145 (U.P), India. Morton, J. 1987. Bael fruit. Fruits of warm climates, Julia F. Orton, Miami, FL. 187-190. Srivastava and Singh, H.K. 1999. Floral biology of Bael (Aegle marmelos) cultivars. Indian Journal of Agricultural Sciences 70 (11):797-798. Wickremasinghe, I.P. 2002. Beli fruit. A publication of the national multipurpose tree research network. Department of Agricultural Biology, Faculty of Agriculture, University of Peradeniya, Peradeniya, Sri Lanka. Annals of Sri Lanka Department of Agriculture. 2006.8:125-135.

MORPHOLOGICAL CHARACTERIZATION AND DISTRIBUTION OF BELI (Aegle marmelos) IN SRI LANKA

K.W. KETIPEARACHCHI, C. KUDAGAMAGE, H.K. KADUPITIYA, T.E.WEERAWARDANE, M.B. WEERASURIYA, D.H.N.W. HETTIGE, N.W.D.A.D. KANNANGARA, A.G.G. KARIYAWASAM and D. BANDARA Horticultural Crop Research Unit 2, Horticultural Crop Research and Development Institute, Gannoruwa, Peradeniya

ABSTRACT

Bael (Aegle marmelos), a member of family Rutaceae, indigenous to India, is a valuable multi-purpose tree crop. Fruits are highly nutritious and almost all the plant parts have medicinal value. Recently, beli has been identified as a potential fruit crop, but improvement and domestication have been neglected. The present study was carried out to explore, collect and characterize germplasm to study the diversity and identify elite germplasm for future crop improvement programs. Sixty two accessions were collected from different parts of the country and morphological characters such as fruit weight, diameter, circumference, peel and pulp thickness, pulp and peel colour, shape, seed number and tree characteristics were studied. Among them, fruit weight (76.6 -1370 g), circumference across poles (7.4 – 43.0 cm) and equator (11.6– 40 cm), diameter (4.5 to 31.5 cm), peel thickness (2 -7 mm), seed number (0- 90) and pulp color (yellow to orange) were prominent variations in fruit characteristics. Geographic locations of beli trees were also recorded using Global Positioning System (GPS). A potential map for beli was developed based on ecological conditions of locations using floramap-GIS software.

KEYWORDS: Beli, Characterization, Distribution, Global positioning system (GPS).

INTRODUCTION

Beli (Aegle marmelos), a member of family Rutaceae, is a valuable multi-purpose tree crop in which all the plant parts are used for medicinal purposes. The fruit is used as a remedy for stomach upsets and the unripe fruit is a traditional Indian remedy for dysentery and diarrhea (http://www.asiafood.org). Protein-rich ripe bael, fruits are eaten by people. Also, they are utilized in preparation of drinks, jams and paints (in Burma), as a substitute for soap, as a source of essential oils and the mucilage of the seed is a good cementing material (http://www.hort.purdue.edu). But still it is considered as an underutilized fruit crop in Sri Lanka.

Beli is a deciduous tree with trifoliate aromatic leaves and the branches sometimes bear long straight spines. The bark of the bael tree is shallowly furrowed and corky. It carries bisexual flowers borne in clusters, sweet scented and greenish white. The calyx has 5 short sepals and the 5 petals are pale greenish white. Stamens are numerous, sometimes coherent in bundles. Ovary is oblong ovoid and the fruits contain numerous seeds arranged in the cells surrounded by slimy transparent mucilage (Misra, 1999). 126 KETIPEARACHCHI et al.

Bael is native to India, but it is found widely in Sri Lanka, Myanmar, Thailand and Indo-China (Parmer and Kaushal 1982). Presently, beli is grown in all parts of the country including wet, intermediate and dry zones of low-mid-up-country regions of Sri Lanka. There are morphological variations among the trees located in different geographical areas. However, a very little attention has been given for selection of superior types from available genetic resources in Sri Lanka.

In India, explorations had been made to collect the variability of bael from various places and a wide range of variability in physico- chemical characters, maturity period and yield components was recorded and based on this information some desirable elite genotypes were isolated and conserved (Bal, 2002). After survey, evaluation and collection of germplasm over years, selection of superior genotypes has been done in India based on the tree characters, fruit characters and yield potentials (Mitra et al., 1999). Commercial varieties of bael such as Mirzapuri, and new varieties such as NB 5 and NB 9 have been developed in India (Chadha, 1995).

The present study was carried out to explore, collect and characterize the bael germplasm in part of Sri Lanka to find out the existing diversity and elite germplasm for future crop improvement programs.

MATERIALS AND METHODS

A baseline survey was conducted through the Agricultural Extension Service of the Provincial Departments of Agriculture, Divisional Secretariats, Mahaweli Extension Officers and through personal communication to identify the potential diversity centers for exploration and collection of the underutilized fruit species. A data collection format was developed for exploration and characterization and to acquire traditional knowledge of these species. Passport data, history of the tree concerned at particular sites, morphological data and fruit characteristics were recorded.

The tree characteristics of stem girth 1 m above the soil surface, height of the plant to the apex along the main stem, canopy diameter and the age of the tree were collected at selected locations. The leaf and fruit samples were characterized in the laboratory. Characterization was carried out on the basis of the IPGRI descriptor for tropical fruits. Ripen or mature fruits were collected and randomly selected for characterization.

Fruit weight, circumference both across the poles and equatorial, peel colour, flesh colour and number of seeds per fruit were tested at Horticultural Research Unit 2 with the collaboration of the Food Research Unit. Fruit shape and the shape of the apex and the base of the fruit were evaluated using the IPGRI descriptor for citrus. Peel and pulp colour of the MORPHOLOGY AND DISTRIBUTION OF BELI 127 fruits were evaluated by using the Royal Horticultural Society Color Chart. The petiole length and length and width of the leaves were measured for the leaf characteristics. The data were analyzed using SPSS version 10 software package. Average fruit weights, average circumference (both equatorial and across the poles), average peel thickness and average number of seeds per fruit were calculated and data were compared for the samples collected from Matale, Kegalle and Polonnaruwa.

Latitude and Longitude (Kandawala Coordinate system) of Beli tree locations were recorded using a Global Positioning System (GPS). Exploration field visits were carried out within and around selected areas with the help of elderly villagers, Grama Niladaris and Agriculture Research and Production Officers attached to Agrarian Service Centers. Distribution maps for each species were developed using the GPS location coordinate data in Arc View GIS software. Potential area probability maps were generated using principal component analysis, probability analysis and mapping procedures in the FloraMap software with inbuilt database of long-term average climatic information.

RESULTS AND DISCUSSION

Sixty two accessions of beli samples were collected mainly from Kegalle, Matale, Polonnaruwa, Kandy, Kurunegala, Matara, Puttalam, and Gampaha districts covering agro-ecological zones of dry zone, intermediate zone and wet zone. Collected samples were characterized for their fruit morphological characters. Among them, fruit weight, circumference across the poles and across the equator, diameter, peel thickness, seed number and pulp color were prominent in variations of the fruit characteristics.

Fruit size and seed number

Fruit morphological characters varied among the samples collected (Plate 1). The size of fruits was studied in terms of circumference (equatorial and across the poles), fruit diameter, fruit weight and peel thickness. The data were statistically analyzed using the SPSS computer package (Table 1).

Plate 1. Variations in fruit morphological characters of beli. 128 KETIPEARACHCHI et al.

Fruit weight ranged from 76.6 g -1370 g while the diameter of the fruits varied from 4.5 cm to 31.5 cm. Circumference of fruits was in a range of 11.6 - 40 cm as equatorial and it was 7.4 - 43 cm as across the poles. Peel thickness varied from 2 - 7 cm. Most of them had filled seeds and number of seeds per fruit ranged from 0-90.

Table 1. Descriptive statistics of fruit characters of beli.

Fruit character Minimum Maximum Mean Std. deviation Equatorial circumference (cm) 11.6 40 26.00 5.80 Circumference across the poles (cm) 7.40 43 26.16 8.00 Fruit Diameter(cm) 4.5 31.5 9.98 5.34 Fruit weight (g) 76.60 1370 350.69 240.56 Peel thickness (mm) 2.0 7.0 3.25 1.02 Number of seeds/fruit 00 90 36 20.62

Relationships between fruit weight vs circumference (both equatorial and across the poles), fruit weight vs peel thickness, fruit weight vs fruit diameter and fruit weight vs number of seeds per fruit were examined. There were positive correlations between fruit weight and the parameters such

as circumference (Figs. 1 and 2) and fruit diameter (Fig. 3). Fruit weight (g) weight Fruit

i u t ) e r h g t F i w g (

1400 1400

1200 1200

1000 1000

800 800

600 600

400 400

200 200

0 0 0 10 20 30 40 50 10 20 30 40 5

Circumference across the poles (cm) Equatorial circumference (cm)

Figure 1. Relationship between fruit weight and Figure 2. Relationship between fruit circumference across the poles. weight and circumference across equator. MORPHOLOGY AND DISTRIBUTION OF BELI 129

i u t ) e r

h g t F i w g (

1400

1200

1000

800

600

400

200

0 0 10 20 30 40 Fruit diameter (cm)

Figure 3. Relationship between fruit weight and fruit diameter.

But, there was no correlation observed between fruit weight and parameters such as peel thickness (Fig. 4) and number of seeds per fruit (Fig. 5).

i u t )

e r

h g t F i w g ( (g) weight Fruit 1400 1400

1200 1200

1000 1000

800 800

600 600

400 400

200 200

0 0 -20 0 20 40 60 80 100 1 2 3 4 5 6 7 8

Peel thickness (mm) Number of seeds per fruit

Figure 4. Relationship between fruit Figure 5. Relationship between fruit weight and peel thickness. weight and no of seeds per fruit.

Variations of average fruit circumference, fruit diameter (Fig. 6), peel thickness and fruit weights (Fig. 7) in 3 districts namely Matale, Kegalle and Polonnaruwa where most of the samples were collected were evaluated. 130 KETIPEARACHCHI et al.

i t r c F r u t c h a r a c e ( m )

35

30

25

20

15

10

5

0 Districts Matale Kegalle Polonnaruwa Average fruit Average circumference Average circumference diameter across equator across poles Figure 6. Variations in Average fruit diameter, equatorial circumference and circumference across the poles in Matale, Kegalle and Polonnaruwa.

Samples collected from Polonnaruwa and Kegalle had larger fruits than from Matale (Fig. 7) in respect of circumference (Fig. 6) and also the peel thickness. But the diameter of the fruits did not show the same pattern due to their different shapes (Fig. 6). ) g (

t h g i e w

t i u r f

600e g a

500r e 400v A 300

200

100

0 Matale Kegalle Polonnaruwa Districts

Figure 7. Variations in average fruit weights of beli in Matale, Kegalle and Polonnaruwa.

Fruit shape MORPHOLOGY AND DISTRIBUTION OF BELI 131

Mainly 4 different fruit shapes (Plate 2) were observed as ellipsoid, spheroid, oblique and oblate according to the IBPGR descriptor for citrus. Most of the fruits were ellipsoid (46.77%) or spheroid (46.77%) in shape. Very few had oblique and oblate shapes (3.22%). Shapes of the fruits varied between the districts Kegalle, Matale and Polonnaruwa (Table 2).

Plate 2. Variations in fruit shapes of beli.

Table 2. Percentages of fruits of four different fruit shapes in each district.

Shape Kegalle Matale Polonnaruwa Other districts Total samples Ellipsoid 61.1 26.66 40.0 57.2 46.77 Spheroid 33.0 66.66 46.66 42.8 46.77 Oblique 5.55 - 6.66 - 3.22 oblate - 6.0 6.66 - 3.22

Shapes of the apices of the fruits were different as convex, necked, truncate or concave while the base of fruits varied as convex, depressed or truncate (Tables 3 and 4). A higher percentage of fruits had convex shaped apexes (90.3%) as well as convex shaped bases (96.66%). Apex of a few fruits was necked (4.83%), truncate (3.22%) or concave (1.61%). Also, few fruits had depressed and truncated bases (1.61%).

Table 3. Percentages of fruits according to the shape of the apex.

Shape Kegalle Matale Polonnaruwa Other districts Total samples Convex 94.44 86.66 86.66 92.85 90.32 Necked 5.55 - 13.33 7.14 4.83 Truncate - 13.33 - - 3.22 Concave - - - - 1.61

Table 4. Percentages of fruits according to the shape of the base.

Shape Kegalle Matale Polonnaruwa Other districts Total samples Convex 100 93.33 93.33 100 96.66 Depressed - - 6.66 - 1.61 Truncate - 6.66 - - 1.61 132 KETIPEARACHCHI et al.

However, further studies on collected accessions are required to conclude about variations in shapes between districts.

Peel color and pulp colour

Groups of peel colour and pulp colour of beli collected are listed in Table 05 after evaluation by using Royal Horticultural Society Colour Chart.

Table 5. Percentage variations in pulp colour of beli.

Colour group Kegalle Matale Polonnaruwa Other districts Total Samples Yellow green - - - 25 8.33 Yellow 13.3 50 14.28 16.66 18.75 Yellow orange 53.3 16.6 28.5 41.66 37.5 Orange 33.3 33.3 14.28 16.66 22.91 Grayed orange - - 42.8 - 12.5

Table 6. Percentage variations in external peel colour of beli.

Colour group Kegalle Matale Polonnaruwa Other districts Total samples Green 43.7 40 21.4 7.14 26.53 Yellow green 12.5 40 14.2 28.57 20.4 Yellow - 20 7.14 35.71 14.28 Yellow orange 25.0 - 42.8 21.42 21.42 Grayed yellow 6.25 - 2.14 7.14 6.12 Grayed orange 12.5 - 2.14 - 6.12

In most of the fruits, pulp colour was yellow-green, yellow- orange or yellow whereas peel colour was green, yellow-green or yellow- orange. Identification of the correct maturity stage of beli is done by using the external peel colour of beli and the smell. However, external peel colour of the ripe beli is uneven. Therefore, errors can occur during evaluating the external peel colour. But, variations in pulp colour of beli can be clearly identified.

Mother tree characteristics

Ninety nine accessions of beli identified in districts Puttalam, Kegalle, Kurunagala, Anuradhapura, Polonnaruwa, Kandy, Matale, Matara and Gampaha were characterized for parameters such as tree height, canopy diameter, main stem girth and the age. The results are listed in Table 7. MORPHOLOGY AND DISTRIBUTION OF BELI 133

Table 7. Variations in mother tree characteristics of beli in different districts.

District No. of Tree height (m) Canopy diameter Girth (m) Age (years) accessions (m) Gampaha 03 9-15 4.3-10 0.63-1.55 30 Kandy 06 9-15 6-9 1.15 20-50 Kegalle 48 8-20 5-20 0.13- 1.1 7-100 Kurunagala 04 6-15 7-20 0.1-0.6 30-70 Matale 14 6-20 4-12 0.5-1.81 25-100 Matara 03 10-12 8-10 0.18-1.2 15-100 Polonnaruwa 07 5-12 5.1-20 0.56-1.52 4-30 Puttlum 04 3-15 3-10 0.6-1.0 10-20 Anuradhapura 10 6-16 3-10 - 8-35

Tree height of the evaluated trees ranged 6 - 20 m while the canopy diameter ranged 3 - 20 m. Stem girth of the trees varied from 0.1 to 0.8 m. There were very older trees aged nearly 100 years.

Leaf characteristics

Beli leaves are trifoliate and aromatic. The leaf widths, leaf lengths and petiole lengths of beli varied (Table 8) in examined samples. Average leaf lengths, leaf widths and petiole lengths were 7.91 cm, 4.6 cm and 1.54 cm respectively. Both upper and lower surfaces of the leaf were in yellow-green or green group.

Table 8. Descriptive statistics of leaf characters of beli.

Leaf character Minimum Maximum Mean Std. deviation Leaf length (cm) 4.50 12.60 7.9133 1.7992 Leaf width (cm) 3.20 8.80 4.6000 1.2978 Petiole length(cm) 1.10 1.80 1.5400 .2608

Distribution of beli

Using 220 tree location coordinates of Beli trees, a probability map for beli was developed using Flora Map GIS software (Fig. 8). Sample areas of higher probability were cross checked by field visits and with actual tree distribution data collected from extension staff by a Grama Niladari division level survey. According to that, a high density of Beli trees was 134 KETIPEARACHCHI et al. reported in the areas shown as high potential in the potential areas map of Beli (Fig. 8).

Figure 8. Probability map of potential areas for occurrences of Beli.

CONCLUSIONS

Fruit and tree characteristics of beli varied among the collected accessions. Fruit weight ranged 76.6 g - 1370 g. while the diameter of the fruits varied from 4.5 cm to 31.5 cm. Circumference of fruits was in the range of 11.6 - 40 cm as equatorial and it was 7.4 - 43 cm as across the poles. Peel thickness varied from 2 - 7 cm. Most fruits were filled with seeds and number of seeds per fruit ranged from 0 - 90. There was a positive correlation between fruit weight and parameters such as circumference and peel thickness while there was no correlation observed between fruit weight and parameters such as peel thickness and seed number. Most of the fruits were ellipsoid (46.67%) or spheroid (46.67%) in shape. Very few had oblique and oblate shapes (3.22%). Shape of the apex of the fruits was different as convex, necked, truncate or concave while the base of fruits varied as convex, depressed or truncate. A higher percentage of fruits had convex shaped apexes (90.3%) as well as convex shaped bases (96.66%). Apex of the few MORPHOLOGY AND DISTRIBUTION OF BELI 135 fruits was necked (4.83%), truncate (3.22%) or concave (1.61%). Also, a few fruits had depressed and truncated bases (1.61%). Tree height of the evaluated trees ranged 6 - 20 m while the canopy diameter ranged 3- 20 m. Stem girth of the trees varied from 0.1 to 0.8 m. Average leaf lengths, leaf widths and petiole lengths were 7.91 cm, 4.6 cm and 1.54 cm respectively.

REFERENCES

Bal, J.S. 2002. Genetic resources of under-utilized fruits in Punjab, ISHS Acta Horticulturae 623: Proceedings of XXVI International Horticultural Congress on Plant Genetic Resources, www.actahort.org. Chadha, K.L. 1995. Status report on tropical fruit species in South Asia, http://www.ipgri.cgiar.org as retrieved on 27 th March 2006. http://www.asiafood.org as retrieved on 19 Mar 2006, Bael fruit, Aegle marmelos from Charmaine Solomon's Encyclopedia of Asian Food, Periplus Editions, 1998, supplied courtesy of New Holland Publishers (Australia) Pty Ltd. Misra, K.K. 1999. Bael , Department of Horticulture, G.B. Pant University of Agriculture and Technology, Pantnagar-263 145 (U.P.) India. http://www.hort.purdue.edu as retrieved on 27 Feb 2006.

Mitra, S. K., C.S. Maity, D. Mani, B. Ghosh, B. Chandra, K. Viswavidyalaya, 1999. Conservation and utilization of genetic resources of bael (Aegle marmelos Correa)- A potential underutilized fruit, Department of Fruits and Orchard Management, Faculty of Horticulture, West Bengal, India. Proceedings of the Symposium on Citrus and Other Subtropical and Tropical Fruit Crops, Metro Toronto Conservation Centre.

Parmar, C. and M.K. Kaushal. 1982. Aegle marmelos Correa. p. 1–5. In: Wild Fruits. Kalyani Publishers, New Delhi, India., http://www.hort. purdue.edu as retrieved on 27 Feb 2006 . Annals of Sri Lanka Department of Agriculture. 2006.8:137-145.

ANTHER CULTURE RESPONSE OF SOME SALT RESISTANT AND SALT SUSCEPTIBLE INDICA RICE VARIETIES

H.M.P.S. KUMARI, U.N.D. SILVA, W.M. ABAYARATHNE, D.S. DE.Z. ABEYSIRIWARDENA, I.W.M.K.N.K. YATAWARA and D.N. SIRISENA Rice Research and Development Institute, Batalagoga, Ibbagamuwa

ABSTRACT

In view of utilizing anther culture in rice improvement programs for developing varieties, anther derived embryos and green plant regeneration ability of rice varieties showing different reactions to salt stress, were studied in different culture media. The rice varieties used in the study were Pokkali, Nonabokra, At 354 and At 401 that were tolerant, Bw 400 that was moderately tolerant and Bg 94-1, Bg 300 and Bg 750 that were susceptible in salt stress. Six different callus induction media (N6) with different combinations and concentrations of 2-4D, Kinetic (KN) and Napthalic acetic acid (NAA) and four different plant regeneration media (MS) with different concentrations of KN, NAA, and BAP were used in the study. Different varieties responded differently to different callus induction and regeneration media. Nonabokra and At 354 did not respond at all for any of the media used. Bg 750 showed the highest response (9%) for callus induction. Pokkali and Bw 400 also showed callus induction responses at acceptable levels (over 3.5%). When KN level was increased from 0.5 to 1.0 mg/l, callus induction response of Pokkali and Bw 400 was improved while that of Bg 750 was ceased. In regeneration Bg 750 and Bg 300 did not respond at all for any of the media while Pokkali responded in all the regeneration media used. Rest of the varieties namely Bw 400, Bg 94-1 and At 401 attained acceptable levels of plant regeneration frequencies ranging from 20.8 to 46.7 in the MS medium with 2 mg/l BAP, 1mg/l NAA and 0.5 mg/l KN for which Pokkali showed the highest response (82.7%). Thus, response to anther culture appeared independent of salt tolerance in rice. However, calli derived plantlets could be successfully established in the field only in Pokkali and Bw 400, which are having improved characters that can be used as breeding lines.

KEYWORDS: Anther culture, Callus formation, Plant regeneration, Rice.

INTRODUCTION

Anther culture is an important biotechnological tool in rice breeding since it offers the possibility of obtaining genetically diverse haploid and homozygous diploid plants within a short time. It may reduce the time needed to reach homozygosity by spontaneous or induced doubling of the haploids chromosome number (Oono, 1975 and Shen et al., 1983).

It also allows early expression of recessive genes and increased selection efficiency as the number of plants required to obtain the desired recombinants is less than the conventional breeding.

The first successful haploid production using anther culture technique was achieved in Datura innoxia (Guha and Maheshwai, 1966). 138 KUMARI et al.

Shortly after this success, the technique was applied to rice (Niizeko and Oono, 1968). The first rice varieties from haploid breeding were released for commercial production in 1976 (Pinghe et al., 1998). At present, the most promising approach for haploid production of rice is microspore culture within or in isolation from the anther. Even though successful application of anther culture in the production of new rice varieties has been reported in China, Taiwan, South Korea, Japan, USA, India and several other countries (Raina and Ifran, 1998), convincing evidence of a general, simple, reproducible method applicable to wide range of varieties or subspecies in indica rice, is still lacking. Therefore, the evaluation of new factors and their manipulation for efficient callusing and green plant regeneration in anther culture of indica rice is still a challenging field. Mondal and Gupta (1997) reported that cold treatment of anthers induced calli formation, because it delayed the senescence of anther wall and facilitated the entry of nutrients for the growth and development of microspores.

Various combinations of auxins and cytokinins in the culture medium have been tested for their effect on both callus induction and plant regeneration in rice. Plant regeneration of calli is enhanced by water stress (Chen, 1986).

In anther culture, the promotive effect of proline suspension cultures has also been reported (Zapata and Abrigo, 1988). The superiority of maltose over sucrose as the carbohydrate source for callus induction in anther culture has been demonstrated (Lentini et al., 1995 and Raina and Ifran, 1998). Spontaneous chromosome doubling was observed in plants obtained from the same callus mass in variety tetep and tulsi (Ranjan et al., 1998). The reasons for spontaneous doubling of chromosome were suggested as endomitosis, irregular meiosis, spindle fusion and nuclear fusion (Oono, 1975 and Shen et al., 1983).

Japonica cultivars are easier to culture than indica cultivars. In addition, genotypic specificity to anther response and green plant regeneration is a more serious constraint in indica than japonica cultivars. Scientists suggested that callus induction ability was inherited as a recessive trait conditioned by a single block of genes. Pinghe et al., (1998) detected 8 independent QTLS involved in anther culturability of rice.

In Sri Lanka there has been tremendous improvement in rice sector through conventional breeding, but the creation of genetic variability and application of advanced technologies to develop varietal resistance for biotic and abiotic stresses had been limited. Utilization of potentially important anther culture technology in rice improvement has not yet been tried. Thus the present study was conducted with the main objective of investigating the anther culture response of selected rice varieties, showing ANTHER CULTURE RESPONSE OF SALT TOLERANT RICE VARIETIES 139 varying response in salt stress (Sirisena and Abeysiriwardhana, 2005). This technique will be applicable to further research activities of salt tolerant line development in rice.

MATERIALS AND METHODS

Selection of explants

Eight popular rice varieties which show varying response to salt stress were selected (Table 1) and grown under natural conditions with regular supply of water and recommended cultural practices during two seasons. Panicles were collected from all these varieties, when the distance between the flag leaf auricle to the penultimate leaf was in between 7-11 cm and microspore stage (late uninucleate) was cytologically detected using heamatoxyline staining.

Table 1. Varietal ranking or grouping for tolerance to salinity based on green house screening test (Sirisena and Abeysiriwardhana, 2005).

Variety Reaction to salinity Traditional Pokkali Tolerant Nonabokra Tolerant Improved At 354 Tolerant At 401 Tolerant Bw 400 Moderately tolerant Bg 94-1 Susceptible Bg 300 Susceptible Bg 750

Pretreatments and sterilization of plant material

Cold treatment was given to the panicles for 7 to 12 days at 10°C. Panicles were sterilized with wiping 70% ethanol and taken to clean air bench. Then open panicles were sterilized with 20% Clorox for 15-20 minutes, followed by sterilization with 70% ethanol for 2 minutes and then washed thoroughly with sterilized distilled water.

Culture media for callus initiation and plant regeneration

Callus initiation from anthers was tested on N6 medium supplemented with different combinations and concentrations of growth regulators (Table 2). The spikeletes were cut at the base and anthers. About one hundred anthers were plated on each petridish containing 15ml of culture medium. For each medium 25 petridishes were used. Inoculated anthers were incubated under dark at 25°C. 140 KUMARI et al.

Partial desiccation of calli and plantlet regeneration

Primary calli of 1-5 mm diameter were subjected to 24 hr water stress condition using oven-sterilized petridishes laid with two layers of Watmann No. 1 filter papers for 24 hours. Then partially desiccated calli were transferred to modified Murashige and skoog (MS) 1962 medium (Table 2) for regeneration and were incubated at 25°C with 16 h light/8 h dark photoperiod.

Table 2. Different concentrations and combinations of sugar types and growth regulators in callus induction (N6) and plant regeneration (MS) medium.

Treatment Sugar type Growth regulators

Sucrose Maltose 2,4 D mg/l KN NAA BAP %- % mg/l mg/l mg/ Callus induction N6-1 6 2 0.5 - - N6-2 6 - 0.5 2 - N6-3 6 2 1 - - N6-4 6 - 1 2 - N6-5 6 2 0.5 2 - N6-6 6 2 1 2 - Plant regeneration

MS1 3 - 0.5 1 1 MS2 3 - 0.5 1 2 MS3 3 - 2 1 0.5 MS4 3 - 2 1 2

Identification of ploidy level and plantlet establishment in soil

Root tops of the regenerated green plants were examined for their ploidy status using aceto carmine stain. Developed plantlets were transferred to sterilized soil and kept in the greenhouse. Seeds collected from tissue cultured plants were screened under field conditions for uniform plant growth and seed setting over them and selected lines were advanced to screen against diseases and salt stress condition.

RESULTS AND DISCUSSION

Differential response with respect to callus induction and green plant regeneration was noticed among the eight cultivars (Table 3). Callus induction frequencies were variable and dependent upon the genotypes used. . Callus induction ANTHER CULTURE RESPONSE OF SALT TOLERANT RICE VARIETIES 141

Anthers of cold treatment of 7 days showed the highest response to callusing and green plant regeneration. Anther viability observed

Table3. Callus induction and plant regeneration potential of different rice varieties.

Variety N6-1 N6-2 N6-3 N6-4 N6-5 N6-6 No. of No. of No. of No. of No. of No. of No. of No.of No. of No. of No. of No. of plated calli anthers calli anthers calli anthers calli anthers calli anthers calli anthers produced plated formed plated forme plated formed plated formed plated formed d Pokkali 484 3 465 6 430 7 494 27 295 - 312 -

BW 400 352 10 314 5 374 9 387 15 325 - 316 -

Bg 300 267 1 275 1 284 - 304 2 245 - 302 -

Bg 750 306 17 244 22 269 18 202 - 224 - 220 -

Bg 94-1 310 3 291 - 285 4 215 - 205 - 196 -

At 354 281 - 290 - 206 - 260 - 254 - 276 -

At 401 395 6 282 11 397 2 123 - 208 8 156 27

Nonabokra 294 40 175 45 244 40 106 44 170 - 165 - 142 KUMARI et al.

up to 12 days with the cold treatment and calli formation favored 7 days treatment than others. Anthers pretreated for 7 days were used for this media test.

The use of different modifications of N6 medium incited different responses (Table 3) but variety x medium interaction was not significant. It ranged from 0% to 17% with respect to the number of anthers cultured. The time required for callus initiation was different in different varieties; callus initiation started as early as 30 days after anther plating. Varieties Pokkali, Bw 400, Bg 750 and Bg 94-1 reached maximum callus formation in between 42 to 50 days. In varieties such as Bg 300 and Nonabokra it was prolonged up to 60 days after anther plating. Microspore derived calli formation was confirmed using microscopic studies. Two types of calli initiated from anther of rice varieties, some were friable and shiny white and others were hard and cream in color. Most of the calli showed fast proliferating types which were eventually turned necrotic with or without regeneration.

Anthers of eight varieties showed differences in response in each media. Pokkali and Bw 400 showed the highest response to medium N6- 4 but for Bg 750 and At 401 the highest response could be observed with medium N6-2. Variety At 401 produced the highest number of calli when cultured on 6 (N6-6) medium while variety At 354 did not produce calli on any of the media tested. Bg 750 showed the highest response in callus formation with media N6-1, N6-2 and N6-3 when compared with all the other varieties. The culture medium supplemented with 2mg/l 2-4D combined with 0.5 mg/l KN (N6-1) and 2mg/l 2-4 D combined with 1mg/lKN appeared to be suitable for four varieties (BW 400, Bg 750, At 401 and Nonabokra). However, the medium supplemented with 2mg/l NAA and 0.5 mg/l KN showed high frequency of callus formation with the variety Bg 750. In contrast to the result reported, the results of the present study showed that 2- 4D can be replaced by NAA. Furthermore, Bg 750, improved variety, having different genetic constituents from Japonica lines, responded well to three culture media than other varieties and none of the varieties responded to media supplemented with 2mg/l 2-4 D and 2mg/l NAA. It may be possible that supply of high auxin concentration may suppress the callus initiation.

Plant regeneration

Regeneration frequencies of different varieties vary with media used. Interaction effect of variety x medium was not significant with respect to plantlet regeneration. Out of eight varieties tested, four varieties viz, Pokkali, Bg 94-1, At 401 and Bw 400 produced green plants on MS2 and the ANTHER CULTURE RESPONSE OF SALT TOLERANT RICE VARIETIES 143

frequency of regeneration was 82.7%, 20.8%, 46.7% and 26.7% respectively. In agreement with this result Ranjan et al. (1998) reported that genotype

Table 4. Plant regeneration efficiency in different MS media.

Variety MS1 MS2 MS3 MS4

No. of Regenerated No. of Regen No. of Regenera No. of Regenerate calli plants calli erated calli ted calli d plants cultured cultured plants cultured plants cultured

Pokkali 17 2(11.7%) 29 24(82. 16 2(12.5%) 41 16(39%) 7%) Bg 750 39 - 26 - 41 - 10 -

Bg 300 7 - 5 - 3 - 7 -

Bg 94-1 7 - 24 5(20.8 17 - 10 - %) At 401 17 - 15 7(46.7 36 - 33 - %) Bw 400 10 - 15 4(26.7 15 - 8 2(25%) %) Nonabokra 12 - 11 - 9 - 9 - 144 KUMARI et al. variation of the rice varieties has an effect mostly on callus induction and plant regeneration. The regeneration potential of variety varied with different modifications of MS medium and MS2 (1mg/l NAA, 2mg/l BAP, 0.5mg/l KN, 3% Sugar, 0.8%Agar) was found to be the best among all modifications (Table 4). High frequency of green (82.7%) and albino (15%) plant regeneration was observed in Pokkali. Direct green and albino shoots were observed on the same callus induction medium from the calli still attached to the anthers.

Calli derived plantlets produced in the varieties (16 plants from Pokkali and 2 plants from Bw 400) were successfully established in the soil. Somatic metaphase analysis from root tips of regenerated green plants of two varieties showed haploid and diploid number of chromosomes. Fertile plants showed doubled number of chromosomes. In first generation (G0) 50% of the plants were sterile and others produced seeds with 85% fertility. In second (G2) generation more then 200 plants were screened in the field and third (G3) generations 3000 plants screened. All the Pokkali plants showed homogeneity, uniformity, seed setting and shorter plant height (73 cm) and smaller seed type compared to that of the mother plant (Table 5). Bw 400 showed homogeneity, uniformity and 100% fertility but plant morphology was the same as mother plant. Results of Pokkali reveal retention of desirable recessive genes and improved protocol developed from this study can be extended to other indica varieties and F1 to improve plant with desirable genetic constituents.

Table 5. Performance of anther derived lines of Pokkali and Bw 400.

Line derived No. of Seed setting Plant height Seed type from anthers plants Pokkali G0 16 85% 65 cm White, Small,Long Pokkali G1 200 100% 73 cm White, Small,Long Pokkali G2 3000 100% 73 cm White, Small,Long Bw 400 G0 2 50% 70 cm Red,large,Round Bw 400 G1 200 100% 95 cm Red,Large,Round Bw 400 G2 3000 100% 95 cm Red,Large,Round

CONCLUSIONS

Variety into medium interaction was not significant in calli induction and plant regeneration of selected indica rice varieties. Even though calli obtained from Pokkali, Bg 94-1, At 401 and Bw 400 could be regenerated on the medium containing 2mg/l BAP with 1mg/l NAA and 0.5m/l KN, only plants derived from varieties of Pokkali and Bw 400 were successfully established in soil. Anther derived breeding lines were obtained from Pokkali with improved characters. Thus anther culture technology can be used in rice improvement program via creating genetic variants and producing double ANTHER CULTURE RESPONSE OF SALT TOLERANT RICE VARIETIES 145 haploids with early expression of recessive genes and shortened breeding cycle.

ACKNOWLEDGEMENTS

The authors wish to acknowledge the financial support received from SL- USDA Cooperative Germplasm Development Project No. 3 and Miss A.P.D.P. Wimasiri who has given much assistance during this study.

REFERENCES

Chen, Y. 1986. The inheritance of rice pollen plant and its applications in crop improvement, Haploids in higher plants in vitro, 118-136 Beijing, China. Guha, S. and S.C. Maheshwari. 1966. Cell division and differenciation of embryos in the pollen grains of Datura in vitro. Nature 212:97-98. Lentini, Z., P. Reyes, C.P. Martinz and W.M. Roca. 1995. Androgenesis of highly recalcitrant rice genotypes with maltose and silvernitrate. Plant Sci. 110: 127-138. Murashige, T. and S. Skoog, 1962. A revised medium for rapid growth and bioassays with tobacco tissue culture. Plant Physiol. 15: 473-497. Niizeki, H. and K. Ono 1968. Introduction of haploid rice plant from anther culture, Proc. Jpn. Acd. 44: 554-557. Mondal, N. and S. Gupta. 1997. Anther culture of an interspecific rice hybrid and selection of fine grain type with submergent tollerence. Plant cell, Tissue and Organ Culture 51:78-82. Oono, K. 1975. Production of haploid plants of rice (Oryza sativa ) by anther culture and their use in breeding. Bull. Natl. Inst. Agric. Sci. (Jpn) ser,D 26: 139-222. Pinghe, L. C., Y. Chen and L. Zhu. 1998. Analysis of quantitative trait loci which contribute to anther culturability in rice ( Oryza sativa L) . Molecular Breeding 4: 165- 172. Raina, S. and T. Ifran. 1998. High frequency embryogenesis and plantlet regeneration from isolated microspores of Indica rice. Plant Cell Report 17: 957-962. Ranjan S., D.K. Sharma and G.Chandal. 1998, Antherculture response of indica rice. Oryza 117-119. Shen J. H., F.M. Li, YQ. Chen and Z. H. Zhang. 1983. Improving rice by anther culture In Cell and Tissue Culture Techniques for Cereal Crop Improvement, Science Press, Beijing. 183-205. Zapata, I.J. and E.M. Abrigo. 1988. Plant regeneration and screening from long term Nacl stress rice callus. Int. Rice Res. News 11: 24-25. Sirisena, D.N. and D.S. de Z. Abeysiriwardena. 2005. Salt water as a medium to screen rice varieties for salinity tolerace.Journal of the Soil Science Society of Sri Lanka. 17: 11-19. Annals of Sri Lanka Department of Agriculture. 2006.8:147-158.

GENETIC DIVERSITY OF INDEGENIOUS COWPEA (Vigna unguiculata (L.) Walp.) IN INDIA

W.D. LESLY1 and M.S. UMA2 1Fruit Crop Research and Development Centre, Kananwila, Horana 2University of Agricultural Sciences, Dharwad, Karnatake,India

ABSTRACT

Loss of genetic diversity has been dramatic for many of the cultivated crop species. An experiment was conducted to study the genetic diversity of indigenous cowpea genotypes. Hundred and sixty nine genotypes including 157 indigenous genotypes, eight exotic lines and four recommended varieties were evaluated during summer 2005 in simple lattice design with two replicates for yield and yield contributing characters to estimate the genotypic coefficient of variation (GCV), phenotypic coefficient of variation (PCV), heritability, genetic mean advance and genetic diversity. Results revealed that high GCV and PCV values were obtained for number of clusters per plant, number of pods/plant, hundred seed weight, harvest index and seed yield/plant while low GCV and PCV obtained for days to flower termination and days to physiological maturity. The highest heritability and genetic mean advance were recorded by 100 seed weight. The highest contribution to divergence (14.75 %) was reported by number of pods/plant followed by harvest index (14.74%). Genotypes were grouped into 46 clusters in which the most divergent clusters were 11 and 42 and the cluster 40 was the highest productive with respect to seed yield. The highest inter-cluster distance of 349.31 was obtained between clusters 11 and 42. Observed genetic diversity in the genotypes can be utilized for any of the crop improvement programs in cowpea.

KEYWORDS: Cowpea, GCV, Genetic mean advance, Genetic diversity, Heritability, PCV.

INTRODUCTION

Cowpea (Vigna unguiculata (L.) Walp.) is one of the most important pulse crops grown in Sri Lanka. It is native to Central Africa and called as vegetable meat due to high amount of protein in grain with better biological value on dry weight basis.

It is the most versatile pulse crop because of its smothering nature, drought tolerant characters, soil restoring properties and multi-purpose uses. Development of cultivars with early maturity and acceptable grain quality has significantly increased the yield and cultivated area (Ehlers and Hall, 1997). Yield being a complex trait, is influenced by many other important yield contributing characters controlled by polygenes and also environmental factors. The overall effect of plant breeding on genetic diversity has been a long standing concern in the evolutionary biology of crop plants (Simmonds, 1962). The loss of genetic diversity has been dramatic for many cultivated species (Wikes, 1983). Better knowledge of the genetic similarity of breeding materials could help to maintain genetic diversity and 148 LESLY AND UMA sustain long term selection gain. Collection and evaluation of germplasm, quantification of the magnitude of variability existing for different characters and classification into groups help in identifying, which would yield greater variability. This enables the breeder to operate selection efficiently.

Keeping these aspects in view, the present study was initiated for evaluating the extent of available genetic variability for different characters in cowpea germplasm collected from different environments which will be of immense practical use for plant breeders to choose the plant of interest for different breeding programs.

MATERIALS AND METHODS

One hundred and sixty nine cowpea (Vigna unguiculata (L.) Walp) germplasm lines were used for the study. Of these lines 157 were indigenous collections of cowpea obtained from the National Bureau of Plant Genetic Resources (NBPGR), Pusa Campus, New Delhi and four check varieties (V-118, C –152, GC-3, Goa Local and Bailhongal Local) obtained from the Department of Genetics and Plant Breeding, University of Agricultural Sciences, Dharwad while eight were exotic lines (EC 394691, EC 394753, EC 394823, EC 394740, EC 394767, EC 394855, EC 394745 and EC 394805). The experiment was conducted during summer 2005 (January to April) season in Botanic Garden of University of Agricultural Sciences Dharwad, India. Dharwad is located in the transitional tract of Karnataka state at 15°13' north latitude, 75°07' east longitudes and at an altitude of 678 m above mean sea level with an average rainfall of about 800 mm. The experimental design followed was Simple Lattice Design with two replicates. Two days before seed sowing recommended inorganic fertilizer was incorporated to the soil at the rate of N -12.5 kg/ha, P2O5 - 25 kg/ha and K2O -12.5 kg/ha. Seeds of each genotype were sown with the spacing of 60 cm x 20 cm in 3 m length rows and used two rows per genotype. One week after sowing extra seedlings were thinned out to maintain the uniform plant density. Irrigation was done as required using sprinklers. Germplasm lines were evaluated to estimate the genetic diversity. Following genetic parameters were used to assess and quantify the genetic variability among the genotypes.

Estimation of variance components

Analysis of variance was carried out for twelve quantitative traits including yield and yield contributing characters of 169 genotypes. Both genotypic and phenotypic coefficients of variability (GCV and PCV), heritability in broad sense was computed as the ratio of genetic variance to the total phenotypic variance and genetic advance as percent of mean and the diversity analysis were estimated. GENETIC DIVERSITY OF COWPEA 149

MSS (genotypes) - MSS (error) M2 - M3

Genotypic variance (σg²) =  =  Number of replications r

M2 - M3

Phenotypic variance (σp²) = σg² + MSS error =  + M3 R σ g Genotypic coefficient of variability (GCV) = x 100 X

σ p Phenotypic coefficient of variability (PCV) = x 100 X Where, σ g = Genotypic standard deviation σ p = Phenotypic standard deviation X = General mean of the character

GCV and PCV values were categorized as low, moderate and high as indicated by Siva Subramanian and Menon (1973). The data were analyzed using Spar 1 statistical package.

Following observations such as plant height, number of primary branches, clusters per plant, days to flower initiation, days to flower termination, days to physiological maturity, pods per plant, pod length, seeds per pod, seed yield per plant, hundred seed weight and harvest index were taken during the period.

RESULTS AND DISCUSSION

Analysis of variance

The results obtained from the ANOVA are listed in the Table 1. All the traits showed highly significant differences among the genotypes. The highest variance was observed for the plant height among the characters studied, while the lowest variance recorded for harvest index. It indicates the presence of sufficient variability for these characters, thus there is a lot of scope for selection. Preeti et al. (2003) reported similar results in studying 32 mungbean genotypes. This can be further confirmed by studying genetic variability components such as GCV, PCV broad sense heritability (h2) and genetic advance. 150 LESLY AND UMA

Table 1. Analysis of variance ( ANOVA) for twelve quantitative characters in 169 genotypes of cowpea.

Mean sum of squares SI. No. Characters Genotype Replication Error 1 Plant height ( cm ) 52.63** 19.69 1.99 2 Days to flower initiation 45.25** 30.75 1.78 3 Days to flower termination 44.81** 17.00 2.07 4 Days to physiological maturity 43.92** 26.25 1.83 5 Number of branches per plant 0.64** 2.53 0.12 6 Number of clusters per plant 9.63** 16.11 0.71 7 Number of pods per plant 38.62** 12.39 1.27 8 Pod length ( cm ) 7.13** 3.07 0.64 9 Seeds per pod 5.05** 1.44 0.63 10 Hundred seed weight ( g ) 19.94** 0.07 0.09 11 Harvest index 0.006** 0.0001 0.00009 12 Seed yield per plant ( g ) 43.64** 3.76 0.79 * Significant at 5% probability level * * Significant at 1 % probability level

Genetic variability parameters

The results on genetic variability parameters of summer 2005 season are presented in Table 2. Genetic diversity is the basic knowledge needed for breeders to improve the crops by adopting appropriate methods of selection based on variability that exist in the materials. It is necessary to partition the total variability into heritable and non-heritable components and further to compute heritability and genetic advances for various metric traits.

Table 2. Variability parameters for 12 characters in cowpea genotypes.

Sl. No. Character GCV (%) PCV (%) h2 (%) GAM (%) 1 Plant height (cm) 11.59 12.04 93.00 22.99 2 Days to flower initiation 7.80 8.12 92.00 15.45 3 Days to flower termination 6.34 6.64 91.00 12.46 4 Days to physiological maturity 6.09 6.35 92.00 12.02 5 Number of branches per plant 11.51 13.85 69.00 19.64 6 Number of clusters per plant 19.53 21.03 86.00 37.37 7 Number of pods per plant 26.97 27.88 94.00 53.75 8 Pod length (cm) 12.52 13.69 84.00 23.63 9 Seeds per pod 11.60 13.15 78.00 21.08 10 Hundred seed weight (g) 28.00 28.13 99.00 57.42 11 Harvest index 24.45 24.82 97.00 50.00 12 Yield per plant (g) 27.57 28.08 96.00 55.75

Both GCV and PCV were observed for all the characters studied during summer 2005 while, all the PCV values were higher than the GCV values (Table 2). The higher PCV and GCV values were obtained for GENETIC DIVERSITY OF COWPEA 151 the number of pods per plant, hundred seed weight, harvest index and seed yield per plant. Rangaiah and Nehru (1998), Selvam et al. (2000) and Vineetha Kumari et al. (2003) reported high GCV and PCV values for pods per plant, hundred seed weight and seed yield per plant in cowpea. Moderate GCV and PCV estimates observed for plant height, while Selvi et al. (1994); Tyagi et al. (1999) and Selvam et al. (2000) showed higher GCV and PCV values for the characters of cowpea. While, low GCV and PCV were obtained for the characters such as days to flower initiation, days to flower termination and days to physiological maturity.

Marangappanawar (1984), Apte et al. (1987) and Patil and Baviskar (1987) also reported low GCV and PCV for the days to maturity in cowpea. The selection is difficult for these characters because of low variation among the genotypes.

GCV together with heritability estimates would give the best information on extent of the advance to be expected from selection. In the present set of material, high heritability and high genetic advance over the mean were exhibited by most of the characters which indicate that the genotypes have high variability, while some traits such as days to flower initiation days to flower termination and days to physiological maturity showed high heritability with moderate percent of genetic mean advance over the mean (GAM %).

The highest heritability was recorded by hundred seed weight and all other characters also exhibited higher level of heritability. The characters such as days to flower initiation, days to flower termination and days to physiological maturity expressed low GCV, PCV and high heritability. Selvi et al. (1998) also recorded high heritability for the above characters.

Genetic diversity analysis

Diversity available in the crop decides the success of any crop improvement program and the assessment of the divergence of the germplasm is essential to know the magnitude of the diversity. D2 statistic has been employed widely to resolve genetic divergence at intervarietal, subspecies and species levels in classificatory problems in crop plants. (Murthy et al., 1967 and Siddique and Swaminathan, 1971). In the present investigation 169 genotypes of cowpea were considered for assessment of nature of genetic diversity between genotypes by cluster analysis. It is indicating that wide diversity is present in the experimental material for majority of the characters. Estimation of degree of divergence between biological population and computation of relevant contribution of different components of genotypes, based on genetic distance and yield potential one can decide the crossing program. 152 LESLY AND UMA

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 1 26.72 183.79 242.47 225.72 71.11 211.92 195.45 147.50 220.89 188.57 254.89 153.79 122.89 222.60 238.49 178.03 2 26.33 137.51 130.71 128.99 164.51 91.82 146.51 168.85 93.68 249.79 178.16 95.43 163.64 187.63 166.22 3 24.89 48.99 205.57 154.54 114.41 203.22 155.09 146.43 277.78 173.21 188.88 236.81 167.26 215.00 4 22.75 185.69 122.02 98.43 181.76 121.89 133.87 262.46 146.69 167.67 228.52 136.53 194.56 5 25.41 172.70 144.43 119.76 183.72 137.16 239.65 135.24 59.41 182.75 205.52 151.32 6 24.59 132.38 127.98 50.84 159.08 180.86 96.97 159.72 241.23 87.85 126.19 7 24.74 165.09 128.73 59.13 257.96 161.19 119.83 165.69 142.62 182.02 8 27.72 139.75 181.77 150.13 112.21 122.01 249.74 165.11 51.78 9 22.36 157.04 187.89 117.34 170.33 240.82 45.97 137.36 10 29.77 269.75 180.88 110.84 113.59 168.93 197.93 11 25.97 184.08 240.69 319.48 203.04 106.11 12 25.55 146.56 254.56 146.47 121.09 13 22.96 163.83 193.36 149.30 14 27.75 248.81 262.07 15 27.47 161.64 16 20.42 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 GENETIC DIVERSITY OF COWPEA 153

17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 1 272.73 196.86 217.75 251.73 184.19 192.93 204.85 228.34 229.84 260.40 203.79 128.46 163.77 292.60 231.64 255.01 2 162.60 114.09 140.69 197.27 49.89 209.52 200.71 149.12 234.88 271.17 99.92 139.89 183.74 183.63 175.34 140.85 3 62.34 179.79 85.46 174.29 156.77 174.69 238.84 239.04 267.35 215.09 193.86 204.85 164.95 100.61 251.50 38.61 4 104.22 168.66 43.32 145.13 151.05 148.10 220.61 234.35 251.39 194.31 188.86 183.02 137.49 143.29 243.94 78.67 5 240.49 148.63 177.27 219.72 133.83 178.37 185.36 194.86 217.14 250.47 162.15 100.49 146.19 263.15 193.91 218.76 6 204.62 188.87 98.17 121.12 180.27 114.61 141.26 252.62 170.12 182.02 212.69 143.74 94.65 239.13 254.03 179.78 7 150.73 100.18 101.77 149.05 121.43 190.26 210.87 219.38 243.91 252.44 170.16 162.69 163.52 178.39 184.20 127.97 8 241.59 205.34 170.15 187.71 141.52 166.08 89.71 185.93 126.21 245.17 157.08 43.43 130.72 267.33 259.09 217.86 9 204.29 187.01 97.19 77.94 184.26 135.98 150.88 255.83 177.64 198.64 215.87 155.30 117.15 239.61 253.79 182.34 10 174.61 51.73 137.08 175.05 124.06 207.37 224.87 219.85 255.68 265.96 173.29 178.47 182.56 197.73 132.61 156.15 11 307.11 288.19 253.31 231.31 246.49 223.41 65.92 275.94 34.55 287.99 255.96 187.07 201.98 328.72 323.86 290.33 12 218.39 207.98 128.93 173.99 188.15 81.80 139.72 254.55 166.60 269.24 218.12 120.85 45.85 248.69 266.67 195.06 13 225.75 124.16 160.22 206.73 100.78 186.22 186.94 171.47 221.83 255.79 134.39 106.85 155.97 249.86 175.96 201.86 14 253.03 71.80 231.17 253.89 184.93 273.90 282.79 257.30 306.98 320.97 222.97 246.27 255.01 267.99 34.05 241.61 15 212.89 196.63 114.17 45.21 201.92 159.64 171.56 269.57 194.11 213.53 231.24 180.14 144.71 247.83 261.43 194.97 16 251.99 220.63 183.37 187.77 161.79 173.87 48.93 202.25 85.49 251.38 175.49 86.81 141.77 277.38 269.83 229.50 17 23.79 202.99 142.39 217.43 178.99 219.77 272.57 255.03 297.57 252.91 212.61 243.43 211.16 46.73 266.32 95.40 18 26.80 171.08 201.51 141.90 231.05 245.73 230.03 274.49 284.86 187.80 201.31 208.65 223.17 89.78 187.47 19 17.19 124.89 160.48 129.02 209.90 240.91 241.72 179.24 196.81 171.29 118.39 181.74 246.89 116.82 20 25.18 210.56 184.19 200.07 275.13 222.31 231.38 237.99 198.90 170.78 252.06 266.63 202.15 21 27.39 219.72 197.65 108.80 231.13 280.15 60.59 133.40 194.52 197.93 195.69 159.32 22 25.88 188.12 278.75 209.85 92.23 245.57 171.46 49.04 252.20 285.10 200.17 23 22.87 234.63 43.10 261.88 210.41 125.74 159.71 296.39 289.28 252.18 24 26.99 261.90 328.04 56.42 176.39 259.42 269.04 263.78 240.83 25 0.00 278.35 241.22 161.87 185.36 319.41 312.04 280.13 26 28.22 300.23 248.74 133.80 283.66 330.54 239.84 27 24.41 148.10 223.65 228.99 231.89 196.07 28 0.00 136.08 268.82 256.49 219.16 29 24.58 242.97 267.19 188.94 30 28.45 280.31 69.69 31 0.00 255.80 32 0.00 33 34 35 36 37 38 39 40 41 42 43 44 45 46

33 34 35 36 37 38 39 40 41 42 43 44 45 46 154 LESLY AND UMA

1 92.68 66.37 205.67 39.09 225.82 144.33 134.11 186.17 250.39 314.62 227.71 228.14 143.47 46.72 2 117.37 238.13 135.11 213.32 241.21 170.08 163.77 38.82 183.18 206.26 174.52 212.48 118.24 160.87 3 197.48 282.36 208.78 265.37 193.89 180.91 177.17 115.16 264.76 128.69 250.90 250.42 185.94 224.33 4 176.37 268.06 197.44 250.07 170.32 155.41 151.36 109.17 259.10 171.84 239.03 232.51 163.44 206.24 5 38.17 129.35 161.01 102.56 214.93 122.98 112.14 131.93 220.78 287.73 188.32 205.10 102.04 52.88 6 167.34 252.91 210.86 235.54 144.76 104.50 99.46 151.13 272.96 268.03 244.76 154.07 131.80 193.31 7 137.37 244.35 132.54 223.18 222.16 155.00 147.74 65.96 248.35 204.17 177.63 222.79 143.14 169.09 8 114.88 188.52 224.29 167.61 203.43 96.04 92.45 149.30 203.64 292.01 252.43 114.41 49.66 141.45 9 178.18 260.61 209.63 243.43 163.36 122.47 119.42 155.34 276.29 266.79 244.31 162.62 143.82 202.45 10 133.26 239.24 81.01 217.61 237.58 175.51 165.85 79.13 252.43 221.04 127.16 235.97 161.69 160.75 11 237.62 280.24 300.42 267.42 247.12 178.38 181.46 249.02 288.19 349.31 322.08 53.17 186.48 251.02 12 135.94 185.53 227.13 171.86 122.79 37.92 37.43 165.61 274.31 276.73 259.46 159.41 118.77 145.75 13 39.81 183.10 139.03 156.01 222.18 134.88 125.46 101.26 199.65 275.27 169.60 201.19 92.09 103.14 14 185.03 268.57 39.06 249.09 198.25 251.11 239.73 166.99 292.69 285.28 41.14 291.36 235.54 198.10 15 200.42 275.34 218.30 259.19 182.09 152.07 149.12 174.79 289.59 272.76 252.17 181.71 170.35 221.25 16 145.48 216.08 237.84 196.96 208.36 107.44 107.73 167.76 218.58 301.39 264.58 68.75 83.38 171.29 17 234.23 308.83 230.09 293.38 235.34 224.89 221.27 142.60 281.33 71.27 270.82 283.49 226.17 256.45 18 147.35 246.84 39.05 225.63 259.03 203.44 192.48 110.17 264.92 243.95 83.57 255.93 187.14 169.27 19 167.94 260.35 198.38 242.31 153.30 139.03 134.75 121.63 263.88 210.54 238.05 222.35 152.99 197.83 20 213.51 287.33 223.39 271.18 204.12 178.86 175.42 185.23 294.82 275.07 257.24 209.23 188.73 234.44 21 116.73 238.86 162.37 212.81 250.53 179.74 174.11 70.29 142.24 218.39 196.02 209.61 112.65 169.59 22 177.31 220.47 248.13 209.47 49.74 113.41 104.63 198.69 297.23 280.30 279.16 204.37 169.08 185.61 23 183.21 235.66 260.78 220.04 218.70 129.51 130.99 200.54 249.34 319.14 285.26 44.01 127.25 199.87 24 175.58 276.53 242.39 250.78 303.42 248.19 243.43 173.29 46.18 283.14 266.52 244.25 164.72 227.29 25 216.88 254.33 287.39 242.54 236.21 159.18 161.19 234.56 274.86 340.54 309.71 48.37 165.52 227.31 26 248.26 282.19 298.69 274.18 55.44 202.08 192.42 262.47 343.99 309.12 326.53 274.66 246.24 254.89 27 143.07 255.58 204.23 229.29 273.09 210.98 206.16 122.27 87.41 246.34 233.61 221.27 131.54 196.49 28 96.29 171.86 220.71 149.29 209.45 104.39 98.29 145.02 194.26 293.62 248.87 151.56 38.89 123.92 29 146.03 194.80 227.72 181.87 89.70 73.20 62.19 171.73 279.27 271.39 260.07 178.42 134.19 155.13 30 258.13 326.21 248.71 311.74 267.02 253.65 250.25 165.29 295.17 36.63 288.27 307.02 252.64 277.51 31 196.73 276.55 57.05 257.48 308.44 263.63 252.11 182.27 300.49 296.73 37.55 297.06 246.47 207.94 32 211.29 294.05 216.11 277.36 219.29 200.31 196.86 118.83 267.33 97.61 257.45 263.69 200.28 237.31 33 0.00 152.70 161.48 125.05 213.63 124.29 114.07 121.77 201.00 283.02 190.48 199.32 87.81 80.58 34 28.99 254.35 33.49 249.25 177.91 169.67 238.56 295.59 346.10 273.34 260.74 192.76 92.53 35 0.00 233.77 274.32 223.32 212.09 138.68 277.56 268.20 50.15 270.02 208.33 179.35 36 0.00 240.28 162.92 154.00 215.17 270.56 332.42 254.11 244.63 168.63 69.09 37 30.53 155.47 147.09 231.31 320.39 293.86 303.88 232.79 207.33 219.97 38 0.00 24.64 157.34 268.12 281.21 255.47 150.35 102.47 135.65 39 0.00 152.12 264.58 277.97 243.75 152.25 97.79 124.32 40 0.00 204.67 189.75 180.28 212.14 122.09 161.76 41 0.00 309.33 302.35 258.32 184.49 252.45 42 0.00 306.08 329.18 278.07 300.77 43 0.00 293.21 238.83 203.25 44 0.00 144.72 220.75 45 0.00 133.07 46 0.00

Based on D2 values, genotypes were grouped into 46 clusters in which most divergent clusters were 11 and 42 and the cluster 40 was the highest productive in respect of seed (Table 3) GENETIC DIVERSITY OF COWPEA 155

Yield showed high harvest index and number of pods per plant. It indicated that wide diversity was present in the experimental material for majority of the characters. Bakiyarani et al. (2000) concluded single plant yield and earliness in flowering together accounted for 80 % of the total genetic divergence of cowpea. Santos et al. (1997) revealed that hundred seed weight and pod length were the most important characters to affect divergence of cowpea.

Among the characters studied, the least contribution to divergence was by 100 seed weight where as Renganayaki and Rangasamy (1991) reported that 100 seed weight, pod length and seed yield contributed the most towards genetic divergence.

The highest inter cluster distance of 349.31 was observed between clusters 11 and 42 followed by clusters 34 and 42 (346.10). The lowest intra cluster distance other than solitary clusters was in the cluster of 19 (17.19). Twenty two clusters in between minimum and maximum values were observed showing different divergence groups while, the maximum intra- cluster distance was observed for cluster 37 (30.53) followed by cluster 10 (29.77). The solitary clusters were 25, 28, 31, 32, 33, 35, 36, 38, 39, 40, 41, 42, 43, 45 and 46 and they showed zero cluster distances.

Mean performance of clusters

Cluster 35 had the highest mean values for days to flower initiation, days to flower termination and days to physiological maturity and also, the cluster 36 showed the lowest means for the same traits indicating that the early maturing genotypes grouped in this cluster. Cluster 32 had greater mean values for pod length and seeds/pod and the cluster 33 had greater mean values for clusters/plant (Table 4). Clusters 40, 41, and 42 had higher mean values for seed yield per plant, harvest index and number of clusters per plant respectively. Cluster 45 had greater mean value for number of pods per plant and the clusters 20, 31 had the highest mean values for hundred seed weight and plant height respectively.

Table 4. Mean performances of different clusters of cowpea genotypes.

Cluster No. X1 X2 X3 X4 X5 X6 X7 X8 X9 X10 X11 X12 156 LESLY AND UMA

20 45.84 58.30 72.40 75.00 4.68 9.08 13.83 14.09 12.32 14.68 0.21 16.98 31 48.85 67.50 80.00 82.50 4.90 9.30 17.90 17.90 15.75 12.05 0.27 22.80 32 46.55 59.00 74.50 77.00 3.40 13.70 22.10 18.08 16.20 12.34 0.14 13.10 33 43.75 60.50 77.00 76.50 3.20 13.75 19.40 16.27 14.40 9.86 0.22 14.62 35 40.85 69.50 81.00 84.00 4.45 9.00 11.15 14.64 11.96 7.73 0.15 12.57 36 45.75 55.50 69.00 72.30 3.90 9.00 10.80 8.25 8.10 10.51 0.25 16.92 40 40.85 62.50 75.50 78.00 4.15 11.50 23.55 13.23 12.60 10.30 0.27 24.53 41 41.65 59.00 74.50 77.50 4.90 10.85 17.10 13.36 11.90 11.76 0.29 21.71 42 43.00 62.00 74.50 76.50 5.45 13.15 20.55 16.85 14.40 12.67 0.22 21.73 45 34.65 59.00 70.50 72.50 4.10 13.00 24.00 15.72 13.09 10.85 0.28 21.21

X1 = Plant height (cm) X7 = Number of pods per plant X2 = Days to flower initiation X8 = Pod length (cm) X3 = Days to flower termination X9 = Number of seeds per pod X4 = Days to physiological maturity X10 = Hundred seed weight (g) X5 = Number of branches per plant X11 = Harvest index X6 = Number of clusters per plant X12 = Seed yield per plant ( g )

Relative contribution to divergence

The contribution of each character towards genetic divergence was estimated (Table 5). It was observed that maximum contribution of 14.75 % was number of pods per plant followed by harvest index (14.74 %), while among the characters studied, the least contribution to divergence was by 100 seed weight.

Table 5. Relative contribution of different traits of cowpea genotypes towards divergence.

Source Traits Times ranked first % Contribution 1 Plant height (cm) 1874 13.20 2 Days to flower initiation 201 1.42 3 Days to flower termination 1948 13.72 4 Days to physiological maturity 198 1.39 5 Number of branches per plant 1910 13.45 6 Number of clusters per plant 185 1.30 7 Number of pods per plant 2094 14.75 8 Pod length (cm) 203 1.43 9 Seeds per pod 1849 13.02 10 Hundred seed weight ( g ) 137 0.97 11 Harvest index 2093 14.74 GENETIC DIVERSITY OF COWPEA 157

12 Seed yield per plant ( g) 1303 9.18

On the basis of diversity study, it is clear that different traits contribute towards yield, and it can be achieved by selecting the diverse genotypes for further breeding programs to improve the seed yield per plant with favorable traits.

CONCLUSIONS

Based on the overall performance, the genotypes displayed high variation for the traits studied with high GCV, PCV, heritability and GAM for the characters viz. number of pods per plant, 100 seed weight, harvest index, and seed yield per plant. The highest contribution to diversity was observed by number of pods per plant followed by harvest index. Cluster 40 was the highest productive with respect to seed yield. Therefore, the diversity of the genotypes can be utilized as a tool to increase variability among the existing genotypes in order to increase the productivity of the crop in any crop improvement program.

ACKNOWLEDGEMENTS

Authors wish to thank the Department of Agriculture and Sri Lanka Council for Agricultural Research Policy for giving the opportunity and for the financial assistance for this postgraduate study. Also, thank the committee members of the study in University of Agricultural Sciences, Dharwad, India for giving valuable guidance throughout the study. The assistance given by the Additional Director, FCRDC Horana, Miss K.D.A. Perera is acknowledged.

