Studies on collar rot of chickpea caused by Sclerotium rolfsii Sacc.

Transcription

1 Studies on collar rot of chickpea caused by Sclerotium rolfsii Sacc. THESIS Submitted to the Jawaharlal Nehru Krishi Vishwa Vidyalaya, Jabalpur In partial fulfillment of the requirements For the Degree of MASTER OF SCIENCE In AGRICULTURE (Plant Pathology) Shantanu T. Jayale Department of Plant Pathology College of Agriculture, Jabalpur Jawaharlal Nehru Krishi Vishwa Vidyalaya, Jabalpur, MP 2014

2 CERTIFICATE - I This is to certify that the thesis entitled Studies on collar rot of chickpea caused by Sclerotium rolfsii Sacc. submitted in partial fulfillment of the requirement for the degree of MASTER OF SCIENCE in Agriculture Plant Pathology of Jawaharlal Nehru Krishi Vishwa Vidyalaya, Jabalpur is a record of the bonafide research work carried out by Shantanu Tryambakrao Jayale under my guidance and supervision. The subject of the thesis has been approved by the Student s Advisory Committee and the Director of Instruction. No part of the thesis has been submitted for any other degree or diploma (Certificate awarded etc.) or has been published/published part has been fully acknowledged. All the assistance and help received during the course of the investigations has been acknowledged by him. (Dr. U. K. Khare) Chairman of the Advisory Committee THESIS APPROVED BY THE STUDENT S ADVISORY COMMITTEE Chairman Dr. U. K. Khare.. Member Dr. J. Bhatt.. Member Dr. S. K. Diwedi.. Member Dr. R. B. Singh..

3 CERTIFICATE II This is to certify that the thesis entitled Studies on collar rot of chickpea caused by Sclerotium rolfsii Sacc. submitted by Mr. Shantanu Tryambakrao Jayale to the Jawaharlal Nehru Krishi Vishwa Vidyalaya, Jabalpur in partial fulfilment of the requirements for the degree of Master of Science in Agriculture in the Department of Plant Pathology has been, after evaluation, approved by the External Examiner and by the Student s Advisory Committee after an oral examination on the same. Place: Jabalpur Date:.. (Dr. U. K. Khare) Chairman Advisory Committee MEMBERS OF THE ADVISORY COMMITTEE Chairman Dr. U. K. Khare.. Member Dr. J. Bhatt.. Member Dr. S. K. Diwedi.. Member Dr. R. B. Singh.. Head of the Department Dr. Smt. Om Gupta Director of Instruction Dr. S. K. Shrivastava

4 Copyright Jawaharlal Nehru Krishi Vishwa Vidyalaya Jabalpur Madhya Pradesh 2014 Copyright transfer Certificate Title of the Thesis: Studies on collar rot of chickpea caused by Sclerotium rolfsii Sacc. Name of the candidate: Shantanu T. Jayale Subject: Plant Pathology Department: Plant Pathology College: College of Agriculture, Jabalpur Year of thesis submission: 2014 Copyright transfer The undersigned Shantanu T. Jayale assigns to the Jawaharlal Nehru Krishi Vishwa Vidyalaya, Jabalpur, Madhya Pradesh, all rights under Copyright Act, that may exists in and for the thesis entitled Studies on collar rot of chickpea caused by Sclerotium rolfsii Sacc. submitted for the award of M.Sc.(Ag.) degree. Date: Place: Jabalpur Major Advisor (Dr. U. K. Khare) (Shantanu T. Jayale)

5 DECLARATION AND UNDERTAKING BY THE CANDIDATE I Shantanu T. Jayale S/o Mr. Tryambakrao R. Jayale Certify the work embodied in thesis Studies on collar rot of chickpea caused by Sclerotium rolfsii Sacc. is my own first hand bonafide work carried out by me under the guidance of Dr. U.K. Khare at Department of Plant Pathology College of Agriculture Jabalpur during The matter embodied in the thesis has not been submitted for the award of any other degree/diploma. Due credit has been made to all the assistance and help. I, undertake the complete responsibility that any act of misinterpretation, mistakes, errors of fact are entirely of my own. I, also abide myself with the decision taken by my advisor for the publication of material extracted from the thesis work and subsequent improvement, on mutually beneficial basis, provided the due credit is given, thereof. Place: Jabalpur Shantanu T. Jayale Date:

6 ABSTRACT Title of the thesis : Studies on collar rot of chickpea caused by Sclerotium rolfsii Sacc. Student s Name Permanent address : Shantanu T. Jayale : C/o. T. R. Jayale, Near Poorva Appartment,Akola Road, Akot, Dist. Akola. (M.H.) Name and address of Major advisor: : Dr. U.K. Khare, Professor, Department of Plant Pathology, JNKVV, Jabalpur (M.P.) Degree to be awarded : Master of Science in Agriculture Year of award of degree : 2014 Major subject : Plant Pathology Total no. of pages in the thesis : 51 Number of words in the abstract : 297 (Dr. U.K. Khare) (Dr. Smt. Om.Gupta) (Shantanu T. Jayale) Signature of Signature of Advisor Head of the Department Signature of Student

