STUDIES ON THE EFFECT OF CHEMICALS, BIOAGENTS AND SEED COAT POLYMERS ON SOIL BORNE DISEASES OF CASTOR AND GROUNDNUT P. RAKESH

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1 STUDIES ON THE EFFECT OF CHEMICALS, BIOAGENTS AND SEED COAT POLYMERS ON SOIL BORNE DISEASES OF CASTOR AND GROUNDNUT P. RAKESH B.Sc. (Ag.) MASTER OF SCIENCE IN AGRICULTURE (PLANT PATHOLOGY) 2016

2 STUDIES ON THE EFFECT OF CHEMICALS, BIOAGENTS AND SEED COAT POLYMERS ON SOIL BORNE DISEASES OF CASTOR AND GROUNDNUT BY P. RAKESH B.Sc. (Ag.) THESIS SUBMITTED TO THE PROFESSOR JAYASHANKAR TELANGANA STATE AGRICULTURAL UNIVERSITY IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE AWARD OF THE DEGREE OF MASTER OF SCIENCE IN AGRICULTURE (PLANT PATHOLOGY) CHAIRPERSON: Dr. G. UMA DEVI DEPARTMENT OF PLANT PATHOLOGY COLLEGE OF AGRICULTURE RAJENDRANAGAR HYDERABAD PROFESSOR JAYASHANKAR TELANGANA STATE AGRICULTURAL UNIVERSITY 2016

3 CERTIFICATE Mr. P. RAKESH has satisfactorily prosecuted the course of research and that thesis entitled STUDIES ON THE EFFECT OF CHEMICALS, BIOAGENTS AND SEED COAT POLYMERS ON SOIL BORNE DISEASES OF CASTOR AND GROUNDNUT submitted is the result of original research work and is of sufficiently high standard to warrant its presentation to the examination. I also certify that neither the thesis nor its part thereof has been previously submitted by him for a degree of any University. Date: Chairperson

4 CERTIFICATE This is to certify that the thesis entitled STUDIES ON THE EFFECT OF CHEMICALS, BIOAGENTS AND SEED COAT POLYMERS ON SOIL BORNE DISEASES OF CASTOR AND GROUNDNUT submitted in partial fulfilment of the requirements for the degree of MASTER OF SCIENCE IN AGRICUTLURE of the Prof. Jayashankar Telangana State Agricultural University, Hyderabad, is a record of the bonafide original research work carried out by Mr. P. RAKESH under our guidance and supervision. No part of the thesis has been submitted by the student for any other degree or diploma. The published part and all assistance received during the course of the investigation have been duly acknowledged by the author of the thesis. Thesis approved by the Student Advisory Committee. Chairman: (Dr. G. UMA DEVI) Professor & University Head Dept. of Plant Pathology, College of Agriculture, Rajendranagar, Hyderabad Co-Chairman : (Dr. R.D. PRASAD) Principal Scientist Plant Pathology IIOR, Rajendranagar Hyderabad Member : (Dr. BHARATI N. BHAT) Associate Professor Dept. of Plant Pathology, College of Agriculture, Rajendranagar, Hyderabad Member : (Dr. K. VIJAYA LAKSHMI) Director (PHM), NIPHM Rajendranagar, Hyderabad Date of final viva-voce:

5 DECLARATION I, P. RAKESH, hereby declare that the thesis entitled STUDIES ON THE EFFECT OF CHEMICALS, BIOAGENTS AND SEED COAT POLYMERS ON SOIL BORNE DISEASES OF CASTOR AND GROUNDNUT submitted to the PROF. JAYASHANKAR TELANGANA STATE AGRICULTURAL UNIVERSITY for the Degree of MASTER OF SCIENCE IN AGRICUTLURE is the result of original research work done by me. I also declare that no material contained in the thesis has been published earlier in any manner. Place : Date : (P. RAKESH) I. D. No. RAM/

6 ACKNOWLEDGEMENTS Guru bramha Gurur Vishnu Guru devo Maheshwaraha Guru saakshat Parabrahma Tasmai sree guravenamaha! At the outset, the time has come for me to gather the words for expressing my gratitude towards all those who helped me in building my career. By the grace of Almighty I have to put all efforts to fulfill the task assigned to me by learned members of the Advisory Committee. Fervently and modesty, I extol the genuine and constant encouragement, immaculate guidance and every suggestive help offered to me by the Chairman of Advisory Committee Dr. G. Uma Devi, Professor and University Head, Department of Plant Pathology, College of Agriculture, Rajendranagar, Hyderabad for her wise counsel, concrete suggestions, her inspiring, meticulous and affectionate guidance, constant help and persistent encouragement during the course of my study and research work which has led to the present investigation to the final shape. I wish to express my esteem gratitude, my deepest admiration and heartful thanks to Dr. R.D. Prasad, Co-Chairman of Advisory Committee, Principal Scientist (Plant Pathology), IIOR, Hyderabad for providing each and every facility, warmer affection and mental support, his sustained interest, caring nature, immense help, constant inspiration and fruitful advice and co-operation. I take it as a great privilege and pride to have an opportunity of working under his unending, inspiring and indomitable spirit and also for his constant encouragement. I deem it my privilege in expressing my deep sense of reverence and gratitude and indebtedness to Dr. Bharathi N. Bhat, Associate Professor, Department of Plant Pathology, College of Agriculture, Rajendranagar, Hyderabad and member of my Advisory Committee for her affectionate guidance, valuable suggestions, careful and reasoned criticism and meticulous attention to the details and also for her constant encouragement. I express my sincere thanks to member of my Advisory Committee Dr. K. Vijaya Lakshmi, Director of Plant Health Management, NIPHM for her kind co-operation, valuable advises during my research work. With immense pleasure, I wish to express my heartfelt respect and affectionate gratitude to my beloved father P. Kondala Rao and mother P. Padmavathi whose

7 everlasting love, unfading faith, incessant inspiration, moral and financial support and blessings kept me enthusiastic throughout my life and molded me to the present position, and whose constant encouragement brings out the best in every one of my endeavors and without which this work could not be completed. I express my wholehearted gratitude to Dr. B. Vidyasagar, Associate Professor, Smt. Prameela Assistant professor, Department of plant pathology, College of Agriculture, Rajendranagar for their insightful, inspiring, scholarly guidance and constructive suggestions during my investigation. I express my sincere thanks to Dr. P. Narayana Reddy, Professor (Retrd.), Dr. P. Kishore Varma, Scientist, Anakapally, M. Suresh, Scientist, Anakapally, Dr. K. Vemana, Senior Scientist, ARS, Kadiri, Dr. Razia Sultana, Associate Professor, Department of Seed Technology, College of Agriculture, Rajendranagar, Hyderabad for helping me during pursuit of my research work. I sincerely thank the technical staff Shailaja, Navaneetha, Aniruddha, Kavitha, Bharathi, Ph.D Scholar, Saida Reddy, T.A, Sai, Dasthagiri and Mahender, High skilled helpers of Plant Pathology Laboratory, IIOR for their generous support, encouragement and technical help during the course of my investigation at IIOR. I am very much beholden and profoundly indebted to my seniors Ramesh Babu, Srinivas, Chandra Sekhar, Ananth, Arut Selvan, Yella Goud, Padmaja, Jalender, Chandrakala, Yamuna and Rajendra Prasad for their help, co-operation, company and support. I am very much beholden and profoundly undebted to my affectionate and beloved friends Srinivas, Sreekanth, Chiranjeevi, Manjunatha, Harsha, Suman and Raju, my juniors Deepak Reddy, Ramakrishna, Ravi, Aditya and Vijay Reddy for their cheerful company, help and co-operation during my course of investigation. I am also thankful to non-teaching staff Department of the Plant Pathology, College of Agriculture, Rajendranagar, Hyderabad for their help during my M.Sc. programme. I feel elated to express my bountiful thanks to those who directly or indirectly helped me in successful completion of thesis work. The financial assistance rendered by PJTSAU, Rajendranagar in the form of fellowship is greatly acknowledged. Date: (P. RAKESH)

8 LIST OF CONTENTS Chapter No Title Page No I II III IV V INTRODUCTION REVIEW OF LITERATURE MATERIAL AND METHODS RESULTS AND DISCUSSION SUMMARY AND CONCLUSIONS LITERATURE CITED

9 LIST OF TABLES Table No. 3.1 Details of Seed coat polymers Particulars 3.2 Details of the treatments used in pot culture studies for Castor Page No. 3.3 Details of the treatments used in pot culture studies for Groundnut 3.4 Details of the fungicides used in bioassay studies under in vitro condition 3.5 Details of the treatments used in pot culture studies for Castor under green house conditions 3.6 Details of the treatments used in pot culture studies for Groundnut under green house conditions 4.1 Effect of different synthetic seed coat polymers and biopolymers on germination and vigour of castor seed 4.2 Effect of different synthetic seed coat polymers and biopolymers on germination and vigour of groundnut seed 4.3 Effect of seed coat polymers on per cent germination and vigour index of castor seed at different storage intervals 4.4 Effect of seed coat polymers on per cent germination and vigour index of groundnut seed at different storage intervals Effect of seed coat polymers on wilt of castor in pot culture studies Incidence of Fusarium oxysporum f. sp. ricini in pot culture of polymer treated castor seed Effect of seed coat polymers on collar rot disease of groundnut in pot culture studies Incidence of Aspergillus niger in pot culture of polymer treated groundnut seed Effect of fungicides on the mycelial growth of Fusarium oxysporum f. sp. ricini under in vitro conditions Effect of biocontrol agents on the mycelial growth of Fusarium oxysporum f. sp. ricini under in vitro conditions

10 Table No. Particulars Effect of fungicides on the mycelial growth of Aspergillus niger under in vitro conditions Page No Effect of biocontrol agents on the mycelial growth of Aspergillus niger under in vitro conditions Effect of seed coating polymers in combination with fungicide and biocontrol agent on the germination and vigour of castor under in vitro conditions Effect of seed coating polymers in combination with fungicide and biocontrol agent on the germination and vigour of castor by Fusarium oxysporum f. sp. ricini inoculation under in vitro conditions Effect of seed coating polymers in combination with fungicide and biocontrol agent on the germination and vigour of groundnut under in vitro conditions Effect of seed coating polymers in combination with fungicide and biocontrol agent on the germination and vigour of groundnut by Aspergillus niger inoculation under in vitro conditions Effect of seed coating polymers in combination with fungicide and biocontrol agent on the germination and vigour of castor under green house conditions Effect of seed coating polymers in combination with fungicide and biocontrol agent on the germination and vigour of castor by Fusarium oxysporum f. sp. ricini infested soil and wilt disease incidence under green house conditions Effect of seed coating polymers in combination with fungicide and biocontrol agent on the germination and vigour of groundnut under green house conditions 4.16 Effect of seed coating polymers in combination with fungicide and biocontrol agent on the germination and vigour of groundnut by Aspergillus niger infested soil and collar rot disease incidence under green house conditions

11 LIST OF PLATES Plate No. Particulars Page No. 3.1 Pure culture of Fusarium oxysporum f. sp. ricini 3.2 Pure culture of Aspergillus niger 3.3 Mass multiplication of Fusarium oxysporum f. sp. ricini on sorghum grains 3.4 Mass multiplication of Aspergillus niger on sorghum grains 3.5 Pure culture of Trichoderma harzianum Th4d 3.6 Pure culture of Trichoderma asperellum TaDOR Castor seed inoculated with Fusarium oxysporum f. sp. ricini 4.2 Groundnut seed inoculated with Aspergillus niger 4.3 Effect of synthetic polymer and biopolymer chitosan on growth of castor 4.4 Effect of synthetic polymer and biopolymer chitosan on growth of groundnut 4.5 Effect of synthetic polymer and biopolymer chitosan on growth of castor at different storage intervals 4.6 Effect of synthetic polymer and biopolymer chitosan on growth of groundnut at different storage intervals Effect of synthetic polymer and biopolymer chitosan treated seed on growth parameters and antifungal activity against wilt of castor in pot culture Effect of synthetic polymer and biopolymer chitosan treated seed on growth parameters and antifungal activity against collar rot disease of groundnut in pot culture

12 Plate No. Particulars Page No. 4.9 Effect of fungicides on the radial growth Fusarium oxysporum f. sp. ricini at recommended concentration under in vitro conditions Effect of fungicides on the radial growth Fusarium oxysporum f. sp. ricini at half the recommended concentration under in vitro conditions Effect of fungicides on the radial growth Fusarium oxysporum f. sp. ricini at above the recommended concentration under in vitro conditions Effect of fungicides on the radial growth Aspergillus niger at recommended concentration under in vitro conditions Effect of fungicides on the radial growth Aspergillus niger at half the recommended concentration under in vitro conditions Effect of fungicides on the radial growth Aspergillus niger at above the recommended concentration under in vitro conditions Effect of biocontrol agents on the radial growth Fusarium oxysporum f. sp. ricini under in vitro conditions Effect of biocontrol agents on the radial growth Aspergillus niger under in vitro conditions Effect of synthetic polymer and biopolymer chitosan in combinations with fungicide and biocontrol agent on growth of castor under in vitro conditions Effect of synthetic polymer and biopolymer chitosan in combinations with fungicide and biocontrol agent on growth of castor against Fusarium oxysporum f. sp. ricini inoculation under in vitro conditions Effect of synthetic polymer and biopolymer chitosan in combinations with fungicide and biocontrol agent on growth of groundnut under in vitro conditions Effect of synthetic polymer and biopolymer chitosan in combinations with fungicide and biocontrol agent on growth of groundnut against Aspergillus niger inoculation under in vitro conditions

13 Plate No. Particulars Page No Effect of synthetic polymer and biopolymer chitosan in combinations with fungicide and biocontrol agent on growth of castor under green house conditions Effect of synthetic polymer and biopolymer chitosan in combinations with fungicide and biocontrol agent on growth of castor against Fusarium oxysporum f. sp. ricini infected soil under green house condition Effect of synthetic polymer and biopolymer chitosan in combinations with fungicide and biocontrol agent on growth of groundnut under green house conditions Effect of synthetic polymer and biopolymer chitosan in combinations with fungicide and biocontrol agent on growth of groundnut against Aspergillus niger infested soil under green house conditions

14 LIST OF SYMBOLS AND ABBREVIATIONS % : Per : at the rate of C : Degree Celcius µg ml -1 : microgram per milliliter µl : microliter CD (p=0.05%) : Critical Difference cm : Centimeter CRD : Completely Randomized Design CV : Coefficient of Variation cv. : Cultivar DAS : Days after sowing e.g. : for example et al. : and others people etc. : and so on g kg -1 : Gram per kilogram g l -1 : Gram per liter g : Gram h : hours ha : Hectare i.e., : that is ISTA : International Seed Testing Association kg ha -1 : Kilogram per hectare kg : Kilogram

15 l : Liter LDPE : Low Density Polyethylene mg l -1 : Milligram per liter mg ml -1 : Milligram per milliliter mg : Milligram ml kg -1 : Milliliter per kilogram ml l -1 : Milliliter per liter ml : Milliliter mm : Millimeter No. : Number ppm : Parts per million psi : Pounds per square inch RH : Relative Humidity rpm : Rotations per minute SE (d) : Standard Error of deviation SE (m) : Standard Error of mean v/v : Volume by volume var. : Variety viz., : Namely w/v : Weight by volume

16 Author : P. RAKESH Title of the thesis : STUDIES ON THE EFFECT OF CHEMICALS, BIOAGENTS AND SEED COAT POLYMERS ON SOIL BORNE DISEASES OF CASTOR AND GROUNDNUT Degree : MASTER OF SCIENCE Faculty : AGRICULTURE Department : PLANT PATHOLOGY Chairperson : Dr. G. UMA DEVI University : PROFESSOR JAYASHANKAR TELANGANA Year of submission : 2016 STATE AGRICULTURAL UNIVERSITY ABSTRACT Castor (Ricinus communis L.) and groundnut (Arachis hypogaea L.) are the two important oilseed crops grown in India. Of the various soil borne diseases, the wilt caused by Fusarium oxysporum f. sp. ricini in castor and the collar rot caused by Aspergillus niger in groundnut are the major diseases. The study was aimed at developing best seed coating combination using seed coat polymers, effective fungicide and potential biocontrol agent for the management of these diseases along with promoting the plant growth. The immediate goal was to determine the influence of both synthetic polymers and biopolymers on the germination and vigour of castor and groundnut. In castor and groundnut among all the treatments, seed treated with 0.25% and synthetic 0.3% were recorded highest seed germination, root length, shoot length, vigour index-i, fresh weight, dry weight and vigour index-ii when compared to other polymers and control. Both castor and groundnut seeds were coated with 0.25% and synthetic 0.3% and stored under ambient conditions to find out the effect of seed coat polymers on castor and groundnut when stored at different storage intervals. The treated seeds were subjected to monthly evaluation for their germination and

17 vigour. Among the two seed coat polymers, 0.25% was significant in maintaining the quality of castor and groundnut seed when compared to control. The effect of polymers was tested on wilt pathogen of castor and collar rot pathogen of groundnut separately by soil inoculation in pot culture studies. The chitosan (0.25%) coated castor and groundnut seeds were grown in F. oxysporum f. sp. ricini and Aspergillus niger inoculated soil respectively there was significant difference in per cent germination, vigou index-i and vigour index-ii with minimum per cent disease incidence of and respectively, when compared to synthetic polymer-ii and control. The efficacy of fungicides with different concentrations and biocontrol agents viz., Trichoderma harzianum Th4d and Trichoderma asperellum TaDOR 7316 were tested against F. oxysporum f. sp. ricini and A. niger separately. Carbendazim against F. oxysporum f. sp. ricini and vitavax+thiram against A. niger recorded cent per cent inhibition at all concentrations tested and Trichoderma harzianum Th4d against F. oxysporum f. sp. ricini and Trichoderma asperellum TaDOR 7316 against A. niger showed maximum inhibition of the pathogen. From the findings the effective fungicide (carbendazim) and potential biocontrol agent Trichoderma harzianum Th4d against F. oxysporum f. sp. ricini and as well as the effective fungicide (vitavax+thiram) and potential biocontrol agent Trichoderma asperellum TaDOR 7316 against A. niger were selected for further studies and made different combinations with polymer, fungicide and biocontrol agents against the both pathogens separately. Studies on the combination of the effective fungicide 0.1% and potential biocontrol agent T. harzianum 1% and seed coat polymers 0.25% and synthetic seed coat 0.3% against castor wilt pathogen Fusarium oxysporum f. sp. ricini both under in vitro and in vivo conditions indicated that chitosan+carbendazim+t. harzianum Th4d combination was effective in reducing the wilt disease incidence in castor. Similarly studies on the combination of the effective fungicide 0.2% and potential biocontrol agent T. asperellum TaDOR 1% and seed coat polymers 0.25% and synthetic seed coat 0.3% against groundnut collar rot pathogen Aspergillus niger both under in vitro and in vivo conditions indicated that chitosan+vitavax+thiram+t. asperellum TaDOR 7316 combination was effective in reducing the collar rot disease incidence in groundnut.