REFERENCES

Apte, U.B., S.A. Chavan and B.B. Jadhav, 1987, Genetic variability and heritability in cowpea. Indian Journal of Agricultural Sciences 57: 596 – 598. Backiyarani, S., N. Nadarajan, C. Rajendran and S. Shanthi, 2000, Genetic divergence for physiological traits in cowpea, (Vigna unguiculata (L.) Walp.). Legume Research 23(2) 114-117. Ehlers, J.D., and A.E. Hall, 1997, Cowpea (Vigna unguiculata L. Walp.) Field Crops Research 53: 187- 204. Murthy, B.R., V. Arunachalam and M.B.L. Saxena, 1967, Catalogue of the world collection of sorghum and pennisetum, Indian Journal of Genetics 27: 1-74. Marangappanavar, L.R., 1984, Genetic diversity gene action and character association in cowpea (Vigna unguiculata (L.) Walp.). Ph.D. Thesis, University of Agricultural Sciences, Bangalore. Patil, R.B. and A.P. Baviskar, 1987, Variability studies in cowpea. Journal of Maharashtra Agricultural Universities 12: 63-66. 158 LESLY AND UMA

Rangaiah, S. and S.D. Nehru, 1998, Genetic variability correlation and path analysis in cowpea. Karnataka Journal of Agricultural Sciences. 11: 927 – 932. Renganayaki, K. and S. R. Rangasamy, 1991, Genetic divergence in Vigna species. Indian Journal of Pulses Research 4(2): 159-164. Santos, C.A.F., E.A. Menezes and F.P. Arauzo, 1997, Genetic diversity in genotypes of cowpea under 2 different environments. Revista Ceres 44: 35-42. Selvam, Y.A., N. Manivannan, S. Murugan, P. Thangavelu and J. Ganeshan, 2000, Variability studies in cowpea (Vigna unguiculata (L.) Walp.). Legume Research 23: 279-280. Selvi, B., P. Rangaswamy, N. Nadarajan, J. Ramalingam and C. Vannirajan, 1994, Genetic variability in cowpea. Annals of Agricultural Research. 15 : 248-249. Siddique, J.A. and M.S. Swaminathan, 1971, Inter specific differentiation in Gossypium arboreum. Indian Journal of Genetics 31 : 55 – 62. Simmonds, N. 1962, Variability in crop plants, its use and conservation. Biol. Rev. 26 : 422- 462. Sivasubramanian, S. and M. Menon, 1973, Heterosis and inbreeding depression in rice. Madras Agricultural Journal 60: 1139. Tyagi, P.C., N. Kumar and M.C. Agarwal, 2000, Genetic variability and association of component characters for seed yield in cowpea (Vigna unguiculata (L.) Walp.) Legume Research 23: 92-96. Wikes, G. 1983, Current status of crop plant germplasm. CRC Crit. Rev. Plant Science 1:133- 181. Annals of Sri Lanka Department of Agriculture. 2006.8:159-166.

INSECTICIDAL PROPERTIES OF DATURA METEL LEAF EXTRACTS ON DIAMONDBACK MOTH (Plutella xylostella L.)

NEEL J. LIYANAGE1, R. CHAUHAN2 and RAM SINGH2 1National Plant Quarantine Service, International Airport, Katunayake 2Department of Entomology, CCS-Haryana Agriculture University, India

ABSTRACT

Leaf extract of Datura metel (Solanaceae) in methanol and subsequent fractions in acetone, chloroform and hexane were evaluated under laboratory conditions against Plutella xylostella (Linnaeus) at CCS Haryana Agricultural University, Hisar, India. Treatments included 2.5, 5.0, 7.5, 10 per cent concentrations of methanol extract and 0.5, 1.0, 1.5, 2.0 per cent concentrations of fractions along with control as water and acetone. In contact toxicity experiment, methanol extract (10.0%) caused maximum larval mortality of 63.34% and 83.34% after 4 and 8 h exposure respectively. Treatments of 10% methanol extract and 2% hexane fraction significantly affected first instar larval survival of 37.78% and 47.78%, larval period of 12.34 and 14.67 days, percent pupation of 22.23% and 38.87%, adult emergence of 0.0% and 21.12% and oviposition of 0 and 28.5 eggs/female of Plutella xylostella respectively.

KEYWORDS: Datura metel, Plutella xylostella, Insecticidal properties.

INTRODUCTION

Plutella xylostella Linnaeus (Lepidoptera: Plutellidae) is an important pest of cole crops throughout the world. It is estimated that about 53 per cent crop loss in marketable yield is due to attack of diamondback moth on cabbage and the losses could be more than 80 per cent under severe infestation (Chelliah and Sirinivasan, 1986). In most cases, use of chemicals to control larval populations has led to a rapid increase in diamondback moth resistance to insecticides (Mehrotra, 1991). This has triggered the exploration of natural pesticides. The annual cost of managing diamondback moth alone is estimated to be US $ 1 billion (Talekar and Shelton, 1993).

Recent research studies have shown that, plant products and their derivatives contain rich source of compounds having insecticidal activities and could be used as botanical insecticides. The genus Datura belongs to the Solanaceae family and consists of fifteen species, out of which three species namely, D. metel, D. stramonium and D. inoxia are medicinal plants. The principal alkaloid in Datura is scopolamine and it has insecticidal value (Solunke and Deshpande, 1991). 160 LIYANAGE et al.

However, available information is inadequate on the relative performance of extracts of Datura metel leaves against Plutella xylostella (L.). The present study was under taken with a view to assess the bioefficacy of Datura metel leaf extracts against contact action and various biological parameters of Plutella xylostella (L.).

MATERIALS AND METHODS

Fresh young leaves of D. metel were collected and allowed to dry under partial shady condition for two weeks. Dried leaves were crushed using mortar and pestle. The crushed leaves were taken in a round bottom flask (5 l) and the solvent methanol was added to it in a volume just sufficient to immerse the bits. Refluxing was done by fitting the flask with a water condenser and boiling the set using a heating mantle for 4-6h. The extract was then filtered out of the flask and was concentrated by distillation process. This refluxing and distillation procedure was repeated thrice. The crude extract obtained is hereafter referred to as methanol extract. A part of this extract was kept for further research and the remaining was further subjected to fractionation.

The methanol extract was mixed with silica gel (column chromatography) in a tray for drying. After 24 hours, the extract was transferred to a glass jar and hexane was poured to fill three-fourths of the jar and was airtight. After 24 hours, hexane was filtered out of the jar and distilled to obtain the concentrated hexane fraction. This process was repeated thrice. As in the case of hexane, the same procedure was followed using chloroform and then acetone (based on their increasing polarity), to obtain the respective fractions.

The experiments were carried out in the laboratories of Department of Entomology and Department of Chemistry and Physics, Chaudhary Charan Singh Haryana Agricultural University, Hisar during 2004 - 2005. The rearing of insects was carried out under laboratory conditions at 24 ± 2ºC. in the B.O.D. incubator. Larvae of P. xylostella were collected from infested cabbage picked from insecticide-free plots in a farmers field. The larvae were reared in glass jars (20 x 15cm) on cabbage leaves till pupation. The adults formed were transferred to another jar (20 x 15cm) provided with cotton swabs soaked in 15 percent sucrose solution. Eggs laid on the cotton swab and muslin cloth were allowed to hatch and culture was maintained for further studies. The methanol extract was tried at 2.5, 5.0, 7.5 and 10.0 per cent concentrations. The fractions in hexane, chloroform and acetone were tried at 0.5, 1.0, 1.5 and 2.0 per cent concentrations. DATURA LEAF EXTRACT ON DIAMOND BLACK MOTH 161

Evaluation for the contact toxicity against the first instar larvae

It was done by the dry film technique adopted by Gupta and Rawlins (1966). One ml of the extract and fraction solution was poured into rimless glass test tubes (25 x 200 mm). The tubes were kept in a hand-rotating wheel and rotated gently at 100 rpm. Angle of the tubes was adjusted in such a way that the solution covered three- fourths of the inner surface of each tube. The process was continued till the solution dried up leaving behind a uniform film of extract on the inner glass surface. For each concentration of the extract, films in three tubes were prepared. Dry film of acetone as control was prepared in a similar manner.

Ten larvae (first instar) of P. xylostella were released gently with a soft camel hair brush keeping three replicates of each treatment. The mouth of the test tubes was plugged with cotton wrapped in muslin. Tubes containing P. xylostella larvae were placed in the B.O.D. incubator at 24 ± 2°C. The larvae were allowed to remain in contact with dry films and mortality was recorded after four and eight hours. Moribund larvae were counted as dead.

Evaluation of the effect on the biology of insects

Twenty, first-instar larvae of P. xylostella were released individually in small Petri dishes (10cm dia), on the treated leaves (dipped in different concentrations of extract (2.5, 5.0, 7.5 and 10.0%), fractions (0.5, 1.0, 1.5 and 2.0%), water and acetone for 30 seconds, and air-dried) along with control and allowed to feed for 48 h. The Petri dishes were kept inside the B.O.D. incubator at 24±2°C. After 48 h surviving larvae were transferred to untreated leaves to record further observations on biological parameters i.e. larval survival, larval period, percent pupation, adult emergence and fecundity. Adults emerging from pupae of a particular treatment were kept batch-wise in glass jars provided with cotton swabs soaked in 15 per cent sugar solution and pairing was allowed for egg laying.

RESULTS AND DISCUSSION

The contact action of methanol extract of D. metel and its fractions against larvae of P. xylostella, four and eight hours after exposure to treatments is described in Table 1. The data revealed that progressive concentrations (2.5 – 10%) of methanol extract resulted in increasing mean larval mortality of P. xylostella which varied from 43.34 to 63.34 per cent and 56.67 to 83.34 per cent after four and eight 162 LIYANAGE et al. hours, respectively. Larval mortality in fractions at different concentrations varied from 23.34 -46.67 per cent in acetone, 33.34 – 53.34 per cent in hexane and 40.00 – 56.67 per cent in chloroform after four hours of exposure. After eight hours of exposure, there was a considerable increase in mortality in all the treatments. It was as high as 76.67, 73.34 and 60.00 per cent in chloroform, hexane and acetone fractions, respectively, each at 2.0 per cent concentration.

Table 1. Contact toxicity of methanol extract and different fractions of Datura metel against first instar larvae of Plutella xylostella (Linnaeus).

Mean larval Mean larval Concentration Treatment mortality after 4h mortality after 8h (%) (%) (%) Methanol 2.5 43.34 (41.15) 56.67 (48.93) extract 5.0 53.34 (46.92) 63.34 (52.77) 7.5 60.00 (50.85) 73.34 (59.00) 10.0 63.34 (52.77) 83.34 (66.14) Acetone 0.5 23.34 (28.77) 36.67 (37.22) fraction 1.0 26.67 (30.99) 43.34 (41.15) 1.5 36.67 (37.22) 53.34 (46.92) 2.0 46.67 (41.15) 60.00 (50.85) Hexane fraction 0.5 33.34 (35.21) 50.00 (45.0) 1.0 36.67 (37.22) 56.67 (48.84) 1.5 46.67 (41.15) 63.34 (52.77) 2.0 53.34 (46.92) 73.34 (59.0) Chloroform 0.5 40.00 (39.14) 53.34 (46.92) fraction 1.0 46.67 (43.07) 60.00 (50.85) 1.5 50.00 (45.00) 63.34 (52.77) 2.0 56.67 (48.84) 76.67 (61.22) Water (Control) 0.00 (0.00) 0.00 (0.00) Acetone 0.00 (0.00) 0.00 (0.00) (Control) SEM ± (1.89) (2.16) CD (P=0.05) (5.39) (10.89) Figures in parentheses denote arcsine-transformed values.

All treatments differed significantly from control. However, 10 per cent methanol extract gave the highest mortality of P. xylostella at both the exposure periods, being 63.34 and 83.34 per cent after four and eight hours of exposure, respectively. The larval mortality in acetone 2% extract was 23.34 and 36.67 per cent after four and eight hours, respectively. This indicated that methanol extract of D. metel was more effective than all other fractions in contact action. No information is available in the literature on contact DATURA LEAF EXTRACT ON DIAMOND BLACK MOTH 163 toxicity of D. metel extracts against P. xylostella. However, extracts of this plant have been reported to possess insecticidal activity against a number of other pests (Solunke and Deshpande, 1991). In the laboratory, methanol extract of D. metel leaves produced 54.74 percent mortality in Rhesala imparata (Kulkarni and Joshi, 1997). D. metel and D. stramonium both at 10 and 1 per cent (w/w) concentrations gave 100 per cent mortality of Sitophilus oryzae (Niber, 1994). Similarly, 5 per cent crude extract of D. metel provided 53.33 per cent mortality of Eutectona machaeralis (Meshram, 1995). Further, Datura leaf extracts have been found to cause moderate to heavy mortalities at the tested concentrations in Spodoptera litura (Murugan et al., 1998).

There were considerable variations in the mean larval survival among different treatments (Table 2). All treatments had significant adverse effect on larval survival over control (acetone). Minimum (37.78%) and significantly lower survival was recorded in 10.0 per cent methanol extract, which was on par with survival in 2.0 per cent hexane fraction (47.78%) and 7.5 per cent methanol extract (47.78%), than the other treatments. Larval survival among different treatments decreased as the concentration of the extract/fractions increased.

There was a prolongation of larval period over the control in treatments involving D. metel leaf extract/fractions. This prolongation was more pronounced at the higher concentrations of the extract/fractions. Maximum larval period was observed in 10 per cent methanol extract (14.67 days), followed by 7.5 per cent methanol extract (13.67), 2.0 per cent chloroform fraction (13.34), 2.0 per cent hexane fraction (12.34), 5.0 per cent methanol extract (12.34) and 1.5 per cent chloroform fraction (12.00) which indicated that these treatments produced significant adverse effect on larval period.

Different fractions/extract also had an adverse effect on the percentage of larvae undergoing successful pupation. As compared to the control, all treatments recorded significantly lower per cent pupation. Minimum and significantly lower percentage (22.23) of larvae was successful in pupating in 10 per cent methanol extract than all other treatments, except 7.5 per cent methanol (28.89). The next superior treatment was chloroform (2.0%) with 36.67 per cent pupation. In the remaining treatments per cent pupation varied between 38.87 and 73.34. Nevertheless, per cent pupation decreased with the increase in the concentration of extract and fractions. 164 LIYANAGE et al.

Table 2. Effect of methanol extract and its fractions of Datura metel on feeding and other parameters of Plutella xylostella (L.) (First instar fed with treated leaves).

Larva Larval Adult Fecundity Concentratio Larval l period Pupation emergenc (No. of Treatment n survival weigh (Days (%) e eggs/female (%) (%) t ) (%) ) (mg) Methanol 2.5 66.67(54.78) 10.67 3.10 46.67 39.99 36.74 extract (43.05) (39.20) 5.0 58.89(50.12) 12.34 2.91 38.89 26.67 28.69 (38.54) (31.04) 7.5 47.78 13.67 2.80 28.89 13.34 8.84 (44.66) (32.43) (21.29) 10.0 37.78 14.67 2.51 22.23 0.00 _ (37.87) (28.09) (0.00) Acetone 0.5 83.34 8.67 3.61 73.34 63.34 60.74 fraction (65.95) (59.01) (52.72) 1.0 76.67 9.34 3.54 66.67 53.34 55.91 (61.13) (54.78) (46.89) 1.5 70.00 9.67 3.42 55.56 43.34 50.79 (56.76) (48.20) (41.14) 2.0 63.34 10.67 3.22 46.67 32.22 44.81 (52.72) (43.05) (34.50) Hexane 0.5 72.23 9.34 3.51 61.12 51.12 45.75 fraction (58.31) (51.42) (45.61) 1.0 63.34 9.67 3.42 52.23 42.22 40.58 (52.76) (46.24) (40.48) 1.5 55.56 11.67 3.32 44.45 31.10 34.83 (48.20) (41.79) (33.83) 2.0 47.78 12.34 3.11 38.87(38.53) 21.12 28.50 (43.69) (27.21) Chloroform 0.5 74.45 9.67 3.41 58.89(50.12) 45.56 43.20 fraction (59.70) (42.42) 1.0 65.56 10.34 3.22 47.78 35.56 39.10 (54.08) (43.67) (36.55) 1.5 57.78 12.00 3.22 39.99 28.89 33.75 (49.49) (39.20) (32.43) 2.0 52.23 13.34 3.02 36.67 17.78 26.45 (46.24) (37.24) (24.76) Water 96.67 8.34 3.82 86.67 85.56 72.84 (Control) (81.45) (68.85) (67.84) Acetone 92.23 8.67 3.81 82.23 73.34 66.64 (Control) (73.85) (65.16) (58.91) SEM± 0.38 0.01 (1.91) (1.69 2.10 (2.52) ) CD 1.09 0.04 (5.45) (4.84 5.99 (P=0.05) (7.19) ) Figures in parentheses denote arcsine-transformed values. DATURA LEAF EXTRACT ON DIAMOND BLACK MOTH 165

All the treatments adversely affected adult emergence in comparison to the control. There was no adult emergence in 10 per cent methanol extract indicating its maximum adverse effect on biological parameters of P. xylostella. Adult emergence was less than 30 per cent in some of the treatments, being 13.34, 17.78, 21.12, 26.67 and 28.89 per cent in 7.5 per cent methanol extract, 2.0 per cent chloroform fraction, 2.0 per cent hexane fraction, 5.0 per cent methanol extract and 1.5 per cent chloroform fraction, respectively. In the remaining treatments, it ranged from 31.10 to 63.34 per cent.

Similarly, as compared to the control, significantly lower fecundity was observed in the case of larvae exposed to methanol extract/fractions. It was inversely related with the concentration of the extract/fractions. Minimum number of eggs was observed in 7.5 methanol extract which was also significantly lower than all the treatments. In all the treatments fecundity declined as compared to the control. However, other treatments producing decline in fecundity were: 2 per cent chloroform fraction, 2 per cent hexane fraction, 5 per cent methanol extract and 1.5 per cent chloroform fraction restricting fecundity to 26.45, 28.50, 28.69 and 33.75eggs/female respectively.

Though no information is available to our knowledge on the effect of D. metel extracts on the biology of P. xylostella, yet biological parameters of various insect species have been reported to be adversely affected by Datura extracts. Rajendran and Gopalan (1978) reported juvenomimetic activity of D. stramonium extract against Dysdercus cingulatus. Leaf powder of D. metel adversely affected adult emergence in Rhizopertha dominica (Jacob and Sheila, 1993). Extracts of various species of plants including neem and Melia azedarach, are known for their growth inhibitory effects in insects. In the present studies, growth inhibitory effects were also noticed in P. xylostella exposed to D. metel extract. This seems to be the first report with respect to P. xylostella (Liyanage, 2005).

CONCLUSIONS

It maybe inferred that leaf extract and fractions of D. metel exhibited adverse effects on various biological parameters of P. xylostella. However, further research is necessary to draw strong conclusion.

ACKNOLEDGEMENTS

The main author is extremely grateful to CARP, Sri Lanka for the financial assistance and Department of Entomology, 166 LIYANAGE et al.

CCS-Haryana Agriculture University, Hisar, India, for constant encouragement and providing laboratory facilities during the study.

REFERENCES

Chelliah, S. and K. Sirinivasan. 1986. Bioecology and management of diamondback moth in India, In Diamondback moth management, Proc. Ist-Int. Workshop, Shanhua, Taiwan Eds., Talekar, N. S. and Griggs, T. D. Asian Veg. Res. and Dev. Centre. Pp. 63-76. Gupta, D.S. and W.A. Rawlins. 1966. Persistence of two systemic carbamate insecticides in three types of soils. Indian J. Ent. 28: 482-493. Jacob, S. and M. K. Sheila. 1993. A note on the protection of stored rice from the Lesser Grain Borer, Rhizopertha dominica (Fabr.) by indigeneous plant products. Indian J. Ent. 55(3): 339 - 340. Kulkarni, N. and K. C. Joshi. 1997. Insecticidal action of some plant extracts against Albizia defoliator, Rhesala imparata Walker (Lepidoptera: Noctuidae). Entomon 22 (2):135-139. Liyanage, N.J. 2005. Studies on insecticidal properties of Datura metel against Plutella xylostella (Linnaeus) and Earias vittella (Fabricius) M. Sc. Thesis, Chaudhary Charan Singh Haryana Agricultural University , Hisar, India. Mehrotra, K.N. 1991. Current status of pesticide resistance in insect pests in India. J. Insect Sci. 4(1): -14. Meshram, P. B. 1995. Evaluation of some medicinal and natural plant extracts against teak skeletonizer, Eutectona machaeralis Walk. Indian Forest 121 (6): 528-532. Murugan, K., N.S. Raja, D. Jeyabalan, N.S. Kumar and S. Sivaramakrishnan. 1998. Evaluation of certain tropical plant extracts for their antifeedant and toxic properties against Spodoptera litura (Fab.). J. Insect Sci. 11(2): 186-187. Niber, B.T. 1994. The ability of powders and slurries from ten plant species to protect stored grains from attack by Prostephanus truncates Horn. (Coleoptera: Bostrichidae) and Sitophilus oryzae L. (Coleoptera : Curculinoidae). J. Stored. Prod. Res. 30 (4): 297-301. Rajendran, B. and M. Gopalan. 1978. Note on Juvenomimetic activity of some plants. Indian J. agric. Sci. 48 (5) : 306-308. Solunke, B.R. and S.V. Deshpande. 1991. Studies on the use of plant products for the control of lemon butterfly larvae. J. Maharashtra Agric.Univ. 16(2): 302-303. Talekar, N.S. and A.M. Shelton. 1993. Biology, ecology and management of the Diamond back moth. Annu. Rev. Entomol. 38: 275 - 301. Annals of Sri Lanka Department of Agriculture. 2006.8:167-173.

IDENTIFICATION OF SUITABLE GROWTH REGULATORS TO INDUCE ROOTING IN GRAPE VARIETY CARDINAL

S. M. NAGAHAWATTA, H. DON SUMANARATHNE and I. M. H. N. PRASAD Grain Legumes and Oil Crop Research and Development Centre, Angunakolapelessa

ABSTRACT

Grape has become a popular fruit crop among the dry zone farmers in the country. Cardinal is a recommended table variety, although its planting material production has been hampered due to the poor rooting nature. Therefore, an experiment was carried out to find out suitable growth regulators to improve the rooting ability. The growth regulators IAA and IBA and 1:1 mixture of IAA and IBA were the three main treatments. Five concentration levels (0, 25, 50, 75 and 100 mg/l) of two growth regulators and the mixture were the five sub treatments. Treatments were replicated five times in split plot design and tested for four seasons. Grape cuttings with 3-4 nodes and 20cm long were dipped in pre-prepared growth regulators as described in treatments for 12 hours. These cuttings were buried in beds of wet coir dust for 14 days. IAA and IBA increased the rooting percentage till the concentration reached up to 75mg/l and declined thereafter. The 1:1 mixture of two growth regulators increased the rooting up to the concentration of 50mg/l. The 1:1 mixture of IAA and IBA is the most suitable growth regulator mixture and its optimum concentration range is 25 to 50mg/l. IAA or IBA also can be used to induce rooting and their optimum concentration range varies from 50 to 75mg/l.

KEYWORDS: Grape, Growth Regulators, Propagation.

INTRODUCTION

Grape (Vitis vinifera L.) was introduced to Sri Lanka in Portuguese period and cultivated in Jafna peninsula and the nearby coastal areas. Consequently, it had been spread to other dry zone areas such as Kalpitiya, Anuradapura, and Hambantota and now it has become a popular fruit crop among the dry zone farmers. At present, the total extent of grapes is around 90ha producing approximately 400mt per year. However, the total imports of fresh grapes in 2004 was 3479mt (value – Rs million 316.) which was eight times greater than the annual production (Anon., 2004).

Grape is generally propagated by rooted cuttings. Winkler (1962) defined a stem cutting as a piece of parent plant that would develop into a new plant when placed under favorable conditions for growth. The matured cuttings (brown in color) taken on pruning of vines should be given wet, cool and dark environment to induce callus formation and rooting. In practice, the cuttings are buried in wet coir dust or sand beds which were prepared under shade. After 2-3 weeks, callus and roots are formed. 168 NAGAHAWATTA et al.

In general, rooting of stem cuttings varies with the varieties. Stem cuttings of some varieties root so rapidly while the others are difficult to root under the same conditions favorable for rooting. Among the varieties recommended in Sri Lanka by the Department of Agriculture, variety Israel Blue gives rapid and variety Muscat MI gives moderately high rooting under favorable conditions. It was observed that, the recommended variety Cardinal gives very low percentage of rooting even under the optimum conditions for rooting. Variety Cardinal is a high yielding, good quality table variety and it performs well in the dry zone area. However, its planting material production has been hampered due to the poor rooting nature of the variety. Therefore, still this variety has not been popularized among the grape growers in the country.

According to Singh and Singh (1968), plant growth regulators can be used to increase the percentage of rooted cuttings and increase the number of roots per cutting. Julliard (1954) reported that stem cuttings of grape treated with IAA, produced abundant roots at the base of cuttings. Sarrisora (1964) also found that stem cuttings of seven grape vine varieties treated with Indole Acetic Acid (IAA), Indole Butric Acid (IBA) and Naptyle Acetic Acid (NAA), rooted better than the control. According to Herath et al. (1978), IBA can be successfully used as a root-promoting substance in grape variety Semillon. Therefore this experiment was conducted to find out suitable growth regulators which would improve the rooting ability of stem cuttings of this variety Cardinal.

In tropical areas, grape vines are pruned twice a year in mid June (June pruning) and in the end of January (January pruning). In general, it was observed that under normal propagation conditions the planting material production is high in the June pruning. Therefore, this experiment was conducted using cuttings of both pruning seasons to determine whether cuttings taking period has an effect on rooting.

MATERIALS AND METHODS

Experiment was conducted in the green house at Grain Legumes and Oil Crop Research and Development Center (GLOCRDC), Angunakolapelessa, over four seasons from June 2002 to July 2004.

Growth regulators Indole Acetic Acid (IAA), Indole Butric Acid (IBA) and 1:1 mixture of above two growth regulators were considered as the three main treatments. Five concentration levels (0, 25, 50, 75 and 100 mg/l) of above three treatments were the five sub treatments. Treatments were replicated five times in split plot arrangement of Randomize Complete Block design (RCBD) and tested for four seasons. GROWTH REGULATORS TO INDUCE ROOTING IN GRAPE 169

Cuttings were taken from 3 year old vines at pruning and selected cuttings of 6-8 mm diameter were trimmed to length of 20 cm, containing 3-4 nodes. In the upper end of the cutting made a straight across cut 1 cm above the node and in the bottom, made a slant cut (450) just below the node. All the cuttings were dipped in fungicide solution Homai (active ingredient Thiophanate Methyl 50% and Thiram 30 %) to avoid fungal contaminations. Prepared the growth regulators in different concentration levels (0, 25, 50, 75 and 100 mg/l) and basal parts of cuttings were dipped in these solutions separately (25 cuttings per one concentration level) and kept for 12 hours. After that bundled cuttings (5 cuttings in each bundle) were labeled and then buried in wet coir dust medium for curing. These coir dust beds were also pretreated with fungicide Homai and watered well at three days interval.

The experiment was completed after 14 days and recorded the rooting percentage and total root length of grape cuttings. Data were statistically analyzed in split plot design and differences between the treatments were tested by ANOVA and followed by mean separation using SAS software package. T-test was used for mean separation in the analysis of pruning time (month).

RESULTS AND DISCUSSION

According to Tables 1 and 2, both growth regulators and their concentrations showed significant increase on the rooting percentage and the total root length. The interaction between two factors was also significant. Therefore, the effect of each growth regulator is considered under each level of concentration.

When considering the growth regulators IAA and IBA separately, with the increase of concentration levels from 0 to 75 mg/l, rooting percentage and total root length increased up to the maximum. However, beyond that level both rooting percentage and total root length decreased (Tables 1 and 2). As shown in Figure 1 in all four seasons, the growth regulator IBA reacted more effectively on rooting of grape cuttings than IAA.

With the 1:1 mixture of IAA and IBA, rooting percentage and total root length reached the maximum at concentration level of 50 mg/l and beyond that, rooting decreased. This pattern was common in all the four seasons.

The maximum rooting percentage varied with the pruning time in which cuttings were collected for planting material production. Cuttings taken from the June pruning recorded significantly greater rooting percentage and total root length over the cuttings taken in January (Table 3). 170 NAGAHAWATTA et al.

Table 1. The effect of growth regulators and their concentration levels on percentage rooting of grape cuttings (variety Cardinal) at GLOCRDC Angunakolapelessa from June 2002 to July 2004.

Main treatments Sub treatments Rooting Percentage* Growth Levels June pruning January pruning regulators mg/l 2003 2004 2003 2004 IAA 00 25.0 d 17.3 d 12.7 d 9.3 e 25 34.7 c 30.0 c 24.3 c 16.7 d 50 43.0 bc 41.7 b 34.3 a 30.0 b 75 57.3 a 57.0 a 40.3 a 40.3 a 100 48.0 a 40.0 b 32.0 b 22.3 c CV % 11.1 5.7 12.6 11.0 IBA 00 24.7 c 16.7 e 12.3 d 12.3 c 25 50.3 b 34.3 d 28.0 c 27.6 b 50 63.0 a 56.0 b 39.0 b 41.0b 75 71.0 a 69.0 a 43.0 a 49.3 a 100 52.7 b 48.0 c 27.0 c 28.7 b CV % 7.8 7.8 5.7 9.6 IAA + IBA 00 27.0 d 19.6 c 10.3 e 11.3 e 25 61.0 b 56.0 b 36.7 c 37.0 c 50 88.1 a 83.0 a 52.7 a 57.0 a 75 79.0 b 65.3 b 43.3 b 52.0 b 100 48.6 c 56.0 b 28.3 d 22.3 d CV % 4.8 10.4 10.3 10.6 *Means followed by the same letter under each main treatment in each column are not significantly different at p = 0.05

Although Harmon (1942) and Weaver (1956) stated that growth regulators had not induced rooting in grape cuttings, this experiment shows that the growth regulators IAA or IBA or the mixture of two can be effectively used to accelerate rooting in this variety Cardinal. Results of the experiments carried out by Sarrisora (1964) for seven grape varieties and Herath et al (1978) for variety Semillon also supported the findings of the present experiment.

The most common and practical method of raising planting material is by rooted cuttings. Rooting is one of the most important factors which directly affect planting material production. In practice, a direct relationship between the rooting percentage and the number of plants produced was observed. Therefore, it is important to use growth regulators to enhance root initiation in cuttings which can increase the planting material production rapidly.

In tropical areas grape vine pruning is carried out twice a year. Vine pruning is presently practiced in mid January and mid June. Usually, cuttings for propagation can be obtained at both of these prunings. GROWTH REGULATORS TO INDUCE ROOTING IN GRAPE 171

Table 2. The effect of growth regulators and their concentration levels on total root length of grape cuttings (variety Cardinal) at GLOCRDC Angunakolapelessa from June 2002 to July 2004. Main treatments Sub treatments Total root length (mm)*

Growth Levels June pruning January pruning regulators mg/l 2003 2004 2003 2004 IAA 00 143 b 137 c 38.3 d 37 d 25 149 b 149 c 74.7 c 82 c 50 264 ab 262 b 133 b 143 b 75 295 a 312 a 181 a 191 a 100 270 ab 266 b 70 c 73 c CV % 31.6 8.5 13.7 8.4 IBA 00 139 c 143 c 31.0 d 35 d 25 487 ab 497 a 92.0 c 96 c 50 513 ab 516 a 157.7 b 162 b 75 606 a 530 a 233.7 a 242 a 100 449 b 382 b 111.3 c 121 c CV % 16.7 5.0 10.8 10.6 IAA + IBA 00 140 d 145 d 34.0 e 32 e 25 658 bc 652 b 152.0 c 150 c 50 753 a 712 a 309.6 a 312 a 75 662 b 610 c 271.3 b 274 b 100 589 c 590 c 125.7 d 133 d CV % 6.9 3.5 4.9 4.5 *Means followed by the same letter under each main treatment in each column are not significantly different at p = 0.05

Table 3. Effect of cuttings taking period on rooting percentage and total root length of grape variety Cardinal over four seasons at GLOCRDC Angunakolapelessa from June 2002 to July 2004.

Observations Rooting percentage* Total root length (mm)*

2003 June pruning 50.06 a 407.8 a January pruning 30.94 b 134.3 b 2004 June pruning 45.84 a 393.5 a January pruning 26.34 b 146.3 b *In each year, column values followed by same letters are not significantly different at p= 0.05 172 NAGAHAWATTA et al.

June 2003 June 2004 100 100

e e g

g 80 a a 80 t t n n e e c c r r 60 e 60 e p p

g g n i n i t

t 40 o 40 o o o R R 20 20

0 0 0 25 50 75 100 0 25 50 75 100 Concentration level mg/l Concerntration level mg/l IAA IBA IAA + IBA IAA IBA IAA + IBA

January 2003 January 2004

100 100

e e g g a t a

80 t

n 80 n e e c r c r e e p 60

60 p

g g n i t n i o 40 t

o 40 o o R R 20 20

0 0 0 25 50 75 100 0 25 50 75 100 Concerntration level mg/l Concerntration level mg/l IAA IBA IAA + IBA IAA IBA IAA + IBA

Figure 1. The effect of growth regulators and their concentration levels on rooting percentage of grape cuttings (variety cardinal) in different seasons at GLOCRDC Angunakolapelessa from June 2002 to July 2004.

According to the results of this experiment, rooting percentage of grape cuttings varies with the pruning time in which cuttings are taken. In general, the rooting of cuttings taken from January pruning is low and accordingly, the planting material production is also low in this season. This may be due to poor growth and low food reserves due to heavy defoliation caused by fungal diseases during the maha rainy period. Also in the maha season, the prevailing cloudy conditions may hamper the photosynthesis process to reserve less food in stems compared to the yala season. Even treating with the growth regulators, the rooting percentage was low compared to the cuttings taken in June.

CONCLUSIONS

Growth regulators of IAA or IBA can be used to induce rooting of grape cuttings in variety cardinal. Concentration range of 50 – 75 mg/l can be used to obtain sufficient rooting. The 1:1 mixture of IAA and IBA was identified as the best combination for propagation of grape variety cardinal. GROWTH REGULATORS TO INDUCE ROOTING IN GRAPE 173

The identified optimum concentration range for the above mixture was 25 - 50 mg/l.

There is a seasonal effect on rooting of grape cuttings. The rooting of grape cuttings taken at January pruning was low, even with the use of growth regulators compared to cuttings taken in January. Accordingly, possibility for the planting material production of grape is high in June.

REFERENCES

Anon., 2004. External trade statistics. Sri Lanka Customs, 2004.

Harmon, F.N. 1942. Influences of IBA on the rooting of grape cuttings. Proc. American Society of Horticultural Science 42: 38- 88.

Herath, H.M.E., R.M.Ariyarathne and E.R.Pinto. 1978. Studies on propagation of grape vine in Sri lanka. Tropical Agriculturist cxxxiv:65-69.

Julliard, B. 1954. The effect of indole B-acetic acid on the rooting of Vitis berlandieri. C R Acad. Science 259: 3356-3359.

Sarrisora, M.M. 1964. The effect of growth substances on root formation in vine cuttings. DOK Lady Akademiya Nauk Arm S S R 39: 53-55.

Singh, R. P. and S.N. Singh. 1968. Effect of nodes on rooting of grapes (Vitis vinifera L.) Tropical Agriculturist cxxiv: 37-45.

Weaver, R. J. 1956. Plant regulators in grape production. California Agricultural Experiment Station Bulletin 752 : 1-26.

Winkler, A. J. 1962. General viticulture. University of California Press, California John, 1970. Annals of Sri Lanka Department of Agriculture. 2006.8:175-181.

NEW TRELLISING SYSTEM FOR BITTERGOURD (Momordica charantia L.)

N. PARARAJASINGHAM Horticultural Crop Research and Development Institute, Gannoruwa, Peradeniya

ABSTRACT

Bittergourd (Momordica charantia) grows fast and the vine elongates rapidly. Hence it is necessary to train the plants on a trellis by removing the lateral branches until the vine reaches the top of the trellis. Horizontal trellising is the traditional practice. However, vertical trellising (fencing) facilitates hand pollination, pesticide spraying and harvesting. The objective of this study was to investigate the possibility of improving yield, fruit quality and net returns through an alternate cost effective trellising system. Experiments were conducted during maha 1999/2000 and yala 2000 at HORDI Gannoruwa to test three trellising systems namely horizontal, inverted V shape and vertical trellising using coir string and steel wire on bittergourd (variety MC43). Vertical trellising resulted in significantly higher yield (13.27 t/ha) when compared to inverted V shape trellising (11.1 t/ha). Fruit quality (weight, length and circumference) and net returns were also higher. However, coir string and steel wire were both equally suitable. Vertical trellising using either steel wire or coir string gave the highest yield (13.56 and 12.98 t/ha) and the highest benefit/cost ratio (1.788 and 1.610). Hence, vertical trellising can be recommended as a cost effective trellising system for bittergourd.

KEYWORDS: Bittergourd, Trellising system, Yield.

INTRODUCTION

Bittergourd (Momordica charantia L.) also known as bitter melon and balsam pear grows well in the tropics and sub tropics (Purnima, 1999). It is cultivated in South East Asian and Caribbean countries, but its origin is not known. (Tindall, 1983). Fruits are used in the preparation of curries and pickles. Seeds are reported to contain toxic alkaloids (Robinson, 1997) and the fruits are high in iron and ascorbic acid contents compared to other cucurbits, (Tindall, 1983). Bitter gourd is a popular vegetable in Sri Lanka mainly due to its medicinal value (Purnima, 1999). It is commonly cultivated in the low country and mid country regions (< 1000m altitude). The annual extent under this crop is about 3662 ha and production is about 26030 t per annum (Anon., 2005). Bittergourd grows fast and the vine elongates within a short period of about two weeks after planting and thereafter the plant sends out lateral stems. Hence it is necessary to train the plants on a trellis by removing lateral branches until the vine reaches the top of the trellis. This gives satisfactory cover of the trellis top and a stronger growth of the main vine (Williams et al., 1991). Although horizontal trellising is the traditional practice, vertical trellising (fencing) is more practical because it facilitates hand pollination, pesticide spraying and even harvesting. However construction of the trellis is costly and accounts for about 14.6% of the cost of 176 PARARAJASINGHAM cultivation (Anon., 2003). Yousuf et al. (2001) have reported that vertical trellising results in better fruit set in Teasle gourd (Momordica dioica roxh) when compared with other types of trellising. This study was conducted to investigate the possibility of improving yield, fruit quality and net returns through an alternate cost effective trellising system.

MATERIALS AND METHODS

Experiments were conducted during maha 1999/2000 and yala 2000 in the mid country wet zone, at Horticultural Crop Research and Development Institute, Gannoruwa on a Reddish Brown latosolic, moderately acidic (pH = 5.2) soil. Mean monthly weather data during the period of the experiment are given in Table 1.Two factors- i.e. three trellising systems (horizontal, inverted V shape and vertical trellis ) with two types of material (coir string and steel wire) were laid out in a Randomized complete block design with 3 replicates. Small plastic bags (10cm diameter and 12.5 cm height) were filled with potting mixture (sand: soil: cattle manure 1:1:1) and a recommended bittergourd variety MC43 was planted (3 seeds per bag). When plants were 10-12 cm in height two seedlings were transplanted in the field at 1.5 m x1.0 m spacing in 4 m x 3 m plots (8 plants per plot) at 15 days after sowing. Well decomposed cattle manure (10t/ha), was applied to all plots one week before planting. At planting 200 kg triple super phosphate, and at one, four and six weeks after transplanting 75kg urea and 60kg muriate of potash per hectare, were applied. Irrigation was done daily using a sprinkler system and manual weeding was practised.

The three trellising systems are shown in Figures 1-3. The top frame of the horizontal trellis was made with steel wire or coir string into a horizontal net like structure. In the inverted V shape system, two stakes were placed in the form of an inverted V and connected with steel wire/coir string, and in vertical trellising, stakes were connected with steel wire/coir string. Two meter long gliricidia stakes (height of the trellis) were used to support the trellis and the vines were carefully trained along the stakes. Side shoots/lateral branches were pruned until the vine reached the top of the trellis and the branches above the trellis were allowed to grow and lateral branches produced below the trellis were pruned.

Downey mildew, gall fly, leaf hopper and fruit fly incidences were observed during both seasons and recommended pesticides were used. Fruits were harvested weekly from about the10th to 15th week after planting. Total fruit yield, weight per fruit, number of fruits per plant, fruit length and circumference were recorded at each harvest. Durability and quantity of the trellising materials used, and time taken to construct the trellis were recorded. TRELLISING FOR BITTERGOURD 177

Steel wire/ coir string

Figure 1. Horizontal trellising system.

Figure 2. Inverted v shape trellising system.

Figure 3. Vertical trellising system.

A – Between rows spacing B – Within rows spacing Height of all three trellising systems was 2 m.

Table 1. Weather data recorded during the experiment.

Season Temperature °C *RH % RF+ **BS ET+ + Max. Min. a.m. p.m. (mm) hour/day (mm) maha 1999/2000 Jan 2000 29.1 18.6 83 71 82.1 5.8 76.9 Feb 2000 30.4 19.8 87 70 177.1 6.0 62.6 Mar 2000 31.1 19.5 84 63 78 6.9 81.8 Apr 2000 31.2 21.1 81 73 255 7.6 81.6 yala 2000 Jun 2000 28.3 22.1 83 76 103.7 4.3 60 July 2000 29.1 21.5 79 68 47 6.9 79.4 Aug 2000 28.1 21.1 83 75 226.4 4.1 59.5 Sep 2000 29.7 20.5 78 74 155.6 6.4 72 178 PARARAJASINGHAM

*RH Relative humidity; a.m. –Reading at 0009 h; p.m.Reading at 1600 h; RF+ Monthly total rainfall;**BS- Bright sunshine; ET++ Monthly evopotranspiration RESULTS AND DISCUSSION

The 1999/2000 maha and 2000 yala data were combined and blocks were nested within the seasons as the experiment consists of three factors namely, three trellising types, two trellising materials and two seasons. Mean yield data for the different trellising systems are given in Table 2.

Table2. Effect of trellising system on mean yield during the two seasons.

Trellising system Yield (t/ha) Maha 1999/2000 Yala 2000 Mean Horizontal Coir string 11.01 12.25 11.63 Steel wire 12.12 13.33 12.73 Mean 12.18 Inverted V Coir string 10.73 11.18 10.96 Steel wire 9.43 12.88 11.16 Mean 11.06 Vertical Coir string 11.44 14.52 12.98 Steel wire 11.38 15.74 13.56 Mean 13.27 Season mean 11.02 13.37

LSD at p=.0.5 Interactions ns Trellising material ns Trellising type 1.15 Season 0.94

CV% 11.1

None of the interactions were significant. Hence, season and trellising material had no influence on trellising type. This means the fruit yield obtained with the different trellising systems was solely due to the trellising type and was not affected by the material used or the season in which the crop was cultivated. Vertical trellising gave a significantly higher mean yield of 13.27 t/ha (Table 2) and this was 20% higher than the yield obtained with inverted V trellising (11.06 t/ha). The traditional horizontal trellising system gave a mean yield of 12.18 t/ha but this was not significantly different to the yield in vertical and inverted V systems.

Yousuf et al. (2001) reported that Tecisle gourd (Momordica dioica roxh) gave the highest yield with vertical rope net trellis, followed by bamboo trellis (horizontal net like structure with bamboo sticks) and upright konchee trellis (bamboo branches placed up right around the plant covering TRELLISING FOR BITTERGOURD 179 area). Chowdhury et al. (1983) reported that type of trellis had a significant effect on cucurbit yield. The better performance of the vertical trellising system could be attributed to the vines in this system being well spread and exposed to sunlight. Hossain et al. (1987) and Islam et al. (1972) reported that vertical rope net trellis in teasle gourd is better than upright konchee and flat bamboo trellis, because the flowers were visible and this makes hand pollination easy.

Mean yield of the trellising material was not significant, thereby indicating that both coir string and steel wire were equally effective (Table 2).

The yala season yield was about 20.9% higher than during maha (Table 2). The low yield during maha was probably due to high evopotranspiration and low rainfall but longer sunshine hours during the fruit development period. (Table 1). Venkateswaralu and Vispears (1987) reported that rice yields increased when solar radiation was high, provided water was sufficient.

Table 3: Fruit characters in different trellising systems. 180 PARARAJASINGHAM

Trellising system Fruit weight (g) Fruit Fruit No. of fruits maha1999/200 lengt circumf per plant 0 yala 2000 h erence maha yala (cm) (cm) Horizontal Coir 86 118 29 15 19 17 string Steel 68 124 29 15 27 15 wire Inverted V Coir 86 109 29 15 18 15 string Steel 83 114 28 14 17 17 wire Vertical Coir 86 131 29 15 20 17 string Steel 86 136 31 16 20 17 wire CV % 9.01 7.2 8.1 12.5 LSD at p=.0.5 Trellising material ns ns ns ns Season 6.424 1.45 0.83 1.53 Trellising type 7.87 ns ns 1.89 Season x Trellising type 11.13 ns ns 2.672 Season x Trellising 13.63 ns ns ns material Season x Tr. type x Tr. 15.74 ns ns 3.78 material * Mean of the season Mean fruit weight, and number of fruits per plant (Table 3) data indicate that there is a seasonal influence on the trellising type. During yala, fruit weight was the highest in the two vertical trellising systems. Mean number of fruits per plant (Table 3) did not vary much in the different treatments.

The cost involved in the different trellising systems was the highest in the horizontal trellising (traditional farmer practice) using coir strings (Table 4).

Table 4. Economic analysis of different trellising systems.

Trellising system Trellisin Cost(Rs/ha Total Crop Net Benefit g ) return return cost other (Rs). (Rs.) ratio operation Horizontal: Coir strings 22733 52864 75597 174495 98898 1.308 Steel wire 21000 52804 73864 190800 116936 1.583 Inverted V: Coir strings 19025 52804 71889 164400 92511 1.287 Steel wire 17350 52804 70222 167400 97178 1.384 TRELLISING FOR BITTERGOURD 181

Vertical: Coir strings 21833 52804 74697 194700 120003 1.61 Steel wire 20100 52804 72964 203400 130436 1.788

All calculations were based on price of in puts, wage rate, price of product as at 1999 Price of one stake Rs 1 Labour wage Rs130/day Price of coir strings 1kg Rs 35 Price of steel wire 1kg Rs 50 Price of bittergourd Rs 15 Steel wire durability >3 seasons Coir strings durability 2 seasons

The vertical trellising system with steel wire gave the highest benefit/cost ratio (1.788) followed by vertical trellising with coir string (1.61) and horizontal trellising with coir strings (1.308).

CONCLUSIONS

The vertical trellising system with either coir string or steel wire is the most cost effective and practical trellising system for bitter gourd. However, considering durability, steel wire is better than coir string.

ACKNOWLEDGEMENTS

The former and present Directors HORDI Dr. G. Jayawardena, Dr. C. Kudagamge and Dr. I.J. de Zoysa, former and present vegetable division heads Ms. B.Wahundeniya and Ms. R Peiris are thanked for their contribution and support during the study period. The staff at HORDI especially Mrs. Bulathgama and Mrs. Wijeratna Menike for the cooperation extended. REFERENCES

Anon., 2005. Ag. Stat. Vol. II. Pocket Book of Agricultural Statistics. Socio-Economic and Planning Centre, Department of Agriculture, Sri Lanka. p 7. Anon., 2003. Cost of cultivation of agricultural crops. Socio-Economic and Planning Centre, Department of Agriculture, Sri Lanka. p 32. Chowdhury, A.R., R.A.Begum and M.A.J .Bhuiyan, 1983. A study on the performance of two cultivars of bitter gourd grown with or without trellis. Bangladesh Hort. 17: 44-45. Hossain, M.A., M.S.A. Faki , M.M.Rahman and M.S.H.Chowdhury, 1987. Fruit setting by artificial and natural pollination in teasle gourd. Bangladesh Hort. 15:40-45. Islam, M,S., R.A.Begum, S.R.Saha, S.M.M.Hossain and M. M. Haque. 1972. Effect of artificial pollination and hormone application on the yield of bottle gourd Lagenaria siceraria L. Bangladesh Hort. 20:47-49. Purnima, J. 1999. Medical and aromatic plant science series No.3. Information Services Centre Industrial Technology Institute, Colombo, Sri Lanka. Pp 1-2. Robinson, R.W. and D.S.Decker-Walters.1997. Cuvurbits.CAB International,U.S.A. 99 p. 182 PARARAJASINGHAM

Tindall,H.D. 1983.Vegetables in the tropics English Language Book Society, Macmillan Hamshire. Pp 179-181. Venkatesawaralu,B. and R.M.Vispears 1987. Solar radiation and rice productivity IRRI research paper series . No.129. International Rice Research Institute, Phlippines. 26p. Williams,C.N., J. Ouzo and W.T.H.Peregrine. 1991.Vegetable Production in the Tropics. Longman Group, U.K Limited. 89p. Yousuf Mian, A. M., M.Rahman, M. A. K. Mian, M.M. Hossain, M.S.Islam and Shahidul. Islam. 2001. Performance of teasle gourd genotypes grown on different trellis. Bangladesh Online Journal of Biological Science 1(5):369-371. Annals of Sri Lanka Department of Agriculture. 2006.8:183-191.

MANAGEMENT OF CORM ROT OF KIRIALA (Xanthosoma sagittifolium (L) SCHOTT) BY BIOLOGICAL CONTROL AGENT, Trichoderma harzianum

R.G.A.S. RAJAPAKSE, J. KAHAWATTA and S.M.I.S.K. SAKALASOORIYA Horticultural Crop Research and Development Institute, Gannoruwa, Peradeniya

ABSTRACT

Kiriala (Xanthosoma sagittifolium) production in the country has suffered heavily in the recent past, due to emergence of corm rot disease. Therefore, studies were conducted to identify causal factor/s and development of control measures for this problem. The causal agents of this disease were identified as Rhizoctonia solani and Pythium species. Disease management mechanism of Trichoderma harzianum was identified as mycoparacitism on fungi. Trichoderma harzianum inoculum was produced by using a sawdust based carrier medium and inoculated into the soil two weeks before and one month after planting. The fungicide, Homai (2 g/l) was used to compare the efficacy of Trichoderma harzianum. The study was repeated for three years in the same field. Results revealed that corm rot symptoms of Kiriala completely disappeared after application of Trichoderma harzianum (500 g inoculum /m-2) and efficacy of the bio control agent was similar to the fungicide treatment.

KEYWORDS: Biological control, Corm rot, Trichoderma.

INTRODUCTION

Kiriala (Xanthosoma sagittifolium (L) schott) is an important crop in wet and intermediate zones of Sri Lanka. Kiriala suckers, which are grown in nursery until 3-5 leaves emerge, are planted in the field throughout the year and are harvested after 8-10 months period. Kiriala production in Kandy, Kegalle, Galle, Gampaha, Kurunagala and Colombo areas have suffered heavily in the recent past due to the emergence of corm rot and has become a serious problem constraining profitable Kiriala cultivation.

Corm rot of Kiriala was first observed in the wet zone areas and since then, this disease has gradually spread into other areas in the country. At present this disease has reached an epidemic level in most of the Kiriala cultivations. This disease tends to occur in fields with poor drainage where water accumulates in the field or on the lower sections of gentle slopes, and causes heavy damage in these places (Nzietchueng, 1984 and Anon., 2004). The main symptoms observed in the infected plants were: poor plant growth, tip of outer side of leaves turning yellow and finally death of the entire leaf and rotting of the corm.

It has been reported that soil-borne fungal pathogens, Pythium species cause corm rot of Kiriala in Cameroon and Gampaha District 184 RAJAPAKSE et al. in Sri Lanka (Hiroshi and Shingo, 2005). However, no studies are recorded on identify the fungal pathogens, which cause corm rot of Kiriala in Sri Lanka. Even though application of fungicides is one of the most effective methods in controlling fungal pathogens associated with diseased plants, it could not effectively control soil-borne diseases due to degradability of fungicides in soil. Therefore, identification of effective control methods for management of corm rot fungi is needed.

Biological control of soil borne diseases is a popular alternative in environment and economical sense because it offers durable and cost effective alternatives to soil applied fungicides (Hornby, 1990). Genus Trichoderma represents widely studied fungi that show antagonistic activity towards soil borne fungal pathogens. Parasitic activity of members of the genus Trichoderma to fungal pathogens such as Rhizoctonia solani, Pythium and Fusarium species has been recorded (Hornby, 1990). This study was undertaken to identify the pathogens, which cause corm rot of Kiriala and efficacy of the bio-control agent, Trichoderma harzianum isolates for the management of corm rot of Kiriala.

MATERIALS AND METHODS

Laboratory investigations Isolation of causal organisms

Isolation of causal organisms was done from diseased Kiriala plants obtained from different localities in Kandy, Kegalle, Gampaha, Galle, Kurunegala, and Colombo districts. Eighteen samples of diseased Kiriala were tested in the laboratory to identify causal agents. Corms of diseased plants were thoroughly washed in water until soil particles were completely removed. Washed corms were placed on a paper for blot drying and then placed in a humid chamber for 5 days for growth and sporulation of fungi. After incubation of corms, mycelia were scraped off from surfaces of rotting corms and observed under the microscope for fungal growth. Mycelia samples of fungal isolates were cultured on potato dextrose agar (PDA) and corn meal agar (CMA) and incubated for 7 days in room temperature. The mycelial growth, conidia and other spore development on culture media were observed. Mycelia and conidial masses were picked from colonies, which were suspected to be different genera of fungi and sub cultured on PDA and CMA media for further purification. Each isolate was identified on the basis of microscopic observations of conidia or spores and mycelial and culture characteristics on media. Pure cultures were maintained on PDA by regular sub-culturing. Pathogenicity of each isolate was tested by wound inoculation on healthy suchers and symptom development. MANAGEMENT OF CORM ROT BY BIOLOGICAL CONTROL AGENT 185

Collection of Trichoderma isolates

The isolates of Trichoderma harzianum were obtained from the Trichoderma culture collection of Horticultural Crop Research and Development Institute, Gannoruwa, Peradeniya. They were inoculated on to PDA and incubated at room temperature for 7-10 days. The fungi growing on the medium were isolated and identified using morphological characters of spores and arrangement of the spores (Rifai, 1969).

Antagonistic activity of Trichoderma against fungal pathogens (in vitro)

The Trichoderma harzianum isolates were inoculated on PDA medium with Fusarium oxysporum, Rhizoctonia solani and on CMA medium with Pythium sp. and incubated for 14 days to test antagonistic reactions. After incubation, growth of Trichoderma and pathogens in each plate was visually and microscopically observed for detection of bio-control ability of Trichoderma harzianum. Selected isolates were used for further bio-control studies in fields. Each treatment was replicated three times and experiment was repeated two times.

Mass culturing of Trichoderma harzianum

A sawdust based carrier medium (Saw dust - 1kg, Rice bran – 100 g, Soy flour – 10 g, Lime – 20 g, MgSO4 – 2 g, K2HPO4 – 0.8 g, Urea - 0.5, KCl – 0.15 g, Glucose 20 g and water 1 l) was prepared and 500 g of this medium was put into polypropylene bags. These bags were autoclaved at 121oC for 20 minutes under 15 Psi (Anon., 2003). Trichoderma harzianum isolates- HORDI 1 and HORDI 2 cultured on PDA were incubated at room temperature for 2 weeks until well sporulated. Conidial suspensions (107 conidia/ml water) were prepared from culture plates of Trichoderma isolates. Ten ml of conidial suspension was added into carrier material of the bags under aseptic condition and incubated for 3-4 weeks period in room temperature. Growth of Trichoderma isolates in carrier material of the bags was observed microscopically. The spore concentrations of Trichoderma in randomly selected bags were estimated by preparing serial dilution and spore counting using a heamocyto meter.

Field Experiments Effect of Trichoderma harzianum on control of corm rot of Kiriala

Experiment was established in year 2003 at Horticultural Crops Research and Development Institute, Gannoruwa and the experiment was repeated two times in the same field in year 2004 and 2005. A location where Kiriala had been cultivated for a long period and later abandoned due to high incidence of corm rot disease was selected as the experimental field. 186 RAJAPAKSE et al.

Experimental procedure followed was RCBD with 4 replications. The treatments used in the experiment are shown in Table 1.

Table 1. Treatment combinations for field studies in years 2003, 2004 and 2005.

Treatments Rate of application T1 - Trichoderma inoculation (HORDI-1) into soil 500 g inoculums/1m2 of plot T2 - Trichoderma inoculation (HORDI-2) into soil 500 g inoculums/1m2 of plot T3 - Homai-corm treatment and soil drenching One litter (2 g/l) per planting hole T4 – Control No Trichoderma or Homai

The causal agent of corm rot of Kiriala in the experimental field was identified as Rhizoctonia solani. Healthy Kiriala suckers at 2-3 leaves stage were transplanted (8 plants/bed) in flat beds of 1.5 m x 3 m size. All agronomic practices were done according to the recommendations given by the Department of Agriculture. Dried cow dung (1 kg/m2) was applied to soil two weeks before planting of Kiriala suckers. Isolates of Trichoderma harzianum were incorporated into the soil by mixing Trichoderma medium with soil 2 weeks before planting (500 g/m2) and then again re-inoculated with soil one month after planting. The average spore concentration in Trichoderma bags was around 106 spores/g of medium. The fungicide treatment was done by dipping corms in a fungicide solution (Homai 2 g/l) for one hour prior to planting in the plots and then again soil drenching (one litter of homai (2 g/l) solution per plant) was done one month after planting. The disease development was observed during crop growth and percentage of diseased plants and yield in each treatment were recorded in each plot. Plants, showing yellowing of outer side leaves, poor plant growth and death of leaves were considered as diseased plants. The corm samples of each treatment obtained from uprooted plants at the end of the season were tested in the laboratory to identify associated pathogens. The data were analysed by ANOVA and mean separation were done by Duncan’s multiple range test.

Determination of survival of Trichoderma under field conditions

The survival of Trichoderma harzianum in the soil was assessed by a serial dilution technique. Four soil samples (10g) from each plot were taken, 3 months after inoculation, and ten fold (10-4, 10-5 and 10-6) dilution series was made. One ml of dilutions from the highest to the lowest were cultured by spreading on the plates of PDA + Streptomycin (0.1%) medium and incubated in room temperature. Two plates were used to culture each inoculation level and numbers of Trichoderma colonies were counted after 5 days of incubation. MANAGEMENT OF CORM ROT BY BIOLOGICAL CONTROL AGENT 187

RESULTS AND DISCUSSION

Identification of pathogens

Different fungal genera were isolated from corm of diseased Kiriala plants in different locations (Table 2). Among them Rhizoctonia sp. was commonly isolated from rotting corms collected from all locations. Pythium was isolated from Kandy, Kegalle and Gampaha areas. Fusarium spp. was isolated only from corm samples obtained from Colombo area. Some of the diseased corms were infected with all above pathogens.

The isolates of fungi were identified by comparison of their colony characters and morphology on PDA and CMA with published data (Anon., 1964, 1967 and 1970). Rhizoctonia species was identified as R. solani and Fusarium species was identified as F.oxysporum. Pythium species could not be identified by microscopic observations and culture characteristics on the media (Table 3). However, Hiroshi and Singo (2005) have reported that P. myriotylum is the causal agent of corm rot of Kiriala in Gampaha area. Isolates of Rhizoctonia sp. and Pythium sp. caused rotting on artificially inoculated corms. Isolates of Fusarium sp. developed only localized rotting but did not develop progressive rotting symptoms when artificial inoculation was done on corms. Inoculation and re-isolation tests confirmed that corm rot of Kiriala (Xanthosoma sagittifolium) is a fungal disease caused by Rhizoctonia solani and Pythium sp.

Table 2. Isolation of fungal pathogens from rotting Kiriala corms.

Sampling areas No. of sample tested Pathogens associated with corm rot Kandy 6 Rhizoctonia solani, Pythium sp. Kegalle 4 Rhizoctonia solani, Pythium sp. Gampaha 2 Fusarium spp, Pythium sp, Rhizoctonia solani Galle 2 Rhizoctonia solani Kurunegala 1 Rhizoctonia solani Colombo 3 Rhizoctonia solani, Fusarium spp.

Antagonism of isolates of Trichoderma against Rhizoctonia, Fusarium and Pythium spp. (in vitro)

The isolates of Trichoderma (HORDI 1 and HORDI 2) inhibited mycelial growth of Rhizoctonia, Pythium and Fusarium spp. on PDA and CMA (Table 4). Microscopic observations revealed that isolates of Trichoderma harzianum suppressed growth of these fungi by cell wall lyses and cell disruptions. Also, coiling of mycelia of Trichoderma around mycelia of Fusarium oxysporum and cell wall lyses of macro conidia were observed. 188 RAJAPAKSE et al.

Table 3. Characteristics of fungal pathogens associated with corm rot of Kiriala.

Pathogen Colony colour Mycelial characters on Microscopic on medium observation medium Rhizoctonia On PDA: Light Mycelium on PDA: show No spores. The solani brown reverse suppressed growth. mature hyphae colony colour: Concentric rings are present. branched at right light brown Margin of colony: smooth. angles (90o) from the main hyphae. Pythium sp. Colony colour: Mycelium on PDA/CMA: Hyphae straight, white. rapid growth, mycelium aseptate & its width copious on medium. in range 5-10 µm. Oospores present. Fusarium white turn brown Mycelial growth not rapid Conidia two types. oxysporum - purple when on medium. Conidia Macro conidia: sickle older. Reverse produced openly within shaped and pointed at colony colour: hyphae and exposed to the the ends, 3-5 cells, purple air. larger length 20- 60µm. Micro conidia: Abundant, Cylindrical, 1-2 cells smaller, 5-10 µm.

Table 4 Suppression of growth of fungi isolated from rotting corms of Kiriala by Trichoderma isolates on PDA.

Trichoderma isolates Rhizoctonia Pythium Fusarium T. harzianum (HORDI-1) + + + T. harzianum (HORDI-2) + + + T. koningi - - + + suppression of fungal growth

Each treatment was replicated three times and experiment was repeated two times.

Effects of Trichoderma on control of corm rot of Kiriala

The mean disease incidence and corm yield of different treatments in year 2003, 2004 and 2005 are given in Table 5. Results in year 2003 revealed that there were significantly low disease incidence and significantly high yield in Trichoderma inoculated and fungicide treated plots (T1, T2 and T3) compared to untreated control plots but differences of disease incidences and yields were not significant among T1, T2, and T3. However, complete disease control could not be achieved by Trichoderma treatments or fungicide treatments in the first Kiriala cultivation.

Results in year 2004 and 2005 revealed that there were no corm rot disease symptoms and there was a significantly (p=0.05) higher yield in MANAGEMENT OF CORM ROT BY BIOLOGICAL CONTROL AGENT 189

Trichoderma inoculated and fungicide treated plots. Laboratory culturing test indicated that incidences of Rhizoctonia and other fungal infection were very low in corms of Kiriala in the treated plots (T1, T2 and T3) compared to control.

Table 5. Incidences of corm rot and yield of Kiriala in different treatments in field experiments conducted in years 2003, 2004 and 2005.

Treatments Field trial 2003 Field trial 2004 Field trial 2005 Disease Yield Disease Yield Disease Yield incidence kg/ incidence kg/plant incidence kg/plant plant T1 - T. harzianum 75 (60.5) b 0.25 a 0 0.96 a 0 1.60 a (HORDI 1) T2 - T. harzianum 67 (55.3) b 0.31 a 0 1.17 a 0 1.41 a (HORDI 2) T3 - Homai (2g/lit) 75 (58.6) b 0.35 a 0 1.08 a 0 1.80 a T4 - Control 100 (90.0) a 0.14 b 100 0.21 b 100 0.37 b In each column, values followed by the same letter are not significantly different at p=0.05 according to Duncan’s multiple range test

Table 6. Survival of Trichoderma in soil of experimental plots after 3 months of inoculation in each year.

Trichoderma Trichoderma spore concentration in soil (cfu/g of soil) isolates Year 2003 Year 2004 Year 2005 Trichoderma 1.5x105 1.2x106 1.6x106 Isolate, HORDI-1 Trichoderma 1.4x105 1.1x106 1.3x106 isolate, HORDI-2

Hornby (1990) reported that mycoparasitic process apparently includes chemotropic growth of Trichoderma, recognition of the host by the mycoparasite, excretion of extra-cellular enzymes and lysis of the host. Elad et al. (1983) reported that Trichoderma produces β-1-3 glucanase, chitinase and cellulase which are potentially capable of degrading the cell walls of Pythium and Rhizoctonia solani. They also reported that in mycoparasitism, the parasite sometimes penetrates the host mycelium by partially degrading the cell wall. In addition to the mycoparasitic activity, Trichoderma spp. may also compete with plant pathogens for nutrients and other requirements (Chet, 1993).

