Choanephora Wet Rot of Cucumber and Its Management
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Sangeeta Choudhary
Thesis
Master of Science in Agriculture (Plant Pathology )
2015
Department of Plant Pathology S.K.N. COLLEGE OF AGRICULTURE, JOBNER-303 329 SRI KARAN NARENDRA AGRICULTURE UNIVERSITY, JOBNER
Choanephora Wet Rot of Cucumber and Its Management
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Thesis
Submitted to the Sri Karan Narendra Agriculture University, Jobner
In partial fulfillment of the requirement for the degree of
Master of Science
In the Faculty of Agriculture
(Plant Pathology)
By
Sangeeta Choudhary
2015
Sri Karan Narendra Agriculture University, Jobner S.K.N. College of Agriculture, Jobner
CERTIFICATE-I
Dated :______2015
This is to certify that Miss Sangeeta Choudhary has successfully completed the comprehensive examination held on 22 April 2015 as required under the regulation for Master’s degree.
(K.S. Shekhawat) Professor & HEAD Department of Plant Pathology S.K.N. College of Agriculture, Jobner
Sri Karan Narendra Agriculture University, Jobner S.K.N. College of Agriculture, Jobner
CERTIFICATE-II
Dated :______2015
This is to certify that the thesis entitled “Choanephora wet rot of cucumber and its management” submitted for the degree of Master of Science in the subject of Plant Pathology embodies bonafide research work carried out by Miss Sangeeta Choudhary under my guidance and supervision and that no part of this thesis has been submitted for any other degree. The assistance and help received during the course of investigation have been fully acknowledged. The draft of the thesis was also approved by the advisory committee on ......
(K.S. Shekhawat) Professor & HEAD (R.P. Ghasolia) Department of Plant Pathology Major Advisor S.K.N. College of Agriculture, Jobner
(S.N. Sharma) DEAN S.K.N. College of Agriculture, Jobner
Sri Karan Narendra Agriculture University, Jobner S.K.N. College of Agriculture, Jobner
CERTIFICATE-III Dated :______2015 This is to certify that the thesis entitled “Choanephora wet rot of cucumber and its management” submitted by Miss Sangeeta Choudhary to Sri Karan Narendra Agriculture University, Jobner, in partial fulfillment of the requirements for the degree of Master of Science in the subject of Plant Pathology after recommendation by the external examiner, was defended by the candidate before the following members of the examination committee. The performance of the candidate in the oral examination on his thesis has been found satisfactory. We therefore, recommend that the thesis be approved.
(R.P. Ghasolia) (S. Godika) Major Advisor Advisor
(B.L. Jat) (K. Ram Krishna) Advisor Director Education Nominee
(K.S. Shekhawat) Professor & HEAD (S.N. Sharma) Department of Plant Pathology Dean S.K.N. College of Agriculture, S.K.N. College of Agriculture, Jobner Jobner
Approved
DIRECTOR EDUCATION S.K.N. Agriculture University, Jobner
Sri Karan Narendra Agriculture University, Jobner S.K.N. College of Agriculture, Jobner
CERTIFICATE-IV
Dated : _____ 2015
This is to certify that Miss Sangeeta Choudhary of the Department of Plant Pathology, S.K.N., College of Agriculture, Jobner has made all corrections/ modifications in the thesis entitled “Choanephora wet rot of cucumber and its management” which were suggested by the external examiner and the advisory committee in the oral examination held on ______2015. The final copies of the thesis duly bound and corrected were submitted on ______2015 and forwarded herewith for approval.
(R.P. Ghasolia) Major Advisor (K.S. Shekhawat) PROFESSOR & HEAD Department of Plant Pathology S.K.N. College of Agriculture, Jobner
(S.N.Sharma) DEAN S.K.N. College of Agriculture, Jobner
APPROVED
DIRECTOR EDUCATION SKNAU, Jobner
ACKNOWLEDGEMENT
In the ecstasy. I express my esteem and profound sense of gratitude to Dr. R. P. Ghasolia, major advisor, Asstt. Professor, Department of Plant Pathology, S.K.N. College of Agriculture, Jobner, for his valuable and inspiring guidance, constant encouragement and keen interest during the course of present investigation and preparation of this research report. I convey the feeling of gratitude to the members of my advisory committee Dr. S. Godika, Professor, Deptt. of Plant Pathology, Dr. B.L. Jat, Professor and Head, Deptt. of Entomology, Dr. K. Ram Krishna, Director Education Nominee for the help rendered during the course of investigation. I am also deep and heartful greatfulness to the Dean, S. K. N. College of Agriculture, Jobner and Dr. K. S. Shekhawat, Head and Professor, Deptt. of Plant Pathology for providing necessary facilities during the course of investigation. I am again ecstatic to express my inherent sense of gratitude to Dr. Mahaveer Singh, Dr. R.L. Mahala, Dr. Rekha Kumawat, Deptt. of Plant Pathology, Dr. O.P. Garhwal, Asstt Professor, Deptt. of Horticulture and all the staff members, S.K.N. College of Agriculture, Jobner whose cooperation made this investigation smooth and easy. Down the memory lane. I am very much thankful to my dear friends Manju, Sukhlal, Jitendra, Vishambar, Anita, Nagar, Sunita my seniors, Sanju di, Arjun Ji, sajjan Ji, suresh Ji, Manoj Ji and my lovely juniors Madhu, Pankaj, Bhagwati, Rajesh, Roshan, Raj kumar, Pramod, Suresh and Rajbala with whom I went hand in hand during my study and research work. My vocabulary falls short to express heartiest regards to my grandmother Smt. Radha Devi, father Sh. Raghunath Prasad Ji, lovely mother Smt. Prem Devi, Elder brother Narendra Mohan, younger brother Dinesh, Bhabhi Sumitra and lovely niece Divya and Nivedita whose consistent encouragement and blessing are beyond my expression that brought me here up to dream without which it could not have been sketched. I am also grateful to Sh. Suresh Yadav of Vimal Computers, Jobner, who typed this manuscript. Last but not the least, a millions of thanks to God the almighty who made me to complete this task and made every job a success for me. Place: Jobner Dated: (SANGEETA CHOUDHARY)
CONTENTS
CHAPTER PARTICULARS PAGE NO. NO.
1. INTRODUCTION ………..
2. REVIEW OF LITERATURE ………..
3. MATERIALS AND METHODS ………..
4. RESULTS ………..
5. DISCUSSION ………..
6. SUMMARY ………..
LITERATURE CITED ………..
ABSTRACT (English) ………..
ABSTRACT (Hindi) ………..
LIST OF TABLES Table Particulars Page No. No. 4.1 Pathogenicity of Choanephora wet rot of cucumber ------4.2 Effect of temperature on mycelial growth of ------Choanephora cucurbitarum 4.3 Effect of relative humidity on mycelial growth of ------Choanephora cucurbitarum at 25 + 10C 4.4 Effect of different media on mycelial growth of ------Choanephora cucurbitarum at 25 + 10C 4.5 Efficacy of salicylic acid on Choanephora wet rot of ------cucumber 4.6 Efficacy of fungicides on mycelial growth of ------Choanephora cucurbitarum at 25 + 10C (in vitro) 4.7 Efficacy of different fungicides against Choanephora wet ------rot of cucumber (in vivo) 4.8 Efficacy of bioagents against Choanephora wet rot of ------cucumber 4.9 Screening of cucumber varieties against Choanephora ------cucurbitarum
LIST OF FIGURES
Figure Particulars Page No. No. 4.1 Effect of temperature on mycelial growth of ………
Choanephora cucurbitarum
4.2 Effect of relative humidity on mycelial growth of ………
Choanephora cucurbitarum at 25 + 10C
4.3 Effect of different media on mycelial growth of ………
Choanephora cucurbitarum at 25 + 10C
4.4 Efficacy of salicylic acid on Choanephora wet rot of ………
cucumber
4.5 Efficacy of fungicides on mycelial growth of ………
Choanephora cucurbitarum at 25 + 10C in vitro
4.6 Efficacy of different fungicides against Choanephora ………
wet rot of cucumber in vivo
4.7 Efficacy of bioagents against Choanephora wet rot of ………
cucumber
4.8 Screening of cucumber varieties against Choanephora ………
cucurbitarum
LIST OF PLATES
Plate Particulars Page No. No. 4.1 Morphological characters of Choanephora cucurbitarum ……