18 INTRODUCTION

19 Chapter I INTRODUCTION Castor (Ricinus communis L.) is native of Ethiopian region of tropical East Africa and cultivated in tropical, subtropical and warm temperate regions of the world. Castor is one of the most important non-edible oilseed crops cultivated in India and is a monopoly in world castor market. The Indian cultivars have about 48% of oil content and 42% of it can be extracted, while the cake retains the rest. The crop is grown in several parts of India in 8.53 lakh ha with a production of lakh tonnes and largely grown in Gujarat, Rajasthan, Andhra Pradesh, Telangana which totally contributes 96% of the total castor seed production in India. In Telangana the crop is grown in an area of 0.83 lakh ha with production of 0.56 lakh tonnes and a productivity of 709 kg ha -1 (Dept.of Agriculture and Co-operation, Telangana, 2015) which is extensively cultivated as a sole crop in Mahaboobnagar, Ranga Reddy, Nalgonda and Khammam districts. Castor is known to suffer from many diseases at different stages of crop growth and about 150 organisms are reported as pathogenic on castor plant. The crop is mainly affected by wilt, seedling blight, Alternaria blight, Cercospora leaf spot, root rot, powdery mildew and also bacterial leaf spot. Among these, the wilt caused by Fusarium oxysporum f.sp. ricini is an important soil borne disease. The extent of yield loss depends on the stage at which the plants wilt which is about 77% at flowering stage, 63% at 90 days and 39% at later stages. The disease assumed serious proportion in all the castor growing areas of India including Telangana state because of long duration survival of the pathogen in the soil and susceptibility of prevalent cultivars. On the other hand, the farmers are not willing to change the age old cropping pattern practice of growing castor after castor without following any crop rotation, which helps in building up of soil inoculum thereby resulting in heavy incidence of wilt disease in all the castor growing areas. High incidence of wilt was reported in the farmer s fields in Gujarat, Andhra Pradesh, Telangana and Karnataka. Maximum incidence (76%) was recorded in Gujarat on hybrid GCH-4. Groundnut (Arachis hypogaea L.) is an important oilseed and ancillary food crop of the world. Groundnut seeds are rich in energy due to its high oil (48-50%) and protein content (25-28%) and carbohydrate (10-20%). A native of South America, groundnut is cultivated in tropical, sub-tropical, and warm temperate regions of the

20 world. India accounts for about 27% of global area and contributes 19% to world groundnut production. India is the second largest producer of groundnut in the world. The crop occupies lakh ha with an annual production of lakh tonnes and a productivity of 1750 kg ha -1 (Oilseeds Division, MoA, 2015). In Telangana, groundnut is mostly grown in Mahaboobnagar, Warangal and Karimnagar districts to an extent of 2.1 lakh ha with an annual production of 3.52 lakh tonnes and mean productivity of 1676 kg ha -1 (Dept.of Agriculture and Co-operation, Telangana, 2015). Groundnut cultivation in India as a rainfed crop is often subjected to significant yield losses annually due to insufficient and uneven distribution of rainfall during the crop growth period, non-availability and inaccessibility of high yielding cultivars and fertilizers to farmers. Of various biotic stresses, soil borne and foliar diseases account for reduced pod yields. Fungal diseases such as collar rot (Aspergillus niger Van Tieghem), stem rot (Sclerotium rolfsi Sacc.), root rot (Rhizoctonia solani Kuhn), dry root rot (Macrophomina phaseolina (Tassi) Goid) and seedling blight (Fusarium oxysporum Schlecht) are causing major havoc in all crop growing areas. Of these, collar rot and stem rot diseases are the major soil borne diseases with significant yield losses annually. Aspergillus niger Van Tieghem causing collar rot disease on groundnut is usually seen during the early stages of crop growth, and often results in seedling mortality at higher rates. This disease was first reported from India by Jain and Nenra (1952). Occasionally, collar rot can continue up to crop harvesting stage resulting in damage to the seed (Gajera et al., 2011). In India, the losses may account to % in terms of seedling mortality due to Aspergillus niger (Ghewande et al., 2002). In most agricultural ecosystems, soil borne plant pathogens can be a major limitation in the production of marketable yields and also more recalcitrant to management and control compared to pathogens that attack the above-ground portions of the plant. Almost all soil borne fungi are necrotrophic. Loss of viability and vigour under high temperature and humid conditions is a common phenomenon in many crop seeds. Seed treatment with fungicide would protect the seeds and young seedlings from many seed borne and soil borne pathogens. Currently seed coating polymers are being used by seed companies along with active ingredients such as insecticides and fungicides. Polymer coating acts as a

21 temperature switch, regulating intake of water by seed coat, the stress imposed by accelerated ageing, which includes fungal invasion and improves the seedling emergence at changing soil moisture regime especially in the sub-optimal range (Scott, 1989; Sherin & Susan John, 2003). The polymer film may act as physical barrier, which has been reported to reduce the leaching of inhibitors from the seed coverings and may restrict oxygen diffusion to the embryo (Vanangamudi et al., 2003). Chitosan is a carbohydrate biopolymer derived from deacetylation of chitin, which is found in the crustacean s shells, insect s cuticle, and cell wall of fungi. The positive charge of chitosan confers the numerous and unique physiological and biological properties with great potential in a wide range of industries such as pharmacology, medicine, and agriculture (Sandford, 1989). Another important attribute of this natural compound is associated with its fungistatic or fungicidal properties against pathogens of various crops (Hadrami et al., 2010). Seeds and seedlings are vulnerable to many soil borne pathogens and they can destroy germinating seeds and young plants, which are relatively tender and lack food reserves to recover from injuries or to survive extended period of stress. Particularly in groundnut, if the seed coat is lost, they will be more prone to the stress as well as diseases. In view of the economic importance of these oil seed crops and soil borne diseases and unsatisfactory control of these soil borne diseases, considerable attention has been given on the detailed studies of development of protection sources and evaluation of new techniques for the management of soil borne diseases in oil seed crops. Hence, the present research work is planned with the following objectives. Objectives of investigation 1. To study the effect of biopolymers and synthetic seed coating polymers on Castor and Groundnut. 2. To study the antimicrobial effect of seed coat polymers on soil borne pathogens of Castor and Groundnut. 3. To test the efficacy of fungicides and bioagents against soil borne pathogens under in vitro conditions. 4. To study the effect of seed coat polymers in combination with effective fungicide and potential bioagent against soil borne pathogens of Castor and Groundnut under green house conditions.

22 REVIEW OF LITERATURE

23 Chapter II REVIEW OF LITERATURE The available literature on the effect of seed coat polymers, different chemicals, bioagents and their compatibility with seed coat polymers to restrict the plant pathogens has been reviewed in this chapter. The review of literature pertaining to this dissertation is presented in the following headings and sub-headings. 2.1 EFFECT OF SEED COAT POLYMERS ON GROWTH AND VIGOUR OF SEED Quality seed is the basis for profitable production and expansion of any crop. Loss of viability and vigour under high temperature and humid conditions is a common phenomenon in many crop seeds especially oil seeds. Polymer coating is used in many seed industries for uniform application of material to seeds. The film formulation consists of mixture of polymer, plasticizer and colorants that are commercially available as ready to use as liquids (Ni, 1997). The polymers used are characterised as either hydrophilic or hydrophobic. Hydrophilic polymers have been used to enhance water uptake (Schneider and Renault, 1997). Hydrophobic coatings have been shown to reduce the water uptake and water movement into seeds (Henning, 1990), as well as to reduce soaking injury (Hwang and Sung, 1991) and imbibitional chilling injury (Priestley and Leopold, 1986; Bulan, 1991; Chachalis and Smith, 2001; Taylor and Kwiatkowski, 2001; Ni, 2001). The reduction of water uptake by hydrophobic polymer coatings, especially the absorption of water from the vapour state (Henning, 1990), may also have a role in improving seed viability by maintaining lower moisture content in uncontrolled storage conditions Synthetic seed coat Polymers Corlett et al. (2014) studied the influence of commercial seed coating polymer with various combinations of calcium, silicon and fungicide to evaluate the vigour and early seedling growth of barley. The results indicated that coating of polymer, fungicide, calcium and silicon can protect the seed against pathogens without affecting the rate of emergence of the barley seedlings while ensuring good seed appearance, adhesion, distribution and coloration.

24 Suma & Srimathi (2014) studied the influence of polymer coating on sesamum seed and seedling quality characteristics. 4 g kg -1 seed has increased the germination to 89% followed by seeds coated with 3 g kg -1 seed (87%). Vinod kumar et al. (2013) studied the effect of seed polymer coating on quality of Pigeonpea. The polymer coated seed showed highest growth parameters viz., germination percentage (83.57%), seedling length (27.52 cm), seedling dry weight (85.17 mg) and vigour index (2810) than untreated redgram seed. The sunflower hybrid RSFH-130 treated with seed coat 5 ml kg -1 gave best results in terms of germination (97%) and vigour index (3377) than control (Shakuntala et al., 2010). Afzal et al. (2009) studied the role of polyamine polymer as priming agent on germination, seedling vigour and anti-oxidative responses of tomato seeds. Priming was achieved by exposing fresh seeds of tomato cultivars Roma and Nagina to 50 mg l -1 Putrescine (Put), 50 mg l -1 Spermine (Spm) and 50 mg l -1 Spermidine (Spd) aerated solutions for 24 h. Priming with Spm and Spd improved seed germination, seedling vigour and enhanced anti-oxidative activity. Zhigang et al. (2008) studied the effect of concentration of super absorbent polymer (SAPs) on the seedling establishment of crops. At 5 and10 cm depth of applying SAPs in soil increased emergence and seedling growth of 3 crops radish, corn and proso. Mixing SAPs with soils increased emergences of corn and proso by 57 times and 24 times respectively at the concentration less than 0.30%. The plant heights were remarkably higher than check for radish at 0.10% concentration and for corn and proso at 0.10 to 0.30% concentrations. John et al. (2005) studied the effect of polymer coating on germination and seedling vigour in Maize cv. Co 1. The optimum dose of seed coating polymer (polykote) was found to be 3 g kg -1 of seed for slurry film coating and was found to be effective in improving physical appearance as well as increased physiological and biochemical parameters of seed. The water required to dissolve polykote for slurry coating was standardized as 5 ml kg -1 of seed. Bittencourt et al. (2005) primed the Mary Washington cultivar of asparagus seeds with PEG 6000 at 25 C. The physiological quality of the seeds was evaluated by standard germination, first count germination, speed of seedling emergence and

25 germination percentage and seedling fresh and dry weights after controlled deterioration test. Primed seeds presented higher germination speed, independently of their initial physiological quality. Li (2002) studied the effects of water holding polymer agent on germination and vigour of Wheat and Corn seeds. The results indicated that polymer had higher effect on the germination per cent and could promote the mean length of the sprout, the mean fresh weight and simplified vigour exponential. Application amount of 4.0 g l -1 was suitable for corn and 6.7 g l -1 was suitable for wheat. Johnson et al. (1999) conducted the laboratory tests for moisture uptake and cold stress germination rates in corn seed which showed that water uptake was significantly reduced in seeds coated with Intelimer polymers compared with uncoated seeds after 48 hours at 10 and 25 C. Water uptake was also much less at 10 C than 25 C. Stand establishment studies in the field showed significant improvements for three of the six hybrids when a polymer seed coating was used. West et al. (1984) studied the effect of several types of seed coating polymers on soybean seeds and the influence of the polymer on water uptake and on maintaining seed quality. One polymer, polyvinylidene chloride, was found to reduce water uptake from a 100% relative humidity environment but increased water uptake from standard germination conditions Biopolymer Biopolymer Chitosan has received much interest for potential wide application in agriculture due to its excellent biocompatibility, biodegradability and bioactivity. This naturally occurring molecule with interesting physiological potential has been getting more attention in recent years. Chitosan enhanced the efficacy of plants to reduce the deleterious effect of unfavorable conditions as well as on plant growth. Chitosan affects various physiological responses like plant immunity, defense mechanisms. Recent studies have shown that chitosan induces mechanisms in plants against various biotic (fungi, bacteria, and insects) and abiotic (salinity, drought, heavy metal and cold) stresses and helps in formation of barriers that enhances plant s productivity. Now-a-day s new approaches and practices are being developed for sustainable agriculture for various crops and vegetables.

26 Chitosan is natural polymer that is applied to crops and also as a seed coating material with the aim of reducing or replacing more costly and environmentally damaging chemicals. With reduced input costs and the potential for increased yields, farmers could gain substantial benefits from these applications of chitosan to crops as foliar spray and seed coating. Chitosan is a one of the most abundant natural amino polysaccharides extracted from the exoskeleton of crustaceans. These substances have a wide variety of applications in agricultural and biotechnological industries. Among the novel families of biological macromolecules, whose relevance is becoming increasingly evident, are chitin and its main derivative, chitosan. Both are the simplest linear polysaccharide composed of α, 1-4 linked D-glucosamine (GlcN) and N-acetyl-D-glucosamine (GlcNAc) with various compositions of these two monomers. Chitin and its derivatives have become a promising alternative treatment due to its natural character, antifungal activity and elicitation of defence responses in plant tissue. Hameed et al. (2014) studied the improvement of seed germination and seedling growth in wheat (Triticum aestivum L.) under osmotic stress induced by Polyethylene Glycol when seeds were primed by chitosan. The final germination percentage (FGP) substantially increased (95%) by chitosan priming treatments compared with the control (80%). It also reduced (74 70 h) the mean germination time (MGT) of the seeds up to 4 h. Under those circumstances, germination energy, vigour and germination index were markedly improved. Liqiang (2014) used different chitosan oligosaccharide solution of concentration (50, 150, 250 and 350 mg l -1 ) with tomato seeds and distilled water as a control and studied their effects on seed germination of tomato. The results showed that the seed germination rate, sprout tomato potential, root fresh weight and germ fresh weight with different concentration of chitosan oligosaccharide solution were higher than those with the control. In 150 mg l -1 of chitosan oligosaccharide solution, tomato seed germination rate, germination index, the root fresh weight, germ fresh weight and vigour index increased by 16.67%, 19.87%, 41.67%, 23.66% and 48.57%, respectively and in 250 mg l -1, tomato seed root growth increased 26.02% whereas in 350 mg l -1, tomato seed germination potential and germ long growth separately increased 36.67% and 26.17% respectively.

27 Hameed et al. (2013) reported that the chitosan priming enhances the seed germination in wheat seeds. All chitosan-priming treatments markedly improved the final germination percentage as compared to non-primed (control) seeds. The germination energy (%) considerably increased by 0.25% chitosan primed seeds, but 0.5% chitosan primed seeds induced the highest increase in the germination index. Mahdavi and Rahimi (2013) reported that the seed treatment with chitosan solutions of 0, 0.01, 0.05, 0.1, 0.2 and 0.5% for 3 hours improves the germination and growth performance of ajowan (Carum copticum). Results indicated that all of chitosan concentrations increased germination percentage, germination rate, seedling vigour index, length and dry weight of hypocotyl and radicle compared to control although, 0.2% chitosan concentration was more effective than other treatments. Mahdavi (2013) studied the effect of chitosan with different concentrations (0, 0.01, 0.05, 0.1, 0.2 and 0.5%) on germination and vegetative growth in Isabgol (Plantago ovata Forsk). Chitosan treatments increased germination percentage, germination rate, length and weight of shoot and root. Plants pretreated with 0.2% chitosan showed highest germination percentage (63.33%), root length (5.45 cm) and shoot length (3.65 cm) in contrast to other concentrations and control (untreated). Al-Tawaha and Al-Ghzawi (2013) primed the lentil seeds with different chitosan concentrations 0, 1, 3 and 6 g l -1, five levels of salinity 50, 100, 200 and 300 mm NaCl, including control were added to the primed lentil seeds. Different variables have been taken including germination percentage, hypocotyl length, radical length, hypocotyl dry weight and radical dry weight. The results indicated that the highest growth parameters were recorded when seed primed with 3 g l -1 chitosan. Ahmed and Rasel (2012) carried out an experiment to investigate the effect of chitosan on growth, yield contributing characters and yield of rice cv. BRRI dhan29. The results revealed that 50 mg l -1 chitosan produced significantly the tallest plant (99.67 cm) and highest TDM (33.46 g), higher number of leaves (73.00), LAI (2.83) and number of total tillers per hill (16.67) at 90 DAT. Length of panicle (28.26 cm), number of grains per panicle (184.67), 1000 grain weight (29.04 g), grain yield (7.05 t ha -1 ), biological yield (17.21 t ha -1 ) and harvest index (40.98%) were noticed from 50 mg l -1 chitosan treated plants at harvest and concluded that almost all of growth, yield characters and yield were found superior in chitosan 50 mg l -1 compared to control.