Corm rot was observed in the first year crop only and thereafter no disease incidence was recorded in Trichoderma inoculated or fungicide treated plots. Chat and Baker (1980) found that minimal effective amount of Trichoderma is about 106 cfu/g of soil. This level can be kept in the soil for several months depending on the type of soil, pH, temperature, amount of Trichoderma applied and the soil moisture condition. The results in Table 6 show that spore concentration of both Trichoderma isolates in the soil after 3 190 RAJAPAKSE et al. months of inoculation drop down to 105 cfu/g of soil in first year. However, spore concentrations of both isolates of Trichoderma increased up to 106 cfu/g due to repeated application of Trichoderma inoculum into soil in second and third years. Complete disease control could be achieved in Trichoderma treated plots in the second and third crops due to the presence of optimum inoculum level in the soil and efficient biological control ability of Trichoderma isolates.

The fungicide treatment (Homai 2 g/l) controlled corm rot up to 25% in the first crop (year 2003) and after that corm rot symptoms did not appear on crops grown in fungicide treated plots as similar as Trichoderma inoculated plots. Application of fungicide is one of the most effective methods in controlling fungal pathogens. Homai (Thiophanate-Methyl 50% + Thiram 30%) is one of the commonly used fungicide that controls soil borne diseases caused by Rhizoctonia, Pythium and Fusarium species (Anon., 1997). Most fungicides are highly degradable in soil. Therefore, fungicide treatment lowered the soil borne disease incidence considerably, but it could not completely prevent fungal population in soil. However, the results in year 2004 and 2005 revealed that repeated applications of fungicides gradually control Rhizoctonia solani and corm rot disease of Kiriala.

CONCLUSIONS

Repeated applications of Trichoderma harzianum isolates (HORDI1 and HORDI 2) and Homai (2 g/l) are equally effective in controlling corm rot disease of Kiriala in the field.

REFERENCES

Anon., 1964. Description of pathogenic fungi and bacteria, CMI, UK, No. Anon., 1967. Description of pathogenic fungi and bacteria, CMI, UK, No. Anon., 1970. Description of pathogenic fungi and bacteria, CMI, UK, No. Anon., 1997. Pesticide recommendations. Department of Agriculture, Peradeniya, Sri Lanka. Anon., 2003. Seasonal report Horticultural Crop Research and Development Institute, Gannoruwa, Peradeniya, Sri Lanka. Anon., 2004. Report of crop coordinator’s meeting, 2004 (Root and Tuber Crop), Department of Agriculture, Peradeniya, Sri Lanka. Chet, I., 1993. Biological control of soil-born plant pathogens with fungal antagonists in combination with soil treatments: Biological control of soil born plant pathogens Ed. D. Hornby, CBA International, 15-26. Chet, I. and R. Baker. 1980. Induction of suppressiveness to Rhizoctonia solani in soil. Phytopathology 70: 994-998. Elad, Y., Y. Henis, P. Boyle and I Chet. 1983. Parasitism of Trichoderma sp. on Rhizoctonia solani and Sclerotium rolfsii. Scanning electron microscope and fluorescence microscope. Phytopathology 73: 85-88. MANAGEMENT OF CORM ROT BY BIOLOGICAL CONTROL AGENT 191

Hiroshi, O., and Y. Shingo 2005. Report of cocoyam root rot caused by Pythium sp. in Sri Lanka and its chemical control. Agrochemicals Japan. Japan Plant Protection Association. No. 86, 19-22. Hornby, D. 1990. Biological control of soil-born plant pathogens. CAB International, Wallingford. 463p. Nzietchueng, S. 1984. Proceeding of the sixth symposium of the international society for tropical root crop. Lima, Peru. 173-188. Rifai, M. 1969. A Revision of the genus Trichoderma. Mycology paper 116. Commonwealth Mycology Institute, Association of Applied Biology, Kew, Surrey, England. 50p. Annals of Sri Lanka Department of Agriculture. 2006.8:193-210.

PLANNING OF FARMER CENTERED EXTENSION APPROACH FOR PADDY FARMERS IN HAMBANTOTA DISTRICT.

W.J.K.V. RANJITH Ministry of Agriculture, Southern Provincial Council, Galle

ABSTRACT

Agricultural extension approaches vary from top down technology transfer to participatory problem solving. When non-technical problems exist, farmer adoption becomes slower, thus technology transfer extension models become less effective. Alternatively, participatory problem solving extension approaches becomes more effective. Therefore, one must study the farming situation beforehand to select a better extension approach for a given area. A survey was conducted during July to September 2005 in the paddy tracts of Provincial area of Hambantota district to record the problems and solutions related to paddy production. The objectives of the study were: (a) to identify problems and farmer suggested solutions, (b) to analyze extension approach of paddy sector during 2004 to 2005, (c) to plan an extension approach based on farmer needs (farmer centered), (d) to compare existing and farmer centered extension approaches using extension dimension methodology and (e) to recommend a suitable extension approach for the situation. The results revealed that the non- technical problems were greatly responsible for limiting paddy production. It showed vast variation of problems among paddy tracts, which proposes application of location specific extension programs for the area. The comparison of existing and farmer centered extension approaches showed that there were differences in the dimensions of objective, scope and direction. Farmer centered extension approach demands more of organizational efforts, material input supply and bottom up dimensions than the existing approach. In other words, farmers wanted an extension program which should have more activities on strengthening farmer organizations, coordination of input supply and more farmer participation in various extension activities.

KEYWORDS: Dimensions, Extension approach, Farmer’s needs assessment, Participatory planning.

INTRODUCTION

An approach is defined as the style of action within the system (Contado, 1990). Agricultural extension services practise different approaches. The dilemma is how successful are our extension approaches to solve farmers’ problems while leading to improved agricultural productivity. There is another argument on how we understand the role of agricultural extension. Chambers (1997) indicated that agricultural extension approaches are varying from technology transfer models to participatory problem solving models. John (1986), Swanson and Claar (1984), Oakley and Garforth (1985) have agreed that extension is basically a technology transfer activity whereas, Roling (1988) saw extension as participatory problem solving intervention. Schwartz (1994) defined extension as an exchange of information about farming. By evaluating all these arguments, Garforth (1996) stated that the 194 RANJITH role of extension depends on the factors that lead to changes in farmers’ behavior and those factors that prevent changes. Farmer adoption of improved technologies does not take place as expected due to numerous field problems. To make extension programs successful, we have to identify those problems and solve them. In other words, we should remove factors which prevent change and make the conditions conducive for adoption. One can conduct field survey to identify farmers’ problems and their solutions with respect to promotion of agricultural production and decide what extension approach should be practised. Similarly, we can analyze present extension strategy and could see whether we do what we ought to do for farmers. In this exercise, extension approaches can be analyzed and compared using Garforth’s (1996) dimension methodology. In this study we are expected to see how compatible the existing extension approach of paddy sector of Hambantota provincial area is with farmer needs and to suggest appropriate characteristics of an extension approach. The specific objectives of the study were: (a) to identify problems and farmer suggested solutions, (b) to analyze extension approach of paddy sector during 2004 to 2005, (c) to plan an extension approach based on farmer needs (farmer centered), (d) to compare existing and farmer centered extension approaches using extension dimension methodology and (e) to recommend a suitable extension approach for the situation.

METERIALS AND METHODS

The problems and solutions of paddy farmers in the track level were recorded by agricultural instructors, after discussions with relevant farmer groups or clusters in the paddy tracts. These group discussions were done during July to September 2005 at all the paddy tracts (762 paddy tracts) of provincial area of Hambantota District. The problems and solutions were grouped into several categories such as extension, research, irrigation, drainage, infrastructure, agronomic, social, marketing and economic. Each category consists of several different problems and solutions. (Appendix1). Other information collected were name of the tract and type of irrigation, paddy extent (ha), number of farmers engaged in farming, average paddy yields in yala and maha. The types of problems and farmer suggested solutions were categorized according to irrigation types (major, minor and rain-fed) and yield levels namely low (less than 69 bu/Ac), medium (70 – 99 bu/Ac) and high (over 100 bu/Ac). The field problems and solutions were coded with numbers and recorded. The summary of farmer suggested solutions were translated into a dimension frame (Appendix 2) during analyzing stage. The extension activities of paddy sector during 2004 and 2005 were recorded from reports of Deputy Director’s office (Extension), Hambantota. All the activities in the paddy sector such as farmer trainings, demonstrations, workshops, related meetings (divisional and district level agricultural meetings), input supply activities etc. were recorded. These PLANNING OF FARMER CENTERED EXTENSION APPROACH 195 activities were arranged into dimension frame (Appendix 3) during analyzing stage.

RESULTS AND DISCUSSION

First part of following discussion covers analyzing farmers’ problems and their solutions in the paddy tract level. The second part of the discussion covers analyzing of existing extension approach using “Dimension Methodology” (Garforth, 1996). Then the farmers’ solutions are arranged in the dimension frame (farmer needed dimensions). Finally these two sets of extension dimensions are compared.

Analyzing problems and solutions

As Figure 1 shows the main problem categories that hinder the paddy production are irrigation, extension, sociological and agronomic. Although marketing is a major problem, farmers had not identified it as they trusted the government paddy purchasing program during that period. Extension problems cover 22% of total number of problems reported by farmers. The data reveal that the most important problems are water shortage (irrigation), seed paddy shortage (extension), weak farmer organizations (sociological) and damage to irrigation channels (irrigation). According to Figure 2, farmers have suggested extension intervention as the most important solution to improve productivity. Other important solutions come under agronomic, and irrigation categories. The data reveal that 30% of solutions fall in extension category. Most important solutions are conducting farmer trainings (extension), promoting organic matter usage as fertilizers (agronomic), repair of irrigation channels (irrigation) and promoting self seed production (extension).

Economic Marketting Sociological y r

o Agronomic g e t a

c Infrastructure

m e l Drainage

b o r

P Irrigation Research Extension

0 0.2 0.4 0.6 0.8 1 1.2 Frequency (average per tract) Figure 1. Occurrence of problems in the paddy tracts. 196 RANJITH

Economic

Marketting

Sociological s n o i Agronomic t u l o s

Infrastructure f o

s e Drainage p y T Irrigation

Research

Extension

0 0.5 1 1.5 Frequency (average per tract) Figure 2. Farmer suggested solutions to promote paddy production.

Figure 3 shows that the farmers in the high yielding tracts identified higher number of problems in the categories of extension and sociological context. Farmers in the medium yielding tracts identified comparatively higher problems in the irrigation and agronomic context.

Economic Marketting Sociological Agronom ic

s Infrastructure m e l

b Drainage o r p

f Irrigation o

s

e Research p y T Extension

0 0.2 0.4 0.6 0.8 1 1.2 1.4 Frequency (average per tract)

Low yielding High yielding Medium yielding

Figure 3. Occurrence of problems in the differently yielding paddy tracts.

Figure 4 shows that the farmers in the medium and higher yielding tracts forwarded more solutions on extension, irrigation and agronomic aspects. Low yielding farmers also have forwarded solutions on same. According to Figure 5, the farmers in the tracts of major PLANNING OF FARMER CENTERED EXTENSION APPROACH 197 irrigation forwarded more problems from extension and sociological aspects. Farmers in the tracts of minor irrigation faced problems in the aspects of irrigation, extension and marketing. Figure 6 shows that the farmers of major irrigation tracks forwarded more solutions in the categories of extension and agronomy. Farmers of minor irrigation and rain-fed tracts need more solutions on the aspects of extension, irrigation, agronomic and sociological aspects. Figure 7 shows the component of agronomic problems such as salinity is the main agronomical problem in the district followed by plant protection problems, soil infertility and iron toxicity. However, farmers see organic matter application as the most important remedial measure (Fig. 8).

Econom ic Marketting Sociological Agronomic

s Infrastructure n o i t

u Drainage l o s

f Irrigat ion o

s

e Research p y

T Ext ension

0 0.5 1 1.5 2

Frequency (average per tract)

Low yielding High yielding Medium yielding

Figure 4. Farmer suggested solutions in the differently yielding tracts.

This may be due to the fact that many farmers experience that organic fertilization improves soil conditions and yield levels and also reduces cost of chemical fertilization. Integrated pest management, growing of tolerant varieties and crop diversification are the other important agronomical solutions (Fig. 8). 198 RANJITH

Economic Marketting

s

m Sociological e l b o

e Agronomic p

f o

Infrastructure s e

p Drainage y T Irrigation Research Extension

0 0.2 0.4 0.6 0.8 1 1.2 1.4

Frequeccy (average per tract)

Major irrigation Minor Irrigation Rain- fed

Figure 5. Occurrence of problems in differently irrigated paddy tracts.

Economic

Marketting Sociological

s n o

i Agronomic t u l

o Infrastructure s

f o

s Drainage e p y

T Irrigation Research

Extension

0 0.5 1 1.5 2 Frequecy (average per tract)

Major irrigation Minor Irrigation Rain- fed

Figure 6. Farmer suggested solutions in the differently irrigated paddy tracts. PLANNING OF FARMER CENTERED EXTENSION APPROACH 199

Delaying cultivation

s Iron toxicity m e l b

o Soil infertility r p

c

i High plant density m o

n Salinity o r g

a Shading

f o

s Poor weed control. e p y

T High pest damage

0 0.05 0.1 0.15 0.2

Frequency (average per tract)

Figure 7. Occurrence of agronomic problems in the paddy tracts.

CropCrop diversification diversification landLand preparation preparation with with rain rain waterwater

s Grow tolerant varieties n Grow tolerence varieties o i t

u Control plant density

l Conyrol plant density o s

f Better land preparation o

s e

p IPM y

T Application of organic matter Improve irrigation Start crop before flood

0 0.1 0.2 0.3 0.4 0.5 0.6 Frequency (average per tract) Figure 8. Farmer suggested solutions for the agronomic problems in the tracts.

Among the number of sociological problems, farmers identify weak farmer organizations as the main problem (Fig. 9), because it retards farmer-to-farmer co-operation and collective efforts resulting in reduced paddy production. Cattle and wild animal damage to paddy crop is another prominent problem in Hambantota district. Shortage of labour, and tenancy are the other important problems (Fig. 9). Even though, there are many sociological problems, farmers give priority and more attention to strengthen farmer organizations (Fig. 10). The reason behind this may be that farmers see this solution as cost effective and achievable compared to other solutions. Control of crop damage by animals is the other important solution. 200 RANJITH

Shortage of capital Small land holdings

s Burning of strow m e l Farmers' low cooporation b o r

p Crop damage by animals

f o

e Digging pits in fields p y

T Tenancy Labour shortage. Weak farmer organizations

0 0.05 0.1 0.15 0.2 0.25 0.3 Frequency (per tract)

Figure 9. Occurrence of sociological problems in the paddy tracts

Figure 11 shows that the important problems in the extension context are shortage of seed paddy, low organic matter application, low technical knowledge and not practising new technology. As shown in Figure 12, conducting farmer training classes is the most important solution in the extension context. Conducting self seed production programs and introducing new technology through demonstrations are the other important aspects in the extension category.

Proper irrigation management

Problem identification and solving

s Promote family labour usage n o i t u l

o Offering tenants' rights s

f o

e

p Strengthan farmer organizations y T Awareness of cattle owners

0 0.05 0.1 0.15 0.2 0.25 0.3 0.35

Frequency (per tract)

Figure 10. Farmer suggested solutions for the sociological problems in the tracts. PLANNING OF FARMER CENTERED EXTENSION APPROACH 201

Excess use of seed paddy

Not adoptingadopting modern modern technology technolg

Poor water management s

m High urea application e l b o

r Low TSP application p

f o

s Excessive use of insecticide e p y

T Shortage of seed paddy

Low organic matter application.

LowLow technical technical knowledge knoledge

0 0.1 0.2 0.3 0.4 Frequency (average per tract) Figure 11. Occurrence of extension problems in the paddy tracts.

Control plant density Timly cultivation

Commercial seed paddy production s n o i

t Self seed production u l o

s Conduct demonstrtions

f o

s Promoting farm machinary e p y Farmer training T Introduce modern technology Introduce short age varieties

0 0.2 0.4 0.6 0.8 1

Frequencies (average per tract) Figure 12. Farmer suggested solutions for the extension problems in the tracts

Putting farmer’s solutions to dimension frame and analyzing existing extension approach

Appendix 1 shows the types of problems and farmers’ solutions in the paddy tracts. Farmer’ solutions were taken as farmer needs. Extension activities could be planned according to them. In other words, this process can be viewed as farmer participatory extension planning. The challenge is that how the extensionist does prepare an extension approach which represents all the farmer needs. This was done as shown in Appendix 2. The eight dimensions (Garforth, 1996) were put in eight columns and the farmer’s solutions as activities were put in the rows. Then the characters of activities were recorded in the respective rows as shown in Appendix 2. One activity 202 RANJITH could be categorized to several dimensions. The total reporting of dimensions (total marks) of all the activities were recorded in the dimension frame as shown in Appendix 3.

Positioning of dimension is done by calculating the proportional distances from the 2 ends as shown in Appendix 3. Accordingly, the "objective" dimension has a total of 33 marks (20+13= 33) from 2 characters in both ends. The distance between 2 ends is 5 spaces (A to E), each space represents 6.6 marks (33/5=6.6). Therefore, the dimension position with respect to transfer of technology character is 3 spaces (20/6.6= 3) from the other end. Therefore, dimension positioning of “objective” is B. Similarly, all other dimension positioning was calculated and the results are given in Appendix 3 which shows the extension approach that most farmers in the district wanted (farmer centered approach). Table 1 shows the final results of analysis in the Garforth’s (1996) dimension frame.

The next part of the analysis was to characterize existing extension approach. Appendix 4 shows the way of recording of all the extension activities in 2004 and 2005. The characters related to extension dimensions were recorded in the respective columns and totaled up. Appendix 5 shows the results of the analysis in the dimension frame and it also shows the details of marks assigned for different characters, the process of calculation and the dimension positioning of existing extension approach. The final dimension positioning is given in Table 1.

Table 1. Dimension positioning of existing extension approach and farmer demanding approach of the paddy sector of Hambantota district.

Dimension A B C D E 1 Enterprise Commodity ☼ ® Whole farm

2 Clientele Target ☼ ® All category households 3 Influence Enforcement ☼® Problem solving 4 Objectives Technology ☼ ® Organization transfer 5 Scale Individual ☼ Group ®

6 Scope Information ☼ ® Material only input also

7 Payment Client pay ☼ ® Free

8 Direction Top down ☼ ® Bottom up

® = Dimension positions of farmer demanding (farmer centered) extension approach ☼= Dimension positions of existing extension approach (2004 to 2005) PLANNING OF FARMER CENTERED EXTENSION APPROACH 203

The results show the dimensions such as enterprise, clientele, influence, scale and payment are similar in both farmer demanding (farmer centered) and existing extension approaches. But, the dimensions of objective, scope and direction are differing from one another. The difference shows that the farmers want the extension program to be more towards organizational, input supply and farmer participatory directions.

CONCLUSIONS

The most important problem categories of paddy tracts are irrigation, extension, marketing, sociological and agronomic issues. It further shows that the problems and solutions vary between tracts. Since the farmer needs are varying among tracts, an appropriate extension program should have location specific activities that solve tract level problems.

The most important field problems are water and seed paddy shortage, weak farmer organizations and damage of irrigation channels. Most important solutions are conducting farmer trainings, promoting organic matter application, strengthening farmer organizations, repairing irrigation channels and promoting self seed production.

The study shows the differences of existing extension approach and farmer demanding extension approach on the dimensions of objective, scope and direction.

The existing extension approach should be changed to match with farmers’ expectations. Therefore, there should be more organizational activities and input supply or input supply coordination activities in the extension approach. Extension planning should be more participatory to match the farmer requirements. Farmer demanding extension model (farmer centered approach in Table 1) is taken as an appropriate extension approach.

ACKNOWLEDGEMENTS

The author thanks all categories of extension staff of Hambantota provincial extension office for conducting the field survey. He especially thanks Hambantota Irrigation Rehabilitation Project (HIRP) for providing funds to conduct the field survey. 204 RANJITH

REFERENCES

Chambers, R. 1997. Whose reality counts, Putting the First Last. International Technology Publication, London. Contado, 1990. Extension approaches, FAO 1990 p 79, Eds. C. Garforth, 1996. Interpretation of Extension, A discussion paper, AERDD, The University of Reading, UK. p10. DDA, 2005. Progress reports of Deputy Director’s office, Department of Agriculture, Hambantota. Garforth, C. 1996. Interpretation of extension, A discussion paper. AERDD, The University of Reading, UK. Pp 12- 21. HIRP, 2005. Problems and solutions of paddy tracts in Hambantota district, Hambantota Irrigation Rehabilitation Project, Deputy Director’s office, Hambantota. John, R. 1986. Investing in rural extension: strategies and goals. Elsevier Applied Science Publication. p159. Oakley, P. A. and C, J. Garforth. 1985. Guide to extension training. Rome, FAO. p20. Roling, N. 1988. Extension science, Information systems in agricultural development. Cambridge University Press, New York. p49. Schwartz, L. A. 1994. The role of the private sector in agricultural extension: economic analysis and case studies. AGREN Network Paper 48, London. ODI. p 2. Swanson, B. E. and J. B. Claar. 1984. Ed. Swanson, B. Agricultural extension: A reference manual. FAO, Rome. p1.

APPENDIX 1

List of problems and solutions identified at participatory farmer need assessment at paddy tract level

Problems

1.0 Extension problems 11,Low technical knowledge, 12 Low organic matter application 13 Shortage of seed paddy, 14 Excessive use of insecticides, 15 Low TSP application 16 High urea application 17 Poor water management 18 Not use modern technology 19 Excessive use of seed paddy.

2.0 Research problems. 21 Low organic matter content in the soils.

3.0 Irrigation and drainage problems. 31 Tank silted 32 Channel silted 33 Amicus got damage 34 Water shortage35 Irrigation channel system is damaged 36 No catchment area/ no irrigation system37 Sluice gate is damaged 38 Improper water management 39 Tank filled with weeds.

4.0 Drainage problem. 41 Low drainage conditions 42 Flood damage 43 River mouth blocked 44 Boggy lands 45 No drainage ditch.

5.0 Infrastructure. 51 No road 52 Roads are damaged 53 Illegal cultivation in tank 54 Channels are not clean (weedy) 55 Land boundaries not clear 56 Sand mining in channels. PLANNING OF FARMER CENTERED EXTENSION APPROACH 205

6.0 Agronomic problems. 61 High pest and disease 62 Poor weed control 63 Shading 64 Salinity 65 High plant density 66 Soil infertility 67 Iron toxicity 68 Delaying cultivation/ staggered cultivation.

7.0 Social and institutional problems. 71 Weak farmer organizations 72 Labour scarcity 73 Rotational land ownership/ tenant problems 74 Digging of soil for removing shells/land filling 75 Cattle/ wild animal damage 76 Farmers not cooperate with officers 77 Straw burning 78 Small land holdings/ part time farmers 79 No capital.

8.0 Marketing problems. 81 Low price for paddy 82 Shortage of agric. machinery 83 Short supply of agro chemicals/inputs.

9.0 Economic problems. 91 Low credit facilities 92 High fertilizer cost 93 High input cost 94 Low profit from paddy.

Solutions

1.0 Extension. 11 Introduce short age varieties 12 Introduce modern technology 13 Awareness program/conduct training class 14 Promote farm machinery usage 15 Organize demonstrations 16 Promote self seed production 17 Commercial seed paddy production 18 Motivate timely cultivation 19 Control plant density.

2.0 Research. 21 Soil testing and fertilization 22 Desalinization.

3.0 Irrigation. 31 Deepen the tank 32 Construct a new tank/ find alternative water sources 33 Repair irrigation channel system 34 Construct a new field channel. 35 Repair anicuit 36 Repair sluice gate 37 Clean the channel 38 Repair the dam 39 Issue water correctly.

4.0 Drainage. 41 Open the river mouth 42 Deepen the drainage ditch 43 Cut drain ditches 44 Fix flood control gates.

5.0 Infrastructure. 51 Repair roads 52 Develop fields 53 Construct new road 54 Fix electric fence 55 Fencing 56 Stop cultivation in the tank area 57 Prune shade trees 58 Mark boundaries of tank/lands 59 Stop sand mining in channels.

6.0 Agronomic. 61 Crop diversification 62 Land preparation with rain water 63 Grow tolerant paddy varieties 64 Row seeding, planting, control plant density 65 Deep ploughing, better land preparation 66 Practice IPM 67 Apply OM 68 Improve irrigation practices 69 Start crop before flood.

7.0 Social changes. 71 Awareness of cattle owners about crop damage 72 Strengthen FO 73 Preserve tenant rights 74 Promote family labour usage, 75 Problem identification and solving 76 Law enforcement on irrigation rule breakers.

8.0 Marketing. 81 Supply inputs 82 Improve paddy marketing.

9.0 Economic. 91 Supply credit facilities 92 Supply tractors to ASCs.

APPENDIX 2 206 RANJITH

Methodology of translating farmer need to extension dimensions (source - HIRP, 2005)

Types of solutions No. of Dimensions recorded from farmer solu Inf Scope Scale Cliente Enterprise participatory need tions Obj lue le assessment record ectiv nc Pay s e e Direction ment Extension Introduce short age Inf & varieties 33 TT Materials Group Target Commodity Top -Bup Free Introduce modern TT- Inf & technology 109 og Materials Indi Target Commodity Bup Free Awareness program/ conduct training class 589 TT Inf only Group All Commodity Top Free Promote farm Inf & machinery usage 17 TT Materials Group Target Commodity Top -Bup Free Organize Inf & demonstrations 67 TT Materials Group Target Commodity Top –Bup Free Promote self seed Inf & production 155 TT Materials Indi Target Commodity Top -Bup Free Commercial seed paddy TT- Inf & production 80 og Materials Group Target Commodity Top -Bup Free Motivate timely cultivation 10 TT Inf only Group All Commodity Top Free Control plant density 3 TT Inf only Indi Target Commodity Top -Bup Free

Research Soil testing and fertilization 62 PS Inf only Indi Target Commodity Top -Bup Free Desalinization 59 TT PS Inf only Indi Target Commodity Top Free Irrigation Deepen the tank 145 PS Target Commodity Free Construct a new tank/ find alternative water sources 18 PS Target Commodity Free Repair irrigation channel system 265 Og PS Target Commodity Free Construct a new field channel. 45 PS Target Commodity Free Repair anicuit 44 PS Target Commodity Free Repair sluice gate 17 PS Target Commodity Free Clean the channel 14 Og PS Inf only Group Target Commodity Top -Bup Free Repair the dam 5 PS Target Commodity Free Issue water correctly 12 PS Target Commodity Free Drainage Open the river mouth 7 PS Target Commodity Free Deepen the drainage ditch 40 Og PS Inf only Group Target Commodity Top -Bup Free Cut drain ditches 15 Og PS Inf only Target Commodity Top -Bup Free Fix flood control gates 1 PS Target Commodity Free Infrastructure Repair roads 17 PS Target Commodity Free Develop fields 1 PS Inf only Target Commodity Free Construct new road 7 PS Target Commodity Free Fix electric fence 2 PS Target Commodity Free Fencing 2 Og PS Inf only Target Commodity Top -Bup Free PLANNING OF FARMER CENTERED EXTENSION APPROACH 207

Stop cultivation in the tank area 9 Og PS Target Commodity Free Prune shade trees 2 TT PS Inf only Target Commodity Top -Bup Free Mark boundaries of tank/ Lands 4 PS Target Commodity Free Stop sand mining in channels 2 PS Target Commodity Free Agronomic TT, Inf & Crop Diversification 67 Og Materials Group Target Commodity Top -Bup Free Land preparation with rain water 12 TT Imp only Group Target Commodity Top -Bup Free Grow tolerant paddy Inf & varieties 71 TT Materials Indi Target Commodity Top -Bup Free Row seeding, planting, control plant density 5 TT Inf only Group Target Commodity Top -Bup Free Deep ploughing, better land preparation 7 TT Inf only Group Target Commodity Top -Bup Free TT, Practice IPM 100 Og Inf only Group All Commodity Top -Bup Free Apply OM 374 TT Inf only Indi All Commodity Top -Bup Free Improve irrigation TT, practices 14 Og Inf only Group Target Commodity Top -Bup Free Start crop before flood 5 TT Inf only Indi Target Commodity Top -Bup Free

Social changes Awareness of cattle owners about crop damage 58 Og PS Group Target Commodity Top -Bup Free Strengthen FO 243 Og Group All Commodity Bup Free Preserve tenant rights 8 PS Promote family labour usage 14 PS Inf only Indi Target Commodity Top -Bup Free Problem identification and solving 17 PS Inf only Group Target Commodity Top -Bup Free Law enforcement on irrigation rule breakers 5 PS Marketing Supply inputs 56 PS Materials Group Target Commodity Free Improve paddy marketing 586 PS All Commodity Free Economic Supply credit facilities 96 PS Indi Target Commodity Free Supply tractors to ASCs 3 PS Target Commodity Free *Total number of solution records 3599

TT- Enf- 20 0 Ind- 10 Free- Total no of marks in the Oga- PS- Inf- 27 Grou- Tar- 44 Com- 50 Top- 27 50 solutions 13 33 Mater- 9 18 All- 6 Who- 0 Bup- 25 Pay- 0

APPENDIX 3 208 RANJITH

Dimension positioning of an appropriate extension approach (farmer centered) for the paddy sector of Hambantota district based on farmer’s need assessment. Dimension A B C D E Total marks Scale position 1 Enterprise Commodity- ® 0- Whole 50/5=10, 50 farm 50 50/10=5=A 2 Clientele Target ® 6 – All 50/5=10, category - 44 household 50 50/10=5=A 3 Influence ® 33 – 33/5=6.6, Enforcement – Problem 33/6.6=5=E 0 solving 33 4 Objectives Technology ® 13 – transfer - 20 Organizatio 33/5=6.6, n 33 20/6.6=3=B* 5 Scale Individual - 10 ® 28/5=5.6, 18 – Group 28 18/5.6=3.2=D 6 Scope Information ® 9 – Supply 36/5=7.2, only -27 materials 36 27/7.2=3.75=B 7 Payment Client pay - 0 ® 50/5=10,50/10=5= 50 – Free 50 E 8 Direction Top down - 27 ® 25- Bottom 52/5=10.4, up 52 25/10.4=2.4=C ® = Dimension position * = Positioning of dimension is done by calculating the proportional distances from the 2 ends as shown in the chart. Accordingly, the "objective" dimension has total of 33 marks (20+13= 33) from 2 characters in both ends. The distance between 2 ends is 5 spaces (=A to E), each space represents 6.6 marks (33/5=6.6). Therefore, the dimension position with respect to transfer of technology character is 3 spaces (20/6.6= 3) from the other end.

APPENDIX 4

Extension dimension of paddy sector in Hambantota district based on the extension activities during 2004 and 2005 (source-DDA, 2005). 2004 Project Activity No. Cost No. of Ext. dimension Program of (Rs) benefici units aries Granary Fertilizer Supply fertilizer 50 34458 50 6.2 *, 6.1, 8.1, 5.2, subsidy 0 4.1, 4.2, 1.1, 2.1, 3.2 (FS) Granary Technology Farmer 30 17563 510 1.1,2.1, 4.1, 5.2, 8.1, transfer trainings(FT) 3.2 Granary Technology Field visits(FV) 151 37960 906 1.1, 2.1, 4.1, 5.1, transfer 6.1, 8.1, 3.2

Criteria based Increase Field days(FD) 12 5197 291 1.1, 2.1, 3.2, 4.1, yield 5.2, 6.1, 8.1 Criteria based Increase FTs 38 15479 608 1.1, 2.1, 3.2, 4.1, yield 5.2, 6.1, 8.1 Criteria based FS Supply fertilizer 40 87958 40 1.1, 2.1, 3.2, 4.1, 5.2, 6.1, 6.2, 8.1 PLANNING OF FARMER CENTERED EXTENSION APPROACH 209

Criteria based Self seed Supply seeds 486 32293 486 1.1, 2.1, 3.2, 4.1, paddy and trainings 5.1, 6.1, 6.2, 8.1, production 8.2 Criteria based IPM IPM- classes 64 15146 896 1.1, 2.1, 3.2, 4.1, 4.2, 5.2, 6.1, 8.2, 8.1 Criteria based IPM Field tours 6 18456 156 1.1, 2.1, 4.1, 5.1, (FTo) 6.1, 8.1 Criteria based IPM IPM- FFS 6 35780 1.1, 2.1, 3.2, 4.1, 4.2, 5.2, 6.1, 8.2, 8.1 Criteria based IPM Field study 4 5018 68 1.1, 2.1, 3.2, 4.1, 5.2, 8.1 Criteria based Problem Workshops 4 24049 126 1.1, 2.1, 3.2, 4.1, solving 5.2, 8.2, 8.1 (PS) workshops General Technology FTs (groups) 545 - 6540 1.1, 2.1, 3.2, 4.1, transfer 5.2, 6.1, 8.1 (TOT) General TOT TOT 1440 - 3200 1.1, 2.1, 3.2, 4.1, (individual) 0 5.1, 6.1, 8.2 General PS PS meetings 1080 - 950 1.1, 2.1, 3.2, 4.1, meetings 5.2, 6.1, 8.1, 8.2 General FG FG formation 60 1150 1.1, 2.1, 3.2, 4.2, formation 5.2, 8.1

2005 Project Activity No. Cost No. of Ext. dimension Program of (Rs) benefici units aries Granary Fertilizer Supply 350 94531 350 6.2 *, 6.1, 8.1, 5.2, 4.1, subsidy (FS) fertilizer 5 4.2, 1.1, 2.1, 3.2 Granary Technology Farmer 442 - 3532 1.1,2.1, 4.1, 5.2, 8.1, 3.2 transfer trainings(FT) Granary Technology Field 160 42127 1120 1.1, 2.1, 4.1, 5.1, 6.1, 8.1, transfer visits(FV) 3.2 Granary Technology Field 43 - 516 1.1, 2.1, 3.2, 4.1, 5.2, 6.1, transfer days(FD) 8.1 Criteria Increase FDs 57 26800 685 1.1, 2.1, 3.2, 4.1, 5.2, 6.1, based yield 8.1

Criteria Increase FTs 112 64000 1568 1.1, 2.1, 3.2, 4.1, 5.2, 6.1, based yield 8.1 Criteria Increase Supply 56 11800 56 1.1, 2.1, 3.2, 4.1, 5.1, 6.2, based yield kuntani 0 6.1, 8.2 Criteria Increase Provide inf. 56 25200 1400 1.1, 2.2, 3.2, 4.1, 5.1, 6.1 based yield Boards Criteria FS Supply 152 27180 152 1.1, 2.1, 3.2, 4.1, 5.2, 6.1, based fertilizer 0 6.2, 8.1 Criteria Self seed Supply seeds 560 53979 560 1.1, 2.1, 3.2, 4.1, 5.1,6.1, based paddy and trainings 6.2, 8.1, 8.2 production Criteria Seed Material 12 84000 120 1.1, 2.1, 3.2, 4.1, 5.1, 6.1, based production supply 6.2, 8.2 Criteria IPM IPM- classes 4 10390 76 1.1, 2.1, 3.2, 4.1, 4.2, 5.2, based 6.1, 8.2, 8.1 Criteria IPM Field tours 4 6344 106 1.1, 2.1, 4.1, 5.1, 6.1, 8.1 based (FTo) 210 RANJITH

Criteria IPM IPM- FFS 7 71304 138 1.1, 2.1, 3.2, 4.1, 4.2, 5.2, based 6.1, 8.2, 8.1 Criteria IPM Field study 28 12150 364 1.1, 2.1, 3.2, 4.1, 5.2, 8.1 based Criteria IPM Form farmer 7 - 138 1.1, 2.1, 4.2, 8.1 based groups (FGs) Criteria IPM FDs 22 12200 242 1.1, 2.1, 3.2, 4.1, 5.2, 6.1, based 8.1, 8.2 Criteria Problem Workshops 4 18900 134 1.1, 2.1, 3.2, 4.1, 5.2, 8.2, based solving (PS) 8.1 workshops General Technology FTs (groups) 575 - 7240 1.1, 2.1, 3.2, 4.1, 5.2, 6.1, transfer 8.1 (TOT) General TOT TOT 1670 - 4325 1.1, 2.1, 3.2, 4.1, 5.1, 6.1, (individual) 0 8.2 General PS meetings PS meetings 1170 - 1320 1.1, 2.1, 3.2, 4.1, 5.2, 6.1, 8.1, 8.2 General FG FG formation 105 1308 1.1, 2.1, 3.2, 4.2, 5.2, 8.1 formation * 1.1 Commodity 1.2 Whole farm 2.1 Target category 2.2 All households 3.1 Enforcement 3.2 Problem solving 4.1Technology transfer 4.2 Organization 5.1 Individual 5.2 Group 6.1 Information only 6.2 Material input also 7.1 Client pay 7.2 Free 8.1 Top down 8.2 Bottom up.

APPENDIX 5

Analyzing dimensions of existing extension approach of the paddy sector of Hambantota district. Dimension Total A B C D E marks Scale position 1 Commodit ☼ 0- Whole 38/5=7.6, Enterprise y- 38 farm 38 38/7.6=5=A 2 Clientele Target ☼ 1 – All category – househol 37/5=7.4, 36 d 37 36/7.4=4.9=A 3 Influence Enforcem ☼ 35 - 35/5=7, 35/7=5 =E ent – 0 Problem solving 35 4 Objectives Transfer ☼ of 8 – technolog Organiza 43/5=8.6, y-35 tion 43 35/8.6=4.1=A 5 Scale Individual ☼ 26 – 36/5=7.2, - 10 Group 36 26/7.2=3.6=D 6 Scope Informatio ☼ 8 – n only Supply -29 materials 37 37/5=7.4, 29/7.4=4 7 Payment Client pay ☼ 39/5=7.8, - 0 39 – Free 39 39/7.8=5=E 8 Direction Top down ☼ 14- - 32 Bottom 46/5=9.2, up 46 32/9.4=3.4=B Annals of Sri Lanka Department of Agriculture. 2006.8:211-217.

TECHNOLOGY FOR “EMBUL” BANANA EXPORT

K.H. SARANANDA and M.W.M.A.G. WIJESUNDARA. Food Research Unit, Horticultural Crop Research and Development Institute, Peradeniya

ABSTRACT

Green and firm fruit of good external appearance and absence of crown rot are the major pre-requisites for banana export. Both field and laboratory simulated studies showed good quality “Embul” banana for export can be produced by bagging after flowering and harvesting them at 11 weeks after flowering. Ripening process of the fruits can be delayed by storing them at 14°C immediately after packaging under modified atmosphere storage using 150 gauge low-density polyethylene bags. Evacuation of bags before sealing showed better extension of storage life at low temperature. If bananas are harvested at 75 days after flowering and packed under vacuum packaging condition, green life can be maintained for more than 21 days with very good external appearance. After the storage period, bananas undergo natural ripening process with similar physico-chemical parameters to that of naturally ripened freshly harvested bananas. If fruits are to be air-freighted, the stage of maturity for harvesting was 80 days after flowering. Good quality harvested bananas must be packed using perforated, 100 gauge low-density polyethylene liner and stored at 14°C immediately after packaging. Low temperature must be continued at least up to the airport, if the air travel duration is one day. If the travel duration exceeds two days or more, bananas must be transported in low temperature containers until they reach their destination. Results of both commercial shipments of air-freight and sea-freight “Embul” bananas to Japan are also included. An additional precaution to control crown-rot during sea-freight has to be taken to control the crown-rot.

KEYWORDS: Air-freight, Crown-rot, Modified atmosphere, Sea-freight, Vacuum packing.

INTRODUCTION

“Embul” banana production in Sri Lanka has come to a saturated level. Further expansion results in price reduction causing a low income to farmers. If bananas are exported, a vacuum in the local market can be created resulting in a reasonable price paid for farmers. Banana export industry in the world is well commercialized with ‘Cavendish’ group of bananas (Scott et al., 1968). However, in Sri Lanka only limited experimental information is available on export of “Embul” bananas (Sarananda and Wijerathnam, 1994 and Sarananda et al., 1996). No commercial export is practiced yet due to risk involved in quality maintenance. Blemish free bananas having a reasonably long green life until they reach the importing country are major requirements during banana export. In addition, control of fungal rot developed at the cut crown surface is another requirement (Sarananda and Wijerathnam, 1994). Technology to produce high quality fruits, delayed ripening and control of crown rot for Cavendish group of bananas has been developed. Ripening process of fruit can be delayed by 212 SARANANDA AND WIJESUNDARA reducing the respiration rate which can be achieved by storing at 13.5°C. Respiration rate can further be lowered down by enclosing in modified atmosphere generated passively using low-density polyethylene bags (Sarananda, 1989a). The maximum storage life, however, can be obtained through vacuum packing. Experimental evidence for the above key areas has been reported for “Embul” banana (Sarananda et al., 1996). However, no commercial scale data are available because the technology has not been used during export. Therefore, this paper describes the results of commercial shipment of “Embul” banana.

MATERIALS AND METHODS

Commercial sea shipment

“Embul” bananas grown in Embilipitiya under irrigation were used for the study. Farmer organization at Mauara was selected and fields with satisfactory banana cultivation were selected for collection of banana. Farmers were made aware about the quality and harvest maturity for export. Polysack bags with 40-60 (1 cm) perforations were used for bagging on tree of newly emerged bunches after removal of inflorescence. Bagging was done to improve the peel quality without blemishes. After bagging, plants were tagged to monitor the harvest maturity. Bunches were harvested at 75 days after bunch emergence. Preliminary studies showed that harvesting bananas at 75 days of maturity delayed the ripening process compared to those harvested at 80-85 days for the local market. Harvested bunches were wrapped individually in foam sheets and bunches were placed vertically as a single layer in a ventilated lorry and transported to the pack house at Mirijjawila.

Bunches were then dehanded using very sharp curved dehanding knife, floral remnants were removed and immediately put into water for washing. Hands were separated into clusters for easy packing. Twisted fingers in hands or clusters were removed using a sharp curved knife. Plastic basins were used for washing. Wash after in the basin was regularly changed to reduce infection at the cut crown surface. Dirt present on fingers was removed during washing and then hands transferred into 1% Alum (Potassium aluminum sulphate) solution to coagulate latex. Alum washed bananas were then taken out and weighed for required quantity of 8 kg per carton. Five ply cartons were used and the carton was lined with 150-gauge low-density polyethylene (LDPE) bag. Banana hands or clusters were then packed inside placing distal end of fingers to the bottom of the box. During the packing rows were separated using a sheet of foam to prevent bruising. Once the box was packed with bananas a low vacuum horse was inserted into the bag and then the bag was evacuated. Bag was then sealed using a rubber band. Packed boxes were immediately transferred into a cold room maintained at 14°C ± 2°C. Packed boxes were stacked as 5 layers on a “EMBUL” BANANA EXPORT 213 standard size pallet and pallets loaded in a 20 foot container, which was also maintained at 14°C ± 2°C. Fruits were exported to Japan. The time of shipment was 26 days. Similarly 10 boxes of bananas harvested at 80 days after bunch emergence were also included for the shipment. Except the maturity difference, the rest of the procedure adopted was similar to that followed for 75 days mature bananas harvested for the sea shipment study.

Commercial air shipment

For air-shipment, bananas from the same field were used. Bagging and tagging were done similar to the previous experiment. Bunches were harvested at 80 days maturity and brought to the packing shed located in the field itself. Bunches were dehanded, floral remnants were removed and washed with water. Bananas were then separated into clusters and washed with 1% alum and fruit weight of 8 kg was weighed and packed in boxes similar to the sea-shipment trial. The bags were lined with perforated polyethylene bags to facilitate air exchange. Boxes were then closed without sealing the bags. Packed boxes were immediately transferred into the refrigerated cargo van, which was maintained at 14ºC ± 2ºC. Sixty-five banana boxes were packed in the refrigerated container and transported to the airport. Bananas were then exported to Japan by air-cargo without temperature control.

Fruits were assessed for ripening using colour index 1 = green, 2 = colour break, 3= more green than yellow, 4= more yellow than green, 5= yellow with green tip, 6= full yellow, and 7= over ripe. Crown rot was also measured using standard crown rot index 0-4. Overall fruit quality together with colour index and crown rot index have been assessed by the Japanese importing company.

RESULTS AND DISCUSSION

Bananas harvested at 75 days after emergence were firm and green when they reached Japan, 26 days after storage at 14° ± 2°C (Table 1). Modified atmosphere generated using low density polyethylene bags has proven to extend the storage life of “Embul” banana even at ambient temperature (Sarananda, 1989b). Low temperature used in this shipment would have further attributed to reduce the respiration rate resulting in long green life. When “Embul” bananas were stored at 13, 5°C under MA conditions, the gas composition developed was 5-7% O2 and 3-5% CO2 (Hewage, 1995). Similar gas composition at the same temperature has increased the storage life of Cavendish bananas (Truter and Combrink, 1990). On the other hand, bananas harvested at 80 days after emergence was less firm and yellow colour development has been initiated. The difference in 214 SARANANDA AND WIJESUNDARA responses of ‘Embul’ banana harvested from the same field may be due to difference in maturity. Since banana belongs to the climacteric group of fruits, at 80 days after flowering, fruits must have reached the pre-climacteric minimum level. When fruits are harvested, washed and enclosed in sealed polyethylene bags, fruits are exposed to high CO2 and low O2 stress. Since mature fruits are sensitive for ethylene production, it would have initiated the ripening process of bananas. Peel colour development up to CI 2.6 is an indication that autocatalytic ethylene production has been initiated. With the ethylene biosynthesis, pectic substances are dehydrated, starch is hydrolyzed to sugars resulting in softening of fruits. Fruits harvested at 75 days after flowering might not have reached the climacteric minimum hence the stress caused due to enclosing in modified atmosphere is not sufficient to initiate auto catalytic ethylene production. Maturity at harvest therefore plays a vital role in extending storage life of “Embul” banana. The mechanism of delaying ripening is mainly due to initiation of ethylene biosynthesis under MA at low temperature (Mapson and Robinson, 1966; Sarananda et al., 1996)

Table 1. Quality parameters of sea freighted ‘Embul’ banana, assessed in Japan upon arrival.

Stage of maturity Firmness Colour index Crown rot index 75 days after emergence Firm 1 0 80 days after emergence Less firm 2.6 0 Colour index: - 1- green, 2-colour break, 3- more green than yellow, 4-more yellow than green, 5- yellow with green tip, 6-fullly ripe and 7- over ripe Crown-rot index: - 0 = no rot, 1 = 1-10% crown is rot, 2 = 11-20% crown is rot, 3 = 21-30% crown is rot, 4 = over 30% of the crown is rot.

Crown rot is caused by a combination of fungi Colletotrichum musae, Fusarium moniliforme, Botryodiplodia theobromae and Verticillium theobromae (Sarananda and Wijerathnam, 1994). Crown rot development normally takes place with the onset of ripening. However, no crown-rot incidence was reported in this consignment. The causes for the cut crown surface infection could be the dehanding knife and wash water. Regular disinfected dehanding knife and regular removal of wash water may have reduced the number of inoculation, which result in no rot development. In addition low oxygen in modified atmosphere would have suppressed the growth of fungi (Follstad, 1996).

Since the ripening process of 80 days mature bananas had been initiated, only 75 days mature bananas were used for ripening with exposure to ethylene gas at low temperature. The results showed that peel colour development was very good giving bright yellow colour. However, the external appearances of the fruits were of poor quality. The reasons for poor quality have been reported as blemishes on the skin due to mechanical “EMBUL” BANANA EXPORT 215 injuries, brown dots due to freckle disease and size variation of fruits in a box. Cavendish group banana requires ethylene exposure at 16-20°C for proper chlorophyll degradation. However, chlorophyll degradation in ‘Embul’ banana does not require those specific conditions described for Cavendish (Sarananda and Wijerathnam, 1994).

Table 2. Peel colour and quality of “Embul” banana after ripening.

Stage of maturity Peel colour Visual quality rating 75 days after flowering CI-6 6.5 Colour index: - 1- Green, 2-Colour break, 3- More green than yellow, 4-More yellow than green, 5- Yellow with green tip, 6-Fullly ripe and 7- Over ripe Visual Quality Index: - 9 - Excellent, 7 – Slight defects, 5 – Moderate defects, 3 – High defects, not saleable and 1 – Not edible

However, ‘Embul’ bananas did respond for commercial ripening treatment. Very high extent of mechanical injuries on the skin of banana may be due to careless packing. Skin of “Embul” banana seems to be much more sensitive than that of ‘Cavendish’. Thin peel with less resistance to mechanical injuries is a varietal character of “Embul” banana. However, careful handling during preparation and packing could reduce the extent of wounds. Very high susceptibility to freckle disease caused by Phylostica musarum is also an inherent disease of “Embul” banana if control measures are not adopted. This problem, however, can be overcome by maintaining the cleanliness in the plantation and bagging bunches soon after emerging of the bunches.

Non-uniformity of size of fruits is an inherent character of “Embul” banana. Fruit size variation from the first to the last hand is gradually reducing in “Embul” bananas. In certain bunches a number of last hands have to be rejected due to under size. One way of overcoming this problem is packing bananas of uniform size in separate boxes. The other alternative is to select varieties, which have greater proportion of hands with uniform size.

The duration to transport banana from Sri Lanka to Japan was approximately 2 days. Although more mature bananas were used for airfreight than those used for sea-freight, ripening process was not initiated (Table 3). This may be due to temperature control immediately after packing and maintaining the same temperature until reaching the airport. Simulated trials, however, showed if refrigerated temperature was not used, ripening process was initiated even 2 days after harvesting. Although bananas are harvested at pre-climacteric minimum stage, low temperature storage alone is sufficient to delay fruits entering the climacteric rise. 216 SARANANDA AND WIJESUNDARA

Table 3. Quality parameters of airfreighted “Embul” banana assessed in Japan .

Physical stage Firmness Colour index Crown rot index Upon arrival (green) Firm I 0 After ripening Soft 6 0 Colour index: - 1- green, 2-colour break, 3- more green than yellow, 4-more yellow than green, 5- yellow with green tips, 6-fully ripe, 7- over ripe Crown-rot index:- 0 = no rot, 1 = 1-10% crown is rot, 2 = 11-20% crown is rot, 3 = 21-30% crown is rot, 4 = over 30% of the crown is rot.

No incidence of crown rot had been observed upon arrival of fruit. The same results were recorded in ripe bananas when they were ripen by exposing to ethylene gas at 18°C. Sanitation followed during dehanding and washing reduced the number of inoculum deposit on cut surface of the crown. This may be the reason for no crown rot development. In addition, very short (2 days) transit time may not be sufficient to initiate the crown rot.

CONCLUSIONS

Based on the results, ‘Embul’ banana could successfully be sea- freighted when fruits were harvested at 75 days of maturity and packed in cartons under vacuum packing and without causing mechanical injuries. By storing bananas at 14°C from the pack house until they reach their destination, ripening process can be delayed for up to 30 days allowing export to any country in the world. Similarly ‘Embul’ bananas can be air freighted using more mature fruits (80 days) if they are packed in cartons as a single layer and transported from the pack house to the airport at 14°C.

ACKNOWLEDGEMENT

Financial support given for commercial export from Export Development Board, Mahae Company and Yobeeda Company are gratefully acknowledged. Field assistance given from Mahaweli Development Authority and its staff is also acknowledged. Tissue cultured bananas supplied by Professor S. Hirimburegama for airfreight is also greatly acknowledged.

REFERENCES

Follstad, M.N. 1996. Mycelial growtht rate and sporulatoin of Alternaria tenuis, Botrytis cinerea, Cladosporium herbarum and Rhizopus stolonifer in low oxygen atmosphere. Phytopathology 56: 1098-1099. Hewage, K.S. 1995. Factors influencing postharvest longevity of ‘Embul’ bananas. PhD Thesis. Wye College, University of London. Mapson, L.W. and J.E. Robinson. 1966. Relation between oxygen tension, biosynthesis of ethylene, respiration and ripening changes in banana fruit. Food Technology 1: 215-225. “EMBUL” BANANA EXPORT 217

Sarananda, K.H. 1989a. Response of 'Embul` bananas to modified atmosphere storage. Tropical Agriculturist 145: 104-113. Sarananda, K.H. 1989b. Response of ‘Embul banana (Musa spp.) to modified atmosphere storage. Tropical Agriculturist 145: 113-119. Sarananda, K.H. and R.S.W. Wijerathnam. 1994. Controlled atmosphere storage of ‘Embul’ bananas. Tropical Agricultural Research 6: 17-22. Sarananda, K.H., H. Wainwright and R. Wijerathnam. 1996. Inhibition of ethylene biosynthesis in modified atmosphere stored bananas. Tropical Agricultural Research 8: 306-314. Scott, K.J., Me W.B. Glasson and E.A. Robberts. 1968. Ethylene absorbent increases storage life of bananas packed in polyethylene bags. Agriculture Gazette, New South Wales, Pp 79-82. Truter, A.B. and J.C. Combrink. 1990. Controlled and modified atmosphere storage of bananas. Acta Horticulturae 275: 631-637. Annals of Sri Lanka Department of Agriculture. 2006.8:219-227.

CLONING OF TRYPSIN INHIBITOR GENE FROM SOYBEAN AND TRANSFORMATION INTO TOBACCO

D.A. SHIRANI1, S. BHAT, R. BHAT2, M.S. KURUVINASHETTI2, P.U. KRISHNARAJ2, H.M. VAMADEVAIAH2 and K. KRISHNAMURTHY2 1Agricultural Research Station, Sita Eliya, Nuwara Eliya 2University of Agricultural Science, Dharwad, India

ABSTRACT

Plant serine protease inhibitors (SPIs) have been found to be effective in developing transgenic crop plants which confer resistance to insects particularly to Lepidoptera. The genes encoding these proteins have less problems, related to codon bias, RNA processing and protein stability. Therefore, a study was initiated to isolate and clone a serine protease inhibitor gene from soybean (Glycine max). Using specific primers, gene encoding kunitz trypsin inhibitor of soybean (654bp) was amplified and cloned into pTZ57R/T vector. The clones were confirmed through PCR amplification and restriction digestion analysis. Further, the clones were sequenced and analyzed in silico. Cloned trypsin inhibitor gene of soybean showed 99 and 100 per cent homology with reported kunitz inhibitor genes of soybean at nucleotide and protein level respectively. Further, to facilitate transformation of crop plants, soybean kunitz trypsin inhibitor gene was sub cloned into a plant transformation vector pHS100 at BamH1 and Xba1 restriction sites. The recombinant clone pHS-SBC1 was mobilized into Agrobacterium tumefaciens LBA4404 by triparental mating. The A. tumefaciens with recombinant clone pHS-SBC1 was used to transform tobacco and the presence of inserts in kanamycin resistant plants was confirmed through PCR with specific primers.

KEYWORDS: Cloning, Soybean, Trypsin inhibitor gene, Plant transformation.

INTRODUCTION

Protease Inhibitors (PIs), which are ubiquitous in nature are one of the prime candidates with highly proven inhibitory activity against insect pests and able to suppress enzymatic activity of phytopathogenic micro organisms and nematodes. The expression of these genes is induced upon mechanical wounding and insect/pathogen attack. Plant protease inhibitors are an ideal choice for heterologous expression as they have no or less problems related to the expression in the transgenic plant. Moreover, the protease inhibitors have practical advantages over other inducible chemicals which are products of complex biochemical pathways. By transferring a single defensive gene from one plant to another either with their own promoter or with constitutive promoters, genetically modified plants can be obtained (Ryan, 1990). Serine class of protease inhibitors is effective against the serine protease in insect gut from many families, particularly Lepidoptera. A large number of plant serine protease inhibitor genes have been cloned and expressed in transgenic plants which confer resistance to lepidopterans and other families of insects (Marchetti et al., 2000; Balestrazzi et al., 2004; 220 SHIRANI et al.

Williams et al., 1997; Leo et al., 1998; Johnson et al., 1989; Qu et al., 2003; Sane et al., 1997; Xu et al., 1996; Gatehouse et al., 1997; Li et al., 1998; Alpteter et al., 1999; Lee et al., 1999 and Cipriani et al., 1998).

The mode of action of these inhibitors on the digestive enzymes of the pest is not always a simple inhibition of proteolytic activity. Broadway and Duffey (1986) have shown that the mode of action of PIs is to cause destructive hyper-production of trypsin coupled with insufficient dietary availability of sulphur-containing amino acids (i.e. methionine) needed for enzyme synthesis. As a result of which, the insects become weak with stunted growth and ultimately die.

Soybean kunitz trypsin inhibitor (SKTI) which belongs to serine class of protease inhibitor with molecular mass of 18-22KDa inhibits both trypsin and chymotrypsin. It is a single polypeptide chain of 181 amino acids including two disulphide bonds (Kunitz, 1946). Using this gene, few transgenic crop plants have been developed.

Transgenic tobacco plants expressing high levels of SKTI have shown greater resistance than cowpea trypsin inhibitor gene (CpTI) which has been widely used in developing transgenics against H. virescens (Gatehouse et al., 1993). Transgenic rice developed with SKTI was found to be resistant against brown plant hopper (Nilaparvata lugenes) (Lee et al., 1999).

Though transgenic crops developed with δ-endotoxin genes from Bacillus thuringiensis are fairly widespread, several reports have shown the possibility of developing resistance against Bt (Tabashnik, 1994 and Michaud, 1997) in the insect populations. In order to delay the resistance, one of the strategies would be the use of alternative genes for developing transgenic plants either singly or in combination with other insecticidal genes.

Considering the fact that plant derived genes efficiently express in the transgenic plants, this study was conducted to clone an alternative insect resistant gene trypsin inhibitor from soybean and to transform it into the model system tobacco.

MATERIALS AND METHODS

The present study was undertaken to clone trypsin inhibitor gene from soybean (Glycine max), sub clone the gene into a plant transformation vector and Agrobacterium mediated transformation of tobacco with cloned trypsin inhibitor gene. The materials used and methods employed are as follows; CLONING OF TRYPSIN INHIBITOR GENE 221

Plant material and PCR amplification

The total DNA of soybean variety JS335 was isolated by following CTAB protocol (Doyle and Doyle, 1987) with some modifications. Complete coding sequence of trypsin inhibitor gene was amplified by PCR using DNA from soybean variety JS335 as template and with a set of gene specific primers (Cipriani et al., 1998) and used for cloning into pTZ57R/T vector and the plant transformation vector pHS100 (BARC, Bombay).

Forward: 5’-ATGAAGAGCACCATCTTC-3' (18 mer) Reverse: 5’-TCACACACTGCGAGAAAG-3' (18 mer)

PCR was carried out in a eppendorf master cycler gradient in 20 µl reaction volume containing 100ng DNA template, 25mM MgCl2, 5pM of each primer, 2.5mM dNTPs, 1X Taq polymarase buffer and 1.0U Taq DNA polymerase (Bangalore Genei) for 40 cycles.

Gel elution of the PCR amplicon

The single sharp amplicon at 650 bp was separated out using sharp sterile scalpel blade and collected in a sterile pre-weighed 2.0 ml microcentrifuge tube. The Qiagen gel extraction kit was used to elute the DNA from the agarose block as described in user manual.

Cloning of PCR The purified PCR fragment of size ~650bp amplicon was ligated to pTZ57R/T cloning vector (2886 bp) as described in InsT/A cloneTM PCR product cloning kit (K1214) form MBI, Fermentas, USA.

For ligation, an optimal molar ratio of ends vector: insert (1:3) was computed and the components of ligation mix were added to 0.5 ml micro centrifuge tube and was incubated at 16°C for 16 hrs. The competent cells of E. coli DH5 were transformed with 10µl of ligation mixture.

The recombinant clones were identified by blue/white assay in the Luria agar plates with Amp50, X-gal, and IPTG after overnight incubation at 37°C. The positive clones were named as pTZ-SB clones.

Confirmation of clones

The confirmation of the presence of cloned fragment in the recombinant vector was done by PCR amplification and through comparative restriction analysis of selected clones and the control vector to ensure the presence of insert with XbaI and BamHI enzymes. 222 SHIRANI et al.

Sequencing of clones

pTZ-SBC3 clone was sequenced using M13 primers employing primer walking technique at Bangalore Genei Private Ltd., Bangalore. The sequence was subjected to analysis using BLAST algorithm available at http://www.ncbi.nlm.nih.gov.

Subcloning into plant transformation vector

The plasmid DNA of pTZ-SBC3 and the plant transformation vector pHS100 was isolated in large quantities and sequentially restricted with XbaI and Bam HI to facilitate directional cloning and these were ligated to get recombinant clones in order to transform the trypsin inhibitor gene into plant. These clones were confirmed by PCR amplification and restriction analysis. The positive clones were named as pHS-SB clones.

Agrobacterium Transformation

The confirmed clones were further mobilized into Agrobacterium tumefaciens strain LBA4404 by triparental mating using pRK2013 as helper plasmid in DH5α. The transconjugants were selected on selection medium. Plasmids were isolated from recombinant Agrobacterium and the presence of trypsin inhibitor insert was confirmed by PCR amplification.

Tobacco transformation

The Agrobaterium containing construct; pHS-SBC1 was used for tobacco (Nicotiana tabacum L. cv. White burley) transformation with the protocol mentioned by Hooykaas and Schilperoort (1992) with some modifications. DNA was extracted from Kanamycin resistant plants and checked through PCR with gene specific primers.

RESULTS AND DISCUSSION

Cloning of trypsin inhibitor gene

In the present study, the amplified ~650bp amplicon was cloned into linearised pTZ57R/T and transformants were screened through Blue-white screening assay. The positive clones were confirmed through PCR (Fig. 1) and restriction analysis (Fig. 2). The sequence was subjected for homology search at nucleotide level and at protein level using BLASTn and BLASTp programs available at http://www.ncbi.nlm.nih.gov. and the cloned 654bp long insert encoding 217 amino acids was found to have 99% homology with reported soybean inhibitor genes. In silico analysis of CLONING OF TRYPSIN INHIBITOR GENE 223 translated amino acid sequence (216 amino acids aligned) showed 100% homology with amino acid sequences of trypsin inhibitor subtype A and Tia Kunitz trypsin inhibitor of soybean.

M 1 2 3 4

1000 bp 600 bp

Figure 1. PCR confirmation of insert in pTZ-SB clones. M- 100bp DNA ladder, 1- positive control (soybean genomic DNA), 2- pTZ-SBCI, 3- pTZ-SBC2 4- pTZ SBC3.

M 1 2 3 4 5

1000 bp 600 bp

Figure 2. Restriction analysis of pTZ-SB clones with Xba1 and BamH1 M- 100bp DNA ladder, 1- vector: pTZ57R/T, 2- PCR product, 3- pTZ-SBC1, 4- pTZ-SBC2, 5- pTZ-SBC3.

The rpsBLAST result showed that the SKTI peptide has a single STI (soybean trypsin inhibitor) domain and has shown 99.4% conservation in the protein. Kunitz (1946) has characterized the trypsin inhibitor gene of soybean as a single polypeptide chain of 181 amino acids including two disulphide bonds. In the deduced amino acid sequence of the cloned SKTI, there were 4 cysteine residues at 64th, 111th, 161st and 170th positions to facilitate intrachain disulphide bridges. It is clear from the deduced amino acid sequence of cloned SKTI that the gene and the peptide are exactly similar to the known soybean kunitz trypsin inhibitor. 224 SHIRANI et al.

Subcloning into plant transformation vector

Sequence result of the pTZ-SBC3 clone showed that the insert was in correct orientation. Therefore, the insert was released from pTZ-SBC3 clone and directionally cloned into pHS100 plant transformation vector (Fig. 3) under CaMV35S constitutive promoter at XbaI and BamH1 sites and transformed E. coli DH5 cells. The confirmed clone pHS-SBC1 was mobilized into Agrobacterium tumefaciens LBA4404 by triparental mating. Plasmid DNA isolated from these transformants showed positive for PCR (Fig. 4) and restriction analysis (Fig. 5). The clone pHS-SBC1 was mobilized into Agrobacterium tumefaciensHind III strain LBA4404 through tri-parental mating using E.coli containing pRK2013 as helper plasmid.

