Int.J.Curr.Microbiol.App.Sci (2017) 6(10): 1243-1257

International Journal of Current Microbiology and Applied Sciences ISSN: 2319-7706 Volume 6 Number 10 (2017) pp. 1243-1257 Journal homepage: http://www.ijcmas.com

Original Research Article https://doi.org/10.20546/ijcmas.2017.610.149

Morphological and Biological Variability of Different Isolates of Paecilomyces lilacinus against Root Knot Nematode in Tomato

B. G. Anusha*, Shripad Kulkarni and S. I. Harlapur

Department of Plant Pathology, College of agriculture science Dharwad, University of Agriculture Science, Dharwad, Karnataka, India *Corresponding author

ABSTRACT

The modern intensive Agricultural practices have considerably raised the output but K e yw or ds created problems like land and environmental degradation and pollution. Besides, this

contamination of food products and pesticidal residue in food has caused much panic

Paecilomyces among the consumers and also the producers. Hence, it is necessary to find out innovative lilacinus , Bioefficacy and and suitable alternative technology like biological control of pests and diseases. Recently, variability . Paecilomyces lilacinus based different formulations are used for the control of nematode diseases, which is economical, ecofriendly and sustainable in the long run. Among the Article Info different bioagents used in the tomato ecosystem, P. lilacinus is extensively used as seed dressing bioagent against many soil borne diseases, however, its usage against foliar Accepted: diseases is limited. Isolates significantly varied with respect to colony characters, 10 September 2017 Available Online: sporulation of the isolate and conidial characters. Based on the colony characters observed 10 October 2017 Tadakoda isolate had initially white colony later it turned to pinkish smooth colony. It produced single, round to oval conidia on phailides which sporulated early.

Introduction

Tomato (Lycopersicum esculantum L.) is one Tomatoes offer significant nutritional of the most popular vegetable crops grown in advantages, including providing a significant the world, next to potato. It is used as a fresh source of dietary lycopene, β-carotene, vegetable and processed and canned as a carotenoids, vitamin C, potassium, fiber, paste, juice, sauce, powder or as a whole color, flavor and antioxidant properties in a (Barone and Frusciante, 2007). The ripe fruits low energy dense food (Britt and Kristin, are good source of vitamin A, B and C which 2011 and Rani and Khetarpual, 2009). add wide varieties of colour and flavour to the food. Several human studies indicated a relationship between a high intake of tomato products and Recently, it started gaining more medicinal a decreased risk of several types of cancer, value because of the antioxidant property atherosclerosis and cardiovascular diseases because of ascorbic acid and lycopene content (Cecilia et al., 2010). Recently, this crop is (Anon., 2000). Hence tomatoes are called as recognized as a model for plant-pathogen poor man’s apple (Rick, 1969). interactions (Arie et al., 2007). 1243

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Many factors operate in successful cultivation cucurbits, potato and chilli etc. These diseases as well as marketing of quality tomatoes of can be managed by cultural, physical, which diseases play an important role. Several chemical and biological methods. Among diseases appear on tomato caused by fungi, these methods biological control is eco- bacteria, viruses, nematodes and abiotic friendly and cheapest method. factors (Balanchard et al., 1992). Among the nematode diseases in tomato, root knot Root knot nematodes devitalize root tips and nematode (Meloidogyne species), sting cause the formation of swellings on roots. nematode (Belonolaimus longicaudatus), These effects not only deprive plants of stubby root nematode (Paratrichodorus and nutrients but also disfigure and reduce the Trichodorus spp.) and lesion nematode market value of many root crops. When (Pratylenchus sp) are important. susceptible plants are infected at the seedling stage, losses are heavy and may result in Root knot nematode Meloidogyne incognita is complete destruction of the crop. Infection on one of the world’s most catastrophic diseases older plants results in slight effects or they resulting in loss at different growth stages of may reduce yield considerably (Agrios, tomato. Root knot nematodes are distributed 1996). throughout the world, especially in areas with warm or hot climates and short or mild winter Above ground symptoms include reduced and they appear in nursery, open field, poly shoot growth with fewer, small, pale green or house and even in green house. yellowish leaves and tend to wilt in warm weather. Blossoms and fruits produced will be Root knot nematode (M. incognita) is the less with poor quality. Characteristic most dominant species accounting for 64 per symptoms of the disease appear on the cent of total population which is widely underground parts of the plants. Infected prevalent inflicting serious loss to tomato fruit plants develop the typical root knot galls that yield (Sasser, 1980). Root knot nematodes are are two to several times larger than the reported to cause losses ranging from 15 to 60 healthy roots. Several infections along the per cent in many vegetable crops such as root give the root a rough, clubbed brinjal, okra, french bean and cowpea (Sasser, appearance. 1980; Krishnappa et al., 1992 and Ravichandra, 2008). Apart from M. incognita, More than 80 species of Meloidogyne have other major nematode species causing been reported from different parts of the diseases include M. javanica, M. arenaria and world and 13 species have been recorded M. hapla. from India. Among them, Meloidogyne incognita, M. javanica, M. arenaria, M. They attack more than 2000 species of plants, graminicola, M. hapla and M. exigua are infecting almost all cultivated plants and economically important. reported to reduce world crop production by five per cent in individual fields loss may be Looking into the severity of root knot even higher (Agrios, 1996). In general, the nematode infestation on various crops and vegetables and pulses are the good hosts farmers inclination towards biocontrol agents whereas, cereal crops are considered to be the for the management of nematode diseases, poor hosts of this nematode. Root knot present study has been taken up with specific nematodes are a serious problem on objectives mentioned below as to study the vegetables, such as brinjal, okra, tomato, variability and bioefficacy of Paecilomyces