28 Shushu et al. (2012) studied the cotton seed germination by soaking the seed in different concentrations (0.1, 0.2, 0.3, 0.4, and 0.5%) of chitosan solution for 12 hours in comparison with seeds in water. Then cultured them for 7 days at room temperature and everyday under natural light at 8 hours. Seed germination rate, germination power, germination index, vigour index were higher than the control. When the concentration was 0.2% the seed germination rate, germination energy, germination index, vigour index were increased by 16.7%, 3.17%, 2.63 and 30.7 respectively. Zeng et al. (2012) used chitosan based bioactive coatings (1, 2, 3, 4 and 5%) for soybean seed protection and also for enhancing the germination and quality of soybean seeds. The chitosan coating increased seed germination (92.5%), germination power (81.9%), germination index (49.7%), plant growth and soybean yield efficiently, especially at a concentration of 5% and the yield was increased by about 20% compared with control. Pingping et al. (2011) studied the effect of chitosan on seed germination and seedling growth of Toona sinensis with standard germination test and sand culture method. The results showed that the seeds germination and seeding growth of T.sinensis was promoted by chitosan. When treated with chitosan at a concentration of 0.20%, the seed germination rate, germination power index, vigour index and the radicle length, seedling height, fresh weight increased by 59.6%, 145.0%, 65.6%, 187.9%, %, 73.8% and 35.5% respectively in contrast with control. Qizhong (2011) studied the effect of chitosan on germination and rooting of wheat. Chitosan at 4-6 mg ml -1 significantly increased the wheat seed germination, sprouts rate, seedling growth and seedling vigour than untreated control at ph, 6.5. Shanhua and Yinglun (2011) studied the maize seed germination by soaking in solution of chitosan. The maize seeds treated with 150 mg l -1 chitosan showed highest seed germination, germination rate, germination potential and the root activity of maize seedlings than untreated control. Weiwei et al. (2011) studied the effects of chitosan seed coating on seed germination and seedling growth in maize. The results indicated that the treatment with chitosan seed coating could improve seed germination percentage, germination index and vigour index whose germination rate even increased 6.9% than control and also height of maize seedling, culm diameter and root length were increased and root-shoot ratio was significantly enhanced.

29 Guan et al. (2009) examined the use of chitosan to prime maize seeds. Although chitosan had no significant effect on germination under low temperatures, it enhanced germination index, reduced the mean germination time, and increased shoot height, root length, and shoot and root dry weights in two tested maize lines. Changmin et al. (2009) studied the effect of chitosan on tomato seed and seedling. Tomato seeds were soaked in different dilutions of chitosan solution which varied from 100 to 600 times. The results showed that the tomato seed germination rate, germination potential, main root length and root activity were higher than the control when treated with water. The chitosan diluted 300 times heavily activated the tomato seed germination but when diluted 400 times it mainly activated the tomato root length and root activity. Boonlertnirun et al. (2008) studied the stimulative effective of biopolymer chitosan on growth and yield performance of rice. Four treatments were done as follows: no chitosan application, seed soaking with chitosan solution, seed soaking and soil application with chitosan solution and seed soaking and foliar spraying with chitosan solution. The results showed that application of chitosan by seed soaking and soil application four times throughout cropping season significantly increased the growth parameters plant height, leaf greenness, tiller numbers per plant, dry matter accumulation and yield parameters like grain yield, panicle numbers per plant, seed numbers per panicle, 1,000 grain weight over the other treatments. Zuji (2008) studied the effect of different chitosan concentrations (0.1, 0.2 and 0.3%) on the sprouting of seeds and physiological and biochemical characteristics of seedlings of Pinus massoniana, Koelreuteria paniculata, Acacia farnesiana, Robinia pseudoacacia, corn and wheat. The results showed that the germination rate, germinating energy, growth of seedlings and vigour index of several seeds are higher than those of the control (untreated). Borkowski et al. (2007) found that spraying tomato plants with chitosan increased the vigour of these plants after some months. Tomato seeds treated with Biochikol 020 PC (chitosan) increased the plant height (142 cm) and fruit yield (649 g) over control after 4 months of growth period. Yingjie et al. (2007) reported the effect of different concentrations (0.1, 0.3 and 0.5%) chitosan on germination of cucumber (Jinchun No.4) Seeds. The results showed that all the three concentrations of chitosan solution could improve germinating

30 percentage, germinating energy, germinating index and vigour index of cucumber seeds than untreated control. Yongxia et al. (2007) treated the soybean seeds with chitosan to evaluate its effects on the germination. The seeds treated with 1.0 ml l -1 chitosan showed the highest germination rate, germination power and fresh weight of single plant, germination index and vigour index of soybean seedlings. Shao et al. (2005) studied the effects of seed priming of maize with acidic (ph, 5.1) and near neutral (ph, 6.4) chitosan solutions on seed germination and physiological characteristics of seedling. The results showed that priming treatments with two acidic chitosan solutions significantly enhanced the energy of germination, reduced mean germination time and increased shoot height and chlorophyll content. Guoyong and Wangou (2004) studied the effect of chitosan seed treatment on bermuda grass seed germination. The results showed that proper chitosan concentration (2%) could increase vigour index and seedling growth, improving germination rate and potential. Ouyang and Langlai (2003) studied the seeds of non-heading chinese cabbage cv. Dwarf hybrid No.1 dressed with chitosan at the rate mg g -1 of seeds and leaf spraying with mg ml -1 increased total fresh weight, leaf area, plant height, root length, soluble protein and soluble sucrose in leaves, while the content of crude fibre decreased. Xianling et al. (2002) indicated that seeds of mulberry cultivar were coated with chitosan solution at 3% prepared from silkworm chrysalises increased the respiration rate of germination seeds, root vigour, chlorophyll, protein content and peroxidase in seedlings as well as nitrate reductase and amylase activities. Yanfeng et al. (2002) studied the effect of various concentrations (0.3, 0.5, 0.7, and 0.9%) chitosan on seed germination and growth parameters in maize. The result showed that germination percentage and germination index and seedling vigour index of the seed treated by 0.5% chitosan was the highest in all growth parameters. Xue et al. (2002) conducted the coating of rapeseed (Brassica chinensis) with small molecular weight chitosan showed positive effects on germination index, growth of seedlings and root length.

31 Ruan and Xue (2002) showed that rice seed coating with chitosan accelerated their germination and improve their tolerance to stress conditions. The results showed that chitosan coating (1.5%) significantly improved germination percentage and activities of β-amylase under normal (H 2 O) or salt stress conditions, and decreased activities of α-amylase under salt stress conditions as compared with the control (noncoating treatment). Ohta et al. (1999) reported that chitosan application to the soil at sowing time remarkably enhanced plant growth and flowered 15 days earlier than the control. Moreover, a greater number and weight of flowers were produced by chitosan application. Chibu and Shibayama (1999) studied chitosan application on early growth of four crops: soybean, lettuce, tomato and rice. The results showed that chitosan at 0.1 and 0.5% increased leaf area, leaf dry weight and leaf length of soybean, lettuce and rice whereas chitosan at 0.1% showed positive effects on leaf area, leaf length and dry weight of tomato. Yan (1998) studied the effect of different concentration of acidified chitosan on tobacco seed germination. The results showed that chitosan solution of 0.25% concentration could improve germinating percentage, germinating index and vigour index of tobacco seeds and stimulate the growth of seedling than untreated control. 2.2 EFFECT OF SEED COAT POLYMERS IN STORAGE Proper seed treatments are needed to maintain the seed quality during storage. The seed deterioration starts right at the field level immediately after the physiological maturity. The seed has to be stored safely so that the viability and vigour is maintained intact. Deterioration of seed during storage leads to different changes at various levels viz., impairment or shift in metabolic activity, compositional changes, decline or change in enzyme activities, phenotypic, cytological changes apart from quantitative losses. Seed deterioration is inevitable and irreversible process but the rate of seed deterioration could be slowed down either by storing the seeds under controlled conditions or by imposing polymer film coating along with seed treatment chemicals. As the controlled condition involves huge cost, seed coating remains the best alternative approach to maintain the seed quality.

32 The polymer film coat provides protection from the stress imposed by accelerated ageing, which includes fungal invasion. The coat is thin, simple to apply, diffuses rapidly and non-toxic to the seedlings during germination. It improves plant stand and emergence of seeds, helps in accurate application of the chemical, reduces chemical wastage and helps to make room for including all required ingredients, protectants, nutrients, plant growth promoters, hydrophobic/hydrophilic substance and oxygen suppliers etc. Verma and Verma (2014) studied the effect of seed coating material on germination and seedling vigour of soybean. All the seed coating treatments of soybean seed showed germination above Minimum Seed certification Standard (>70 %) germination up to 6 months of storage. At the end of the 8 th month of storage, polymer 3 ml kg -1 showed the germination (71.2%) and vigour index (8510) which was significantly higher than control (66.2% and 7854). Kaushik et al. (2014) evaluated the seed quality of maize with polymer film coating in storage by treating the seeds with polymer. The results revealed that, the seed treatment with 9 ml kg -1 of seed attained superior growth parameters like germination percentage (82.15%), root length (11.55 cm), shoot length (17.51 cm) and vigour index (1439) after 6 months of storage than the untreated control seed. Ambika et al. (2014) reported the storability of polymer coated CORH 3 hybrid rice seeds. The seeds coated with Quick roots polymer recorded higher germination of 79% at ninth month of storage when compared to untreated control seeds (71%). Rettinassababady and Ramanadane (2014) studied the seed quality status of polymer coated Bt cotton (Gossypium spp.) during storage under coastal environment. The seeds were coated with synthetic polymer 3 ml kg 1 diluted in 5 ml of water kg 1 seed) and observations on percentage seed germination, seed moisture content and seed infection were recorded at bimonthly intervals upto 4 months. The results of standard germination test on treated and untreated stored seeds indicated that seeds treated with polykote polymer (85%) excelled over the untreated seeds. Shakuntala et al. (2014) studied the influence of polymer coating and storage on quality of sunflower seeds. The storability of sunflower (RSFH-130) was improved due to the treatment with polymer seed 5 g kg 1 of seeds which derive higher germination percentage (75.4%) and vigour index (2417) than the untreated seed after 14 months of storage.

33 Rettinassababady et al. (2012) studied the effect of polykote seed coating on per cent seed germination during storage in hybrid paddy KRH 2. The seed coating with polymer 4 ml kg -1 of seed gave highest germination percentage after 4 months of storage than the untreated control. Basavaraj et al. (2008) conducted an experiment on the effect of polymer film coating on storability of onion seeds. The treatment of seed coating with 12 ml kg -1 recorded higher germination, vigour index, dry weight of seedlings and lower seed infection and electrical conductivity as compared to control throughout the storage period. Manjunatha et al. (2008) studied the effect of seed coating on seed quality in chilli during storage. Seed coating with 3, 5, 7 g kg -1 of seed showed that treatment with 7 g kg -1 of seed bestowed highest germination, vigour index of the initial, 3 rd, 6 th, 9 th and 12 th month than other polymer concentration treatments and untreated control. Kunkur et al. (2007) studied the effect of seed coating with 3, 4, 5 g kg -1 of seed on seed quality in cotton during storage. The polymer treatment with 5 g kg - 1 of seed bestowed the highest germination and vigour index of the initial, 3 rd, 6 th and 9 th month than other polymer concentration treatments and untreated control. Giang and Gowda (2007) studied the influence of seed coating with synthetic polymers (Polykote TM and Littles Polykote W Yellow) on seed quality and storability of hybrid rice. The seed coated with Littles Polykote W Yellow stored in polythene bag recorded higher germination (71%) than the seed coated with Polykote TM (70%) and untreated (66%) seed at the end of the 10 th month of storage. 2.3 ANTIMICROBIAL ACTIVITY OF SYNTHETIC AND BIOPOLYMERS Seed treatments consist of chemical or biological substances that are applied to seeds to control infection by plant disease-causing organisms. Seed coating provides protection during the critical germination and vigour establishment stages but the seeds and emerging seedlings are unable to protect themselves from invasive pathogens. Further the polymer coat also provides protection from the stress imposed by accelerated ageing, including fungal invasion which serve as the first line of defence for

34 seeds and seedlings besides improving seed germination, seedling emergence, seedling establishment and plant vigour. Among the usage of these polymers, biopolymers will give better protection over the synthetic polymers in the view of protection against the plant pathogens. Mostly the synthetic polymers are known to enhance the growth promotion because they form thin oily film around the seed surface and act as barrier for direct contact between water and seed surface. In this way slow absorption of water into the seed by synthetic polymer activates the enzymes for the seed germination and seedling establishment but is not associated in triggering the defence mechanisms within the seed and cannot cure the seeds from already established pathogen infections. The chemical groups present in the synthetic polymer do not easily intermingle with the natural plant defence chemicals which are present within the plant. Whereas the biopolymers are complete analogues with some minor modifications of the natural defence chemicals present in the plant and sometimes easily activate the defending mechanisms along with growth promotion in plants. Leuba and Stossel (2016) demonstrated the antifungal effect of chitosan in field experiments. A marked reduction of root rot in beans and of vascular wilt in radishes, both caused by Fusarium spp., were observed subsequent to addition of chitosan to soil. Chitosan amendment suppressed total fungal population and stimulated lytic and antibiotic producing microorganisms such as actinomycetes. Al-Hetar et al. (2011) investigated in vitro antifungal activity of chitosan against Fusarium oxysporum f. sp. cubense race 4 (FocR4) the causal agent of banana wilt. Chitosan at all concentrations tested reduced the hyphal growth of FocR4 on potato dextrose agar media and recorded maximum inhibition of 76.36% at 8 mg ml -1. Chitosan inhibited the sporulation of FocR4 by a maximum of 96.53% at 8 mg ml -1 chitosan and 100% inhibition for spore germination was recorded at all concentrations tested. Paulin et al. (2011) determined the protective effect of chitosan in maize seedlings subjected to abiotic stresses which facilitated the infection with fungi such as Aspergillus flavus and Fusarium moniliforme and consequently increased the production of mycotoxins. Three treatments were tested (a negative control, a positive control, and a group coated with chitosan solution) under four abiotic stresses conditions since their germination stage: drought, moisture, acid ph and alkaline ph.

35 Positive effect was observed in seeds treated with chitosan or stressed with acidic ph in dimensions of seedlings and there was no fungal growth. Li et al. (2008) studied the antifungal activities of chitosan in vitro with different concentrations against Aspergillus niger. The results showed that chitosan with 0.1% concentration gave complete inhibition (100%) of Aspergillus niger. The effects of chitosan on Aspergillus niger and hyphal ultrastructure were examined to gain more information on its mode of action. The ultrastructure morphology investigated by transmission electron microscopy results indicated that chitosan acts on Aspergillus niger by inhibiting the growth of sporules. The fluorescein isothiocyanate labelled chitosan observation has elucidated the antifungal activity of chitosan mainly by inhibiting the DNA to RNA transcription. So the antifungal activity of chitosan towards Aspergillus niger was the combined effect of the above two observations. Guerrero et al. (2008) investigated the toxic effect of chitosan on important root pathogenic and biocontrol fungi (nematophagous, entomopathogenic and mycoparasitic). The results showed that the root pathogen (Fusarium oxysporum f. sp. radicis-lycopersici) and mycoparasitic fungi Pochonia chlamydosporia were more sensitive to chitosan than nematophagous and entomopathogenic fungi. Juan et al. (2008) studied the effects of chitosan treatment and inoculation on dry rot in tubers and slices of potato. The results showed that chitosan treatment at 0.25% significantly reduced the lesion diameter of potato inoculated with Fusarium sulphureum and it also increased the activities of peroxidase and polyphenoloxidase, and the contents of flavonoid compounds, lignin in tissues and ß-1,3-glucanase. Yongxia et al. (2007) treated soybean seeds with chitosan to evaluate the induced resistance to Fusarium oxysporum. The results showed that the seeds treated by 1.0 ml l -1 chitosan had germination rate, germination power, fresh weight of single plant, germination index, vigour index and resistance in treated soybean seeds had better effect reaching 65.45%. Burrows et al. (2007) conducted a growth enhancement study with black-eyed peas exposed to the different acidified chitosan treatments. A similar anti-fungal experiment with peanut seeds infested with Penicillium was conducted as well. Observations on plant height, stem diameter and leaf counts were recorded biweekly for 4 months. The results suggested that seeds pre-treated with the acidified (0.5% HCl)

36 chitosan had the best overall growth and gave better results for fungal elimination than the commercial fungicide captan. Photchanachai et al. (2006) investigated the effects of chitosan on the growth of Colletotrichum spp. that caused anthracnose disease in chilli. Seeds treated with chitosan solution for 60 min placed on a wet paper surface inoculated with a Colletotrichum spore suspension showed reduced fungal infection. Chitosan treatment, particularly at 0.8%, also increased seedling survival to 77% whereas, without chitosan, about 33%. The lignin content of seedlings obtained from chitosan treated seeds was higher than that without chitosan treatment. Chitosan was evaluated for growth inhibition of Fusarium oxysporum, Penicillium digitatum and Rhizopus stolonifer under in vitro. Except for P. digitatum sporulation, chitosan at 1.5% onwards inhibited both mycelial growth and sporulation. The sporulation of all these fungi were inhibited when treated with 3%. F. oxysporum was the most sensitive to chitosan because even at 1.5%, it showed 100% inhibition (Banos and Lopez, 2004). Barka et al. (2004) evaluated the potential of chitosan both to stimulate plant development and to induce protection from Botrytis cinerea in Vitis vinifera L. plantlets. The presence of 1.75% (v/v) chitogel in the culture medium was the optimal concentration for in vitro grapevine plantlet growth, as determined by measurements on enhancement of root and shoot biomass. Chitogel reduced the development of Botrytis cinerea and induced cytological alterations to the pathogen. When challenged with the fungus, a significant decrease in disease incidence was observed in plants growing on medium supplemented with chitogel. Hassni et al. (2004) studied the effect of chitosan on the growth and morphology of Fusarium oxysporum f. sp. albedinis (Foa), the causal agent of bayoud disease and its ability to elicit a defence reaction against this fungus in date palm roots. Chitosan injected into roots at three concentrations (0.1, 0.5 and 1 mg ml -1 ), elicited peroxidase (PO) and polyphenoloxidase (PPO) activity and particularly at a concentration of 1 mg ml -1, increased the level of phenolic compounds which led to an accumulation of nonconstitutive hydroxycinnamic acid derivatives, known to be of great importance in date palm resistance to bayoud. The broad spectrum antimicrobial activity of a synthetic peptide polymer D 4 E 1 completely inhibited the growth of all fungi studied ranged from 4.67 to 25 μm.