7 Acknowledgement The author of this manuscript praises the omniscient and Almighty God who has enabled me to achieve this seemingly invincible task. Words cannot express my profound sense of gratitude and thanks to most esteemed guide and chairman of Advisory Committee Dr. U. K. Khare [Professor], Department of Plant Pathology, JNKVV, Jabalpur. I was extraordinarily fortunate in having Dr. U. K. Khare as my guide. With his enthusiasm, his inspiration, and his great efforts to explain things clearly and simply, he helped to make thesis fun for me. Throughout my thesis writing period, he provided encouragement, sound advice, good teaching, good company, and lots of good ideas. He taught me how to express my ideas. He showed me different ways to approach a research problem and the need to be persistent to accomplish any goal. I would have been lost without him. It is an immense pleasure to express my heartfelt thanks and gratitude to the members of my advisory committee, Dr. Jayant Bhatt, Professor, Department of Plant Pathology, Dr. S. K. Diwedi, Professor, Department of Plant Physiology and Dr. R.B. Singh, Professor, Department of Mathematics and Statistics, J.N.K.V.V, Jabalpur for their valuable suggestions and timely help during the course of this investigation. I wish to express deep sense of gratitude to Dr. V. S. Tomar, Honorable Vice-Chancellor, JNKVV, Jabalpur, Dr. S.S. Tomar, Director Research Services, honorable Director of Instruction Dr. O. P. Veda and honorable, Dean, Dr. R.V. Singh, for providing necessary facilities for my thesis work. I wish to express my grateful and sincere thanks to Dr. (Smt.) Om Gupta, Professor and Head- Department of Plant Pathology, J.N.K.V.V., Jabalpur for her valuable suggestions with constant motivation, encouragement and timely help. I am especially and grateful thank her for valuable teaching during the period of my post graduation. I express my deep sense of gratitude to Dr. S.P. Tiwari, Dr.R.K. Varma, Dr. S. Nema, Dr. M.S Bhale, Dr. (smt.) Usha Bhale, Dr. J. Bhatt, Dr. A.R. Wasnikar and Dr. Sanjay Kumar, Department of Plant Pathology, College of

8 Agriculture, JNKVV, Jabalpur for their infinite favour with which they encouraged me during the period of research work. I am very thankful to Ku. Madhuri Mishra madam for providing necessary facilities and support during course of investigation, without whom my practical knowledge would have been incomplete. I express my sincere thanks to my seniors Ashish Kale, Anand Dapkekar, Kamlesh Pawar, Ramesh Amule, Tushar Nanekar, Jyoti Tiwari and Amrita Tiwari. I am indebted to my colleagues for providing a stimulating and fun environment to learn and grow. My appreciation and thanks to my colleagues and friends Ku. Pushpanjali Wagh, Ku. Rashmi Singh, Kamlesh Patel, Milind Deshbhratar, Arvind Jaware and Saurabh Bhalekar and my affectionate Juniors Akhilesh, Maruti, Om Prakash, Vineet, Kratika, Sumita, Monika, Gaurav, Ashwin, Vaibhav, Kunal, Krishna and Ravishankar for their enduring help and encouragement. I have no words to express my sincere gratitude for Amol, Rujuta, Rakhi, Rushikesh, Ravikumar, Sachin who in their own elusive way were there for me in the hours of the need during the course for instilling in me a spirit of hope, struggle, determination and affection. Wish them all the best. Where would I be without my family? My father Shri. Tryambakrao R. Jayale and mother Sau. Kastura Tryambak Jayale, deserve special mention for their inseparable support and prayers. I must mention that the unbounding affection, encouragement, blessing and untiring efforts of my family members brought me to success. I hope that my research should always be useful and serve good purposes for all humankind. Finally, I would like to thank everybody who was important to the successful realization of thesis, as well as expressing my apology that I could not mention personally one by one. Place: Jabalpur Date: (Shantanu T. Jayale)

9 LIST OF CHAPTERS Chapter Title Page 1 Introduction Review of Literature Material Methods Results Discussion Summary, Conclusion and Suggestions for further work References Vita

10 LIST OF TABLES Table No. Title Page No. 1 Pathogenicity test of Sclerotium rolfsii against chickpea 2 Growth of Sclerotium rolfsii inhibited by different species and local isolates of Trichoderma in dual culture method under in-vitro conditions 3 Effect of seed treatment by different species and local isolates of Trichoderma on germination, pre and post emergence mortality of chickpea caused by Sclerotium rolfsii 4 Effect of different temperature on radial growth of Sclerotium rolfsii 5 Effect of different ph on radial growth of Sclerotium rolfsii 6 Inhibitory effect of medicinal plant leaf extracts on mycelial growth and sclerotial production of Sclerotium rolfsii 7 Effect of fungicides against Sclerotium rolfsii in invitro condition 8 Effect of fungicidal treatment on germination, pre and post emergence mortality of chickpea seeds caused by collar rot pathogen in pot condition

11 LIST OF FIGURES Figure No. Title Page No. 1 Growth of Sclerotium rolfsii by different spp. and local isolates of Trichoderma in dual culture method under in vitro conditons. 2 Effect of seed treatment by different species and local isolates of Trichoderma on germination, pre and post emergence mortality of chickpea caused by Sclerotium rolfsii. 3 Effect of different temperatures on radial growth of Sclerotium rolfsii. 4 Effect of different ph levels on the growth of Sclerotium rolfsii 5 Inhibitory effect of medicinal plant leaf extracts on mycelial growth and slcerotial production of Sclerotium rolfsii. 6 Effect of fungicidal seed treatment on germination pre and post mergence mortality of chickpea seeds caused by collar rot pathogen in pot condition

12 LIST OF PLATES S. No. Plates Page in between 1 Symptoms on chickpea seedling and vegetative growth of Sclerotium rolfsii 2 Pathogenecity assay on chickpea seedling and colonization of Sclerotium rolfsii 3 Inhibitition of vegetative growth of Sclerotium rolfsii by Trichoderma spp. 4 Suppressive activity in soil for Sclerotium rolfsii as indicated by reduced pre and post emergence mortality. 5 Effect of different temperatures on radial growth of Sclerotium rolfsii 6 Effect of different ph and different medicinal plant leaf extract on radial growth of Sclerotium rolfsii 7 Efficacy of different fungicides on radial growth of Sclerotium rolfsii 8 Efficacy of different fungicides on seed germination, pre and post emergence mortality

13 LIST OF SYMBOLS Symbol Abbreviation Stand At the rate of % Per cent ± Plus or minus % Percentage 0 C Degree centigrade avg average WP Wettable powder WG Wettable granules sp species Conc concentration S. rolfsii Sclerotium i.e. that is viz., namely et al. co- workers fig figure CD Critical difference cm Centimeter ha Hectare Hrs Hours mt Million tonnes ml Mililiter mm Milimeter g Gram mg Milligram NS Non significant SEm Standard error of mean ppm Parts per million Temp Temperature PDI percent disease incidence PDA Potato dextrose agar