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Int.J.Curr.Microbiol.App.Sci (2017) 6(10): 1243-1257 lilacinus isolates and its bioefficacy against An intensive survey was carried out to know the root knot nematode. the appearance of root knot nematode disease and prevalence of the P. lilacinus in different Materials and Methods crops in Belgaum, Bagalkot, Haveri, Gadag and Dharwad districts of northern Karnataka. Present investigations were carried out in Samples of both healthy and root knot kharif season during 2013-2014 at the Main infested soil were collected for isolation of P. Agricultural Research Station (MARS), lilacinus. Each sample was taken separately in University of Agricultural Sciences, and polythene bags and tied with a rubber band Dharwad. Laboratory experiments were and labeled immediately. Information conducted at the Institute of Organic Farming, pertaining to the locality, crop history, stage College of Agriculture, University of of the crop, etc. was also noted. Samples of Agricultural Sciences, Dharwad, and soil and roots were analysed on the day of Karnataka. Various studies were undertaken collection or after keeping them for a few with the objectives such as, isolation of days under refrigerated conditions. The soil Paecilomyces sp. and maintenance of pure was mixed thoroughly and 200 cc of soil was culture, studies on mass production of processed following Cobb’s sieving and Paecilomyces and their shelf life, in vitro and decanting method (Cobb, 1918) followed by in vivo evaluation of talc based formulation of modified Baermann’s funnel method the bioagent Paecilomyces against root knot (Schindler, 1961). nematode. Details of the materials used and the methodology adopted during the course of Isolation of Paecilomyces lilacinus investigation are presented in this chapter. The procedure given by Ramakrishna (1989) General procedure was followed to isolate P. lilacinus from the collected soil sample. The techniques of serial Glassware and cleaning dilution and purification were carried out as suggested by Sankaram (1961). The Potato For all the laboratory studies Corning and dextrose agar media was used for isolation of Borosil glassware were used. The glass ware Paecilomyces lilacinus. were kept submerged overnight in the cleaning solution prepared by dissolving 60 g Composition of media of potassium dichromate (K2Cr2O7) and 60 ml of concentrated sulphuric acid (H2SO4) in one Potato Dextrose Agar litre of distilled water. Then, they were washed with vim powder followed by rinsing Potato Dextrose powder: 20 g several times in running tap water and finally Agar-agar: 20 g used when needed for laboratory studies. Distilled water : 1000 ml pH: 7.0 Sterilization Twenty gram potato dextrose powder and All the glassware used in the studies were agar-agar were dissolved in 800 ml of sterilized in an autoclave at 1.1 kg cm2 distilled water. The volume was made up to pressure for 20 min. and then dried in a hot air 1000 ml with distilled water and medium was oven at 60°C. Media were sterilized at 1.1 sterilized at 1.1 kg per cm2 pressure for 15 kg/cm² pressure for 15 min. min.