EcoRI

RB LB Nos- r Nos- npt-II (Kan ) 35-S 35S AMV SKTI UidA-npt-II Nos- P T T Xba BamH I I i Figure 3. T-DNA cassette of pHS100 plant transformation vector containing SKTI gene.

M 1 2 3 4 5 6

1000 bp 600 bp

Figure 4. PCR confirmation of SKTI insert in pHS-SB clones M- 100bp DNA ladder, 1 – pHS-SBC1, 2- pHS-SBC2, 3- pHS-SBC3, 4-pHS-SBC4, 5-pHS-SBC5, 6- positive control (pTZ-SBC3). CLONING OF TRYPSIN INHIBITOR GENE 225

M 1 2 3 4 5 6 7

1000 bp 600 bp

Figure 5. Restriction analysis of pHS-SB clones with Xba1 and BamH1 M- 100bp DNA ladder, 1- vector: pHS100, 2-PCR product, 3-pHS-SBC1, 4-pHS-SBC2, 5- pHS-SBC3, 6-pHS-SBC4, 7-pHS-SB5.

Tobacco transformation

The Agrobacterium, with the construct of pHS-SBC1 was used to transform tobacco by following protocol from Hooykaas and Schilperoort (1992). The surviving green shoots which showed resistance to kanamycin at 200µg/ml were rooted with selection and transferred to sterilized peat. DNA was isolated from putative transformants and checked for the presence of insert. More than 80% of the checked plants were PCR positive for the gene (Fig. 6).

M 1 2 3 4 5 6 7 8 9 10

1000 bp 600 bp

Figure 6. PCR confirmation for the presence of SKTI insert in tobacco M-100bp DNA ladder, 1-negative control (untransformed tobacco), 2- positive control (pHS-SBC1), 3 to 10-positive plants. 226 SHIRANI et al.

CONCLUSIONS

Serine protease inhibitor genes have proved to be very important in developing transgenic crop plants either singly or in combination with other insecticidal genes like cry genes of Bacillus thuringiensis, lectins and other protease inhibitor genes. In the present study, soybean trypsin inhibitor gene was cloned to a plant transformation vector and tobacco (the model system) was transformed. Expression of the gene in the plant system has to be confirmed before deployment in the transgenic plant development. Since the gene does not have introns, the expression can also be tried in the prokaryotic system. ACKNOWLEDGEMENT

The authors wish to express their gratitude to CARP, Sri Lanka for funding this research project under long term training program.

REFERENCES

Alpteter, F., I. Diaz, H. Mcauslane, K. Gaddour, P. Carbonero and I.K. Vasil, 1999. Increased insect resistance in transgenic wheat stably expressing trypsin inhibitor CMe. Molecular Breeding 5:53-63.

Balestrazzi, A., M. Confalonieri, M. Odoardi, V. Ressegotti, G. Allegro, A. Tava and D. Carbonera, 2004. A trypsin inhibitor cDNA from a novel source, Snail Medic (Medicago scutellata L.): Cloning and functional expression in response to wounding, herbivore, jasmonic acid and salicylic acid. Plant Science 167:337-346. Broadway, R.M. and S.S. Duffey, 1986. Plant proteinase inhibitors: Mechanism of action and effect on the growth and digestive physiology of larval Heliothis zea and Spodoptera exigua. Journal of Insect Physiology 32: 827-833.

Cipriani, G., D. Michaud, F. Brunelle, A. Golmirzaie and D.P. Zhang, 1998. Expression of soybean proteinase inhibitor in sweet potato. CIP Program Report (1997- 98): 271–277. Doyle J.J. and J.L. Doyle, 1987. Isolation of plant DNA from fresh tissue. Phytochemistry Bulletin 19:15-38. Gatehouse, A.M.R., G.M. Davidson, C.A. Newell, A. Merryweather, W.D.O. Hamilton, E.P.J. Burgess, R.J.C. Gilbert and J.A. Gatehouse, 1997. Transgenic potato plants with enhanced resistance to the tomato moth, Lacanobia oleracea: growth room trials. Molecular Breeding 3:49-63. Gatehouse, A.M.R., Y. Shi, K.S. Powell, C. Brough, V.A. Hilder, W.D.O. Hamilton, C. Newell, A. Merryweather, D. Butler and J.A. Gatehouse, 1993, Approaches to insect resistance using transgenic plants. Philosophical Transactions of the Royal Society, London 342:279-286. Hooykaas, P.J.J. and Schilperoort, 1992. Agrobacterium and plant genetic engineering. Plant Molecular Biology 19:15-38. http://www.ncbi.nlm.nih.gov. CLONING OF TRYPSIN INHIBITOR GENE 227

Johnson, R., J. Narvaez, G. An and C. Ryan, 1989. Expression of proteinase inhibitor I and II in transgenic tobacco plants: Effects on natural defense against Manduca sexta larvae. Proceedings of National Academic Science, USA 86:9871- 9875.

Kunitz, M., 1946. Crystalline Soybean Trypsin Inhibitor. Journal of General Physiology 29:149-154. Lee, Soo In, Lee, Sung-Ho, Koo, Ja Choon, Chun, Hyun Jin, Lim, Chae Oh, Mun, Jeom Hee, Song, Yoo Han, Cho, and Moo Je, 1999. Soybean Kunitz trypsin inhibitor (SKTI) confers resistance to the brown plant hopper (Nilaparvata lugens Stael) in transgenic rice. Molecular Breeding 5(1):1-9. Leo, F.D., M.A. Bonade-Bottino, L.R. CecI, G. GalleranI and L. Jouani, 1998. Opposite effects on Spodoptera littoralis larvae of high expression level of a trypsin proteinase inhibitor in transgenic plants. Plant Physiology 118:997–1004. Li, Y.E., Z. Zhu, Z.X. Chen, X. Wu, W. Wang and S.J. Li, 1998. Obtaining transgenic cotton plants with cowpea trypsin inhibitor. Acta Gossypii Sinica 10: 237-243.

Marchetti, S., M. Delledonne, C. Fogher, C. Chiaba, F. Chiesa, F. Savazzani and A. Giordano, 2000. soybean Kunitz, C-II and PI-IV inhibitor genes confer different levels of insect resistance to tobacco and potato transgenic plants. Theoretical and Applied Genetics 101:519-526. Michaud, D., 1997, Avoiding protease-mediated resistance in herbivorous pests. Trends Biotechnology 15: 4–6. Qu, L. J., J. Chen, M. Liu, N. Pan, H. Okamoto, Z. Lin, C. Li, D. Li, J. Wang, G. Zhu, X. Zhao, X. Chen, H. Gu and Z. Chen, 2003. Molecular cloning and functional analysis of a novel type of Bowman-Birk inhibitor gene family in rice. Plant Physiology 133:560–570. Ryan, C. A., 1990. Protease inhibitors in plants: genes for improving defenses against insects and pathogens. Annual Review of Phytopathology 28:425-449. Sane, V.A., P. Nath, Aminuddin and P.V. Sane, 1997. Development of insect-resistant transgenic plants using plant genes: Expression of cowpea trypsin inhibitor in transgenic tobacco plants. Current Science 72(10):741-747. Tabashnik, B.E., 1994. Evolution of resistance to Bacillus thuringiensis. Annual Review of Entomology 39:47–79. Williams, D.L., W.C. Kain and R.M. Broadway, 1997. Isolation and characterization of a serine proteinase inhibitor cDNA (Accession No. U18995) from cabbage. Plant Physiology 114:747. Xu, D.P., Q.Z. Xue, D. Mcelroy, Y. Mawal, V.A. Hilder and R. Wu, 1996. Constitutive expression of a cowpea trypsin inhibitor gene, CpTi, in transgenic rice plants confers resistance to two major rice insect pests. Molecular Breeding 2:167-173. Annals of Sri Lanka Department of Agriculture. 2006.8:229-238.

USE OF LEAF COLOUR CHARTS TO DETERMINE NITROGEN REQUIREMENT AND YIELD RESPONSE OF RICE VARIETIES

D.N. SIRISENA, W.M.A.D.B. WICKRAMASINGHE and R.A.N.D. RANATHUNGE Rice Research and Development Institute, Batalagoda, Ibbagamuwa

ABSTRACT

An experiment was conducted at the Rice Research and Development Institute, Batalagoda to find out the variation in leaf colour of rice varieties with the application of nitrogen and its relation to nitrogen response. Four rice varieties, Bg 300, Bg 305, Bg 250 and Bg R1, as sub plots and four nitrogen levels, 0, 50, 100 and 150 Kg N/ha, as main plots were used with split plot arrangement. Leaf Colour Chart (LCC) readings were taken on uppermost fully expanded leaves at 2 week intervals. Nitrogen response of rice varieties was determined on the basis of grain yield at different N levels. It was observed that LCC readings of nitrogen treated rice plants were always higher than the untreated plants and all the varieties showed significant increase in leaf colour up to 100kg N/ha and thereafter increment was not significant or colour reduction was observed. As seen in the LCC readings, yield response of all rice varieties were only up to 100 kg N/ha and thereafter there was no yield increase due to application of nitrogen. Variety Bg 350 recorded the highest yield followed by Bg 300, Bg 250 and Bg R1. There was a reduction in leaf colour of some rice varieties (Bg 300, Bg 305, Bg 250) at high level of N (150 kg N/ha) and as a result there was a reduction in grain yield of the same rice varieties. Relationships between LCC readings across the growth duration and grain yields of all varieties were positive but the highest relationship (r2 > 0.70) between grain yield and LCC readings was recorded at 4th and 8th weeks after sowing. Change of LCC readings at 4 weeks after sowing with increasing N levels and change of grain yield of varieties at each N level showed a similar trend. According to the above results, it can be suggested that under local conditions leaf colour changes of rice with the N application is a better indicator of N requirement and readings taken at 4 weeks after sowing are highly related to yield response to added N. Therefore, measurement of leaf colour at 4 weeks after sowing using LCC as a tool can be used as a better indicator to screen rice varieties which respond to N fertilizer.

KEYWORDS: Leaf colour charts, Nitrogen response, Rice.

INTRODUCTION

Application of high rate of nitrogen fertilizer has become a common practice in Sri Lankan paddy cultivation because urea is available at subsidised prices. Under present cultivation system of rice, nitrogen recovery is about 30% and as such large amounts of applied nitrogen are lost from the system as ammonia gas and pollute the environment (Sirisena et al., 2001). In addition, uptake of high nitrogen by rice plants increases the bio-mass, decreases the crop yield due to lodging and susceptibility to diseases (Yoshida, 1981). Therefore, management of nitrogen is one of the most important criteria in rice cultivation in Sri Lanka to meet the rice requirement of the country with minimum environmental pollution. Introduction of rice varieties 230 SIRISENA et al. responding to N-fertilizer and application of nitrogen on demand driven basis are the important two criteria to suit the above conditions. At present N response of rice is determined through yield measurements at increased N- level and there is no method developed so far to find out the optimum N levels under local conditions.

Because leaf-N content is closely related to photosynthetic rate (Peng et al., 1995) and biomass production (Kropff et al., 1993), it is a sensitive indicator of the dynamic changes in crop-N demand within a growing season. Therefore, measurement of leaf N content at different N levels is a good indicator to determine the N response of rice. The direct measurement of leaf-N concentration by laboratory procedures is laborious, time consuming, and costly. Such procedures have limited use as a diagnostic tool to determine N response because of the extensive time delay between sampling and obtaining results.

Sirisena et al. (2002) did some experiments under local conditions with Minolta SPAD 502 meter and reported that leaf colour measurement with SPAD meter can effectively be used to determine the N responsiveness of rice. Sirisena et al. (2003) further reported that SPAD meter results can also be used to cut down N application by ¼ when N is applied on the basis of leaf colour.

The high price of SPAD meter limits its use by individual income-poor farmers. Another simple, quick, and nondestructive method for estimating leaf N status is a LCC. There are several types of LCC developed for determining leaf greenness in rice around the world (Yang et al., 2003). The most common ones are those developed by the International Rice Research Institute (IRRI, 1996). Unlike the SPAD, which measures light absorption, LCC measures leaf greenness and the associated leaf N by visually comparing light reflection from the surface of leaves.

Even though LCC has been tested for real-time N management in the farmers' fields in several other countries (Balasubramanian et al., 1999), limited information is available on the accuracy of LCC in estimating leaf colour changes and N response of rice varieties under Sri Lankan conditions. Therefore, this paper reports the observations made in the change of leaf colour with the N application under local conditions and how LCC can be used as a tool to determine nitrogen response of rice varieties.

MATERIALS AND METHODS

An experiment was conducted at the Rice Research and Development Institute, Batalagoda in maha 2005/2006 with the objective of identifying variation in leaf colour of rice varieties with the application of LEAF COLOUR CHARTS AND NITROGEN RESPONSE OF RICE 231 nitrogen and its relation to nitrogen response. Experimental site was Plinthudults in Kurunegala soil series. Soil pH is 6.5 and Olsen’s P and exchangeable K contents are at 9 ppm and 70 ppm, respectively. Experimental design was split plot with four replicates. Nitrogen (0, 50, 100, and 150 kg/ha) was designated as main plot and cultivar (Bg 300, Bg 305, Bg 250 and BgR1) was designated as subplots. Bg 300, Bg 305 and Bg 250 were recommended varieties and Bg R1 was an elite breeding line. Pre-germinated seeds were sown as 100 kg seed paddy per hectare. Fertilizer N in the form of urea was split-applied, with 6% at basal, 26% at two weeks after sowing, 44% at 4 weeks after sowing and 24% at 6 weeks after sowing. All plots received 45 kg P2O5/ha and 20 kg K2O/ha and 1 kg Zn/ha at the time of sowing. All basal fertilizers were uniformly broadcasted manually before sowing and incorporated to the soil. At 6 weeks after sowing additional 20 kg K2O/ha was added to all plots. Pests and diseases were intensively controlled using chemicals to avoid yield losses. Fields were flooded 7 days after sowing and a floodwater depth of 5-10 cm was maintained until 14 d before harvest when fields were drained. The LCC, which has been developed by International Rice Research Institute consisting of six green strips showing increasing greenness with increasing number, was used to take the leaf colour readings. Twenty disease-free rice plants were randomly selected in the plot, and the colour of the youngest fully expanded leaf of the selected plant was compared by placing its middle part on top of the color strip in the LCC. LCC readings were taken before fertilizer application and continued up to 50% flowering stage at two weeks intervals. Each plot was harvested at maturity, grains were air dried for a period of 2-3 weeks to bring down the moisture content to14% and then weighed.

Data were analyzed following analysis of variance (SAS Inst., 1982), and means were compared by DMRT. Relationships of LCC scores to grain yield were determined by regression analysis using data from LCC scores across the growth duration and grain yield.

RESULTS AND DISCUSSION

As shown in Figure 1, LCC readings increased with the application of nitrogen fertilizer. Changing pattern of green colour across the growth duration was similar in all varieties. Analysis of leaf colour readings of rice varieties at different N levels showed significant increase in LCC score with the application N fertilizer. It was observed that irrespective of the varieties, difference of LCC scores between zero and 50 kg N/ha was vary much higher and comparatively lower between 50 and 100 kg N/ha. Difference of LCC scores between 100 kg N/ha and 150 kg N/ha was not as high as earlier. 232 SIRISENA et al.

5 6 Bg 300 Bg R1 4.5 5 4

3.5 s 4 s e e 3 r r o o c c

s 2.5

S 3

C C 2 C C

2 L L 1.5

1 1

0.5 0 0 2 4 6 8 10 2 4 6 8 10 Time (weeks after sowing) Time ( weeks afetr sowing)

6 5 Bg 305 Bg 250 4.5 5 4 3.5 s e s 4 r e 3 r o o c s c

2.5

S 3

C 2 C C C

2 L 1.5 L 1 1 0.5 0 0 2 4 6 8 10 2 4 6 8 10 Time (weeks after sowing) Time (Weeks after sowing)

0 kg N /ha 50 kg N/ha 100 kgN/ha 150 kg N/ha

Figure 1. Difference of LCC scores of rice varieties after application of different rates of nitrogen fertilizer.

All the varieties showed positive and linear relationship between nitrogen levels and LCC scores throughout the growth period. Regression coefficient of Bg 305 ranged from 0.64 to 0.95 while that for Bg 300, Bg 250 and Bg R1 varied from 0.36 to 0.87, 0.26 to 1.00 and 0.10 to 0.98, respectively (Table 1). LEAF COLOUR CHARTS AND NITROGEN RESPONSE OF RICE 233

Table 1. Regression coefficient (r2) between nitrogen levels and LCC scores at different N levels.

Variety Regression coefficient (r2) between N levels Vs LCC scores 2 WAS 4 WAS 6 WAS 8 WAS 10 WAS Bg 300 0.36 0.82 0.87 0.62 0.80 Bg 305 0.64 0.95 0.86 0.86 0.89 Bg 250 0.26 0.88 0.96 0.75 1.00 Bg R1 0.10 0.94 0.97 0.75 0.98

As suggested by Singh et al. (2002) and results appearing in Figure 1, the critical LCC score for the timing of N topdressing, however, can be determined on the basis of LCC scores across the growth duration. Under local conditions at 4 weeks after sowing, leaf colour of the N zero plots and plots receiving 50 Kg N/ha is around 3 and as such N has to be applied at this time even if the leaf colour score is around 3. At 6 weeks after sowing, leaf colour of all the N levels had gone close to 4.5 even in the N zero plots. At 8 weeks after sowing, average leaf colour was above 3.5. As such critical LCC readings at 2 and 4 weeks should be around 3.0 and at 8th week should be around 3.5. Leaf colour readings at 6 weeks after sowing should be around 4.5 to have a better rice production. Singh et al. (2002) reported that N management based on the critical LCC reading of 4.0 with LCC-IRRI helped avoid over-application of N to rice crop. It is seen in this experiment that under local conditions LCC reading to determine N requirement is between 3.5 and 4.5. Results of this experiment agreed with the findings of Balasubramanian et al. (1999) that readings of leaf color chart can be tested for real-time N management. It has also been suggested that different threshold LCC values may have to be used for different varietal groups (Balasubramanian et al., 2000) but it is not observed here because all the varieties performed similarly. Therefore, to attain maximum yield with minimum N losses, experiments have to be undertaken to find out the minimum N requirement to achieve the above critical leaf colour readings.

6 ) a h / 4 t (

d l 2 e i Y 0 Bg 300 Bg 305 Bg 250 BgR1 Variety

0 kg N/ha 50 kg N/ha 100 kg N/ha 150 kg N/ha 234 SIRISENA et al.

Figure 2. Grain yield of rice obtained with the application of different rates of N fertilizer.

Figure 2 shows that grain yield of rice in all varieties increased with the application of nitrogen. All rice varieties had higher N response to first 50 kg N/ha but comparatively lower response to the second 50 kg N/ha and least to 3rd 50 kg N/ha. All the varieties had a little reduction in yield at 150 kg N/ha.

Relationship between grain yield and pooled values of LCC readings across growth stages is shown in Figures 3, 4, 5 and 6. It is clear from these results that better linear relationships between LCC scores and grain yield were obtained at 4 and 8 weeks after sowing and r2 > 0.75.

y = 1.261x + 1.0935 6 r 2 = 0.7598 5 4 3 2 1 0 2 2.5 3 3.5 4

Leaf colour scores

Figure 3. Relationship between LCC scores pooled data and grain yield of rice grown under different N levels at 4 weeks after sowing.

y = 0.6946x + 1.7618 6 R2 = 0.3858 5 )

a 4 h / t (

3 d l e

i 2 Y 1 0 2 2.5 3 3.5 4 4.5 5 5.5 Leaf colour scores

Figure 4. Relationship between LCC scores pooled data and grain yield of rice grown under different N levels at 6 weeks after sowing. LEAF COLOUR CHARTS AND NITROGEN RESPONSE OF RICE 235

y = 0.9847x + 1.8942 6 r 2 = 0.7339 5 )

a 4 h / t ( 3 d l e i

Y 2 1 0 2 2.5 3 3.5 4 Leaf colour scores

Figure 5. Relationship between LCC scores pooled data and grain yield of rice grown under different N levels at 8 weeks after sowing.

y = 0.7108x + 1.7263 6 R2 = 0.3912 5 )

a 4 h / t ( 3 d l e i 2 Y 1 0 2 2.5 3 3.5 4 4.5 5 5.5 Leaf colour scores

Figure 6. Relationship between LCC scores and grain yield of rice grown under different N levels at 10 weeks after sowing.

Figure 7 shows the changes of LCC scores at four weeks after sowing and changes of grain yield irrespective of the varieties. It is clear from the results that there is no increase in yield beyond 100 kg N/ha and improvement of green colour with N application showed the similar trend.

d 6 l

e r i 5 a y u a

b o ) n l 4 a i a c a o a h r c

/ 3 b

t g f ( / c

a 2 s e e r L 1 o

c 0 s Yield Leaf colour scores Time after sowing

0 kg N/ha 50 k g N/ha 100 kg N/ha 150 kg N/ha 236 SIRISENA et al.

* Same letters on the column are not significantly different by DMRT (P=0.05) Figure 7. Average leaf colour scores of rice varieties at 4 weeks after sowing and average grain yield of rice varieties at different N levels.

Table 2. Yield response and leaf colour changes at 4 weeks after sowing of four rice varieties at different N levels.

Varieties Grain yield (t/ha)* LCC scores at 4 weeks after sowing* 0kg 50 Kg 100 kg 150 kg 0kg 50 Kg 100 kg 150 kg N/ha N/ha N/ha N/ha N/ha N/ha N/ha N/ha Bg 300 3.54a 4.57a 5.02a 4.64ab 2.07b 2.87a 3.57a 3.37a Bg 305 3.97a 4.88a 5.34a 5.12a 2.07b 2.90a 3.97a 3.60a Bg 250 3.37a 4.74a 4.98a 4.57ab 2.87a 2.95a 3.80a 3.30a Bg R1 3.37a 3.78b 3.87b 3.92b 2.27b 2.55b 2.67b 3.15a *Means within a column followed by same letter are not significantly different by the DMRT (p=0.05)

Results appearing in Table 2 show that there is a change of grain yield of varieties with the application of nitrogen at 50 kg N/ha and 100 kg N/ha and change of leaf colour with the same levels of N at 4 weeks after sowing showed the similar trend. It is clear from the results that leaf colour changes and yield changes of Bg 300, Bg 305, Bg 250 and Bg R1 were similar at all the N levels. At 150 kg N/ha there was a reduction in yield in Bg 300, Bg 305 and Bg 250. Similarly there was a reduction in leaf colour of the same varieties as well. Application of nitrogen enhances the leaf colour in rice varieties but application of high level of N enhances the production of new tillers (Yoshida, 1981) and this may reduce the leaf colour as well as the grain yield of rice. It is clear from these results that if there is a variation in leaf colour among varieties there is a variation in yield as well. Therefore, leaf colour is a good indicator to identify the yielding ability of rice varieties at different rates of N fertilizer. Therefore, determination of leaf colour with the application of N fertilizer can be used to determine the optimum level of nitrogen of rice crops.

CONCLUSIONS

Irrespective of the varieties, LCC readings are linearly increased with the application of nitrogen fertilizer but at high level of nitrogen there is a reduction in both leaf colour as well as the grain yield of rice. As such LCC reading is a good indicator to determine N requirement of rice varieties under local conditions. The critical levels of LCC readings change according to the growth stage of the crop. At the early and latter stages, it should be around 3.5 but at middle it should be 4.5. Similarity was LEAF COLOUR CHARTS AND NITROGEN RESPONSE OF RICE 237 observed in changing grain yield with the application of nitrogen and changing LCC readings at 4th week after sowing. Therefore, LCC scores at 4 weeks after sowing can be used to screen the rice varieties which respond to added N.

REFERENCES

Balasubramanian, V., A.C. Morales, R.T. Cruz, and S. Abdulrachman. 1999. On-farm adaptation of knowledge -intensive nitrogen management technologies for rice system. Nutr. Cycling Agroecosyst. 53:59–69. Balasubramanian, V., A.C. Morales, R.T. Cruz, T.M. Thiyagarajan, R. Nagarajan, M. Babu, S. Abdulrachman, and L.H. Hai. 2000. Adaptation of the chlorophyll meter (SPAD) technology for real-time N management in rice: A review. Int. Rice Res. Notes 25(1):4–8. IRRI. 1996. Use of leaf color chart (LCC) for N management in rice. Crop Resour. Manage. Network Technol. Brief 2. IRRI, Manila, Philippines Kropff, M.J., K.G. Cassman, H.H. Van Laar, and S. Peng. 1993. Nitrogen and yield potential of irrigated rice. Plant Soil 155/156:391–394. Peng, S., R.C. Laza, F.V. Garcia, and K.G. Cassman. 1995. Chlorophyll meter estimates of leaf area based nitrogen concentration of rice. Commun. Soil Sci. Plant Anal. 26:927–935. SAS Institute. 1982. SAS user's guide: Statistics. 4th ed. SAS Inst., Cary, NC. Singh, B., Y. Singh, J.K. Ladha, K.F. Bronson, V. Balasubramanian, J. Singh, and C.S. Khind. 2002. Chlorophyll meter– and leaf color chart–based nitrogen management for rice and wheat in northwestern India. Agron. J. 94:821–829. Sirisena D.N., D.B. Wickramasinghe, and L S Silva. 2001. Fate of nitrogen fertilizer applied to wetland paddy soils. Annals of the Sri Lanka Department of Agriculture 3: 231-236. Sirisena, D.N., W.M.A.D.B. Wickramasinghe, W.M.W. Weerakoon, and R. A. N. D. Ranatunge. 2002. Determining nitrogen response of rice through chlorophyll meter technique. Annals of the Sri Lanka Department of Agriculture 4: 424- 428. Srisena, D.N., W.M.A.D.B. Wickramasinghe, W. M. W. Weerakoon, and D. Kumaragamage and S T Bandara, 2003. Evaluation of leaf N based nitrogen fertilizer management in irrigated transplanted rice Annals of the Sri Lanka Department of Agriculture 5 : 233-241. Yang, W., S. Peng, J. Huang, A.L. Sanico, R.J. Buresh and C. Witt, 2003. Nitrogen management using leaf color charts to estimate leaf nitrogen status of rice. Agronomy Journal 95:212-217. Yoshida, S. 1981. Fundamentals of rice crop science. International Rice Research Institute, Los Banos, Philippines 135p. Annals of Sri Lanka Department of Agriculture. 2006.8:239-249.

EVALUATION OF PROPAGATION TECHNIQUES OF GUAVA (Psidium guajava L.)

M.K. THANTIRIGE, C.K.D. WELLALA and M.S. KARUNARATNE Fruit Crop Research and Development Centre, Horana

ABSTRACT

Expansion of guava cultivation in Sri Lanka is mainly affected by non- availability of quality planting materials in sufficient quantities. Guava is commonly propagated by patch budding method. This method is not very successful due to certain drawbacks such as long holding periods in nursery, low success rate and slow recovery period after budding. Therefore, alternative propagation techniques were evaluated at the Horticultural Farm, Bandaragama and Fruit Crop Research and Development Centre, Horana during November 2003-May 2005. Results indicated that stool (mound) layering with use of an auxin (3000 ppm 1 Naphthyl acetic acid, Secto) was found to be successful in producing well rooted planting materials within 3-4 weeks with a high success rate of 96%. In contrast to this method air layers took 6-7 weeks with a success rate of 60%. Attempts were also made on chip budding method on young root-stocks of 6-8 and 9-12 months old using deleafed bud chips resulted in success rates of 73% and 56% respectively. Unlike patch budding, this technique can be practised easily as it does not require the peeling ability of budding materials. Patch budding performed on 6-8 months old stock plants required 21 days for the bud recovery with a success rate of 73 %, whereas for much older plants it was 45 days with 76 % success. Similarly, wedge grafting and inarching were also shown to be successful on 9-12 months old root-stocks kept for 21 days with 69 % and 83 % success rates; on 13-15 old stock plants it was 23% and 96% and kept for 45 days after grafting respectively. Top working was successful for guava with 67% bud take and set fruits by 6-8 months after budding. This technique helps replacing of undesirable trees with recommended varieties. Thus, to increase the efficiency of multiplication rate of guava, stooling can be successesfully adopted. Also in bud grafting, deleafed chip and patch methods and for shoot grafting, wedging could be adopted.

KEYWORDS: Chip-bud grafting, Inarh, Stooling, Wedge-shoot grafting.

INTRODUCTION

Guava known as an excellent source of Vitamin C is one of the popular fruit among Sri Lankans. In the past, crop was confined to backyards of almost all homesteads. With the release of four high yielding varieties of high quality in 2001 along with dissemination of technical knowledge, a gradual transformation of its cultivation from home levels to small-scale commercial plantings has taken place. This increased the demand for quality planting material. In addition, the concept of enhancing the productivity of fruit crops in small farms of Sri Lanka was initiated under Small Farm Development Programme by the Ministry of Agriculture. Among several crops assigned under this programme in order to increase planting material production and distribution among farmers, guava is also included. 240 THANTIRIGE et al.

Guava can be propagated through patch or modified forkert methods of budding, (Gorakh-Singh and Pandey, 1998; Aulakh, 1998), air layering (Bhagat et al., 1999), cleft grafting, stem cuttings (Ramiraz, 1999) and stooling (Pandey, 1996; Saroj and Pathak, 1995). In Sri Lanka guava is generally propagated by patch method of bud grafting, which has certain drawbacks. To root-stocks plants to come into growth dimensions required for grafting, a nursery stage of 12-14 months is usually taken.

Furthermore, the ability to peel off the bark of both stock plant and scion shoot is one of the important prerequisites for bud grafting. Also, budded plants take another 5-6 months period for shoot formation prior to field establishment. In some instances, even though patch bud is taken, a subsequent shoot recovery from bud growth either delays, retards or does not occur. Due to such reasons, success rate of the conventional method of propagation becomes considerably low, about 30-50%. Consequently planting material production reduces drastically. As such, it is important to overcome the existing shortcomings in patch bud grafting, and to develop efficient alternative propagation techniques of bud and shoot grafting and rooting of cuttings.

Thus, with the objective of developing efficient propagation techniques of stooling, wedge grafting inarching and top working a series of experiments was carried out at the Horticultural Farm, Bandaragama and the FCRDC, Horana during 2003-2005 period.

MATERIALS AND METHODS

Experiment I. Propagation of guava through stooling

The experiment consisted of propagation techniques of stooling, along with air layering (marcotting) as the control. It was arranged in a RCBD having 30 plants per treatment using variety Horana Red with 3 replications at the FCRDC, Horana.

For stooling, underneath shoots (45-60 cm in length) of the mother plants were ringed (0.5-1.0 cm in width) and applied with a rooting hormone containing 3000 ppm NAA (Secto). Then shoots were earthed up using a 15-20 x15-20 cm sized layer of wet coir dust placed around the mother plant. A similar set of shoots was stooled without addition of hormone. Shoots were watered when required. For air layering, shoots of 45-60 cm long were ring barked (0.5-1 cm in width). The ringed portion was wrapped tightly with 300 gauge opaque polythene placing wet coir dust with and without adding the rooting hormone used for stooling. GUAVA PROPAGATIONS TECHNIQUES 241

Ten of each stooled and air-layered shoots were detached from mother trees at 3, 6 and 9 weeks after the treatment and the percentage success was recorded. Then those were established in pots containing a mixture of coir dust, top soil, well-rotted cow dung and sand (1:1:1:1) and were kept in a non-mist propagator for 2 weeks and another week for hardening. The percentage survival was also recorded 3 weeks after each establishment.

Experiment II. Effect of deleafing on success of chip and patch bud grafting techniques

The experiment was carried out in a RCBD with 5 replications having 10 uniformly grown seedlings per treatment at the Horticultural Farm, Bandaragama. Two sets of guava seedlings (var. Pubudu) of 6-8 months old (diameter of 0.5-0.7 cm) for chip budding and 9-12 months old (diameter of 0.8-1.0 cm) for patch bud grafting were used as root-stocks, each consisting of 50 plants. Fifteen days prior to bud grafting, scion sticks of mother plants (var. Pubudu) were deleafed leaving ½ of the leaf stalk and 2 apical leaves intact. Seedlings to be used for patch budding were sprayed with 1 % urea solution at 15 days before grafting for easy separation of stem bark away from wood when budding. Two methods of bud grafting, patch and chip were done. For chip budding, greenish brown colour (partially matured) stem chips (2-2.5 cm in length) each having well-grown bud with chip of wood attached and for patch budding, brown colour (well-matured) easily separable bark patches (1cm x 2cm) each with a well-grown bud were used. Number of bud-take was recorded at 2 months after bud grafting.

Experiment III. Effect of age on root-stock on the success of four techniques of propagation

The experiment was carried out in a RCBD with 3 replications at FCRDC, Horana. Guava (var. Pubudu) seedlings of 6-8 months old (stem diameter of 0.6-0.75 cm) and 9-12 months old (stem diameter 0.8-1.8 cm) and 13-15 months old (stem diameter 1.2-1.8 cm) having 20 uniformly grown plants per treatment were used as root-stocks.

When seedlings of each category were suitable for bud and shoot grafting, techniques of chip, patch, wedge and inarch (in-situ grafting) were done. For chip budding, green colour well grown bud with a piece of wood attached was taken. Two weeks before chip and patch bud grafting, scion shoots were deleafed. One percent urea solution was given as a foliar spray for stock plants at 2 weeks prior to patch budding to facilitate peeling off bark from wood. The scion wood used for chip (2-2.5 cm in length), patch (1 cm x 2 cm) budding and shoot (wedge) grafting (15-20 cm in length) was uniform and extracted from well maintained mother plants of variety Pubudu. Before inarching, stock plants were uprooted and repotted in sealed pots using 242 THANTIRIGE et al. wet coir dust. These were inarched on to 45-60 cm long shoots of well- maintained 3 mother plants. Wedge-grafted plants covered with 100 gauge transparent sealed polythene bags served as single propagators. Ten each of inarched plants were severed from mother plants at 21 days and 45 days after inarching. Then those were repotted in 25 x 30 cm pots and kept in non-mist propagator for 2 weeks. The percentage success was recorded.

Removal of polythene strip on 10 plants of each chip and bud graft and uncovering of wedge grafts were done at 21 and 45 days after bud/shoot grafting. The success of graft take was observed at 2 months after grafting.

Experiment IV. A study on possibility of top working of guava

Ten years old trees with low yielding, poor quality fruits established in FCRDC, Horana were headed back. Out of 10-15 new shoots formed, leaving 5-7 well-grown uniform shoots the rest were cut off. When they were in 0.8-1.0 cm girth, patch budded using deleafed (2 weeks before patch budding) shoots of variety Pubudu. Thirty days after patch budding, bud union was uncovered and percentage bud success was recorded at 2 months after top working. The number of fruits formed on new shoots was also observed.

All data were analyzed by using the SAS package.

RESULTS AND DISCUSSION

Propagation through stooling

Stooling tested along with air (aerial) layering (as the control) indicated with addition of NAA, a success rate of 96% with numerous well- developed roots at 3 weeks after stooling (Table 1). Whereas, air layers treated with NAA emerged with only 1-2 poorly developed roots by 3 weeks. However, by 6 weeks after treatment air layers also showed 60 % success in rooting. In stooling, an ample space of 15-20 x15-20 cm of moist coir dust was provided on the soil surface to develop the initiated root system without having any constraint. Whereas in air layering, only a restricted amount of rooting medium is supplied for the root growth. Furthermore, roots developed in layers receive penetrated sunlight through polythene wrapper whereas stools are deprived of sunlight. These may be causes for delay in root emergence and subsequent root growth in air-layers, while early root emergence and densely grown root system in stools. GUAVA PROPAGATIONS TECHNIQUES 243

NAA growth hormone treated stools formed roots at 3 weeks after the treatment, while untreated stools initiated only a few roots with low number of rooted stools by 6 weeks. Similarly in air layers also NAA increased rooting ability. Both stools and air layers had a survival rate of 100% at 6 weeks after stooling and 9 weeks after layering respectively (Table 1). Therefore, rooting hormone plays an important role in emergence and development of root system.

Table 1. Effect of application of NAA on % success of rooting of stooled and air layered shoots of guava (mean of 10 shoots).

Technique of propagation Time period after propagation (weeks) % success % survival 3 6 9 6 9 12 Air layering (NAA) 0 b 60 b 100 - 100 90 Air layering (untreated) 0 b 50 c 60 - - - Stooling (NAA) 96 a 100 a 100 100 100 80 Stooling (untreated) 0 b 13 d 65 - - - CV % 4.93 8.67 Values followed by the same letter in each column are not significantly different at P=0.05.

Propagation of guava by means of stooling has been investigated with the use of growth regulators such as NAA, IBA and paclobutrazol at various concentrations to improve root formation by several researchers. Chaudharu et al. (1994) revealed that application of 4500 ppm IBA + 400 ppm NAA enhanced rooting percentage (100% after 60 days) and survival percentage (80% after 90 days).

In another study on response of IBA and NAA to rooting of pineapple guava (variety Nikitski) rooting and survival were the highest (72% and 76% respectively) with the application of 1000 IBA in stooling (Pandey, 1996).

Using 2500 ppm paclobutrazol in lanolin paste significantly increased rooting, survival percentage number of primary and secondary roots in stools of 2 guava varieties of guava in Rajasthan, India (Singh, 1998). The highest percentage rooting and the best root development were obtained on the stools treated with 4500 ppm IBA and grown in polythene bag containing a medium of soil, sand and cow dung mixture. Roots were visible 45-60 days after stooling (Bhagat et al., 1998; Dod et al., 1998).

Results of the current experiment are consistent with these findings and showed that shoots dipped in NAA produced well-developed root system in relatively a short time period of 21 days after stooling. Stooled plants were ready to field transplant after another 2 weeks in the non-mist 244 THANTIRIGE et al. propagator and 1 week left in sun light for hardening. Hundred percent survival rate was shown 6 weeks after stooling (Table 1).

In contrast to stooling, air layers with added rooting hormone (NAA) took 9 weeks to detach from the mother plant with a 100% success rate (Table 1). Layers usually take another 2-2.5 months to recover for field planting (Sharma et al., 1991; Tomar et al., 1999). Considering the time factor in comparison to layering, stooling can easily be adopted to propagate guava to hasten the planting material production. It was reported that stooling was standardized for faster multiplication of guava. It can be successfully practised twice a year, getting 30-40 stools/plant/year. This technique has been popularized as a commercial propagation method in India and replaced tedious methods of inarching and air layering which were popular in the recent past (Anon., 1999).

Deleafing and bud graft success of chip and patch methods

Defoliation of scion shoots (leaving only 2 leaves at the apex with ½ of leaf petiole intact) fifteen days prior to bud grafting indicated a significantly higher bud take of both chip and patch methods than that of undefoliated control (Fig. 1). Remaining portion of petiole abscised making bud to swell, by the time of bud grafting. Such swollen buds form better unions and grow readily after grafting. This consequently enhanced the number of successful bud grafts at 2 months after grafting. Thus, deleafing can be adopted to increase bud success in chip and patch methods. Khattak et al. (2002) reported that apex excision of bud stick made better bud sprouting in Pakistan.

90 a a 80

) 70 %

( 60

e

t 50 a r 40 b s s b e 30 c c

u 20 S 10

0 Chipping Patching Bud grafting techniques

Deleafed Undeleafed

Figure 1. Effect of deleafing on success of chip and patch bud grafting techniques Bars with the same letter are not significantly different at p=0.05. GUAVA PROPAGATIONS TECHNIQUES 245

Comparing the conventional patch bud method with the chip method, no significant difference was observed in the bud success when deleafed. Khattak et al. (2002) also showed a bud take of 73% in chip method.

Patch budding is best done when the rind of both stock plant and scion shoot including bud is easily separable from the wood, which largely influences the graft success. In addition, good vegetative growth potential as found in well-maintained parent scion materials and young actively growing root-stocks is an important prerequisite for graft success. Apart from agronomic maintenance practices, ability of peeling off of budding materials is also partially dependent on climatic parameters where parent plants are grown. Therefore, patching cannot be performed when it is required. In comparison to patch budding, chip separation of peel from wood is not required in chip budding since a bud containing the chip is taken along with the piece of wood attached. Similarly removal of a chip portion is done on stock plant. Thus, despite the peeling, quality of budding materials for chipping can be maintained easily.

Age of root-stock and percentage success of bud shoot grafting techniques Chip bud grafting

Chip budding on root-stocks of 3 different age groups indicated that there is a significant difference in bud takes, at 21 and 45 days after grafting (Fig. 2). Out of root-stocks of three ages tested, graft success was found to be the highest (73%) in chip budding of the youngest root -stocks (6- 8 months old) and bud union can be uncovered at 21 days after grafting (Fig. 2). Thus, with the maturity of root-stock increased, the number of successful grafts decreased. Jaffe (1970) also reported that green chip budding can be readily accomplished when tissues are tender.

According to Browse (1992), chip bud grafting can be carried out at any time of the year provided that well matured buds are available and as it provides a greater cambial contact between root-stocks and scion than does shield (patch) method. 246 THANTIRIGE et al.

120 a 100 a a ) a % a a ( 80 a

e b a t

a a r 60 s s e c

c 40

u b a S b 20 b b b c b c b b b b b 0 21 Days 45 Days 21 Days 45 Days 21 Days 45 Days 21 Days 45 Days

Wedging Chipping Patching Inarching Days after grafting

6-8 Months 9-12 Months 13-15 Months

Bars with same letter are not significantly different at p=0.05.

Figure 2. Effect of age of the root-stock on four techniques on the success of propagation of guava.

Patch bud grafting technique

Performance of patch bud method with the age of root-stocks indicated that, a significant increase in percentage success (73%) was observed on moderately mature root-stocks than those of either tender or well matured, at 21 days after grafting (Fig. 2). The reasons for the lowest success rate recorded on tender root-stocks may possibly be the inadequate maturity of stock and scion portions for patch budding. A higher success rate (76%) was obtained when patch budding was done on aged root-stocks (13-15 months old), kept for 45 days, for bud recovery (Fig. 2). Aulakh (1998) also found a bud success of 95% with the use of 12 months old seedlings. Investigation of relative efficiency of patch and forkert budding indicated that patch was more successful and the best time for budding was months of July and August (Gorakh-Singh and Pandey, 1998).

Wedge-shoot grafting

Shoot grafting using wedge method on 3 age types of root- stocks showed a significant difference in graft take at 21 and 45 days after grafting (Fig. 2). The success rate (69%) of graft was recorded on 9-12 months old, moderately aged stock plants at 21 and 45 days after grafting. Whereas, the lowest rate was given on the youngest (6-8 months old) stocks in both time periods (Fig. 2). This suggests that tender shoots used as scion wood for young stock plants might have been subjected to higher moisture GUAVA PROPAGATIONS TECHNIQUES 247 loss resulting in lower number of successful grafts. Wedging performed on the oldest root stocks (13-15 months old) had a higher graft success (83%) when kept for 45 days, showing mature shoots need much time to form union. Previous findings are in support of observations made in this study that there is a stock-scion interaction with the age of stock. Ramiraz et al. (1999) reported that guava can be successfully propagated through cleft and side veneer grafting, which have similarities with wedge grafting technique in the present experiment.

Inarching

Similar nature of graft take was observed in inarching as in the wedge method (Fig. 2). In inarching, a largely grown well-mature twig is grafted, while it is on the parental tree. It generally takes more time period for uniting the inarched union. As such, inarched shoots have shown good success rates (96% and 93%) when inarched onto well-matured (13-15 months old) and moderately aged (9-12 months) stocks at 45 days after inarching. (Fig. 2). However, the minimum success rates were shown on root-stocks on all three ages at 21 days after inarching.

Even though inarching was shown to be a laborious technique, it can be adopted when largely grown plants are required as planting materials. Consequently, it may be possible to obtain early yields on inarched plants.

Top working of guava

Results indicated that top working was successful for guava with 67% bud take. Top worked plants initiated flowers and set fruits (4-10/ plant) by 6-8 months after budding (Table 2). By utilizing the established root structure of the undesirable tree, a new tree can be developed readily and with less effort when compared to replanting a young plant. Findings of Lyannaz (1994) confirmed these results that 95% success rate was achieved on cultivar Beamount and onset of production was earlier than replanting a new plant.

Table 2. Success rate (%) of top working of guava. Tree No. No. of shoots No. of bud No. of fruit patch budded/tree take set / plant 1 7 5 10 2 8 5 8 3 7 4 7 4 6 5 4 Total 20 19 % success 67% 248 THANTIRIGE et al.

CONCLUSIONS

Among several propagation techniques evaluated for guava, stooling was found to be the most promising. In contrast to bud grafting technique, this approach constitutes a rapid multiplication, which saves a considerable amount of time and maintenance cost.

Guava can also be propagated by deleafed chip bud and wedge grafting on root-stocks of 6-11 and 9-15 months old respectively. Chip budding and wedge grafting can be performed as and when required.

Efficiency of patching can also be improved by deleafing scion shoots. Furthermore, results revealed that bud and shoot grafting done on relatively aged (12-15 months) root stocks needed 45 days for satisfactory graft take.

Also, inarching can be adopted effectively to develop well- grown large sized planting materials. In addition, top working can also be applied in the rehabilitation of unwanted guava trees, which provides a fast way of changing tree canopy with a desired variety without replanting.

REFERENCES

Anon., 1999. Propagation and root-stock studies. A report on significant research achievements in fruit crops. Indian Agric. Research Institute:13-15. Aulakh, P.S. 1998. Standardization of patch budding time in guava under rain fed condition in the lower foothills Shiwalik of Punjab. Progressive-Horticulture 30 (3/4): 221-222. Browse, P.C. 1992. Plant propagation Eds. C. Brickell and K.A. Beckett, Michelin House, London: 184-185. Bhagat, B.K., B.P. Jain and C. Singh, 1999. Success and survival of intergene grafts in guava (Psidium guajava L), J. of Research (1): 79-81. Bhagat, B.K., B.P. Jain, C. Singh and B.M. Chowdhary, 1998. Propagation of guava (Psidium guajava L.) by ground layering. J. of Res. Birsa Agric. University, India 10(2): 209-210. Chaudharu, S.M., U.T. Desai and P.N. Kale. 1994. Effect of growth regulators on stooling in guava under semi arid conditions, J. of Maharashtra Agric. University 19(3): 458-459. Dod, V.N., S.G. Bharad, B.J. Jadhas, P.S. Joshi, P.M. Kulkarni. 1998. Seasonal variation in rooting of ground layers by polybag method in guava (Psidium guajava L.) J. of Soils and Crops 8 (2): 182-184. Gorakh- Singh and D. Pandey. 1998. Effect of time and method of budding on propagation of guava (Psidium guajava L.). Annals of Agric. Research 19(4): 445-448. Jaffe, A. 1970. Chip grafting guava cultivars. Plant Propagator 16 (2): 6. GUAVA PROPAGATIONS TECHNIQUES 249

Khattak, M.S., M.N. Malik, M.A. Khan, 2002. Guava propagation through chip budding. Pakistan J. of Agric. Res. 17(2): 178-181. Lyannaz, J.P. 1994. On site top working of guava trees, Fruits-Paris 49 (5/6):448-449. Pandey, G. 1996. Response of IBA and NAA on rooting of Pineapple guava. Advances in Horti. and Forestry 5:75-79. Ramiraz, M., A. Urdaneta and M. Marin. 1999. Grafting and cuttings from guava under very dry tropical forest. J. of Revista. 16: 36-42. Saroj, P.L. and R.Y. Pathak. 1995. Role of nurse shoot in propagation of guava through stool layering. Indian-Forester 121 (12): 1172-1173. Sharma, R.S., T.R. Sharma and R.C. Sharma. 1991. Influence of growth regulators and time of operation on root age of air layering in guava (Psisdium guajava Linn.) cv. Allahabad safeda, Orissa J. of Horti. 19 (1-2): 41-45. Singh, D.K. 1998. Regeneration of guava (Psidium guajava L.) cultivars by stoolings with the aid of paclobutrazol. Annals of Agric Research 19 (3): 317-320. Tomar, K.S., B.S. Gurjar and R.S.S. Tomar. 1999. Responses of different concentrations of IBA and NAA on root age and growth of air layers of guava. (Psidium guajava Linn.) cultivar Gwarlio- 27, Advances-in-Plant Sciences 12 (2): 535-538. Annals of Sri Lanka Department of Agriculture. 2006.8:251-262.

COMPARISON OF GROWTH AND YIELD PERFORMANCES OF TOMATO (Lycopersicon esculentum mill.) VARIETIES UNDER CONTROLLED ENVIRONMENT CONDITIONS

W.M.K.B. WAHUNDENIYA1, R. RAMANAN1, C. WICKRAMATUNGA1 and W.A.P. WEERAKKODI2 1Horticultural Crop Research and Development Institute, Gannoruwa, Peradeniya 2Faculty of Agriculture, University of Peradeniya.

ABSTRACT

Controlled Environment Agriculture (CEA) is one of the modern techniques, which has the potential to meet the challenges faced by tomato producers in the country. Presently numbers of imported tomato varieties including hybrids are available in the market and seeds of these varieties are very expensive. Greenhouse vegetable growers use these varieties without having a proper knowledge on the performances under controlled environment conditions. Therefore, there is an urgent need to evaluate these tomato varieties under controlled environment conditions. This study was conducted to evaluate the performance of the tomato varieties under controlled environment conditions and to select promising varieties under CEA conditions at HORDI, Gannoruwa. Fifteen tomato varieties in three-replicates were evaluated. These varieties were grown hydroponically using Albert’s solution for fertigation under drip irrigated grow bag culture using coir dust and partially burnt paddy husk as the medium. Vegetative, reproductive and quality characters were recorded and were analyzed. According to the results, measured growth parameters were significantly different. The varieties Big beef, Ninja, Volcano and Alambra F1 performed well and produced higher yields. These varieties had more or less the same yield and quality characters and showed indeterminate growth habit.

KEYWORDS: Controlled environment, Hybrid varieties, Hydroponics.

INTRODUCTION

Vegetable production is highly seasonal and mostly weather dependent in Sri Lanka and the extents available have been fully exploited. As a result there is a glut during favorable seasons and scarcity during unfavorable seasons. This has created unrealistic high prices of these commodities in unfavorable seasons. Meanwhile the expanding export demand for quality vegetables is another aspect to be considered.

There is a great potential in Sri Lanka to adopt CEA for high value vegetables. According to the available records, total land area in Sri Lanka under CEA is approximately 10 ha. Most of these lands belong to farmers with smallholdings and vegetables cultivated are tomato, bell pepper, salad cucumber (Weerakkody et al., 2001). 252 WAHUNDENIYA et al.

By adopting several techniques independently or in combination with each other, it is possible to bring about off-season production of vegetables under controlled environment conditions with the use of new vegetable varieties and improved technologies. The concept of CEA was developed to minimize the influence of undesirable environmental conditions on crop production, and it provides new alternatives and economic opportunities in vegetable production.

Tomato is one of the most popular and market potential vegetables grown under CEA in Sri Lanka. In 2002 the total production of tomato was 44,974 t and extent of cultivation was 5,936 ha with an average yield of 7.58 t/ha (Department of Census and Statistics, 2003). According to the customs reports, in 2003 the country had imported 1.24 t and exported 1.54 t. Sri Lanka imports annually about 488 t processed products of tomato worth of 52 million rupees.

The consumer demand is for high quality, moderate sized, red coloured tomato with high firm fruit, good appearance and good taste. Growers prefer high yielding, higher weight, indeterminate growth habit, and resistance to pest and disease. The solution to these requirements is to find suitable varieties for growing under CEA.

Presently large numbers of imported tomato varieties including hybrids are available in the market, and seeds of these varieties are sold at exorbitant prices. Greenhouse vegetable growers use these varieties without knowing the performance of these under CEA. Therefore, the objectives of this research were to evaluate the growth and yield performance of different tomato varieties including hybrids under CEA conditions and to select promising varieties for CEA cultivation at commercial level. The findings will help the grower to overcome the problems inherent to varieties and at the same time achieve increased yields with high quality, typical to this system of agriculture.

MATERIALS AND METHODS

The research was conducted at Horticultural Crops Research and Development Institute (HORDI), Gannoruwa under controlled environment conditions. Fifteen tomato varieties namely N-83, HRD-2, N-17, N-139, N-162, Lapsigede, Ninja, Volcano, Bigbeef, Redboy, Heatmaster, Makis, Credo, Alambra F1, Thilina were included in the experiment. The experimental design was Randomized Complete Block Design and replicated three times. The plot size was four plants. GROWTH AND YIELD PERFORMANCES OF TOMATO VARIETIES 253

Nursery management

The nursery was established in styrofoam seedling trays inside the plant house. Seeds treated with Thiram were sown. Application of Albert’s solution at the concentration of 1g/l commenced when seedlings were at two true leaf stage. Vigorously grown seedlings were transplanted maintaining one seedling in each bag when they were four weeks old.

Media preparation and filling of grow bags.

Coir dust and partially- burnt paddy husk mixed at the ratio of 2:1 were used as growing media and were filled into black polyethylene bags (30cm x 35cm). Before filling the bags, holes with 10mm diameter were punched at the lower one-third of the bag to facilitate drainage. The fungicide Homai was applied to the media in the bags and kept for two days before transplanting the seedling. Silver colour polyethylene was laid as a mulch to prevent emergence of weeds and soil born diseases.

Crop management

Plants were arranged in a double row system and space between two double rows was 60 cm and within a double row was 45cm. Plants were arranged 45cm apart in each row to give 0.2025 m2 of space per plant.

Fertilizer application was done through drip irrigation. Following concentrations and volumes of Albert’s solution were applied depending on the growth stage of tomato.

Table 1. Concentration and volume of fertilizer applied during the crop cycle.

Growth stages Concentration of Volume (ml) Albert’s solution (g/l) Seedling 2 100 Growing 2 200-250 Flowering 3 200-250 Bearing 1* 4 250 Bearing 2** 4 400 * Have two clusters ** More than two clusters

The pH of the nutrient solution was maintained between 5.8-6.5 while Electrical Conductivity (EC) ranged between 1.5-2.5 dS/mm or 3.25-4.5 mS/cm. Required amount of water was given as split application through drip 254 WAHUNDENIYA et al. irrigation and fertigation. The drip irrigation system comprised of arrow drippers. The flow rate of a dripper was 1 l/hr. Plant training was started 2 weeks after transplanting. Plants were trained as a single stem by removing suckers when they were about 5 cm high during early in the day at 3-5 days intervals. Branches were removed by using a disinfected pair of scissors. Three weeks after transplanting staking was done to support the main stem. Then the single stem was trained vertically using a string fixed to the overhead wires. The strings were fixed to the bottom of each branch with the help of a piece of 2cm PVC pipe ring.

Evaluation of plant growth and yield parameters

Vegetative growth, and yield components of tomato were evaluated. Vegetative growth parameters measured included plant height, cumulative leaf number, internode length and growth habit. Reproductive growth parameters, measured were number of inflorescences per plant, number of flowers per cluster, number of fruits per truss, days taken for 50% flowering and fruit maturity and stigma position in relation to anthers at full anthesis, Total yield, marketable yield and total number of fruits harvested per plant were also recorded.

Fruit quality parameters

Fruit quality parameters were studied using fully ripe fruits at second harvest. Basically average fruit weight (g), fruit width (cm) and fruit length (cm) were recorded. Then pericarp thickness (mm) from equatorial section of the fruit was also determined. Skin colour of ripe fruit, firmness of ripe fruit (Firmness was measured by pericarp thickness and locule number), number of fruits having blossom-end-rot (BER) and radial cracking were also recorded. External colour of ripe fruit, pH of raw juice squeezed from ripe fruit, total soluble solids (brix value), citric acid percentage, moisture content and number of seeds per fruit were also recorded.

RESULTS AND DISCUSSION Plant height

Plant height differed significantly among the treatments at maturity stage; the lowest plant height was recorded in the variety, Redboy (Table 1). The highest plant height was observed with variety, Alambra F1, but it was not significantly different from the varieties, Volcano, Big beef and Ninja. Up to 3 weeks after transplanting, uniform stand was observed and that could be an after effect of the proper nursery management such as low root damage at transplanting. GROWTH AND YIELD PERFORMANCES OF TOMATO VARIETIES 255

Cumulative leaf number

The highest number of leaves was observed in the variety, Lapsigede (Table 1). The lowest number of leaves were recorded in the variety, Redboy and it was not significantly different from the varieties N-139, N-83, N-17, N-162, Makis, Heatmaster and Credo. As reported by Kinet and Peet (1986) if only a few leaves form before floral initiation, assimilate supply may be insufficient to support flower and fruit development (Kinet and Peet, (1986). At the stage of 50% flowering, Thilina had the highest number of leaves per plant (16), and Makis had the lowest number of leaves per plant (11).

Internode length

Treatment difference in terms of internode length was significant (Table 2). Variety, N-139 had the highest internode length while variety, HRD-2 had the lowest internode length when compared with the other varieties. High internode length increases the plant height and as a result crop management may be difficult. Mutual shading of leaves due to shorter internode length may reduce the net assimilation rate.

Table 2. Variation of vegetative growth parameters of varieties.

Variety Plant height Cumulative leaf Internode Growth habit at maturity number at length (cm) (cm) maturity (cm) N-83 113 cde 21de 8 ab Indeterminate N-17 101 def 21de 6 c Indeterminate N-162 120 b 24de 7 bc Indeterminate Lapsigede 139 b 30a 4 de Indeterminate Ninja 173 a 29ab 6 dc Indeterminate Volcano 178 a 28ab 6 c Indeterminate Bigbeef 171 a 28ab 6 c Indeterminate Heatmaster 101 def 20e 7 bc Indeterminate Alambra 185 a 28ab 7 bc Indeterminate F1 HRD-2 97 ef 27abc 4 e Determinate N-139 95 ef 18e 8 a Determinate Redboy 93 f 18e 6 c Determinate Makis 117 cd 20de 6 c Determinate Credo 112 cde 19e 6 cd Determinate Thilina 146 b 25bc 6 c Semi determinate 256 WAHUNDENIYA et al.

CV 7 8 10 - Pr>F 0 0 0 - Growth habit

Five varieties among 15 varieties were observed as determinate type, one was semi determinate and the others were indeterminate (Table 2). In case of indeterminate type, only two or three inflorescences, separated by one or more leaves, are continuously initiated at leaf intervals varying with varieties. Indeterminate tomato’s vegetative growth and reproductive development are thus proceeding concomitantly during the greatest part of plant life. A strong competition between developing leaves and apical meristems influences both the earliness of harvest and the total yield (Hussey, 1963).

Number of inflorescences per plant

Table 3 shows mean inflorescence number at maturity. Number of inflorescences at maturity varied from 4-10 among varieties and the difference was significant. N-162 had the highest number (10) and Heatmaster, Redboy and Credo (5) had the lowest number.

Table 3. Variation of reproductive growth parameters among treatments.

Variety Number of Number of flowers / Number of inflorescences / plant cluster fruits / truss N-83 9a 8bcde 6def N-17 7bcd 8bcde 7ab N-162 10a 9bc 6def Lapsigede 7bcd 10ab 7ab Ninja 6cde 8bcde 5g Volcano 6cde 9bc 6bcde Bigbeef 6e 7f 5g Heatmaster 5e 7ef 5g Alambra F1 6cde 7ef 6def HRD-2 8bc 7def 6def N-139 7bcd 8bcde 6def Redboy 5e 7ef 6bcde Makis 6cde 9bc 8a Credo 5e 9bc 7ab Thilina 6cde 9bc 7ab

CV 15 11 12 Pr>F 0 0 0 Means followed by the same latter are not significantly different at P=0.05 GROWTH AND YIELD PERFORMANCES OF TOMATO VARIETIES 257

Number of fruits per truss

Makis gave the highest number of fruits per cluster and Ninja gave the lowest number of fruits. The failure of flowers to produce fruits may occur in both determinate and indeterminate cultivars, becoming a limiting factor for the yield (Table 3). It is advisable to maintain a range of 4-6 fruits per cluster to reduce the variation of average fruit weight. Number of fruits per truss also affects fruit shape and size (Sharma, 2002).

Time of 50% flowering

Twelve varieties started flowering at three weeks after transplanting. But varieties Heatmaster and Credo took 4 weeks from transplanting. Significant differences were observed among the fifteen tested varieties. The check variety Thilina took the longest period to flower (35 days after transplanting) and varieties N –83, N-17, N-139 and Redboy took the shortest period of 30 days after transplanting. Longer the time taken for flowering, broader the uneconomic period of the crop, but produce much healthier plants that could prolong the bearing period.

Time of maturity

Number of days taken to maturity is significantly different. Varieties N-17 and N-139 took the shortest period for maturity (7 weeks) from transplanting, and variety Thilina had taken the longest period to maturity (10 WATP). Most of the varieties had taken only 8 weeks to mature from transplanting.

Yield parameters

Table 4 gives the yield variation among the varieties. Total fruit yield was significantly low in variety Lapsigede and it could be due to the lowest average fruit weight. The highest yield (2.66 kg/plant) was given by variety Big beef. Except varieties Ninja and Volcano, all the other varieties gave yields less than 2 kg/plant.

The data clearly showed that there were significant differences in marketable yield among varieties. Significantly higher marketable yield was obtained with Bigbeef (2.28 kg/plant) which is an indeterminate type variety and yield could be obtained for a long period. Varieties Heatmaster, Credo, Redboy, Almbra Fl, Ninja, N-139, N-17 and HRD-2 gave considerably higher marketable yields. The variety Lapsigede was inferior in yield to the check variety Thilina and this could be due to small fruit size. 258 WAHUNDENIYA et al.

Variety Heatmaster and Ninja gave the lowest number of fruits per plant (16) while Lapsigede gave the highest number of fruits per plant (57). However, its average fruit weight was very low and as a result it gave only about 1 kg per plant.

Table 4. Yield parameters of tomato varieties.

Variety Total fruit yield per Marketable yield Total number of plant (Kg) per plant (kg) fruits per plant N-83 1de * 1cd 23def N-17 1cde 1cd 31cd N-162 1cde 1cd 43b Lapsigede 1e 1d 57a Ninja 2bc 1cd 16f Volcano 2ab 2ab 26de Bigbeef 2a 2a 21ef Heatmaster 1cde 1cd 16f Alambra F! 1bed 1abc 23def HRD-2 1cde 1cd 38bc N-139 1cde 1cd 29cde Redboy 1cd 1bc 21ef Makis 1de 1cd 29cde Credo 1bed 1cd 27de Thilina 1bed 1cd 24def CV 18 20 17 Pr>F 0 0 0 * Means with same letter are not significantly different.

Average fruit weight and size

Bigbeef had the highest fruit weight and it was not significantly different from Ninja. Maximum average fruit length of 10.84 cm and maximum fruit width of 6.96 cm were found in variety Ninja. Except those two varieties Volcano, Heatmaster and Alambra FI produced fruits with more than 100g. The lowest average fruit weight was observed with variety Lapsigede (20g) and it was more or less similar to cherry type. Fruit size was less important for processing purpose, but it was important for table purpose. According to Resh (2003), fruit size determines the consumer preference.

Quality evaluation of fruits

Brix value

Total soluble solids, which was composed of all fruit components except water and volatile compounds was estimated by refractive index and reported as Brix. In this study it varied from 4.0-6.2, Variety Lapsigede had the highest total soluble solids content (Brix=6.2), which is a cherry type GROWTH AND YIELD PERFORMANCES OF TOMATO VARIETIES 259 tomato. The varieties Thilina, Redboy and Alambra Fl 1 had the lowest total soluble solid content (Brix=4) (Table 4). Hence Brix value appeased to be a determinant of varieties for processing industries. Moisture percentage of fruits varied from 88.3 - 93.4

Table 5. Chemical composition of fruits.

Variety Moisture pH of juice Brix value Percentage of Sugar: acid content (%) citric acid ratio N-83 89 4 5 0.94 5.53 N-17 90 4 6 0.79 7.59 N-162 90 4 4 0.97 4.64 Lapsigede 91 4 6 0.83 7.47 Ninja 89 4 5 0.80 6.25 Volcano 93 4 4 0.92 4.46 Bigbeef 91 4 4 0.89 5.17 Heatmaster 92 4 4 0.82 5.24 Alambra Fl 93 4 4 0.93 4.30 HRD-2 89 4 4 0.98 4.90 N-139 90 4 4 0.95 4.74 Redboy 92 4 4 0.78 5.13

Makis 90 4 5 0.83 6.02 Credo 88 4 5 0.97 5.15 Thilina 93 4 4 0.93 4.30

Acidity percentage

The acidity percentage as citric acid of tested varieties ranged from 0.78-0.98 and the variation in pH was from 4.1-4.4. The variety HRD-2 had a citric acid percentage of 0.98 and pH of 4.4. The fruit pH affects the time required for heating to achieve sterilization of processed commodity. Longer time is required as the product pH increases. Therefore, pH values above 4.5 are generally unacceptable for processing purposes. The varieties Volcano, Alambra Fl, Thilina and Credo had a pH of 4.3 which is acceptable for processing purpose and all tested varieties recorded pH of < 4.5 (Table 5).