4.2 Pathogenicity test …….
4.3 Symptoms of Choanephora wet rot on cucumber plants ……
4.4 Effect of temperature on mycelial growth of Choanephora ……. cucurbitarum 4.5 Effect of relative humidity on mycelial growth of …… Choanephora cucurbitarum at 25 + 10C 4.6 Effect of different media on mycelial growth of ……. Choanephora cucurbitarum at 25 + 10C 4.7 Efficacy of fungicides on mycelial growth of …… Choanephora cucurbitarum at 25 + 10C (in vitro)
1. Introduction
Cucumber (Cucumis sativus L.) is known as “Kheera” in Hindi. It is a popular and widely cultivated summer vegetable in India which belongs to family Cucurbitaceae. It is one of the important vegetable crops which supply edible product and fibre. Cucumber is used as salad or as vegetable and as desert fruit specially in Rajasthan. In India, cucumber is commonly grown in
Andhra Pradesh, Karnataka, Assam and Rajasthan, generally towards the riversides. Rajasthan state provides the maximum potential for the production of cucumber because of its agro-climatic conditions that are best suited for their growth and yield. According to De Candole (1967) cucumber is an indigenous vegetable of India. Purseglove (1967) has suggested that all cultigens (Cucumis sativus) originated from northern India where the related Cucumis hardwicki Royle occurs as wild, although this might be a
“weedy” form of Cucumis sativus, which has escaped from cultivation.
Cucumber has been cultivated in India for at least three thousand years (Rai and Yadav, 2005).
Cucumber is an annual deep-rooted crop with tendrils and hairy leaves. The plants may have an indeterminate, determinate or a compact plant habit. The compact growth habit consists of plants with shorter internode length than plants with indeterminate or determinate growth habit.
Several flowering habits exist in cucumbers. Most cultivars are monoecious i.e. with separate male and female flowers in the same plant. Besides the cucumber, other important cucurbitaceous crops are watermelon (Citrullus vulgaris Schard), muskmelon (Cucumis melo L.), pumpkin (Cucurbita moschata Duch), summer squash (Cucurbita pepo L.), ridge gourd (Luffa acutangula L.), sponge gourd (Luffa cylindrica Roem), bottle gourd [Lagenaria siceraria (Mol) Standl] and bittergourd (Momordica charantia L.) etc.
“Kheera” is eaten raw with salt and pepper or as salad with onion and tomato. The pulp of the fruit is used in making mask cakes. The fruit is said to have cooling effect and prevent constipation and is useful in checking jaundice. Cucumber is rich in vitamin B and C as well as in minerals such as calcium, phosphorus, iron and potassium.
The nutritive value of cucumber per 100 g of edible portion is as follows: S. No. Particulars Quantity (Per 100 g) 1. Moisture 96.3 g 2. Protein 0.4 g 3. Fat 0.1 g 4. Minerals 0.3 g 5. Fibre 0.4 g 6. Carbohydrate 2.5 g 7. Calcium 10 mg 8. Phosphorus 25 mg 9. Iron 1.5 mg 10. Thiamine 0.03 mg 11. Niacin 0.2 mg 12. Vitamin-C 7 mg 13. Energy 13 K Cal (Rai and Yadav, 2005)
In world, China is a leading country in cucumber production with an
area of 1052.5 million ha, with production of 45546.2 million tonnes and yield ( 43.3 ton/ha), followed by Iran, Turkey, Russia, USA (Anonymous,
2011).
In India, cucumber production is 678.15 million tonnes with an area of
43.28 million hectares. It is mostly grown in Andhra Pradesh (7.89 million
ha), Karnataka (6.82 million ha), Assam (6.59 million ha), Odisha (2.46
million ha) and Rajasthan (2.27 million ha) states. In Rajasthan, it is mainly
cultivated in Alwar, Bharatpur, Ganganagar, Bhilwara, Sawai Madhopur,
Jaipur and Dausa districts. In Rajasthan, total area under cucumber
cultivation is 2.34 million ha with production of 8.91 million tonnes.
(Anonymous, 2014).
Cucumber is a warm season crop but it is also grown in summer and
rainy season. It requires 18 °C minimum temperature for seed germination
and 20-30 °C for growth and development of plant. It requires sandy to loam
soil for early and good crop. Cucumber fruits become ready for first
harvesting in about 60-70 days after sowing. Under protected cultivation, the
optimum production of cucumber is in the slightly acidic to neutral soils (i.e.
pH 5.5 to 7.0) with a spacing of row to row 120 cm and plant to plant 20 cm.
However, a number of fungal diseases which have been reported to
cause heavy losses to the crop are given below.
S.No. Disease Causal organism Reference 1 Anthracnose Colletotrichum lagenarium Rao (1966) 2 Powdery Erysiphe cichoracearum Dc Khandelwal mildew Sphaerotheca fuligenea (Schl.) (1974) Poll. 3 Downy Pseudoperonospora cubensis Singh (1985) mildew (Berk.and Curt.) Rost. 4 Wilt Fusarium oxysporum f.sp. Jhamaria and cucumerinum (Owen) Patel (1971) Synder and Hansen 5 Leaf blight Alternaria cucumerina Khandelwal and (Ellis and Everhart) Elliott Prasada (1970) 6 Leaf spot Alternaria alternata Vakalounakis and Malathrakis (1988) 7 Damping off Pythium aphanidermatum Dwivedi and (Edson) Fits Tandon (1976) 8 Fruit rot and Choanephora cucurbitarum Yu and Ko (1997) leaf blight
In Japan, Choanephora cucurbitarum causes fruit rot, flower and leaf
blights on okra, squash, pumpkin, pepper, pea and bean including cucumber
(Yu and Ko, 1997). Choanephora cucurbitarum was also reported on brinjal
(Kwon and Hyeong, 2005), chilli (Mian and Meah, 1981), cowpea
(Naimuddin et al., 1999), Amaranthus (Roy and Deka, 1989), Boerhavia
diffusa (Sharma et al., 2011), Withania (Saroj et al., 2012) and okra
(Hussein and Ziedan, 2013) in India and abroad.
Abdel-Motaal et al. (2010) reported that young fungal colony is
colorless, as it aged, it becomes white to yellowish brown and has aseptate
hyphae with irregular branching. The monosporous sporangiophores are
long, slender and branched at the apex with monosporous sporangiola on
each branch. The monosporous sporangiola are sub-spherical to ovoid in
shape while sporangiospores are dark brown, fusiformis or elliptical to ovoid
with hair-like appendages on both ends. Choanephora cucurbitarum is a facultative saprobe that belongs to Zygomycota, order-Mucorales and family-
Choanephoraceae (Umana et al., 2014).
Choanephora cucurbitarum causes dieback and fruit rot symptoms on chilli (Mian and Meah, 1981). It causes typical symptoms of the soft rot on eggplant fruits started with water-soaking and dark-green lesions. Usually whitish mycelium and monosporous sporangiola are produced on the lesions
(Kwon and Hyeong, 2005). It also causes water soaked lesions on leaves and stems that progressed to a wet rot on Withania somnifera (Saroj et al.,
2012). Okra plants exhibit blossom blight, chlorosis and wet rot of young fruits (Hussein and Ziedan 2013). Initial symptoms begin as reddish purple spots at the tip of flowers and expanded to encompass entirely on Chinese hibiscus (Park et al., 2014). It also leads to dark grey flower spots that often coalesced to blighting of flowers under conditions of high humidity on periwinkle (Holcomb,1998).
However, due to this pathogen, disease incidence in cow pea has been reported up to 15.4 per cent (Naimuddin et al., 1999). Twenty to thirty per cent disease incidence has also been observed on pepper (Blazquez,
1986). In okra, disease severity was observed up to 63.93 per cent (Siddiqui et al., 2008). Hussein and Ziedan (2013) recorded 50 per cent yield loss due to Choanephora cucurbitarum in okra.
Crop loss was estimated to be 20 per cent in a diseased plot of okra in Malaysia (Huan and Jamil, 1975). Crop loss due to Choanephora cucurbitarum was estimated 7 to 20 per cent in cowpea in Nigeria (Oladiran,
1980). The literature search revealed that no work has been done on the proposed wet rot of cucumber (Cucumis sativus L.) caused by Choanephora cucurbitarum in India. Therefore, the present study was under taken with following aspects.
i. Isolation, purification, pathogenicity and identification of wet rot
pathogen.
ii. Effect of temperature, RH and media on growth of Choanephora
cucurbitarum.
iii. Induction of disease resistance in cucumber against
Choanephora cucurbitarum.
iv. Management through fungicides, bio agents and host resistance.