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Collection of cultures and maintenance of separate the individual eggs. The known Meloidogyne incognita nematodes number of eggs of root knot nematodes were put in a watch glass and different Root knot infected tomato plants were concentrations of Paecilomyces and other collected from the farmer’s fields during the material as per the treatment were added to survey in northern Karnataka in polythene these watch glasses and incubated for 96 hrs bags and kept in the freezer. Root portion was and microscopic observation on number of carefully removed from the soil and washed eggs hatched was recorded and percentage gently under running tap water. Egg masses was calculated. were picked and kept for hatching in watch glass with water. After 12-24 hours, hatched Juvenile mortality test juveniles were used to inoculate tomato grown in sterilized soils and (1:1) mixture in Freshly hatched, 50 active juveniles were greenhouse. These plants served as culture counted in a counting dish using a stereo plants. After completion of 3-4 generations of binocular microscope and were carefully the nematode, the plants were depotted transferred to individual vials containing five carefully. The root system was washed free of ml of each of the different Paecilomyces soil, the knots containing egg masses were isolates culture filtrates with same used to get inoculum of the nematode for concentrations. further studies. Each treatment was replicated three times and Extraction of nematodes arranged in completely randomized design and incubated at 28±2ºC. Observations were Soil samples of 200 g was washed thoroughly recorded at 12, 24 and 48 hours after exposure and processed using combined Cobb’s sieving period and per cent mortality was calculated. and Bauermann’s funnel method. The root Known number of root knot nematode knot nematode and other plant parasitic juveniles was added in a watch glass and nematodes present in the suspension were different concentrations of Paecilomyces and identified to genus level by observing other materials as per the treatment were also different morpho anatomical characters. Their added to these watch glasses. These watch number present in the suspension was glass were incubated for 96 hrs and four determined by taking the average number of replication maintained observation on nematodes present in five different one juvenile mortality was recorded and mililitre aliquot of nematode suspension. percentage was calculated.

In vitro studies on bioefficacy of In vivo studies on bioefficacy of Paecilomyces lilacinus Paecilomyces lilacinus

Hatching inhibition test Roll towel method

Egg masses of M. incognita were collected Bioefficacy of Paecilomyces and other from culture plants maintained in nematology treatments were carried out based on the net house of the Department of Plant seedling vigour index using the standard roll Pathology, Agricultural College Dharwad. towel method (Anon., 1993). Hundred treated Egg masses were picked up and treated with tomato seeds were kept over the presoaked NaOCl (1%) to dissolve the egg matrix and to germination paper.

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The seeds were held in zigzag position by districts of northern Karnataka viz., Dharwad, placing another presoaked germination paper Belgaum, Bagalkot, Haveri and Gadag. strip and gently pressed. The polythene sheet along with seeds were then rolled and Symptomatology incubated in growth chamber for seven days. Three replications were maintained for each Root knot nematodes devitalize root tips and treatment. Seeds soaked in sterile water cause the formation of swellings on roots. served as control. Root and shoot length of Above ground symptoms include reduced individual seedlings were measured and the shoot growth with fewer, small, pale green or germination percentage of seeds was also yellowish leaves and tend to wilt in warm recorded. weather. Blossoms and fruits produced will be less in number with poor quality. The vigour index was calculated by using the Characteristic symptoms of the disease appear formula as described by Baki and Anderson on the underground parts of the plants. (1973). Infected plants develop the typical root knot galls that are two to several times larger than Vigour index (VI) = Seedling length (Mean the healthy roots. Several infections along the root length x mean shoot length) x roots give the roots a rough, clubbed Germination % appearance.

Ten seedlings were taken randomly from each Meloidogyne incognita nematodes were treatment and their seedling length was identified based on morphological character recorded. such as slender stylet, vermiform and pointed tail tip of posterior region. Results and Discussion Isolation and identification of bioagent The results of the present investigation on Paecilomyces lilacinus Mass production of Paecilomyces lilacinus and bioefficacy against root knot nematode Isolation of bioagent caused by Meloidogyne incognita infecting tomato conducted at Department of Plant The isolation of P. lilacinus from the infected Pathology, College of Agriculture, UAS, and healthy soil sample was made as Dharwad and laboratory experiments described in Material and Methods. The pure conducted at Institute of Organic farming culture obtained was again subcultured on UAS, Dharwad during kharif 2013-14 are potato dextrose agar slants and kept in the presented here under. refrigerator at 5°C for further studies.

Survey for the root knot nematode The Pure culture of Paecilomyces lilacinus infestation in tomato and presence of was again subculture on malt agar, CTAB+ Paecilomyces lilacinus Oat meal agar. The growth of P. lilacinus was good in both media compared to other. Roving survey was undertaken during kharif, 2013 to assess presence of nematophagous Identification of bioagent fungi i.e. P. lilacinus and observation recorded on incidence of root knot nematode The culture was identified based on mycelia in major tomato, chilli and brinjal growing characters like septate mycelium, spore

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Int.J.Curr.Microbiol.App.Sci (2017) 6(10): 1243-1257 morphology and the spore size. The conidia Dharwad-1 (PL-2) and Dharwad-2 (PL-3) were hyaline, single celled, oval to round isolates had similar colony and conidial shaped, and produced on phialides. Based on characters. Initially they had white coloured these characters the bioagent was identified as colonies later they turned to pinkish colour Paecilomyces lilacinus with single round to oval conidia produced on phailides. They differed slightly with respect Morphological diversity of different to sporulation i.e. Dharwad-1 sporulated isolates of Paecilomyces lilacinus earlier compared to Dharwad-2 isolate.