14 INTRODUCTION Chickpea (Cicer arietinum L.) is an important pulse crop and belongs to family Leguminose, ranking third after dry beans (Phaseolus vulgaris L.) and dry peas (Pisum sativum L.) (Dhar and Gurha, 1998). Chickpea (Cicer arietinum L.) is the third most important food legume in the world grown in 8.57m ha with 7.35 million ton production.it is grown in over 45 countries in all continents of the world. It provides a high quality protein to the people in developing countries. Pulses play an important role not only from economical point of view but also in their nutritional value. Chickpea is valued for their nutritive seeds with high protein content % after dehulling (Hulse, 1991) carbohydrate 61.5%, fat 4.5% and vitamin 2.44%. Chickpea is grown in tropical, subtropical and temperate regions. The major chickpea growing countries are India, Pakistan and Turkey in Asia, Ethiopia in Africa, California and Washington state in the U.S.A., Mexico and Australia (F.A.O. 1994).The total cultivated area of chickpea in the world is about 8.57 m.hac. and annual world production is 7.35m. tonnes. The average productivity is estimated to be around 858 kg/ha (AFO 2001). In Madhya Pradesh, it ranks first in both, area (2.9 m.hac.) and production (2.6 m.tonnes) and the average productivity is estimated to be 931 kg / hac. (Anon., ) Chickpea is attacked by air borne, seed borne and soil borne diseases (Chattopadhyay et. al., 2001). More than 75 pathogens have been reported to infect chickpea (Nene et. al., 1989). In 1892, Peter Henry Rolfs first published a description of a new disease on tomato, from Florida and recorded losses up to 70 per cent, later Saccardo named the fungus Sclerotium rolfsii in 1911, which continues to be a problem in a variety of crops when conditions are warm, humid and rainy. Presently this disease occurs at all over the world in equatorial zone between 45 0 N and S latitude. 1

15 Later in 2000, Ansari et. al. (2000) showed that S. rolfsii is one of the serious polyphagus pathogen causing diseases in as many as 500 plant species. Maurya et. al.(2008) reported that collar rot (S. rolfsii) of chickpea (Cicer arietinum) is one of the devastating soil borne diseases of fungal origin, due to which per cent yield losses are recorded annually according to severity of disease. Looking to the importance of the crop and the collar rot disease that is prevalent in the chickpea growing areas of state it was thought desirable to manage the disease biologically with following objectives: Confirmation of pathogenecity of S. rolfsii on chickpea. To study the effect of various parameters on disease development. Management of collar rot disease. 2

16 REVIEW OF LITERATURE The available literature pertaining to research investigation is reviewed critically under the following major heads: 2.1 History and taxonomy of the fungus Rolfs (1892) for the first time recorded a tomato blight disease caused by Sclerotium rolfsii Sacc., in Florida and recognised the small, round sclerotia as the outstanding morphological characteristics of the organism. The name Slerotium rolfsii was given by Saccardo (1911), who recognised the fungus as an imperfect form. Curzi in 1932 proposed the generic name Corticium for the fungus, based on the studies of perfect stage in pure culture. West (1947) suggested that the generic name may be changed to Pellicularia, since it was characterised by aerolate hymenia, short celled, stout hyphae and right angled branching of mycelia. However, according to Taebot (1973), the basidial stage of Sclerotium rolfsii is a species of Athelia in corticiaceae. Aycock (1966) reported that hyphae have clamps in the form of forks and hooks or H-like connections. 2.2 Isolation and purification Sulladmath et al. (1975) reported that oat (Avena sativa) grown in experimental plots, were severely affected by S. rolfsii at the flag leaf stage. The affected plants indicated root-rot symptoms mark by dark brown discolouration at the collar region, covered by a white cottony mycelial growth. On uprooting of such plants, the stem breaks easily at ground level. In advanced conditions, numerous sclerotia were seen on the collar region. Isolation from the infected portions yielded Sclerotium rolfsii Sacc. Siddaramaiah et al. (1978) reported that about one per cent of the niger plants wilted in 1978 at Botanical Garden of College of Agriculture, Dharwad. Minute examination of the wilted plants showed that collar portion of the plants were sunk and covered with white mycelial mat and large number of sclerotial bodies around the collar portion. Heavily infected plant died within a week. Repeated isolation of the infected portion of the plant yielded the fungus Corticium rolfsii. 3

17 Chattopadhyay et al. (2001) found soil borne fungal pathogens, namely Sclerotium rolfsii, Macrophomina phaseolina and Fusarium oxysporum f.sp. ciceri causing collar rot, dry root rot and wilt disease, respectively, were associated with chickpea during a survey of plots in West Bengal during January-March 1991 and These organisms were isolated singly or in association. Wilt was the most destructive. Combined artificial inoculation with F. oxysporum f.sp. ciceri and M. phaseolina had a synergistic effect on wilt development as evidenced by the large number of plants wilted when compared to single inoculation with either fungus. Collar rot caused by S. rolfsii was also greatly enhanced in the early stages when the fungus was mixed inoculated with M. phaseolina. In the later stages, however, combined inoculation with S. rolfsii and M. phaseolina resulted in reduced disease severity compared to inoculation with S. rolfsii alone. Padole et al. (2009) studied incidence of collar rot in 15 to 45 days old crop which ranged from 5-30% in 51 locations surveyed nearby Jabalpur. Investigations on variations in 51 isolates of Sclerotium rolfsii showed considerable variations with regards to cultural and morphological characters on PDA and grouped into three pathotypes. The pathogenicity test showed the isolates to vary in number of days taken to initiate plant mortality and 100% mortality 2.3 Pathogenecity test Sulladmath et al. (1975) reported that for testing pathogenecity, Sclerotium rolfsii was grown on corn-meal-sand medium for a week and mixed into the top layer of sterilized soil filled into six inches diameter clay pots. Typical root rot symptoms were observed days after sowing. The organism was re-isolated from such infected plants. Siddaramaiah et al. (1978) reported that Niger (Guyzotia abyssinica Cass) is one of the important oilseed crop normally grown in all types of soil. They observed that about 1 per cent of niger plants were wilted. Collar rot infection was caused by Corticium rolfsii Curzi. The pathogenecity was proved by sowing 50 seeds, artificially inoculated with 20 days old Corticium sp. culture and the same quantity of the seeds were sown in sterilized soil as control. Out of 50 seeds, 40 seeds germinated and 10 seeds recognised ungerminate. The fungus started infection after the third day of seed germination and all the 40 seedlings were infected within a week, causing post emergence death. 4