Number of locules per fruit 260 WAHUNDENIYA et al.

Ninja and Heatmaster had the highest locule number (6) but Thilina, Makis and Lapsigede had the lowest number (2) (Table 5). Higher locule number increases fruit firmness (Sharma, 2002). Pericarp thickness

Significantly higher thickness was found in Alambra Fl and the lowest thickness in Lapsigede, which was not significantly different from Heatmaster. N-83, HRD-2, N-17, N-139 and N-162 had pericarp thickness ranging from 5.58 - 5.92 mm. Varieties Alambra Fl, Credo, Makis, Redboy, Bigbeef and Volcano had the pericarp thickened 7 mm (Table 5). Thick pericarp is a useful character as far as post-harvest handling is concerned in transportation. So, it could be an added advantage to have a thicker pericarp when selecting tomato varieties.

Firmness of ripen fruits

Heatmaster had soft fruits. N-83, N-139 and Ninja varieties had fruits of medium firmness. All the other varieties gave solid fruits (Table 6). Most consumers prefer firm fruits. Sharma (2002) reported firmness of fruit as indispensable for transportation and it adds to shelf life. The salad making quality of the fruit is determined by variety firmness and it is also related to high pericarp thickness: locule ratio.

Table 6. Variation of quality characters.

Variety Locules Pericarp Ratio of pericap Firmness number thickness (mm) thickness / locule No. N-83 4 5 ef 1 Medium N-17 3 5 ef 1 Firm N-162 3 5 ef 1 Firm Lapsigede 2 5 f 2 Firm Medium Ninja 6 6de 1 Firm Volcano 3 7 243 - Bigbeef 5 8 ab 1 Firm Heatmaster 6 5 f 0 Soft Alambra Fl 4 8 a 2 Firm HRD-2 3 5 ef 1 Firm N-139 4 5 ef 1 Medium Redboy 3 8 ab 2 Firm Makis 2 7 cd 3 Firm Credo 3 7 be 2 Firm Thilina 2 6 cd 3 Firm

CV - 7 - „ Pr>F - 0 - - GROWTH AND YIELD PERFORMANCES OF TOMATO VARIETIES 261

Means with the same letter are not significantly different at P=0.05

Correlation between yield, vegetative and reproductive growth parameters

Significant correlation was found between yield and plant height (r = 0.52), cumulative leaf numbers and marketable yield (r = 0.31). Marketable yield was negatively correlated with number of flowers per cluster, number of fruits per truss (r = -0.39), indicating that the low number of fruits per truss had given high yields.

However, no significant correlation was found between internode length and inflorescence number per plant. Significant correlation was found between average fruit weight and average pericarp thickness (r= 0.48).

Physiological disorders

Radial cracking

Fruit cracking of the tested varieties ranged within 0-8%. A significant difference among the varieties in terms of crack resistance was observed. HRD-2, N-162 and Alambra Fl were resistant to cracking. Level of cracking was the lowest in varieties Thilina, N-83, N-139 and Makis. The highest cracking percentage was observed in Ninja, Bigbeef and Lapsigede. Peet (1992) considered characteristics of radial crack susceptible cultivars large fruit size, low skin strength, thin skin, thin pericarp, shallow cutin penetration and fruits not shaded by foliage. The problems are most often seen in the large, multilocular (Beefsteak type) cultivars grown by greenhouse tomato growers and growers of other types. Here, Volcano, Ninja and Bigbeef had the large size, multi locular and few fruits in plants. But, Lapsigede had small fruits and high number per plant. This variety had low pericarp thickness.

Blossom-end-rot (BER) of the fruits

None of the tested varieties showed the resistance to BER. Credo recorded the highest percentage of BER and Thilina Alambra F1, Makis, Redboy, N-162, and HRD-2 showed the lowest percentage. External factors affecting BER are water availability and relative humidity. Genetic factors that influence the susceptibility of individual cultivars include large fruit size, a rapid rate of cell enlargement and perhaps the inability of the vascular system to transport calcium to the distal end of the fruit (Ho et al., 1993). 262 WAHUNDENIYA et al.

In this experiment Bigbeef, Ninja and Alambra F1 had the highest percentage of BER due to above reasons. Adams and Ho (1993) reported low day time humidity, especially when accompanied by high temperature and high light, increases the transpiration so proportionately more calcium goes to the leaves than fruit.

CONCLUSIONS

Indeterminate type varieties Ninja, Volcano and Bigbeef, can be recommended for controlled environment conditions based on growth, yield and fruit quality characteristics. However, Bigbeef, Ninja, and Volcano are more susceptible to fruit cracking and blossom end rot than the other varieties. Cherry type tomato variety Lapsigede had the highest total soluble solid content and it is more suitable for processing industry.

REFERENCES

Adams, P. and L.C. Ho.1993. Effects of environment on the uptake and distribution of calcium in tomato and on the incidence of blossom-end-rot. Plant and Soil 154:127-132. Department of Census and Statistics. 2003. Annual Report. Colombo, Sri Lanka. Ho, L. C., R. Belda, M. Brown, J. Andrews and P. Adoms. 1993. Uptake and transport of calcium and the possible causes of blossom-end-rot in tomato. J. Exp. Bot. 44:50 9-18. Hussey, G. 1963. Growth and development in the young tomato. II. The effect of defoliation on the development of the short apex. J. Exp. Bot. 14:26-33. Kinet, J.M. and M.M. Peet. 1986. Tomato. In Wien, H.C. (Ed), Physiology of vegetables crop, CAM Publication. London, Pp. 204-247. Peet, M.M. 1992. Fruit cracking in tomato. Hort. Technology 2:216-223. Resh, H. M. 2003. Hydroponic food production. 6th edition. Wood Bridge Press Publishing Company, California. Sharma, J.P. 2002. Crop production technology for cold arid region. Kalyani Publishers. New Delhi. Pp. 124-135. Weerakkody, W.A.P., L.H.P. Gunarathne and B.C.N. Peiris. 2001. Controlled Environmental Agriculture (CEA) in Uva Province in Sri Lanka. Final Year Project Report, Faculty of Agriculture, University of Peradeniya, Peradeniya. Annals of Sri Lanka Department of Agriculture. 2006.8:263-272.

EFFECT OF CYTOKININS AND BRASSINOSTEROID WITH GIBBERELLIC ACID ON YIELD AND QUALITY OF THOMPSON SEEDLESS GRAPES

A.J. WARUSAVITHARANA1 and T.B. TAMBE2 1Fruit Crop Research and Development Centre, Kananwila, Horana 2All India Co-ordinated Research Project on Grapes, Mahatma Phule Agriculture University, Maharashtra, India.

ABSTRACT

The effects of Cytokinins and Brassinosteroid (BR) with Gibberellic Acid (GA3) on yield and quality attributes were investigated in the Department of Horticulture, Mahatma Phule Agriculture University, Maharashtra, India during 2004- 2005. The experiment was laid out in Randomized Block Design (RBD) with 3 replications and 10 treatments. After October pruning (forward pruning) vines and bunches at specific stage of development were treated with respective plant growth regulators as per the treatments. i.e. GA3, BR, N-( 2-chloro-4 pyrydyl) N- phenyl urea (CPPU) and Benzyladenine (BA). Application of GA3 at 10 ppm at pre-bloom stage, 15 ppm at initiation of flowering, 25 ppm at 3 to 4 mm berry diameter stage in combination with 1 ppm BR, BA at 10 ppm twice at 4 leaf and berry setting stage, respectively and last application of GA3 at 25 ppm at 7 to 8 mm berry diameter stage was found better for maximum berry diameter (19.60 mm), berry length (21.58 mm), berry weight (3.36 g), number of berries per bunch (131.78), number of bunches per vine (15.41), bunch weight (447.85 g) and yield (6.79 kg/vine, 15.09 MT/ha) with attractive grayish yellow colour berries. This treatment (T 10 ) also produced berries with considerable amount of Total Soluble Solid (TSS) (22.41 0B) and acidity (0.59 %) at the time of harvesting. The highest TSS (23.43 0B), lowest acidity (0.53 %), highest TSS : acidity ratio (44.31) and minimum days to harvest (132 days) were recorded in vines under control. The application of GA3 in combination with BR and BA was found to be effective for cell elongation and cell division, which led to increase berry size, yield and quality of Thompson Seedless grapes.

KEYWORDS: Benzyladenine, Brassinosteroid, CPPU, GA3, Grape, Quality.

INTRODUCTION

Grape (Vitis vinifera L.) is a widely grown fruit crop in the world. It is cultivated under temperate, sub-tropical as well as tropical climates of the world, over an area of 7,674,237 ha with a production of about 65,486 Lakh t (FAO, 2004). Considering the demands for grapes in both the national and international markets, it is obvious that good quality grape will fetch a more premium price in the market. High production of better quality grapes depends on various horticulture practices, cultural practices and plant growth regulators. Among the different growth regulators GA3, BA, CPPU and BR play a significant role in increasing the productivity and improving the quality of grapes. Gibberellic Acid (GA3) stimulates both cell division and cell elongation (Randhawa, 1971). Cytokinins or cytokinin like substances, which promote cell division and increase cell expansion along the lateral axis, can 8 WARUSAVITHARANA AND TAMBE increase the berry size (Krishnamoorthy, 1981). Bassinosteroids are a new group of plant hormones with a regulatory function in cell elongation and cell division (Clouse and Sasse, 1998). At present, table grape growers tend to increase the dose of plant growth regulators and repetitions of application to increase the berry size, which may disturb the berry quality. Therefore, stage of application and concentration of these plant regulators are to be standardized for better yield and quality. Hence present investigation was undertaken with the objectives of studying the effect of cytokinins and brassinosteroid with GA3 on yield and quality of grapes and to standardize the application of cytokinins and BR with GA3 in grapes.

MATERIALS AND METHODS

The present investigation was conducted in the Department of Horticulture, Mahatma Phule Agriculture University, Maharashtra, India, during 2004 - 2005. Three years old Thompson Seedless vineyard, spaced at 3.0 x 1.5 m and trained on bower system was used for this study. The experiment was laid out in randomized block design with three replications and ten treatments given in Table 1. Three experimental vines were maintained under each treatment with guard rows on each side. Plant growth regulators were sprayed, at the time of 4 leaf stage, pre bloom stage and initiation of flowering, while in case of 50 per cent cap fall and later stage of cluster development, clusters were dipped in the solution of respective plant growth regulators, as per treatments. Cultural operations were performed in accordance with standard commercial production practices for table grapes in this region. The observations on diameter, length, weight of berry, number of berries per bunch, number of bunches per vine, bunch weight, yield and quality parameters were recorded during the investigation. The TSS content was determined by using the refractometer and acidity was measured by titrating with 0.1 N NaOH. Statistical analysis was done by using methods as suggested by Panse and Sukhatme (1985).

RESULTS AND DISCUSSION

Effect on berry attributes

The application of cytokinins and BR with GA3 had significant influence on diameter, length and weight of berry and number of berries per bunch (Table 2). The highest diameter of berry (19.60 mm), length of berry (21.58mm) and weight of berry (3.36g) were recorded in treatment with application GA3 at 10 ppm at pre-bloom stage, 15 ppm at initiation of flowering, 25 ppm at 3 to 4 mm berry diameter stage in combination with 1 ppm BR, 25 ppm GA3 at 7 to 8 mm berry diameter stage, and BA at 10 ppm twice at 4 leaf and berry setting stage, (T10) than all the other treatments. It was followed by application of GA3 at 10 ppm at pre-bloom stage, 15 ppm PLANT GROWTH REGULATORS ON GRAPES 9 at initiation of flowering, 25 ppm at 3 to 4 mm berry diameter stage in combination with 1.5 ppm CPPU, 25 ppm at 7 to 8 mm berry diameter stage, and BA at 10 ppm twice at 4 leaf and berry setting stage (T7). This treatment recorded 19.03 mm of berry diameter, 21.00 mm of berry length, and 3.28 g of berry weight. GA3 was the only treatment that had little effect on berry size and weight of berry.

Table 1. The details of treatments.

Trt. No. Plant Stage of application and concentrations (ppm)* growth 4 PB IF 50 BS Berry Berry regulator LS % diameter diameter s CF (3 to (7 to 4mm) 8mm)

T1 (Control) GA3 - 10 15 25 25 - 40

T2 GA3 - 10 15 25 25 - 40 BA 10 ------

T3 GA3 - 10 15 25 25 - 40 BA - - - - 10 - -

T4 GA3 - 10 15 25 25 - - BA 10 - - - 10 - -

T5 GA3 - 10 15 - - 25 25 CPPU - - - - - 2 -

T6 GA3 - 10 15 - - 25 25 CPPU ------2

T7 GA3 - 10 15 - - 25 25

BA 10 - - - 10 - - CPPU - - - - - 1.5 -

T8 GA3 - 10 15 - - 25 25

BR - - - - - 1 -

T9 GA3 - 10 15 - - 25 25

BR ------1

T10 GA3 - 10 15 - - 25 25

BA 10 - - - 10 - - BR - - - - - 1 - * 4 LS = Four leaf PB = Pre-bloom IF = Initiation of flowering CF = Cap fall BS = Berry setting 10 WARUSAVITHARANA AND TAMBE

The increase of berry size was due to the plant growth regulators applied in treatment T10 viz., GA3, BA and BR which were responsible for cell division and cell elongation. These effects of GA3, BA and BR were reported by Randhawa (1971), Tambe (2001) and Mitchell et al. (1970), respectively. Enlargement of berry size leads to increase in berry weight. Furthermore, increment in invertase activity and hexose content by GA3 (Francisco and Gomez, 2000), induction of protein synthesis by cytokinins (Coombe, 1976), activation of protein synthesis (Vladimir et al., 2000 and Sivakumar et al., 2002), nucleic acid synthesis (Vladimir et al., 2000) and translocation of sugar from source to sink (Sivakumar et al., 2002) by application of BRs may be responsible for the increase of size and berry weight. These results indicate that the combined application of GA3 with BA and BR have synergistic effect on berry growth. Similar work was also reported by Shikhamany (2001) and Tambe (2002).

In the present study, treatment T10 recorded significantly highest number of berries per bunch (131.78) over rest of the treatments. The increased number of berries might be due to increased fruit set and decreased fruit drop. Effects of combined application of GA3 with BA, CPPU and BR on higher berry setting were also confirmed by several authors (Liu, 2002; Stephen et al., 2003 and Vladimir et al., 2000).

Effect on yield

The data presented in Table 2 revealed that the yield attributes i.e. number of bunches per vine, weight of bunch and yield were significantly affected by the application of cytokinins and BR with GA3.

In the present investigation the highest number of bunches per vine (15.41) and maximum yield (6.79 kg/vine; 15.09 t/ha) were recorded in treatment T10 i.e. application GA3 at 10 ppm at pre bloom stage, 15 ppm at initiation of flowering, 25 ppm at 3 to 4 mm berry diameter stage in combination with 1 ppm brassinosteroid, 25 ppm at 7 to 8 mm berry diameter stage, and BA at 10 ppm twice at 4 leaf and berry setting stage, respectively. It was at par with treatment T7, which recorded 15.36 bunches per vine and yield of 6.57 kg/vine (14.60 t/ha). The weight of bunch (447.85 g) was found to be significantly high in the treatment T10 over rest of the treatments. Bearing increase in treatment T10 may be due to maximum flowering, higher fruit set, carbohydrate accumulation and maximum leaf area.

More or less similar findings were reported by Tambe (2002). The reason for higher fruit set was same as for number of berries per bunch and this is in conformity with results reported by Liu (2002), Stephen et al. (2003) and Vladmir et al. (2000). PLANT GROWTH REGULATORS ON GRAPES 11

Table 2. Effect of cytokinins and brassinosteroid with GA3 on berry, yield and

quality attributes of Thompson seedless grapes.

Treatment Diameter Length Weight Number of Number of Weight Yield Yield TSS Acidity TSS: Days to No. of berry of berry of berry berries per bunches of bunch per vine per ha (0 B) (%) acidity harvest (mm) (mm) (g) bunch per vine (g) (kg) (MT) ratio

T1 15.90a 17.69a 2.64a 100.38a 12.78a 285.99a 3.68a 8.18a 23.43g 0.53a 44.31f 132.00a

T2 16.17b 17.88b 2.67a 103.24ab 12.83ab 295.90b 3.80ab 8.44ab 22.87e 0.57bc 40.14C 134.33d

T3 17.43e 19.45f 3.03d 117.36d 14.30e 368.50f 5.23e 11.62e 22.54d 0.57bc 39.59b 136.11e

T4 16.58c 18.38c 2.76b 107.46bc 13.11bc 315.58c 4.16bc 9.24bc 22.38c 0.60de 37.41b 139.21g

T5 18.21g 20.21h 3.16e 122.65e 15.10fg 405.49h 6.10f 13.55f 21.58b 0.63f 34.29a 142.30i

T6 16.90d 18.89d 2.87c 110.92c 13.40c 337.25d 4.53cd 10.07cd 21.69b 0.62ef 35.13a 140.67h

T7 19.03h 21.00i 3.28f 127.90f 15.36gh 428.73i 6.57g 14.60g 21.34a 0.63f 34.01a 144.20j

T8 17.95f 19.99g 3.13e 120.15de 14.98f 391.50g 5.79f 12.87f 23.39g 0.55ab 42.67e 132.67b T 16.98d 18.98e 2.91c 112.10c 13.78d 348.27e 4.80d 23.12f 0.56b 41.39d 133.31c 9 10.67d

T10 19.60i 21.58j 3.36g 131.78f 15.41h 447.85j 6.79g 15.09g 22.41c 0.59cd 38.17b 137.20f SE ± 0.061 0.028 0.014 1.716 0.101 0.569 0.136 0.302 0.042 0.007 0.412 0.113 CD at 5% 0.180 0.084 0.040 5.096 0.299 1.691 0.404 0.896 0.126 0.022 1.222 0.335

Means with same letter are statistically not significant at P=0.05 12 WARUSAVITHARANA AND TAMBE

It was reported that, 6 BA after absorption was transported and finally accumulated in shoot tips and roots (Zhang and Huang, 2002) and was responsible for the formation of flower buds (Krieken et al., 1989). Furthermore, cytokinins enhanced the mobilization of nutrient and amino acids to treated sites and it may also be beneficial for higher bearing capacity due to increase of carbohydrate accumulation (Letham, 1969)

Application of cytokinins i.e. BA and CPPU might be nullified by the effect of GA3 which adversely affects flowering. BR is also responsible for flowering (Rao et al., 2002) and exogenous application of cytokinins in combination with GA3 helps to reduce Absisic acid concentration in the ovule, stimulating cell division, cell growth and increasing sink activity (Bangerth, 2004), which may contribute to considerable number of bunches per vine at harvest.

Maximum bunch weight and yield were recorded in treatment T10 might be due to significant increase in berry size, berry weight and also related with proper nutritional supply to the developing bunches associated with photosynthetic capacity, translocation of photosynthates to the bunch and reduced berry drop. Higher flowering and higher fruit set are also responsible for maximum yield. The findings of this result are in close agreement with those reported by Anon. (1998) and Tambe (2002). Enhanced translocation of photosynthates and nutrients into treated site in the application of BR was also reported by Vladimir et al. (2000) and Sivakumar et al. (2002). The photosynthetic capacity was enhanced by BA (Sivakumar and Virendra, 2000) and BR (Vladimir et al., 2000).

BR increased the level of ingredients, which are essential for synthesis of sugar, carbohydrates and nucleic acid in grapes (Anonymous, 1998).

All the yield-contributing attributes were influenced by the application of BR, BA and CPPU. Thus, conformity of reason for yield increase is same as for number of bunches per vine and average weight of bunch. Sivakumar et al. (2002) reported BR application increased yield due to enhanced carbohydrate fixation, soluble protein and effective transportation of metabolites.

Tambe (2002) observed maximum yield by the application of GA3 with BR at 1 ppm at 3-4 mm berry diameter stage. Vladimir et al. (2000) stated that BRs increase the crop yield due to enhancement of photosynthetic capacity and translocation of products. This helps to increase considerable amount of yield. PLANT GROWTH REGULATORS ON GRAPES 13

The application of 3 ppm CPPU at 3 mm berry size with GA3 gave the highest yield per vine (Ranpise et al., 1999). Yield increase by application of cytokinins might be due to increase in the nutrient and carbohydrate supply i.e. increase in sink activity. Importance of assimilates in fruit growth is well documented. Bangerth (2004) pointed out that application of cytokinins with GA3 increased the sink activity and the application of BRs induced the sucrose transport (Petzold et al., 1992).

Effect on maturity and quality attributes

The quality attributes such as TSS, acidity, TSS : acidity ratio and days to harvest were significantly influenced by application of cytokinins and BR with GA3.

The highest value of TSS (23.43 0B), and the lowest acidity (0.53 %) were observed in control treatment, which was at par with treatment 0 0 T8 (23.39 B, 0.55%, respectively). The lowest TSS (21.34 B) was given in treatment T7 whereas; the highest acidity (0.63 %) was obtained in treatments T7 and T5, which was at par with treatment T6 (0.62%).

Higher TSS and lower acidity might be due to hastening of ripening because of high stimulation of ethylene production in only GA3 treated bunches. GA3 was reported to have increased the production of ethylene (Weaver and Singh, 1978). There was a progressive decrease in total acidity with an advancement of maturity.

The observations of increased TSS content and decreased acidity percentage by GA3 treatments are in agreement with those reported earlier by Uarma (1991) and Singh et al. (1994). However, Ben (1990) observed lower percent TSS and higher percent acidity by repeated GA3 applications. In present investigation, the lowest TSS and higher acidity were obtained in treatment T7 (GA3 with CPPU + BA). This could have been due to delaying in maturity. Significantly more days were required to reach the maturity stage (144.20 days) in treatment T7 as against the rest of the treatments. This treatment delayed harvesting of grapes by about 12 days as compared to control to attain optimum TSS. This might be due to delaying of ripening, because of reduced ethylene production.

Ranpise et al. (1999), Bhujbal et al. (2002), and Michele and Dokoozlian (2004) noted that CPPU+GA3 application delayed fruit maturation.

The data regarding TSS: Acidity ratio revealed that the highest TSS: acidity ratio (44.31) was recorded in the control. The treatment T7 14 WARUSAVITHARANA AND TAMBE

recorded minimum TSS: Acidity ratio (34.01), which was at par with T5 (34.29) and T6 (35.13).

GA3 reduced fruit set accounting for better nutrition of the remaining berries and higher TSS: Acidity ratio (Singh and Singh, 1980). On the other hand, Bindra et al. (1980) concluded that more acidity associated with high yield and maximum TSS: Acidity hastened the ripening.

It was obvious that the reduced acidity with an increase in the total soluble solids resulted in increased TSS: Acidity ratio. These findings are in conformity with results reported earlier by Singh and Singh (1980), Singh et al. (1994) and Uarma (1991). BRs treatment enhanced TSS: Acidity ratio, which was confirmed by the findings of Tambe (2002). The lowest TSS: Acidity ratio was registered in treatment T7, due to delaying maturity.

CONCLUSIONS

The present investigation indicated that application of 10 ppm GA3 at pre-bloom stage, 15 ppm at initiation of flowering, 25 ppm at 3 to 4 mm berry diameter stage in combination with 1 ppm BR, 25 ppm at 7 to 8 mm berry diameter stage, and BA at 10 ppm twice at 4 leaf and berry setting stage was the most effective for obtaining higher yield with good quality grapes.

REFERENCES

Anon., 1998. Brassinos: Organic grape booster, A technical leaflet on Brassinosteroid. p 5. Bangerth, F. K. 2004. Internal regulation of fruit growth and abscission. Acta Hort. (ISHS). 636: 235-248. Ben, Y. T. 1990. Effects of gibberellin treatments on ripening and berry drop from Thompson Seedless grapes. Am. J. Enol. Vitic. 41 (2): 142-146. Bhujbal, B. G., D. B. Ranawade and K. B. Jagtap. 2002, Effect of CPPU on quality of grapes CV. Tas-A-Ganesh. J. Maharashtra Agric. Univ. 27 (1): 013-014. Bindra, A. S., K. V. Sanghvi and S. S. Brar. 1980. Relationship between vine growth, bearing and fruit quality in grapes. Acta Bot. Indica. 8 (2): 206-209. Clouse, S. D. and J. M. Sasse. 1998. Brassinosteroids, Essential regulators of plant growth and development. Annu. Rev. plant physiol. Plant Mol. Biol. 49: 427-451. Coombe, B. G. 1976. The development fleshy fruits. Ann. Rev. Plant Physiol. 27: 507-528. FAO, 2004. Statistical data of Area, Yield, Production, Imports and Export. www.fao.org/faostat.fao.org Francisco, J. P. and M. Gomez. 2000. Possible role of soluble invertase in the Gibberellic Acid berry sizing effect in sultana grape. Plant growth Regulation 30 (2): 111-116. Krieken, W. M. V. de., F. C. Anton, J. M. S. Marinus and J. W. George. 1989. cytokinns and flower bud formation in vitro in Tobacco. Plant Physiol. 92: 565-569. PLANT GROWTH REGULATORS ON GRAPES 15

Krishnamoorthy, H. N. 1981. Plant growth substances including applications in agriculture. Tata Mc Graw-Hill Publ. Co. Ltd., New Delhi. p. 214. Letham, D.S. 1969. Cytokinins and their relation to other phytohormones. Bio Science 19: 309-316.

Liu., J. L. 2002. Effect of 6 BA and GA3 on the fruit growth and development of Fujiminori grape variety. China Fruits 5: 20-21. Michele, M. and N. Dokoozlian. 2004. CPPU increases berry size and delays maturation and colour development of table grapes. Am. J. Enol Vitic. 55 (3): 309. Mitchell, J. W., Mandava, N. B., Worley, J. F., Plimmer, J. R. and Smith, M. V. 1970. Brassins; a new family of plant hormones from grape pollen. Nature 225: 1065-1066. Panse, V. G. and P. V. Sukhatme. 1985. Statistical methods for agricultural workers ( 4 th Edn.). ICAR Publ., New Delhi. p. 347. Petzold, U., S. Peschel, I. Dahse and G. Adam. 1992. Stimulation of source applied 14 C- sucrose export in Vicia faba plants by brassinisteroids, GA3 and IAA. Acta Bot. Neerl. 41: 469-479. Randhawa, G. S. 1971. Gibberellins. pp. 82-99. In Use of plant growth regulators and Gibberellins in Horticulture, ICAR pub. New Delhi. Ranpise, S. A., B. T. Patil and T. K. Ghure. 1999. Effect of forchlorfenuron (CPPU) and gibberellic acid on physico-chemical properties of Thompson seedless grapes. J. Maharashtra Agric. Univ. 24 (3): 249-251. Rao, R. S. S., B. V. Vardhini, E. Sujatha and S. Anuradha. 2002. Brassinosteroids - A new class of phytohormones. Current Sci. 82 (10): 1239-1244. Shikhamany, S. D. 2001. Grape production in India. Expert consultation on “Viticulture (grape production) in Asia and the Pacific. Bangkok, Thailand. 2-4 May 2000. Grape production in the Asia Pacific Region. Singh, I. S. and H. K. Singh. 1980. Timing of application of gibberellic acid on quality of “Delight”grapes. Indian J. Agric. Sci. 50 (4): 334-337.

Singh, S., I. S. Singh and D. N. Singh. 1994. Effect of GA3 on ripening and quality of grapes (Vitis vinifera L.). The Orissa J. Hort. 22 (1-2): 66-70. Sivakumar, T. and N. Virendra. 2000. Effect of benzyladenine on photosynthesis and pigment concentration in the flag leaf and ear of wheat and triticale. Indian J. Plant Physiol. 5(4): 354-357. Sivakumar, R., G. Pathmanaban, M.K. Kalarani, M. Vanangamudi and P.S Srinivasan. 2002. Effect of foliar application of growth regulators on bio-chemical attributes and grain yield in Pearl Millet. Indian J. Plant Physiol. 7(1): 79-82. Stephen, J. S., D. A. Marshall and B. J. Sampson. 2003. Response of Muscadine grape (Cv.

rotundifolia) Michx to the growth regulators CPPU and GA3. Hort. Sci. 38 (3): 699. Tambe, T. B. 2001. Growth regulator in grapes. In “Physiology and Bio-chemistry in Fruit Crops”. MPKV Publi. 18: 117-121. Tambe, T. B. 2002. Effect of Gibberellic Acid in combination with Brassinosteroid on berry size, yield and quality of Thompson Seedless grapes. J. Maharashtra Agric. Univ. 27 (2): 151-153. Uarma, S. K. 1991. Effect of dipping flower clusters in Giberellic Acid on fruit set, bunch and berry size, yield and fruit quality in grapes (Vitis vinifera L.). Indian J. Agric. Res. 25 (1): 54-58. 16 WARUSAVITHARANA AND TAMBE

Vladimir, K., Z. Vladimir and D. G. Aede, 2000. Twenty yrs of BRS: Steroidal plant hormones warrant better crops for the XXI century. Annals of Botany 86 (3): 441-447. Weaver, R. J. and I. S. Singh. 1978. Occurrence of endogenous ethylene and effect of plant regulators on ethylene production in the grape vine. Am. J. Enol. Vitic. 29: 282- 285. Zhang, P. and W. D. Huang. 2002. Transportation and distribution of 6 BA in grape. J. Fruit Sci. 19 (3): 153-157. Annals of Sri Lanka Department of Agriculture. 2006.8:273-279.

PHYSODERMA BROWN SPOT DISEASE IN HYBRID MAIZE

W.A.P.G. WEERARATNE and J. A.V.J. JAYASINGHE Field Crop Research and Development Institute, Mahailluppallama

ABSTRACT

A brown spot disease has been reported from hybrid maize cultivation at Galenbidunuwewa area for the first time during maha 2005/06 season. The high disease incidence (>50%) was observed in the highly susceptible variety ‘Jumbo’. Brown spot lesions first appeared as very small, round to oblong, yellowish spots on the leaf blade, sheaths and stalks. Spots on leaf midribs were circular, chocolate brown to reddish brown in colour while lesions on the laminae continued as yellowish spots. In severe infections, these lesions coalesced to form large irregular, angular blotches resulting in the weakened stalks frequently breaking at the infection centers. On the basis of microscopic observations and the pathogenicity test, the pathogen was identified as ‘Physoderma maydis’. This identification was confirmed by comparing it with CMI description chart No. 753. This is the first reported incidence of Physoderma brown spot in hybrid maize in Sri Lanka. Heavy rains that prevailed during November to January coupled with susceptible hybrid varieties seemed to have favored development and spread of the disease. Usually, leaf spot caused by Helminthusporium spp is the prominent disease in maize during maha season. However, Physoderma brown spot will be another potential threat for maize cultivation in Sri Lanka in the future.

KEYWORDS: Leaf spot disease, Maize, Physoderma maydis.

INTRODUCTION

Maize is the second most important grain crop after rice in Sri Lanka; it is used as human food as well as animal food. More than fifty fungal pathogens are known to infect maize throughout the world but only a few of them were reported in Sri Lanka (Shurtieff, 1980). Among them, sheath blight and Helminthusporium are the two diseases reported in Sri Lanka infecting maize. However, these two diseases did not appear economically significant since they could be controlled successfully by adopting correct agronomic practices.

An attempt is being made to increase maize production in Sri Lanka by cultivation of hybrid maize varieties. As a result, importation of hybrid maize seeds has increased in recent years. There is a potential of introduction of new pathogens to the country with imported hybrid maize seeds. It has been reported that the farmer fields of hybrid maize at Galenbidunuwewa area have been affected by a leaf spot disease during maha 2005/2006 season. It attacks leaf blades, sheaths and stalks and the high disease incidence (>50%) was observed in affected hybrid varieties. The causal organism was not identified and, therefore, it was important to find out the causal organism before formulating control measures for this disease. The 18 WEERARATNE AND JAYASINGHE disease symptoms on affected plants, the causal organism and its pathogenicity were studied in detail in this experiment.

MATERIALS AND METHODS

Microscopic observation of sporangia

Disease symptoms were studied in both hybrid varieties ‘Jumbo’ and ‘Pacific’. Seeds and infected plant parts were collected from farmer fields at Galenbidunuwewa area for pathogen isolation. Dusty pustules were picked from infected plant tissues and crushed on a drop of distilled water on slide and observed for sporangia of pathogen under the compound microscope. Further, thin sections of infected leaf and midrib tissues were also observed directly under the compound microscope for the identification of the pathogen.

Blotter test

Seeds collected from susceptible variety “Jumbo” were surface sterilized by immersing them 2-3 minutes in 70% Ethanol. Twenty seeds were incubated on water moistened blotters in Pyrex sterilized Petri dishes (95 mm) at room temperature (25°C ±) under normal day light conditions for 7 days. Fungi developing from contaminated seeds were identified microscopically from their growth characteristics and identification confirmed by examining slide preparation with a compound microscope. A minimum of 200 seeds were tested.

Pathogenicity test

The inoculation techniques were carried out according to the methods explained by Lal and Chakrawarti, (1977). Ten plants of ‘Jumbo’ variety were raised in autoclaved soil in clay pots in the green house and inoculations were made by pouring 2 ml of sporangial suspensions (approximately 20,000/ ml) into the whorls of 15 day old maize plants. Humidity was maintained in the whorls after every inoculation. The inoculation was repeated 3 times at intervals of 3 days. Another ten plants inoculated with sterile distilled water were used as the control treatment. Disease symptoms were observed and recorded one month after inoculation.

RESULTS AND DISCUSSION

Disease symptoms observed in the field

The symptoms appeared in late December, 2005 when the crop was at the reproductive stage. The high disease incidence (>50%) was PHYSODERMA BROWN SPOT DISEASE IN HYBRID MAIZE 19 observed in “Jumbo” variety while less incidence (10% - 20%) was observed in ‘Pacific’ variety. Brown spot lesions first appear as very small, round-to- oblong, yellowish spots on the leaf blade (Plate 1). The spots may occur in bands across the leaf blade. Symptoms are most prominent in the leaf midrib area (Plate 2). Infected tissues turn to chocolate brown to reddish brown and merge to form large blotches with an irregular, angular appearance. Cells of infected tissues disintegrate exposing dusty pustules (brown blisters). Under favorable conditions, severe stalk rot will develop at nodes beneath the leaf sheath and yield reduction may have occurred by the lodging of infected plants.

Microscopic observation of sporangia

Microscopic observation revealed large numbers of round shaped brown sporangia (resting spores) as well as corn midrib cells. The sporangia were identified by comparison of their colour, shape and size with published data (Walker, 1983; Shurtieff, 1980). The shape and colour of the sporangia were similar to those of Physoderma maydis described in the CMI description chart No. 753. Walker (1983) reported that sporangia produced in pustules in infected leaf tissues are brown, flattened on one side with a circular cap or lid and 18-24 x 20-30 cm and it requires water on leaf surfaces and relatively high temperatures (23 to 30°C) for germination and release of 20-50 zoospores. Zoospores move in water on leaf surfaces, and attack young maize tissues within the leaf whorl, forming infectious hyphae. The resulting mycelium enters mesophyll or parenchyma cells forming large vegetative structures. The development of symptoms and the germination of new sporangia occur approximately 6-20 days after infection, completing the disease cycle (Hebort and Keiman, 1958).

Blotter test

In blotter test, elongated mycelia that lack cross walls, large number of round shape, brown sporangia and zoospores were observed. These structures were similar to that of sporangia of Physoderma maydis reported in maize. This suggests that this fungus could be associated with seeds collected from ‘Jumbo’ hybrid maize plants.

Pathogenicity test

After one month of inoculation, typical symptoms of the leaf spot developed on the inoculated plants which were identical to those observed in the field. But, all the control plants remained healthy. The pathogen was reisolated from infected plants and observed mycelial characteristics. 20 WEERARATNE AND JAYASINGHE

According to disease symptoms, pathogenicity test and microscopic observation of sporangia, the pathogen was identified as “Physoderma maydis (Miyabe)” and its pathogenicity was established. This identification was further confirmed by comparing it with CMI description chart No. 753 (Walker, 1983). Physoderma is a lower fungus which belongs to class phycomycetes and sub class chytridiomycetes. Sporangia of the fungus are released from infected pustules, disintegrating corn debris, and soil and are carried to susceptible plants by air currents, insects splashing rain or flowing water and humans (Broyles, 1962; Walker, 1983).

Development of the disease is favored by high relative humidity (73- 90%) and temperature of 23-30°C in susceptible plants (Walker, 1983). The weather conditions during November to January were favourable for the development and spread of the disease. The relative humidity range of 70-92%, mean temperature of 23-30°C and heavy rains received (104- 330 mm) during November to January coupled with highly susceptible hybrid varieties `Jumbo` and `Pacific` seemed to have favored the disease development in this particular field.

In Sri Lanka, there may be a potential to cause heavy losses by this disease when the susceptible hybrid maize varieties are cultivated during the maha season. This is the first report of brown spot caused by Physoderma maydis of maize cultivation from Sri Lanka. Physoderma is known to remain viable in the soil, as sporangia, for about 3 years (Walker, 1983). Therefore, crop rotation and ploughing infected corn debris back into the soil after harvest will reduce the infection and the use of resistant varieties is also important to control the disease. Use of pathogen free planting material is also essential in commercial cultivation in the future. It is important to carryout awareness programme for seed importers regarding the risk of introducing new pathogens to the country with the imported seeds, and to follow the quarantine regulation strictly.

CONCLUSIONS

Brown spot of hybrid maize has been identified as a fungal disease caused by Physoderma maydis (Miyabe) in Sri Lanka. Affected ‘Jumbo’ maize seeds were contaminated with fungus Physoderma maydis. Further research on epidemiological studies, screening of fungicides and selection of resistant varieties in controlling the disease needs to be carried out. PHYSODERMA BROWN SPOT DISEASE IN HYBRID MAIZE 21

Plate 1. Small yellowish spots on corn leaf blades.

Plate 2. Chocolate brown blotches on midrib of corn leaf. 22 WEERARATNE AND JAYASINGHE

ACKNOWLEDGEMENTS

The support given by Mr. MP Mahindhapala, Deputy Director (Provincial Council), Research Officers RM Ranaweera Banda, Mr. KM Karunarathna and Mr. KGS Senevirathne during the study period is highly appreciated.

REFERENCES

Broyles, J.W. 1962. Penetration of meristematic tissues of corn by Physoderma maydis. Phytopathology 52:1013-1018. Hebort, T.T. and A.Keiman. 1958. Factors influencing the germination of the resting sporangia of Physoderma maydis. Phytopathology 48: 102-106 Lal, B.B. and B.P. Chakrawarti. 1977. Resistance of maize varieties and lines to Physoderma maydis, causal organism of brown spot of maize in Udaipur. India. Plant Disease Reporter 61(12): 1080-1081 Shurtieff, M.C. 1980. Physoderma brown spot. Compendium of corn diseases. 105p. The American Phytopathological Society. USA. Walker, J.C. 1983. Physoderma maydis. CMI Descriptions of pathogenic fungi and Bacteria . No. 753. The Commonwealth Mycological Institute, Kew, Surrey,England. Annals of Sri Lanka Department of Agriculture. 2006.8:281-294.

INDUCTION OF RESISTANCE TO Alternaria solani IN TOMATO BY CHEMICAL INDUCERS

W.A.R.T. WICKRAMAARACHCHI1 and P. NARAYAN REDDY2 1Regional Agricultural Research and Development Center, Aralalaganwila 2Department of Plant Pathology, College of Agriculture, Acharya NG Ranga Agricultural University, Rajendranagar, Hyderabad, India

ABSTRACT

Foliar spray of different concentrations of salicylic acid (SA), benzothiadiazole (BTH) and 2, 4-dinitrophenol (DNP) were evaluated for induction of resistance to Alternaria solani (Early blight) in tomato under glass house conditions. The highest disease control (64.65 percent) was achieved with the two applications BTH 5 mM, followed by the two applications of BTH 10 mM (55.17 percent) when challenge inoculation was done five days after inoculation. Two applications of BTH 5 mM with challenge inoculation one day after treatment and two applications of DNP 10 mM with challenge inoculation 5 days after treatment gave comparatively higher disease control of 53.46 percent and 51.72 percent respectively. The highest overall mean percent disease control (44.99 percent) was recorded with the application of BTH 5 mM followed by BTH 10mM (42.92 percent) and DNP 10 mM (36.23 percent) which were equally effective in inducing resistance. Lower concentrations of chemical inducers tested were less effective than higher concentrations. A booster application was required to induce acceptable level of resistance when lower concentrations were used. All these chemical inducers tested required more than one day lag period to induce the plant defense system against A. solani. Foliar application of SA was less effective in inducing resistance in tomato against A. solani. However, prophylactic application of BTH and DNP can be effectively used for the management of tomato early blight.

KEYWORDS: Alternaria solani, Benzothiadiazole, Early blight, Induced resistance, Salicylic acid, Tomato, 2,4-dinitrophenol.

INTRODUCTION

Early blight in tomato (Lycopersicon esculentum Miller) caused by Alternaria solani (Ellis and Martin) Jones and Grout, is one of the most common and destructive foliar diseases causing yield losses ranging from 25 -100 percent (Datar and Mayee, 1981 and Sohi, 1984).

Control of early blight has been accomplished primarily by application of fungicides and using resistant cultivars (Spletzer and Enyedi, 1999). Many tomato breeding lines and cultivars with measurable level of resistance are of late maturity, or the level of resistance is less than sufficient under most epidemic conditions (Maiero et al., 1990). Further, breeding for resistance against early blight has little scope due to lack of single gene resistance, complex pattern of inheritance (Spletzer and Enyedi, 1999) and difficulties in the screening of plants for resistance and transferring resistance genes across genotypes (Foolad et al., 2000). Therefore, cultivation of 26 WICKRAMAARACHCHI AND REDDY resistant varieties does not offer a practical solution to control disease. Currently, management of the disease relies principally on foliar application of fungicides (Mills et al., 2002). Frequent and abusive use of fungicides cause a number of problems such as adverse effects on the environment, consumers and non-target organisms (Pharand et al., 2002) and emergence of resistant pathogens due to their high selection pressure (Yamaguchi, 1998).

The disadvantages associated with such measures have provided an incentive to develop more economical, environmentally sound and reliable methods of disease control. A potential alternative to application of fungicides for managing early blight of tomato may be the concept of Systemic Acquired Resistance (SAR) mediated through certain natural and synthetic chemical compounds with no direct antibiotic effects on the pathogen. SAR involves expression of a set of genes including those encoding pathogenesis related (PR) proteins and enzymes (Sticher et al., 1997). Some SAR gene products have direct microbial activity or are closely related to classes of antimicrobial proteins. The resistance induced by chemical treatment can be very effective (Gorlach et al., 1996) and may be a commercially useful broad-spectrum plant protection strategy that is stable, long lasting and environmentally benign (Frey and Carver, 1998). A potential for chemically mediated disease control could be based on compounds that would induce the SAR responses. Chemical inducers include natural compounds such as salicylic acid (Frey and Carver, 1998) unsaturated fatty acids (Cohen et al., 1991) and synthetic molecules such as 2,6-dichloroisonic acid (Durner and Klessing, 1995), benzo (1,2,3) thiadiazole-7-carbothioic acid-s-methyl ester (Gorlach et al., 1996 and Tosi et al., 1999) and 2,4- dinitrophenol (Elad, 1993).

The present investigation was undertaken to find out the possibility of inducing systemic acquired resistance in tomato to early blight disease by chemical inducers viz salicylic acid (SA), benzothiadiazole (BTH) and 2,4-dinitrophenol (DNP).

MATERIALS AND METHODS

Experiments were conducted at the Department of Plant Pathology, Acharya NG Ranga Agricultural University, Hyderabad, India, in 2002. Seeds of tomato cultivar, Pusa Early Dwarf (PED) were used and plants were grown in the glasshouse under normal temperature and light conditions. Plants were irrigated daily and fertilized weekly with a commercial preparation of NPK mixture (14:14:14).

Pathogen and preparation of inoculum CHEMICALLY INDUCED RESISTANCE IN TOMATO 27

The pathogen was isolated from naturally infected tomato leaves showing typical early blight symptoms under aseptic conditions using tissue segment method (Aneja, 2001). The culture was purified by single spore isolation described by Tuite (1969) and identified as A. solani based on morphological characteristics described by Ellis (1971). Since A. solani seldom produces conidia under laboratory conditions, a standard-mycelial spore suspension was prepared as previously described by Dhiman and others (1980) for mass inoculation of tomato plants.

Challenge inoculation

Induced resistance was detected via challenge inoculation of tomato foliage by spray inoculation with mycelial spore suspension of A. solani (104 infective propagules/ml) followed by incubation in a moist chamber for 48 h in the dark. Two days after challenge inoculation, plants were transferred to glasshouse bench from moist chamber. Plants were regularly observed for disease symptoms. Control consisted of plants treated with distilled water and subsequently challenge inoculated with A. solani.

Assessment of induced resistance

Assessing the area of lesions determined induced resistance. The level of induced resistance was relative to symptoms on control plants treated with water, instead of chemical inducers. Seven days after challenge inoculation, maximum development was usually evident, and data were therefore obtained 7 days after challenge inoculation.

Disease severity

Disease severity of early blight was recorded using 0-4 scale (Rajagopal and Vidyasekaran, 1982; Devanathan and Ramanujam, 1995).

Numerical rating Disease intensity (percent leaf area blighted)

0 Infection free or nearly so 1 Trace to 25 2 26-50 3 51-75 4 76-100

Percentage disease index (PDI) was computed using the formula:

Sum of numerical ratings x 100 PDI = Total no. leaves assessed x Maximum disease rating 28 WICKRAMAARACHCHI AND REDDY

Foliar application of SA, BTH and DNP

An experiment was designed in the glasshouse to determine if Systemic Acquired Resistance (SAR) could be elicited in tomato by foliar application of SA, BTH and DNP. All inducers were applied on foliage as sprays. SA was tested at 1 mM, 3 mM and 5 mM while BTH and DNP at 1 mM, 5 mM, 10 mM in 0.01% Tween 20. Thirty-day old tomato plants were divided into two sets. First set sprayed twice with chemical inducers on 30th and 40th day. Then plants were sub divided again into two sets from which one set was challenge inoculated one day after second chemical spray (41st day) and other set was challenge inoculated five days after second chemical spray (45th day). The second set of plants were spray inoculated with chemical inducers only once at 40th day. One set of these plants was challenge inoculated one day after chemical spray (41st day) and the other set five days after the treatment (45th day). Plants sprayed with 0.01% Tween 20 served as control and plants sprayed with mancozeb (2000 ppm) served as the check. Three replications were maintained for each treatment. The severity of early blight was rated on 0-4 point scale 7 days after challenge inoculation.

Experimental design and statistical analysis

The experiment was conducted in a completely randomized design (CRD) with 3 replications. Data were analyzed statistically using ANOVA and mean separation carried out using the minitab statistical software. When necessary, appropriate transformations of the values were performed to normalize data prior to performing ANOVA. All data expressed as percentage were angular (arcsine) transformed before analysis.

RESULTS AND DISCUSSION

The results on the effect of foliar application of SA, BTH and DNP at different concentrations and number of applications on induction of resistance against Alternaria solani are presented in Table 1.

It is evident from the results that mean percent disease index was the lowest (13.54) in fungicide Mancozeb at 2000 ppm (Control). Similarly the highest mean percent disease reduction over control (82.64) was observed in Mancozeb 2000 ppm, in comparison to all chemical inducer treatments. CHEMICALLY INDUCED RESISTANCE IN TOMATO 29

Table 1. Effect of foliar spray of salicylic acid, benzothiadiazole and 2,4- dinitrophenol on induction of resistance in tomato against A. solani.

Per cent disease Index of early blight Per cent disease reduction over control S. Chemicals/ One application Two applications One application Two applications Mean Mean No. concentrations Challenge inoculation Challenge inoculation Challenge inoculation Challenge inoculation 1 DAT 5 DAT 1 DAT 5 DAT 1 DAT 5 DAT 1 DAT 5 DAT 1. SA 1 mM 72.91 74.30 65.97 68.05 70.31 7.89 (15.33) 7.53 (12.75) 6.27 15.51 9.30 (58.71) (59.69) (54.34) (55.60) (57.08) (11.87) (23.09) (15.76) 2. SA 3 mM 70.83 60.41 58.33 63.19 63.19 10.52 24.34 16.83 21.55 18.31 (57.42) (51.03) (49.86) (52.68) (52.75) (17.65) (29.42) (22.36) (27.48) (24.23) 3. SA 5 mM 66.66 56.94 56.25 54.86 58.68 15.78 28.69 19.80 31.89 24.04 (54.78) (49.00) (48.62) (47.80) (50.05) (23.01) (32.32) (25.67) (34.31) (28.83) 4. BTH 1 mM 71.52 59.02 54.37 43.33 56.84 9.64 (17.22) 26.08 23.76 46.20 26.42 (57.81) (50.23) (46.99) (40.91) (48.99) (30.47) (28.99) (42.81) (29.87) 5. BTH 5 mM 69.44 40.27 32.63 28.47 42.70 12.28 49.56 53.46 64.65 44.99 (56.47) (39.37) (34.73) (32.18) (40.69) (20.22) (44.74) (47.01) (53.57) (41.39) 6. BTH 10 mM 61.11 41.66 36.11 36.11 43.75 22.80 45.21 48.51 55.17 42.92 (51.44) (40.18) (36.91) (36.90) (41.36) (28.36) (42.24) (44.14) (47.98) (40.68) 7. DNP 1 mM 70.13 64.58 56.94 52.08 60.93 11.40 19.13 18.81 35.34 21.17 (56.96) (53.52) (49.00) (46.19) (51.42) (18.96) (25.66) (25.52) (36.37) (26.63) 8. DNP 5 mM 66.66 54.16 47.22 43.75 52.95 15.79 35.50 32.67 45.69 32.41 (54.75) (47.40) (43.40) (41.39) (46.73) (23.25) (36.52) (34.72) (42.51) (34.25) 9. DNP 10 mM 63.19 50.00 45.13 38.88 49.30 20.17 37.39 35.64 51.72 36.23 (52.68) (45.00) (42.20) (38.57) (44.61) (26.48) (37.63) (36.60) (45.99) (36.67) 10. Mancozeb 2000 17.36 14.58 9.02 (17.31) 13.19 13.54 78.07 81.73 87.13 83.62 82.64 ppm (24.50) (22.31) (21.21) (21.33) (62.20) (64.84) (69.16) (66.22) (65.61) 11. Control 79.16 79.86 70.13 80.55 77.43 - - - - - (62.96) (63.38) (56.89) (63.96) (61.80) Mean 64.45 54.16 48.29 47.50 53.60 18.58 32.29 31.17 41.03 30.77 (53.50) (47.37) (43.66) (43.40) (46.98) (22.97) (32.42) (31.46) (38.21) (31.26) SEm+ CD (P < 0.05) SEm+ CD ( < 0.05) Chemicals and concentrations 1.080 3.033 1.687 4.737 No. of applications plus challenge inoculation 0.651 1.829 1.017 2.857 Interactions 2.160 6.067 3.374 9.475 Figures in parentheses are angular transformed values; DAT = Days after treatment. 30 WICKRAMAARACHCHI AND REDDY

This is in conformity with many other workers who reported that 0.2 per cent Mancozeb effectively controlled early blight of tomato (Rajagopal and Vidhyasekaran, 1983; Mathur and Shekhawat, 1986; Ganeshan, 2000).

Induced resistance by means of synthetic inducers in some cropping systems is a suitable strategy to utilize the plant’s natural defense responses to control pathogens (Sauerborn et al., 2002). The resistance induced by chemical treatment can be very effective (Gorlach et al., 1996) and may be commercially useful (Frey and Carver, 1998). Application of SA to susceptible plants can enhance resistance to pathogens by inducing a wide range of defense genes encoding the PR proteins (Murphy et al., 2000). Treatment with BTH has been shown to result in decreased disease incidence in different plant species (Friedrich et al., 1996). DNP, a potent C2H4 inhibitor, was differentially effective in controlling the diseases of various hosts (Elad, 1993).

The degree of protection from chemical inducers depended on concentration and number of applications and the time interval between chemical inducer spray and challenge inoculation. All concentrations of SA, BTH and DNP showed significantly lower mean percent disease index over control. The highest disease control (64.65 percent) was achieved with the two applications of BTH 5 mM followed by two applications of BTH 10 mM (55.17 percent) when challenge inoculation was done five days after treatment application. Further, maximum mean percent disease reduction was recorded with BTH 5 mM (44.99) followed by BTH 10 mM (42.92). BTH 1 mM showed significantly lower mean percent disease control of 26.42 compared to higher concentrations of BTH (Figure 1). Many workers have demonstrated the efficacy of foliar spray of BTH as a chemical inducer. Gorlach and others (1996) reported that foliar spray of wheat with BTH 30 g/ha led to an average of 35 percent reduction in disease symptoms of brown rust and Septoria leaf spot. In contrast to present findings, Hijwegen and Termorshuizen (2000) demonstrated that aerial applications of low concentration of Bion® (BTH) 0.1 and 0.2 mM/l delayed Verticillium wilt in eggplant caused by Verticillium dahliae. However, these findings are in conformity with Kaur and Kolte (2001) who reported that wet seed treatment of mustard with BTH at 500 mg/l significantly reduced foliar infection of Albugo candida whereas BTH 10 mg/l, 50 mg/l and 250 ml/l had no significant effect on foliar infection.

Mean percent disease reduction in treatments receiving two sprays of chemical inducers and challenge inoculation one day after second spray (31.17) and one spray of inducers and challenge inoculation after 5 days (32.29) were not significant. However, two sprays of inducers followed by challenge inoculation with A. solani, 5 days after second spray of chemical CHEMICALLY INDUCED RESISTANCE IN TOMATO 31 inducers were found to be significantly higher (41.03). Second application of chemicals did not give any cumulative effect of resistance if plants were challenge inoculated one day after second spray (31.17). However, challenge inoculation 5 days after second spray gave significantly higher reduction (41.03) in percent disease reduction over control implying that induction required a lag period. Single application of BTH followed by challenge inoculation with Alternaria solani one day after treatment had the lowest per cent disease reduction at 1 mM (9.64), 5 mM (12.28) and 10 mM (22.80) in comparison with challenge inoculation at 5 days after chemical treatment with 1 mM (26.08), 5 mM (49.56) and 10 mM (45.21) concentrations of BTH. Similarly two applications of BTH followed by challenge inoculation with A. solani 5 days after chemical treatment gave better control over challenge inoculation one day after the treatment (Figure 1).

The findings of present study reveal that a lag period of 1-5 days was required for the expression of resistance. Gorlach et al. (1996) showed that BTH as a foliar spray provided protection in wheat against powdery mildew caused by Erysiphe graminis tritici for an entire growing season and also effective protection could be achieved by pre-treatment of plants with BTH 4-7 days before pathogen inoculation. Similarly, present results are consistent with proposed activity of BTH as a plant activator (Ciba- Geigy, 1995) in which a lag period of about 4-7 days was suggested to build up SAR in plants by BTH. Tosi et al. (1999) also demonstrated that CGA 245704 (BTH) applied as a soil drench at the rate of 100 mg/kg soil was more effective in protecting sunflower plants against downy mildew caused by Plasmopara helianthi 3 days before inoculation than 1 day before or 1 day post-inoculation, confirming the need for a lag period between the application of activator and protection. In contrast to the present findings, Ishii et al. (1999) reported that cucumber anthracnose caused by Colletotrichum laginarium was suppressed with BTH even when plants were inoculated 3 h after treatment with BTH and they found rapid expression of peroxidase, chitinase and other resistance related genes in leaves several hours after treatment with chemical. Similarly, Kaur and Kolte (2001) reported obtaining 60.9 per cent protection against Plasmopara helianthi in sunflower when BTH was applied one day post-inoculation.

Foliar spraying of tomato plants twice with SA 1 mM, BTH 1 mM and DNP 1 mM and challenge inoculation with A. solani 5 days after second chemical inducer treatment showed significantly higher disease reduction compared to the corresponding values with challenge inoculation one day after two applications of chemical inducers. In other concentrations of chemical inducers, the differences between corresponding values were not significant. Thus SA 1 mM, BTH 1 mM and DNP 1 mM required a booster application to achieve desired level of resistance against A. solani. Matheron (2002) reported that reduction of stem cankers caused by Phytophthora 32 WICKRAMAARACHCHI AND REDDY capsici on pepper plants after four applications of BTH was significantly greater than on plants treated with one application of BTH.

In the present investigation, application of DNP 10 mM led to 36.23 mean per cent disease reduction which was comparable with mean protection achieved by BTH 5 mM (44.99) and BTH 10 mM (42.92). Further, the degree of protection by DNP 10 mM was statistically on par with protection by DNP 5 mM (Fig. 1). These results suggest that DNP could be used to induce resistance in tomato against early blight as effective as BTH. Higher concentration of DNP induced resistance in tomato against A. solani as foliar spray of tomato once with DNP 5 mM and DNP 10 mM when challenge inoculated 5 days after treatment, showed significantly higher per cent disease control of 35.50 and 37.39 respectively than challenge inoculation one day after treatment (15.79 in DNP 5 mM and 20.17 in DNP 10 mM). It is evident that DNP 5 mM and DNP 10 mM also required 5 days lag period to induce resistance effectively. There is no earlier record regarding the induction of resistance in tomato against A. solani by DNP, which involves the biosynthesis of ethylene, a plant hormone.

The findings of Song and Zheng (1998), that foliar spray of cotton seedlings with ethylene biosynthesis inhibitor (2,4-DNP) resulted in the reduction of Fusarium wilt caused by Fusarium oxysporum f. sp. vasinfectum are in agreement with present findings. Kumar (2001) also showed that the exogenous application of 10 mM 2,4-DNP induced local resistance in castor against Botrytis ricini whereas no significant effect of 1 mM 2,4-DNP was observed against the disease.

Ethylene (C2H4) promotes development of diseases in various hosts and spraying inhibitors of ethylene biosynthesis onto hosts enables control of such diseases (Biles et al., 1990). The effect of C2H4 was demonstrated in systems like Diplodia natalensis on lemon, Helminthosporium sativum on barley, Botrytis cinerea on strawberry, tomato, cucumber, pepper, French bean and black current (Elad, 1993). 1- aminocyclopropane-1-carboxylic acid (ACC) is the immediate precursor of C2H4 production in higher plants, DNP an inhibitor of electron transfer and oxidative phosphorylation effectively inhibits ACC conversion to C2H4 leading to reduction of C2H4 (Apelbaum et al., 1981). DNP a potent uncoupler of oxidative phosphorylation reduced ATP levels 98 and C2H4 production 88 percent in apple-tissue slices at 0.1 mM concentration (Apelbaum et al., 1981). CHEMICALLY INDUCED RESISTANCE IN TOMATO 33

Figure 1. Effect of foliar spray of salicylic acid, benzothiadiazole and 2,4-dinitrophenol on induction of resistance in tomato against Alternaria solani.

In the present study, higher concentration of DNP such as DNP 5 mM, DNP 10 mM were more effective than DNP 1 mM (Figure 1). In contrast, Elad (1993) reported that DNP significantly inhibited the grey mould caused by B. cinerea on tomato by 70 percent over control at lower dosage of 0.1 mM whereas lesser reduction of 37 percent in 10 mM DNP. But, reduction of grey mold in bean is in agreement with present results. Similarly, Kumar (2001) observed significantly higher disease control of grey mold of castor with foliar application of DNP 10 mM than DNP 1 mM.

The results of the present investigation (Figure 1) reveal that SA was less effective in inducing resistance in tomato against A. solani compared to BTH and DNP. The percent disease control with application of SA at 1 mM (7.89) and 5 mM (15.78) and challenge inoculation with A. solani one day after treatment were comparable with SA 1 mM (7.53) and SA 5 mM (28.69) followed by challenge inoculation 5 days after treatment respectively. However, treatment of tomato plants once with SA 3 mM and challenge inoculation 5 days after treatment showed significantly higher disease control (24.34) compared to the SA 3 mM followed by challenge inoculation one day after treatment (10.52). Thus it implies that 5 days lag period was required for SA 3 mM to induce resistance in tomato plants. Contrary to present findings, Cai et al. (1996) reported that maximum reduction of rice blast disease was achieved by foliar spray of SA 0.01 mM but not by 10 mM. Resistance 34 WICKRAMAARACHCHI AND REDDY induced shown to last for 15 days after treatment with chemicals and booster treatment after 2 or 5 days extends the period of persistence of induced resistance.

In the present investigation, foliar application of SA induced comparatively less degree of resistance. These results are consistent with observations by Spletzer and Enyedi (1999) who reported that foliar application of SA on tomato was an ineffective method for induction of resistance against A. solani. Systemic resistance in tomato against A. solani is induced by providing 200 µM SA solution to the root system directly in hydroponically grown tomato. By contrast, in cereals induction of resistance to powdery mildew by exogenous SA treatment required application at relatively high concentration of 15 mM for maximum effect in barley and 10 mM for a detectable level in wheat (Frey and Carver, 1998).

Further, application of SA 5 mM showed slight phytotoxic effect in tomato plants suggesting that immediate resistance induction was partly due to tissue damage as foliar application of SA 5 mM and challenge inoculation one day after treatment recorded higher percent disease reduction (15.78) compared to lower concentrations of SA. Phytotoxicity of SA is well known (Kessmann et al., 1994). Spletzer and Enyedi (1999) noticed that root application of 500 µM SA in tomato exhibited a loss of leaf turgor and wilting.

These results are in agreement with the findings of Frey and Carver (1998) who reported that exogenous application of 0.15 mM SA did not induce systemic resistance against Erysiphe pisi in pea whereas 15 mM SA solution was phytotoxic. No apparent damage was caused by SA 1.5 mM suggesting that resistance was not due to tissue damage. Induced resistance persisted at least 15 days after treatment. Possible direct toxicity of SA 5 mM against A. solani in the present study is in conformity with the findings of Kumar (2001) who reported that SA 10 mM showed 74 percent inhibition in radial growth of Botrytis ricini (Grey mould of castor) whereas 27.11 percent inhibition by SA 1 mM in vitro. Similarly SA 1000 µg/ml has completely arrested the radial growth of Alternaria helianthi while percent inhibition of radial growth was 10.48 and 10 µg/ml in vitro (Ratnam, 2001). Leeman et al. (1996) reported that effect of SA on growth of hyphae from germinated micro conidia of the Fusarium wilt pathogen of radish were concentration dependent. At 0.1 mg/ml SA had no significant effect while 1 mg/ml of SA significantly reduced the growth and at 10mg/ml hyphal growth was completely inhibited. CHEMICALLY INDUCED RESISTANCE IN TOMATO 35

It was evident from a preliminary trial that foliar spray of SA 10 mM was moderately phytotoxic to tomato plants (30-day old) under glasshouse condition showing necrotic symptoms and withering of leaves within 12-18 hours after spraying. The highest concentration of SA used in the present study (SA 5 mM) was found to be slightly phytotoxic to tomato plants showing slight necrosis of leaf margins and slight withering of plants observed 18-24 h after spraying (data not shown).

CONCLUSIONS

Foliar spray with salicylic acid (SA), benzothiadiazole (BTH) and 2,4-dinitrophenol (DNP) at different concentrations were evaluated for induction of resistance in tomato against A. solani. The highest disease control (64.65 percent) was achieved with the two applications BTH 5 mM followed by the two applications of BTH 10 mM (55.17 percent) when challenge inoculation was done five days after inoculation. Two applications of BTH 5 mM with challenge inoculation one day after treatment and two applications of DNP 10 mM with challenge inoculation 5 days after treatment gave comparatively higher disease control of 53.46 percent and 51.72 percent respectively. Maximum mean percent disease reduction over control was recorded with BTH 5 mM (44.99) followed by BTH 10 mM (42.92) and DNP 10 mM (36.23), which were equally effective in inducing resistance in tomato against A. solani. Lower concentrations of chemical inducers were less effective than higher concentrations and required a booster application to induce acceptable level of resistance. All the three chemical inducers required more than one day lag period to induce the plant defense system against A. solani. Foliar spray of SA was less effective in inducing resistance in tomato against A. solani.