Diversity in cultural and morphological Mudhol (PL-6) and Masankatti (PL-7) characters of P. lilacinus was studied on isolates had similar colony characters, conidia potato dextrose agar media at room produced were similar and sporulation pattern temperature 27 ± 1o C as described in was same. Initially they had white coloured “Material and Methods” and the results colonies later on turned to pinkish to greyish obtained are presented in table 1. colour, single round to oval conidia were produced on phailides. Both the isolates During the present study various P. lilacinus sporulated little late compared to when isolates were coded based on their similarity. isolates from other districts. Tadakod isolate was coded as PL-1, Dharwad-1 as PL-2 and these two isolates Bioefficacy of isolates of Paecilomyces were similar colony characters. Although lilacinus against root knot nematode Bagalkot isolate was similar to them its colony colour was greyish and it was coded as Artificial inoculation of M. incognita PL-5. Bailahongal, Dharwad-2 and nematode to tomato plant was done as Masankatti isolates sporulated late hence, explained in “Material and Methods”. they were coded as PL-4, PL-3 and PL-7 respectively. Symptoms appeared after completion of life cycle of nematodes such as yellowing of leaf Isolates significantly varied with respect to foliage, stunting of plant and galling on the colony characters, sporulation of the isolate roots. and conidial characters. Egg hatching inhibition test Based on the colony characters observed Tadakoda isolate had initially white colony From the root knot infected plants eggs were later it turned to pinkish smooth colony. It separated from the egg masses. Different produced single, round to oval conidia on isolates of P. lilacinus were tested against phailides which sporulated early. these eggs. Hatching was significantly reduced in all the treatments with different Bailhongal (PL-4) and Bagalkot (PL-5) concentrations of Paecilomyces isolates. isolates had similar characters such as initially Hatching of M. incognita eggs increased with white coloured colonies later turning to decrease in concentration of P. lilacinus pinkish to greyish colour with single, round to isolates. The interaction between different oval conidia on phailides. With respect to isolates and time intervals was significant and sporulation they differed slightly. Sporulation it was indicated that an increase in was late in Bailhongal isolate where as it was concentration tend to modify the effect of early in Bagalkot (PL-5) isolate. other in a significant manner.

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Different isolates of P.lilacinus were tested significantly increased the mortality of for their bioefficacy against root knot juveniles (Table 3). nematodes through egg hatching inhibition in different time interval such as 12, 24 and 48 Minimum mortality was observed with 12hr hours. The culture filtrates of P. lilacinus at incubation and maximum mortality with 48 hr different time intervals significantly inhibited incubation with all the isolates. The mean per the hatching of eggs (Table 2a). cent juvenile mortality has increased from 12 hr to 48 hr incubation (Fig 1b). Inhibition was minimum with 12hr incubation and it was maximum with 48 hr incubation The highest mean juvenile mortality was with all the isolates. The mean per cent egg recorded with Mudhol isolate (75.77 %). hatching inhibition has increased from 12 hr Even it had 80.33 per cent mortality at 48 hr to 48 hr incubation. The highest mean egg incubation followed by Tadakoda isolate hatching inhibition was recorded with Mudhol (66.66%). Bagalkot isolates had moderate isolate (89.14 %). Even it had 90.26 per cent mean juvenile mortality of 64.32 per cent and inhibition at 48 hr incubation. Tadakoda and Dharwad-1 and Dharwad-2 isolates had mean Bagalkot isolates had moderate mean juvenile mortality of 58.30 and 59.09 per cent inhibition of 81.45 and 81.64 per cent respectively over the control and these respectively over the control and these isolates were on par with to each other. isolates were on par with each other (Fig 1a) Minimum mortality of 52.55 per cent was The lowest inhibition was observed with observed with Bailhongal isolate followed by Dharwad-2 and Masankatti isolates with Masankatti isolate with 55.66 per cent 74.33 per cent and 74.88 per cent inhibition mortality over the control and these isolates over the control and these isolates were on par were on par with each other (Table 2b and 3). with each other. In presence of Dharwad-2 and Masankatti isolates root knot nematodes In vivo studies on bioefficacy of showed maximum per cent egg hatching Paecilomyces lilacinus (25.67 and 25.12 %) and both are on par to each other indicating their poor performance. Roll towel method

However minimum mean per cent egg Various bioagents such as P. lilacinus, T. hatching was observed with Mudhol isolate harzianum, Lecanicillium lecanii with (10.86 %) followed by Tadakoda and different concentrations along with treated Bagalkot isolates (18.55 and 18.54 %) and check such as Carbofuran were used to study both are on par with each other indicating their effect on tomato growth parameter viz., their superiority (Table 2a). Per cent germination, Shoot length (cm), Root length (cm) and also seedling vigour index Juvenile mortality test through roll towel method (Table 4).