18 Roy (1977) tested pathogenecity of Sclerotium rolfsii on pea (Pisum sativum L.), cauliflower (Brassica oleracea var capitata L.) and Arum (Colocassia sp.). Rotting in all the cases, was more than 50 per cent in four to five days except Colocassia sp. Haware and Nene (1978) obtained 100 per cent germination in 42 inoculated chickpea checks, 50 per cent of the seeds, in the inoculated treatment, failed to germinate. The causal fungus initiated the rolling of these seeds and the seed surfaces were covered with white mycelial mat in soil. Germinated seedlings were killed within 7 days after emergence. Symptoms were evident as yellowing of leaves followed by death of the seedlings. The affected roots showed abundance of superficial white fluffy mycelium. A semiwatery decay at collar region was evident. 2.4 Efficacy of various antagonists against S. rolfsii Agrawal et al. (1977) reported that Trichoderma harzianum was highly antagonistic to Sclerotium rolfsii causing collar rot of lentil. Filtrate of this organism also checked the growth of Sclerotium rolfsii on potato dextrose agar. Trichoderma harzianum could check the mortality of lentil caused by Sclerotium rolfsii under pot condition. The culture of Trichoderma sp. was more effective when used with the seeds as compared to that used in soil. Wells et al. (1972) showed that Trichoderma harzianum effectively controlled Sclerotium rolfsii on blue lupines, tomatoes and peanuts. Under natural field conditions, the application of T. harzianum inoculum over the plants onto the soil suface was highly effective in reducing Sclerotium rolfsii damage to tomato plants. Backman and Rodriguezkabana (1975) reported significant reduction in Sclerotium rolfsii damage and increase in yield over the three year test period by application of Trichoderma harzianum. Matti and Sen (1985) tested the improvement in the efficacy of controlling Sclerotium rolfsii Sacc. through the use of combination of nitrogenous fertilizers and an antagonistic isolate of Trichoderma harzianum Rifai. Bicici et al. (1994) reported that Trichoderma harzianum suppressed Corticium rolfsii upto 71.4 % if applied with 2 kg/m 2 perlite + maize meal culture to potted plants. Jagdeesh and Geeta (1994) reported that a wheat 5

19 bran and biogas manure mixture (1:1) stimulated growth and multiplication of biological control agent Trichoderma harzianum which suppressed Sclerotium rolfsii and increased seedling emergence of groundnut. Mukhopadhyay (1987) reported that Trichoderma sp. have long been known as effective antagonist against plant pathogenic fungi. Trichoderma sp. are known to inhibit the growth of Sclerotium rolfsii on lupine, tomato, peanut, bean, cotton, sugar beet and lentil, Rhizoctonia solani on bean, cotton, tomato, strawberry, peanut and chickpea, Pythium aphanidermatum on tobacco and sugar beet, Pythium sp. on pea, tomato and brinjal, Fusarium oxysporum f. sp. ciceri on chickpea. Anilkumar and Pandurange (1989) reported the possible use of Trichoderma harzianum Rifai, a mycoparasite for control of Sclerotium rolfsii affecting sunflower. Trichoderma harzianum effectively killed the colonized Sclerotium rolfsii at the both levels tried. Elad et al. (1982) reported that Trichoderma harzianum excreted B-1, 3- glucanase and chitinase into the medium, when grown on leminarin and chitin respectively, or on cell walls of the pathogen Sclerotium rolfsii, as sole carbon source. Trichoderma harzianum also showed high activity of both enzymes, produced when mycelium of Sclerotium rolfsii, Rhizoctonia solani and Pythium aphanidermatum in soil was applied. This phenomenon was correlated with the ability of each of the Trichoderma isolates to control the respective soil borne pathogens. Gaikwad and Kapgate (1990) reported that the spore suspention of T. harzianum and P. pinophilum prevented the germination of sclerotia of S. rolfsii under in-vitro conditions. Sariah-meon (1991) examined in-vitro hyphal interaction between S. rolfsii and T. harzianum. Thin hyphal branches grow towards and along those of S. rolfsii forming several short branches and coil. Asghari and Mayee (1991) reported that application of T. harzianum and soil drenching of 0.2% carbendazim reduce the population of S. rolfsii in soil and increased the population of T. harzianum. Mukhopadhyay et al. (1992) reported that combined application of biological agents and fungicides as seed treatment, first with Gliocladium 6

20 virens and then with 0.1% carboxin was effective in controlling the Sclerotium rolfsii in chickpea, lentil and groundnut. The bio-agents i.e. T. harzianum showed its parasitic or mycoparastic activity against S. rolfsii when it was applied as an alternative or in combination with sub-lethal doses of chemical fungicide immediately after soil solarisation in greenhouse test (Ordentlich and Chet,1989 and Mehata et al., 1995). Sugha et al. (1993) studied the conidial coating of the antagonists Trichoderma viride, T. harzianum on seeds and found the significant reduction in seedling mortality (47-65%) of chickpea as compared with the untreated control under in-vitro conditions. Correa et al. (1995) tested the Trichorzianines A and B obtained from Trichoderma harzianum on the mycelial growth of Sclerotium rolfsii. Bioassays showed that the Trichorzianines possessed different inhibitory activity. Bioassays also showed that Trichorzianines inhibited the mycelium of Trichoderma harzianum. Mathur and Sarbhoy (1978) tested five fungi viz. Trichoderma viride, Trichoderma harzianum, Aspergillus flavus, Fusarium oxysporum and R. bataticola known to have some antagonistic properties. Trichoderma harzianum and Trichoderma viride were showing antagonism against Sclerotium rolfsii under laboratory conditions. Mukhopadhyay (1995) stated that the seed treatment with bio-agents viz. Trichoderma harzianum and Gliocladium virens in different crops like chickpea, lentil, groundnut, tomato and cauliflower increased the value of the seed to improve plant growth and plant productivity, to protect a wide range of soil borne pathogens viz. Sclerotium rolfsii, Rhizoctonia solani and Fusarium oxysporum. A talk based formulation of T. viride used for dry seed treatment of the plants recorded improved growth and higher population of T. viride throughout the growing period indicating its high rhizosphere competence (Jayarajan and Ramkrishnan, 1995). Benhamou and Chet (1996) studied the interaction between Trichoderma harzianum and sclerotia of the soil borne plant pathogen Sclerotium rolfsii by SEM and TEM to assess the potential role of enzymatic hydrolysis in the antagonistic process. SEM investigations revealed that 7