37 Microscopic analysis of D 4 E 1 effects on fungal morphology of Aspergillus flavus and R. solani revealed abnormal hyphal growth and discontinuous cytoplasm. After 8 h of exposure to 25 μm D 4 E 1, A. flavus spore germination was reduced by 75% (Rajasekaran et al., 2001). Laflamme et al. (2000) studied the effect of chitosan in vitro on the growth, morphology and ultrastructure of Cylindrocladium floridanum, Cylindrocarpon destructans, Fusarium acuminatum and Fusarium oxysporum. Chitosan was found to reduce the radial growth, induced the morphological and ultrastructural alterations of all the fungi studied with some differences. Chitosan treatment (2-8 mg ml -1 ) of wheat seeds significantly improved seed germination and vigour in two cultivars (Norseman and Max) of spring wheat by controlling seed borne Fusarium graminearum infection was reduced to >50% at higher chitosan treatments compared to the control. Results indicated that chitosan controlled seed borne F. graminearum infection and increased the resistance in seedlings by stimulating the accumulation of phenolics and lignin. Chitosan also inhibited fungal transmission to the primary roots of germinating seedlings (Reddy et al., 1999). Zhang and Quantick (1998) studied the effects of chitosan coatings (1.0 and 2.0% w/v) in controlling the decay of strawberries and raspberries and maintaining their quality. Chitosan coating proved almost as effective as the fungicide TBZ in controlling the decay of berries stored at 13 C caused by Botrytis cinerea and Rhizopus spp. Lafontaine and Benhamou (1996) investigated the potentiality of chitosan for controlling Fusarium crown and root rot of greenhouse-grown tomato caused by Fusarium oxysporum f. sp. radicis- lycopersici (FORL). The amendment of plant growth substratum with chitosan at concentrations of 12.5 or 37.5 mg l -1 significantly reduced plant mortality, root rot symptoms and yield loss attributed to FORL. Maximum disease control was achieved with chitosan at 37.5 mg l -1, when plant mortality was reduced by more than 90% and fruit yield was comparable with that of non-infected plants. Benhamou et al. (1994) applied chitosan at concentrations ranging from 0.5 to 1 mg ml -1 as seed coating and substrate amendment prior to infection with the fungus Fusarium oxysporum f. sp. radicis-lycopersici. A combination of seed coating and substrate amendment was found to significantly reduce disease incidence as judged by the decreased number of root lesions and the healthier appearance of the root system.

38 Although seed treatment alone could induce a delay in symptom development. Examination of the root tissues at sites of fungal penetration revealed that a pretreatment with chitosan was always associated with the expression of plant defense reactions. Benhamou and Theriault (1992) applied chitosan at concentrations ranging from 0 5 to 2 mg ml 1 to tomato plants prior to inoculation with the root pathogen, Fusarium oxysporum f. sp. radicis-lycopersici. It was found to markedly reduce the number of root lesions caused by the fungus, and to drastically increase the formation of putative physical barriers in infected root tissues. The effect of chitosan on the induction of host cell reactions was observed at an optimal effect at 2 mg ml 1. Effect of chitosan coating on decay of strawberry fruits was investigated by Ghaouth et al. (1992). Freshly cut strawberry fruits were inoculated with spore suspensions of Botrytis cinerea and Rhizopus stolonifer and subsequently coated with chitosan solutions (10 and 15 mg ml -1 ). After 14 days of storage, decay caused by B. cinerea (30%) and R. stolonifer (32.1%) was markedly reduced by chitosan coating (15 mg ml -1 ) than the untreated control (72.7% and 78%). Chitosan was very effective in inhibiting spore germination, germ tube elongation, and radial growth of B. cinerea and R. stolonifer in culture. Further they found that chitosan induced cellular leakage of amino acids and proteins in Botrytis cinerea and Rhizopus stolonifer, causing morphological changes in R. stolonifer. The ultrastructural study showed that chitosan caused deep erosion of the cell wall as well as increasing the cell wall thickness. Kendra and Hadwiger (1984) characterized the antifungal effect of chitosan on Fusarium solani f. sp. pisi and F. solani f. sp. phaseoli and their ability to elicit pisatin formation in immature pea pods. The antifungal and pisatin inducing abilities were shown to increase as the polymer size increased. Chitosan and its derivatives showed maximal activities in both antifungal action and pisatin induction, while chitin and chitin derivatives showed no antifungal activity and only weak pisatin formation activity. These findings showed that high molecular weight chitosan fragments are more active in both antifungal and pisatin formation activity than the intermediate and low molecular weight fragments.

39 2.4 SCREENING OF FUNGICIDES IN VITRO Use of chemicals for the management of soil borne diseases has been reported for various soil borne plant pathogens. Several researchers studied the efficacy of these fungicides against collar rot pathogens under in vitro conditions Screening of fungicides against Fusarium spp. in vitro Species of Fusarium are responsible for a variety of seedling diseases including seed rots, pre and post-emergence damping-off, wilts, root rot, and late season dampingoff (Carey and Kelley, 1994; Pawuk, 1978). Seed-borne contamination is believed to be the primary source of Fusarium spp., (Fraedrich and Dwinell, 1996). Several disease management strategies are available e.g. cultural, biological control, resistant cultivars, crop rotation and chemical control (Kamal et al., 2009). Resistant cultivars are the most effective measure of controlling Fusarium wilt (Beckman 1987; Amini 2009), but new races of the pathogen appear to overcome resistance genes in currently grown cultivars (Tello-Marquina and Lacasa, 1988). Dar et al. (2013) conducted in vitro study of nine fungicides carbendazim, hexaconazole, thiophanate methyl, triadimefan, metalaxyl, mancozeb, captan, copper oxychloride and chlorothalonil against Fusarium oxysporum f. sp. pini. Among these fungicides maximum inhibition in mycelial growth and spore germination was observed in carbendazim (10, 20, 30, 40 ppm) followed by other fungicides. Mamza et al. (2012) evaluated the effect of six fungicides at three rates i.e. one and half, one and half of recommended rates (1.5x, 1.0x and 0.5x mg a.i/ml) on the spores of Fusarium pallidoroseum isolated from castor (Ricinus communis) in vitro. Benomyl, benomyl+thiram and tricyclazole completely inhibited sporulation at 1.5x, 1.0x and 0.5x mg ml -1. Nisa et al. (2011) tested the in vitro inhibitory effect of fungicides (carbendazim, hexaconzol, bitertanol, myclobutanil, mancozeb, captan and zineb) on mycelia growth and spore germination of Fusarium oxysporum. However, the hexaconozole at highest concentration (1000 ppm) caused highest reduction of mycelial growth (8.80 mm) followed by carbendazim (9.40 mm), bitertanol (18.60 mm) and myclobutanil (20 mm) at the same concentration.

40 Jamil and Kumar (2010) evaluated five fungicides viz., carbendazim, mancozeb, maneb, thiram and ziram against four Fusarium spp. found in the phyllosphere of foliage plants. Carbendazim showed a broad spectrum of fungitoxic activity against four species of Furasium. Amini and Sidovich (2010) evaluated the effect of benomyl, carbendazim, prochloraz, fludioxonil, bromuconazole and azoxystrobin for their efficacy against Fusarium oxysporum f. sp. lycopersici under in vitro. Seven different concentration (0.0001, 0.001, 0.01, 0.1, 1, 10, 100 μg ml -1 ) were used for assessment of their inhibitory activities against the pathogen through mycelial growth inhibition on PDA. Prochloraz and bromuconazole were the most effective fungicides against the pathogen in vitro followed by benomyl and carbendazim. Allen et al. (2004) evaluated the effect of benomyl, difenoconazole, hydrogen dioxide, mancozeb, and thiabendazole against four species of Fusarium for their ability to inhibit the pathogen. Fusarium spp. did not grow on benomyl and mancozebamended media. Only F. solani grew on thiabendazole and hydrogen dioxide amended media. Kopacki and Wagner (2006) tested ten fungicides in vitro for their effectiveness to inhibit the linear growth of three isolates of Fusarium avenaceum. The most effective fungicides were difenaconazole, carbendazim and flusilazol while the least effective were mancozeb, chlorothalonil and captan. Bardia and Rai (2007) evaluated five fungicides namely, carbendazim, thiophanate methyl, chlorothalonil, captan and carboxin at 1, 10, 20, 50, 100, 200 and 500 μg ml -1 concentrations using poisoned food technique for the management of wilt of cumin (Cuminum cyminum) caused by Fusarium oxysporum f.sp. cumini. On the basis of in vitro studies carbendazim was the best treatment in inhibiting the growth of the pathogen. The effect of mixture of metamidoxime and copper oxychloride on F. oxysporum f.sp. lycopersici was tested in vitro and the results showed that these fungicides had a strong synergistic effect and could be used as a basis for a new product to control tomato diseases (Nedelcu and Alexandri, 1995). Chemical control for Fusarium wilt of castor was attempted for the first time by Cristinzio (1946). The use of seed treatment as a preventive measures was highlighted.

41 Fungicides including benomyl, captafol, imazalil, thiram, and prochloraz-mn, provided inconsistent control of Fusarium crown and root rot on tomatoes, leaving problematic residues in fruit tissues (Marois and Mitchell, 1981; Jarvis, 1988; Hartman and Fletcher, 1991) Screening of fungicides against Aspergillus spp. in vitro Nathawat and Partap (2014) conducted the evaluation of fungicides against collar rot of groundnut caused by Aspergillus niger van Tiegham. Results revealed that all the systemic fungicides were capable of inhibiting the growth of the test fungus at different concentrations as compared to check. Tebuconazole and propiconazole proved to be the most effective in inhibiting cent per cent growth of the test fungus at all the concentrations (100, 250, 500, 750 and 1000 ppm). Saharan and Singh (2014) tested the efficacy of fungicides under in vitro against A. niger showed that propiconazole, carbendazim and carboxin completely inhibited the mycelial growth up to 100 per cent at 200, 500 and 1000 ppm concentration, respectively. Captan and thiram were less effective as they inhibited and per cent of fungal growth, respectively at higher concentration of 1000 ppm. Mahato et al. (2014) conducted an in-vitro experiment to determine the effect of different fungicides on radial colony growth of Sclerotium rolfsii Sacc. following poison food technique in PDA medium. One systemic (carbendazim), three contact (mancozeb, copper oxychloride, chlorothalonil) and three combination of systemic and contact fungicides (Vitavax power, Krilaxyl gold, Curzate) were evaluated against S. rolfsii in laboratory. Viavax Power (95%) was the best fungicide to restrict the fungal growth effectively followed by Krilaxyl gold (94.04%), chlorothalonil (93.55%), mancozeb (90.66%) and Curzate (87.77%). Manu et al. (2012) tested the efficacy of fungicides against Sclerotium rolfsii causing foot rot disease of finger millet under in vitro conditions. Out of four combination products viz., Vitavax power, Avatar, Merger and Nativo the combination of carboxin 37.5%+thiram 37.5% (Vitavax power) inhibited the growth of at all the five concentrations tested. Anand et al. (2010) evaluated six fungicides for their antifungal activity against Aspergillus niger, the causative agent of crown or collar rot disease in groundnut. Out

42 of six fungicides tested, better results were obtained with carbendazim (30 μg ml -1 ) and hexoconazole (75 μg ml -1 ). 2.5 SCREENING OF BIOCONTROL AGENTS IN VITRO The usage of chemical compounds is a widely applied method to control soilborne diseases, but these have adverse effects on the environments affecting the beneficial functions of microorganisms living in the soil and root ecosystem (Harman et al., 2004). Biological control has been advanced as an eco-friendly alternative to synthetic fungicides, and remarkable success has been achieved by utilizing antagonistic microorganisms. For example, Trichoderma species are common saprophytic fungi found in the soil and many studies have focused on their ability to reduce the incidence of the disease caused by plant pathogenic fungi (Elad, 2000; Freeman et al., 2004; Dubey et al., 2007). The Trichoderma species are useful avirulent plant symbionts that play an important role especially in controlling soilborne fungal pathogens. Commercial biological products based on the Trichoderma species are manufactured and marketed worldwide as biofungicide, plant biostimulant and soil fertilizers for use on a wide range of crops. The use of these products for the management of diseases is ecofriendly, economical and also practical for improving soil health Screening of biocontrol agents against Fusarium spp. in vitro Altinok and Erdogan (2015) examined the Trichoderma harzianum under in vitro against the pathogenic strains of Fusarium oxysporum spp. Trichoderma harzianum showed significant inhibition of mycelial growth in the pathogenic strains of F. oxysporum and the maximum inhibition were recorded when the T. harzianum strain T16 was used (72.69%). Bardia and Rai (2007) tested the fungal antagonists for the management of wilt of cumin (Cuminum cyminum) caused by Fusarium oxysporum f. sp. cumini. On the basis of in vitro studies, Trichoderma harzianum isolate I6 was the best treatment in inhibiting the growth (64.4 %) of the pathogen. Bashar and Chakma (2014) tested seven soil fungi viz. Aspergillus flavus, A. fumigatus, A. niger, A. terreus, Penicillium spp., Trichoderma harzianum and T. viride associated with the rhizosphere, non-rhizosphere and rhizoplane of brinjal plants to observe their antagonistic potential against the test fungi Fusarium oxysporum and F.