Different isolates of P. lilacinus were tested In this method, the P. lilacinus with 6 g/kg for their bioefficacy against root knot seed concentration showed more than 82 per nematodes through juvenile mortality in cent seed germination and produced higher different time interval such as 12, 24 and 48 shoot (9.07) and root length (7.17 cm) of hour interval. The culture filtrates of P. tomato seedlings with enhanced vigour index lilacinus at different time interval (291.47%) after seven days. The maximum

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Int.J.Curr.Microbiol.App.Sci (2017) 6(10): 1243-1257 vigour index of 1331.60 was recorded in P. respectively. Carbofuran 3 g/kg treated lilacinus @ 6 g/ kg treated seeds with seedlings also showed good vigour index maximum shoot and root length of 9.07 and (1051.2), shoot (8.27 cm) and root length 7.17 cm, it increases shoot and root length by (6.13 cm) with 123.48 and 97.7 per cent 145 and 132 per cent over the control increase over the control.

Table.1 Morphological variability of Paecilomyces lilacinus

Isolate Districts Isolates Colony characters Sporulation Conidial Character codes Tadakoda PL-1 Initially white turn Dharwad to light pink, Dharwad -1 PL-2 smooth Early sporulation Initially white turn Bagalkot Bagalkot PL-5 to Pink greyish colour smooth Single,round to oval Dharwad Dharwad -2 PL-3 Initially white conidia & production Belagavi Bailhongal PL-4 coloured later turn of phailides Late sporulation to pink colour Bagalkot Mudhol PL-6 smooth Initially white turn Haveri Masankatti PL-7 to pink to greyish Late sporulation smooth

Table.2a Bioefficacy of the isolates of Paecilomyces lilacinus on root knot nematode egg hatching inhibition

Per cent egg hatching inhibition/Time (hr) Per cent egg Isolates 12 24 48 Mean hatching 72.01 82.30 89.95 81.45 18.55 Tadakoda (PL-1) (58.10)** (64.94) (71.52) (64.49) (25.51) 71.77 81.22 82.30 78.81 21.19 Dharwad -1 (PL-2) (57.91) (64.32) (65.12) (62.59) (27.41) 68.40 74.63 77.56 74.33 25.67 Dharwad -2 (PL-3) (56.10) (59.76) (61.72) (59.56) (30.44) 73.03 79.0 80.26 77.79 22.21 Bailhongal (PL-4) (58.71) (62.73) (63.62) (61.88) (28.12) 73.56 82.53 86.30 81.64 18.54 Bagalkot (PL-5) (59.06) (65.29) (68.28) (64.63) (25.50) 81.16 86.21 90.26 89.14 10.86 Mudhol (PL-6) (64.28) (68.20) (71.81) (70.76) (19.24) 71.60 75.06 76.93 74.88 25.12 Masankatti (PL-7) (57.80) (60.04) (61.29) (59.92) (30.08) Mean 73.07 80.14 83.36 78.85 20.31 S.Em.± CD at 1% Isolates (I) 1.21 4.87 Time interval 0.79 3.19 I x Time interval 2.11 8.45

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Table.2b Mortality of juveniles of root knot nematode by Paecilomyces lilacinus isolates

Per cent juvenile mortality /Time (hr) Isolates 12 24 48 Mean 62.33 66.66 71.00 66.66 Tadakoda (PL-1) (52.14)* (54.74) (57.41) (54.74) 53.33 58.26 63.33 58.30 Dharwad -1 (PL-2) (46.91) (49.77) (52.73) (49.89) 54.66 59.33 63.3 59.09 Dharwad -2 (PL-3) (47.68) (50.40) (52.73) (50.00) 49.33 52.66 55.66 52.55 Bailhongal (PL-4) (44.61) (46.52) (48.25) (46.85) 59.66 63.66 69.66 64.32 Bagalkot (PL-5) (50.57) (52.93) (56.58) (53.58) 71.66 75.33 80.33 75.77 Mudhol (PL-6) (57.85) (60.25) (63.70) (61.20) 53.00 55.33 58.66 55.66 Masankatti (PL-7) (46.72) (48.06) (49.92) (48.75) Mean 57.71 61.60 65.99 61.79 S.Em ± CD at 1% Isolates (I) 0.82 2.46 Time interval 0.53 1.61 I x Timeinterval 1.42 4.27

Table.3 Bioefficacy of different isolates of Paecilomyces lilacinus against root knot nematode