2. Review of Literature
2.1 Isolation, purification, pathogenicity and identification of
wet rot pathogen:
In Bangladesh, Mian and Meah (1981) noticed dieback and fruit rot symptoms on chilli plants by artificial inoculation of injured leaves, side shoots and fruit of 9 week old plants with Choanephora cucurbitarum in the field.
Roy and Deka (1989) reported that infection of Amaranthus hypochondriacus starts at the top of the shoot as a brownish discoloration that spreads downwards to the stem and leaves. Choanephora cucurbitarum was identified from affected parts and its pathogenicity was confirmed in
Assam. They also established that infection occurs primarily from soil borne inoculum and in traces (0.5%) through seed borne inoculum.
Holocomb (1998) reported dark grey spots on flowers often coalesced and led to blighting of flowers under conditions of high humidity on all of 18 periwinkle cultivars at Burden Research Plantation, Louisiana, USA in
September 1997. The white cottony growth and black spore masses of fungus was recovered from diseased tissue and identified as Choanephora cucurbitarum.
Kagiwada et al. (2010) observed leaf and stem rot of ice plant in a hydroponic greenhouse in Japan and the causal agent was confirmed as
Choanephora cucurbitarum. Abdel-Motaal et al. (2010) isolated fungus from floral rot of Egyptian henbane (Hyoscyamus muticus), pathogenicity proved and identified as
Choanephora cucurbitarum (Berkeley & Ravenel) Thaxter. In Japan, this new disease was named as Choanephora rot of Egyptian henbane. They observed that initially fungal colony was colorless, as it aged, it became white to yellowish brown and had aseptate hyphae with irregular branching.
They also noted that monosporous sporangiophores were long, slender and branched at the apex with monosporous sporangiola on each branch.
The monosporous sporangiola were sub-spherical to ovoid in shape while sporangiospores were dark brown, fusiformis or elliptical to ovoid with hair- like appendages on both ends.
Abdel-Motaal et al. (2010) noted symptoms like wilting and rotting of flowers, leaves and stems due to Choanephora cucurbitarum on henbane in
Japan under greenhouse conditions. They also reported that disease gradually increased over 8 days after inoculation with a conidial suspension.
They also noted that fungal hyphae and abundant sporangiophores with fertile sporangial heads were clearly seen on all the infected parts of the plant.
Sharma et al. (2011) first reported twig blight of Boerhavia diffusa caused by Choanephora cucurbitarum from Jaipur, Rajasthan.
Saroj et al. (2012) proved pathogenicity of Choanephora cucurbitarum on Withania somnifera by artificial inoculation with spray of an aqueous spore suspension containing 106 spore /ml and symptoms appeared after 7 to 10 days.
Hussein and Ziedan (2013) reported that okra plants were exhibited blossom blight, chlorosis and wet rot of young pods. Isolation from diseased tissue of young pods on PDA revealed several fungal isolates. Among them, white aerial mycelial growth that later changed in to light yellow coloured colony was identified as Choanephora cucurbitarum (Berk. & Ravenel)
Thaxter.
Park et al. (2014) observed blight on Chinese hibiscus in a commercial flower nursery in Seoul, Korea. Initial symptoms were appeared as reddish purple spots at the tip of flowers and expanded to encompass entire flowers and associated fungus was identified as Choanephora infundibulifera. This is thought to be the first report of Choanephora infundibulifera on Hibiscus rosa-sinesis in Korea.
Ravat et al. (2015) found rice sample infected with sheath rot during survey in west Bengal, India. The causal pathogen was identified as
Choanephora cucurbitarum based on cultural, morphological and pathogenicity analysis. This is thought to be the the first record of
Choanephora cucurbitarum on rice in West Bengal, India.
2.2 Effect of temperature, relative humidity and media on
growth of Choanephora cucurbitarum
2.2.1 Temperature: Kuo et al. (1999) found that optimum temperature for mycelial growth and germination of sporangiospores of Choanephora cucurbitarum was between 25 to 35 ºC, whereas the optimum temperature for sporangia and sporangiola was 25 ºC and 15 ºC, respectively.
Abdel-Motaal et al. (2010) evaluated growth of C. cucurbitarum at different temperature levels (15, 20, 25, 30 and 37 °C) on PDA for 7 days.
The best vegetative growth was recorded at 25 °C followed by 20 °C, at which the colony diameter was maximum and both mycelial and sporangia formation were abundant.
2.2.2 Humidity:
Kwon and Hyeong (2005) noted soft rot on fruits of eggplant caused by Choanephora cucurbitarum in the experimental field at Gyeongnam
Agriculture Research and Extension Services in Korea. The inoculated plants were placed in a humid chamber with 100 per cent RH at 30 ºC for 24 hrs and returned to the greenhouse until end of experiment. Typical symptoms of the soft rot appeared at 8 days after inoculation on fruits of eggplant.
Hussein and Ziedan (2013) inoculated the pods of okra with
Choanephora cucurbitarum and kept them in plastic bags at 95 per cent RH for seven days in growth chambers at 25 ºC or 12 ºC. After seven days of inoculation, wounded pods were completely rotted and the infected tissue fully covered with mycelia. Park et al. (2015) recorded higher disease on Hosta plantaginea by inoculating with Choanephora cucurbitarum in greenhouse at 80 per cent RH in Korea.
2.2.3 Media:
Kuo et al. (1999) reported a newly documented disease, namely
Choanephora wet rot of lima bean caused by Choanephora cucurbitarum at
Chiayi, Taiwan in Augus,t 1998. Potato dextrose agar and Czapeck’s dox yeast agar were found most favourable for mycelial growth of the fungus.
Mishra and Mishra (2012) used different media viz. PDA, oat meal agar, peptone-dextrose-rose bengal agar and malt extract agar for isolation of
Choanephora cucurbitarum from eggplant and observed good growth on
PDA.
2.3 Induction of resistance in cucumber varieties against
Choanephora cucurbitarum:
Transplanting of cucumber seedlings:
Ramamoorthy et al. (2001) reported that mixtures of different PGPR strains have resulted in increased efficacy by inducing systemic resistance against several pathogens attacking the same crop. Seed treatment with
PGPR causes cell wall structural modifications and biochemical/physiological changes leading to the synthesis of proteins and chemicals involved in plant defence mechanism. Lipopolysaccharides siderophores and salicylic acid are major determinants of PGPR mediated induced systemic resistance.
Sharma et al. (2002) conducted an experiment during summer 1998 in Palampur, Himachal Pradesh, India, to determine the effect of growing cucumbers in plastic bags in a greenhouse and after hardening, transplanting them to the field. They transplanted cucumber seedlings outside the greenhouse after 2-8 days of exposure, recorded significantly higher yields than direct sowing and immediate transplanting of seedlings.
Kone et al. (2009) evaluated the effect of DL-3-aminobutyric acid
(BABA), 2,6-dichloro isonicotinic acid (INA), Saver (a.i. salicylic acid), syrup
(nutrient supplement) and acibenzolar-S-methyl (ASM) at concentrations ranging from 25 to 2000 µg/ ml on mycelial growth, zoospore germination and sporangium production of Phytophthora capsici on squash. INA and
Saver reduced mycelial and sporangial production at 100 µg/ml or zoospore germination at 500 and 1000 µg /ml. They also reported that all the products applied as a soil drench or foliar spray at 25 or 50 µg/ml significantly reduced disease severity on squash.
2.4 Management stratiegies
2.4.1 Management through fungicides:
Chahal and Grover (1974) reported that zineb, mancozeb, ziram and thiram were the most effective in controlling Choanephora cucurbitarum on chilli. Tripathi et al. (1978) concluded that leaf blight and tip rot of sunhemp caused by Choanephora cucurbitarum were effectively controlled by thiram,
Ferbam, Blitox-50 and ziram.
Mian and Meah (1981) evaluated Vitavax (carboxin) at 5000 ppm that completely inhibited fungal growth of Choaenophora cucurbitarum while
Brassicol (quintozene) at 1000 ppm arrested it.
Oikawa et al. (1986) tested 9 compounds in vitro and found that mancozeb, triazine and triflumizole gave some control but were ineffective in vivo, possibly owing to their poor adhesion.
Hammouda (1988) reported that mancozeb, dinocap and thiabendazole were found effective in reducing infection against
Choanephora cucurbitarum in the southern region of Oman (Dhofar).