21 hyphae of Trichoderma harzianum grew abundantly on the sclerotial surface forming a dense branched mycelium that appeared to establish contact with the outer host cells through thin mucilage Prasad et al. (1999) reported that isolates of Trichoderma and Gliocladium spp. inhibited mycelial growth (54.9 to 61.4%) and suppressed the sclerotial production (31.8 to 97.8%) of Sclerotium rolfsii under in-vitro conditions. Biswas and Sen (2000) tested 11 isolates of Trichoderma harzianum, out of these isolates of Groundnut field and Potato field reduce stem rot incidence of groundnut significantly, when delivered as seed dressing (30 to 50%) or through direct soil application (72 to 83%) in the pot trials. Biswas and Sen (2000) tested 11 isolates of Trichoderma harzianum. Out of these, the isolates of Wheat field, Groundnut field and Potato fields were effective against stem rot of Ground nut caused by S. rolfsii. Patel and Anahosur (2001) reported that Trichoderma harzianum showed mycoparasitic property by overgrowing, or ceasing the mycelial growth and reducing the sclerotial production of S. rolfsii under laboratory conditions. Trichoderma harzianum and Bacilus subtilis population in the spermosphere gradually increased after 6 th day of sowing, then decrease the association of S. rolfsii and Rhizoctonia bataticola. It is suggested that this increase and decrease was due to the antagonistic activity of T. harzianum and B. subtilis. Dutta and Das (2002) observed 61.5, 59.1 and 57.2 per cent inhibition in mycelial growth of S. rolfsii by T. harzianum, T. viride and T. koningii, respectively. They also found reduction in sclerotial production by all the antagonists. Prasad et al. (2003) found T. harzianum (44.1%) isolate to be superior over T. viride (39.1%) isolate in suppressing the colony diameter of S. rolfsii the causal agent of collar rot of cauliflower. Dutta and Das (2002) revealed that soil application of Trichoderma spp. reduced the disease incidence and increased the yield. Thus Trichoderma spp. was antagonistic or effective in controlling the collar rot of tomato incited by S. rolfsii under in-vitro conditions. The conidial coating of the 8

22 antagonists viz. T. viride, T. harzianum and Gliocladium roseum on seeds significantly reduced seedling mortality (i.e. 47 to 65%) of chickpea compared to untreated control (Sugha et al., 1993). As regards bio-control of soil borne fungal pathogens like Rhizoctonia bataticola, R. Solani, Sclerotium rolfsii and Phytophthora parasitica by employing antagonistic fungi like Trichoderma viride, T. lignorum, Aspergillus niger and Gliocladium fimbriatum, valuable contributions were made by Garrett (1980), Mall and Mall (1980), Freeman (1981) and by Mukerji (1983) Physiological effect Effect of temperature Singh and Gandhi (1991) reported that the incidence of disease caused by Sclerotium rolfsii was 100% at 30ºC in treated seedlings of guar, while at 25ºC and 35ºC temperature seedling mortality was 95 and 83% respectively, therefore 30ºC was found to be the optimum temperature for the growth of Sclerotium rolfsii. Tu et al. (1991) reported that the percentage germination of sclerotia increased when the sclerotia were incubated under dry condition for 3 day at 20ºC and then remoistened and placed at 28ºC. They found that the sclerotia germinated best at 20-28ºC on soil plates. Hari et al. (1991) reported that the maximum radial growth of Sclerotium rolfsii was observed at 30ºC at 72 hours of incubation followed by 25ºC at 96 hours of incubation and they found that the 25-30ºC temperature was the best for the growth of Sclerotium rolfsii collar rot of groundnut. Prasad et al. (1986) obtained best mycelial growth at 30 ± 0.5ºC whereas 25 ± 0.5ºC for the sclerotial formation Effect of ph Mathur and Sinha (1968) observed that the infection of S. rolfsii in guar was maximum (54.2%) at ph 6.6 and in gram (89.6%) at ph 5.7. Alkaline condition reduced the disease in both the crops. Singh and Gandhi (1991) reported that maximum mortality of guar seedling was observed at ph 6.1 and an increase in ph to 8.4 significantly reduced disease incidence of Sclerotium rolfsii. Kulkarni and Kulkarni (1998) 9

23 found the maximum saprophytic activity of Sclerotium rolfsii at ph level of 6.0 followed by 5.5, 6.0 and 8.4. Hari et al. (1991) reported the radial growth of Sclerotium rolfsii causing collar rot of groundnut at ph range of 2 to 9 but the maximum growth was at ph 6. Prasad et al. (1986) found best mycelial growth of S. rolfsii at ph 5.0 while sclerotial formation at ph Effect of medicinal plant leaf extracts Plant extracts of 13 medicinal plants were tested for their anti-fungal activity against S. rolfsii, the causal agent of collar rot of chickpea. Out of these plant extracts, the Dioscorea bulbifera gave 85 per cent control against S. rolfsii (Daya and Ram, 1992), under in-vitro conditions. Hanthegowda and Adiver (2001) tested different plant extracts against S. rolfsii. Among different plant extracts, 1:20 dilution of Parthenium hysterophorus, Polyalthia longifolliua and Azadirachta indica significantly inhibited the mycelial growth of S. rolfsii. 2.7 In-vitro effect of fungicide against Sclerotium rolfsii The first report was on southern blight of peanuts caused by Sclerotium rolfsii can be controlled with PCNB, as stated by Cooper, The poisoned food technique was followed for in vitro studies on the effect of certain fungicides on Sclerotium rolfsii. The best inhibition was showed by Calixin, Vitavax, Duter, Ferbam, Ceresan wet, Sandoz seed dressing 6334 and Brassicol which allowed no growth of fungus (Chauhan, 1978). Randon et al. (1995) reported that the carbendazim (Bavistin), Kasumin and Tecto used at five concentrations under in-vitro conditions were the most effective in inhibiting mycelial growth and sclerotia formation at low concentration. Toorray et al. (2007) evaluated seven fungicides (each at 1000, 1500, 2000 ppm) against Sclerotium rolfsii under in-vitro condition. Complete inhibition of growth of S. rolfsii was recorded by Captan, Thiram, Mancozeb, Hinosan (edifenphos), Antracol where as Cholrothalonil showed partial inhibition at low concentration. Bavistin (carbendazim) did not show much 10