Isolates Per cent egg hatching inhibition Per cent juvenile mortality Tadakoda (PL-1) 81.45 (64.49)* 66.66 (54.74) Dharwad-1 (PL-2) 78.8 (62.58) 58.30 (49.89) Dharwad-2 (PL-3) 74.33 (59.56) 59.09 (50.00) Bailhongal (PL-4) 77.78 (61.88) 52.55 (46.85) Bagalkot (PL-5) 81.64 (64.63) 64.32 (53.58) Mudhol (PL-6) 89.14 (70.76) 75.77 (61.20) Masankatti (PL-7) 74.88 (59.92) 55.66 (48.75) S.Em.± 0.70 0.82 CD at 1% 2.98 2.46

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Table.4 Effect of Paecilomyces lilacinus on plant growth parameters by roll towel method

Per Per Per cent Shoot cent Root cent Per cent Seedling increase Conc. Germination length increase length increase increase in vigour in Treatment (g/kg (%) (cm) in shoot (cm) in root germination index at vigour seed) at length at 7 length over control 7 days index 7days over days over over control control control Paecilomyces 2 70.33 40.66 6.0 62.16 4.87 57.09 764.40 124.48 lilacinus Paecilomyces 4 75.00 50.00 7.20 94.59 5.83 88.06 977.25 187.43 lilacinus Paecilomyces 6 82.00 64.00 9.07 145.00 7.17 131.2 1331.60 291.47 lilacinus Trichoderma 2 66.00 32.60 5.07 37.02 4.20 35.00 611.82 79.95 harzianum Trichoderma 4 70.00 40.00 6.03 62.97 5.03 62.25 774.20 127.70 harzianum Lecanicillium 2 66.00 32.00 6.00 62.16 4.40 41.19 686.40 101.88 lecanii Carbofuran 3 73.00 46.00 8.27 123.0 6.13 97.70 1051.20 209.12 Control - 50.00 _ 3.70 _ 3.10 _ 340.00 _ S.Em ± 1.05 012 0.19 6.76 CD at 1% 3.15 0.36 0.57 27.93

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The P. lilacinus @ 2g/kg seed resulted in Among the different bioagents used in the 70.33 per cent germination and there was tomato ecosystem, P. lilacinus is extensively 62.16 per cent increase in shoot length used as seed dressing bioagent against many (6.00cm) and 57.09 per cent increase in root soil borne diseases, however, its usage against length (4.87cm), vigour index was 764.40 and foliar diseases is limited. it increased by 124.48 per cent over the control and it was on par with Trichoderma Among several nematodes of economic harzianum @ 4 g/kg seeds treated with importance, root knot nematodes are most Trichoderma harzianum at 4g/kg seed had widely studied and are commonly found 70.00 per cent seed germination with 6.03cm involved in synergistic interactions with wilt shoot and 5.03 cm root length with vigour inducing fungi and bacteria. index of 774.20 after seven days compared to control. Microbial control of soil borne plant diseases is economically viable and environmental Least germination of 66 per cent, least shoot friendly method aimed at sustainable and root length of seedlings with less vigour Agriculture. Among the micro-organisms, index of 611.82 was observed in treatment bioagent are having great promise with the involving Trichoderma harzianum @ 2 g/kg dual advantage of plant growth promotion and followed by Lecanicillium lecanii @ 2 g/kg plant disease suppression. Recently, P. seed with 66.00 per cent germination, shoot, lilacinus spp. has received greater attention of root length of 6.00 and 4.40 cm respectively the scientists working towards management of with seedling vigour index of 686.40. plant parasitic nematodes. Hence, in the Untreated control produced seedling with present study, an attempt was made to isolate, shoot length of 3.70 and root length of 3.10 to screen the different isolates and to study cm and seedling vigour index was 340 (Table the bioefficacy and antagonistic effect of P. 4). lilacinus against Meloidogyne incognita and to study their effect against plant growth Tomato (Solanum lycopersicum Mill) is one parameters through different methods in of the most important crops and out ranks all tomato. other vegetables accept the potato crop in popularity and value in the world. The present investigations were carried out at the Departments of Plant Pathology and The modern intensive Agricultural practices Institute of Organic Farming, Agriculture have considerably raised the output but College of Dharwad during 2013-14 and the created problems like land and environmental results obtained are discussed here. degradation and pollution. Besides, this contamination of food products and pesticidal In general, root knot disease caused by residue in food has caused much panic among Meloidogyne incognita is one of the major the consumers and also the producers. Hence, constraints to Agriculture. Very less it is necessary to find out innovative and information is available with respect to the suitable alternative technology like biological occurrence and prevalence of P. lilacinus in control of pests and diseases. Recently, tomato growing areas of northern Karnataka. Paecilomyces lilacinus based different Hence, a survey was undertaken to assess the formulations are used for the control of occurrence of P. lilacinus in solanaceous nematode diseases, which is economical, crops in Haveri, Belgaum, Dharwad, Gadag ecofriendly and sustainable in the long run. and Bagalkot districts.