43 solani. Out of seven soil fungi T. harzianum was found most effective to control the growth of both the test fungi. Dar et al. (2013) conducted in vitro study with seven bioagents namely: Trichoderma harzianum, Trichoderma viride, Gliocladium virens, Lacaria laccata, Boletus edulis, Suillus placids and Russula lutea by using dual culture and cultural filtrate techniques for their effect on the inhibition of mycelial growth and spore germination of Fusarium oxysporum f. sp. pini. In dual culture maximum mycelial growth inhibition was recorded in T. harzianum followed by filtrates of bioagents. Eshghi et al. (2015) investigated the biocontrol potential of Trichoderma harzianum isolate against Fusarium solani isolates that cause dry rot of potato. The antagonistic capacity of the T. harzianum isolate was tested using dual culture and volatile metabolites assays. In the dual culture assay, T. harzianum significantly reduced the growth of F. solani isolates at different time points compared to the control treatments. Tapwal et al. (2015) screened two Trichoderma species, (T. viride and T. harzianum) against five seed borne phytopathogens (Curvularia lunata, Fusarium oxysporum, Alternaria alternata, Colletotrichum gloeosporioides and Rhizoctonia solani) by dual culture technique. Both antagonists have exerted inhibitory effect on the growth of selected seed borne phytopathogens to a varied extent. Kucuk and Kivanc (2004) studied the interactions between Trichoderma harzianum strains and some soilborne plant pathogens (Gaeumannomyces graminis var. tritici, Fusarium culmorum and F. moniliforme) on PDA medium. All T. harzianum strains tested produced a metabolite that inhibited the growth of plant pathogenic fungi on PDA medium. Mahmood et al. (2015) tested the biological control agents in vitro against chickpea wilt pathogen by dual culture technique. Biocontrol agents with Foc showed that Pseudomonas fluorescens had more (70.94%) mycelial growth inhibition of pathogen (Foc) with over control. Trichoderma harzianum was proved to be second best followed by Rhizobia spp. and Bacillus subtilis with 63.95%, 60.79% and 57.68% growth inhibition respectively. Sharfuddin and Mohanka (2012) assessed the indigenous potential of bio-agents and their antagonistic potential against Fusarium oxysporum f. sp. lentis. They worked

44 out with nineteen isolates of Trichoderma were isolated and these were ascribed to three species namely: Trichoderma harzianum (Th), Trichoderma viride (Tv) and Trichoderma koningii (Tk). The isolate Th-5 caused maximum inhibition (82.8%) followed by Th-7 (82.3%), Tv-2(79.2%), Tv-18 (74.4%) and Tk-9 (71.0%). Souna et al. (2012) tested the effect of Trichoderma harzianum on mycelial growth of Fusarium oxysporum f. sp. albedinis (Foa) in dual culture. The antagonistic effect of Trichoderma harzianum revealed that it has inhibited mycelial growth of the pathogen by more than 65% compared to the control when incubated for four days at 25 C. Moreover, beyond this period and after six days, T. harzianum invades and also sporulates on Fusarium oxysporum f. sp. albedinis colonies revealing its high mycoparasitism. Sundaramoorthy and Balabaskar (2013) evaluated the efficacy of the native isolates of Trichoderma species to promote the growth and yield parameters of tomato and to manage Fusarium wilt disease under in vitro and in vivo conditions. Under in vitro conditions, the results revealed that Trichoderma harzianum (ANR-1) isolate was found to effectively inhibit the radial mycelial growth of the pathogen by 53% when compared to all other isolates Screening of biocontrol agents against Aspergillus spp. in vitro Lone et al. (2012) tested the potential efficacy of Trichoderma harzianum against the Aspergillus niger. T. harzianum caused the maximum growth inhibition in A. niger (75%) followed by C. spherospermum (72.2%) and F. oxysporum (25%) at the specific temperature and ph. Nathawat and Partap (2014) evaluated of biocontrol agents against collar rot of groundnut caused by Aspergillus niger van Tiegham. Results revealed that Trichoderma harzianum (81.4 %) showed strong antagonistic effect, followed by T. viride (77.4%). Rajasekharam and Reddy (2013) tested the antagonistic effect of ten morphologically different isolates of Trichoderma spp. The results revealed that all the ten bioagents tested in vitro by dual culture technique against A. niger significantly inhibited the mycelial growth of the test pathogen over untreated control. Castillo et al. (2011) determined the antagonism effect of Mexicans Trichoderma strains on S. sclerotiorum and S. cepivorum under in vitro. The results revealed that T. asperellum (T1 and T11) were the most efficient species with the

45 highest antagonistic effect against S. sclerotiorum ( %) and S. cepivorum ( %). Asad et al. (2014) investigated the in vitro ability of three different isolates of Trichoderma (T. asperellum, T. harzianum and Trichoderma spp.) against soil borne plant pathogen Rhizoctonia solani. The results showed that the T. asperellum was more effective and consistent in inhibiting growth of the pathogen. Joeniarti et al. (2014) tested the in vitro antagonistic activity of two T. asperellum isolates (TK & TS) against the plant pathogen Phytophthora infestans by using dual culture method. The results showed that the antagonistic activity of the two T. asperellum isolates against P. infestans was 5.08% (TK) and 16.37% (TS). Paica (2014) assessed the in vitro efficacy of Trichoderma asperellum isolate Tdal12 against important pathogens of corn (F. oxysporum, F. graminearum and Aspergillus flavus). Trichoderma asperellum Tdal12 strain showed a strong antagonism in vitro against strains of Fusarium oxysporum (0.48), F. graminearum (0.39) and A. flavus (0.71). Prabhakaran et al. (2015) tested the in vitro efficacy of Trichoderma asperellum (Ta10) against the Pyricularia oryzae and Bipolaris oryzae which was effective in inhibiting the mycelial growth of P. oryzae and B. Oryzae, 100% and 97.7% respectively. 2.6 EFFECT OF SEED COATING TREATMENTS UNDER GREEN HOUSE CONDITIONS Seed treatment is the cheapest and safest method to control seed and soil borne diseases which protects the seed, seedling and subsequent stages of the crop from the disease causing organisms. The method is simple in execution and inexpensive in application. Seed treatment has resulted in significant reduction in losses caused by a variety of diseases with enhancement of quality and quantity of yields (Dharmvir, 1968 and 1981).

46 2.6.1 Evaluation of antagonistic activity of fungicides as seed coating treatments against Fusarium spp. in pot culture Kamdi et al. (2012) studied two fungicides against Fusarium oxysporum f.sp. ciceri causing chickpea wilt. Field studies found that carbendazim seed 2g kg -1 seed gave minimum wilt incidence (26.38%) and maximum yield (13.47 qt ha -1 ). Bagga and Sharma (2006) evaluated five fungicides viz., Bavistin, Benomyl, Topsin-M, Tilt and Emisan as seedling treatment, for controlling foot-rot disease of basmati rice seedlings under natural infection and after artificial inoculation in the field. Seedling treatment with Bavistin and 0.1% each for 6 and 8 h respectively, were very effective. Omar et al. (2006) evaluated the antagonistic activity of carbendazim as a seed coating against Fusarium oxysporum f.sp. radicis-lycopersici, the causal organism of fusarium crown and root rot of tomato. Carbendazim reduced disease symptoms by over 50% when used at >50 µg ml -1, but had little effect at lower concentrations. 10 µg ml -1, significantly reduced disease symptoms by 77% when compared with artificially inoculated controls. Poddar et al. (2004) evaluated the antagonistic activity of four systemic fungicides carbendazim, propiconazole, thiophanate-methyl and tebuconazole. Carbendazim caused maximum growth inhibition (90%) of the pathogen. Cromey et al. (2001) used the fungicides (tebuconazole, carbendazim, and azoxystrobin) against Fusarium head blight (FHB) of wheat. High levels of Fusarium was recorded in harvested grain in untreated. Levels of both Fusarium and resulting mycotoxins were substantially reduced following treatment with tebuconazole and carbendazim but were not affected by treatment with azoxystrobin. De and Chaudhary (1999) conducted a field experiment to assess the effect of seed treatment using fungicides against wilt disease caused by Fusarium oxysporum f. sp. lentis in lentil. The results revealed that carbendazim (Bavistin) exhibited maximum wilt control (56%) and increase the yield (92%). Howell et al. (1997) assessed the efficacy of fungicides as the seed treatment against cotton seedling disease pathogens in the field under different soil and ambient environmental conditions. The seed treatment with carboxin-pcnb and carboxin-pcnb

47 plus metalaxyl generally resulted in better seedling stands than metalaxyl alone and the untreated controls Evaluation of antagonistic activity of fungicides as seed coating treatments against Aspergillus spp. in pot culture Mohanty and Mohapatra (2015) conducted a trial on the integrated management of collar rot of groundnut indicated that seed dressing with 2g kg -1 alone was superior to all other treatments in their efficacy in reducing collar rot disease, maintenance of crop stand and realisation of increased pod yield. Srinivasan and Kannan (2013) evaluated the antagonistic effect of four fungicides (Pyraclostrobin Vitavax, Mancozeb, Carbendazim) against Aspergillus niger causing collar rot in groundnut. Among them Pyraclostrobin 20% WDG at 1.5 g kg -1 as seed treatment was found most effective over the other treatments which increased the germination percentage and reduced disease incidence in the seedlings as compared to control and other fungicides. Johnson and Subramanyam (2010) conducted field experiments on evaluation of different seed treating fungicides against soil borne diseases of groundnut. Seed treatment with tebuconazole resulted in minimum collar rot (9.1%) followed by hexaconazole+captan (10.1%) and carboxin+thiram (13.5%) compared to other treatments. Ozer and Koyco (1998) evaluated four fungicides (benomyl, thiram, prochloraz and tebuconazole) for their effectiveness against Aspergillus niger Van Tiegham on onion seed in vivo condition. Thiram, prochloraz and benomyl+thiram mixture at all dosages stimulated the rate of germination of seed as compared with control. Thiram significantly reduced the percentage of pre emergence dampingoff when the soil was infested with the pathogen. However when seeds were infested with A. niger, prochloraz significantly reduced the percentage of post emergence dumping off Evaluation of antagonistic activity of Trichoderma as seed coating treatments against Fusarium spp. in pot culture Chemical seed treatment effectively controls seedborne diseases of cereals, oil seeds and other important crops. However, certain limitations and environmental disadvantages have been associated with the use of chemical fungicides. This has

48 increased the demand for alternatives and coating seeds with antagonistic microorganisms may be such an alternative. Nayaka et al. (2010) tested the antagonistic activity of T. harzianum against Fusarium verticillioides is one of the most important fungal pathogens in maize causing both pre and post-harvest losses and also capable of producing Fumonisins. Formulations of T. harzianum were effective at reducing the F. verticillioides and fumonisin infection and also increasing the seed germination, vigour index, field emergence, yield and thousand seed weight in comparison with the control. Poddar et al. (2004) tested the antagonistic effect of three isolates of Trichoderma harzianum from rhizosphere (TH-1) and non-rhizosphere (TH-2 and TH- 3) soils of chickpea field. TH-1 exhibited highest antagonistic activity against Fusarium oxysporum f.sp. ciceri which caused wilt of chickpea. Mousseaux et al. (1998) grew Douglas-fir seedlings through T. harzianum inoculated medium before growing into a mixture of T. harzianum and F. oxysporum inoculated medium, mortality was reduced about 50%. Although contamination by resident Fusarium occurred, subsequent root colonization was significantly reduced in T. harzianum amended growing medium. Howell et al. (1997) assessed the efficacy of Trichoderma as the seed treatment against cotton seedling disease pathogens in the field under different soil and ambient environmental conditions. Trichoderma virens G-6 shows the highest control of disease incidence and seedling survival (56.1%) Evaluation of antagonistic activity of Trichoderma as seed coating treatments against Aspergillus spp. in pot culture Gajera et al. (2015) screened groundnut varieties grown in non-infested soil (T1), pathogen infested soil (Aspergillus niger) (T2) and seed treatment with Trichoderma viride JAU60 challenged with fungal pathogen (T3), showed significant differences in per cent disease incidence of collar rot in pot culture. Seed treatment (T3) of T. viride reduced approximately 51 to 58% disease incidence in different groundnut varieties. Gajera et al. (2011) tested the antagonistic effect of 12 isolates of three Trichoderma strains (T. virens, T. viride and T. harzianum) against the collar rot disease-causing fungus A. niger. The five varieties of groundnut grown in normal (T1),

49 sick A. niger infested soil (T2) and sick + Trichoderma viride 60 (seed treatment) (T3) in pot culture showed significant differences in the per cent of disease incidence of collar rot, up to 15 days after sowing (DAS). Trichoderma viride seed treatment (T3) reduced 51.6% of the disease incidence in susceptible varieties and 58.1% in tolerant varieties at 15 DAS, in A. niger infection (T2) in pot culture studies. Kishore et al. (2001) tested antagonistic activity of Trichoderma isolates by seed treatment against A. niger under green house conditions. The Trichoderma harzianum and T. viride were effective in reducing the pre-emergence (15.1 & 10.7%) and post emergence (25.3 & 20.9%) collar rot infection when applied as seed treatment compared with control. Raju and Murthy (2000) investigated the efficacy of T. harzianum and T. viride as seed coating against A. niger on groundnut. The inoculation of seeds with A. niger reduced the germination percentage to 36% compared to 68% in the control. However, seed germination increased by inoculating seeds with T. harzianum (80%) and T. viride (76%). T. viride recorded greater inhibition of A. niger growth (51.07%) than T. harzianum (46.41%). Collar rot infection was reduced to 25.67% by T. harzianum and 26.67% by T. viride (70% in the control). 2.7 COMPATABILITY OF FUNGICIDES AND BIOCONTROL AGENTS Attempts have been made to reduce the dose of fungicide as seed dresser to control the disease by combination of seed treatment with tolerant bioagent. Highly effective strains which are compatible with fungicides are required. Unfortunately, most of the wild isolates of Trichoderma harzianum are highly sensitive to carbendazim, a commonly used fungicide as seed treatment in chickpea. Genetic modification of biocontrol agents through mutation by physical and chemical means has been used to produce biocontrol agents with greater tolerance to toxicants, enhanced antagonistic potential and improved survival in the agro-ecosystems (Mukherjee and Mukhopadhyay, 1993). Kamdi et al. (2012) studied the combined antagonistic effect of fungicides and Trichoderma viride against Fusarium oxysporum f. sp. ciceri causing chickpea wilt. Field studies found that T. viride + carbendanzim gave minimum wilt incidence and maximum yield followed by T. viride +thiram.

50 Devi and Prasad (2009) studied the collor rot incidence in groundnut was reduced by the application of Trichoderma viride as seed treatment along with fungicides. In pot culture experiment, the combined effect of seed treatment with T. viride and captan resulted in significant reduction of collor rot. Combination of antagonist and fungicide also improved the growth parameters like length of the plant, biomass and yield besides decreasing the disease incidence. De and Chaudhary (1999) conducted a field experiment to assess the effect of seed treatment using fungicide combination with different biocontrol agents against wilt disease caused by Fusarium oxysporum f. sp. lentis in lentil. Combination of Bacillus subtilis and carboxin (vitavax) showed 66 per cent wilt control and 145 per cent increase in yield, whereas in separate combinations with Gliocladium virens, Trichoderma harzianum and T. viride, they reduced wilt incidence by 79 per cent and increased yield by 140, 224 and 241 per cent, respectively. Howell et al. (1997) assessed the biocontrol efficacy of the fungus Trichoderma virens in combination with fungicides against cotton seedling disease pathogens in the field under different soil and ambient environmental conditions. The treatment of cotton seed with T. virens+metalaxyl generally resulted in greater inhibition of disease incidence, greater seedling stands than those in untreated controls and equal to those of the fungicide control. 2.8 COMPATABILITY OF FUNGICIDES AND BIOCONTROL AGENTS WITH SEED COAT POLYMERS Seed coating are the known to improve the physiological quality of seeds and crop yield (Sampaio & Sampaio, 1994). Polymers give additional protection to the seeds, acting against pathogens, ensuring greater safety during handling and combined with the fungicide and biocontrol agents treatment, they can increase the germination of seeds for lots of low vigour, seedling establishment and reduction of disease incidence. Considering the large number of factors and interactions involved in the technique of coating, it is necessary to maintain continuous and extensive studies on the use of polymers (Sampaio & Sampaio, 1994). Vinod kumar et al. (2014) conducted both laboratory and field studies on effect of seed polymer coating in combination with fungicide and pesticide on growth and yield of pigeonpea. The results revealed that, 5 ml kg -1 seeds+deltamethrin

51 ml kg -1 seeds+vitavax 3 g kg -1 seed was found to be significantly superior with respect to growth and yield parameters viz., plant height, number of primary branches, number of secondary branches, number of pods per plant, pod yield per plant, seed yield per hectare as compared to untreated control. Mohamedy and Kareem (2014) studied the combined effect of Trichoderma harzianum and chitosan against Fusarium oxysporum f. sp. radicis-lycopersici (Forl) which causes Fusarium crown and root rot (FCRR) in tomato was assessed in vivo. Under greenhouse conditions, application of T. harzianum and chitosan (1 g l -1 ) as root dipping treatment combined with chitosan (0.5 g l -1 ) as foliar spray has reduced FCRR incidence and severity by 66.6 and 47.6%, respectively. The results from this study showed the possibility of using combined treatments based on T. harzianum and chitosan commercially as an approach for controlling FCRR on tomato. Junior et al. (2012) evaluated the effect of polymer coating and fungicide on seed quality of soybean. The seed film coating was performed using two commercial brands of polymers, LABORSAN and LANXESS in doses of 2.0 and 3.5 ml kg -1, with and without mixture of the fungicide carbendazim + thiram. The results indicated that the polymer and fungicide were higher in the accelerated aging test, with the highest percentage of germination of soybean seeds in relation to the control. Bays et al. (2007) evaluated the effect of soybean seed cv. BRS 153 coating on seed quality and seedling performance. By using three doses of micronutrients (1, 2 and 4 ml kg -1 ) seeds, Derosal Plus (carbendazim+thiram) and a polymer (Laborsan Red Solid Pam) under green house conditions. The results revealed that coating soybean seeds with micronutrients, fungicide and polymer provided better uniformity in adherence, distribution and coloration to the seeds, greater standing establishment, seedling emergence and vigour. Benhamou et al. (1998) tested the combining potentiality of Bacillus pumilus (PGPR strain SE 34) in combination with chitosan, for inducing defence reactions in tomato plants inoculated with the vascular fungus, Fusarium oxysporum f. sp. radicislycopersici. Ultrastructural investigations revealed that a substantial increase in the extent and magnitude of the cellular changes induced by B. pumilus was observed when chitosan was supplied to bacterized tomato plants.

52 MATERIAL AND METHODS

53 Chapter III MATERIAL AND METHODS This chapter includes all the materials used and methods adopted in the investigation and the techniques are detailed under respective headings. 3.1 LOCATION OF WORK The present experiments were carried out in the Department of Plant Pathology at College of Agriculture, Rajendranagar and Plant Pathology Laboratory, Indian Institute of Oilseeds Research (IIOR), Rajendranagar, Hyderabad, Telangana. 3.2 LABORATORY TECHNIQUES The general laboratory techniques followed in the present study were those described by Aneja (2001), Nene and Thapliyal (1993) and Dhingra and Sinclair (1995) for preparation of media, sterilization, isolation and maintenance of fungal cultures, with slight modifications wherever necessary. 3.3 MATERIAL AND METHODS Glassware The glassware used in the present study were Petri plates (90 mm), conical flasks (250, 500, 1000 and 2000 ml) and test tubes of Borosil make Cleaning of glassware Glassware were first washed with a detergent, then cleaned with tap water and finally placed in cleaning solution of the following composition. Potassium dichromate : 60 g Concentrated sulphuric acid : 60 ml Distilled water : 1000 ml The glassware were kept in the cleaning solution for 24 h and then thoroughly washed with running tap water before its final cleaning with distilled water and dried Sterilization of glassware Glassware like Petri plates, test tubes, pipettes etc., were wrapped in butter paper and sterilized in hot air oven at 180ºC for one hour. Media were sterilized at 15 psi for 20 minutes in an autoclave.