24 inhibition at all concentrations. In in-vivo evaluation of chickpea with the 7 fungicides (captan at 3 g, thiram at 3 g, Bavistin at 3 g, mancozeb at 3 g, Hinosan at 1 g, Antracol at 3 g and Kavach at 3 g/kg) and two biological control agents (Trichoderma harzianum and T. viride, each at 4 g/kg) against S. rolfsii, captan, Kavach and thiram showed some reduction in preemergence mortality, while both the biological control agents did not show protection against pre-emergence mortality due to S. rolfsii. 11

25 MATERIAL AND METHODS The materials used in the course of present studies and methods followed are given below: A. MATERIAL 1. General Borosil make glassware s were used throughout the investigation. These were cleaned with detergent powder followed by rinsing in tap and distilled water. Glassware s were then sterilized in hot air oven at 180 o C for 2 hrs. The inoculation needle and other metallic instruments were sterilized by dipping them in alcohol and heating over flame. All precautionary measures were applied to sterilize the inoculated in chamber. The culture media were sterilized in an autoclave at 1.05 kg pressure per square inch (15 lbs psi) for 20 min at o C. The soil and sand were sterilized at 2.10 kg pressure per square inch (15 lbs psi) for two hours for two consecutive days. The roots of diseased plants were thoroughly washed with tap water and cut into pieces. Root pieces of samples were surface sterilized by dipping them in 1:1000 mercuric chloride solution for 1-2 minutes followed by three washing in sterilized water. The petriplates containing the media were stored for 24 hrs before use to avoid the possibility of contamination. After isolation culture of isolates were maintained on PDA medium for further investigations. Earthern pots were sterilized by dipping them in 5 percent CuSO 4 solution followed by rinsing with water before use. 2. Test pathogen Sclerotium rolfsii the causal organism of chickpea collar rot. 3. Culture Media The following media were used during the course of investigation, and the composition of media are given below. 12

26 1. Potato dextrose agar : Peeled potato slices g Dextrose - 20 g Agar Agar - 20 g Distilled water ml 2. Potato Dextrose Broth: Peeled potato slices g Dextrose Distilled water - 20 g ml For potato extraction peeled and sliced potato was taken and boiled in 500 ml water. Then it was strained through muslin cloth. Agar-agar was washed with water and taken in 500 ml of water then heated until it melted fully. Melted agar agar was poured in potato extract and dextrose was added and the solution was finally made-up to one litre. The media was poured in conical flask and sterilized in an autoclaved. In PD broth similar procedure as described above was employed only agar agar was not added in potato dextrose broth. Statistical analysis The data of the experiments conducted in the laboratory, pots and fields were subjected to statistical analysis. (B) METHODS 3.1 Collection, isolation, purification and identification of pathogen Collection Infected plants which showing collar rot symptoms were collected during the month of October to December 2012 from chickpea fields in JNKVV, Jabalpur. Samples were brought in to the Dept. of Plant Pathology for isolation and further studies Isolation of pathogen Collected plant samples of diseased plants were thoroughly washed in water and allowed to dry on blotter paper. From each sample, small pieces were cut with the help of sharp blade then these infected root pieces were 13

27 surface sterilized with mercuric chloride (0.1%) solution, followed by three washing in sterilized water. Three small pieces of roots were kept on the poured PDA plates and incubated at 25+2 o C for 5-7 days Purification and identification of the associated pathogen The cultures of Sclerotium rolfsii were purified by sub-culturing it through single hyphal tip method and maintained by mass transfer on PDA slants at 25+2 o C for further studies. After purification, isolates of S. rolfsii were identified by observing the colony characters and sclerotia formation. The morphology and cultural characters of fungus were studied. The objective material were grown on potato dextrose agar medium and maintained at 25+1 o C temperature. 3.2 Testing of pathogenecity by different inoculation methods Soil inoculation The sterilized soil was mixed with Sclerotium rolfsii which was isolated from diseased plants. The mixture was placed in earthen pots. Chickpea seeds (JG-315) were surface sterilized with 0.1% mercuric chloride for one minute followed by three washings with sterilized water. Ten seeds were placed in one earthen pot and three replications were maintained. These pots were kept in a net house. Proper isolation was maintained to avoid other pathogens. Observations on germination, pre and post emergence mortality were recorded. No soil treatment with test fungus was treated as control Seed inoculation Chickpea seeds were surface sterilized with 0.1% mercuric chloride for one minute followed by three washings with sterilized water and seeds were dressed with test fungi, treated seeds were sown in sterilized soil in earthen pots. Proper isolation was maintained. No seed dressing with test fungus was used as control. Observations were recorded on seven days after sowing Seedling inoculation Sterilized soil was taken in earthen pots, chickpea seeds which were surface sterilized were sown on earthen pots in growth chamber. The test fungus was inoculated on seven days old seedlings at collar region. Proper 14