Gunasekera and Shantichandra (1989) noted that triadimenol, vinclozolin and bitertanol were most effective in inhibiting spore germination and mycelial growth of Choanephora cucurbitarum.
Gunasakera and Shantichandra (1990) observed that triadimenol, bitertanol and vinclozolin reduced the incidence of Choanephora cucurbitarum on Psophocarpus tetragonolobus (winged bean) in field trials in
Sri Lanka in 1986. The maximum pod yield was obtained with triadimenol and was associated with decreased disease incidence.
Panja (1999) concluded that captan 50 per cent (2.0 g/litre), ziram 27 per cent (1.5 ml/litre) and copperoxychloride with 50 per cent (3.0 g/litre) completely inhibited the mycelial growth and sporulation of Choanephora cucurbitarum and improved yields of Capsicum annum in West Bengal.
2.4.2 Management through bioagents:
Ikediugwu et al. (1994) reported that isolates of Bacillus subtilis from soil and Ogili (a local food condiment prepared from watermelon seeds) controlled Choanephora shoot disease of Amaranthus hybridus in the greenhouse. Both strains of Bacillus subtilis inhibited mycelial growth and conidial germination.
Emoghene and Okigbo (2001) observed a considerable zone of inhibition between the pathogen Choanephora cucurbitarum and the isolates of Xanthomonas compestris, Erwinia herbicola and Bacillus subtilis on PDA plates. Sixty seven per cent of Amaranthus hybridus showed disease symptoms when it was inoculated with X. compestris or E. herbicola while
33.30 per cent developed the shoot disease when Bacillus subtillus applied.
Siddiqui et al. (2008) concluded that disease severity was lowest in plants treated with Trichoderma-fortified RST extracts (9.56%) with a disease index of 1.00. There was a reduction of 85.04 per cent in
Choanephora wet rot severity on okra treated with Trichoderma-fortified RST extracts during 12 weeks of assessment in the field, which was comparable to the conventional fungicide Dithane M-45, suggesting that application of extracts produced from well matured compost fortified with biocontrol agent could be an alternative control strategy. Alamri et al. (2012) evaluated the ability of Rhizoleen-T and
Rhizoleen-B to suppress the root disease of cucumber caused by Fusarium oxysporum and Rhizoctonia solani and induction of the plant defence system. They increased the surviving percentage of the treated plants to 98 per cent and reduced the mean disease rating to 1.
Bhat et al. (2014) reported that seeds of Withania treated with
Azotobacter showed moderate germination followed by combined treatment of Azotobacter and Trichoderma which showed very high germination.
Elsharkawy et al. (2014) reported the efficacy of Bacillus subtilis,
Pseudomonas fluorescens, Derxia gummosa and Trichoderma harzianum to control powdery and downy mildew on cucumber plants under green house conditions. Spray of cucumber plants with suspension of these bioagents significantly reduced severity of both mildew as well as increased fruit yield compared to the control. Bacillus subtillus recorded highest reduction of area under disease progress curve (AUDPC) being 175.0 as compared to 536.55 in the control for powdery mildew, while Trichoderma harzianum recorded highest reduction being 241.15 as compared to the control 513.45 for downy mildew.
2.4.3 Management through host resistance:
Naimuddin et al. (1999) observed seedling blight of pigeon pea caused by Choanephora cucurbitarum in experimental plots at the Indian
Institute of Pulse Research, Kanpur, Uttar Pradesh, India. Disease incidence under field conditions was recorded on 148 genotype of pigeon pea. Amongst them, 103 genotype were disease free and 45 were infected.
Disease incidence in these 45 genotype varied between 3.8 to 15.4 per cent.
Wehner et al. (2000) were evaluated 851 cultigens, breeding lines and plant introduction in the field against gummy stem blight pathogen
(Didymella bryoniae) of cucumber in North Carolina. The most resistant breeding lines and PI accession were PI 200815, PI390243, LJ90430,
PI279469 and PI432855. The most resistant cultivars were Homegreen2,
Little John, Transamerica and Poinsett 76. The most susceptible cultigens in the study were PI288238, PI357843, PI357865 and PI167134. Two popular cultivars Calypso and Dasher II were found moderately resistant in North
Carolina.
Ajibade and Amusa (2001) investigated 71 lines of cowpea in humid zone of South Western Nigeria and found that 85 per cent lines had
Choanephora pod rot.
Siddiqui et al. (2008) categorized the okra pods using 0-5 disease rating scale, 0 per cent disease incidence as free or immune, 1-5 per cent disease incidence as resistant, 6-15 per cent disease incidence as moderately resistant, 16-50 per cent disease incidence as moderately susceptible, 51-95 per cent disease incidence as susceptible, more than 95 per cent disease incidence as highly susceptible.
3. Material and Methods
3.1 Collection of sample, isolation, purification, pathogenicity
and identification of the pathogen.
3.1.1 Collection of diseased plants:
As information received from the horticulturists about a disease appeared on cucumber plants grown in polyhouse at the Department of
Horticulture, SKN College of Agriculture, Jobner (Jaipur), a visit was made to know the problem during Zayad, 2014. Watery appearance on stem near soil line, rotting of roots & stems and whitish growth of fungus on affected part were observed. Samples of affected cucumber plants were collected in paper bags, brought to the laboratory and stored in refrigerator for further studies.
3.1.2 Isolation:
For isolation of the pathogen, small pieces of the affected root and stem were cut along with healthy tissues. These pieces were surface sterilized by dipping in 0.1% sodium hypochlorite solution for 1-2 minute.
After three consecutive washings with sterilized distilled water, the pieces were transferred to autoclaved potato dextrose agar (200 g potato extract +
20 g dextrose + 20 g agar + 1000 ml water) medium in Petriplates and incubated at 25±1ºC. After 3 days of incubation, mycelial growth emanating from bits was transferred aseptically to fresh PDA slants with the help of sterilized inoculating needle and reincubated for next 3 days to obtain further mycelial growth and sporulation for their purification.
3.1.3 Purification:
Purification of the pathogen was done by single spore and hyphal tip technique.
3.1.3.1 Single spore technique:
For purification, spores were obtained from culture slant after 10 days of incubation and were suspended in sterilized distilled water. The dilution of suspension was adjusted such that in one loopful, 5-10 spores could be counted under the low power objective of the microscope. One ml of above suspension was spread in Petriplates containing 20 ml sterilized plain agar medium. After 12-24 hours of inoculation, the germinating spores were located under the microscope and marked with the help of dummy objective and then transfered to PDA slant and kept in BOD for further growth. The culture was maintained by periodical transfer on PDA slants for further studies. 3.1.3.2 Hyphal tip technique:
The method was same as described earlier (3.1.3.1) except instead of single spore, hyphal tip was marked and transferred on PDA.
3.1.4 Pathogenicity:
Cucumber seeds were sown in pots, containing sterilized soil + FYM in 3:1 ratio. The pathogenicity of the pathogen was tested under pot conditions by seed, soil and seed-cum-soil inoculation techniques (Kataria and Grover, 1976 and Radhakrishna and Sen,1985).
3.1.4.1 Seed inoculation technique:
Apparently healthy surface sterilized seeds of cucumber variety Fumiko-10 (Sakata Pvt. Ltd.) were taken. The seeds were rolled on 7 days old sporulating culture of Choanephora cucurbitarum thriving on PDA contained in Petriplates. Inoculated seeds were sown in 30 cm diameter earthen pots (Pre-sterilized pots having autoclaved soil) at the rate of 5 seeds/pot with four replication. The uninoculated apparently healthy seeds served as check. These pots were kept in cage house and watered regularly as and when required.
3.1.4.2 Soil inoculation technique:
Prior to sowing, pots (30 cm diameter) were sterilized with copper sulphate solution and filled with sterilized soil + FYM (Soil:FYM=3:1; sterilized at 1.045 kg/cm2 for one hour for three consecutive days). These pots were inoculated with inoculum, multiplied on sorghum grains @ 20 g/pot. Five apparently healthy and surface sterilized cucumber seeds (Fumiko-10) were sown in each pot with four replications. Surface sterilized seeds sown in uninoculated sterilized soil, served as check. These pots were kept in cage house and watered regularly as and when required and maintained under identical conditions.