54 3.3.2 Sterilization of other instruments Inoculation needles, forceps, cork borers and blades were sterilized by passing through the flame after dipping in alcohol Equipment and apparatus used BOD incubators were used for incubating cultures at different temperatures. The cultures were stored in a refrigerator. Haemocytometer was used for recording spore count and adjusting the spore concentration. Samples were weighed using a single pan electronic balance. The other equipment and apparatus used in the present investigation for various experiments included inoculation needles, earthen pots, microscope, etc Plastic ware Polythene bags (LDPE) of 250 gauge thickness and 18 x 27 size were used to sterilize the soil and also to cover the whole pot along with the plant to maintain humidity. Eppendorf tubes, Pipette, Pipette tips (100 µl, 1000 µl) for preparation of polymer stock solutions Chemicals The chemicals used were of analytical and laboratory grades. Streptomycin sulphate was added to the medium to avoid bacterial contamination. Formaldehyde 10% solution was used to fumigate the laminar airflow chamber. Sodium hypochlorate 1% was used to surface sterilization of the castor and groundnut seed and commercial formulations of fungicides were used in all experiments. Fungicides were stored at 25ºC in the dark to maintain and preserve their biocidal activity Culture media used 1. Potato Dextrose Agar (PDA) Peeled and sliced potatoes : g Dextrose : 20.0 g Agar agar : 20.0 g Distilled water : ml Preparation of culture media A) Potato Dextrose Agar (PDA) Potato Dextrose Agar (PDA) was used for the isolation of the pathogen from the infected plant and maintenance of fungal cultures. The medium was prepared by boiling

55 200 g of peeled and sliced potatoes in 500 ml of water for about 30 minutes. In another container 20 g of agar was melted in 500 ml of water. Both of these were filtered through a muslin cloth into a third container and dextrose was added to the filtrate. The final volume was made up to one liter by adding required water and the ph was adjusted to 6.0 before autoclaving by using 1N HCL or 1N NaOH. The medium was autoclaved at 15 psi (121.6 ºC) for 20 minutes. 3.4 ISOLATION AND MAINTENANCE OF THE PATHOGENS Isolation and maintenance of the Castor wilt pathogen (Fusarium oxysporum f. sp. ricini) Isolation of the test fungal pathogen was made from infected plant roots of castor wilt which were collected from the IIOR, Rajendranagar, Hyderadad. The infected roots were thoroughly washed under running tap water and transferred to blotting paper. They were cut into 0.20 cm thick pieces and surface sterilized with 1% sodium hypochlorite solution for 1 minute followed by three washings with sterile distilled water and were placed on Petri plates containing PDA medium. The plates were incubated at 25-28ºC for 4 to 5 days. The fungal growth emerging from diseased root pieces were picked up and the culture was further purified by single spore isolation method (Hansen, 1926) and incubated at 28±2ºC for 7 to 8 days. The pure culture of the pathogen was maintained on PDA medium by periodical transfers (Plate 3.1) Identification of the isolated fungus The isolated fungus was identified based on the colony characters and spore measurements with the help of relevant monograph (Booth, 1971), illustrated books and CMI descriptions Isolation and maintenance of the Collar rot pathogen (Aspergillus niger) The infected collar portions of the diseased plants were washed under running tap water to remove adhering soil particles and other plant debris. The infected collar portion was cut with sterilized blade into small pieces of 1 cm and was surface sterilized by dipping in 1% sodium hypochlorite solution for 1 minute followed by three washings with sterile distilled water before plating them onto PDA. The plates were incubated at 28±2ºC for 4 to 5 days. Fungal growth emerging from root bits was directly transferred onto the PDA with help of a sterilized needle. The culture was further purified by single spore isolation method (Hansen, 1926) and incubated at 28±2ºC for 6 to 7 days. The

56 pure culture of the pathogen was maintained on PDA medium by periodical transfers (Plate 3.2) Identification of the isolated fungus The isolated fungus was identified based on the colony characters and spore measurements with the help of relevant monograph (Onions, 1966), illustrated books and CMI descriptions. 3.5 MORPHOLOGICAL STUDIES Castor wilt pathogen (Fusarium oxysporum f. sp. ricini) Twenty milliliter of sterilized PDA medium was poured in sterilized Petri dish and allowed for solidification. Discs of a 5 mm in diameter of Fusarium oxysporum f. sp. ricini from 7-day-old culture maintained on potato dextrose agar plates were taken out with the help of a cork borer and placed at the centre of Petri dishes containing PDA medium. After inoculation, Petri dishes were incubated at 28±2 C. The diameter of the test fungus was recorded in millimeters in two directions at right angles to each other, and then average colony diameter in millimeters was calculated and recorded. The growth was measured at an interval of 24 hours, till it covered the entire plate. The culture was stained with 0.1% lactophenol cotton blue and observed for the presence of microconidia, macroconidia and chlamydospores, using a compound microscope Microconidia Presence of microconidia was observed at 25±1ºC. Conidia size was measured with the help of an ocular micrometer after calibrating the microscope Macroconidia Length and breadth of 100 conidia of test fungus for each of three replications were measured 15 days after culturing at 25±1ºC Chlamydospores The mode of chlamydospores production viz., solitary, pairs, chains and the size of the spores were recorded 25 days after incubation Collar rot pathogen (Aspergillus niger) Twenty milliliter of sterilized PDA medium was poured in sterilized Petri dish and allowed for solidification. Discs of a 5 mm in diameter of test fungus (Aspergillus niger) from 7-day-old culture maintained on potato dextrose agar plates were taken out

57 with the help of a cork borer and placed at the centre of Petri dishes containing PDA medium. After inoculation, Petri dishes were incubated at 28±2 C. The diameter of the test fungus was recorded in millimeters in two directions at right angles to each other, and then average colony diameter in millimeters was calculated and recorded. The growth was measured at an interval of 24 hours, till it covered the entire plate. The culture was stained with 0.1% lactophenol cotton blue and observed for the presence of conidia, using a compound microscope. 3.6 SEED MATERIAL Castor: GCH-4, procured from IIOR, Rajendranagar, Hyderabad. Groundnut: K-6 (Kadiri-6), procured from ARS, Kadiri, Anathapuram, districts. 3.7 PATHOGENICITY TESTS Castor wilt Pathogenicity test was carried out using seeds of susceptible castor variety, GCH-4 in a rolled paper towel which was kept in growth chamber at 25±2ºC. Before inoculation, the seeds were surface sterilized with 1% sodium hypochlorite solution for 1 minute followed by three washings with sterile distilled water Preparation of inoculum Inoculum of the pathogen was prepared by using seven-day-old culture of F. oxysporum f. sp. ricini grown on PDA medium. Conidia of the pathogen were harvested by flooding sporulating cultures with sterile distilled water and gently scraping the surface with a sterile needle and put in sterilized water and shaken well, so that the spores were dislodged. The resultant suspension was filtered through a muslin cloth and the conidial concentration was adjusted to 10 6 ml -1 conidia by using a haemocytometer (Pathak, 1984) Inoculation About 0.1 ml of the conidial suspension (10 6 ml -1 conidia) of test fungus (F. oxysporum f. sp. ricini) was inoculated on each seed present in paper towel and rolled it carefully without displacing the seeds from their position Incubation The inoculated seeds were placed in a growth chamber at 28±2ºC temperature and above 90% humidity to facilitate the infection process and observed for development of typical symptoms of the disease.

58 Mass Multiplication of Pathogen Sorghum grains were used for the mass multiplication of the pathogen (Gupta and Kolte, 1982). Sorghum grains were soaked in water overnight. Later, the excess water was removed and soaked grains were transferred into conical flasks ( g) were sterilized in an autoclave at 121ºC at 15 psi for 20 minutes. The flasks were allowed to cool at room temperature and inoculated with 5-6 pathogen culture discs of 5 mm from actively growing 7-day-old culture of F. oxysporum f. sp. ricini to the filled sorghum grains (200 g) at aseptic conditions and thoroughly mixed up. The inoculated conical flasks were incubated in growth room at 28±2ºC for 10 days (Plate 3.3) Collar rot Pathogenicity test was carried out using seeds of susceptible groundnut variety K-6 (Kadiri-6) in a rolled paper towel and kept in growth chamber at 25±2ºC. Before inoculation, the seeds were surface sterilized with 1% sodium hypochlorite solution for 1 minute followed by three washings with sterile distilled water Preparation of inoculum Inoculum of the pathogen was prepared by using seven-day-old culture of Aspergillus niger grown on PDA medium. Conidia of the pathogen were harvested by flooding sporulating cultures with sterile distilled water and gently scraping the surface with a sterile scalpel and put in sterilized water and shaken well to dislodge the spores. The resultant suspension was filtered through a muslin cloth and the conidial concentration was adjusted to 10 6 ml -1 conidia by using a haemocytometer (Pathak, 1984) Inoculation About 0.1 ml of the conidial suspension (10 6 ml -1 conidia) of test fungus (Aspergillus niger) was inoculated on each seed present in rolled paper towel and rolled it carefully without displacing the seeds from their position Incubation The inoculated seeds were placed in a growth chamber at 25±1ºC temperature and above 90% humidity to facilitate the infection process and observed for development of typical symptoms of the disease.

59 Mass Multiplication of Pathogen The test pathogen was mass multiplied on sorghum grains (Gupta and Kolte, 1982). Sorghum grains were soaked in water overnight. Later, the excess water was removed and soaked grains were transferred into 500 ml 200 g and autoclaved at 15 lb psi (121ºC) for 20 min. The flasks were allowed to cool at room temperature and inoculated with 5 mm discs of actively growing 3-4-day-old culture of Aspergillus niger. Five to six discs per flask were added and the flasks were later incubated in growth room for 10 days at 28±2ºC (Plate 3.4). 3.8 SEED COAT POLYMERS Seed coat polymers of synthetic, commercial biopolymer and chitosan were used in the present study to carry out the experiment. The details of synthetic seed coating polymers and biopolymers are given below (Table 3.1). Table 3.1 Details of seed coating polymers Polymer Trade Name Obtained From Synthetic Polymer-I Seed Polymer Synthetic Polymer-II Seed Coat Synthetic Polymer-III Fleck Lite Plus Red Polymer Commercial Bioploymer Biopolymer Chitosan - M/s. Reliable corporation, Chennai (India) Mahendra Overseas Manufacturers, Gandhinagar (India) Centor India, Hyderabad (India) Centor India, Hyderabad (India) Sri Biotech Laboratories Pvt Ltd, Hyderabad (India) In the preliminary studies, tested the effect of synthetic polymers and commercial biopolymer with different concentrations (0.1%, 0.2%, 0.3%, 0.4% and 0.5%) and two concentrations (0.2% and 0.25%) with chitosan, from that synthetic polymer-ii (0.3%) and chitosan (0.25%) were used for further studies. 1. Synthetic Seed Coat Polymers (0.3%) Seed Coat Polymer : 3 ml Seed : g

60 2. Biopolymer (0.25%) Chitosan : 2.5 g Distilled Water : ml Acetic Acid 1% : 5 ml Seed : g Preparation of Polymer solution Preparation of Synthetic Polymer solution Synthetic seed coat polymer solution was prepared by mixing 3 ml of polymer solution in 5 ml of sterile distilled water in clean, dry Eppendorff tube (10 ml) by pipette Preparation of Chitosan solution For the preparation of chitosan solution, 2.5 g of chitosan was weighed, mixed with water and to this 5 ml of 1% acetic acid was added to dissolve chitosan Seed coating with Synthetic Polymer Prior to coating initial seed moisture content, germination percentage, seedling dry weight and seedling vigour index were recorded. One hundred gram of seeds was taken in a polythene bag and added 3 ml kg -1 of seeds. The polythene bag was closed tightly trapping air in it to form of a balloon then polythene bag was vigorously shaken till the seeds are uniformly coated, later the treated seeds were spread on a sheet under the shade and dried completely. The dried seeds were used for sowing (Shakuntala et al., 2010) Seed coating with Biopolymer Chitosan Seed coating with chitosan was done as per the procedure of Paulin et al. (2013). One hundred grams of the clean, dry seeds were dipped in a solution of acetylated 2.5 g kg -1 of seeds in a conical flask and kept on magnetic stirrer about 12 h at 100 rpm (25ºC). After 12 h of incubation, the uniformly coated seeds were spread on a sheet under the shade and dried completely. The dried seeds were used for sowing. 3.9 EFFECT OF BIOPLOLYMERS AND SYNTHETIC SEED COATING POLYMERS ON CASTOR AND GROUNDNUT Two hundred seeds of castor (40 g) and groundnut (80 g) were taken for each treatment separately and treated with synthetic polymer (3 ml kg -1 ) and biopolymer

61 chitosan (2.5 g kg -1 ). The treated seeds were tested for their germination by rolled paper towel method (ISTA). The seeds were placed in rows in between two layers of moistened towel. Later the towels were rolled up along with polythene sheet to retain the moisture and incubated at constant temperature and humidity in an incubator at 25±1ºC and 95% RH. Proper check was maintained for each treatment. The seed quality parameters viz., germination percentage, vigour index were recorded and calculated using the following formula. Germination Percentage Where, GP = D 100% E D = Number of germinated seeds on the seventh day E = Number of total seeds investigated Vigour Index Vigour index-i = % Germination x Seedling growth (shoot length + root length) Vigour index-ii = % Germination x Seedling Dry matter 3.10 EFFECT OF SEED COATING POLYMERS ON SOIL BORNE PATHOGENS OF CASTOR AND GROUNDNUT A pot experiment was carried out at IIOR, Rajendranagar, Hyderabad. Black polythene planting bags were filled with one kilogram sterilized soil and inoculum of Fusarium oxysporum f. sp. ricini and Aspergillus niger mass multiplied on sorghum grains was mixed with soil 2 g kg -1. The seeds of castor and groundnut coated with 2.5 g kg -1 seed and synthetic 3 ml kg -1 seed separately and were sown in sterilized soil previously mixed with the inoculum of the Fusarium oxysporum f. sp. ricini and Aspergillus niger pathogen. Each experiment was conducted in completely randomized block design with six treatments and replicated five times. Data on per cent disease incidence, germination, seedling vigour, growth parameters like root length, shoot length and number of leaves were recorded. The details of treatments are mentioned in Table 3.2 and Table 3.3.

62 Table 3.2 Details of treatments used in pot culture studies for Castor Treatment No. T1 T2 T3 T4 T5 T6 Treatment Seed coating with Synthetic polymer-ii (3 ml kg -1 seed) Seed coating with Synthetic polymer-ii (3 ml kg -1 seed) + soil application with Fusarium oxysporum f. sp. ricini (2 g kg -1 soil) Seed coating with chitosan (2.5 g kg -1 seed) Seed coating with chitosan (2.5 g kg -1 seed) + soil application with Fusarium oxysporum f. sp. ricini (2 g kg -1 soil) Uncoated seed Uncoated seed + soil application with Fusarium oxysporum f. sp. ricini (2 g kg -1 soil) Table 3.3 Details of treatments used in pot culture studies for Groundnut Treatment No. T1 T2 T3 T4 T5 T6 Treatment Seed coating with Synthetic polymer-ii (3 ml kg -1 seed) Seed coating with Synthetic polymer-ii (3 ml kg -1 seed) + soil application with Aspergillus niger (2 g kg -1 soil) Seed coating with chitosan (2.5 g kg -1 seed) Seed coating with chitosan (2.5 g kg -1 seed) + soil application with Aspergillus niger (2 g kg -1 soil) Uncoated seed Uncoated seed + soil application with Aspergillus niger (2 g kg -1 soil) 3.11 EVALUATION OF FUNGICIDES AGAINST Fusarium oxysporum f. sp. ricini and Aspergillus niger IN VITRO The following seed treatment fungicides were evaluated against Fusarium oxysporum f. sp. ricini and Aspergillus niger under in vitro conditions by poisoned food technique (Vincent, 1927).