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Rhizosphere soil samples of tomato crops However, rootknot nematode incidence was were collected and processed to isolate P. not there in Kaginele, Nagnur and Motebenur lilacinus which is known to support all types villages of Byadgi taluka. of fungi. Seven isolates were obtained from the rhizosphere of tomato plants around Main In Gadag district, in none of the locations P. Agricultural Research Station, Dharwad and lilacinus was found. Whereas, rootknot other districts of northern Karnataka. nematode incidence was observed in Gajendragada and Lakkundi villages. The findings of the present study revealed that the P. lilacinus was more prevalent in Similar type of observation were made by Dharwad district followed by Bagalkot Patil et al., (2003) while working with district. Root knot nematode incidence was medicinal crop such as Coleus, Long pepper noticed in all the tomato, chilli and brinjal and Ashwagandha. Overall prevalence of growing locations surveyed. nematophagous fungi and disease incidence observed in northern Karnataka from In Belgaum district, P. lilacinus was found in flowering to fruiting stage. The prevalence of Nayanagar villages of Bailhongal taluka the nematophagous fungi was more in which had rootknot nematode incidence also. Dharwad district followed by Bagalkot district. Similar studies were conducted by In Munvali and Belavadi villages only root Archana (2012) and she isolated P. lilacinus, knot nematode incidence was observed Aspergillus spp from M. incognita egg whereas, it was not found in Inchal, masses. Overall 35 locations were surveyed Sankeshwar, and Yamakanmaradi locations. and soils from these locations were used to isolate P. lilacinus. However, isolation was In Dharwad district, at three locations viz. possible only from seven locations. MARS dharwad, Devar hubballi and Tadakoda villages both P. lilacinus and root In the present study, all the seven isolates of knot nematode were observed. However, at P. lilacinus were further examined for Hebsur village only rootknot nematode was nematicidal activity against M. incognita. observed which indicated natural occurrence Similar studies have been conducted by of bioagent at places wherever root knot various workers (Owino and waudo 1996: nematode was prevailing. Chen et al., 2000: Imran et al., 2001) who have randomly screened Paecilomyces In Bagalkot district, P. lilacinus was isolated lilacinus for their antagonistic activity against from Gaddankeri cross and Mudhol soils nematodes and evaluated them for their plant whereas, rootknot nematode incidence was growth promoting activity also (Goswami et found in Gaddankeri cross and Karadi Village al., (2006) and Kumar et al., (2012) of Bagalkot district. At Jamankandi, Lokapur, Ilkal and Kandgal villages of Bagalkot district The findings of the present study revealed that rootknot nematode incidence was not the P. lilacinus was prevalent in one or other observed during the survey. locations of all the districts surveyed. But overall occurrence was more in Dharwad In Haveri district, P. lilacinus was found only district. The present survey also indicated the in Masankatti village of Hangal taluka prevalence of M. incognita in different crops whereas, root knot nematode was observed in in all the districts surveyed. Invariably Masankatti, Akkialur and Kakol villages. Paecilomyces was isolated from all the

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Int.J.Curr.Microbiol.App.Sci (2017) 6(10): 1243-1257 location where root knot incidence was maximum egg hatching inhibition at 48 hr observed indicating its natural occurrence. incubation and there was increase in juvenile mortality when incubated for 48 hr. Similar, Isolates obtained were identified through studies were conducted by Ghazala et al., microscopic observation and they were (2004) and they also reported significant egg characterised into different groups based on hatching suppression in root knot nematodes the colony characters, sporulating habits and by P. variotii. Further, they reported that conidial characters. Almost all had similar hyphae of P. lilacinus and P. variotii strains characters of the P. lilacinus explained by penetrated into the eggs of root knot Samson (1975). Initially colonies were of nematode and destroyed them (Fig. 3a and white colour later on they turned to pink 3b). colour with smooth edges sometimes they turn to greyish colour and differ with Reduction in hatching, viability of juveniles respected to sporulation. In case of Tadakoda and eggs of M. incognita are induced by (PL-1), Dharwad-1 (PL-2) and Bagalkot (PL- secondary metabolite such as protease, 5) isolates early sporulation was observed paecilotoxin and serine protease which are whereas Bailhongal (PL-4), Dharwad-2 (PL- produced by P. lilacinus (Peter et al., 1995). 3) Mudhol (PL-6) and Masankatti isolates The bioagent infecting eggs of the root knot (PL-7) late sporulation type was observed. nematode Meloidogyne incognita suggested the involvement of lytic enzymes such as Regarding conidial characters they did not protease which binds to nematode eggs. They differ much and produced single, round to also concluded that the serine protease might oval conidia attached to phailides in long play a role in penetration of fungus through chain. The phailides were possessing swollen the egg shell of nematode (Fig. 3c and 3d). base with distinct neck. Similar observations were made by Bainer (1907) while working The juvenile mortality and egg hatching with root knot nematode management in inhibition of M. incognita observed in the tomato crop. present study might be due to antibiosis. Similar observation were recorded by Zaki In the present study, in vitro bioassay with (1994) he opined that the fungal filtrates of P. culture filtrates of different isolate of P. lilacinus inhibited the hatching of M. javanica lilacinus strains at similar concentrations eggs at all the concentrations used when revealed that egg hatching inhibition with an exposed for 48-72 hours (Fig. 4) increase in exposure period as well as concentration. Meloidogyne incognita References juveniles and eggs were highly vulnerable to the Paecilomyces strains. Among the seven P. Agrios, G. N., 1996, Plant Pathology. 4th lilacinus strains tested, all the strains showed Edition, Academic Press, New York, significantly higher antinematode action. America, p. 812. Ahuja, S., and Arora, J. S., 1980, Paecilomyces lilacinus significantly inhibited Susceptibility of flowering annuals to the hatching of M. incognita eggs and Mudhol root nematodes (Meloidogyne isolate was more effective in reducing egg incognita) and control of plant parasitic hatching when compared to control. nematodes. Trop. Pest Managt, 26: 293- Inhibition increased with the increase in the 295. concentration and Mudhol isolate showed Amala, U., 2012, Mass multiplication of