ACKOWLEDGEMENTS

The financial support given by Sri Lanka Council for Agriculture Research Policy (CARP) for the postgraduate degree program (M.Sc.) is highly acknowledged.

REFERENCES

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SCREENING TECHNIQUE FOR RESISTANCE TO COLLAR ROT OF COWPEA INCITED BY Sclerotium rolfsii

W.A.R.T. WICKRAMAARACHCHI, G.G. SIRIYAWATHI and H.P.D. SUMANASINGHE Regional Agricultural Research and Development Center, Aralaganwila

ABSTRACT

The collar rot of cowpea caused by Sclerotium rolfsii Sacc. is one of the most serious biotic constraints to yield. The growing of resistant varieties is the most practical and economical way to get rid of the disease. Sources of resistance for the disease are identified by screening of large number of cowpea lines using a reliable screening method. Screening under natural field infection which is being presently used was not repeatable due to differences of inoculum densities in the soil. Hence, attempts were made to develop an effective and efficient screening method for finding out suitable resistance sources. Series of experiments were conducted to find out suitable substrates (carrier material) for the mass multiplication of S. rolfsii and the most suitable plant age to achieve the desirable level of infection. Rice bran sand medium was found to be the most suitable substrate for mass multiplication of S. rolfsii. Six varieties of cowpea were inoculated with rice bran + sand medium at 3 different ages and percent collar rot incidence was taken weekly for 3 times. Forty five days old plants as well as week after inoculation showed high variation of collar rot incidence. New screening technique formulated consists of several parameters such as mass multiplication of S. rolfsii on rice bran sand medium for 7 days, inoculation of 45-day-old cowpea plants with mass multiplied inoculum at the rate of 50g per 1m length of cowpea row, scattered paddy straw along rows to enhance fungal growth, assessment of collar rot incidence week after inoculation and determination of resistant reaction based on percent collar rot incidence.

KEYWORDS: Collar rot, Cowpea, Sclerotium rolfsii, Screening technique, Rice bran sand medium.

INTRODUCTION

Cowpea (Vigna unguiculata (L.) Walp is the second most important grain legume crop in Sri Lanka. Seedlings as well as mature cowpea plant mortality due to collar rot incited by Sclerotium rolfsii Sacc. is one of the most destructive diseases in cowpea in dry zone of Sri Lanka where grain legume cultivations are more prevalent (Wijethilaka, 2003). Disease incidence can go up to 20 percent under intensive cultivation in sand rich soils. Sclerotium rolfsii is the most abundant and severe in sandy or sandy loam soils (Mehan et al., 1995).

Various options are available for the management of collar rot of cowpea such as crop sanitation, crop rotation, control of moisture, soil amendments, biological control and genetic resistance (Mehan et al., 1995). Cultural control measures are not much effective under high inoculum pressure in the soil along with favorable environmental conditions. Crop 40 WICKRAMAARACHCHI et al. rotation does not offer practical option for the management of this disease since the pathogen has broad host range. Several soil fungi, bacteria and mycorrhizal fungi highly antagonistic to S. rolfsii have been identified in India and Philipines (Prasad and Rangeshwaran, 2001; Cuevas et al., 2001 and Paningbatan, 1994). However, such biological strategies are yet to be developed in Sri Lanka. Being a soil borne pathogen, it is not economical to control S. rolfsii through soil drenching with fungicides. Further, no effective fungicides are available against S. rolfsii. Limited availability and narrow spectrum of control with chemicals discourage farmers adopting chemical control measure.

Breeding for resistance against collar rot has little scope due to the difficulty in the screening of accessions and lines owing to unavailability of an efficient and reliable screening technique (Rajapakse, personal communication). Evaluation of resistance to collar rot has been based on field experiments under natural inoculum level. Field screening technique with known inoculum densities and suitable age of the host plants is required for more precise results. However, progress in developing such techniques has been limited. The present investigation was undertaken to find out suitable substrate for mass production of S. rolfsii and to find out the correct age of plants to achieve desirable infection to be adopted as an effective screening method.

MATERIALS AND METHODS

Experiments were conducted at the research field at Regional Agricultural Research and Development Center (RARDC), Aralaganwila located in Low Country Dry Zone (DL2b) of Sri Lanka.

Isolation of the pathogen

A few infected cowpea plants having white luxurious mycelial growth and abundant sclerotial bodies of the fungus at the collar region and the root region were collected from the research field at RARDC, Aralaganwila. The fungus was isolated by plating such sclerotial bodies on potato dextrose agar (PDA) plates. The culture was purified by hyphal tip culture method and maintained on PDA at 25°C.

Mass production of Sclerotium rolfsii inoculum

Several substrates (carrier materials) which are readily available in the area were tested for their ability to support the growth of the pathogen. Series of laboratory experiments were conducted using six substrates viz. rice straw, parboiled rice grains, parboiled sorghum seeds, rice bran + sand medium, maize meal + sand medium and cowdung + sand SCREENING TECHNIQUE FOR COWPEA COLLAR ROT 41 medium to find out the best substrate for mass production of Sclerotium rolfsii. Chopped straw, rice and sorghum were soaked in tap water overnight. Chopped straw was drained out and kept in autoclavable polyethelene bags. Soaked rice and sorghum seeds were boiled for 20-30 min until seeds get spilt. Rice bran was mixed with clean sand at the rate of 1:1 (W/W) and little water to make rice bran +sand medium. Similarly maize meal + sand medium was prepared using maize flour. Cowdung was mixed with sand to prepare cowdung + sand medium. All substrates in autoclavable polyethelene bags were autoclaved for 30 min at 121°C for 2 consecutive days. Each bag containing 100 g of autoclaved (sterilized) carrier material was inoculated with 5 discs of S. rolfsii taken from growing edge of one week old culture. Then all bags were incubated in an incubator at 25°C. Mycelial growth was observed periodically until any one of the bags was fully covered with white mycelium. Percent mycelial growth was calculated based on how much of media covered with mycelium. The experiment was laid out as Complete Randomized Design (CRD) with 4 replicates.

Inoculation of cowpea plants at different ages

Six varieties of cowpea (MICP-01, MICP-02, MICP-03, MI-35, Dhawala and Waruni) were separately sown during December, 2005 – January 2006 in the field at 15 days interval to achieve 3 sets of plants of different ages. There were 3 separate sets of plants with 15 days old, 30 days old and 45 days old in the field after 45 days. All plots of plants at different ages were uniformly inoculated with mycelia of pathogen grown on rice bran + sand medium at the rate of 50g per 1m of cowpea row according to the method described by Mehan et al. (1995). Inoculum was applied to the soil surface around the base of cowpea plants. Paddy straw was scattered along the rows as a mulch to enhance the fungal growth on soil. Inoculated plots were regularly irrigated to maintain the soil moisture at the field capacity.

Assessment of collar rot incidence

Collar rot incidence was assessed 3 times 1 WAI (Weeks After Inoculation), 2 WAI and 3WAI by counting dead and wilted plants showing S. rolfsii sclerotia and/or mycelial growth at the collar region of the cowpea plants. Percentage incidence was computed from the total number of collar rot infected plants in a plot.

Experiments were laid out as RCBD and data were analyzed as 2 factor factorials. (Varieties vs WAI and varieties vs age of crop). 42 WICKRAMAARACHCHI et al.

RESULTS AND DISCUSSION

Mass production of Sclerotium rolfsii

Six different types of substrates were evaluated for their ability to support the growth of S. rolfsii. The highest growth was observed on rice bran sand medium (95.00%) followed by parboiled rice grains (81.67%). However, differences observed among these two treatments were not significant (P=0.05) suggesting both rice bran + sand and parboiled rice media equally supported the growth of S. rolfsii (Table 1). Mehan et al. (1995) reported that re-hydrated autoclaved groundnut shells, sorghum grains and oat meal were ideal for mass multiplication of S. rolfsii isolated from groundnut. Most of the bags with rice bran + sand medium were fully covered with the mycelium within 7 days from inoculation. The inoculum has also been multiplied on sorghum sand, wheat sand or grain sand mixtures (Asghari and Mayee, 1991).

Table 1. Percent mycelial growth of S. rolfsii on different substrates 7 days after inoculation.

Trt. No. Substrates Pcercent mycelial growth 1 Rice straw 11.67 bc 2 Parboiled rice grains 81.67 a 3 Parboiled sorghum grains 9.33 bc 4 Rice bran sand medium 95.00 a 5 Maize meal sand medium 21.00 b 156 Cowdung sand medium 2.33 c LSD (P=0.05) 11.79 CV% 17.54 Values are mean of 4 replicates; Values with same letter in the column are not significantly different at P=0.05

Cowdung sand medium did not support the growth of pathogen. Cowdung may have inhibitory effect on growth of the pathogen. Fungal growth in cowdung sand medium was confined to the agar discs inserted for inoculation. Maize sand medium, rice straw and parboiled sorghum showed poor mycelial growth of 21.00, 11.67 and 9.33 percent respectively. Even though maize meal sand medium showed poor mycelial growth in this experiment, Anahosur (2001) used similar kind of maize sand medium for multiplication of S. rolfsii. Further sterilized sand maize meal medium (1 maize meal: 9 sand W/W) was used to produce inoculum of S. rolfsii pathogenic to groundnut (Biswas and Sen, 2000). According to Mehan et al. (1995), sorghum was one of the ideal grains for mass multiplication of S. rolfsii. However, sorghum did not show promising results in this experiment. Exceptionally high moisture retention in parboiled sorghum grains may have led to poor fungal growth. SCREENING TECHNIQUE FOR COWPEA COLLAR ROT 43

Field inoculation of S. rolfsii

Since interactions between varieties and WAI are not statistically significant, mean data of percent collar rot incidence of different varieties at different ages are presented in Table 2. There are no published data regarding the resistant reactions of recommended cowpea varieties available in Sri Lanka against collar rot. However, MI-35, Dhawala and Waruni varieties used in this experiment were ranked respectively as moderately susceptible, highly susceptible and highly resistant against collar rot based on the long term performances under research fields and farmer fields in Sri Lanka (Wasala, personnel communication).

Table 2. Percent collar rot incidence of different cowpea varieties at different ages.

Trt.No. Varieties Percent collar rot incidence 15 days old plants 30 days old 45 days old plants plants 1 MICP-01 59.18 a 58.75 ab 39.44 b 2 MICP-02 46.37 bc 31.90 c 41.16 b 3 MICP-03 59.97 a 44.59 bc 39.27 b 4 MI-35 45.87 bc 36.49 c 62.98 a 5 Dhawala 41.95 c 70.43 a 76.36 a 6 Waruni 53.47 ab 38.54 c 28.08 b LSD (P=0.05) 10.61 17.92 15.09 CV% 17.12 31.61 26.01 Values are average of collar rot incidence taken at 3 different dates ( 1week, 2 weeks and 3 weeks) from inoculation; Values with same letter in the column are not significantly different at P=0.05

According to Table 2, 45-day-old plants showed considerable variation of percent collar rot incidence over varieties. The highest range of collar rot incidence (from 28.06 to 76.36%) was recorded in 45-day-old plants compared to 30-day-old plants (from 31.90 to 70.43%) and 15-day-old plants (41.95 to 59.97%). The lowest percent disease incidence of 28.06 was recorded in variety Waruni when inoculatation was done on 45 day old plants. These observations were in accordance with long term field experiences. Thus inoculation of 45-day-old plants would be a better option to exploit the inherent resistance of varieties or lines to be tested. Percent collar rot incidence among varieties tested reflected the prior information of resistant reactions known for MI-35, Dhawala and Waruni in 45-day-old plants. The lowest collar rot incidence (28.08) was recorded in Waruni which is said to be highly resistant. Similarly the highest collar rot incidence (76.36) was recorded in Dhawala which is said to be highly susceptible. 44 WICKRAMAARACHCHI et al.

Table 3. Percent collar rot incidence of different cowpea varieties at different time periods from the field inoculation.

Tr. No. Varieties Percent collar rot incidence 1 WAI 2 WAI 3 WAI 1 MICP-01 30.75 b 58.97 ab 67.64 ab 2 MICP-02 25.35 c 38.33 d 55.75 b 3 MICP-03 21.85 d 56.92 abc 65.06 ab 4 MI-35 29.11 b 51.01 bcd 65.89 ab 5 Dhawala 43.43 a 69.61 a 75.48 a 6 Waruni 21.94 d 40.54 cd 57.62 b LSD (P=0.05) 2.96 17.65 16.26 CV% 31.98 27.71 20.78 Values are average of collar rot incidence taken at 3 different ages of plants; Values with same letter in each column are not significantly different at P=0.05; WAI=Weeks After Inoculation

Data in Table 3 show that collar rot incidence recoded at 1, 2 and 3 weeks after inoculation (WAI) differed considerably. Percent collar rot incidence increased with increasing age. High infection was observed at 3 WAI ranging from 55.75 to 75.48 percent. Therefore, inherent resistance could not be traced. However, inherent resistance could be exploited at 1 WAI and observations recorded reflected the long term field performances of recommended varieties of cowpea.

CONCLUSIONS

Rice bran + sand medium and parboiled paddy are better substrates (carrier materials) for mass multiplication of S. rolfsii compared to maize meal + sand medium, rice straw, parboiled sorghum and cowdung + sand medium. However, rice bran being a by product of rice milling industry and readily available at a low cost, can be recommended to be used as a substrate for mass multiplication of S. rolfsii.

The proposed screening technique consists of several parameters. Forty five day cowpea plants grown in the open field are inoculated with mass multiplied S. rolfsii inoculum on rice bran sand medium for one week at the rate of 50g per 1m length of cowpea row. Paddy straw is scattered along the rows to enhance fungal growth on soil. Soil moisture is maintained at field capacity to avoid the desiccation of growing fungi. Varietal differences in resistace to collar rot are estimated by assessing the percent collar rot incidence week after inoculation of pathogen. SCREENING TECHNIQUE FOR COWPEA COLLAR ROT 45

ACKOWLEDGEMENTS

Guidance and encouragement extended by Dr SHSA de Silva, Deputy Director (Research), Regional Agricultural Research and Development Center, Aralaganwila is highly acknowledged.

REFERENCES

Anahosur, K.H. 2001. Integrated management of potato Sclerotium wilt caused by Sclerotium rolfsii. Indian Phytopathology 54(2):158-166. Asghari, M.A. and C.D. Mayee. 1991. Comparative efficiency of management practices on stem and pod rots of groundnut. Indian Phytopathology 44:328-332. Biswas, K.K. and C. Sen. 2000. Management of stem and pod rot of groundnut caused by Sclerotium rolfsii through Trichoderma harzianum. Indian Phytopathology 53(3):290-295. Cuevas, V.C., A.M. Sinohin and E.A. Arro. 2001. Efficacy of Trichoderma spp. As biocontrol agent of Sclerotium rolfsii Sacc. Philipine Agricultural Scientist 84(1): 35- 42. Mehan, V.K., C.D. Mayee, T.B. Brenneman and D. McDonald. 1995. Stem and pod rot of groundnut, Information bulletin no. 44. ICRISAT, India. 23p. Paningbatan, R.A. 1994. Trichoderma species for the biocontrol of sweet pepper stem rot (Sclerotium rolfsii Sacc.). Philipine Phytopathology 30(1):16-25. Prasad, R.D. and R. Rangeshwaran. 2001. Biological control of root and collar rot of chickpea caused by Sclerotium rolfsii. Annals of Plant Protection Sciences 9(2): 297- 303. Wijethilaka, L.C. 2003. Biological control of collar rot in cowpea (Vigna unguiculata (L.) walp.). Annals of the Sri Lanka Department of Agriculture 5:299-315. Annals of Sri Lanka Department of Agriculture. 2006.8:303-311.

EVALUATION OF DIFFERENT DOSAGES OF SELECTED FUNGICIDES AGAINST TOMATO EARLY BLIGHT DISEASE

W.A.R.T. WICKRAMAARACHCHI, H.P.D. SUMANASINGHE and G.G. SIRIYAWATHI Regional Agricultural Research and Development Center, Aralaganwila

ABSTRACT

Early blight caused by Alternaria solani is one of the major fungal diseases of tomato (Lycopersicon esculentum Mill.). Control of this disease has been primarily accomplished by foliar application of chemical fungicides especially during the maha season. Exploration of the efficacy of new fungicides and new formulations of already recommended fungicides is highly prioritized. Different dosages of Chlorothalonil 500 g/l SC (Daconil SC) and Copper hydroxide 57.6% DP (Champ DP) were evaluated with Copper oxichloride 50%WP (Copperdoz 50% WP) and Mancozeb 80% WP (Manzate) as check fungicides against tomato early blight disease during wet season under natural conditions of infection in the open field. Spraying was started 40 days after transplanting and continued further five times at ten day intervals. Percent Disease Index (PDI) was regularly recorded at ten day intervals just before each sprayings. Marketable yield was recorded. Plots treated with Chlorothalonil 30 ml/10 l (1500 ml/ha) showed the highest yield and present disease index was comparable with plots treated with Chlorothalonil 35ml/10l (1750 ml/ha). Thus Chlorothalonil at 30ml/10l (1500 ml/ ha) was found be the most effective and economical in controlling tomato blight. None of the dosages of Copper hydroxide 57.6% (Champ DP) performed better. Therefore, foliar spray of chlorothaloni 500 g/l SC at the rate 30ml/10l (1500 ml/ha) is recommended for management of tomato early blight.

KEYWORDS: Alternaria solani, Chlorothalonil, Copper hydroxide, Early blight, Fungicides, Tomato.

INTRODUCTION

Tomato (Lycopersicon esculentum Mill.) production is hindered by damage due to pests and diseases (Fontem et al., 1999). Early blight caused by Alternaria solani (Ellis and Martin) Jones and Grout, is one of the most severe fungal diseases of tomato (Fontem et al., 1999). High soil moisture and high atmospheric humidity are favorable for the development and the spread of the early blight (Arjunan et al., 1999). Early blight is a common disease of tomato all over India (Rangaswami and Mahadewan, 1999). Early blight disease is a common leaf spot disease in the USA (Spletzer and Enyedi, 1999). Tomato early blight disease can cause a reduction in yield from 50 to 80 percent under epidemic conditions (Mathur and Shekhawat, 1986).

Alternaria solani, a nectrophic pathogen can cause disease on foliage (leaf blight), stem (stem rot) and fruit (fruit rot) resulting in severe 48 WICKRAMAARACHCHI et al. damage during all stages of tomato plant development (Nash and Gardner, 1988; Spletzer and Enyedi, 1999). Control of early blight diseases has primarily been accomplished by the application of chemical fungicides, long crop rotations, using resistant cultivars and sterilization of seed beds either with steam or fumigants (Spletzer and Enyedi, 1999). Long crop rotations are not economically feasible in profit oriented intensive commercial cultivation of tomato under limited extent of lands available. Sufficient early blight resistance is not known within the cultivated species of tomato (Nash and Gardner, 1988).

Currently management of early blight disease principally relies on foliar application of chemical fungicides (Keinath et al., 1996) in spite of problems and adverse effects (Pharand et al., 2002 and Singh et al., 2000). Effective and safer formulations of fungicides enable the active ingredient to exert its full biological activity with consideration of environmental hygiene and product safety (Schmidt and Grupp, 1993). Suspension Concentrations (SC) and Dry Pill (DP) are examples for such improved formulations of fungicides that are increasingly safer and effective (Anon., 2002).

Calendar based application of fungicides at 7-10 days interval beginning soon after transplanting and continued through harvest effectively control the early blight of tomato (Mills et al., 2002). Therefore, evaluation of safe and effective formulations of new fungicides for the control of this disease is highly prioritized. The present study was planned to find out the efficacy of different concentrations of certain fungicides against early blight disease of tomato.

MATERIALS AND METHODS

Experiments were conducted at the research field of Regional Agricultural Research and Development Centre, Aralaganwila situated in the Low Country Dry Zone (DL2b) of Sri Lanka during maha season 2005/2006. Different dosages of new formulations of two fungicides viz. Chlorothalonil 500g/l SC (Daconil 500 SC) and Copper hydroxide 57.6%DP (Champ DP) were field evaluated for their efficacy against early blight of tomato. Copper oxichloride 50% (Copperdoz 50% WP) and Mancozeb 80% WP (Manzate) were used as check fungicides. Dilutions, rate of applications and spraying schedule of fungicides are shown in Table 1.

Twenty one day old seedlings of tomato (var. T-245) were transplanted on 1m X 3m field plots at recommended spacing of 80cm X 50cm during second week of November 2005. Each treatment was replicated 3 times in a randomized complete block design. Plants were grown in the field under natural conditions. All the cultural practices including manuring, FUNGICIDES AGAINST TOMATO EARLY BLIGHT 49 fertilization, irrigation, weeding, staking were adopted according to the recommendation given for tomato by Department of Agriculture, Sri Lanka. Table 1. Fungicides, dilutions, rate of applications and their spaying schedule.

Trt. Common Trade Dilution Rate Spraying schedule No. name name of 40 50 60 70 80 90 Appli D D D D D D n AP AP AP AP AP AP 1 Chlorothalo Daconil 25 ml/10 l 1250       nil 500g/l 500 SC ml/ha SC 2 Chlorothalo Daconil 30 ml/10 l 1500       nil 500g/l 500 SC ml/ha SC 3 Chlorothalo Daconil 35 ml/10 l 1750       nil 500g/l 500 SC ml/ha SC 4 Copper Champ 20 g /10 l 1000       hydroxide DP g/ha 57.6% DP 5 Copper Champ 30 g /10 l 1500       hydroxide DP g/ha 57.6% DP 6 Copper Copper 40 g / 10 l 2000g       oxichloride doz /ha 50% WP 50%WP 7 Mancozeb Manzate 20 g / 10 l 1000g       80% WP /ha 8 Control - - -       (water spread) DAP = Days After Planting;  = Denotes spraying of fungicide on the respective date

Fungicides spraying was commenced just after appearance of typical early blight symptoms (40 DAP) and continued for further 5 times at 10 days interval by using a hand operated knapsack sprayer with corn type nozzle. Control plots were similarly sprayed with water. Early blight disease development was recorded regularly at 10 days intervals for 5 times using 0-4 points scale (Wickramaarachchi, 2005) as shown below.

0 = Infection free or nearly so 1 = Trace to 25% of leaf area blighted 2 = 26-50% of leaf area blighted 3 = 51-75% of leaf area blighted 4 = 76-100% of leaf area blighted Percent Disease Index (PDI) was computed by using following formula given by Wickramaarachchi (2005).

PDI = Sum of numerical ratings of plants x 100 50 WICKRAMAARACHCHI et al.

Total number of plants assessed x maximum rating

At the end of the experiment, area under disease progress curve (AVDPC) was estimated on the basis of PDI using the following formula (Chandra et al., 2001), where Si = PDI at the end of 10 days, i,k = number of successive evaluation, d=interval between i and i-1.

k AUDPC = ∑ i=3 ½ (si+si-1) X d

Harvesting was started 8 weeks after planting and continued for 4 weeks with 10 picks. Total marketable yield (t/ha) was calculated based on yield of net harvested area of the plots. Standard analysis of variance (ANOVA) and mean separation were performed using MSTATC 6.0 statistical software package

RESULTS AND DISCUSSION

Foliar application of fungicides for the effective and efficient management of tomato early blight has been demonstrated by many scientists (Keinath et al., 1996 and Dillard et al., 1997). Fungicide spray was commenced at 40 DAP when typical early blight symptoms (Bose et al., 1980) just appeared in the field. Kapsa and Osowski (2003) observed the early blight symptoms on potato 41 days after planting.

Data presented in Table 2 clearly show that average PDI and AUDPC have given an estimate of disease development. The highest average disease severity of 62.5 % was recorded in untreated plots. All the dosages of different fungicides tested showed significantly lower disease severity than control plots. Keinath et al. (1996) noticed that all plots receiving fungicides had less blighted foliage than the non-sprayed control plots in an experiment treated with fungicides to control early blight. The PDI of non-sprayed control plots reached up to 96.51 percent at latter part of growing season (Fig. 1) suggesting the requirement of effective fungicide treatments even late in the season. A study revealed that destruction of potato plants reached up to 97.5 present at the end of the growing season (Kapsa and Osowski, 2003). The highest value for AUDPC (1822.78) was also reported in non-sprayed control plots. Copper containing fungicide treatments such as 20g/10l of copper hydroxide 57.6% DP, 30g/10l of copper hydroxide 57.6%DP and copper oxichloride 50% WP showed significantly higher average PDI 48.29%, 48.63% and 50.94% respectively than other fungicides. Even though copper fungicides are recommended for the control of early blight on tomato (Anon., 1997), tested dosages of copper fungicides in this experiment did not exhibit satisfactory control of early blight. Washing of copper fungicides and prolonged application intervals (10 days) may lead to the efficacy of copper FUNGICIDES AGAINST TOMATO EARLY BLIGHT 51 fungicides. Further, concentrations and application intervals of copper hydroxide 57.6% DP tested may not be sufficient enough to control the disease under high disease inoculum pressure. According to the technical information published by Nufarm, USA for Champ DP, 1.55kg/ha of chemical should be applied at 7 days intervals when conditions are highly favorable for infection (Anon., 2002). Saucassani and Rho (2002) reported that copper hydroxide effectively controlled the grapevine downy mildew at the lowest copper rate.

Table 2. Effect of fungicides on average percent disease index (Ave PDI), area under disease progress curve (AUDPC) and total yield.

Trt. Fungicides Dosage Ave AUDPC Total No. PDI yield (t/ha)

1 Chlorothalonil 500g/l SC 25 ml/10 l 37.46 c 1036.04 c 14.79 cd

2 Chlorothalonil 500g/l SC 30 ml/10 l 23.27 d 649.13 d 20.42 a

3 Chlorothalonil 500g/l SC 35 ml/10 l 19.75 d 545.67 d 20.10 ab

4 Copper hydroxide 57.6% DP 20 g /10 l 48.29 b 1359.59 b 8.96 ef 5 Copper hydroxide 57.6% DP 30 g /10 l 48.63 b 1364.95 b 10.63 def

6 Copper oxichloride 50% WP 40 g / 10 l 50.94 b 1451.91 b 11.46 de 7 Mancozeb 80% WP 20 g / 10 l 34.55 c 943.71 c 15.94 bc

8 Control (water spread) - 62.58 a 1822.78 6.67 f LSD (P=0.05) 5.53 190.80 4.32 CV% 7.76 8.62 18.11

Values are mean of 3 replicates; Values with same letter in the column are not significantly different at P=0.05

Spray of chlorothalonil 500 g/l at the rate of 35 ml/10 l gave the lowest average PDI of 19.75 followed by chlorothalonil 500 g/l 30 ml/10 l (23.27). However, these values were on par and superior over other treatments. 25 ml/10 l of chlorothalonil 500 g/l was not as effective as 30ml/10l and 35 ml/10 l of same chemical. It is evident that disease severity decreased with increased concentration of chlorothalonil. Increasing concentration of daconil from 16 ppm to 31 ppm decreased cucumber downy mildew from 33% to 17% (Anon., 1990). Efficacy of chlorothalonil 25 ml/10 l was found to be on par with manocozeb 80%WP 20 g/10 l exhibiting 37.46% and 34.55% PDI values respectively. Mancozeb 80%WP 20 g/10 l has already been recommended for the control of early blight in tomato (Anon., 1997). 52 WICKRAMAARACHCHI et al.

AUDPC was the lowest in plots treated with chlorothalonil 35ml/10l at 10 days interval followed by chlorothalonil 30 ml/10 l with 545.67 and 649.13 respectively. Since these two values are statistically on par, chlorothalonil 30 ml/10 l was the highest effective and the lowest possible concentration tested. Keinath et al. (1996) reported that area under the disease progress curve for early blight of tomato was the lowest with weekly application of fungicides. Chlorothalonil applied at 10 days interval for early blight of tomato resulted in the highest benefit per ha compared to the 7 and 14 days interval (Byrne et al., 1997). Chlorthalonil being a contact fungicide has advantageous of low selectivity of pathogen and low risk of resistance compared to the systemic fungicides in long term application (Anon., 1990). It is evident that soluble concentration, new formulation of chlorothalonil used in this experiment, has more advantages. According to the technical information of Daconil published by SDS Biotech KK, Japan, preventive activity of SC formulation of daconil is better than WP formulation. Further, SC formulation shows better rain fastness over WP formulation. In another experiment, foliar application of 31.1 ppm concentration of SC formulation of daconil showed lower disease severity of 16.7% of cucumber downy mildew compared to the 75% with same concentration of WP formulation (Anon., 1990).

100

80

60 I D P 40

20

0 50th day 60th day 70th day 80th day 90th day Time

C hlo ro thalo nil 25g/10m l C hlo ro thalo nil 30g/10m l C hlo ro thalo nil 35g/10m l C o pper H ydro xide 20g/10l C o pper H ydro xide 30g/10l C o pper o xichlo ride M a n c o z e b C o n t r o l

Figure 1. Early blight disease development in different fungicides under field conditions.

Very less deposits (stains) were visible on tomato fruits treated with chlorolnalonil compared to copper oxychoride and mancozeb treatments (Data not shown). Chlorolnalonil SC formulation hand very less deposits on fruits compared to other WP formulations (Anon., 1990). High efficacy of chlorothalonil against early blight of tomato may be due to inherent effectiveness of active ingredients as well as novel characteristics of the new FUNGICIDES AGAINST TOMATO EARLY BLIGHT 53 formulation. Resistance of daconil to wash off after application due to excellent surface sticking characteristics of formulation and low water solubility of the active ingredient contribute to high efficacy of chlorothalonil SC against many pathogens even during wet seasons with frequent rains (Anon., 1990).

In addition to early blight, chlorothalonil possesses broad spectrum of activity against many fungal pathogens associated with tomato such as Phytopthara infestans (Late blight), Stemphylium solani (Gray leaf spot), Cladosporium fulvnm (Gray leaf mold), Septoria lycopersici (Leaf spot) and Colletotrichum phomoides (Anthracnose) (Anon., 1990). Preventive application of chlorothalonil against early blight may automatically prevent the crop loss from above diseases too.

Total marketable yield of tomato in different fungicide treatments are also shown in Table 2. Efficacy of fungicides leads to reducing the disease severity and reflected in corresponding yields. The lowest yield of 6.67 t/ha was recorded in control plots followed by copper hydroxide 20 ml/10 l (8.96 t/ha) and 30 ml/10 l (10.63 t/ha). These two yield values are on par with control. Even without any fungicide application, control plots gave 6.67 t/ha yield. This is because early blight systems first appeared in this particular experiment only 40 days after planting in the field. The highest yield of 20.42 t/ha was recorded with the application of chlorothalonil 30ml/10 l followed by 20.10 t/ha with 35 ml/10 l of chlorothalonil. However, differences in yield between the above two treatments were not significant. According to the crop techno-guide of DOA, Sri Lanka, properly maintained tomato cultivation yields 20-30 t/ha with 10-20 picks.

CONCLUSIONS

The rate of disease development (given as AUDPC) and average percent disease index (Ave. PDI) are measures for estimates of efficacy of fungicides. Foliar application of certain fungicides tested can effectively be used for the management of early blight disease of tomato. Increasing concentration of chlorothalonil increases the efficacy against early blight. However, chlorothalonil 500g/l SC at the rate of 30 ml/10 l (1500ml/ha) is the optimum concentration for the management of disease. Chlorothalonil 500g/l SC at the rate of 30ml/10l can be sprayed at 10 days intervals as preventive measures to manage early blight of tomato. Chlorothalonil has less ability to develop resistant pathogens and leave less deposit (stains) on harvested fruits. All dosages of copper hydroxide 57.6% (Champ DP) are not much effective against early blight of tomato during wet season. Tomato yield increase from 6.67 t/ha to 20.42 t/ha (200% yield increase) with the application of chlorothalonil 500 g/l SC at the rate of 30ml/10l (1500 ml/ha). 54 WICKRAMAARACHCHI et al.

ACKOWLEDGEMENTS

Authors wish to thank Dr SHSA de Silva, Deputy Director (Research), Regional Agricultural Research and Development Center, Aralaganwila for his encouragement and the field staff attached to Division of Plant Pathology for their assistance.

REFERENCES

Anon., 1990. Technical information on Daconl ® 500. SDS Biotech KK, Japan. Anon., 1997. Pesticide recommendations, Department of Agriculture, Sri Lanka. 76 pp. Anon., 2002.Technical information on Champ DP, Nufarm Americas, Inc, USA. Arjunan, G., G. Karthikeyan, D. Dinakaran and T. Raguchander. 1999. Disease of horticultural crops. Department of Plant Pathology, Tamilnadu Agricultural University, Coimbatore, India. 399 pp. Bose, T.K., M.G. Som and J. Kabir. 1980. Vegetable crops. Naya Parkash, Calcutta, India. Byrne, J.M., M.S. Hausbeck, R.X. Latin. 1997. Efficacy and economics of management strategies to control anthracnose fruit rot in processing tomatoes in the Midwest. Plant Disease 81(10):1167–1172. Dillard, H.R., S.A. Johnston, A.C. Cobb and G.H. Hamilton. 1997. An assessment of fungicide benefits for the control of fungal diseases of processing tomatoes in New York and New Jersey. Plant Disease 81(6):677–681. Fontem, D.A., Y.D. Gumedzoe and Nono- R. Womdim. 1999. Biological constraints in tomato production in the western highlands of Cameroon. Tropicultura (Belgium). 16-17 (2):89-92 Kapsa, J. 2003. Usefulness of fungicides with various mode of actions in the protection of potato crops. Journal of Plant Protection Research (Poland) 43(2):191–198. Kapsa, J. and J. Osowski. 2003. Efficacy of some selected fungicides against early blight (Alternaria Spp) on potato crops. Journal of Plant Protection Research (Poland) 43(2):113–120. Keinath, A.P., V.B. DuBose and P.J. Rathwell. 1996. Efficacy and economics of three fungicide application schedules for early blight control and yield of fresh market tomato. Plant Disease 80(11):1277-1282. Mathur, K. and K.S. Shekhawat. 1986. Chemical control of early blight in kharif sown tomato. Indian Journal of Mycology and Plant Pathology 16(2):235-236. Mills, D.J., C.B. Coffman, J.R. Teasdale, K.L. Everts and J.D. Anderson. 2002. Factors associated with foliar disease of staked fresh market tomatoes grown under differing bed strategies. Plant Disease 86:356-361. Nash, A.F. and R.G. Gardner. 1988. Tomato early blight resistance in a breeding line derived from Lycoprrsicon hersutum PI 126445. Plant Disease 72:206–209. FUNGICIDES AGAINST TOMATO EARLY BLIGHT 55

Pharand, B., O. Carisse and N. Benhamou. 2002. Cytological aspects of compost–mediated induced resistance against Fusarium crown and root rot in tomato. Phytopathology 92:424-438. Rangaswami, G. and A. Mahadevan. 1999. Diseases of crop plants in India, 4 th edition. Prentice Hall of India Private Limited, New Delhi, India. 536 pp. Saneassani, G.P. and G. Rho. 2002. Evaluation of various copper fungicides effectiveness in grapevine downy mildew management. Atti–delle–Gioruate– Fitopatologiche (Italy). 2:303–308. Schmidt, U. and B. Gruup. 1993. The distribution problems in pesticides. Courier Agrochem by Bayer. 93(1):12-17. Singh, N., R.K. Grewal and K.K. Chahal. 2000. Dissipation of mancozeb residues in tomato. Pestology 24(9):28-30 Spletzer, M.E. and A.J. Enyedi. 1999. Salicylic acid induces resistance to Alternaria solani in hydro phonically grown tomato. Phytopathology 89:722-727 Wickramaarachchi, W.A.R.T. 2005. The effect of rhizobacteria on increasing plant growth and inducing systemic resistance in tomato against early blight disease. Annals of the Sri Lanka Department of Agriculture 7:309–325. Annals of Sri Lanka Department of Agriculture. 2006.8:313-327.

BASELINE STUDY ON PUBLIC AWARENESS ON CROP WILD RELATIVES

A. WIJESEKERA1, A. HERATH2 and J. ILLANKOON3 1Crop Wild Relatives Project, Horticulture Crop Research and Development Institute, Gannoruwa, Peradeniya 2Department of Export Agriculture, Gatambe, Peradeniya 3Extension and Training Center, Peradeniya

ABSTRACT

Crop wild relatives (CWR) are an important Plant Genetic Resource for improving agricultural production and for maintaining sustainable agro-ecosystems. Sri Lanka has recognized that its national biodiversity conservation program planning, decision-making and implementation frameworks need substantial improvements in order for effective conservation and use of CWR. This paper presents the current status of public awareness relating to CWR. The study was based on a survey of a sample of multiple stakeholder categories associating with CWR. Several indicators assessed the awareness of CWR. The study revealed that many stakeholders have inadequate awareness about various subjects related to CWR. Even the stakeholders who are directly involved in CWR related activities, including protected area managers have low awareness. It is concluded that well-planned public awareness programs and capacity improvement are urgent needs to support national CWR conservation programs.

KEYWORDS: Crop Wild Relatives, Public Awareness, In situ Conservation.

INTRODUCTION

Wild relatives of crop plants, which include progenitors of crops as well as other species closely related to them, constitute an increasingly important resource for improving agricultural production and maintaining sustainable agro-ecosystems (Azzu and Collette, 2005). Sustainable conservation and use of crop wild relatives (CWR) are essential elements for increasing food security, thereby eliminating consumption poverty, and maintaining the environment. The natural populations of many CWR are increasingly at risk. They are threatened by habitat loss through destruction and degradation of natural environment, deforestation or their conversion to other uses. Examples are wild relatives of fruits, nuts, industrial crops (e.g. mango, rubber, coffee) and cereals etc.

Conservation and sustainable use of CWR has been recognized as a key element in the implementation of the convention on biological diversity to which Sri Lanka is a signatory (Secretariat of the Convention on Biological Diversity, 2005). Conservation and sustainable use is a relevant activity in Global Environment Facility (GEF) Operational Program #13 on Conservation and Sustainable Use of Biological Diversity important to agriculture (para. 19.iii). The promotion of in situ conservation of CWR and 58 WIJESEKERA et al. other wild plants for food production constitutes one of the 20 agreed activities of the FAO Global Plan of Action (FAO, 1996).

Sri Lanka has recognized that its national biodiversity conservation program planning, decision-making and implementation frameworks need substantial improvement in order for effective CWR conservation and sustainable use to occur (Ministry of Forestry and Environment, 1999). To achieve this objective, the Government of Sri Lanka is collaborating with the International Plant Genetic Resources Institute to implement a five year project on ‘In situ Conservation of Crop Wild Relatives through Enhanced Information Management and Field Application’ (UNEP/GEF Project, 2004). This multi-country, multi-stakeholder project is being implemented in Sri Lanka by the Department of Agriculture in collaboration with Biodiversity Secretariat of the Ministry of Environment and Natural Resources. National CWR conservation action plans, identifying priorities and programs for CWR conservation, do not yet exist in Sri Lanka, although some work has been carried out by Jayasuriya and Vaughan (2003). The limited development of conservation work on CWR is reflected in the insufficient awareness that exists among decision makers and the general public, of the importance of this resource and the need to maintain and use them.

It is imperative that decision makers and the general public have an adequate awareness about CWR and their in situ conservation for formulating effective conservation plans and sustainable use of CWR (UNEP/GEF Project, 2004). This study assessed the current status of public awareness in Sri Lanka on CWR and related issues. The specific objectives were to assess: (a) awareness about and use of crop wild relatives, (b) awareness of CWR-targeted conservation activities and policies, and (c) ways of disseminating information on CWR related issues and activities.

METHODOLOGY

The study was based on a sample survey of multiple stakeholders having some association with CWR. The sample had several categories of stakeholders namely (a) Policy makers (national and district level – 150 respondents); (b) Research scientists (ecological/conservation, social, plant breeders – 84 respondents); Protected area managers (37 respondents); (c) Extension workers and NGOs (273 respondents); (d) Local communities (361 respondents); (e) Students at higher education level (102 respondents); and (f) Students at primary education level (447 respondents). Each of these categories have a few stakeholder groups - for example there are 22 stakeholder groups in the Policy Makers Category; four groups in research scientist category and so on as listed in Table 1. A group consists of stakeholders with similar professional characteristics. There are 65 such PUBLIC AWARENESS ON CROP WILD RELATIVES 59 stakeholder groups divided among the categories listed above. The final sampling unit is the respondent in each stakeholder group – for example, there are 51 respondents in the Divisional Secretary stakeholder category. As such there are 1454 respondents distributed in the 65 stakeholder groups (Table 1).

Table 1. Major stakeholder categories, groups in each category and the number of respondents.

Major categories Stakeholder groups Respondents in in each category each category 1. Policy makers 22 150 2. Research scientists 4 84 3. Protected area managers 7 37 4. Extension workers and NGOs 10 273 5. Local communities 16 361 6. Students at higher education level 4 102 7. Students at primary education level 2 447 Total 65 1454

The sample is well distributed with respect to the geographical coverage of the stakeholders in the country. If the stakeholders are employed, then their district of employment is used as the relevant district to refer to the survey information or if unemployed the residential district is used. The study has covered 19 districts out of 26, distributed in eight provinces. Except Vavuniya, the districts in the Northern and Eastern Provinces were not included in the study. The largest sample (n=344) was from Anuradhapura and the lowest (1) from Matara. Kandy (283), Kurunegala (200), and Polonnaruwa (106) also had large samples.

The list below summarizes the indicators that were used in the study to assess the public awareness. Information required to derive these indicators was obtained from a set of questionnaires, designed for each stakeholder category.

1. Awareness assessment of the stakeholders about CWR. This is based on several variables namely:

a. General awareness of CWR b. Level of awareness of CWR rated from the lowest (scoring 1) to the highest (scoring 5) c. Awareness of importance of CWR d. Uses of CWR e. Prevalence of CWR in the region that the respondents live

2. Uses of CWR in various ways corresponding to the respondents’ regular activities 3. Awareness on the conservation of CWR 60 WIJESEKERA et al.

4. Sources of information about CWR and the relative importance of such sources 5. Awareness of the issues that many CWR are being extinct 6. Awareness of the importance of conserving CWR 7. Reasons for conserving CWR 8. Awareness of programs and projects that are implemented to conserve CWR

RESULTS AND DISCUSSION

General awareness on CWR

The general awareness was assessed by asking whether or not the respondents have heard of CWR in Sri Lanka. The survey indicated what the CWR were, whether they were wild relatives of crops, wild plants or weedy crops. Table 2 presents the results averaged over major categories of stakeholders. Respondents in the research scientist category have the best awareness while primary education category has the lowest.

Table 2. Respondents in each stakeholder category having the awareness of CWR (%).

Stakeholder category Aware Unaware Not Sample sure size (n) Protected area managers 86 8 5 37 Policy makers 69 17 14 150 Research scientists 100 0 0 84 Community 61 18 20 361 Extension workers and NGOs 77 10 13 273 Student at higher education level 92 6 2 102 Primary education 41 27 32 447 Grand sample 64 17 19 1454

The study assessed the depth of the awareness, using a knowledge rating, in addition to the general awareness. The lowest knowledge was represented by “0” rating and the highest by “5”. The respondents were asked to circle the appropriate number depending on their level of the knowledge about CWR. Table 3 summarizes the results.

Respondents’ awareness was further examined by questioning their understanding about the importance of CWR. The respondents, who answered positively, were asked to explain the importance of CWR. Table 4 summarizes the responses averaged around the stakeholder categories. About half of the respondents in the total sample were aware of the importance of CWR. Students and teachers in the primary education category had the lowest PUBLIC AWARENESS ON CROP WILD RELATIVES 61 awareness about the importance of CWR. Research scientists had the best awareness on CWR.

Table 3. Respondents in each stakeholder category who ranked (0 - poorest to 5 - highest) their knowledge about CWR (%).

Stakeholder category Rating Sample 0 1 2 3 4 5 size Protected area managers 3 16 59 16 3 3 37 Policy makers 3 59 31 4 1 2 150 Research scientists 5 26 36 21 7 5 84 Community 14 23 29 17 10 7 361 Extension workers and NGOs 7 39 31 14 7 2 273 Student at higher education level 10 36 35 10 6 3 102 Primary education 17 28 26 21 6 2 447 Grand sample 11 32 30 16 7 4 1454

Table 4. Response of stakeholders on their awareness about the importance of CWR (%).

Stakeholder category Aware Unaware Not sure Sample size (n)

Protected area managers 68 14 19 37 Policy makers 54 26 20 150 Research scientists 90 7 2 84 Community 52 28 20 361 Extension workers and NGOs 58 22 21 273 Student at higher education level 72 17 12 102 Primary education 28 38 34 447 Grand sample 50 27 23 1454

The level of awareness about the importance of CWR varies considerably among the stakeholder categories and groups. For instance, less than 50% of the respondents in Veterinary Officers and District Forest Officers groups were aware of the importance of CWR. In the policy makers’ category too, a notable number of stakeholders had very poor awareness of CWR. Of all stakeholder groups, policy makers’ category was the poorest in awareness.

Stakeholders involved in national planning, protected area management, and the extension service and also the community were tested on their awareness of the regional prevalence of CWR. Their awareness will be useful for planning and also conservation of CWR. Only about half of the 62 WIJESEKERA et al. overall sample knew that there were CWR in their regions/residential areas. Out of those who were not aware, policy makers predominated as in previous cases (Table 5).

General awareness of the stakeholders about CWR was assessed using four main indicators, namely overall general awareness, knowledge rating, awareness of the importance of CWR and awareness on regional prevalence of CWR. A composite knowledge score was constructed combining these four indicators in order to assess the overall knowledge level of the stakeholders and also to rank the stakeholders with the best knowledge to the worst. The composite knowledge score was constructed by allocating scores for each indicator.

Table 5. Stakeholder’ awareness on the prevalence of CWR in their work/residential regions (%).

Type of stakeholders Aware Unaware Not sure Sample size Protected area managers 51 8 41 37 Policy makers 31 23 46 150 Research scientists 62 26 12 84 Community 43 27 30 361 Extension workers and NGOs 36 20 17 273 Grand sample 42 26 32 747

90%

80%

70% y r o

g 60% e t a C

e h

t 50%

r o f

e r o

c 40% S

e g a r

e 30% v A

20%

10%

0% Primary Policy Makers Community Extension Protected Area Student at Research Education Workers & Managers Higher Scientists NGOs Education level

Figure 1. Average knowledge score of the stakeholder categories (%). PUBLIC AWARENESS ON CROP WILD RELATIVES 63

A stakeholder got 10 scores if answered positively for the indicator on general awareness of CWR or no score for negative answers. The level of awareness of CWR, the second indicator, was rated from the lowest (1) to the highest (5). A stakeholder got increasing scores for increasing rating, i.e. 1 score for rating 1; 2 scores for rating 2 and 5 for rating 5 etc. The third indicator, awareness on the importance of CWR, provided 10 scores for those who answered positively and 0 otherwise. The last and fourth indicator was on the awareness on prevalence of CWR in the region that the respondents lived. If a respondent answered positively, 10 scores were given or 0 otherwise. The maximum and minimum score that any respondent could obtain according to this scoring system was 35 (i.e. 10, 5, 10 and 10) and 0 respectively. The average composite score of each stakeholder category, expressed as a percentage of the total achievable score of 35, is presented in Figure 1. The research scientists got the highest score whereas the students at primary grades got the lowest. Policy makers and the extension workers who are involved in CWR related activities had barely adequate awareness. The respondents in the policy stakeholder category with the lowest awareness include environmental officers too.

Use of CWR in stakeholders’ regular activities

This indicator assesses whether different stakeholders use CWR knowingly or unknowingly in their regular work. It is expected that most of the stakeholders in categories such as the policy makers, extension workers, researchers, protected area managers and university students use CWR in their regular work. The respondents were asked during the survey whether or not CWR were featured in their regular work, which were planning, research, advisory or educational. The results are summarized in Table 6. Only about one quarter of the respondents in the overall sample were aware of CWR being included in their regular work. Ironically only about half of protected area managers knew that CWR was in one way or another related to their regular work. This clearly shows the lack of adequate awareness on CWR among the stakeholders.

Table 6. General awareness of stakehlder categories on whether CWR is featuring in their regular work (%).

Type of stakeholders Aware Unaware Not sure Sample size Protected area managers 41 16 43 37 Policy makers 19 72 9 150 Research scientists 43 55 2 84 Community 28 52 20 361 Extension workers and NGOs 18 29 53 273 Grand sample 25 47 28 905 64 WIJESEKERA et al.

The responses of stakeholders in the protected area manager category were further analyzed in order to assess their in-depth awareness about the subject, as they are one of the key stakeholder categories in dealing with CWR. Only about half of the protected area mangers’ category had the awareness about the existence of CWR in the protected areas. Forest and wild life officers were totally unaware of the presence of CWR in the protected areas. Those stakeholders in this category who were aware of having CWR in the protected areas under their purview were further questioned on three issues: (a) about the conservation status of CWR; (b) type of CWR being conserved; and (c) having a management plan to conserve important flora and fauna including CWR. Only 19% answered positively to the first issue, 20% had not responded and the balance 2% answered negatively. It is noteworthy that none of the Forest Officers, Veterinary Officers and Wild Life Assistant Director were aware of the conservation status of CWR in protected areas. The responses for second issue were also very poor. A large percentage (70%) of the stakeholders mentioned above were unaware of the type of CWR being conserved. Only about 20% mentioned that some CWR were indigenous species to a particular protected area while the balance 10% was of the opinion that some of the CWR were extinct. About 30% of the stakeholders responded positively to the third issue, which was on the management plans and another 30% reported saying that there were no management plans in protected areas. The results indicate that the awareness of protected area mangers category on CWR related issues is grossly inadequate.

The policy makers’ awareness was very poor on CWR being featured in agriculture or biodiversity policies, policy strategies and or any implementation plans of agriculture and biodiversity conservation. Only about 8% in this category were positive about CWR being addressed in these national policies. Analysis of individual groups in the policy makers’ category revealed that Provincial Agriculture Secretaries and a few of Divisional Secretaries were aware about the subject. Further, some of the district, divisional and municipal environmental officers who are involved in environmental activities were also totally ignorant about the issue. The lack of overall awareness could be due to the fact that they were not aware of what the CWR were and therefore they could not comment on their inclusion in policy documents.

The study attempted to elicit researchers’ practices in including CWR as a major or minor component in their regular research programs. They were also asked to list the type of CWR being used. About half of the group use CWR in their research work. A majority uses CWR as a small component of their research and only 6% of them use CWR as a major component. The most widely used CWR in research were wild tomato species PUBLIC AWARENESS ON CROP WILD RELATIVES 65 followed by wild okra species. The main uses were DNA finger printing, variety evaluation, and development of pest and disease resistant host plants. Traditionally communities use CWR for various purposes. It was revealed that about 28% of the respondents in the community category use CWR for various purposes including ornamental, timber, scion for budding and grafting, food, soil conservation, and shade. Predominant usages were ornamental and food purposes. Respondents in the community category indicated the easiness to find CWR using a rank which varied from 0 (most difficult to find) to 5 (easiest to find) and Figure 2 presents the results. The results clearly indicate that the users of CWR could find them easier than the non-users (Fig. 2).

30%

25% y t i n u

m 20% m o c

e h t

n i

s 15% t n e d n o p s e

R 10%

f o

%

5%

0% Most difficult to 1 2 3 4 Easiest to find find Scale of easiness to find (0 - most difficult)

Users of CWR Non-uses of CWR Figure 2. Stakeholders in the community category indicating the degree of difficulty to find CWR in their living areas (%).

Availability of CWR in living areas of respondents could influence the use. The survey, however, did not assess whether the uses were influenced by the easiness to reach CWR. The survey also inquired the community whether the availability or the abundance of CWR had been changing over time. As shown in Figure 3, a large percentage asserts that the availability of known CWR in their vicinity has decreased over time. Non- users of CWR also had the same response, and some of them said CWR were not available at all.

Reasons given by the community category for reducing the availability of CWR include forest clearance, lack of conservation attention, negative impact of development activities on CWR such as urbanization, road constructions etc., lack of proper conservation plans, over usage of CWR for 66 WIJESEKERA et al. various purposes such as timber and fire wood, as food crops etc., lack of awareness about conservation of CWR; and also adverse effects of environmental pollutions. However, these responses could well be based on prejudice of respondents rather than on their own careful observations.

The CWR and agricultural biodiversity are referred to in various national policies and strategies. The study assessed whether the respondents in the extension workers and NGO category were aware of this reference. Further, the study inquired whether extension programs focused on issues such as conserving biodiversity, agricultural diversity and conserving CWR. About 18% of the respondents in the extension category, mainly Agricultural Instructors, Technical Assistants, and Development Officers of the Department of Agrarian Development, have the awareness on conservation aspects mentioned above and CWR being referred in national policies. However, only 4% of the respondents in this category were able to highlight specific policies targeted at conservation aspects of CWR. One such policy that was referred was the Forest Ordinance. The balance was unaware of any national policy or policy strategies that address CWR conservation. Further, only 20% mentioned that extension programs focused on conservation related subjects. It revealed that the respondents in this category could not comprehend the connection between contents of extension programs and the subject of conservation.

70%

60% R W C

f o

e 50% c n a d n a b a 40% g n i n o i t n e

m 30%

s t n e d n o p

s 20% e R

f o

% 10%

0% Increased availability No change Decreased availability Not available

CWR Users CWE Non-users

Figure 3. Changes in availability of CWR during the last five years (% users and non- users of CWR).

Whether or not school curricula had included conservation subjects and also CWR was assessed using the relevant responses of the PUBLIC AWARENESS ON CROP WILD RELATIVES 67 primary and secondary education categories. According to the responses, aspects regarding biodiversity conservation and use were mentioned in the curricula. The higher education category too confirmed the inclusion of these subjects in curricula.

Information sources on CWR and biodiversity

Dissemination of information on all aspects of conservation and sustainable utilization of biological resources is a dire need. The available sources of information are electronic media (internet, television, radio); print media (news papers, technical bulletins and journals, pamphlets and posters); meetings; NGOs; and extension systems (protected area managers or protected area information centers, extension workers, field days etc.). The study assessed the popularity of information sources among the respondents using a ranking system. The degree of importance of a source to gather information was ranked from 0 - least important to 5-most important. An information source is claimed to be popular and important if the majority of the respondents selected it as well as allocated higher ranks to it. Figure 4 presents the percentage of stakeholders that selected and ranked information sources. It was found that all stakeholders used television and papers as the most popular media methods for getting conservation related information (Fig. 4).

80%

70%

60% s

t 50% n e d n o p

s 40% e R

f o

% 30%

20%

10%

0%

r s t s n s s n o s e y e O e o g r ti i r V th a rn G g i n te e d e T D e N a s ti s ll a ap O t an en e o u R P ld In t e p B ie m x m l F a E a e ic ar n t h ro ec P T Sources of Information

Rank 0 Rank 1 Rank 2 Rank 3 Rank 4 Rank 5 Figure 4. Stakeholders’ ranking of information sources according to the importance of the sources for getting conservation related information (%). 68 WIJESEKERA et al.

Technical bulletins, radio, newspapers, and television have also been selected by a larger number of stakeholders as sources of conservation information. This indicates that these sources could be effectively used as media methods to disseminate information on CWR.

Conservation of CWR

Public awareness on CWR conservation is critically important to implement conservation programs, particularly in situ conservation. The public awareness about the importance of conservation and conservation programs that are implemented in the country was assessed through four indicators: (a) awareness of the extinction of CWR; (b) awareness of the importance of conserving CWR; (c) reasons for conserving CWR; and (d) awareness of programs and projects that are implemented to conserve CWR.

About half of the respondents in the total sample were aware of the threat of extinction of CWR. However, there is a wide variation in the awareness. The respondents in the primary education category showed a low level of awareness whereas the protected area mangers category had a higher level (Table 6). In addition, the policy makers and extension workers categories, who are directly involved in protecting biodiversity including CWR, were aware of the fact that wild varieties are in the verge of being extinct.

Table 6. Awareness of the stakeholder categories about extinction of CWR (%).

Type of stakeholders Aware Unaware Not Sample size sure Protected area managers 84 3 14 37 Policy makers 78 3 19 150 Research scientists 71 14 14 84 Community 53 5 41 361 Extension workers and NGOs 78 4 18 273 Primary education 26 8 66 447 Grand sample 54 6 40 1352

Responding to the importance of conserving CWR, researchers mentioned that CWR were useful for future plant breeding and crop improvement, preserving important genetic traits in plants and maintaining biodiversity. In the community and the primary education categories, about 20% of the stakeholders were aware of the reasons listed above as important for conserving CWR. In addition, further 20% had mentioned a variety of other reasons for supporting CWR conservation. These include having benefits for future generations, having the genetic base required to the country, being useful as medicinal plants, having an economic importance, conserving PUBLIC AWARENESS ON CROP WILD RELATIVES 69 biodiversity, providing rich nutrients as food crops, and being useful in facing a famine or food insecurity situations etc..

The study also assessed the stakeholders’ awareness about conservation programs that are available in the country. About half of the total sample was unaware of any conservation program (Table 7). Among the stakeholders, research scientists had the highest level of awareness of this issue while the primary education category had the lowest.

Table 7. Stakeholders in all categories who have the awareness about conservation programs available in the country (%).

Type of stakeholders Aware Unaware Not Sure Sample size Protected Area Managers 32 49 19 37 Policy Makers 21 65 13 150 Research Scientists 57 29 14 84 Community 26 11 63 361 Extension Workers and NGOs 37 30 33 273 Primary Education 17 10 73 447 Grand Sample 27 23 50 1347

The CWR related issues and corresponding public awareness

Discussions in the previous sections focused on the findings of public awareness on various CWR related issues. The level of awareness varies across the categories of stakeholders and also varies across different issues. In order to provide a birds-eye view of this two-way variation, a matrix was developed summarizing the CWR related issues on one axis, stakeholder categories on the other axis and the awareness level in the body of the matrix. Table 8 presents the matrix. The key CWR related issues listed in the matrix are in situ and ex situ conservation; featuring CWR in regular work of various stakeholders; availability of conservation programs; regional importance of CWR; awareness of food crops related to CWR; awareness of new crop varieties; importance of CWR; awareness of CWR extinction; and general awareness of CWR. Percentages of stakeholders in each category who positively responded to each issue are presented in the body of the matrix in Table 8.

Highlighted cells of the matrix represent the issues and the stakeholders needing higher emphasis in training and capacity building interventions of a CWR conservation program. For instance, if more cells are shaded in a row of the matrix, that issue needs to be the focus of capacity enhancement program. Further, if a stakeholder category column has more shaded cells, that category of stakeholders should be the focus for training programs. 70 WIJESEKERA et al.

Accordingly, in situ and ex situ conservation; featuring in regular work of various stakeholders; availability of conservation programs; regional importance; and awareness of new varieties are important issues; whereas policy makers, community, extension and NGO officers, and primary education are such stakeholder categories requiring emphasis on training programs.

Table 8. Stakeholders in each category that positively responded for the awareness of CWR related issues (%).

CWR Related subjects Stakeholder Categories

s s s s y e n n t t l r r o o i s O p e e i i i n t t t k g G m u a a n a a a N c c e m n

s i m u u

a d c m d d d y s n

e e o m c n

a i h

r l a C y a c s r e r o e r r h a r P G e a g a k e m

i i r s d h r e

o e t t P R w a c

t e n t n o o i e r s d n P u t e t S x l e E v e l In situ and ex situ conservation 65 21 64 17 37 1 20 Featuring in regular work 41 19 43 28 18 25 Availability of conservation programs 32 21 57 26 37 17 27 Regional importance of CWR 51 31 62 43 36 42 Awareness of food crops related to CWR 70 59 48 41 70 26 45 Awareness of new crop varieties 51 37 83 70 35 47 Importance of CWR 68 54 90 52 58 72 28 50 Awareness in CWR extinction 84 78 71 53 78 26 54 General awareness of CWR 86 69 100 61 77 92 41 64

CONCLUSIONS

This study assessed the current situation of public awareness of CWR and a number of other aspects related to CWR. Focus was mainly on their general understanding of CWR, usages of CWR, the abundance or lack of it, conservation needs and about the conservation programs of CWR. The study consistently indicated that the awareness about many aspects relating to CWR is grossly inadequate among the stakeholders. It is noteworthy that many of the stakeholders who are directly and decisively involved in CWR related work such as protected area managers, policy makers, and extension personnel need awareness improvement in the CWR related subject areas. The assessment of the baseline indicators provided guidelines to prioritize PUBLIC AWARENESS ON CROP WILD RELATIVES 71 capacity development programs of the stakeholders both in terms of type of stakeholders and nature of subjects. The overall conclusion of this study is that public awareness creation is an essential and urgent requirement. Depth of the awareness and clarity of the awareness relating to CWR, and also the connections of CWR with food crops and other usages are key areas in which capacity improvement is required. ACKNOWLEDGEMENTS

This survey was conducted with the financial assistance from UNEP/GEF funded ‘In-situ Conservation of Crop Wild Relatives through Information Management and Field Application Project’ implemented in Sri Lanka by the Department of Agriculture. The International Plant Genetic Resources Institute, Rome, Italy, provided the framework questionnaire for the survey. Authors appreciate the support rendered by all the respondents.

REFERENCES

Azzu, N and L. Collette. 2005. Addressing the conservation and sustainable utilization of CWR: The international policy context. In Book of Abstracts, First International Conference on Crop Wild Relative Conservation and Use. Eds. Kell and Scholten, University of Birmingham. 129 pp. FAO, 1996. Global Plan of Action for the Conservation and Sustainable Utilization of the Plant Genetic Resources for Food and Agriculture and the Leipzig Declaration. Food and Agriculture Organization of the United Nations. 63pp Jayasuriya, A.H.M. and D.A. Vaughan. 2003. Conservation and Use of Crop Wild Relatives. Proceedings of the joint Department of Agriculture, Sri Lanka and National Institute of Agrobiological Sciences, Japan, Workshop held on 3rd February 2003. 164 pp. Ministry of Forestry and Environment. 1999. Biodiversity Conservation in Sri Lanka: A Framework for Action. Ministry of Forestry and Environment, Battaramulla, Sri Lanka. 126 pp. Secretariat of the Convention on Biological Diversity. 2005. Handbook of the Convention on Biological Diversity Including its Cartagena Protocol on Biosafety, 3rd ed Montreal, Canada. 1493 pp. UNEP/GEF, 2004. In situ Conservation of Crop Wild Relatives through Enhanced Information Management and Field Application. United Nations Environment Program/Globle Environment Facility, Project Document. 188 pp. Annals of Sri Lanka Department of Agriculture. 2006.8:329-336.

STATUS OF SELECTED CHEMICAL CHARACTERISTICS IN CITRUS GROWING SOILS OF THE LOWER UVA REGION OF SRI LANKA

J.D.H. WIJEWARDENA Regional Agricultural Research and Development Centre, Makandura

ABSTRACT

An investigation was conducted to find out the soil fertility characteristics of major citrus growing soils of the Lower Uva region of Sri Lanka. Hundred and twenty soil samples were randomly collected at a depth of 0-15 cm from citrus growing soils in the Moneragala district. Organic matter content, pH, extractable P (Olsen) and exchangeable K in soils were determined. Majority of the soils in citrus growing lands (67%) showed pH in the range of 6-7. Since the majority of the soil samples (98%) had pH more than 5, there is no need of liming for citrus in the region. Majority of the soils (96%) had organic matter content below 3% and 81% showed extractable P below 25 mg/kg. Also, 82% of the samples showed exchangeable K more than 75 mg/kg. These results indicate that soils of the citrus growing lands in the Lower Uva region are low in organic matter and extractable P. On the other hand, majority of the soils in the region contain a reasonable amount of exchangeable K. These findings demonstrate the importance of proper soil fertility management in citrus growing lands in the region to achieve a high citrus crop yield. In this regard, the applications of chemical fertilizers particularly P as well as organic manures is rather important. The results of this study indicate that the low P status as well as low organic matter content may be crucial factors for declining crop productivity in citrus of the Lower Uva region of Sri Lanka.

KEYWORDS: Citrus growing soils, Exchangeable K, Extractable P, Lower Uva region, Organic matter, pH.