63 Table 3.4 Details of the fungicides used in bioassay studies under in vitro condition S. No. Fungicide Recommended Dosages (ppm) 1. Carbendazim Mancozeb Thiram Tebuconazole Carbendazim + Mancozeb Vitavax + Thiram Control (Without fungicide) Evaluation of fungicides against F. oxysporum f. sp. ricini and Aspergillus niger under laboratory conditions Six seed treatment fungicides viz., carbendazim, mancozeb, thiram, tebuconazole, carbendazim+mancozeb, vitavax+thiram (Table 3.4) at recommended, half-recommended and above-recommended dosages were evaluated separately against wilt pathogen, Fusarium oxysporum f. sp. ricini and collar rot pathogen Aspergillus niger under in vitro conditions by poisoned food technique (Vincent, 1927). For each treatment, 100 ml of potato dextrose agar was taken in 250 ml conical flask and sterilized in an autoclave. To the sterilized medium, fungicide was added at lukewarm temperature and mixed thoroughly by shaking to obtain the above mentioned concentrations. The poisoned medium was equally distributed in the Petri plates and allowed to solidify. Four replications were maintained for each treatment. Discs of 5mm diameter of the actively growing test fungal cultures were cut with sterilized cork borer separately and transferred to the centre of the poisoned medium in each of the Petri plates. Similarly, control was maintained by placing 5 mm discs of test fungal culture in centre of the plates containing the medium without fungicide. All the Petri plates were incubated at 25+1ºC in BOD incubator. The diameter of fungal colony was measured in each of the treatment when the fungal colony growth in control plate was full. The colony diameter inhibited in fungicide treated plates as compared to control was taken as a measure of fungitoxicity. Per cent inhibition over control was calculated by following the equation (Vincent, 1927):

64 I = C T x 100 C Where, I = Per cent inhibition of mycelial growth C = Radial growth of pathogen in control (mm) T = Radial growth of pathogen in treatment (mm) Evaluation of Trichoderma asperellum TaDOR 7316 and Trichoderma harzianum Th4d against Fusarium oxysporum f. sp. ricini and Aspergillus niger in vitro The pure cultures of the Trichoderma asperellum TaDOR 7316 and Trichoderma harzianum Th4d were obtained from Plant Pathology laboratory, IIOR (Plate 3.5 & 3.6) and the cultures of Trichoderma strains were maintained on PDA medium by regular sub culturing. Trichoderma asperellum TaDOR 7316 and Trichoderma harzianum Th4d were screened separately for antagonism against Fusarium oxysporum f. sp. ricini and Aspergillus niger under in vitro conditions on potato dextrose agar (PDA) medium by following dual culture technique (Morton and Strouble, 1955). Twenty milliliter of sterilized luke warm potato dextrose agar (PDA) medium was aseptically poured into 90 mm diameter sterilized Petri plates. Five mm discs of Trichoderma asperellum TaDOR 7316 and Trichoderma harzianum Th4d culture and the test pathogen was cut with a sterilized cork borer from the edge of seven-day-old cultures and was placed on the solidified medium opposite to each other separately. Three replicates were maintained for each treatment. Suitable control was maintained by placing only the pathogen on culture medium. The plates were incubated at 25+1ºC. Petri plates were observed daily for recording antagonistic interactions between the pathogen and biocontrol agent. The per cent inhibition (I) of the test pathogen was calculated when the growth of the pathogen was full in the control plates by using the formula as given below. I = C T x 100 C Where, I = Per cent inhibition of mycelial growth C = Radial growth of pathogen in control (mm) T = Radial growth of pathogen in treatment (mm)

65 3.12 EFFECT OF SEED COAT POLYMERS IN COMBINATION WITH EFFECTIVE FUNGICIDE AND POTENTIAL BIOCONTROL AGENT AGAINST CASTOR WILT AND GROUNDNUT COLLAR ROT PATHOGENS IN POT CULTURE Seed coating with Synthetic Polymer One hundred grams of castor (GCH-4) and groundnut (K-6) seeds each was taken separately in polythene bag and a 3 ml kg -1 of seeds in a polythene bag. The polythene bag was closed tightly trapping air in it to form of a balloon then polythene bag was vigorously shaken till the seeds were uniformly coated, later the treated seeds were spread on a sheet under the shade and dried completely. The dried seeds were used for sowing (Shakuntala et al., 2010) Seed coating with Biopolymer Chitosan One hundred grams of the clean, dry seeds are dipped in prepared acetylated 2.5 g kg -1 of seeds in a conical flask and kept on magnetic stirrer about 12 h at 100 rpm (25ºC) (Paulin et al., 2013). After 12 h of incubation, the chitosan coated seeds were spread on a sheet under the shade and dried completely. The dried seeds were used for sowing Seed coating with polymers and Fungicide For the seed coating manual method of coating is adopted by using transparent plastic bags with a capacity of 1L (Junior et al., 2012). Hundred grams of polymer coated seed (both synthetic and chitosan treated) were taken in a plastic bag and then fungicide (without water) was applied directly over the seed. After that they were shaken for 3 minutes. Subsequently, seeds were dried at room temperature for 1 hour Preparation of Trichoderma talc formulation Trichoderma spp. were grown on molasses-soy medium (Prasad and Rangeshwaran, 2000) by inoculation of 2 ml of spore suspension from sporulating cultures on potato dextrose agar slants to 100 ml of medium in 250 ml Erlenmeyer flasks. Inoculated flasks were kept in shaking incubator for 3 days at 200 rpm and allowed to sporulate in incubation room for 2 days. The five-day old inoculum was homogenized in a blender and mixed with talc powder (1:2 v/w). The powder formulation was air-dried by spreading as thin layer in a clean room for 2 days to reduce moisture to 8-10% and carboxymethyl cellulose (CMC) as sticker was 5 g kg -1

66 formulation at the end. The colony forming units (cfu) were determined by plating serial dilutions of homogenized suspensions on TSM Seed coating with polymers, fungicide and Trichoderma For the seed coating manual method of coating is adopted by using transparent plastic bags with a capacity of 1L (Junior et al., 2012). Hundred grams of polymer coated seed (both synthetic and chitosan treated) were taken in a plastic bag and then fungicide (without water) and Trichoderma were applied directly over the seed. After that they were shaken for 3 minutes. Subsequently, seeds were dried at room temperature for 1 hour EFFECT OF SEED COAT POLYMERS IN COMBINATION WITH EFFECTIVE FUNGICIDE AND POTENTIAL BIOCONTROL AGENT AGAINST CASTOR WILT PATHOGEN (Fusarium oxysporum f. sp. ricini) UNDER GREEN HOUSE CONDITIONS The castor seed coated with synthetic polymer and chitosan were again coated with effective fungicide 1 g kg -1 seed) and potential antagonist (Trichoderma harzianum 10 g kg -1 seed) against Fusarium oxysporum f. sp. ricini which were obtained during poisoned food technique and dual culture with the following treatments. The seeds of castor were imposed with the following treatments and sown in pots inoculated with the soil borne pathogen Fusarium oxysporum f. sp. ricini (2 g kg -1 soil) which was mass multiplied on sorghum grains earlier. The experiment was conducted in completely randomized block design with twelve treatments and replicated three times. Data on per cent disease incidence, germination, seedling vigour, growth parameters like root length, shoot length and number of leaves were recorded. The details of treatments are mentioned in Table 3.5.

67 Table 3.5 Details of the treatments used in pot culture studies for Castor under green house conditions Treatment No. T1 T2 T3 T4 T5 T6 T7 T8 T9 T10 T11 T12 Treatment Seed coating with synthetic polymer-ii (3 ml kg -1 seed) Seed coating with chitosan (2.5 g kg -1 seed) Seed coating with carbendazim (1 g kg -1 seed) Seed coating with Trichoderma harzianum Th4d (10 g kg -1 seed) Seed coating with carbendazim (1 g kg -1 seed) + Trichoderma harzianum Th4d (10 g kg -1 seed) Seed coating with synthetic polymer-ii (3 ml kg -1 seed) (T 1 ) + carbendazim (1 g kg -1 seed) (T 3 ) Seed coating with synthetic polymer-ii (3 ml kg -1 seed) (T 1 ) + Trichoderma harzianum Th4d (10 g kg -1 seed) (T 4 ) Seed coating with synthetic polymer-ii (3 ml kg -1 seed) (T 1 ) + carbendazim (1 g kg -1 seed) (T 3 ) + Trichoderma harzianum Th4d (10 g kg -1 seed) (T 4 ) Seed coating with chitosan (2.5 g kg -1 seed) (T 2 ) + carbendazim (1 g kg -1 seed) (T 3 ) Seed coating with chitosan (2.5 g kg -1 seed) (T 2 ) + Trichoderma harzianum Th4d (10 g kg -1 seed) (T 4 ) Seed coating with chitosan (2.5 g kg -1 seed) (T 2 ) + carbendazim (1 g kg -1 seed) (T 3 ) + Trichoderma harzianum Th4d (10 g kg -1 seed) (T 4 ) Uncoated seed (Control) 3.14 EFFECT OF SEED COAT POLYMERS IN COMBINATION WITH EFFECTIVE FUNGICIDE AND POTENTIAL BIOCONTROL AGENT AGAINST GROUNDNUT COLLAR ROT (Aspergillus niger) PATHOGEN UNDER GREEN HOUSE CONDITIONS The groundnut seed coated with synthetic polymer and chitosan were again coated with effective fungicide Vitavax Power 2 g kg -1 seed) and potential antagonist (Trichoderma asperellum TaDOR 10 g kg -1 seed) against Aspergillus niger which were obtained during poisoned food technique and dual culture

68 with the following treatments. The seeds of groundnut were imposed with the following treatments and sown in pots inoculated with the soil borne pathogen Aspergillus niger (2 g kg -1 soil) which were mass multiplied on sorghum grains earlier. The experiment was conducted in completely randomized block design with twelve treatments and replicated three times. Data on per cent disease incidence, germination, seedling vigour, growth parameters like root length, shoot length and number of leaves were recorded. The details of treatments are mentioned in Table 3.6. Table 3.6 Details of the treatments used in pot culture studies for Groundnut under green house conditions Treatment No. T1 T2 T3 Treatment Seed coating with synthetic polymer-ii (3 ml kg -1 seed) Seed coating with chitosan (2.5 g kg -1 seed) Seed coating with Vitavax Power (2 g kg -1 seed) T4 Seed coating with Trichoderma asperellum TaDOR 7316 (10 g kg -1 seed) T5 T6 T7 Seed coating with Vitavax Power (2 g kg -1 seed) + Trichoderma asperellum TaDOR 7316 (10 g kg -1 seed) Seed coating with Synthetic polymer-ii (3 ml kg -1 seed) (T 1 ) + Vitavax Power (2 g kg -1 seed) (T 3 ) Seed coating with Synthetic polymer-ii (3 ml kg -1 seed) (T 1 ) + Trichoderma asperellum TaDOR 7316 (10 g kg -1 seed) (T 4 ) Seed coating with Synthetic polymer-ii (3 ml kg -1 seed) (T 1 ) + Vitavax T8 Power (2 g kg -1 seed) (T 3 ) + Trichoderma asperellum TaDOR 7316 (10 g kg -1 seed) (T 4 ) T9 T10 T11 Seed coating with chitosan (2.5 g kg -1 seed) (T 2 ) + Vitavax Power (2 g kg -1 seed) (T 3 ) Seed coating with chitosan (2.5 g kg -1 seed) (T 2 ) + Trichoderma asperellum TaDOR 7316 (10 g kg -1 seed) (T 4 ) Seed coating with chitosan (2.5 g kg -1 seed) (T 2 ) + Vitavax Power (2 g kg -1 seed) (T 3 ) + Trichoderma asperellum TaDOR 7316 (10 g kg -1 seed) (T 4 ) T12 Uncoated seed (Control)

69 3.15 STATISTICAL ANALYSIS The data obtained in different experiments were statistically analyzed following completely randomized block design (CRD) as per the procedures suggested by Snedecor and Cochran (1967) and Panse and Sukhatme (1978). The data pertaining to percentage were angular transformed wherever necessary.

70 RESULTS AND DISCUSSION

71 Chapter IV RESULTS AND DISCUSSION The results of the experiments conducted in the present investigation are presented hereunder along with critical examination and discussion with the available literature. 4.1 ISOLATION OF THE PATHOGEN AND PROVING THE PATHOGENICITY Isolation of castor wilt pathogen (Fusarium oxysporum f. sp. ricini) The infected roots of wilt affected castor plants were brought to the laboratory, surface sterilized and isolated on the potato dextrose agar (PDA) medium. The pure culture of the fungus was obtained and purified by single spore isolation method and maintained on PDA medium (Hansen, 1926) Pathogenicity test The pathogenicity of the fungus was established by following Koch s postulates. The pathogenicity test was conducted on a rolled paper towel as described in materials and methods The conidial suspension (10 6 conidia ml -1 ) of test fungus (F. oxysporum f. sp. ricini) 0.1 ml was inoculated on each castor seed (GCH-4). Initial symptoms were obtained 15 days after inoculation (Plate 4.1) it was observed that young seedling at two-three leaf stage exhibited discolouration of hypocotyl and loss of turgidity with or without change in colour. Dange et al. (2006) observed that young plants were severely attacked, which exhibit gradual yellowing of apical leaves, shrivelling with marginal necrosis and dried completely. The pathogen was re-isolated from the infected seeds and roots onto potato dextrose agar medium and the morphological characters were compared with the original culture which was similar in all aspects Isolation of groundnut collar rot pathogen (Aspergillus niger) The pathogen causing collar rot of groundnut was isolated from the infected collar portions of the diseased plants on potato dextrose agar (PDA) medium. The pure culture of the fungus was obtained and purified by single spore isolation method and maintained on PDA medium (Hansen, 1926).

72 4.1.4 Pathogenicity test The pathogenicity of the fungus was established by following Koch s postulates. The pathogenicity test was conducted on a rolled paper towel as described in materials and methods The conidial suspension (10 6 conidia ml -1 ) of test fungus (A. niger) 0.1 ml was inoculated on each groundnut seed (K-6). Initial symptoms were obtained 10 days after inoculation. The seeds are covered with black masses of spores and internal tissues of seed become soft and watery (Plate 4.2). The emerging young seedling showed circular brown spots on the cotyledons which later spreads to the hypocotyl and stem. Brown discoloured spots appeared on collar region. The affected portion became soft and rotten, resulting in collapse of the seedling. Matloob and Juber (2014) conducted the pathogenicity test on groundnut and observed significant reduction in per cent seed germination with increased disease incidence and severity. The pathogen was re-isolated from the infected seeds and isolated on potato dextrose agar medium and the morphological characters were compared with the original culture which was similar in all aspects. 4.2 EFFECT OF BIOPOLYMERS AND SYNTHETIC SEED COATING POLYMERS ON CASTOR AND GROUNDNUT SEED A total of three different synthetic polymers (synthetic polymer-i, synthetic polymer-ii, synthetic polymer-iii), one commercial biopolymer with different concentrations (0.1, 0.2, 0.3, 0.4, and 0.5%) and natural biopolymer chitosan (0.2% and 0.25%) were used for seed coating to evaluate their effect on castor and groundnut seed. The data on germination, seedling vigour, growth parameters like root length, shoot length were recorded and presented in table 4.1 and 4.2. Perusal of the data indicated that in general biopolymers were found to be superior in enhancing the germination, seedling vigour, growth parameters when compared to synthetic polymers in both castor and groundnut Castor In castor among all the treatments seed coated with 0.25% recorded highest seed germination (97.40%), root length (18.76 cm), shoot length (14.76 cm), vigour index-i ( ), fresh weight (19.24 g), dry weight (1.76 g) and vigour index- II (171.74) (Table 4.1) when compared to synthetic polymer I, II, III, commercial biopolymer and control (Plate 4.3).

73 Among the synthetic polymers higher germination was obtained in synthetic polymer-ii at 0.3% (92.80%) which was on par with biopolymer at 0.1% (92.40%) followed by synthetic polymer-i at 0.1% (91.80%). Whereas the maximum root length (18.00 cm) was recorded with synthetic polymer-i at 0.1% followed by synthetic polymer-ii at 0.3% (15.92 cm) and synthetic polymer-iii with a root length of cm at 0.1%. However maximum shoot length (13.92 cm) was recorded by biopolymer coated seed with 0.4% concentration followed by 0.3% (13.68 cm) and 0.1% (13.30 cm) of synthetic polymer-ii. Maximum vigour index-i was obtained in synthetic polymer-ii at 0.3% ( ) followed by biopolymer at 0.1% ( ) and 0.2% ( ). In case of fresh weight there is a variation among treatments, maximum fresh weight of g and g was recorded in synthetic polymer-ii at 0.3% and 0.2% concentration respectively which was on par with biopolymer at 0.1% (28.28 g). Maximum dry weight was obtained in synthetic polymer-ii at 0.3% (5.75 g) followed by 0.1% (5.22 g) and 0.4% (5.07 g) concentration. Seed coated with synthetic polymer- II at 0.3% recorded maximum vigour index-ii (496.16) followed by 0.2% and 0.1% of the same polymer which recorded and respectively (Table 4.1) Groundnut A similar trend was observed in groundnut. Among all treatments the highest germination (91.80%), root length (15.94 cm), shoot length (12.60 cm), vigour index-i ( ), fresh weight (39.76 g), dry weight (6.12 g) and vigour index-ii (562.24) (Table 4.2) are recorded with chitosan (0.25%) than synthetic polymer-i, II, III, commercial biopolymer and control (Plate 4.4). Among the synthetic polymers the maximum germination recorded when coated with synthetic polymer-i at 0.1% (87.60%) and 87.00% at 0.2% and synthetic polymer- II with a germination of 86.20% at 0.3%. However there is a variation among treatments with regard to root length, maximum root length of cm was observed in synthetic polymer-ii at 0.3%, root length of cm at 0.4% and cm at 0.1% when treated with biopolymer. Similarly the shoot length was maximum (9.06 cm) when treated with synthetic polymer-ii at a concentration of 0.3%, followed by biopolymer at 0.1% (8.10 cm). Treatments 0.3% and synthetic 0.2% were on par with each other recording a shoot length of 7.72 and 7.70 cm respectively. The vigour index-i was maximum ( ) when treated with synthetic polymer-ii at 0.3%, followed by the same polymer at 0.4% with a vigour index of and synthetic polymer-i with at 0.1% concentration. Synthetic polymer-ii recorded fresh