3.1.4.3 Seed-cum-soil inoculation technique:
Prior to sowing, pots (30 cm diameter) were sterilized with copper sulphate solution and filled with sterilized soil + FYM (Soil:FYM=3:1; sterilized at 1.045 Kg/cm2 for one hour for three consecutive days). These pots were inoculated with inoculum, multiplied on sorghum grains @ 20 g/pot. The seeds were rolled on 7 days old sporulating culture of fungus thriving on PDA contained in Petriplates. Inoculated seeds were sown in inoculated pots at the rate of 5 seeds/pot with four replication. Surface sterilized seeds sown in uninoculated pots served as check. These pots were kept in cage house and watered regularly as and when required and maintained under identical conditions.
Observation on seed germination was recorded 10 days after sowing and post emergence mortality was recorded up to 30 DAS in all above three experiments. The fungus was reisolated from artificially inoculated plants and resulting culture was compared with original one to confirm the identity of the fungus.
3.1.5 Identification of the pathogen:
The isolated fungus tentatively identified as Choanephora sp. on the basis of morphological characters. The culture was also sent to ITCC, Division of Plant Pathology, IARI, New Delhi for further confirmation or identification of fungus. The fungus was identified as Choanephora cucurbitarum with ID No. 9574.14.
3.2 Physio-pathological studies:
All the glasswares were thoroughly cleaned and rinsed with distilled water. Chemicals of analar grade were used. Five different synthetic and semi-synthetic media were prepared by weighing the different constituents of each medium and autoclaved at 1.045 kg/cm2 for 20 minutes.
In all three physiopathological experiments, inoculation was done with 5 mm diameter bit taken from 7 days old fungal culture and incubated at 25±1ºC (except for temperature study) for 3 days. The each experiment, under physio-pathological study was arranged in completely randomized design (CRD) with four replications.
3.2.1 Effect of temperature on mycelial growth:
Effect of temperature on mycelial growth of Choanephora cucurbitarum was studied in vitro. Twenty ml of sterilized PDA was poured in each sterilized Petriplate. Inoculation was made with 5 mm disc of 7 days old culture of Choanephora cucurbitarum with the help of sterilized cork borer and incubated at different levels of temperature viz. 20, 25, 30, 35 and 40 ºC for 3 days. Observations on mycelial growth was recorded after 3rd day of incubation.
3.2.2 Effect of relative humidity on mycelial growth:
To study the effect of relative humidity on mycelial growth of Choanephora cucurbitarum, five different levels of relative humidity i.e. 60, 70, 80, 90 and 100 per cent were maintained by using the concentrate sulphuric acid and sterilized distilled water in different proportion in glass desiccators according to the method suggested by Buxton and Mellanby (1934). The composition of the acid solution used was as follows.
RH (%) Stock solution (ml) Distilled water (ml)
60 374.0 396.0
70 348.0 510.3
80 294.0 640.0
90 161.0 712.0
100 0.00 Only distilled water
Petriplates containing PDA medium were inoculated with 5 mm disc of 7 days old culture of Choanephora cucurbitarum with the help of sterilized cork borer. Inoculated Petriplates were immediately accommodated in glass desiccators containing mixture of sulphuric acid and distilled water in required proportion and incubated at 25±1ºC for 3 days. Observations on mycelial growth was recorded after 3rd day of incubation.
3.2.3 Effect of media on mycelial growth:
Growth on solid media was determined by measuring the colony diameter along with the two diagonals passing through the center of colony by excluding initial diameter (5 mm) of bit. Five solid media whose composition is given below were taken for in vitro studies. Petriplates having sterilized medium were inoculated with 5 mm disc of mycelial growth with the help of sterilized cork borer and incubated at 25±1ºC in incubator for 3 days. Observations on mycelial growth (radial growth) was taken after 3rd day of incubation. Medium giving best radial growth of mycelium used for further studies.
Composition of different media used during the study.
S.No. Medium Constituents Quantity 1. PDA Agar-agar 20.00 g Dextrose 20.00 g Peeled-potato 250.00 g Distilled water 1000 ml
2. Czapeck’s Dox Agar medium Agar-agar 15.00 g Sucrose 30.00 g Distilled-water 1000 ml Dipotassium-phosphate 1.00 g Magnesium 0.50 g Potassium chloride 0.50 g Sodium nitrate 2.00 g Ferrus sulphate 0.01g 3. Oat meal medium Agar-agar 20.00 g Glucose 20.00 g Oat meal 20.00 g Distilled water 1000 ml
4. Corn meal medium
Agar-agar 20.00 g
Corn meal 20.00 g
Glucose 20.00 g
Distilled water 1000 ml Martin’s medium 5. Agar-agar 15.00 g Dextrose 10.00 g Peptone 5.00 g Distilled water 1000 ml Rose Bengal 1 part in 3000 part of media Potassium-dihydrogen 1.00 g phosphate Magnesium sulphate 0.50 g Streptomycin 30.00 mg
3.3 Induction of disease resistance in cucumber:
Seedlings of Karan and Cucumber-CS-404 varieties of cucumber were raised in protrays containing sterilized potting mixture in the cage house for 3 weeks and watered regularly. The seedlings were drenched overnight with salicylic acid in protrays at different concentrations (50,100,150 and 200 ppm) before transplanting. After overnight drenching, the plugs were shifted in to artificial inoculated microplots. The inoculum multiplied on sorghum grains was added @5 g/seedling in pits before transplanting.
3.4 Management of wet rot of cucumber through fungicides, bio agents and host resistance:
3.4.1 In vitro efficacy of fungicides:
Efficacy of five fungicides was tested against mycelial growth of Choanephora cucurbitarum by poisoned food technique. Required quantity of each fungicide was added aseptically to 100 ml of sterilized PDA medium in 150 ml flask separately so as to get concentration of 100, 300, 500 and 700 ppm. The flasks were shaken several times to ensure proper and uniform distribution of the fungicides. The medium was poured separately in sterilized Petriplates and allowed to solidify. Medium without fungicides served as check. Each plate was inoculated with 5 mm diameter mycelial bit of the fungus. Inoculated plates were incubated at 25±1ºC for 3 days. The linear growth of test fungus was recorded and per cent growth inhibition was calculated by Vincent’s (1947) formula.
C-T Per cent growth inhibition= ------x 100 C whereas,
C=Diameter of the colony in check (Average of both diagonals)
T=Diameter of colony in treatment (Average of both diagonals) The following fungicides were used during the experimentations.
S.No. Trade name of fungicide Common name
1. Indofil-M-45 Mancozeb
2. Antracol Propineb
3. Companion Carbendazim+ Mancozeb
4. Steam Captan + Hexaconazole
5. Moximate Cymoxanil + Mancozeb
3.4.2 In vivo efficacy of fungicides:
The experiment was carried out in earthen pots (9×12 inches) in cagehouse. Prior to sowing, pots were sterilized with copper sulphate solution and filled with sterilized soil + FYM (soil:FYM=3:1; sterilized at 1.045 kg/cm2 for one hour for three consecutive days). The inoculum multiplied on sorghum grains for one week used as soil and seed- cum- soil application. Pots were inoculated @ 20 g/pot.
Fungicides were applied through following three methods:
3.4.2.1 Seed application:
Seed inoculation was done by rolling seeds in Petriplates containing 7 days old inoculum. The pots were covered with polythene bags and kept for 24 hours in cage house. Apparently healthy surface sterilized seeds were used as check. The seeds were treated with fungicides @ 2 g/kg seeds. These treated seeds were separately sown in pots.
3.4.2.2 Soil application:
For soil application, the upper 5 cm layer of soil in each pot was thoroughly mixed with inoculum @ 8% (w/w). Fungicidal solution was drenched @ 2g /litre per pot. The pots were covered with polythene bags and kept for 24 hours in cage house. Apparently healthy surface sterilized seeds were used as check.
3.4.2.3 Seed-cum-soil application:
For this experiment, inoculation and fungicidal application were as above (3.4.2.1 & 3.4.2.2). Four replications were maintained for each treatment. The pots were watered as and when required. Observations were started after 15 days of sowing and continued up to 60 days to note the wet rot incidence. Per cent wet rot incidence was calculated by following formula.
No. of rotted plants % wet rot incidence = ------× 100 Total no. of plants 3.4.3 In vivo efficacy of bioagent:
3.4.3.1 Seed application :
Apparently healthy surface sterilized seeds of cucumber were coated with Trichoderma harzianum, T. viride @ 4 g/kg seed and Pseudomonas fluorescens @ 8 g/kg seed. Seed inoculation and observation were taken as described earlier (3.4.2.1). Fungus inoculated pots without treatment were served as check.