INTRODUCTION

The Lower Uva region of Sri Lanka refers to the area situated within the Intermediate Zone of the low country region of the Uva Province. The administrative district that comes under this region is Moneragala. The mean air temperature ranges from 23°C to 35°C and the average annual rainfall from 900 to 1150 mm. Immature Brown Loam (IBL- Inceptisols) is the predominant soil of this region (Panabokke, 1996). In general, land holdings in the area are rather small and vary from 0.25 to 0.5 ha (Bandara, 1997).

The Lower Uva region has been traditionally known as the main producer of citrus in Sri Lanka and was known as the citrus valley of the country. A few decades ago, citrus crops were grown extensively in this region by clearing jungles and it affected considerably the soil depth and natural soil fertility. The reported extent under citrus cultivation in Sri Lanka during the mid 1940’s was about 4000 acres (Dissanayake, 1997). In the late forties it was reported that citrus cultivation in Sri Lanka declined due to 74 WIJEWARDENA damage caused by various diseases (Soyza, 1997). This condition aggravated due to poor management of citrus trees as well as citrus growing lands in the region (Wijewardena, 1997). Citrus cultivation in the region however survived without adopting proper fertilizer application, irrigation and any erosion control measures. As a result, the soil fertility gradually declined owing to low nutrient retention capacity of IBL soils (Bandara, 1997; Wijewardena, 1997).

As many other fruit crops, citrus is also sensitive to soil fertility and it requires a high level of management practices for healthy growth and satisfactory crop yields. It has been reported that citrus is very sensitive for imbalanced nutrient supply and often subjected to deficiencies of major, secondary and micro nutrients which occur very widely throughout the country (FAO, 1983 and FAO, 1984). However, the fertilizer requirement of citrus cultivars is very complicated by its perennial nature. Hence, it is essential to fertilize citrus properly and maintain the soil fertility in good condition to obtain satisfactory yields.

However, there are hardly any reported chemical characteristics in soils of the citrus growing lands of the Lower Uva region of Sri Lanka. Therefore, the objective of this study was to investigate some selected soil fertility characteristics such as soil pH, organic matter, extractable P and exchangeable K of citrus growing soils in the Moneragala district.

MATERIALS AND METHODS

Hundred and twenty composite soil samples were randomly collected at a depth of 0-15 cm from 120 citrus growing farmer fields of the Lower Uva region. Soil samples were air dried ground and sieved through a 2 mm sieve. Soils were analysed for pH (1:2.5 ; H2O:soil) by glass electrode method (Mc Lean, 1982), extractable P by Olsen’s method (Olsen et al., 1954) and exchangeable K by extracting with 1M ammonium acetate at pH 7 (Carson, 1980). Exchangeable potassium was determined by Flame Photometer. Organic matter content was determined by Walkey – Black method (Nelson and Sommers, 1982).

RESULTS AND DISCUSSION

Results were grouped according to the critical limits for soil described by previous workers and publications namely Soil pH by FAO (1984); organic matter by Panabokke and Nagarajah (1964); exchangeable K and extractable P by the Macaulay Institute for Soil Research (1985). CHEMICAL CHARACTERISTICS IN CITRUS GROWING SOILS 75

Soil pH

Results showed that of the soil samples collected, only 2% had pH values below 5, which is considered as strongly acidic. In addition, 23% samples had pH between 5-6, which could be considered as moderately acidic with the mean of 5.7. However, majority of soils (67%) had pH between 6-7 (slightly acidic) with the mean of 6.2, which could be considered as the suitable pH for citrus growth. Furthermore, only 8% samples had pH over 7 with the mean value of 7.5 (Table 1).

Table 1. Range of soil pH in the lower Uva region.

pH No. of sites Range Mean <5 (Strongly acidic) 2 (2)* 4.8 - 4.9 4.9 (0.05)** 5-6 (Moderately acidic) 28 (23) 5.0 - 6.0 5.7 (0.33) 6-7 (Slightly acidic) 81 (67) 6.0 - 7.0 6.2 (0.26) >7 (Alkaline) 9 (8) 7.1 - 8.0 7.5 (0.30) * Numbers within parentheses denote % of sites, ** Numbers within parentheses denote standard deviation values

High pH of soils of the citrus growing lands in this region may be due to low rainfall in the area. This situation in the citrus gardens in this region is suitable for crop growth as well as for optimum nutrient uptake by the crops (FAO, 1984). The soils of the citrus growing lands in the Lower Uva region have high pH compared to the upper Uva region (Wijewardena et al., 1996 and Wijewardena, 1999). If the soil pH is over 5, Department of Agriculture (DOA) does not recommend lime application for any crop grown in Sri Lanka (DOA, 1995). Hence the soils of citrus growing lands in the Lower Uva region are suitable for citrus cultivation without addition of soil amendments such as lime or dolomite. These results suggest that soil pH is not a limiting factor for citrus production in the region.

Organic matter

The results showed that almost all soil samples (96%) contained organic matter between 1-3% which is considered as low and the mean value is 1.9%. Only 2% of the samples showed a medium value between 3-5% with a mean of 3.4%. Furthermore, only 2% of the samples showed organic matter content over 5% with a mean of 5.2% which could be considered as high (Table 2). 76 WIJEWARDENA

The low organic matter content in citrus soils may be due to high decomposition rate owing to high temperature prevailing in the region as the decomposition rate of organic matter increases with increasing temperature (Kononova, 1966). In addition, the application of organic manure is not a common practice among citrus growers in the region (Wijewardena, 1997), hence the low organic matter content of soils in this region could be expected. In highly weathered tropical soils, organic matter can play a favourable role by increasing the cation exchange capacity due to ionization of carboxyl and other active groups.

Table 2. Range of soil organic matter in the lower Uva region.

Organic matter (%) No. of sites Range (%) Mean (%)

<1 (Very low ) 0 (0)* 0 0 (0)** 1-3 (Low) 115 (96) 1.1- 2.9 1.9 (0.52) 3-5 (Medium) 3 (2) 3.2 – 3.7 3.4 (0.22) >5 (High) 2 (2) 5.1 – 5.3 5.2 (0.10) * Numbers within parentheses denote % of sites, ** Numbers within parentheses denote standard deviation values

The beneficial effect of organic matter on soil aggregate stability is well known (Mapa and Gunasena, 1995). Hence, the low organic matter content in these soils reduced the aggregate stability resulting in more soil erosion and low water retention capacity. These soils can become unproductive due to deterioration of soil structure, loss of topsoil by erosion and depletion of plant nutrients. Hence, the results indicate that the application of organic manure would be an important soil fertility management practice to increase the citrus crop production in the region.

Extractable P

Results indicate that 51% of the sites had extractable P content less than 10 mg/kg which is considered as very low content of extractable P with the mean value of 6.6 mg/kg extractable P. In addition, 30% soil samples had extractable P between 10 and 25 mg/kg which could be considered as low content of extractable P with the mean value of 15.2 mg/kg. Similarly, 15% sampled sites had extractable P between 25-75 mg/kg which is considered as the moderate value of soil extractable P with the mean value of 43.2 mg/kg. In addition, only 4% of the sampled sites had extractable P content over 75 mg/kg, which is considered as the high content of extractable P with the mean value of 105.1 mg/kg (Table 3).

Table 3. Range of soil extractable P in the lower Uva region.

Extractable P (mg/kg) No. of sites Range Mean (mg/kg) (mg/kg) < 10 (Very low ) 61(51)* 1.2 – 9.9 6.6 (2.0)** CHEMICAL CHARACTERISTICS IN CITRUS GROWING SOILS 77

10 – 25 (Low) 36 (30) 10.2 – 22.4 15.2 (3.8) 25 – 75 (Moderate) 18 (15) 25.5 – 67.2 43.2 (14.1) > 75 (High) 5 (4) 76.2 – 152.6 105.1(29.8) *Numbers within parentheses denote % of sites, ** Number within parentheses denote standard deviation values

As in many other upland soils in Sri Lanka, P could be considered as the most limiting plant nutrient (Wijewardena, 1994; Kumaragamage et al., 1999 and Kumaragamage et al., 2005), even in citrus growing soils in the Lower Uva region. The low P availability in soils of the citrus growing lands may be related to low P fertilizer used by farmers in this region.

In general, use of organic manure is also not a common practice among citrus growers in this region. These factors indicate the poor soil fertility management practices followed by citrus growers in the Lower Uva region of Sri Lanka. In addition, a majority of farmers use fertilizer mixtures, which are containing rock phosphates as a source of P. Due to low acidity prevailing in these soils the solubility of rock phosphate may be limited as previously reported by Khasawneh and Doll (1978) and Hammond et al. (1986). In addition, citrus crops remove an appreciable amount of P from the cultivated fields (Kemmler and Hobt, 1986).

Immature Brown Loam soils are coarse textured and thus have high infiltration rates. Similarly, leaching of some plant nutrients could be expected due to the low organic matter content in these soils. However, due to poor management of soils in many citrus plantations in this region, topsoil has been eroded badly. Hence, soils of citrus growing lands in the Lower Uva region are poor in soil fertility. Since, majority of citrus growing soils in this region is low in extractable P, addition of high grade P fertilizers could be important for soil fertility management practice (Wijewardena, 2000). It indicates that the citrus growers in this region should follow the proper P fertilizer application to their crops to achieve a satisfactory crop yield. In this regard, the application of high-grade P fertilizers such as triple superphosphate would be useful. The Department of Agriculture in 1990 changed its policy of recommending fertilizer mixtures and introduced the straight fertilizers for different crops. It gives more flexibility in adjusting the amount of nutrients to suit both the crop and soil. Such practices should be adopted in citrus crop sector as well to maintain a reasonable status of available P in soil. In addition, the application of organic manure as well as adopting soil conservation methods is important in soil fertility management practices in citrus cultivations.

Exchangeable K 78 WIJEWARDENA

The results indicate that only 2% of the samples have exchangeable K less than 40 mg/kg which could be considered as very low concentration of exchangeable K in soil with the mean value of 30 mg/kg. In addition, only 15% of the soil samples showed exchangeable K between 40-75 mg/kg which is considered as low concentration of exchangeable K with the mean value of 58.9 mg/kg. However, majority of soils had (52%) exchangeable K concentration between 75-200 mg/kg with the mean value of 128 mg/kg. This category could be considered as moderate level of exchangeable K for crop growth. Similarly, 31% of the samples had exchangeable K concentration over 200 mg/kg which could be considered as high in exchangeable K with the mean value of 278 mg/kg (Table 4).

Table 4. Range of soil exchangeable K in the Lower Uva region.

Exchangeable K (mg/kg) No. of sites Range Mean (mg/kg) (mg/kg) < 40 (Very low ) 3 (2)* 16 – 39 30 (10.0)** 40 – 75 (Low) 18 (15) 43 – 74 58.9 (9.4) 75 – 200 (Moderate) 62 (52) 82 – 199 128 (37.0) > 200 (High) 37 (31) 203 – 483 278 (71.6) * Numbers within parentheses denote % of sites ** Numbers within parentheses denote standard deviation values

These results indicate that soils in many of the citrus growing lands contained reasonable amount of exchangeable K in the Lower Uva region. Under these circumstances, the majority of soils in the citrus growing lands are in a position to supply K for satisfactory crop growth. The moderate to high exchangeable K in these soils may be favourable for citrus crop growth. Wijewardena and Amarasiri (1993), and Wijewardena (1996) reported low response to K fertilizers in the Upper Uva region even with high K feeding crops such as potato and upcountry vegetables. The results indicate that soils in the Uva province can be considered as rich in exchangeable K. However, citrus could be considered as a high K user (Kemmler and Hobt, 1986). Hence, it should be noted that the appropriate rate of K fertilizer application should be done to maintain the optimum soil K concentration in soil and obtain high yields of citrus crops from the region. CONCLUSIONS

Most of the citrus growing soils in the Lower Uva region have pH in the range of 6-7 indicating that soils in the citrus growing lands have suitable soil pH for crop growth and optimum soil nutrient availability. Hence, liming is not required immediately. Majority of the soils have organic matter content below 3% indicating that the soils are low in organic matter. Hence, applications of organic manures is rather important to achieve high productivity from citrus crop. In general, majority of the sampled sites showed low concentration of extractable P. Hence, application of P fertilizers is rather important to achieve high yields and use of high grade P fertilizers is CHEMICAL CHARACTERISTICS IN CITRUS GROWING SOILS 79 more suitable to obtain high yields than the use of rock phosphate sources due to low acidity in the soils. Majority of soils of the citrus growing lands show moderate to high concentration of exchangeable K. Based on the overall results, it could be suggested that adoption of proper chemical and organic fertilizer use and systematic soil conservation methods could improve the soil fertility status and increase the citrus crop productivity in the Lower Uva region of Sri Lanka. ACKNOWLEDGEMENTS

The author expresses his sincere appreciation to Mr. U.B. Dissanayake, Research Officer in-Charge of the Agriculture Research Station Moneragala for assisting in soil sampling and Mrs. J.B.K. Kannangara Research Assistant, Regional Agricultural Research and Development Centre, Bombuwela for undertaking soil analysis.

REFERENCES

Bandara, T.M.J. 1997. Land suitability assessment for citrus cultivation. In Decline of Citrus Cultivation in Sri Lanka. History, Causes, Recommendations. Task Force on Fruits, Ministry of Agriculture Lands and Forestry, 32-33. Carson, P.L. 1980. Recommended potassium test. In Recommended chemical soil test proceedings for the North Central Region. W.C. Dahuke. Ed. North Central Region. Publication No. 221, North Dakota Agric, Expt. Sta USA Bull 499. Dissanayake, U.B. 1997. The history of citrus decline in Sri Lanka. In Decline of Citrus Cultivation in Sri Lanka. History, Causes, Recommendations. Task Force on Fruits, Ministry of Agriculture Lands and Forestry, 1-3. Department of Agriculture, 1995. Soil –test based fertilizer recommendations. A Technical Working Document for Soil Scientists of the Department of Agriculture, Peradeniya, Sri Lanka 30p. FAO, 1983. Micronutrients. Fertilizer and Plant Nutrient Bulletin No.7, FAO, Rome 82 p. FAO, 1984. Fertilizer and Plant Nutrition Guide. Fertilizer and Plant Nutrient Bulletin No.9, FAO, Rome 172 p. Hammond, L.L., S.H. Chien and A.U. Mokwunye, 1986. Agronomic value of unacidulated and partially acidulated phosphate rocks indigenous to the tropics. Advances in Agronomy 4: 89-140. Kemmler, G. and H.Hobt 1986. Potash a Product of Nature. K+S fertilizers from the Federal Republic of Germany. pp.112. Landwirtschaftliche Forschungsanstalt, Buintehof. Khasawneh, E.F. and D.C.Doll 1978. The use of phosphate rock for direct application to soils. Advances in Agronomy 30:159-206. Kononova, M.M. 1966. Soil Organic Matter, Its Nature and Its Role in Soil Formation and in Soil Fertility. Pergamon Press, Oxford, U.K. 450p. Kumaragamage, D., H.B. Nayakekorale and L.P. Vidhana Arachchi, 1999. Risk and limitations of the wet zone soils. In Soils of the Wet Zone of Sri Lanka. Morphology, Characterization and Classification. Ed. R.B. Mapa, S. 80 WIJEWARDENA

Somasiri and S. Nagarajah. Special Publication No.1, Soil Science Society of Sri Lanka, 139-159. Kumaragamage, D., K.M.A. Kenderagama and H.B. Nayakekorala. 2005. Risk and limitations of intermediate zone soils. In Soils of the Intermediate Zone of Sri Lanka. Morphology, Characterization and Classification. Ed. R.B. Mapa, A.R. Dassanayeke and H.B. Nayakekorale. Special Publication No.4, Soil Science Society of Sri Lanka, 165-190. Macaulay Institute for Soil Research and Scottish Agricultural Colleges 1985. Advisory Soil Analysis and Interpretation. Bulletin-1, 13pp. Mapa, R.B. and H.P.M. Gunasena 1995. Effect of alley cropping on soil aggregate stability of a tropical Alfisol. Agroforesry Systems 32: 237-245. Mc Lean, E.O. 1982. Soil pH and lime requirement. In Methods of Soil Analysis. Part 2. Chemical and Microbiological Properties (2nd Ed.). Eds. A.L. Page, R.H. Miller and D.R. Keeney. Amer. Soc. of Agron. Madison, Wisconsin, USA. Nelson, D.W. and L.E. Sommers. 1982. Total Carbon, Organic Carbon and Organic Matter. Methods of Soil Analysis, Part 2. Amer. Soc. Agron. Madison, Wisconsin. USA. 577 pp. Olsen, S.R., C.V. Cole, F.S. Watanabe and L.A. Dean 1954. Estimation of extractable phosphorus in soils by extraction with sodium bicarbonate, USDA Circ. No 939 Washington D.C. USA. Panabokke, C.R. 1996. Soils and Agro – Ecological Environment of Sri Lanka. Natural Resources Series No 2. Natural Resources Energy and Science Authority, Colombo, Sri Lanka. 220p. Panabokke, C.R. and S. Nagarajah 1964. The fertility characteristics of the Rice- growing soils of Ceylon. Tropical Agriculturist CXX: 3-28. Soyza, I.J. de. 1997. Citrus diseases in Sri Lanka. In Decline of Citrus Cultivation in Sri Lanka. History, Causes, Recommendations. Task Force on Fruits, Ministry of Agriculture Lands and Forestry, 16-22. Wijewardena, J.D.H. 1994 Response of potato and vegetable crops grown sequentially to phosphorus application in upcountry intermediate zone. Tropical Agriculturist 149:1-11. Wijewardena, J.D.H. 1996. Effect of potassium and magnesium fertilizers on vegetable cultivation in the up country intermediate zone. Proc. 52nd Annual Session, Sri Lanka Assn. Adv. Sci. Part1: 50-51. Wijewardena, J.D.H. 1997. Importance of Nutrient Management in Citrus Cultivation in Sri Lanka. In: Decline of Citrus Cultivation in Sri Lanka. History, Causes, Recommendations. Task Force on Fruits, Ministry of Agriculture Lands and Forestry, 27-31. Wijewardena, J.D.H. 1999. Nutrient availability in the rice soils of upcountry intermediate zone. Sri Lankan Journal of Agricultural Sciences 36:63-69. Wijewardena, J.D.H. 2000. Sustainable plant nutrient management in intensive vegetable growing lands in the upcountry of Sri Lanka. Journal of the Soil Science Society of Sri Lanka 12:1-13. Wijewardena, J.D.H. and S.L. Amarasiri 1993. Response of vegetable crops in a cropping sequence to K and Mg fertilizers in the up country intermediate zone. Tropical Agriculturist 149: 13-22. CHEMICAL CHARACTERISTICS IN CITRUS GROWING SOILS 81

Wijewardena, J.D.H., U.W.S.P. Yapa and S.M.K.G. Yatagama 1996. Status of selected chemical characteristics of vegetable growing lands in the upcountry intermediate zone. Proc. 52nd Annual Session, Sri Lanka Assn. Adv. Sci. Part 1: 49-50. IN VITRO PLANT REGENERATION IN A MALE STERILE LINE OF CHILLI (Capsicum annuum L.)

K.N. KANNANGARA1, S.S. GOSAL2 and D.S. KHURANA3 1Field Crop Research and Development Institute, Maha Illuppallama 2Department of Plant Breeding, Genetics and Biotechnology,PunJab Agricultural University, Ludhiana,India 3Department of Vegetable Crops, PunJab Agricultural University, Ludhiana,India

ABSTRACT

An efficient method for in vitro plant regeneration of a male sterile line of chilli (Capsicum annuum L.) was developed. Cotyledons, cotyledonary nodes and shoot apex explants were excised from 12, 18, 24 and 30 day old in vitro grown seedlings and cultured on Murashige and Skoog (MS) basal medium supplemented with different concentrations and combinations of Benzyl aminopurine (BAP), Kinetin (KIN) and Indole acetic acid (IAA). The highest number of adventitious shoots were formed directly, without any intervening callus, on the explants on MS medium containing 9.0 mg/l BAP + 2.0 mg/l KIN + 1.0 mg/l IAA after 5 weeks of culture. The process of induction, proliferation and elongation of shoots was completed on the medium containing high concentration of BAP. Explants from 18 and 24 day-old-seedlings produced more multiple shoots than explants from 12 and 30 day-old seedlings. Cotyledon explants (either wounded or un-wounded) exhibited better regeneration capacity and produced more multiple shoots than cotyledonary nodes and shoot apex explants. The axillary shoots with few leaf primodia were subsequently cultured in medium containing 9.0 mg/l BAP + 2.0 mg/l KIN + 1.0 mg/l IAA for shoot multiplication. By incorporating Indole buteric acid (IBA) into the ½ MS rooting medium, 100% of the shoots produced roots. The rate of survival of plants during hardening was 86%. The tissue culture approach appears highly relevant to multiplication of male sterile line for further use in hybrid seed production.

KEYWORDS: Capsicum annuum L., Cotyledons, In vitro, Male sterile, Micropropagation, Plant regeneration, Shoot multiplication.

INTRODUCTION

Chilli (Capsicum annuum L.) is an important commercial spice grown as an annual vegetable all over the world. The pods are gaining popularity in houses not only for their uniqueness but also because they are low in sodium, cholesterol free, low in calories and high in vitamins A and C (Danise, 2003). Though India stands first in chilli cultivation covering 45 per cent of the world hectarage, its productivity is quite low (less than 1 t/ha) as compared to USA, China, South Korea, Taiwan and some other countries (Peter, 1998 and Madhavi et al., 2002). The main reason for low productivity is the use of open pollinated varieties with minimal use of hybrid varieties. Recent experience provides the superiority of F1 hybrids in chillies for earliness, high productivity, high fruit weight and dry recovery. Development of hybrids by manual emasculation and hand pollination is a tedious process that involves high cost. Exploitation of heterosis economically depends on the development of new techniques such as genetic male sterility (GMS), which leads to cheap hybrid seed production. The use of genetic male sterility controlled by a recessive gene can greatly help in making F1 hybrid, because tedious and costly hand emasculation of individual flower bud can be avoided (Patel et al., 2001). This technique also increases the purity of F1 seed, as there is no self-pollination (Madhavi et al., 2002). However, the major bottleneck in the seed production of GMS based hybrids is the female parent of genetic male sterile line, having 1:1 ratio of male fertile: male sterile plants which suggests currently practiced rouging of male fertile plants at the anthesis by observing each and every individual. Tissue culture technology has contributed immensely to the advancement of agriculture for more than five decades. In vitro techniques for different crops e.g. tobacco, petunia, potato, sugar cane, carrot, rice, citrus and strawberry etc., have already been standardized primarily due to the ease of theses crops to manipulate. However, chilli spp. have remained recalcitrant to in vitro manipulation and have lagged far behind in the application of tissue culture techniques. If the production of male sterile plants can be done through tissue culture, the tedious rouging out can be avoided and seed yield per unit area can be increased. Clonal propagation of male sterile lines may ensure the requirement of male sterile plants in the field. It may facilitate 100% genetically pure hybrid plant stand as well as utilization of male sterile plants in other hybridization programs. The tissue culture approach appears highly relevant to multiplication of male sterile lines for further use in hybrid seed production (Gupta et al., 1998 and Gupta et al., 2002).

MATERALS AND METHODS

A study was conducted in the Department of Vegetable Crops in collaboration with the Department of Plant Breeding, Genetics and Biotechnology, Punjab Agricultural University during 2004- 2005 to develop a protocol for in vitro plant regeneration in a genetic male sterile line of chilli, ms 12.

Plant material

Seeds of chilli male sterile line, “ms12”, obtained from the Department of Vegetable Crops, PAU, were sterilized in 75% commercial bleach, Ala (5% sodium hypochlorite) for 20 minutes and then rinsed three times in sterile distilled water under a laminar flow hood. Sterilized seeds were cultured in culture tubes (25 x 150 mm, Borosil) containing 15 ml of half-strength MS basal medium (Murashige and Skoog, 1962) supplemented with 3% sucrose adjusted to pH 5.8 before adding 0.8% (W/V) agar and autoclaved at 121°C for 20 min. They were placed at 25±2°C and incubated with a photoperiod of 16hrs. After germination cotyledons, wounded cotyledons, cotyledonary nodes and shoot apices excised from 12, 18, 24 and 30 day old, in vitro- grown seedlings were used as explants.

Culture media and conditions

The explants were cultured on medium containing 3% (W/V) sucrose and supplemented with growth regulators. The media were adjusted to pH 5.8 by using 1 N NaOH and 1 N HCl before adding 0.8% (W/V) agar. About 15 ml and 50 ml of the media were then dispensed in culture tubes (25 x 150 mm, Borosil) or 200ml jars respectively and closed with cotton plugs before autoclaving at 121°C for 20 min. Different combinations and concentrations of BAP, Kinetin and IAA were tested for shoot initiation. Different treatments used for establishment of cultures (both liquid and solid) were as follows.

Medium Abbreviation MS + BAP 1.0 mg/l + KIN 1.0 mg/l + IAA 0.5 mg/l - MS1 MS + BAP 3.0 mg/l + KIN 2.0 mg/l + IAA 1.0 mg/l - MS2 MS + BAP 6.0 mg/l + KIN 4.0 mg/l + IAA 1.0 mg/l - MS3 MS + BAP 7.5 mg/l + KIN 3.0 mg/l + IAA 1.0 mg/l - MS4 MS + BAP 9.0 mg/l + KIN 2.0 mg/l + IAA .0 mg/l - MS5 MS + BAP 10.0 mg/l + KIN 5.0 mg/l + IAA 1.0 mg/l - MS6 MS + BAP 10.0 mg/l + KIN 7.5 mg/l + IAA 1.0 mg/l - MS7

There were 7 treatments replicated thrice in a completely randomized design. The number of shoot buds developing on the explants were recorded after 5 weeks of culture and the effect of shoot induction media was evaluated.

Shoot proliferation and elongation of shoots

After induction of shoots from the initial cultures, shoots were excised separately and transferred to solid and liquid MS media supplemented with various concentrations of BAP, KIN, IAA and GA3 for shoot proliferation and elongation.

Medium Abbreviation MS + BAP 3.0 mg/l + KIN 2.0 mg/l + IAA 1.0 mg/l -MM1 MS + BAP 5.0 mg/l + KIN 3.0 mg/l + IAA 1.0 mg/l - MM2 MS + BAP 6.0 mg/l + KIN 4.0 mg/l + IAA .0 mg/l - MM3 MS + BAP 7.5 mg/l + KIN 3.0 mg/l + IAA 1.0 mg/l - MM4 MS + BAP 9.0 mg/l + KIN 2.0 mg/l + IAA 1.0 mg/l - MM5

MS + GA3 5.0 mg/l - MM6

There were 6 treatments replicated thrice in a completely randomized design. The concentration of cytokinins in the shooting medium was gradually reduced before transferring the shoots to the rooting medium. The number of shoots developing on the explants were recorded after 2 and 3 weeks of culture and the effect of shoot multiplication media was evaluated. Induction of rooting

The in vitro derived shoots were separated and transferred to the rooting medium for root initiation. The half strength MS medium supplemented with IAA, NAA and IBA were tested in following combinations.

Medium Abbreviation ½ MS (Solid) - MR1 ½ MS (Liquid) - MR2 ½ MS + IAA 1.0 mg/l + NAA 1.0 mg/l (Solid) - MR3 ½ MS + IAA 1.0 mg/l + NAA 1.0 mg/l (Liquid) - MR4 ½ MS + IBA 1.0 mg/l + NAA 1.0 mg/l (Solid) - MR5 ½ MS + IBA 1.0 mg/l + NAA 1.0 mg/l (Liquid) - MR6 ½ MS + NAA 2.0 mg/l (Solid) - MR7 ½ MS + NAA 2.0 mg/l (Liquid) - MR8 ½ MS + IBA 2.0 mg/l (Solid) - MR9 ½ MS + IBA 2.0 mg/l (Liquid) - MR10 ½ MS + IBA 2.0 mg/l + 2.0 g/l Charcoal (Solid) - MR11 ½ MS + IBA 2.0 mg/l (one week) - ½ MS (Solid) - MR12

The experiment was conducted in a completely randomized design with two replications. Data on percentage of rooting and the length of roots were recorded after 3 weeks.

Hardening and transplanting of rooted plantlets

Plantlets with well developed roots were carefully transferred from the culture tubes into a trough of running water for two hours to remove the traces of agar and other constituents of the culture medium adhering to plantlets. These plantlets were hardened by placing the root system on moist cotton with ½ MS salts for three days under high light condition in side the incubation room. The plantlets were then transferred to plastic pots (5 cm in diameter) containing surface soil. The pots were kept covered with polythene sheet to maintain a high relative humidity for six days in a glass house before exposing them to ambient temperature for ten days. Plants were then transferred to the 25-30 cm diameter clay pots containing top soil and farm yard manure at the ratio of 1:1.

Statistical analysis

All the shoot induction and shoot proliferation experiments were set up in a completely randomized design and each treatment had 3 replicates. The data on the shoot regeneration percentage, number of days taken to shoot initiation and number of multiple shoots regenerated on explants were analyzed using 2 factor factorial experiment in Complete Ramdomized Design (CRD). Root induction experiments were set up in a completely randomized design and each treatment had 2 replicates. Data on the percent shoots responded, number of days to rooting and number of roots per shoot were analyzed using one-way analysis of variance.

RESULTS AND DISCUSSION

Response of different explants to establishing/initial culture media

Plant regeneration via organogenesis involves removing plant tissues such as hypocotyls or cotyledons from germinating seeds or seedlings and placing them on tissue culture media to induce differentiation and development of organs and plantlets. Except anther culture, it is generally shown that chilli is recalcitrant to in vitro plant regeneration from somatic explants and genetic transformation has been limited (Kang et al., 1998). Establishment of initial cultures on liquid media completely failed. Callus formation was observed initially (7-10 days after culturing) at the base of the explants but all the explants turned brown later on and no shoot initiation was observed in liquid cultures at all the levels of BAP and KIN. Distorted and shriveled explants were seen in cultures with 12 and 18 day old explants. Shoot initiation and regeneration were the only success of the explants cultured on solid media.

As illustrated in Table 1, significantly higher rate of shoot regeneration was shown in 24 day old cotyledonary nodes followed by 18 day old cotyledonary nodes. There is not much difference for shoot regeneration among the wounded and un-wounded cotyledons but 24 day old explants of both wounded and un-wounded cotyledons showed better response followed by 18 day old explants. It was observed that all these explants cultured on 9.0 mg/l BAP concentration performed well followed by 7.5 mg/l BAP and 6.0 mg/l BAP respectively. The best interaction of media X age of the explant was observed in MS5 media with 24 day old both wounded and un-wounded cotyledons followed by MS5 media with 18 day old cotyledonary nodes and shoot apices, respectively. There have been several reports of successful plant regeneration from adventitious buds in capsicum. Embryos, seedlings and vegetative plant parts have been used as explants, where cotyledons and hypocotyls were the most effective for organogenesis (Sanatombi and Sharma, 2006).

Response of wounded and un-wounded cotyledon explants was very poor on the lowest and the highest BAP concentrations. Except cotyledonary nodes, other explants did not respond to the MS7 medium which was having the highest BAP and KIN concentrations and most of the cultures were browned and finally died. The regeneration protocols consisted of adenine type cytokinins, commonly Benzyl aminopurine (BAP), with or without auxin (Pundeva et al., 1998). Table 1. Shoot regeneration from wounded cotyledons, un-wounded cotyledons, cotyledonary nodes and shoot apex explants cultured on different media.

Mediu Age of the explant (days) m Wounded cotyledons Un-wounded cotyledons Cotyledonary nodes Shoot apices 12 18 24 30 mean 12 18 24 30 mean 12 18 24 30 mean 12 18 24 30 mean MS1 4.00 6.00 8.00 4.00 5.50 5.00 6.00 5.00 4.00 5.00 27.77 50.00 73.63 40.27 47.92 12.5 50.00 57.00 43.07 40.64 0 MS2 12.0 22.95 41.67 16.70 23.33 6.60 31.93 38.90 12.6 22.51 48.60 70.91 79.17 54.24 63.23 36.1 54.29 54.17 34.73 44.83 0 0 3 MS3 18.0 31.93 47.20 23.60 30.18 16.70 27.80 45.83 18.0 27.1 63.94 79.13 86.10 55.57 71.19 38.8 47.23 48.68 37.50 43.07 0 7 7 MS4 18.0 40.27 57.00 31.93 36.80 26.40 31.97 55.57 26.4 35.08 54.18 84.73 87.50 66.74 73.29 38.8 75.00 57.09 47.20 54.54 0 0 7 MS5 20.8 69.47 77.77 44.43 53.12 25.00 66.73 68.13 38.8 49.68 66.70 95.83 94.43 72.23 82.30 37.5 83.33 77.80 48.68 61.83 0 7 0 MS6 8.07 30.57 27.80 26.40 23.21 5.40 18.00 26.60 31.9 20.48 19.47 48.60 56.97 40.27 41.33 8.33 9.72 40.34 30.57 22.24 3 MS7 4.00 6.00 8.00 4.00 5.50 5.00 6.00 5.00 4.00 5.00 9.72 19.47 18.07 18.07 16.33 4.00 6.00 6.60 5.00 5.42

Mea 12.1 29.60 38.20 21.58 12.87 26.92 35.00 19.4 41.48 64.10 70.84 49.63 25.1 46.51 48.82 35.25 n 2 1 7 LSD (p=0.05 6.14 6.89 7.12 7.42 ) 4.64 5.21 5.38 5.61 Media 12.28 13.79 14.23 14.84 Age 29.56 35.77 15.38 23.29 M X A CV Table 2. Number of days taken for shoot regeneration from wounded cotyledons, un-wounded cotyledons, cotyledonary nodes and shoot apex explants cultured on different media.

Medium Age of the explant (days)

Wounded cotyledons Un-wounded cotyledons Cotyledonary nodes Shoot apices 12 18 24 30 mea 12 18 24 30 mean 12 18 24 30 mea 12 18 24 30 mean n n MS1 35.00 33.00 32.00 35.00 33.75 35.00 33.00 32.00 35.00 33.75 14.00 12.33 11.50 13.33 12.71 12.00 11.33 11.33 12.64 11.83

MS2 27.00 29.00 25.67 28.5 27.54 29.00 29.67 27.33 30.00 29.00 12.33 12.00 12.33 12.33 12.25 11.67 11.33 10.67 11.83 11.38

MS3 25.00 28.67 26.00 28.00 26.92 27.33 29.33 27.33 30.00 28.50 12.00 11.33 11.50 11.83 11.67 12.33 9.33 10.33 11.67 10.92

MS4 27.00 28.33 24.67 26.33 26.51 28.00 28.67 28.00 29.33 28.50 10.33 10.50 9.67 11.33 10.46 10.00 9.00 9.00 10.00 9.50

MS5 28.00 26.33 24.67 26.00 26.25 27.67 28.00 26.33 28.00 27.50 8.67 7.33 8.00 9.17 8.29 10.00 8.33 8.67 8.67 8.92

MS6 29.00 23.33 22.67 25.33 25.08 27.00 24.67 25.33 26.67 25.92 7.83 7.33 7.67 8.33 7.79 10.00 7.83 8.14 8.83 8.71

MS7 35.00 33.00 32.00 35.00 33.75 35.00 33.00 32.00 35.00 33.75 7.83 6.33 7.33 7.83 7.33 14.00 13.33 13.00 13.33 13.42

Mean 29.43 28.81 26.81 29.17 29.86 29.48 28.33 30.57 10.43 9.60 9.71 10.57 11.43 10.07 10.17 11.00

LSD (p=0.05) 1.05 0.67 0.78 0.64 Media 0.79 0.51 0.59 0.48 Age 2.10 1.35 NS NS M X A 4.49 2.79 9.51 7.32 CV Annals of Sri Lanka Department of Agriculture. 2006.8:91-106.

Table 3. Number of shoots per explant regenerated from wounded cotyledons, un-wounded cotyledons, cotyledonary nodes and shoot apex explants cultured on different media.

Mediu Age of the explant (days)

m Wounded cotyledons Un-wounded cotyledons Cotyledonary nodes Shoot apices 12 18 24 30 mean 12 18 24 30 mean 12 18 24 30 mean 12 18 24 30 mean

MS1 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.53 2.58 2.20 1.66 1.99 1.60 1.75 2.19 2.00 1.89 MS2 5.50 8.70 10.20 3.50 1.97 2.80 7.00 8.60 4.20 5.65 1.58 3.26 2.68 2.10 2.41 1.70 2.19 2.44 2.25 2.15

MS3 6.00 12.00 13.00 3.50 8.63 4.83 9.00 10.20 3.50 6.88 2.02 3.50 3.43 2.27 2.81 1.78 2.22 2.61 2.19 2.20

MS4 5.00 14.50 15.50 5.87 10.22 4.70 12.50 12.00 4.60 8.44 2.08 3.80 4.05 2.62 3.14 1.92 3.22 2.86 2.58 2.64

MS5 7.00 18.00 20.00 8.00 13.25 3.33 17.00 17.00 5.23 10.64 2.66 4.88 4.67 3.05 3.82 2.30 2.97 3.14 2.47 2.72

MS6 2.00 5.00 7.50 2.90 4.35 3.50 5.00 5.00 3.67 4.29 1.00 2.17 2.05 1.83 1.76 1.05 1.88 1.35 1.20 1.37

MS7 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00

Mean 3.93 8.60 9.74 3.68 3.02 7.50 7.83 3.31 1.70 3.03 2.87 2.08 1.62 2.18 2.23 1.96 1.96

LSD (0.05) 1.30 0.88 0.29 0.18 Media 0.99 0.67 0.22 0.14 Age 2.61 1.76 0.57 0.36 M X A 24.57 19.88 14.43 11.12 CV 92 KANNANGARA et al.

Days taken for shoot initiation

With regard to the days taken for shoot bud initiation from the different explants, wounded and unwounded cotyledonary explants showed a similar pattern that of more than 4 weeks for shoot initiation. According to the results obtained cotyledonary nodes were found to be the best expalnt for early shoot initiation. Days taken for shoot bud initiation was less with the increase of cytokinin in the medium (Table 2). Induction of shoot buds and their subsequent proliferation is a function of cytokinins such as 2ip, kinetin and BAP of which BAP has been found the most effective (Chen and Evans, 1990). When considering the age of the explants, the least number of days were taken by the18 and 24 days old cotyledonary nodes respectively. Shoot initiation in shoot apices showed a similar pattern to that of cotyledonary nodes. Also, it was observed that the high concentration of cytokinins was detrimental to the explants irrespective of their age.

Number of shoots regenerated per explant

The MS medium supplemented with 9.0 mg/l BAP + 2.0 mg/l KIN + 1.0 mg/l IAA gave rise to the highest number of shoots/explant in wounded cotyledons (Plate 1) followed by MS medium supplemented with BAP 7.5 mg/l + KIN 3.0 mg/l + 1.0 mg/l IAA in wounded cotyledons and unwounded cotyledons. But, the numbers of shoots per explant were reduced when increasing the concentration of above cytokinins in all the explants. The results clearly showed that 24 days old wounded cotyledons were the best explant generating more number of shoots per explant followed by 18 days old wounded cotyledonary explants, 24 days old un-wounded cotyledonary explants and 18 days old un-wounded cotyledonary explants respectively. The number of shoots regenerated was increased with the increase in BAP and KIN concentrations in the media up to a level and beyond that it was detrimental to all the types of explants. The increase in BAP concentration up to optimum level results in increase in mean shoot number due to increase in the activity of cell division (Skoog and Armstrong, 1970). It was observed that there were more number of shoots per explant in MS5 medium (BAP 9.0 mg/l + KIN 2.0 mg/l) followed by MS4 (BAP 7.5 mg/l + KIN 3.0 mg/l + 1.0 mg/l IAA) and MS3 (BAP 6.0 mg/l + KIN 4.0 mg/l + 1.0 mg/l IAA) respectively (Table 3). Sanatombi and Sharma (2006) observed up to a maximum of 7 buds and 5 buds from shoot apex explants cultured on BAP 44.4 uM in chilli cultivars of Meitemorok and Haomorok, respectively.

Shoot multiplication and elongation

BAP in the culture medium has been known to cause proliferation of adventitious in many plants (Murashige, 1974). The elongation growth of leafy buds was severely inhibited in the continuous presence of high concentrations of cytokinins, and frequently the buds became quite thick, ill-defined and vitreous (Binzel et al., 1996). IN VITRO PLANT REGENERATION IN CHILLI 93

Table 4. Shoot multiplication in different media after 14 days and 21 days of culture.

Medium Number of shoots per explant 14 days after sub-culturing 21 days after sub-culturing 1st Sub- 2nd Sub- 3rd Sub- Average 1st Sub- 2nd Average culture culture culture culture Sub-culture MM1 1.58 1.75 1.50 1.61 MM2 2.33 2.75 2.58 2.55 1.65 1.25 1.45 MM3 3.25 3.06 2.88 3.06 2.30 2.00 2.15 MM4 4.08 3.88 4.00 3.99 2.20 1.80 2.00 MM5 4.23 4.00 3.88 4.04 1.80 1.20 1.50 Mean 3.09 3.09 2.97 3.05 2.03 1.81 1.65

Average number of shoots per explant in sub-culturing was showed decreasing trend in subsequent culturing. It was observed that there were 3.09 shoots/explant during first and second sub- culturing and 2.97 shoots/explant in third sub-culturing irrespective of the media used (Table 4). Maximum number of shoots were obtained in MM5 medium (4.04 shoots/explant) (Plate 2.) followed by MM4 medium (3.99 shoots/explant). Fourteen days interval of sub-culturing was found to be better than the twenty one days interval. Dying of shoots was observed in 21 days duration of sub-culturing which led to the low number of shoots per explant cultured. All the cultures failed in MM6 media which were having GA3 5 mg/l.

Rooting of in vitro raised shoots

It is evident from the data presented in Table 5, that percent shootlets responding to root initiation on ½ MS medium varied with the addition of different auxins. Hundred percent root initiation was observed in all the shootlets cultured on MR12 solid medium for one week and subsequently transferred to the ½ MS hormone free medium 2.0 mg/l of IBA indicates that it is the best treatment for root initiation. The time taken for root initiation was minimum (11.66 days) in (MR12) medium followed by MR11 medium (16.00 days). Delay in rooting and callus formation at the base of the explant was observed in media supplemented with IAA.

Number of roots per shoot

The highest number of roots per shoot was observed in MR12 medium (7.50 roots/shoot) followed by MR9, MR11 (5.00 roots/shoot) and MR5, MR7 (4.00 roots/shoot). It was clearly observed with the present studies that those rooting media supplemented with IBA were responsible for the increase of number of roots per shoot. 94 KANNANGARA et al.

Plate 1. Clusters of shoot buds regenerated from cotyledons cultured on MS medium supplemented with BAP 9.0 mg/l + KIN 2.0 mg/l + IAA 1.0 mg/l. (Magnification 1 X 1).

Plate 2. Shoot multiplication 14 days after sub-culturing on MS medium supplemented with BAP 9.0 mg/l + KIN 2.0 mg/l + IAA 1.0 mg/l. (Magnification 1 X 1 IN VITRO PLANT REGENERATION IN CHILLI 95

Table 5. Effect of different rooting media on rooting of in vitro generated shoots.

Medium Per cent Days to Number of Root length* responded rooting roots/shoot size MR1 27.77 20.30 2.50 Short MR3 27.80 20.00 3.00 Short MR5 41.67 19.66 4.00 Medium MR7 36.10 20.00 4.00 Short-Medium MR9 63.90 17.00 5.00 Medium MR11 55.57 16.00 5.00 Medium-Long MR12 100.00 11.66 7.50 Long * Length of the roots Size category of roots Less than 3 cm Short 3 cm to 6 cm Medium Above 6 cm Long

Length of the roots

It was observed that NAA and IAA caused callusing at the cut end. Culturing the shoots on half strength MS medium supplemented IBA 2.0 g/l for one week and then transferring to half MS medium without growth hormones (MR12) was found to be the best medium for root elongation. More than 6cm long roots obtained from the same medium followed by MR11 medium (½ MS Supplemented with IBA 2.0 mg/l + charcoal 2.0 g/l) were having medium-long (3-6 cm in length) size roots.

Hardening and field establishment of rooted plantlets

Hardening the plantlets 3 days prior to the transfer to soil on moist cotton with ½ MS salts was found to be more effective than that of hardening plantlets on moist cotton with water. Eighty six per cent survivals were observed with ½ MS salts and 75% survivals were observed with simple water treatments. Ninety two percent survivals were observed three weeks after planting the hardened plantlets in soil. Keeping the potted plants under high humidity during initial six days helped a lot to obtain the high survival after planting (Plate 3). Plantlets having 4-5 well developed leaves performed well after putting them on soil. Plants showed vigorous growth, well developed branches and attained maturity early and displayed the features of male sterility.

Table 6. Per cent survival after hardening and field establishment.

Hardening Hardening treatment No. of plantlets Number of Percent hardened plantlets survived survival ½ MS Salt 30 26 86.66 Water 24 18 75 Plant establishment Number of plantlets established Number of plantlets survived Per cent survival 26 24 92 The technique of micropropagation has witnessed a major success in its application for large scale propagation of the rare and elite germplasm. The tissue culture derived plants are usually true to type, vigorous and free from diseases. At the same time, the in vitro multiplication can be carried out throughout the year, irrespective of the season. Thus, increasing the rate of propagation manifolds as compared to 96 KANNANGARA et al. conventional methods. In the present investigation, an attempt has been made to solve the problem associated with maintenance and multiplication of male sterile line via micropropagation

In micropropagation studies, explants of genetic male sterile line were cultured on MS medium supplemented with different combination and concentration of phytohormones. Explants from younger seedlings (18 -24 days old) responded the best on MS medium supplemented with BAP 9.0 mg/l + KIN 2.0 mg/l + IAA 1.0 mg/l followed by MS medium supplemented with BAP 7.5 mg/l + KIN 3.0 mg/l + IAA 1.0 mg/l.

It was observed that 24 days old wounded cotyledons were the best explants generating multiple shoots per explant followed by 18 days old wounded cotyledonary explants, 24 days old unwounded cotyledonary explants and 18 days old unwounded cotyledonary explants respectively.

More shoot proliferation was obtained on MS medium supplemented with BAP 9 mg/l + KIN 2 mg/l + IAA 1 mg/l. The number of days kept for shoot proliferation also was found to be an important factor in shoot multiplication. It was observed that number of shoots per sub-culturing cycle decreasing with the increasing of time duration. Sub-culturing after every fortnight period was observed to be more successful than extending the period for three weeks because dying of shoots was observed when extending the culturing period. There was decreasing trend in number of shoots per explant in successive sub- culturing.

Plate 3. Properly hardened male sterile chilli plants ready for field planting. Hundred percent shootlets responded to the root initiation, less number for rooting and lengthy roots resulted with the MR12 solid medium (½ MS medium supplemented with IBA 2.0 mg/l one week and subsequently transferred to ½ MS hormone free medium). Therefore, IBA 2.0 mg/l has been found to be the best for root initiation.

Hardening the plantlets 3 days prior to the transfer to soil on moist cotton with ½ MS salts was found to be more effective than that of hardening plantlets on moist cotton with water. Eighty six per cent survivals were observed with ½ MS salts and 75% survivals were observed with simple water treatments. IN VITRO PLANT REGENERATION IN CHILLI 97

CONCLUSIONS

The present investigation indicated that the technique of micro-propagation can be utilized for rapid and true to type multiplication of male sterile lines. However, long term experiments covering a large number of media with different concentrations of growth regulators (BAP and KIN) etc. are required to measure the rate of multiplication up to the level when it can be used for commercial purpose. The media used to elongate the multiple shoot buds developed from cotyledons should be further standardized to obtain more number of shoots than the number of shoots obtained.

ACKNOWLEDGEMENTS

The authors are grateful to Sri Lanka Council for Agriculture Research Policy (CARP) for the financial assistance provided for the post graduate degree programme (M.Sc.). Also the staff of Department of Vegetable Crops and Department of Plant Breeding, Genetics and Biotechnology of the Punjab Agricultural University, Ludhiana, India are highly appreciated for providing valuable guidance and assistance throughout the study.

REFERENCES

Binzel, M.L., N. Sankhla, S. Joshi and D. Sankhla 1996. Induction of direct somatic embryogenesis and plant regeneration in pepper (Capsicum annuum L.). Plant Cell Reports 15: 536-540. Chen, Z. and D.A. Evans. 1990. General techniques of tissue culture in perennial crops. Pp 22-61 In Handbook of Plant Cell Culture. Danise Coon 2003. Chile peppers: Heating up Hispanic foods. Food Technology 57(1): 39-43. Gupta, Ch.G., N.Lakshmi and T. Srivalli. 1998. Micropropagation studies on a male sterile line of Capsicum annuum L. at Nagarjuna University. Capsicum and Eggplant Newsletter 17: 42-45. Gupta, R. K., M.S. Sharma., N. Kour., R.Kour and P.S. Andotra. 2002. Field evaluation of micropropagated male sterile lines in Tomato (Lycopercicon esculentum Mill.). Proc. International Conference on Vegetables, Bangalore. p 147. Kang, G.Q., M.T. Azzimonti., R. Marino and G. Nervo. 1998. Factors affecting regeneration and transformation in Capsicum spp. Proc. Xth EUCARPIA Meeting on Genetics and Breeding of Capsicum and Eggplant, Avigon, France. p 220. Madhavi, R. K., A.T. Sadashiva., H.V. Satyanarayana., R.H.S. Narayana and M. Chinna Babu. 2002. Standard heterosis of the hybrids developed using CGMS lines in Chilli (Capsicum annuum L.) Proc. Internatuional Conference on Vegetables, Bangalore. p 51. Murashige, T. and F. Skoog. 1962. A revised medium for rapid growth and bioassays with tobacco tissue culture. Physiol Plantarum 15: 413-438. Murashige, T. 1974. Plant propagation through tissue culture. Ann. Rev. Plant Physiol. 25: 135-66. Pundeve, R., N. Simeonova and Z. Etimova. 1998. Morphogenetic potential of several Capsicum genotypes cultivated in vitro. Proc. Xth EUCARPIA Meeting on Genetics and Breeding of Capsicum and eggplant, Avigon, France. p 223. Patel, J.A., M.J.Patel., A.S. Bhanvadia., R.R. Acharya and M.K. Bhalala. 2001. Extent of natural cross pollination with GMS lines in chilli (Capsicum annuum L.). Capsicum and Eggplant Newsletter 20: 35-37. Peter, K. V. 1998. Recent advances in chilli breeding. Indian Spices 35: 3-5. Sanatombi, K. and G. J. Sharma. 2006. In vitro regeneration and mass multiplication of Capsicum annuum L. Journal of Food, 98 KANNANGARA et al.

Agriculture & Environment 4 (1): 205-208. Skoog, F and D.J. Armstrong. 1970. Cytokinins. Ann. Rev. Plant Physiol. 21:359-384. Vandana Kumar and Athul Kumar. 2002. Tissue culture of vegetable crops for enhancing production in hilly areas. International Conference on Vegetables, Bangalore. p 146. Annals of Sri Lanka Department of Agriculture. 2006.8:337-340.

SHORT COMMUNICATION

RATOON CROP MANAGEMENT TOWARDS YIELD IMPROVEMENT OF BRINJAL (Solanum melongena L.)

S. ARULMAGESWARAN and I.M.N. CHANDRASIRI Extension and Training Centre, Peradeniya

INTTRODUCTION

Brinjal (Solanum melongena L.) is a popular annual crop in Sri Lanka and South Asian countries (Rajapaksha, 1998). It is one of the nutritious food crops for humans and heals many disorders such as diabetes, high blood pressure, cough and loss of appetite (Choudhury and Kalda 1960) and diversified colour and shape of the fruit helps to fetch good market demand (Tomar et al., 1996). It is a hardy plant compared to other vegetables grown in Sri Lanka. It can therefore be successfully grown in very dry areas under rainfed or with minimum irrigation facilities. The plant can be kept more than one year in productive manner by pruning. At present every farmer has to consider the economics of crop production because of increased cost of inputs and higher demands for the produce in the market (Stansel, 1977). There are various practices to reduce cost of cultivation and ratoon crop management is one of the possible ways to reduce cost of cultivation and maximize economic yield (Tindall, 1983). Therefore, the present experiment was designed to investigate the management of branches in ratoon brinjal plants with mulch and without mulch in relation to maximizing yield from unit land.

MATERIALS AND METHODS

The experiment was conducted at the School of Agriculture, Kundasale from September 2003 to August 2004 (maha 2003/04 to yala 2004). The soil at experimental site was immature brown loam (Panaboke, 1996). The maximum mean daily temperature of site was 32.8°C and a total of 879.8 mm rainfall was received during the experimental period. Brinjal cultivar Lena iri seedlings were planted. They were cut back after first crop was completed and allowed to develop second crop. The cutting was done 15 to 20 cm above soil level and arranged the following treatments: Two branches (T1), Three branches (T2), Natural branch growth (Control-T3), Two branches with mulch (T4), Three branches with mulch (T5) and Control with mulch (T6). They were managed as recommended by Department of Agriculture (DOA, 1990). The experimental design was 3 x 2 factorial arrangement in Randomized Complete Block Design (RCBD) with four replicates. Economically important parameters such as plant height at 82 ARULMAGESWARAN AND CHANDRASIRI maturity (PH), number of fruits per plant (NF), leaf area index at the time of 50% flowering (LAI), fruit length (FL), fruit diameter (FD), fruit weight (FW) and yield per hectare (YD) were recorded from both fresh and ratoon crops. The data were analyzed by using the statistical package SAS and correlation analysis was also performed.

RESULTS AND DISCUSSION

Table 1. Correlation matrix of observations.

LA LAI YD FW PH FD FL

NF 0.95** 0.96** 0.36* -0.73 -0.75 -0.22 -0.19 FL -0.16 -0.16 -0.09 0.15 0.36 0.31 FD -0.25 -0.25 0.016 0.25 0.61 PH -0.65 -0.65 0.18 0.9 FW -0.58* 0.57 * 0.36* YD 0.56* 0.56* LAI 0.99** * , ** ; significant at p=0.05 and 0.01 level, respectively

The correlation coefficients between studied parameters are presented in Table 1. The leaf area (LA), Leaf area index (LAI), Number of fruits (NF) and Fruit weight (FW) had positive and significant association with yield (YD).

25

21 20 ) a

h 15 / t (

d l e i a Y 10 b bc b *d bc

5

0 Main crop 2 branch 3 branch control (2 branch +) (3 branch +) (control +) mulch mulch mulch

Figure 1. Mean yield (t/ha) of main and ratoon crops. (* Duncan multiple range test (DMRT) results; Coloumns with same letters are not significantly different at p=0.05).

Number of branches had direct effect on yield of ratoon crop, increasing number of branches from two to three in ratoon mulched crop led to yield increase from 7.9 to 9.9 t/ha (Fig. 1). Non-branch managed ratoon crop in control treatment with mulch and without mulch produced 7.5 and 8.2 t/ha, RATOON CROP MANAGEMENT OF BRINJAL 83 respectively. The fresh crop produced 21 t/ha. This was about three times higher than ratoon crop.

Leaf area, leaf area index and fruit characteristics

The leaf area of ratoon crops significantly varied among treatments at p=0.05. Among ratoon crops, control treatment with mulching produced the highest leaf area of 11830 cm2. This was less than the leaf area of fresh crop.

3.5 3.21

3

2.5 a a 2 b c I b A L

1.5 d*

1

0.5

0 Main crop 2 branch 3 branch control (2 branch +) (3 branch +) (control +) mulch mulch mulch

Figure 2. Mean leaf area index (LAI) of main and ratoon crops. (*DMRT results; Coloumns with same letters are not significantly different at p=0.05).

Table 2. The mean value of fruit length, diameter, weight and leaf area of main and Ratoon crop.

Treatment Fruit length Fruit Avg.fruit Leaf area (cm) diameter (cm) weight per plant (grams) (cm2)

Main crop 21.9 6.3 132.8 17365 T1 13.8 ab 3.7 a 75 c 7134 c T2 14.2 a 3.4 bc 80.8 a 9588 b T3 12.3 c 3.4 bc 58.1 d 11514 a T4 13.4 ab 3.3 c 84.5 b 9646 b T5 12.6 c 3.6 a 86.4 b 10374 b T6 13.9 ab 3.6 a 61.5 d 11830 a Values in a column with same letters are not significantly different at p=0.05

Increasing branch number from 2 to 3 led to increasing leaf area in both mulched and non-mulched plants (Fig. 2). Mulching had direct positive effect on leaf area. Optimum leaf area of plant balanced 84 ARULMAGESWARAN AND CHANDRASIRI photosynthesis and led to enhanced yield (Yadav et al., 1974). Plants with three branches and mulching produced optimum leaf area and gave the highest yield level of 9.9 t/ha. Mean number of days required to 50 percent flowering was 58 days in fresh crop and 31days in ratoon crop. Leaf area (LA), leaf area index (LAI), average fruit length (FL) and diameter, and average fruit weight (FW) are presented in the Table 2.

CONCLUSIONS

It can be concluded that on the average ratoon crop provided 1/3 yield of main crop (Stansel, 1977). The management of ratoon crop enhanced yield level more than non-branch managed ratoon crops. Management of 3 branches with mulch produced optimum leaf area and dominated over 2 branch managed and control ratoon crops in respect of yield. The low yield in ratoon crop may be due to physiological aging of plants. The beneficial advantages of ratoon crops are coming to flowering early and providing considerable yield without doing much effort on land. Further, physiological study of ratoon crop with cost-benefit analysis would reveal more information about cultivation of ratoon crop.

ACKNOWLEDGEMENTS

Authors wish to express sincere gratitude to Mr. K.G.Sriyapala, Principal/Assistant Director of Agriculture of School of Agriculture, Kundasale and other staff for their assistance provided to carry out this experiment successfully.

REFERNCES

D.O.A. 1990. Crop recommendation Technoguide. Department of Agriculture, Sri Lanka. Pp:46-50. Choudhry, B. and T.S. Kalda, 1960. A vegetable of the masses. Indian Horticulture 12:21- 23. Stansel, J. 1977. Cost cutting fertilization and rationing technologies. Field day of the Texas A and M Agricultural Research and Extension Center at Beaumont, July 1977. Panabokke, C.R. 1996. Soil and agro-ecological environments of Sri Lanka. Natural Resource, Energy and Science Authority of Sri Lanka. Rajapaksha, U.J. 1998. Traditional food plants in Sri Lanka. Hector Kobbekaduwa, Agrarian Research and Training Institute, Colombo. p:480. Tindall, H.D. 1983. Vegetables in the tropics. The Macmillan Press Ltd., London and Basingstoke. Pp:281-319. Tomar, B.S., K.A. Kalda, K.A. Balakirishna and S.S.Gupta. 1996. Explaining nutritive potential of brinjal. Indian Horticulture 40 4:32. Yadav, M.S., K.I. Mehara and M.L. Magoon. 1974. Genetic variability and correlation of a few quantitative characters in the pasture grass, Cenchurs ciliaris. Indian Forester 100:512-17. Annals of Sri Lanka Department of Agriculture. 2006.8.341-344.

SHORT COMMUNICATION

EFFECT OF DIFFERENT MULCHING MATERIALS ON GROWTH AND DEVELOPMENT OF HYBRID SWEET CORN(Zea mays L.) VARIETY, HONEY SWEET.

S. ARULMAGESWARAN, I.M.N. CHANDRASIRI and K.G. WASANTHA Extension and Training Centre, Peradeniya

INTRODUCTION

Maize is the second important cereal crop in Sri Lanka next to rice. Most of the farmers in Sri Lanka cultivate inbred and hybrid varieties for fresh cob and animal feed. Sweet corn is the green maize of world commerce but various other types are available. It is produced for human consumption either as fresh or processed products. However, developing countries including Sri Lanka have paid little attention to cultivation of sweet corn (Akintunde, 1987). Low yield in maize is attributed to various factors such as moisture stress, poor crop management practices, low soil fertility, pest and disease incidence etc. Moisture stress has been a major cause of yield reduction in corn as it grows in dry zone under rain-fed conditions (Satchithananthan and Bandara 2000). Almost all growth stages are often vulnerable to moisture stress and have shown yield reduction very often under dry conditions (Heisey and Edmeades, 1998). It affects leaf development (Wolfe et al., 1988), photosynthesis and grain filling of maize (Muschow, 1988). Mulching is one of the cultural practices to overcome the above problem. The commonly used non-live mulching materials are rice straw, crop residues, cut grass, paddy husk and polythene mulch. Each mulching material has its merits and demerits. The advantages of polythene mulch have been known as early as 1950’s (Spice, 1959) and it helps to increase nutrient absorption (Moore, 1989) and yield (Jayasinghe and Goonasekera, 1993). This experiment was designed to find out the suitable mulching material for hybrid sweet corn production and to study the effect of different mulching materials on growth and development of hybrid sweet corn.

MATERIALS AND METHODS

The study was conducted in maha 2005/06 in the School of Agriculture, Kundasale in the Intermediate Zone (IM3a) on immature brown loam soil (Panabokke, 1996). Mean annual rainfall is greater than 1400 mm and mean atmospheric temperature ranges from 25° to 32°C. The treatments were white polythene (T1), black polythene (T2), control (T3) and paddy straw (T4). The treatments were laid out as Randomized Complete Block Design (RCBD) with four replications. The hybrid sweet corn variety used in 86 ARULMAGESWARAN et al. this experiment was honey sweet and crop was managed in accordance with the recommendations of the Department of Agriculture for inbred maize (DOA, 1990). Growth and yield parameters were studied and data collected were subjected to ANOVA by using SAS. The Duncan Multiple Range Test (DMRT) was also carried out for mean comparison.

RESULTS AND DISSCUSION

1020 a

1010

1000 b 990 b*

t 980 n a l p / 2 m 970 c

A

L 960 c

950

940

930

920 White polythene Black polythene Control Paddy straw Treatments Figure 1. The mean leaf area per plant with different mulching materials. (*DMRT results; Coloumns with same letters are not significantly different at P=0.05).

600

b a 500 c* d

) 400 s m m

m o

a p r g (

t

h 300 g i e W

200

100

0 White polythene Black polythene Control Paddy straw Treatments Cob weight with husk Cob weight after removal of husk EFFECT OF MULCH ON SWEET CORN PRODUCTION 87

Figure 2. The mean cob weight with husk and without husk. (*DMRT results; Coloumns with same letters are not significantly different at P=0.05).

Growth and yield characteristics of maize with different mulches showed significant difference. The mean leaf areas per plant for different mulch materials are shown in Figure 1. The mean leaf area of treatments were significantly different at p=0.05. Straw mulched plants produced larger leaves and leaf area. The leaf area of white polythene mulched plants did not differ significantly from control plants.

Mulching treatments had significant effect on yield and yield components. The mean weights of cob with husk and without husk are shown in Figure 2.

After removal of husk, the mean weight of cob was higher (375.9 g) in straw mulched plants and did not differ significantly from control plants (370 g). The black polythene mulched plants produced the lower cob weight (341.67 g) than white polythene mulched plants (356 g). Other yield related parameters studied in this experiment are shown in Table1. There were significant differences among yield and yield components at p=0.05. The straw mulched plants gave the highest yield of 31.5 t/ha whereas yield of control and white polythene mulched plants were not significantly different at p=0.05. The black polythene mulched plants produced the lowest yield of 30.2 t/ha.

Table 1. The mean values of yield related studied parameters.

Yield components White Black Control Paddy straw polythene polythene mulch Cob length 20 d 20.6 c 21.7 b 22.3 a Cob circumference 17.6 b 17.5 b 17.6 b 18 a Number of grains/row 18 a 18.33 a 17.6 b 18.6 a Number of grains/column 38.2 c 41 b 41.3 b 42.1 a Yield (t/ha) 30.2 b 29.3 c 30.8 b 31.5 a Means in a column with same letters are not significantly different at p=0.05.

CONCLUSIONS

The experiment revealed that mulching of sweet corn had significant effect on yield. Paddy straw mulch provided suitable condition for growth of sweet corn whereas frequent addition during growth period was important. Black polythene mulch induced growth during vegetative phase but not much contributed to yield increase. Further studies on the effect of mulch on soil properties and evaluation of mulch for consecutive season are important to recommend the suitable mulching material for sweet corn. 88 ARULMAGESWARAN et al.

ACKNOWLEDGEMENTS

Authors wish to thank to Mr.K.G.Sriyapala, Principal/Assistant Director of Agriculture at School of Agriculture, Kundasale for encouragement and necessary support to complete this study successfully.