28 control was maintained where the test fungi were not inoculated. Observation on the mortality of seedlings was recorded seven days after inoculation. 3.3 Antagonistic effect of Trichoderma spp on S. rolfsii The pure cultures of Trichoderma viride and Trichoderma harzianum were obtained from Department of Plant Pathology, College of Agriculture, Indore. The culture of Trichoderma viride, Trichoderma harzianum were also obtained from Department of Plant Pathology, Marathwada Uniersity of Agriculture, Parbhani (Maharashtra). The Jabalpur isolate of Trichoderma spp. from mushroom beds and paddy straw beds were also used. The antagonistic activity of these isolates against Sclerotium rolfsii was evaluated Dual culture technique Different isolates of Trichoderma spp. viz. Trichoderma viride and Trichoderma harzianum (Indore isolate), Trichoderma viride and Trichoderma harzianum (Parbhani isolate) and Trichoderma spp. isolated from mushroom beds and paddy straw beds (Jabalpur isolate) were screened for their antagonistic activity by dual culture method (Movton and Stroube, 1955) on potato dextrose agar medium in petriplates. An amount of 20ml potato dextrose agar was poured in sterilized petriplates, after solidification of media 4 days old pure culture of the antagonists and the pathogen were inoculated separately at the same time. A five mm disc was taken from the margin of young vigorously growing cultures and placed in petriplates at the opposite points from each other. The plates were incubated in the BOD incubator at C. All the isolates of Trichoderma spp. were tested for their effectiveness against the pathogen. Observations on the zone of inhibition between the antagonists and the test pathogen were recorded after 4 and 7 days of inoculation with the help of a scale. The mean of three replications was calculated and expressed in mm in each case. The percentage inhibition of growth of test pathogen in presence of Trichoderma spp. was calculated over control as under. Per cent growth inhibition = Growth of test pathogen in control plate - Growth of test pathogen in presence of Trichoderma spp. Growth of test pathogen in control plate X100 15

29 3.3.2 Pot culture technique (in vivo) The experiment was conducted to find out an effective antagonist enhancing germination and minimizing mortality of seedlings. Earthen pots were filled with inoculated soil. The soil was inoculated with test fungus Sclerotium rolfsii grown on soil maize medium. Seed of susceptible cultivar of chickpea JG-315 were treated with different species and isolates of Trichoderma spp. Six seeds were sown in each pot. Untreated seeds sown in the inoculated soil served as check. Three replications were maintained. Observations on germination, pre and post emergence mortality were recorded. 3.4 Physiological studies The following physiological studies were conducted: Growth of test fungus at different temperature The experiment was conducted to find out the most suitable temperature for mycelial growth. The Petriplates poured with potato dextrose medium were inoculated with the 5 mm disc of the pathogen of a 4 days old culture. The Petriplates were incubated at 15 C, 20 C, 25 C, 30 C and 35 C. Three replications were maintained for each treatment and observations for mean radial growth were recorded at 4 and 8 days after inoculation Growth of Sclerotium rolfsii at different ph The effect of different ph ranging from 4, 5, 6, 7, 8 and 9 on the growth of the pathogen was studied on potato dextrose agar medium. The ph of the medium was adjusted before autoclaving with the help of digital ph meter, by addition of 0.1N HCL. and 0.1N NaOH. Twenty ml of medium was poured in the Petriplates. The Petriplates were inoculated with 4 days old pure culture of Sclerotium rolfsii and were incubated in BOD incubator at C. Three replications were maintained for each treatment and observations for radial growth after 3, 5 and 7 days interval were recorded. 16

30 3.5 In-vitro evaluation of medicinal plant leaf extract on mycelial growth of Sclerotium rolfsii Antifungal activity of different medicinal plant leaf extracts were studied under in vitro condition taking plant leaf dextrose agar medium. The following medicinal plants viz., Japanese pudina. Shikakai (Acacia coneini), Kalmegh (Andrgraphis panioulaki), Sinduri (Bixa orellana), Vantulsi (Ocimum basil'icum), Ashwagandha (Withania. sominifera), Nirgudi (Vitex negando), Khamar, were used and PDA was used for control. The preparation of leaf extract medium was same as PDA medium, 20 gm leaves of each medicinal plant were taken in 100 ml water and boiled till it became softened. Softened medicinal plant leaves were curshed in pastel and mortar and then extract was filtered. Two gm of dextrose and two gm Agar- Agar were mixed in filtered leaf extracts and volume was made up to 100 ml and then sterilization was done by autoclaving at 15 lbs pressure for 20 minutes. To avoid bacterial contamination a little amount of streptomycin sulphate was added at the time of pouring of the medium. In each plate 20ml medium was poured in sterilized Petriplates and allowed to solidify. A 5 mm disc from 4 days old culture of test fungus was placed in the centre of medium. Three replications were maintained in each treatment along with a control. The inoculated petriplates were then incubated in the BOD incubator at C and observed 12 days after incubation and sclerotia were counted 12 days after incubation. 3.6 Evaluation of fungicides against Sclerotium rolfsii Poisoned food technique (in-vitro) Seven fungicides were tested in-vitro against Sclerotium rolfsii. These are Kitazen, Captan, Bavistin, Mancozeb, Hinosan, Kavach and Antracol. 100 ml potato dextrose agar medium was sterilized in conical flask of 150 ml capacity. Seven fungicides were separately incorporated aseptically in molten PDA to make 500 and 1000 ppm concentration. The amended medium was then poured in sterilized petriplates. Five mm disc of test fungus was cut with the help of sterilized cork borer from the margin of 4 days old culture and then placed centrally in petriplates. The disc was placed in inverted position to 17

31 allow the contact of fungus with medium. The inoculated petriplates were incubated in the BOD incubator at 27 ± 2ºC and the colony diameter of the pathogen was measured after 4 and 8 days of incubation with the help of scale in mm. percent growth inhibition under the influence of of different fungicides was calculated Pot culture technique (in-vivo) The experiment was conducted to find out the most effective chemical for obtaining maximum emergence and minimum seedling mortality. The pots of 10 cm diameter were filled with infested soil. Seeds of sclerotial rot susceptible cultivar i.e. JG-62 was treated with Kavach, Antracol, Mancozeb, Kitazin, Captan, Hinoson and 0.20%. Six seeds were sown in each pot and untreated seeds sown in infested soil served as check. Three replications were maintained. Observations on germination, pre-emergence and post-emergence mortality were recorded. 18