3.4.3.2 Soil application: Inoculum multiplied on sorghum grains was added @ 20 g/pot whereas bioagents, multiplied on FYM for 10 days were added @10 g /pot and rest were same as 3.4.2.1. Fungus inoculated pots without treatment served as check.
3.4.3.3 Seed-cum-soil application: For this experiment, inoculation, bioagent application and observations were same as described at 3.4.3.1 & 3.4.3.2. The pots were watered regularly. Observations were started after 15 days of sowing and continued up to 60 days to note wet rot incidence.
3.4.4 Host resistance:
Six cultivars/varieties of cucumber were screened against Choanephora cucurbitarum. Inoculum multiplied on sorghum grains was placed in pots at 5 cm depth @ 20 g/pot to increase the disease pressure. Observations were started after 15 days of sowing and continued up to 60 days to note wet rot incidence. The cultivars/varieties were categorized according to their reaction against the disease as per criterion of Siddiqui et al. (2008).
Category Per cent disease incidence (PDI)
Free (immune) 0
Resistant 1-5
Moderately resistant 6-15
Moderately susceptible 16-50
Susceptible 51-95
Highly susceptible > 95
4. Results
4.1 Collection, isolation, purification, pathogenicity and identification of the pathogen.
4.1.1 Collection, isolation and purification of the pathogen:
Diseased plants were collected from polyhouse of Department of Horticulture, SKN COA, Jobner during Zayad, 2014 and brought to the laboratory. The fungus was isolated on PDA from infected tissues of rotted root and stem of cucumber under aseptic conditions (Plate 4.1). The culture was purified by single spore and hyphal tip technique.
4.1.2 Pathogenicity:
The pathogenicity of Choanephora cucurbitarum was proved under pot conditions by seed, soil and seed-cum-soil inoculation techniques. Initial symptoms as rotting of seedlings were developed in 7 days while, typical disease symptoms appeared on all the inoculated plants after 15 to 20 days as watery appearance on rotted stem & roots, white aerial mycelial growth and shredding of affected parts. Later on symptoms were also appeared on flower buds, petioles & fruits. The flower buds & petioles were blighted and fruits were rotted. The re-isolation from artificially infected plants again yielded a Choanephora sp. thus fulfilling Koch’s postulates. Maximum disease incidence was observed found in seed-cum-soil inoculation technique (72.00 %), followed by soil inoculation (68.00 %) and minimum incidence was found in seed inoculation (64.00 %) technique (Table 4.1, Plate: 4.2).
Table : 4.1 Pathogenicity of Choanephora wet rot pathogen of cucumber Inoculation Germination (%)* Disease incidence (%)* technique Soil inoculation 80.00 68.00
(63.43) (55.55) Seed inocualtion 75.00 64.00
(60.00) (53.13) Seed cum soil inocualtion 70.00 72.00
(56.79) (58.05) Control 90.00 0.00
(71.57) (0.00) SEm+ 2.04 1.90 CD (p = 0.05) 6.30 5.85 *Average of four replications Figures given in parenthesis are angular transformed values
4.1.3 Identification of the pathogen:
The isolated fungus was identified on the basis of morphological characters (Plate 4.1). The fungus produced white aerial mycelial growth that turned pale yellow. The mycelia were hyaline & non-Septate. Sporangiophores bearing sporangiola were erect, hyaline and unbranched. Sporangia were multispored, spherical, initially white to yellow and pale brown to dark brown at maturity. On the basis of the cultural as well as morphological characteristics, the fungus was identified as a Choanephora Sp. The culture was sent to ITCC, Division of Plant Pathology, IARI, New Delhi for further confirmation or identification of fungus. The fungus was identified as Choanephora cucurbitarum with ID No. 9574.14.
4.2 Physio-pathological studies:
4.2.1 Effect of temperature on mycelial growth:
The entire microorganisms grow under certain range of temperature within which a minimum, optimum and maximum temperature could be located.
It is evident from the data (Table: 4.2, Plate: 4.4, Figure: 4.1) that the fungus grew at all the temperature range of 20 0C to 40 0C. Maximum mycelial growth of the fungus was observed at 25 0C (90 mm) and 30 ºC (90 mm) at 3rd day of incubation and found at par with 35 ºC (89.25 mm). A gradual decrease in mycelial growth was observed at 20 0C (75.25 mm). Minimum mycelial growth (35.56 mm) of the fungus was observed at 40 0C.
Table :4.2 Effect of temperature on mycelial growth of Choanephora cucurbitarum S.No. Temperature (OC) Mycelial growth (mm)* 1 20 75.25
2 25 90.00
3 30 90.00
4 35 89.25
5 40 35.56
SEm+ 1.26
CD (p = 0.05) 3.89
* Average of four replications
4.2.2 Effect of relative humidity on mycelial growth:
To evaluate the effect of atmospheric moisture, the fungus was exposed directly to different levels of relative humidity viz. 60, 70, 80, 90, 100 per cent and incubated at 25±10C for 3 days. It was observed that all the five humidity levels include the growth of Choanephora cucurbitarum.
Perusal of data (Table: 4.3, Plate: 4.5, Figure: 4.2) showed that maximum mycelial growth (90 mm) of Choanephora cucurbitarum was observed at 100 and 90 per cent relative humidity and found at par with 80 per cent (89.00 mm) relative humidity. A significantly decrease in mycelial growth was observed at 70 per cent (81.25 mm) relative humidity. Minimum mycelial growth (76.00 mm) was observed at 60 per cent relative humidity.
Table: 4.3 Effect of relative humidity on mycelial growth of Choanephora cucurbitarum at 25 + 10C S.No. Relative humidity Mycelial growth (mm)* (%) 1 60 76.00 2 70 81.25 3 80 89.00 4 90 90.00 5 100 90.00 SEm+ 1.31 CD (p = 0.05) 4.03 * Average of four replications
4.2.3 Effect of media on mycelial growth:
To find out a suitable medium for mycelial growth of Choanephora cucurbitarum, five different media were tested. Perusal of data (Table: 4.4, Plate: 4.6, Figure: 4.3) revealed that potato dextrose agar medium was significantly superior in supporting maximum mycelial growth (90.00 mm). This was followed by Martin’s medium (36.66 mm), corn meal agar (9.5 mm) and Czapeck’s Dox agar (8.45 mm). Mycelial growth of the fungus was not recorded on oat meal agar.
Table : 4.4 Effect of different media on mycelial growth of Choanephora cucurbitarum at 25 + 10C S.No. Medium Mycelial growth (mm)* 1 PDA 90.00 2 Corn meal agar 9.50 3 Oat meal agar 0.00 4 Czapeck’s dox agar 8.45 5 Martin’s medium 36.66 SEm+ 0.54 CD (p = 0.05) 1.68 * Average of four replications
4.3 Induction of disease resistance in cucumber :
Salicylic acid (50, 100, 150 & 200 ppm) was tested as inducer of systemic resistance against wet rot in cucumber varieties. Minimum disease incidence ((Table: 4.5, Figure: 4.4) was found at 200 ppm of salicylic acid. Karan and Cucumber-CS-404 varieties of cucumber showed minimum disease incidence (8.90 % and 15.75 %, respectively) at 200 ppm concentration as compared to control (60.00-66.66%). Other concentrations were also found significantly superior over control.
Table : 4.5 Efficacy of salicylic acid on Choanephora wet rot of cucumber S.No. Concentration Disease Incidence (%)*
Karan Cucumber CS-404 1 50 ppm 50.00 58.36 (45.00) (49.81) 2 100 ppm 33.33 33.33
(35.26) (35.26) 3 150 ppm 20.75 25.00
(27.10) (30.00) 4 200 ppm 8.90 15.75
(17.36) (23.38) 5 Control 60.00 66.66 (50.77) (54.73) SEm+ CD (P = 0.05)
Variety (V) 0.55 1.53 Concentration (C) 0.45 1.25 V x C 1.10 3.06 * Average of four replications Figures given in parenthesis are angular transformed values
4.4 Management strategies:
4.4.1 In vitro efficacy of fungicides against Choanephora cucurbitarum:
In vitro efficacy of five fungicides viz., Companion, Antracol, Indofil-M- 45, Moximate and Steam were tested at 100, 300, 500 and 700 ppm against Choanephora cucurbitarum. The data (Table: 4.6, Plate: 4.7, Figure: 4.5) revealed that all the fungicides tested, significantly inhibited the mycelial growth of Choanephora cucurbitarum as compared to check. Antracol and Companion were found significantly superior in inhibition of mycelial growth at 700 ppm (100 %) followed by Moximate (93.88 %). As the concentration of fungicides increased, the inhibition of mycelial growth was also increased and maximum inhibition was observed at 700 ppm concentration. Fungicides and concentration interaction was also found significant. Steam was recorded as least effective in inhibition of mycelial growth of the fungus.