REFERENCES

Akintunde, A.Y. 1987. Yield potentials, nitrogen use efficiencies and economics of green cob production of single, three way and double cross hybrid maizes in south- west Nigeria. M.Sc thesis. Nigeria. University of Ibaden. D.O.A., 1990. Crop recommendation Techno guide. Department of Agriculture, Sri Lanka. Pp:46-50. Heisey, P.W. and G.O. Edmeades, 1998. Maize production in drough-stressed environments. Technical option and research resource allocation. Pp:1-5. Jayasinghe, A.P.R and K.G.A. Goonasekera, 1993. Influence of polyethylene film mulch on the yield of irrigated chilli in the dry zone of Sri Lanka. Trop.Agri.Res. 5:41-49.

Moore. F. 1989. Black polythene, CO2-9%. Yield boost. Ag. Consultant. p:16. Muschow, R.C.1988. Effect of nitrogen supply on the comparative productivity of maize and sorghum in a semi-arid tropical environment. Field Crop Research 18:1-16. Pannabokke, C.R. 1996. Soil and Agro-ecological environments of Sri Lanka. Natural Resource, Energy and Science Authority of Sri Lanka. Satchithanantham, S. and D.C. Bandrara, 2000. Physiological response of maize (Zea mays L.) to the interactive effects of nitrogen fertilizer and water regimes. Ed. S.P. Nissanka .Trop.Agri.Res: 72:22-32. Spice, H.R. 1959. Polythene film in horticulture Chap.10 Faber and Faber Publication, London.Pp:131-138. Wolfe, D.W., D.W. Henderson, T.C. Hsiaso and A. Alvino, 1988. Interactive water and nitrogen effects on senescence of maize 11. Photosynthetic decline and longevity of individual leaves. Agro.J. 80:865-870. Annals of Sri Lanka Department of Agriculture. 2006.8:345-349.

SHORT COMMUNICATION

EFFECT OF INSECTICIDES IN CONTROLLING RICE LEAF FOLDER - Cnaphalocrosis medinalis (Guenee)

C.M.D. DHARMASENA Rice Research and Development Institute, Batalagoda, Ibbagamuwa*

INTRODUCTION

Yield loss caused by insect pest in rice is around 10 - 20% (Kudagamage, 2000). Of the six major insect pests identified, Rice Leaf Folder (RLF) causes a considerable yield loss of 5 - 25% in infested fields (Kudagamage, 2000). Sixty percent of the Sri Lankan farmers applies insecticides to control RLF (Nugaliyadde et al., 2000). Rice Gall Midge (RGM), Brown Plant Hopper (BPH) and Yellow Rice Stem Borer (YSB) are the other important insect pests at the vegetative stage of the rice crop. Of the six insecticides recommended by the Department of Agriculture for the control of RLF, Chlorpyrifos is popular among farmers because of its ability to kill insect pests quickly. However, over-use of Chlopyrifos over a long period of time in the past has led to development of resistant strains to target and non- target pests (Nugaliyadde et al., 2000). Therefore, the present study was carried out to evaluate effective as well as safer insecticides against RLF and all the other major insect pests at the vegetative stage.

MATERIALS AND METHODS

A field experiment was conducted at the Rice Research and Development Institute (RRDI) Batalagoda, Ibbagamuwa in 2005 yala season. The rice seedlings (Variety BG 94/1) were transplanted in experimental field with a plot size of 17 m2 and 15 X 10 cm spacing. All agronomic practices, except pesticide application were carried out according to the recommendation of the Department of Agriculture (DOA). Experimental Design was Randomized Block Design with four replications. Four insecticides, namely Azadirachtin 0.3 g/l EC at 3000 ml/ha, Dinotofuran 20 % WP at 1000 ml/ha, Ethiprol 100 g/l EC at 500 g/ha and Carbosulfan 200 g/l SC at 1500 ml/ha were evaluated against RLF and other major insect pests, taking Tebufenozide 200 g/l F at the rate of 750 ml/ha as the standard check and the untreated control plots as the control. In addition, to the field experiment at RRDI, Batalagoda, these insecticides were evaluated in the farmer’s field at Moragolla, Ibbagamuwa, where RLF population was high in the same season. 90 DHARMASENA

*Present address: Office of the Deputy Director of Agriculture (Extension), Badulla First insecticide application was carried out when the RLF infestation was visible in the field with few rolled leaves and the second insecticide application was carried out 10-12 days later. Numbers of predators (Spiders, Carabid beetles and Dragon flies) were recorded using two sweep net counts one day before and two days after each insecticide application. One cycle of sweep was considered as one sweep. Pre-spray count was taken from one corner and the post-spray count was taken from the opposite corner. Total number of leaves and RLF damaged leaves were counted five days after the second insecticide application taking five randomly selected hills per plot and percentage number of leaf damage was calculated in the field at RRDI, Batalagoda. Similarly, percentage of leaf area damage due to RLF was calculated in the same hills, taking only the last three leaves of each tiller.

However, in the farmer’s field at Morogolla, only the total number of hills damaged due to RLF were counted and percentage numbers of damage hills were calculated. Furthermore, total number of white heads per plot was counted at both fields. Yield data were collected at both fields. Yield data were recorded taking 3 m2 areas at the center of each plot.

Some sets of data were transformed according to the need. Data were analyzed using ANOVA and means were compared using LSD at 0.05 probability level. MSTAT computer package was used to analyze the data.

RESULTS AND DISCUSSION

Percentage of damaged leaf number as well as percentage of damaged leaf area in the field at RRDI, Batalagoda, were significantly low in plots treated with Dinotofuran 20% WP, with 24.9% and 12.9% respectively, compared to 41.9% and 27.1% respectively in the control plots (Table 1). Furthermore, 227 RLF damaged hills per plot treated with Dinotofuran in the field at Moragolla was significantly lower than 381 damaged hills per plot in the control plots. Results show that Dinotofuran 20% WP was as effective as Tebufenozide 200 g/l F; the present recommendations of DOA (Anon., 1997) are with 18.7% number of leaf damage and 11.2% leaf area damage at RRDI, Batalagoda and 168 RLF damaged hill per plot at Moragolla.

Mean number of white heads per plot at Batalagoda (271) and Moragolla (247) in plots treated with Dinotofuran 20% WP were significantly lower than 588 and 528 white heads per plot in the control plots respectively (Table 2). Furthermore, results show that Carbosulfan 200 g/l SC (340 whiteheads per plot) was as effective as Dinotofuran 20% WP (247 white heads per plot) in controlling YSB at Moragolla. EFFECT OF INSECTICIDES ON RICE PESTS 91

Table 1. Effect of five different insecticides in controlling rice leaf folder damage at Batalagoda and Moragolla in 2005 yala season.

Treatment RRDI Batalagoda Moragolla % of leaves % of leafarea Mean number of damaged by damaged by hills with RLF RLF* RLF * damage ** Control 41.9 a 27.1 a 381 a (0.95) (0.49) (2.58) Ethiprol 100 g/l EC 35.4 a 21.9 a 319 ab (0.72) (0.40) (2.40) Carbosulfan 200 g/l SC 33.6 a 19.7 ab 306 ab (0.71) (0.36) (2.39) Azadirachtin 0.3 g/l EC 32.6 ab 24.9 a 217 ab (0.70) (0.46) (2.20) Dinotofuran 20% WP 24.9 b 12.9 bc 227 b (0.48) (0.23) (2.19) Tebufenocide 200 g/l F 18.7 b 11.2 c 168 b (0.33) (0.2) (2.11) CV% 27.8 27.1 14.6

LSD(0.05) 0.41) (0.16) 0.38

* Figures in the parantheses are arcsin values ** Figures in the parantheses are log values

Table 2. Effect of five different insecticides in controlling yellow stem borer in rice at Batalagoda and Moragolla in 2005 yala season.

Treatment Batalagoda Moragolla Mean no. of whiteheads Mean no. of whiteheads per plot per plot Ethiprol 100 g/l EC 588 a 528 a Azadirachtin 0.3 g/l EC 544 a 538 a Control 511 ab 505 a Tebufenocide 200 g/l F 485 ab 506 a Carbosulphan 200 g/l SC 418 b 340 b Dinotofuran 20% WP 271 c 247 b CV% 16.8 22.3 LSD(0.05) 119 149

Nevertheless, carbosulfan 200 g/l SC was less effective at Batalagoda (418). However, Carbosulfan 200 g/l SC was found to be effective in comparison with the control at Moragolla (505 white heads per plot) though the same treatment was not effective at Batalagoda (418 white heads per plot) in controlling YSB. Probably the biotic and environmental factors found in both locations may be different in controlling YSB. Other major pests at the vegetative stage, namely, BPH and RGM were not found in high numbers to 92 DHARMASENA cause significant damage to the crop. Average yield per plot in plots treated with dinotofuran 20% WP as well as tebufenocide 200 g/l F at RRDI, Batalagoda (5.65 t/ha) and Moragolla (6.21 t/ha) were significantly higher than 4.73 t/ha and 4.8 t/ha in the control plots respectively (Table 3).

Table 3 Yield performance under different insecticide treatments at Batalagoda and Moragolla in 2005 yala season.

Treatment Batalagoda Moragolla t/ha t/ha Dinitofuran 20 WP 5.65 a 6.21 a Tebufenocide 200 g/l F 5.43 ab 6.47 a Carbosulphan 200 g/l SC 5.16 bc 4.99 b Azadirachtin 0.3 g/l EC 4.90 c 5.18 b Control 4.73 c 4.80 b Ethiprol 100 g/l EC 4.36 c 4.78 b

CV% 6.14 14.5 LSD(0.05) 0.7 1.78

Number of predators, namely Spiders, Carabid beetles and Dragon flies found at two days after the first spray in plots treated with carbosulphan 200 g/l SC (0.8) was significantly lower than 1.8 per sweep net in the control (Figure 1). Furthermore, 0.9 predators per sweep net in plot treated with tebufenocide 200 g/l F was significantly lower than 2.3 in the control one day before the second spray. Nevertheless, number of predators found in dinotofuran 20% WP treated plot was not significantly different from those of theFig. 1 control Effect of five plots different at insecticideany stage. treatments on major predators in rice at Batalagoda in 2005 yala season

3

LSD(0.05) = 1.1

2.5 LSD (0.05) = 0.99 LSD (0.05) = 1.1

t LSD (0.05) = 0.89 e n 2 p e e

w Azadirachtin s /

s Tebufenocide r o t Ethyiprol a 1.5 d

e Dinotofuran r p Carbosulphan f o

r Control e b

m 1 u N

0.5

0 1st pre spray 1 st post spray 2nd pre spray 2nd post spray Stage of the environment

Figure 1. Effect of five different insecticide treatments on major predators in rice at Batalagoda in 2005 yala season. EFFECT OF INSECTICIDES ON RICE PESTS 93

Dinotofuran 20% WP is a new furanicotinyl insecticide, belonging to neonicotinid group. According to the Environment Protection Agency in USA, this insecticide is an organophosphorus alternative with reduced risk status. Acute mammalian toxicity is low with LD50 value as high as 2000 mg/Kg for Rat oral as well as dermal.

Present study shows that dinotofuran 20% WP at the rate of 1000 g/ha was as effective as tebufenocide 200 g/l SC at the rate of 750 ml/ha in controlling RLF and minimize yield losses significantly. Furthermore, there is a possibility of using this novel insecticide for control of YSB as well. Moreover, the novel insecticide found to be less toxic to major predators found in both locations. However, it is necessary to evaluate this insecticide with insecticides recommended for the control of YSB to confirm the result of the present study.

CONCLUSIONS

Denotofuran 20% WP at the rate of 1000 g/ha could be used for the control of Cnaphapalocrosis medinalis in rice.

REFERENCES

Anon., 1997. Pesticide recommendations, Department of Agriculture, Peradeniya, Ministry of Agriculture and Lands. 76p. Kudagamage, C. 2000. Present status and future direction of pest management of food crops In seminar of pest control in the next millennium, Eds Rohan Rajapaksa and Ratnasekara, 1-20. Nugaliyadde, L., D.M.N. Disanayake and J. Mitrasena. 2000. Advances of pest and disease management of rice in Sri Lanka, Annals of the Sri Lanka Department of Agriculture. 2: 402 – 422. Annals of Sri Lanka Department of Agriculture. 2006.8:351-354.

SHORT COMMUNICATION

BIOLOGY OF PLANT HOPPER, Hansenia glauca. Kirby INFESTING JAMAN, WATER APPLE AND GUAVA

L.D.GALANIHE Horticulture Crop Research and Development Institute, Gannoruwa, Peradeniya

INTRODUCTION

Jaman (Madan), Sizigium cumini and water apple (pini-jambu), Syzigium samarangense are not cultivated on commercial scale and are under- utilized. All parts of jaman tree have medicinal value and fruits are edible. The juicy fruits of water apple are popular among children. In India, many pests were reported to infest jaman and water apple (Nayar et al., 1981; Chanduwat, 1990 and Arya, 1993). However, there are no published reports about the pest prevalent in Sri Lanka.

Leaves of jaman and water apple trees at Gannoruwa, were observed to be heavily infested with a plant hopper which had been first collected from Peradeniya, Sri Lanka in 1900 on Eugenia malaecensis, and was identified as Hansenia glauca Kirby (Hemiptera: Fulgoroidea) and later on Eugenia jambos. The specimens were deposited in the insect reference collection of the Department of Agriculture (DOA) with reference nos.2003, 4112, 6708. There is no published information available on this pest either in Sri Lanka or elsewhere. Therefore, present study was carried out to investigate the biology of the pest.

MATERIALS AND METHODS

This study was conducted at the Horticulture Crop Research and Development Institute (HORDI) Fruit Orchard Unit 1 at Gannoruwa during the period 2004 to 2006.

Laboratory study

Ten jaman leaves containing freshly laid egg masses were collected from the field and brought to the laboratory. The nymphs that emerged from each egg mass were transferred separately into ten petri dishes at the rate of 10 nymphs per petri dish each lined with damp filter paper containing an immature, uninfested jaman leaf. The petri dishes were placed in the laboratory at 30°C±1°C, 70-90% RH. The insects were observed daily under microscope and changes in life stages were recorded. Insects were transferred to fresh leaves as and when necessary. 96 GALANIHE

Field study

Thirty freshly laid egg masses on the three host plants in the field were labeled. These egg masses were observed twice a week and records taken at hatching, larval development and adult emergence.

RESULTS AND DISCUSSION

Biology

Eggs: Are translucent, white, and cylindrical and laid on the lower surface of the leaf in groups of 20 to 60 (average 44) in parallel lines (Plate 1). Oval shaped egg masses are covered with white waxy scales. Under laboratory conditions, the incubation period varied from 3-7 days while in the field it unusually took a long time (20-64 days). This phenomenon needs to be studied further.

Nymphs: There are five nymphal instars in the life cycle. First instar nymph is white, bare bodied with long legs and prominent black eye spots. The abdomen is upturned. During the development of the nymphal instars (Plate 2), they exude white, waxy exudates in the form of abdominal filaments on their body until white feathery filaments cover the entire body. With the development their body colour changes to yellow and orange. Last instars (Plate 3) are light greenish white. Thorax is large and broader than the abdomen and covered by the developing wing pads. At the end of the abdomen there are thick, short and long filaments. The nymphs mostly stay in an aggregated colony concealed within their casted waxy filaments (Plate 4). In the laboratory, duration of the nymphal period ranged from 15- 21 days. In the field it ranged from 30-45 days.

Adults: Adult (Plate 5) is a medium size insect with bluish white wings, triangular in shape and moth like in appearance. It has two pairs of wings covered with pure white powdery substance. Their broad triangular wings are folded to form a steep roof over the body. Wingspan is 10 -15 mm. with an average of 12.6 mm. The adult plant hoppers were mostly associated with the leaves having the nymph masses. They were also seen resting in rows along the twigs and on large veins of the leaf among the nymphs (Plate 6). They suck sap from leaf veins, immature twigs and also from the developing flowers and fruits. BIOLOGY OF PLANT HOPPERS 97

Plate 1. Egg masses of plant hopper Plate 2. Early instar nymphs

Plate 3. Last instar nymph Plate 4. Waxy mass of nymphs

Plate 5. Adult of the plant hopper Plate 6. Adults resting in rows

Host plants

Both adults and nymphs were found feeding on jaman, water apple, guava and chinese guava. Although guava and chinese guava were infested with egg masses and early instars of nymphs, later instars and adults could not be seen abundantly. All developmental stages were found infesting heavily on jaman and water apple trees indicating that jaman and water apple are the main host plants of this pest.

Damage

Feeding of the large number of nymphs on the leaves caused yellowing of leaves and the damaged leaves eventually dropped. Black sooty moulds grow on the upper surfaces of leaves and on flowers and fruits due to honey dew secreted by the insects. Adults and nymphs suck sap from flowers 98 GALANIHE and fruits of water apple. Due to their feeding habit, the damaged young fruits dried and dropped off. Mature fruits were malformed. This resulted in the reduction of yield and fruits were unmarketable.

CONCLUSIONS

The insect pest that damages jaman, water apple and guava having bluish white triangular wings covered by a powdery substance and resembling a medium sized moth is the plant hopper, Hansenia glauca Kirby. Wings are held like a steep roof on the body while resting. The adults characteristically sit in rows along the twigs of the trees while resting. Cylindrical, white, translucent eggs covered with white waxy scales are laid in oval shaped masses on the lower surface of leaves. There are five nymphal instars in the life cycle. White, feathery waxy filaments cover the entire body of the nymph. They are active and mostly in an aggregated colony covered by their casted skins and feed on leaf sap. The damaged leaves turn yellow and eventually drop. Honey dew secreted by the insects results in growth of sooty mould on the leaves, flowers and fruits. Adults and nymphs suck sap from developing fruits resulting in the damaged fruits being malformed or to dry up and fall. Jaman and water apple are the main host plants of the pest.

ACKNOWLEDGEMENTS

The author gratefully acknowledges the technical assistance provided by Mr. Zameer Ahmed, Miss. Ruwanthi Jayasinghe and Miss.Anusha Vithana.

REFERENCES

Arya, A. 1993. Tropical fruits. Diseases and pests. Kalyani Publishers. New Delhi. 208p. Chanduwat, B.S. 1990. Arid fruit culture. Oxford and IBH Publishing Co. Pvt. Ltd. New Delhi. 208p. Nayar, K.K., T.N. Ananthakrishnan and B.V. David. 1981. General and applied entomology. T.M.H.Publ. Co.Ltd. New Delhi. In A. Arya. 1993. Tropical fruits. Diseases and pests. Kalyani Publishers. New Delhi. 208p. Annals of Sri Lanka Department of Agriculture. 2006.8:355-359.

SHORT COMMUNICATION

RICE MILLING QUALITY, GRAIN FILLING AND YIELD INFLUENCED BY FERTILIZER MANAGEMENT

R. F. HAFEEL, D. N. SIRISENA, E.M. R. D. EDIRISINGHE and D. M. N. DISSANAYAKE Rice Research and Development Institute, Batalagoda, Ibbagamuwa

INTRODUCTION

Grain yield alone does not satisfy the producer, millers or consumers as market value of rough rice is based, to a larger extent, on milling quality and milling out-turn. Therefore, attention has been focused on improving grain yield and milling quality of rice. Milling quality is defined as the head rice recovery after milling (Brorsen et al., 1984). Many studies have been conducted on the factors that affect milling quality, such as genetic (Jongkaewwattana et al., 1993; Nangju and De Datta, 1970), field management (Yoshida, 1981; Leesawatwong et al., 2004)) and environmental conditions during crop growth (Henderson, 1954; Yoshida and Hara, 1976). Milling quality of rice may depend mainly on the rate of grain filling as compact grain structures show less breakage than loosely packed structures. There are many factors that contribute to grain filling during plant growth. According to Li and Senadeera (1991), rate of grain filling is mainly controlled by additive gene effect and has very high heritability. On the other hand, Seetanum and Datta, (1973); Wickramasinghe (1987) and Wopereis- Pura et al. (2002) reported that rate of grain filling can be controlled by fertilizer management. With the available information it could be understood that grain yield of rice can be increased with the selection of high yielding rice varieties along with high rates of fertilizer application, but understanding the effect of these factors on the rate of grain filling and milling quality of rice varieties remains unclear. As such, the objective of this study was to determine the effects of fertilizer management practices on grain yield, rate of grain filling and milling quality parameters of some cultivated rice varieties.

MATERIALS AND METHODS

A field trial was established in yala 2001 and continued in maha 2001/2002 with three popular rice varieties (Bg 403, Bg 352, Bg 300) and three fertilizer management practices. The practices were fertilizer recommendation of the year 2000 (F1), 1998(F2) and no fertilizer (Fo) as control. F1, included, 6 t/ha straw, 1 t/ha glyricidia, 5 t/ha cow dung and 625 kg/ha burnt paddy husk with 140 kg N/ha, 45 kg P2O5/ha , 45 kg K2O/ha and 1 kg ZnO/ha applied as urea, super phosphate, muriate of potash and ZnSO4 100 HAFEEL et al.

respectively. F2 included above levels of urea, super phosphate and muriate of potash but no organic matter was added. The treatments were replicated thrice and arranged in split plot design with varieties as main plots and fertilizer levels as sub plots. All field practices were alike for all the plots except fertilizer management practices. Panicles were tagged when approximately 5% of spikelets had dehisced anthers. Five tagged panicles were taken randomly from the day of tagging to 36 days after tagging at 4-days interval. The panicles were oven dried at 48°C for 3 days. Filled or unfilled grains, of each panicle were removed by hand, counted and 1000 grain weight recorded. At the physiological grain maturity, each treatment was harvested and grains were shade dried for 2-3 weeks to bring down the moisture content to13% and weighed. Samples were collected, de-hulled and milled in a Mc Gill sample mill for one minute and milling quality parameters such as brown rice, total milled rice and head grain percentage were quantified. These parameters were tested according to Cruz and Kush (2000).

RESULTS AND DISCUSSION

Mean yield of all varieties significantly increased with fertilizer application (treatments F1 and F2) compared to control (F0) irrespective of season (Table1).

Table 1. Grain yield of the rice varieties at three fertilizer levels.

Yala Maha Treatmen Bg 403 Bg 352 Bg 300 Bg 403 Bg 352 Bg 300 t F1 6.9a 6.5a 6.0a 6.5a 6.2a 6.0a F2 6b 6.3b 5.5b 5.5b 5.3b 5.0b F0 3.5c 3.23c 3.1c 3.2c 3.2c 3.0c Means with similar letters in each column are not significantly different

Comprising higher N P K level as well as organic manure, F1 recorded the highest mean yield (6.4 t/ha in yala season) followed by F2 (5.9 t/ha) and F0 (3.3 t/ha) irrespective of varieties and season. The combined effect of N, P, and K fertilizer with organic manure could be the reason for higher yield obtained with F1. In addition, rate of grain filling was also higher in the plots treated with fertilizer than the untreated plots (Figs.1 and 2). As a result the rate of grain filling may have also contributed to the high yields in the fertilized plots. According to Jennings et al. (1979), rate of grain filling is not a varietal character but depends mostly on environmental factors. Similarly the mean rates of grain filling in the fertilized plots were significantly higher than that in the control plots (Figs.1 and 2). As suggested by Wickramasinghe (1987), application of nitrogen increases grain filling rate while application of potassium decreases it. This may be a reason for grain filling rates in F1 and F2 not being significant even with application of high FERTILIZER MANAGEMENT FOR RICE GRAIN QUALITY 101 fertilizer levels. In addition, grain yield is not only decided by the rate of grain filling but also by the number of grains per panicle (Jennings et al., 1979). As such, the rate of grain filling alone cannot be taken as a factor that decides grain yield in rice.

In comparison to control plots, head rice recovery values were higher than the plots treated with fertilizer F1 and F2 practices irrespective of the season (Table 2). According to Japanese Food Agency (1998) application of N fertilizer can enhance photosynthetic capacity during grain filling period leading to an increase in head rice yield. In addition, Sajwan et al. (1990) found that grain protein content, head rice yield, and hardness increased with increasing N rates. As suggested by Leesawatwong et al. (2004), application of high rates of N may increase protein N content of rice together with corresponding decrease in the percentage of endosperm breakage. There was a significant effect of fertilizer practices on milling quality of rice irrespective of season, but between two fertilizer treatments there was no significant difference (Table 2).

22 %

e t a

r 17

g n i l l

i 12 f

n i a

r 7 G 2 0 5 10 15 20 25 30 35 40 Days after 5% flowering

F1 F2 F0

Figure 1. Mean grain filling pattern of three rice varieties under different fertilizer management during yala 2001.

22

17

12

7

2 0 5 10 15 20 25 30 35 40 Days after 5% flowering

F1 F2 F0

Figure 2. Mean grain filling pattern of three rice varieties under different fertilizer management during maha 2001/02. 102 HAFEEL et al.

Table 2. Percent brown, total milled and head rice of different rice varieties during yala 2001 and maha 2001/02.

Treatment Brown rice % Total milled rice % Head rice% Yala Maha Yala Maha Yala Maha F1 80.50a 79.62a 76.33a 74.22a 66.28a 68.76a F2 80.63a 80.01a 75.89a 74.12a 67.00a 65.19a F0 79.47b 78.90b 74.40b 71.46b 63.30b 59.29b Means with similar letters in each column are not significantly different

CONCLUSIONS

Application of the year 2000 fertilizer recommendation increased rice yields significantly without reduction in milling quality parameters. Therefore, the above recommendation increases rice production while fulfilling the needs of the producer, miller and consumer.

REFERENCES

Brorsen, B.W., W.R. Grant and M.E. Rister. 1984. Economic values of rice quality factors. Texas Agriculture Experiment Station. Progress report 4202, p26. Cruz, N.D. and G.S. Kush. 2000. Rice grain quality evaluation procedures. In aromatic rices. Eds. R. Singh, U.S. Singh and G. S. Kush. Oxford and IBH Publishing Co. Pvt. Ltd. New Delhi. Pp 15-28. Henderson, S. M/1954. The causes and characteristics of rice checking. Rice Journal 57:16- 18. Japanese Food Agency. 1998. Rice inspection technology. The Food Agency, Ministry of Agriculture, Forestry and Fisheries. Jennings, P.R., W.R. Coffman, H E Kauffman. 1979. Rice Improvement. International Rice Research Institute. Manila, Philippines. 79 – 97. Jongkaewwattana, S.S. Geng, D.M. Brandon and J.E. Hill 1993. Effect of nitrogen and harvest grain moisture on head rice yield. Agronomy Journal: 85.1143-1146. Leesawatwong, M.S., J.J. Kuol, B. Dell and B. Rerkasem. 2004. Nitrogen fertilizer alters milling quality and protein distribution in head rice. 4th International Crop Congress. Li, G.F. and D. Senadera. 1991. Variability and genetics of rate and duration of grain filling in rice (Oryza sativa L.) Sabrao Journal 23(1):29-40. Nangju, D. and S. K. De Datta. 1970. Effect of time of harvest and nitrogen level on yield and breakage in transplanted rice. QGRONOMY Journal 62: 468-474. Sajwan, K.S., D. I. Kaplan, B.N. Mittra and H.K. Pande. 1990. Applied Agricultural Research. 5(3):198-204. Seetanum, W. and S.K. De Datta (1973). Grain yield, milling quality and seed viability of rice as influenced by time of nitrogen application and time of harvest. Agronomy Journal 65:390-394. FERTILIZER MANAGEMENT FOR RICE GRAIN QUALITY 103

Wikramasinghe, W.M.A.D.B. 1987. Preliminary studies on sink-source relationship of some rice varieties under different fertilizer regimes. Proceedings of rice research workshop on 16-17 June 1987 at Central Rice Breeding Station, Batalagoda, Ibbagamuwa, Sri Lanka Pp 169-189. Woperesis-Pura, M.M., H. Watanabe, J. Moreira and M.C. Wopewresis. 2002 Effect of late nitrogen application on rice yield, grain quality and profitability in the Senegal river valley. European Journal of Agronomy 17:191-198. Yoshida, S. 1981. Fundamentals of rice cropscience. International Rice Research Institute. LosBanos, Phillipines. Yoshida, S. and Haraa. 1076. Effect of air temperature and light grain filling of indica and japonica rice under controlled environmental condition. Soil Science and Plant Nutrition 2.93-107. Annals of Sri Lanka Department of Agriculture. 2006.8:361-364.

SHORT COMMUNICATION

ASSESSMENT OF QUALITY PARAMETERS OF SEED PADDY PRODUCED IN THE SOUTHERN PROVINCE OF SRI LANKA

T.JAYASENA SILVA and Y.M.H.LIYANAGE Seed Testing Laboratory, Bata-Ata

INTRODUCTION

Seed paddy production is a profitable venture. The country needs 4.4 million bushels of seed paddy per year. Out of which 15% is supplied as certified seed through various sectors by the multiplication program of the Department of Agriculture (Department of Agriculture, 2004). There is a high possibility of varietal purity deterioration with increase in number of cycles of multiplication. Certain limits have been imposed to regulate the quality standards of seed paddy.

MATERIALS AND METHODS

Each paddy sample represents a lot. Maximum lot weight for paddy was 25000kg. Sampling officers draw samples according to the rules of International Seed Testing Association (ISTA, 2004) and send a composite sample to the laboratory with a separate sample for moisture determination. A request form is also sent along with detailed description of the crop.

Whole number of samples received from private seed growers during the selected period in each district is included in the study. After the testing procedure in the laboratory, sample acceptance or rejection is determined by quality compared with the standards. Rejected percentage for each category was calculated depending on number of total rejected samples of respective district, irrespective of other causes for rejection.

In seed testing, mixing of other distinguishable varieties, noxious weed seeds, damaged seeds and other crop seeds were considered. The maximum number permissible for 500 grams for these factors is 100, 5,200 and 0 respectively for certified seed. Appearance and smell also should be at acceptable levels.

Germination test determines the maximum germination potential of a seed lot. Seed moisture content is determined by constant temperature oven method. 106 JAYASENA SILVA AND LIYANAGE

Laboratory test results of 5 seasons during the period maha 2002/03 to maha 2004/05 were considered for this study.

RESULTS AND DISCUSSION

A sample was rejected even if one factor concerned for quality certification exceeded the limit. Seed paddy from Galle district showed the highest rejection in all seasons. It was much higher than in other two districts and always over 50%. In Matara district the total sample rejection was lower than in Hambantota district except in yala 2003 when it was between 30-40%. Sample rejection in Hambantota was around 50% (Figure 1).

100

80

60

40

20

0 1 2 3 4 5 Season

Galle Hambantota Matara

Figure 1. Percentage samples rejected in Galle, Hambantota and Matara districts during maha 2002/03 to maha 2004/05.

Seed sample rejection due to moisture content showed more or less the same trend for each district. Hambantota being a drier area has an unusual high percentage of rejection due to moisture. Samples received from government farms in the same area were accepted for moisture, (unpublished data) showing farmer ignorance about drying seed lots properly (Figure 2).

Seed sample rejection due to weed seeds was comparatively low in Matara district. Galle district has shown the same trend but total rejection was much higher. Seasonal influence on weed population may contribute to weed seed production which finally affects the quality of the lot. Both districts fall under the wet zone and hence have similar climatic conditions.

Seed sample rejection due to weeds at Hambantota district showed an increasing trend throughout the period. Rotational water issues and sub optimal weed controlling may be the reason for increased weed population in Hambantota. Special attention must be paid for weed control by seed growers in Hambantota to avoid sample rejection (Figure 3). SEED PADDY QUALITY ASSESSMENT 107

80 %

60 n o i

t 40 c e j

e 20 R 0 1 2 3 4 5

Seasons

Galle Hambantota Matara

Figure 2. Percentage samples rejected due to moisture during maha 2002/03 to maha 2004/05.

80

60

40

20

0 1 2 3 4 5 Seasons

Galle Hambantota Matara

Figure 3. Percentage samples rejected due to weed seeds during maha 2002/03 to maha 2004/05.

Rejection of seed samples due to poor germination was identical in Galle and Matara districts. High seasonal influence was seen in both districts. Bad weather conditions prevailing in the wet zone during harvesting periods might cause low germination. Low percentages of rejection due to poor germination in Hambantota could be due to favorable weather conditions that prevailed during harvesting (Figure 4).

80 60 40 20 0 1 2 3 4 5 Seasons

Galle Hambantota Matara

Figure 4. Percentage seed sample rejections due to poor germination during maha 2002/03 to maha 2004/05.

Seed lot rejection due to other distinguishable varieties (ODV) was higher in Galle and Matara districts compared to Hambantota district. 108 JAYASENA SILVA AND LIYANAGE

Mixing of other varieties could be due to various reasons but small holdings in Galle and Matara districts with different varieties could be a major reason for high percentage of ODV (Figure 5).

80 %

60 n o i 40 t c e

j 20 e R 0 1 2 3 4 5 Seasons Galle Hambantota Matara

Figure 5. Percentage seed sample rejections due to other distinguishable varieties during maha 2002/03 to maha 2004/05. CONCLUSIONS 50-80% of seed lots in the certified seed program in Galle district have been rejected mainly due to high number of ODV weed seeds and moisture contents. This could be minimized by intensive farmer training, using proper weed control methods and using proper post-harvest operations. 40-50% of seed lots in the certified seed program in Hambantota district have been rejected mainly due to high number of weed seeds and moisture content. This could be minimized by using proper weed control methods, drying seed lots properly and using proper machinery for threshing. 30-50% of seed lots in the certified seed program in Matara district have been rejected mainly due to high number of ODV and high moisture contents. This could be minimized by intensive farmer training on seed production and proper drying of seed lots. Seed lot rejection due to poor germination was seen in yala seasons in Galle and Matara districts. Hence special attention is required in these districts to minimize rejections. ACKNOWLEDGEMENTS The author wishes to thank Dr. K.K.S. Fernando, Director SC&PP and Dr.K.A.Mettananda, Deputy Director SCS, Gannoruwa, for the encouragement given to carry out this work. REFERENCES

Department of Agriculture, 2004. Administration report. Peradeniya. Pp 184-185. International Seed Testing Association (ISTA) Edition, 2004. Chapters 3, 4, 5, 8, and 9. Annals of Sri Lanka Department of Agriculture. 2006.8:365-370.

SHORT COMMUNICATION

MOTHER BULB STORAGE OF BIG ONION (Allium cepa L.) FOR SEED PRODUCTION DURING THE YALA SEASON.

K.A. METTANANDA Seed Certification Service, Gannoruwa, Peradeniya

INTRODUCTION

Unavailability of good quality seeds of recommended varieties in adequate quantities is a major constraint to increasing productivity of big onion in Sri Lanka. At present, local production of true seeds is limited to small scale self seed level during December to April. Further expansion is not possible due to environmental constraints. Studies conducted locally have proved the possibility of undertaking medium to large scale production of true seeds during yala season. Mother bulbs for yala season seed production have to be selected from the previous yala harvest in October and stored until April the following year, nearly seven months. However, heavy losses could be expected when onions are stored under ambient conditions in the tropical region for long periods (Vendenburg and Lentz, 1973; Brice et al., 1997). Presently no suitable technologies are available to minimize these losses.

This investigation was undertaken at Pelwehera under REAP/CIC big onion seed project during 2004 - 2005 to quantify storage losses of mother bulbs of recommended varieties under ambient conditions and to identify the underlining reasons so that suitable strategies could be adopted to minimize losses during storage.

MATERIALS AND METHODS

Mother bulbs of big onion cultivars Rampur, Pusa red, and Agri found light red, produced by farmers in Kimbissa and Dambulla areas using locally produced seed during 2004 yala season were placed in a conventional type store shelf (1m x 6m) to a thickness of 7 – 8 cm. Separate sections of 1m x 6m were assigned to each variety and five such shelves were used for replications so that treatments could be in a RCBD with five replications. Perforated separations, 60cm x 60cm x 15cm (ht) were used at the middle of each section to collect data on storage losses. Data on sprouting and rotting were collected from each variety/replicate at three weeks interval.

Daily mean RH and temperature were also measured. Data were statistically analysed using “Minitab” (Ver. 13.1). 110 METTANANDA

RESULTS

Data on total storage losses in each variety due to sprouting and rotting from 6 to 24 weeks are given in Figure 1.

100

80

60

40

20

0 6 9 12 15 18 21 24 Weeks in storage

Rampur AFLR Pusa red Rampur Y= 2.99x -0.88 Linear (AFLR) Linear (Pusa red) Linear (Rampur) Pusa red Y= 2.7x -0.40 AFLR Y= 2.42x -1.04

Figure 1. Trend lines for total storage loss between 6 to 24 weeks storage period.

Variety Agri found light red had low total storage loss compared to Rampur which was about 62% for 24 weeks storage period (P<5%). Total storage losses of varieties Pusa red and Rampur were similar and around 70 – 78% during 24 weeks storage period (Figure 1).

Data on percentage of bulbs removed due to rotting in each variety at three weeks interval from 6 to 24 are given in Figure 2.

50

40

30

20

10

0 6 9 12 15 18 21 24 Weeks in storage

Rampur AFLR Pusa red Pusa red Y=1.6x +2 Linear (AFLR) Linear (Rampur) Linear (Pusa red) Rampur Y=1.74x - 0.88 AFLR Y=1.25x -1.0

Figure 2. Trend lines for cumulative storage loss due to rotting during 24 weeks storage period.

Variety Agri found light red had the lowest storage loss due to rotting contributing only about 30% to total storage loss (P<1%). Storage loss MOTHER BULB STORAGE FOR YALA SEED PRODUCTION 111 due to rotting in varieties Rampur and Pusa red were similarly high, contributing around 45% of the total storage loss (P<1%) (Figure 2). Data on percentage of bulbs removed due to sprouting at three week intervals from 6 to 24 are presented in Figure 3.

50

40

30

20

10

0 6 9 12 15 18 21 24 Weeks in storage

Rampur AFLR Pusa red Rampur Y=1.2x + 0.6 Linear (Pusa red) Linear (AFLR) Linear (Rampur) AFLR Y=1x + 2.0 Pusa red Y=0.85x + 0.27 Figure 3. Trend lines for cumulative storage loss due to sprouting during 24 weeks storage period.

Lowest loss due to sprouting was recorded in Pusa red, contributing only 22% of the total storage loss at 24 week storage period. In the other two varieties, sprouting contributed up to 30% of the total storage loss at the end of 24 week storage period (Figure 3).

Data on storage loss due to sprouting and rotting in each sampling would help to identify possible reasons that would have contributed to these losses. Data on storage loss of each variety at each sampling due to rotting and sprouting are given in Table 1. Losses due to sprouting had accelerated by 9th week, in variety Rampur which corresponded to mid December 2004 and was maintained at a higher level thereafter. Minimum temperature during this period was around 24°C (Table 1). Losses due to rotting and sprouting in variety Pusa red and Agri found light red had accelerated by 12th week which corresponded to end January, 2005 and was maintained at a higher level thereafter. Minimum temperature and RH during this period were 20 – 25°C and 82 - 90% respectively (Table 1). 112 METTANANDA

Table 1. Percent storage loss due to rotting and sprouting at each week of different varieties during 24 weeks storage period.

Weeks in Rotting Sprouting storage Rampur AFLR Pusa red Rampur AFLR Pusa red 0 0 0 0 0 0 0 3 0.79 0.78 1.82 0 0 0 6 0.7 1.9 4.57 1.27 2.65 0.94 9 9 2.74 3.2 9.31 2.28 2.76 12 11.76 10.2 14.7 5.61 10.15 8.1 15 5.65 5.32 7.64 5.79 3.95 4.45 18 8.4 9.2 15.38 7.43 9.08 4.94 21 19.98 10.9 9.32 4.94 4 3.68 24 21 9.76 11.4 10.4 6.27 8.73

DISCUSSION

Storage losses varied in the different cultivars and this could be due to genetic and dry matter content variation (Tanaka et al., 1985; Patil and Kale, 1985). Accelerated sprouting and rotting losses after December could be due to low night temperature and high RH. As reported by Brice et al. (1997), and Uzo and Currah (1995), the ideal temperature and RH to minimize sprouting and rotting losses in onion are 26 – 30°C and 60 – 75% respectively. However, the temperature and RH levels during December – April period at the study location had been 20 – 24°C and 80 – 90%, respectively leading to high sprouting and rotting losses. This is also supported by the findings of Mettananda and Edirimanna (2001) who recorded high sprouting losses at Sita- eliya and Rahangala where minimum environmental temperature is between 15 – 20°C. According to Aoba (1985), onion bulbs in storage at 20°C resumed elongation of the sprout leaves and initiation of new leaves at 40 – 45 days after harvest, depending on the cultivar. Some other workers had reported that sprouting increased up to a temperature of 16 – 22°C and was inhibited at 30°C, and sprouting was not affected by atmospheric humidity (Tanaka et al., 1985). High rotting losses could be due to activation of storage fungi at high RH.

CONCLUSIONS

Storage loss up to 75% can be expected during a period of six months when mother bulbs are stored under ambient conditions for yala season seed production. Variety Rampur is not suitable for storage under ambient conditions for yala season seed production because there is upto 75% storage losses. Variety Agri found light red has comparatively better shelf life and therefore, could be recommended to store for yala season seed production with only 60% storage loss. Low night temperature (20 -25°C) and high RH (80 - 90%) during December to February period appeared to be the major reasons for high storage losses. MOTHER BULB STORAGE FOR YALA SEED PRODUCTION 113

Use of a low cost heating system to maintain in-store temperature at around 28-30°C would be useful to reduce losses due to sprouting and rotting. Further studies are needed to test some low cost heating methods such as rice husk burners and the frequency of heating in order to obtain satisfactory results.

Further, 25-40% of the bulbs that remained at the end of the storage period under ambient conditions would be valuable genetic material with good storability. Therefore, one could expect a genetic drift towards good storability by using the remaining bulbs for seed production and selecting mother bulbs produced from same seeds for progressive seed production and repeating the process for several years. However, a systematic study should be conducted to prove this assumption.

AKNOWLEDGEMENTS

Author wishes to thank the Director and staff of REAP, Matale, for collaborative support to conduct this valuable study. Special thanks to Mr. Priyanga Dematawa, Manager, CIC, Seed Farm, Pelwehera for providing required facilities to conduct this study and Mr. V.R. Dingiribanda and T.G.R.S. Premathilaka, Field Executives, CIC, Seed Farm, Pelwehera for their help in collecting required data. Thanks are also due to Mr. Jayalal Chandrasiri., RO, at the Progress Monitoring Unit of the DOA for the help in statistical analysis of the data. The contribution of Miss. K.S.K. Thibbotuwawa, Technical Assistant to prepare this manuscript, is also gratefully acknowledged.

REFERENCES

Aoba, T., 1985. On bulb formation and dormancy in onion iii. On the process of sprouting in stored onion. Journal of Japanese Society of Horticultural Science 24:199 -200. Brice, J., L. Currah, A. Malins and R. Bancroft. 1997. Onion storage in the tropics: A practical guide to methods of storage and their selection. Chatham, UK: Natural Resource Institute. Mettananda, K.A. and E.R.S.P. Edirimanna. 2001. Effect of environmental factors on shelf life of b'onion. Tropical Agriculturist 152: 1-8. Patil, R.S. and P.N. Kale., 1985. Correlation studies on chemical composition and keeping quality of some onion cultivars. J. Maharashtra Agriculture University (India) 10:154-157. Tanaka, M., K. Chee, and S. Komochi. 1985. Studies on the storage of autumn harvested onion bulbs. I. Influence of storage temperature and humidity on the sprouting during storage, Research Bulletin of Hokkaido National Horticultural Research Station 141: 1-16. 114 METTANANDA

Uzo, J.O. and L.Currah. 1995. Cultural systems and agronomic practices in tropical climates. In. Onions and Allied crops. volume II: Eds. H.D. Rabinowitch and J.L. Brewster. Pp 49 – 62, CRC press Boca Raton, Florida. Vendenburg, L. and C.P. Lentz. 1973. Effect of relative humidity, temperature and length of storage on decay and quality of potato and onion. Journal of food science 38:81-89. Annals of Sri Lanka Department of Agriculture. 2006.8:371-377.

SHORT COMMUNICATION

PLANT PATHOGENS INTRODUCED TO SRI LANKA THROUGH IMPORTED SEED POTATO (Solanum tuberosum L.)

R.G.A.S. RAJAPAKSE1, R. EKANAYAKE1, R.K. RANATHUNGA1, R.N.I. PERERA1, R.D.S.S. WIJESEKARA1, I.A. EKNELIGODA2, S.A.M.R. ABEKOON3 1Horticultural Crop Research and Development Institute, Gannoruwa, Peradeniya 2Seed Certification and Plant Protection Center, Gannoruwa, Peradeniya 3Seed Certification Center, Sita Eliya

INTRODUCTION

Sri Lanka imports 25% of the total seed potato requirement from Holland, Germany, France, Poland, Australia, New Zealand and United Kingdom. De Silva et al. (2002) reported that many fungi and bacteria that are potential pathogens on local crops are introduced to the country through imported fruits. Consequently it is possible that many plant pathogens, insect and weed species enter through seed potato. The importation of 5000 to 6000 t every year and subsequent direct planting in the soil will aggravate this situation. The Plant Quarantine Act No. 35 of Sri Lanka has been introduced to prevent the entry of such alien species to the country through imported commodities. Quarantine restrictions are imposed on seed potato imports considering the danger of introducing certain pathogens (Anon., 1999). This paper reports the pathogens detected in seed potato lots imported to the country during the period 2003 to 2006, with a view to draw the attention of scientists on the contaminated flora in imported commodities.

MATERIALS AND METHODS

Sampling

Samples were collected from seed potato lots of different varieties imported into the country through seaport. Ten percent of the boxes/bags of seed potato were randomly collected from each lot and the condition of the tubers visually examined. Sample of 300 tubers was randomly collected from each box/bag and all samples mixed and finally 100 tubers collected randomly for further studies. Each sample was dispatched to the laboratory for detection of pathogens by laboratory and greenhouse studies. Another sample of 100 tubers was collected for field studies conducted at the post quarantine unit in Sita Eliya. 116 RAJAPAKSE et al.

Laboratory investigation

Hundred tubers collected from each sample were checked for pathogens and disease symptoms visually and microscopically. Some tubers were kept in humid chambers and incubated for 2 - 4 days to observe microscopically the associated pathogens of the tubers. Laboratory tests were conducted for the detection of fungi, bacteria and viruses associated with the peel and internal tissues of tubers. Pieces of periderm and internal tissues of 25 tubers were cultured on Potato Dextrose Agar (PDA), PDA + 1% Streptomycin and Malt Extract Agar and incubated for 4 - 5 days at room temperature. Fungal pathogens were identified by comparison of morphological characteristics and microscopic observations of fungi with published data (Hooker, 1981). Bacteria were distinguished by comparison of disease symptoms, colony characters on media, staining and microscopic observations with published data (Hooker, 1981).

Greenhouse studies

Randomly selected tubers of seed potato lots were planted under greenhouse conditions to detect any disease development due to latent infection of pathogens including viruses. Shoots of plants were used to detect potato virus X, S, Y, M, and leaf roll virus by enzymes linked immunosorbent assay (ELISA).

Field studies

Hundred tubers were grown in the field at post quarantine unit, Sita Eliya to observe performance of potato varieties and detection of disease development due to latent infection.

RESULTS AND DISCUSSION

Diseases on tubers were basically identified by comparison of visual observations of disease symptoms, culture characters on artificial media and microscopic observations of pathogen morphology (Table 1) with published data (Hooker, 1981). Some samples of seed potato lots, imported each year had no fungal or bacterial disease symptoms on surface or in internal tissues. However, most of seed potato lots frequently showed symptoms of fungal diseases such as black scurf caused by Rhizoctonia solani, silver scurf caused by Helminthosporium solani, dry rot caused by Fusarium species and actinomycetes diseases such as common and netted scab caused by Streptomyces species (Tables 2 and 3). Fungal disease viz. rubbery rot caused by Geotrichum candidum was rarely observed in seed potato lots in year 2003. However, imported seed lots were highly infected with Geotrichum candidum PATHOGENS IN IMPORTED SEED POTATO 117 in the recent past (Table 2). Another severe disease i.e. bacterial soft rot caused by Erwinia carotovora was detected in some imported seed lots (Table 2). Erwinia survives in potato tubers as latent infections and as a result soft rot epidemics can occur in the potato cultivation (Hooker, 1981), and such epidemics were reported in potato cultivations of imported varieties Kondor and Granola in Mandaram Nuwara and Keppetipola area in years 2004 and 2005, respectively.

Table 1. Disease symptoms and characteristics of major fungal pathogens associated with imported seed potato.

Disease Disease symptoms Morphology and Culture Associated characters of pathogen patho gen Black Black colour sclerotia Mycelium septate, dark Rhizoctonia scurf develop superficially on brown and hyphae large (7- solani surface. 10 µ in diameter). Mycelia show right angle branching.

Silver Small brown localized Mycelia hyaline, septate, Helminthosporium scurf spots. Affected areas have branched and turn brown solani silvery sheen if the surface with age. is wet. Un-branched conidiophores are septate. Conidia have 2- 8 septa and length 20 – 50 µ, colour dark brown.

Fusarium Periderm wrinkling over Mycelia septate and Fusarium species dry rot rotted tissues and in branched. Colony colour; internal cavities. Cavities white, pink, purple or brown lined with white gray according to species. mycelia. Conidia produced openly within hyphae. Conidia two types. Macro conidia - sickle shaped, 3-6 cells, larger in size, length 25-60 µ. Micro conidia - abundant, cylindrical, 1-2 cells, smaller

Common Angular corky scabs Gray colour filamenteus Streptomyces scabs appear on the surface. mycelia, size about 1µ. scabies Black pigmentation surrounding the colony. Spore size about 0. 5µ. Gram staining - ve. Netted Brown lesions have net Colourless filamenteus Streptomyces Scabs like structures on the mycelia produce spores, species surface spore size about 0.5µ. Gram staining + ve. 118 RAJAPAKSE et al.

Table 2. Diseases observed in internal tissues and on periderm of tubers and their frequency in seed potato lots.

Disease and pathogen Disease frequency % variation of disease of lots in each year* infected tubers of lots in each year 200 200 200 200 200 2004 2005 2006 3 4 5 6 3 Black scurf caused by 96 96 102 81 1-24 1-24 1-60 1-23 Rhizoctonia solani Silver scurf caused by 94 91 97 84 3-75 1-100 2-100 1-68 Helminthosporium solani Dry rot caused by 53 64 34 21 1-9 1-44 1-60 1-9 Fusarium species Rubbery rot caused by 1 3 9 15 2 1-21 1-22 1-100 Geotrichum candidum Common and netted scab 75 92 155 79 1-76 1-100 1-100 1-74 Caused by Streptomyces spp. Bacterial soft rot caused by 2 5 1 3 1-3 1-33 57 1-6 Erwinia carotovora

* No. of tested seed potato lots =244

Morphological and cultural characteristics on media and microscopic observations of pathogens indicated that although there were no disease symptoms on tubers, many fungi and Streptomyces spp. were associated with periderm of potato tubers as surface contaminants. Some fungi viz. Rhizoctonia solani, Helminthosporium solani. Fusarium spp., Alternaria spp., Geotrichum candidum, Aspergillus spp. and actinomycetes viz. Streptomyces spp. were frequently observed on tubers of tested seed lots (Table 3).

Some seed potato lots were not released for cultivation when disease incidences mentioned in quarantine regulation exceeded the permitted limits or due to other diseases that were severely damaging the tubers. Among the varieties imported to the country, over 60% included varieties like Granola and Desiree. Imported seed potato variety Desiree frequently showed scab symptoms, while Granola and Lyra showed high incidence of silver scurf, black scurf and dry rot. For example, of 1028 t. of total imports of variety Desiree, 343 t. of seed were refused release for cultivation in year 2005 due to high incidence of scab disease which exceeded permit limits of scab disease. Even though diseases were present on or inside tubers, most of the seed potato lots were released for cultivation in each year because their disease incidences PATHOGENS IN IMPORTED SEED POTATO 119 did not exceed permit limits or they were not considered as important quarantine diseases.

Table 3. Organisms isolated on periderm tissues and internal tissues when cultured on different media and frequency of isolation of each pathogen from seed potato lots in years 2004 and 2006.

Fungi and bacteria isolated from tissues Frequency of pathogen found in lots * Year 2004 Year 2006 (244)* (229)* Rhizoctonia solani 187 163 Helminthosporium solani 160 97 Fusarium species, F. solani and F. roseum 203 70 Alternaria alternata and A. solani 102 16 Stemphylium species 19 3 Sclerotium species 28 2 Botrytis species 9 4 Macrophomina species 44 2 Geotrichum candidum 41 48 Aspergillus species 88 186 Pennicilium species 24 2 Trichoderma species 26 23 Pythium species 6 2 Colletotrichum species 9 7 Rhizopus species 4 - Streptomyces species 144 116 * No. of tested seed potato lots

It took 3-4 weeks for virus indexing of each seed potato lot by ELISA but they need to be released within 5 days after inspection. Therefore, virus standard of phytosanitary certificates issued by authorities of producing countries were considered to grant permission for cultivation. However, virus indexing was done at HORDI to re-check and compare virus incidences in seed lots. This indicated that there were one or more harmful viruses such as potato viruses X, S, Y, M and leaf roll virus in tubers of most of the imported seed potato lots (Table 4). These viruses cause severe crop losses in some crops of family Solanaceae, Chenopodiaceae, Compositae, Asteraceae and Fabaceae (Agrios, 1997; Hooker, 1981). It has been reported that diseases such as potato mop-top virus and powdery scabs caused by Spongospora subterranse are not tested in local laboratories, but occur in seed potato producing countries especially in Europe. Field observations indicated that out of 244 lots, 40 lots had virus symptoms under the field conditions (Table 5). These results indicate that, it is possible many fungi, bacteria and viruses such as Rhizoctonia solani, Helminthosporium solani. Fusarium spp., Alternaria spp. Stemphylium spp., Sclerotium spp., Phoma sp., Botrytis sp., Macrophomina sp., Geotrichum candidum, Pythium spp., Colletotrichum sp., are introduced to the country through imported seed potatoes even though 120 RAJAPAKSE et al. foreign authorities certify that these seed potato lots are free from pathogens or within permitted limits of quarantine regulations of Sri Lanka.

Table 4. Viruses detected by ELISA of imported seed potato lots in years 2003, 2004, 2005 and 2006.

Virus detected in shoots Disease frequency in seed potato lots Year 2003 Year 2004 Year 2005 Year 2006 (53)* (207) * (195)* (227)* Potato virus X 11 41 41 74 Potato virus S 3 40 60 12 Potato virus Y 1 38 47 20 Potato virus M 6 33 66 20 Potato leaf roll virus 14 51 33 32 * Number of tested seed potato lots

Table 5. Visual observation of diseases on growing plants of different seed potato lots under field conditions at Sita Eliya in year 2004.

Disease observed Frequency of disease Disease severity of lots of seed potato lots No. of severe lots No. of mild lots Fusarium wilt 4 - 4 Black leg 1 - 1 Mosaic symptoms (virus) 33 11 22 Leaf roll symptoms (virus) 7 1 6 * No.of tested seed potato lots =244

Streptomyces species (actinomycetes), bacteria and viruses detected in tubers were recognized as pathogens on potato as well as many other crops (Hooker, 1981). For example, Geotrichum candidum, the causal agent of sour rot of oranges and other fruits can infect several crops (Holliday, 1980). Leaf spots and leaf blight caused by Alternaria solani and Botrytis species were common diseases responsible for the considerable yield reduction in the vegetables such as tomato, leeks and Capsicum in up-country regions (Anon., 1990). Nursery disease damping-off and root rot can be caused by Rhizoctonia solani, Sclerotium spp., Fusarium solani and other Fusarium species can cause serious diseases of solanaceae and fabaceae crops and it has been reported severe crop losses occur in most of the areas in rainy seasons. Bacterial pathogen, Erwinia carotovora causes soft rot to more than 44 species including cabbage and carrot (Shekhawat and Chakrabarti, 2000) and may severely damage cultivation in up and mid country areas. It was reported that Streptomyces spp. (actinomycetes) is pathogenic on carrot, beet and radish and fungal pathogens such as Aspergillus spp., and Penicillium spp., pathogenic mainly on fruits such as papaya and grains of pulses (Agrios, 1997; Shekhawat and Gadewar, 2000). In the tropical countries, disease development and such losses due to diseases may be higher because of the favourable weather conditions for the microorganisms throughout the year PATHOGENS IN IMPORTED SEED POTATO 121

(Holliday, 1980). Since these pathogens are recorded in the country but importation of large quantities of disease infected seed potatoes may increase in inoculum density of pathogens in the local soil and cause diseases of crops. Therefore, it is necessary to pay attention to avoid invasion of unwanted pathogens into the country through imported seed potatoes.

REFERENCES

Agrios, G.N. 1997. Plant Pathology, CAB International, London. 172-316. Anon., 1999. Permit to import plants under plant protection act. No. 35 of 1999. Department of Agriculture, Sri Lanka. 15-16. De Silva, R.S.Y., A.R.W.M.M.U.M. Amarakoon and P.H. Nilangika, 2002. Plant pathogens in fruits imported into Sri Lanka. Annals of Sri Lanka Department of Agriculture 4: 419-423. Holliday, P. 1980. Fungus diseases of tropical crops. Cambridge University Press, UK P 60. Hooker, W.J. 1981. Compendium of potato diseases. American Phyto pathological Society 27-74. Shekhawat G.S. and S.K. Chakrabarti, 2000. Soft rot. Disease and pest of potato, Ed. S. M. Paul Khurana, Central Potato Research Institute, Shimla, India 27-29. Shekhawat G.S. and A.V. Gadewar, 2000. Common scab. Disease and pest of potato, Ed. S.M. Paul Khurana, Central Potato Research Institute, Shimla, India 30-32. Annals of Sri Lanka Department of Agriculture. 2006.8:379-381.

SHORT COMMUNICATION

TSUNAMI EFFECTS ON SOIL SALINITY AT VADAMARACHCHI AREA IN THE JAFFNA PENINSULA.

S.VIJAYARATNAM, B.BALASINGHAM and S.KULENDRAN Agricultural Research Station, Thirunelvely, Jaffna

INTRODUCTION

Due to the Tsunami of 26 December 2004, large extents of agricultural lands in the coastal areas were affected by sea water intrusion. The island has never experienced a devastation of this magnitude before. The disaster has claimed nearly 35,000 human lives and over 5,50,000 were rendered homeless (Central Bank of Sri lanka, 2004). The seawater intrusion was around 2-3 kilometers from the coast, damaging most of the crops. After fisheries, the agricultural sector was the hardest hit by the disaster. This refers not only to farm fields, but also to the home gardens of farmers, villagers, fishermen with vegetables and fruits. These home gardens supplied the household nutritional needs. A total of 92.2 ha paddy, 47.8 ha other field crops, 0.6 ha fruits and 0.4 ha vegetables were completely damaged in Jaffna (Anon., 2004). Therefore, a study was conducted to determine salinity levels of soils and well water affected by the Tsunami disaster in Vadamarachchi area of the Jaffna district.

MATERIALS AND METHODS

Soil and water samples were collected at two months interval from nine locations for a period of twelve months commencing January 2005. Soil samples were analyzed for electrical conductivity and pH. Electrical conductivity was measured using EC meter and pH using pH meter. Two soil samples were collected from non-affected area for comparison. Soils were collected at two months interval from January, 2005 to December 2005 at depth of 15-25 cm from affected vegetable growing lands. Water samples were collected from the wells of the same cropping areas for analysis.

RESULTS AND DISCUSSION

Electrical Conductivity (EC) measurements indicate high salt concentration in soils and water. EC and pH of the soil samples measured are given in Tables 1 and 2. The same parameters of water samples are given in Tables 3 and 4. 124 VIJAYARATNAM et al.

Table 1. Electrical conductivity (dS/m) of soil samples.

Location January March May July September November Thumpalai 13 4 6 6 6 5 Point pedro 14 7 2 2 1 2 Puloly west 19 12 5 3 3 2 Polykandy 7 3 2 2 2 1 Thampasiddy N.A 3 1 1 2 2 1 Thikkam 9 1 1 2 2 2 Viyaparimoolai 18 12 15 18 7 2 Thondamanaru 11 2 5 2 2 1 Thikkam N.A 2 1 1 2 2 1

Table 2. pH of soil samples.

Location January March May July September November Thumpalai 8.2 8.9 8.5 8.2 8.5 8.7 Point pedro 8.3 8.1 8.6 8.1 8.6 8.8 Puloly west 8.2 8.3 8.8 8.5 7.8 8.9 Polykandy 7.7 8.4 8.3 7.8 8 8.2 Thampasiddy N.A 8.2 8.4 8.4 8.2 8.3 8.3 Thikkam 7.9 8.7 8.7 8.3 8.7 8.1 Viyaparimoolai 8.3 8.5 8.3 8 8.6 9.2 Thondamanaru 8.3 9 8.5 8.1 8.3 8.2 Thikkam N.A 8.4 8.4 8.5 7.9 8.5 -

Results revealed that EC of soils collected from Tsunami affected areas high (7-19 dS/m) in January and low (1-5 dS/m) in September (Table 1). This indicates the soils in affected areas contained high quantities of salts at the beginning and decreased by end of the sampling time. Based on the standards developed by USDA (1954), the salinity levels just after Tsunami showed high value and decreased to lower values later. The onset of maha rains in September resulted in a decrease in E.C, (Table 1) presumably as a result of the salts being flushed out beyond the rooting zone.

The lowest and highest pH values recorded were 7.4 - 9.2 respectively. These pH values agreed with the recommended pH range 6.5 - 9.0.

Water samples

Water samples had high EC values and ranged between 2.2- 12.6 dS/m. based on the standards developed by Nagaraja et al. (1988) the majority of well water samples in the tsunami affected area can be categorized as high salinity water. The EC of the well water fluctuates with time. This is due to high temperature and leaching of salts from soils to water. IMPACT OF SEA WATER ON SOILS AND WATER 125

Table 3. Electrical conductivity (dS/m) of water samples.

Location January March May July September November Thumpalai 9.1 9.1 7.9 7.2 7 7.5 Point pedro 4.7 5.6 6.2 5.8 5.6 6.2 Puloly west 7.4 6.2 7 7.1 6.9 9.4 Polykandy 2.4 2.3 2.9 3.1 3.4 2.9 Thampasiddy N.A 0.9 1.4 1.6 1.7 1.2 1.2 Thikkam 5.4 5.2 5 4.7 4.5 6.1 Viyaparimoolai 12 12.2 12.6 12.5 12 11.1 Thondamanaru 2.2 2.1 3.3 3.6 3.6 2 Thikkam N.A 1.6 1.7 2.1 2.4 2.7 2

Table 4. pH of water samples

Location January March May July September November Thumpalai 7.9 7.7 7.9 7.6 7.8 7.6 Point pedro 7.8 7.8 7.9 7.6 8 7.4 Puloly west 7.9 8.1 8.2 7.9 8 7.7 Polykandy 8 8.1 8.2 7.6 7.5 7.7 Thampasiddy N.A 9 8.5 8.5 8 7.6 7.8 Thikkam 8.3 8 8 7.4 7.5 7.5 Viyaparimoolai 7.4 7.4 7.7 7.6 7.5 7.4 Thondamanaru 7.7 7.9 8 7.6 7.5 8.2 Thikkam N.A 8.4 8.4 8 7.8 7.6 7.4 Recorded pH values ranged between 7.4 and 8.5. The pH of the water in irrigation tanks which are situated in the dry zone of Sri Lanka is over 7.0 (Amarasiri, 1973). Similarly, Nagaraja et al. (1988) reported high pH values in well water in Jaffna. CONCLUSIONS

The electrical conductivity of water and soil was high just after the Tsunami and decreased to low levels with time. The well water in the tsunami-affected areas contains high quantity of salts. If high salinity water is used for irrigation it would adversely affect vegetable crop cultivation. Therefore appropriate remedial measures should be taken to rehabilitate the water sources. REFERENCES

Anon., 2004. Report of the district secretary on the Tunami effect in Jaffna district. Amarasiri, S.L. 1973. Water quality of major irrigation tanks in Sri Lanka. Trop.Agric. 129:19-25. Central Bank of Sri Lanka. 2004. Annual report, Central Bank of Sri Lanka. Nagaraja, S., B.N. Emerson, V. Abeykoon and S. Yogalingam. 1988. Water quality of some wells in Kilinotchchi with special reference to nitrate pollution. Trop. Agric. 144:61-78. 126 VIJAYARATNAM et al.

USDA. 1954. Diagnosis and Improvement of Saline and Alkali Soils. Agriculture Hand Book No. 60 P-9.