32 RESULTS The experimental findings of various studies conducted on Sclerotium rolfsii Sacc. are given below: 4.1 Isolation, purification and identification of Sclerotium rolfsii Sacc. from infected chickpea plants and its Symptomatology Symptomatology The symptoms of collar rot of chickpea caused by Sclerotium rolfsii are follows. Collar rot of chickpea (Sclerotium rolfsii) is very widely prevalent disease and can cause considerable loss to plant stand when soil moisture is high and of nearly 30 C at sowing time in the form of pre-emergence mortality. Drying plants whose foliage turns slightly yellow before death scattered through the field in an indication of collar rot infection. The disease generally appears within 2 weeks after sowing. Young seedlings collapse but older seedlings turns yellow and may dry without collapsing. The younger seedlings exhibit clear rotting at collar region. The rotten portion and surrounding of soil of the infected plant is often covered with white mycelial strands of the fungus. Sometimes rapeseed like sclerotia can be observed attached to mycelial strands around the collar of the effected seedlings and soil. Affected seedlings usually occur in small patches scattered in the field. The infected plants were collected from the field for isolation and purification of the causal fungus in the laboratory, followed by identification of the fungus Morphological characters The mycelium of the pathogen is septate and hyaline with conspicuous branching at acute angles. The well developed mycelium is in cord-like strands. The hyphae have clamps in the form of forks and hooks or H-like connections (Aycock, 1966). The young growing mycelial mass, in vivo and in vitro, is snow white with a silky-lustre. Sclerotia are at first white, becoming light brown to dark brown at maturity. They are sub spherical, the surface being finely wrinkled or pitted, sometimes flattened, commonly mm in diameter. On the basis of morphological characters, the pathogen was identified as Sclerotium rolfsii. 19

33 4.1.3 Testing the pathogenicity by soil infestation method Sterilized seeds were sown in earthen pots (containing infested soil) to test the effect of the fungus on germination per cent and pre and post emergence mortality. The data presented in Table 1 reveals that the germination of seed was only 50 percent in infested soil, while highest germination i.e. (100%) was recorded in control (un infested). Pre emergence mortality of 50% was observed in S. rolfsii infested soil as compared to no mortality in control. Similarly, the 100% post emergence mortality was recorded in the test fungus infested soil as compared to no mortality in control. Table 1: Pathogenicity test of Sclerotium rolfsii against chickpea Treatment * Mortality (%) Germination (%) * Preemergencemergency Post- S. rolfsii infested soil Control *Average of three replications 4.2 Effect of bio agents on Sclerotium rolfsii Growth of Sclerotium rolfsii inhibited by different species and local isolates of Trichoderma in dual culture under in vitro conditions The data on inhibition of Sclerotium rolfsii by Trichoderma spp. is presented in Table 2. The result indicates that the bio-control agents reduced the growth of S. rolfsii. Maximum growth inhibition (25.1%) of Sclerotium rolfsii was observed with Trichoderma spp. (PSJ) followed by T. viride (Indore) (22.7%), T. harzianum (Parbhani) (17.6%) and least inhibition was recorded with T. viride (Parbhani) (2.9%) at 3 days after inoculation. Highest inhibition of S. rolfsii after 5 days inoculation, was recorded with Trichoderma spp. (PSJ) (60.8%) followed by Trichoderma spp. (MBJ) (49.1%), T. harzianum (Indore) (49.0%), T. viride (Indore) (42.2%) and lowest inhibition was observed with T. viride (Parbhani) (40.2%). At 7 days after inoculation, the maximum inhibition was also recorded with Trichoderma spp. (PSJ) (68.6) then, T. harzianum (Indore) (58.3%), Trichoderma spp. (MBJ) (56.7%), and the least inhibition was observed with T. viride (Parbhani) (50.0%). 20

34 Sr. No. 1. Table 2: Growth of Sclerotium rolfsii inhibited by different species and local isolates of Trichoderma in dual culture Bioagents Trichoderma harzianum (I) 2. Trichodrma Viride (I) method under in-vitro conditions Sclerotium rolfsii Redial growth * (mm) 3 DAI 5 DAI 7 DAI Trichoderma Inhibition (%) Sclerotium rolfsii Trichoderma SPP. Inhibition (%) Sclerotium rolfsii Trichoderma spp. Inhibition (%) Trichoderma Harzianum (P) Trichodrma Viride (P) Trichodrma spp.(mbj) Trichoderma spp. (PSJ) Control *Average of three replications (I) = Indore isolate (P) = Parbhani isolate DAI = Days after inoculation (MBJ) = Isolated from mushroom beds, Jabalpur (PSJ) = Isolated from paddy straw, Jabalpur 21

35 4.2.2 Effect of seed treatment on mortality of seedlings by Sclerotium rolfsii The results of seed treatment with Trichoderma spp. and their effect on germination, pre and post emergence mortality caused by Sclerotium rolfsii were recorded and presented in Table 3. Table 3: Effect of seed treatment by different species and local isolates of Trichoderma on germination, pre and post emergence mortality of chickpea caused by Sclerotium rolfsii Mortality (%) * Sr. Isolates of Germination* Average Preemergencemergence Post- No. Trichoderma (%) mortality (%) 1. Trichoderma harzianum (I) (51.4) (38.4) (36.7) 2. Trichoderma viride (I) (62.1) (27.8) (38.8) 3. Trichoderma harzianum (P) (48.2) (41.7) (38.4) 4. Trichoderma viride (P) (44.9) (44.9) (36.7) 5. Trichoderma spp. (MBJ) (41.7) (48.2) (44.9) 6. Trichoderma spp. (PSJ) (38.4) (51.4) (41.7) 7. Control (38.4) (51.4) (89.3) SEm ± CD (p=0.05) *= Average of three replication Values in parenthesis shows Arc sine transformation (I) = Indore isolate (P) = Parbhani isolate (MBJ) = Isolated from mushroom beds, Jabalpur (PSJ) = Isolated from paddy straw, Jabalpur There was significant difference in germination of seeds treated with some isolates of Trichoderma and control. Maximum germination of chickpea (77.7%) was observed when the chickpea seeds were treated with T. viride (Indore) followed by T. harzianum (Indore). However, the germination was least (38.8%) when the seeds were treated with Trichoderma spp. (PSJ) which was equal to seed germination (38.8%) in control. The pre emergence mortality in chickpea also differed significantly in two treatments. It was Significantly least (22.2%) in seeds 22