4.4.2 In vivo efficacy of fungicides:
Three methods of fungicidal application were screened against Choanephora wet rot of cucumber. Results (Table: 4.7, Figure: 4.6) showed that seed-cum-soil application method of fungicide application was found most effective in reducing disease incidence over seed and soil application methods. Seed-cum-soil application methods of fungicide application was observed most effective in disease reduction. Antracol was found most effective in reducing disease incidence (75.00 %) followed by Moximate (58.35 %), Companion (50.00 %) and Indofil M-45 (44.00). Steam was observed at lowest (25.00 %) limit.
Table : 4.6 Efficacy of fungicides on mycelial growth of Choanephora cucurbitarum at 25 + 10C (in vitro) S.No. Fungicides Inhibition of mycelial growth (%)* 100 ppm 300 ppm 500 ppm 700 ppm Mean 1 Antracol 49.86 59.00 72.78 100.00 70.41 (44.92) (50.19) (58.56) (90.00) (57.05) 2 Companion 23.47 31.52 72.07 100.00 56.77 (28.97) (34.15) (58.11) (90.00) (48.89) 3 Indofil-M-45 21.67 39.72 68.75 77.50 51.91 (27.74) (39.07) (56.02) (61.69) (46.09) 4 Moximate 25.13 42.36 71.53 93.88 58.23 (30.08) (40.61) (57.76) (75.88) (49.73) 5 Steam 20.69 34.72 64.31 75.00 48.68 (27.05) (36.10) (53.32) (60.02) (44.24) 6 Control - - - - - SEm+ CD (p= 0.05) Fungicide (F) 0.38 1.07 Concentration (C) 0.31 0.87 F x C 0.77 2.14 * Average of four replications Figures given in parenthesis are angular transformed values
Table : 4.7 Efficacy of different fungicides against Choanephora wet rot of cucumber (in vivo) Fungicides Soil application Seed application (Coating) Seed cum soil (Drenching) application (Coating and drenching) Dose PDI* % disease Dose PDI* % disease PDI* % disease (g/l) reduction (g/kg) reduction reduction over control over control over control Antracol 2.0 25.00 65.51 2.0 31.25 54.54 18.75 75.00
(29.85) (33.84) (25.65)
Companion 2.0 50.00 31.03 2.0 56.25 18.18 37.50 50.00
(45.00) (48.61) (37.74)
Indofil-M-45 2.0 56.25 22.41 2.0 62.50 9.09 50.00 44.00
(48.62) (52.32) (45.00)
Moximate 2.0 37.50 48.27 2.0 50.00 27.27 31.25 58.35
(37.73) (45.00) (33.84)
Steam 2.0 62.50 13.79 2.0 65.75 4.36 56.25 25.00
(52.33) (54.32) (48.63)
Control - 72.50 - - 68.75 - 75.00
(58.51) (56.11) (60.07)
SEm+ 1.47 1.61 1.01
CD (p = 0.05) 4.52 4.97 3.11 * Average of four replications, PDI = Per cent disease incidence Seed applicaton of Antracol also gave higher disease reduction (54.54 %) followed by Moximate (27.27 %), Companion (18.18 %) and Indofil-M-45 (9.09 %) whereas steam was found least effective (4.36 %). Results of soil application were found intermediate between seed and seed- cum-soil application methods in reducing disease incidence.
4.4.3 Efficacy of bio-agents against Choanephora cucurbitarum:
Efficacy of four bioagent were tested against wet rot of cucumber by applying through seed, soil and seed-cum-soil application methods. Seed- cum-soil application of bioagents was found highly effective in reducing disease incidence over control (Table: 4.8, Figure: 4.7). Trichoderma harzianum + Pseudomonas fluorescens was found significantly superior in reducing disease incidence (66.66 %) over control followed by Trichoderma harzianum (48.63%) and Trichoderma viride (31.50%) whereas, Pseudomonas fluorescens (16.66 %) was found least effective against Choanephora cucurbitarum.
4.4.4 Host resistance:
Six varieties/cultivars of cucumber were screened (Table: 4.9, Figure: 4.8) against Choanephora cucurbitarum under artificial inoculation conditions. None of the entry was found immune or resistance to wet rot. However, amongst these cultivars, Almgir-CT-380 and Rohini were categorized as moderately susceptible and rest were come under the category of susceptible or highly susceptible.
Table : 4.8 Efficacy of bioagents against Choanephora wet rot of cucumber Bio-agents Soil application Seed application Seed-cum soil application PDI* % disease PDI % disease PDI* % disease reduction over reduction over reduction control control over control Trichoderma harzianum 50.00 31.03 56.25 18.18 37.50 48.63 (44.99) (48.61) (37.73) Trichoderma viride 62.50 13.79 63.75 7.27 50.00 31.50 (52.28) (53.03) (45.00) Pseudomonas fluorescens 65.75 9.31 64.00 6.90 62.50 16.66 (54.25) (53.17) (52.28) Pseudomonas fluorescens + 37.50 48.27 50.00 27.27 25.00 66.66 Trichoderma harzianum (37.74) (45.00) (30.00) Control 72.50 - 68.75 - 75.00 (58.41) (56.01) (60.03) SEm+ 1.01 1.32 1.00 CD (p = 0.05) 3.10 4.08 3.09 * Average of four replications, PDI = Per cent disease incidence Figures given in parenthesis are angular transformed values
Table : 4.9 Screening of cucumber varieties against Choanephora cucurbitarum S.No. Variety Disease incidence Host Reaction (%)* 1 Alamgir-CT-380 33.33 Moderately susceptible (35.22) 2 Cucumber CS-404 66.66 Susceptible (54.81) 3 Fumiko-10 68.00 Susceptible (55.64) 4 Karan 60.00 Susceptible (50.80) 5 Rohini 20.00 Moderately susceptible (26.54) 6 Local 53.33 Susceptible (46.92) SEm+ 0.86 CD (p = 0.05) 2.66
PDI-0=Immune, PDI-1-5%=Resistant, PDI-6-15=Moderately resistant, PDI- 16-50= Moderately susceptible, PDI-51-95=Susceptible, PDI > 95 Plant dead *Average of four replications, Figures given in parenthesis are angular transformed values
5. Discussion
Cucumber (Cucumis sativus L.) is known as “Kheera” in Hindi. It is a popular and widely cultivated summer vegetable in India. It belongs to family
Cucurbitaceae. It is one of the important vegetable crops which supply edible product and fibre. Cucumber is used as salad or as vegetable and as desert fruit specially in Rajasthan. In India, cucumber is widely grown in Andhra
Pradesh, Karnataka, Assam and Rajasthan, commonly towards the riversides (Anonymous, 2014). Rajasthan state provides the maximum potential for the production of cucumber because of its agro-climatic conditions are best suited for their growth and yield.
The crop suffers from number of diseases caused by phytopathogenic fungi and other pathogenic organisms. Amongst all diseases of the cucumber, wet rot of cucumber incited by Choanephora cucurbitarum, is one of the important disease and a big constraint in successful cultivation of this crop.
In the present study, the isolation of the pathogen was made from rotted roots and stems of cucumber. The culture was purified by single spore and hyphal tip technique. On the basis of cultural and morphological characters, the fungus was tentatively identified as Choanephora sp. The identity of fungus was further got confirmed from ITCC, IARI, New Delhi (ID
No. 9574.14) as Choanephora cucurbitarum.
The pathogen was found pathogenic when inoculated artificially to cucumber plant under pot house conditions. The characteristic symptoms of wet rot appeared after 15-20 days of inoculation. Infection in the beginning was typically in the form of water soaked lesions on stem. Roots of the diseased plants showed brownish water soaked lesions, watery appearance
& shredding of rotted parts. Flower buds & petioles were blighted & fruits were rotted. Choanephora cucurbitarum is a plant pathogenic fungus causing fruit rots, wet rot, flower rot and leaf blight on a variety of plants including squash, pumpkin, pepper, pea and bean (Kacharek et al., 2003).