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entomopathogenic fungus, textural properties. J. Sci. Food Agric., Paecilomyces lilacinus with solid 90(10): 1665-1672. substrate. J. Biopest., 5 (2):168. Chandarkala, S., Gangopadhayay and Godara, Anonymous, 1993, Proceedings of the S. L., 2013, Studies on shelf life of international seed testing association, Trichoderma spp and Pseudomonas international rules for seed testing. Seed fluorescence in different formulating Sci. Technol., 21: 25-30. materials. Pl. Dis. Res., 28 (1): 53-57 Anonymous, 2000, Ann. Rep. (2000-01) El-Shanshoury, A. E. R., El-Sayed, S. A., Asian vegetable research development, Mahmoud, Y. A. G. and Khalefa, D. Taiwan, p. 110. M., 2005, Evaluation of Pochonia Bainer, G., 1907, Mycotheque de 1’ecole de chlamydosporia, Paecilomyces lilacinus Pharmacie, XI. Paecilomyces genere and Arthrobotrys dactyloides as nouveae de Mucedinees. Bull. biocontrol agents for Meloidogyne Trimmest. Soc. Medit., 16: 135-136. incognita under greenhouse condition. Baki, A. A., and Anderson, J. D., 1973, Pakistan. J. Bio. Sci., 8: 1511-1516. Vigour determination in soybean seed Enrique, C., Barker, K. B. and Nelson, L. A., by multiple criteria. Crop Sci., 13: 630- 1989, Growth of isolate of 633. Paecilomyces lilacinus and their Balanchard, D., Lecoq, H. and Pitrat, M., efficacy in biological control of 1992, A colour atlas of cucurbit Meloidogyne incognita on tomato. J. diseases: Observation, Identification Nematol., 21(2): 164-172. and control. Wiley and sons, New Ghazala, S. M., Ghashira, B. A. and Dabaj, K. York, p: 304. H., 2004, Effect of some fungal isolates Cadioli, M. C., Santiago, D. C., Oliveira, A. on egg-hatching inhibition of root-knot D. D., Paes, V. D. S., Arieira, G. D. O. nematodes Meloidogyne spp. under and Baida, F. C., 2009, Effect of laboratory conditions. Proc. Eighth isolates of Paecilomyces lilacinus on Arab. Cong. Pl. Pro, 12-16 El-Beida, the development of coffee plantations Libya, p. 133 and on the population of Meloidogyne Gintis, B. O., Morgan, J. G. and Rodriguez, paranaensis Ciencia. Agrotechologia, K. R., 1983, Fungi associated with 33: 713-720. several developmental stages of Cecilia, A. S., Evelina, A. T. and Lilia, M. A., Heterodera glycine from an Alabana 2010, Processing of tomato: impact on soybean field soil. Nematropica, 13: in vitro bioaccessibility of lycopene and 221-223.

How to cite this article:

Anusha, B.G., Shripad Kulkarni and Harlapur, S.I. 2017. Morphological and Biological Variability of Different Isolates of Paecilomyces lilacinus Against Root Knot Nematode in Tomato. Int.J.Curr.Microbiol.App.Sci. 6(10): 1243-1257. doi: https://doi.org/10.20546/ijcmas.2017.610.149

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