In present investigation, seed, soil and seed-cum-soil inoculation techniques were used to prove the pathogenicity. Highest disease incidence was found in seed-cum-soil inoculation technique. Symptoms appeared as white mycelial growth on rotted parts, watery appearance, shredding of affected parts etc. However, to our knowledge incidence of this pathogen on cucumber has not been reported so far. Thus, Choanephora wet rot of cucumber caused by C. cucurbitarum is a new report from India. Similarly, such symptoms were observed on eggplant and Withania somnifera (Kwon and Hyeong, 2005 and Saroj et al., 2012).
Nutrition plays an important role in growth and sporulation of the fungus. In order to determine basal medium for growth and sporulation of
Choanephora cucurbitarum, five different solid media were tested. The potato dextrose agar medium was supported maximum mycelial growth
(90.00 mm) and it was followed by Martin’s medium (36.66 mm). Similarly,
Kuo et al. (1999) and Mishra and Mishra (2012) found good growth on
PDA. Temperature is one of the important factor for the growth and sporulation of an organism which also influences the occurrence and development of disease and most of the organisms grow between 0 to 42 ºC
(Wolf and Wolf, 1947). Results of temperature studies showed the maximum growth (90.00 mm) at 25 ºC and 30 ºC, and least mycelial growth (35.56 mm) was found at 40 ºC temperature. Optimum temperature 25 ºC and 30
ºC for mycelial growth of Choanephora cucurbitarum was also observed by
Kuo et al., (1999) and Abdel-Motaal et al., (2010).
In the present investigation it was observed that pathogen
Choanephora cucurbitarum grew and sporulated efficiently at 80 to 100 per cent relative humidity, whereas, decline was observed at lower humidity levels. Maximum growth and sporulation of Choanephora cucurbitarum was also observed best at 90 to 100 per cent relative humidity by earlier workers
(Kwon and Hyeong, 2005, and Hussein and Ziedan, 2013).
Disease resistance in cucumber varieties was induced by salicylic acid at varying concentrations. Minimum disease incidence was found at 200 ppm concentration of salicylic acid in Karan (8.90%) and cucumber-CS-404
(15.75%) varieties. Similarly, Waheed et al. (2014) proved that 200 ppm concentration of salicylic acid was found effective against Fusarium wilt in tomato.
In in vitro test, all the fungicides namely Antracol, Steam, Indofil-M-
45, Moximate and Companion were tested at 100, 300, 500 and 700 ppm concentration and inhibited the mycelial growth of Choanephora cucurbitarum. Antracol and Companion gave maximum inhibition of mycelial growth at 500 and 700 ppm. Moximate effectively inhibited growth of fungus at 500 and 700 ppm concentrations. Steam was found least effective in mycelial growth inhibition. Although there was increase in growth inhibition with the increase in concentration. Because of these, chemicals either inhibit the germination, growth and multiplication of the pathogen or are directly toxic. Our observations are in conformity to George and Girija (2015) as they evaluated the effectiveness of carbendazim and mancozeb (Companion) against Choanephora cucurbitarum.
Results of field experiment indicated that all the fungicides tested against Choanephora wet rot of cucumber were found effective in reducing disease incidence over untreated check. Antracol showed maximum
(75.00%) disease control followed by Moximate (58.33%), when used as seed dresser (@ 2 g/kg seed) and soil drenching (@ 2 g/lit) than other fungicides used. Hammouda (1988) reported that mancozeb, dinocap and thiabendazole were found effective in reducing infection against
Choanephora cucurbitarum in the Southern region of Oman (Dhofar). Chahal and Grover (1974) also reported that zineb, mancozeb, ziram and thiram were the most effective in controlling Choanephora cucurbitarum on chilli.
To circumvent pollution hazard due to injudicious use of agrochemical and also to avoid development of resistance in pathogenic fungi to commonly used fungicides, use of bio control agents for the management of plant diseases has increased in recent years. Bio-control agents are proved to be very effective especially against soil borne pathogens. In present investigation, Trichoderma harzianum, T. viride and Pseudomonas fluorescens were tested under field conditions. T. harzianum + Pseudomonas fluorescens was found more effective with 66.66 per cent disease control followed by T. harzianum (48.63%). P. fluorescens and T. viride were found least effective. Our observations are in conformity to
Siddiqui et al. (2008) as they reported that disease severity was lowest in plants treated with Trichoderma-fortified RST extracts (9.56%) with a disease index of 1. There was a reduction of 85.04 per cent in Choanephora wet rot severity on okra treated with Trichoderma-fortified RST extracts during 12 weeks of assessment in the field.
Six Varieties/cultivars of cucumber were screened against
Choanephora cucurbitarum under artificial inoculation conditions. None of the entry was found immune or resistance to wet rot. Although, among these cultivars, Almgir-CT-380 and Rohini were categorized as moderately susceptible and rest were come under the category of susceptible or highly susceptible. Ajibade and Amusa (2001) investigated 71 lines of cowpea in humid zone of South Western Nigeria and found that 85 per cent lines had
Choanephora pod rot.
6. Summary
Wet rot, an important disease of cucumber caused by Choanephora cucurbitarum was observed in polyhouse of Department of Horticulture,
SKN, COA, Jobner (Jaipur) during Zayad, 2014.
The isolation of the pathogen was made from rotted roots and stems of cucumber. The culture was purified by single spore and hyphal tip techniques and identified as Choanephora cucurbitarum. However, to our knowledge incidence of this pathogen on cucumber has not been reported so far. Thus, Choanephora wet rot of cucumber caused by C. cucurbitarum is a new report from India.
The pathogen was found pathogenic when inoculated artificially to cucumber plants. The characteristic symptoms of wet rot appeared after 15-
20 days of inoculation. Infection in the beginning was typically in the form of water soaked lesions on stem. Roots of the diseased plants showed brownish water soaked lesions.
Seed, soil and seed-cum-soil inoculation techniques were used to prove the pathogenicity. Highest disease incidence was found in seed-cum-soil inoculation technique.
Maximum mycelial growth of fungus was recorded at 25 0C temperature, 100 per cent relative humidity and on PDA.
Among the concentrations of salicylic acid, 200 ppm concentration was found effective to induce disease resistance in cucumber varieties. Studies on the relative efficacy of fungicides in in vitro and in vivo,
Antracol was found most effective followed by Moximate in both the conditions whereas steam was found least effective.
Studies on bio control agents in pot house conditions, Trichoderma harzianum + Pseudomonas fluorescens was found most effective followed by Trichoderma harzianum alone. Pseudomonas fluorescens and
Trichoderma viride were found least effective.
Six cultivars of cucumber were screened against Choanephora cucurbitarum under artificial conditions. None of the entry was found immune or resistant to Choanephora wet rot.
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Choanephora Wet Rot of Cucumber and its Management
Sangeeta Choudhary* Dr. R.P. Ghasolia** (Research Scholar) (Major Advisor) Abstract Wet rot, an important disease of cucumber caused by Choanephora cucurbitarum was observed in polyhouse of Department of Horticulture, SKN, COA, Jobner (Jaipur) during 2014. The isolation of the pathogen was made from rotted pieces of cucumber, pathogenicity proved and pathogen identified as Choanephora cucurbitarum. However, to our knowledge incidence of this pathogen on cucumber has not been reported so far. Thus, Choanephora wet rot of cucumber caused by C. cucurbitarum is a new report from India.
Maximum mycelial growth was observed at 25 ºC temperature,100 per cent relative humidity and on potato dextrose agar medium.
Drenching of salicylic acid @ 200 ppm was found effective in inducing disease resistance in cucumber varieties.
In vitro, Antracol and Companion gave maximum inhibition of mycelial growth at 700 concentration. When tested as seed dressing and drenching under pot conditions, Antracol was also observed to be most effective in reducing the disease incidence.
Studies on bio-control agents Trichoderma harzianum + Pseudomonas fluorescens was found most effective followed by Trichoderma harzianum alone.
Six cultivars of cucumber were screened against Choanephora cucurbitarum under artificial conditions. None of the entry was found immune or resistant to Choanephora wet rot
* A post graduate student, Department of Plant Pathology, S.K.N. College of Agriculture, Jobner, (Jaipur). ** Thesis submitted in partial fulfillment of the requirement for the degree of M. Sc. (Ag.) in Plant Pathology under the supervision of Dr. R. P. Ghasolia, Asstt. Professor, Deptt. of Plant Pathology, S.K.N. College of Agriculture, Jobner.
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