74 weight and dry weight of g and 5.75 g respectively at a concentration of 0.3% and a fresh weight of g and 5.22 g dry weight at 0.2%. Biopolymer at a concentration of 0.1% recorded fresh weight of g whereas dry weight of 5.07 g was recorded by synthetic polymer-ii at 0.4%. Among all the treatments, maximum vigour index-ii was recorded by synthetic polymer-ii at 0.3% (496.16), 0.2% (442.78) and 0.1% (398.05) (Table 4.2). From the above study it was found that chitosan a natural polymer was superior among all the treatments which showed significant effect on germination, reduced the mean germination time and increased shoot length, root length, and dry weights. Similar results were obtained by Liqiang (2014) in tomato. The tomato seed treated with 150 mg l -1 of chitosan showed highest germination rate, germination index, root fresh weight, germ fresh weight and vigour index than the untreated control. This enhanced effect may be due to the relative permeability of the plasma membrane which increased the concentrations of soluble sugars and proline, and enzymes like peroxidase and catalase activities (Basavaraj et al., 2008). Zhou et al. (2002) reported that when peanut seeds were soaked in chitosan, exhibited an increased rate of germination. Vinodkumar et al. (2013) while working with pigeonpea, used polymer coated seed and found highest germination (83.57 %), seedling length (27.52), seedling dry weight (85.17 mg) and vigour index (2810) than untreated seeds. Similarly Shakuntala et al. (2010) treated sunflower hybrid RSFH-130 with seed coat 5 ml kg -1 found best results in germination (97 %) and vigour index (3377) than control. The results of the present study also indicated that the synthetic polymers had a beneficial effect on castor and groundnut enhancing per cent germination, seedling vigour I and II. 4.3 EFFECT OF SEED COAT POLYMERS ON GROWTH AND VIGOUR AT DIFFERENT STORAGE INTERVALS In the previous experiment, it was found that synthetic polymer-ii at a concentration of 0.3% and 0.25% were best among all treatments. Hence the present study was carried out to find the effect of seed coat polymers on castor and groundnut when stored at different storage intervals. The castor and groundnut seed of one kilogram each were coated with 0.25% and synthetic 0.3% and stored in polythene bags at room temperature for six months. Hundred gram seeds each of castor and groundnut were drawn every month and studied for their effect

75 on germination, vigour index and growth parameters. The experiment was terminated at 6 th month after drawing the samples. The results are presented in Table 4.3 and 4.4. In the present investigation irrespective of the treatments, the per cent germination, vigour index-i, II and growth parameters declined progressively with increasing storage intervals Castor Among the two seed coat polymers chitosan 0.25% was significantly superior in maintaining the quality of castor seed when compared to control (Table 4.3). The per cent germination decreased from first month onwards (96.00% to 84.20%) when treated with 0.25% but the rate of reduction in germination percentage from the beginning of the storage period till the end of 6 th month of storage was observed lesser in seeds treated with chitosan compared to synthetic polymer-ii treated and untreated seeds (Plate 4.5). However when treated with synthetic polymer-ii the per cent germination gradually decreased upto 3 rd month (93.20% to 83.20%) with no change in germination in the 4 th month (83.20%) but there after there was a decrease in per cent germination from 5 th to 6 th month (78.20% to 73.80%). The root length and shoot length of castor seedling when coated with polymers were observed throughout the storage period. There was not much change in root length from 1 st month (18.80 cm) to 4 th month (18.24 cm) when coated with chitosan but reduced in the 5 th (17.66 cm) and 6 th month (17.10 cm). Similar trend was observed for shoot length. In the initial month the shoot length was cm and at 6 th month, it was cm. The per cent germination, root length, shoot length, vigour index-i, vigour index-ii of castor seed coated with 0.25% at the beginning were 96%, cm, cm, and respectively and after 6 months of storage observed values were 84.20%, cm, cm, and , respectively. The vigour index-i ( to ) and II ( to ) of castor seed coated with 0.25% also declined with the storage periods when compared to control. Initially not much variation was observed in 1 st to 2 nd of month of storage but gradually it decreased. Similarly root length (16.06 cm to 13.34cm), shoot length (14.06 cm to cm), vigour index-i ( to ) and II ( to ) of castor seedlings gradually decreased with storage when coated with synthetic 0.3%,

76 though not much variation in the above parameters was observed when sampled from 3 rd and 4 th month of storage. Castor seed coated with synthetic 0.3% recorded the per cent germination (93.20%), root length (16.06 cm), shoot length (14.06 cm), vigour index-i ( ), vigour index-ii (155.44) at the beginning and after 6 months of storage observed values were 73.80%, cm, cm, and , respectively. The untreated castor seed recorded 87% germination, root length of cm, shoot length of cm, vigour index-i of , vigour index-ii of at the beginning and after 6 months of storage observed values were 69.40%, cm, 9.82 cm, and respectively Groundnut The groundnut seed when coated with chitosan 0.25% and stored for 6 months was significantly superior in maintaining the quality of seed when compared to seed coated with synthetic polymer-ii and control. The chitosan coated seed showed gradual decrease in germination (91.60% to 80.60%). In the initial month the per cent germination was 91.60% and later reduced to 89.20%, 87.40%, 85.80% and 83.80% upto 4 th month with no change in 5 th month (83.40%) and further decreased in the 6 th month (80.60%). However the synthetic 0.3% coated seed showed gradual decrease in germination with a significant variation in every month. Initially the synthetic polymer-ii coated seed showed good per cent germination but later on rapid decrease in per cent germination was observed with rapid deterioration than the uncoated seed from 1 st month to 6 th month of storage (Plate 4.6). Regarding root length and shoot length of groundnut seedling, the chitosan coated seed showed higher root length (15.78 cm) and shoot length (12.28 cm) initially later on with storage, the seedling length gradually decreased and recorded a root length of cm and shoot length of cm at the end of 6 th month. However the synthetic polymer-ii coated seed recorded lesser root length than the untreated seed throughout storage period except initial drawing of sample but the shoot length was higher than the control and decreased gradually in storage. The vigour index-i and vigour index-ii were high from initial month of storage to 6 th month when groundnut seed was coated with 0.25% but gradually it

77 decreased. In case of synthetic polymer-ii coated seed the vigour index I vigour index II were lower than uncoated seed throughout 6 months of storage. The per cent germination, root length, shoot length, vigour index-i, vigour index-ii of groundnut seed coated with 0.25% at the beginning were 91.60%, cm, cm, and respectively and after 6 months of storage observed values were 80.60%, cm, cm, and respectively. Similarly groundnut seed coated with synthetic 0.3% recorded per cent germination of 85.80%, root length (14.80 cm), shoot length (9.24 cm), vigour index-i ( ), vigour index-ii (480.63) at the beginning and after 6 months of storage observed values were 66.60%, cm, 6.98 cm, and respectively. The untreated groundnut seed recorded the 85.60%, cm, 8.64 cm, , of per cent germination, root length, shoot length, vigour index-i, vigour index-ii respectively at the beginning and after 6 months of storage observed values are 67.00%, cm, 6.88 cm, and respectively. Maintenance of seed quality during storage period is important not only for crop protection but also to the maintenance of seeds because of their constant threat and genetic erosion. Results of the present study also showed that castor and groundnut seeds coated with chitosan as well as synthetic polymer-ii exhibited superiority in maintaining the seed quality throughout the storage period. The results obtained in present study are in accordance with Kaushik et al. (2014). The maize seeds treated with 9 ml kg -1 of seed showed high germination (82.15%), root length (11.55 cm), shoot length (17.51 cm) and vigour index (1439) after 6 months storage than the untreated control seed. The synthetic polymer act as a good growth promoter but chitosan being a biopolymer contain glucosamine and its analogues of glucosamine compounds which are present in plant cell wall as well as in seed coat (Hadrami et al., 2010), it enhanced the efficacy of seed to reduced the deleterious effect of unfavorable conditions whatever prevailing during storage and to keep safe the various physiological responses occurring in seed during germination and promote the growth by easily activating the natural and complex processes of germination. The effect of chitosan treatment on storage life of castor and groundnut seed is a new information observed from the present study.

78 4.4 EFFECT OF SYNTHETIC AND BIOPOLYMERS ON Fusarium oxysporum f. sp. ricini and Aspergillus niger An experiment was conducted to test the effect of synthetic and biopolymers on castor wilt and collar rot of groundnut. One kilogram of sterilized soil was weighed and filled in black polythene planting bags. Inoculum of Fusarium oxysporum f. sp. ricini and Aspergillus niger mixed in the sterilized soil separately. Castor and groundnut seed were coated with synthetic 0.3% and 0.25% and sown in the inoculated bags separately. Uninoculated bags for each treatment served as control. Data on per cent germination, vigour index-i, II and growth parameters were recorded at 30 days after sowing and results are presented in Table and Castor Data in Table indicated that there was a significant increase in per cent germination (95.20%), vigour index-i ( ), vigour index-ii (197.96) and growth parameters when the castor seeds were treated with chitosan alone when compared to control. But a significant difference was not observed in per cent germination (88.20%), vigour index-i ( ), vigour index-ii (99.10) and growth parameters when treated with synthetic polymer-ii compared to control (Plate 4.7). However when chitosan and synthetic polymer-ii coated castor seeds were grown in Fusarium oxysporum f. sp. ricini inoculated soil there was significant difference in per cent germination when compared to control. The chitosan treated seed recorded germination of 84.60% while in synthetic polymer-ii coated seed the germination was when compared to control. Similarly the vigour index-i and vigour index-ii of castor seed coated with chitosan was and respectively and with synthetic polymer-ii, it was and respectively. The per cent disease incidence of Fusarium oxysporum f. sp. ricini was also calculated (Table 4.5.2) and it was observed that chitosan treated seed was highly significant (34.60%) in reducing the disease incidence when compared to synthetic polymer-ii (63.60%) Groundnut Data in Table indicated that there was significant difference in per cent germination of groundnut seeds (89.40%) and also in case of vigour index-i ( ), vigour index-ii (166.87) and growth parameters when the groundnut seeds were treated

79 with chitosan alone when compared to control. But there was no significant difference in per cent germination (84.60%) but some degree of significant difference was observed in vigour index-i ( ), vigour index-ii (122.70) and growth parameters when treated with synthetic polymer-ii compared to control (Plate 4.8). However when the chitosan coated groundnut seeds were grown in Aspergillus niger inoculated soil, there was significant difference in per cent germination when compared to control. The chitosan treated seed recorded highest germination of 75.80% and lowest disease incidence of 68.80% (Table 4.6.2). Similarly the vigour index-i and vigour index-ii of groundnut seed coated with chitosan was and respectively. Whereas total seed rotting (100%) and inhibiting germination was recorded in both synthetic polymer-ii coated and untreated seeds of groundnut. The synthetic polymer-ii coating also did not act as barrier against the collar rot pathogen and recorded the complete seed rotting as like as untreated seed infected with pathogen. The synthetic polymer forms a thin oily film around the seed surface, act as barrier for direct contact between water and seed surface and enhanced the growth of plants. The chemical groups present in the synthetic polymer do not easily intermingle with the natural plant defence chemicals which are present within the plant. The biopolymer chitosan easily activates the defending mechanisms by lignifications of tissues along with growth promotion in plants. From the present investigation it was identified that the biopolymer chitosan is superior over the synthetic polymers in defending against the plant pathogens. Yongxia et al. (2007) observed highest germination rate, germination power, fresh weight of single plant, germination index, vigour index and resistance in soybean seeds treated by 1.0 ml l -1 chitosan against Fusarium oxysporum and observed a disease incidence upto 65.45%. Li et al. (2008) showed antifungal activities of chitosan (0.1%) in vitro with complete inhibition (100%) of Aspergillus niger. Similar observations were made by Burrows et al. (2007) who suggested that chitosan treatment resulted in best plant growth and showed antifungal activity. 4.5 SCREENING OF FUNGICIDES IN VITRO Screening of fungicides against Fusarium oxysporum f. sp. ricini in vitro The efficacy of six fungicides was tested in vitro by poisoned food technique and the results are presented in Table All the fungicides were effective in inhibiting

80 mycelial growth of Fusarium oxysporum f. sp. ricini to varying degrees. Significant difference was observed among the fungicides in inhibiting the mycelial growth of the pathogen. Of all the fungicides tested, complete inhibition (100%) of growth of pathogen over control was observed in carbendazim treatment at all three concentrations tested (Table 4.7.1). The fungicides tebuconazole and combination fungicides carbendazim+mancozeb and vitavax+thiram were on par with carbendazim at recommended (2000 and 2000 ppm) and above recommended (2500 and 2500 ppm) concentrations. Mancozeb was least effective at all concentrations in inhibiting the growth of the pathogen (Plate 4.9, 4.10 and 4.11). Similar results were obtained by Dar et al. (2013) who tested the sensitivity of Fusarium oxysporum f. sp. pini to carbendazim, hexaconazole, thiaphonate methyl, triadimefon, metalaxyl, mancozeb, captan, copper oxychloride, chlorothalonil and reported the effectiveness of carbendazim against F. oxysporum f.sp. pini. The sensitivity of four Fusarium spp. to carbendazim, mancozeb, maneb, thiram and ziram was studied by Jamil and Kumar (2010) who reported the broad spectrum of fungitoxic activity of carbendazim against four species of Furasium Screening of fungicides against Aspergillus niger in vitro The efficacy of six fungicides was tested in vitro by poisoned food technique and the results were presented in Table The results indicated that majority of fungicides were effective in inhibiting mycelial growth of Aspergillus niger to varying degrees. Significant difference was observed among the fungicides in inhibiting the mycelial growth of the pathogen. Of all the fungicides tested, complete inhibition (100%) of growth of pathogen over control was observed in vitavax+thiram treatment at all three concentrations tested (Table 4.8.1). The fungicides tebuconazole and combination fungicide carbendazim+mancozeb were on par with the vitavax+thiram at recommended (1000 and 2000 ppm) and above recommended (1500 and 2500 ppm) concentrations. Mancozeb was least effective at all concentrations in inhibiting the growth of the pathogen (Plate 4.12, 4.13 and 4.14). Similar results were obtained by Mahato et al. (2014) who tested the sensitivity of Sclerotium rolfsii to carbendazim, mancozeb, copper oxychloride, chlorothalonil, vitavax+thiram, metalaxyl+mancozeb, curzate and reported the effectiveness of

81 vitavax+thiram against Sclerotium rolfsii. Nathawat and Partap (2014) reported that all the concentrations of tebuconazole were effective against Aspergillus niger. The fungicide carbendazim for Fusarium oxysporum f.sp. ricini and vitavax+thiram for Aspergillus niger at recommended dose were taken to carry out further studies in combination treatments with seed coat polymers. 4.6 SCREENING OF BIOCONTROL AGENTS IN VITRO Screening of Trichoderma spp. against Fusarium oxysporum f. sp. ricini in vitro Two Trichoderma spp. viz., T. harzianum Th4d and Trichoderma asperellum TaDOR 7316 obtained from the Plant Pathology Laboratory, IIOR, Rajendranagar were screened for their antagonistic potential against Fusarium oxysporum f. sp. ricini by dual culture method. The perusal of the data presented in Table indicated that Trichoderma harzianum Th4d was highly effective (82.44%) in inhibiting the mycelial growth of Fusarium oxysporum f. sp. ricini. The present investigations are in agreement with the findings of Altinok and Erdogan (2015) who tested the efficacy of Trichoderma harzianum against the pathogenic strains of Fusarium oxysporum under in vitro conditions. The maximum mycelial growth inhibition (72.69%) was recorded with Trichoderma harzianum strain T16 against pathogenic strains of F. oxysporum. The effectiveness of Trichoderma harzianum was established by Bardia and Rai (2007) who tested against Fusarium oxysporum f. sp. cumini (Plate 4.15) Screening of Trichoderma spp. against Aspergillus niger in vitro Two Trichoderma spp. viz., T. harzianum Th4d and Trichoderma asperellum TaDOR 7316 were screened for their antagonistic potential against Aspergillus niger by dual culture method. The perusal of the data presented in Table indicated that Trichoderma asperellum TaDOR 7316 was highly effective (72.00%) in inhibiting the mycelial growth of Aspergillus niger (Plate 4.16). The results were in accordance with Castillo et al. (2011) who tested the efficacy of Trichoderma asperellum against S. sclerotiorum and S. cepivorum under in vitro conditions and reported the strong antagonistic potential of Trichoderma asperellum.

82 Similarly Paica (2014) also found antagonistic effect of Trichoderma asperellum against F. oxysporum, F. graminearum and Aspergillus flavus under in vitro conditions. 4.7 EFFECT OF SEED COAT POLYMERS, FUNGICIDE AND BIOCONTROL AGENT IN VITRO Effect of seed coating polymers in combination with fungicide and biocontrol agent against castor wilt in vitro Combination of effective fungicide 0.1% and potential biocontrol agent T. harzianum 1% and seed coat polymers 0.25% and synthetic seed coat 0.3% were tested against castor wilt pathogen Fusarium oxysporum f.sp. ricini under in vitro conditions and data presented in Table 4.9 and The treatment T13 (chitosan+carbendazim+t. harzianum Th4d) was highly significant in increasing the per cent germination (98.00%) and vigour index-i ( ) and vigour index-ii (210.32) when compared to control (germination %, vigour index-i , vigour index-ii ) under uninocualted conditions (Plate 4.17). Similar trend was observed with treatment T12 (chitosan +T. harzianum Th4d) germination of 94.60% and vigour index-i of and vigour index-ii of Synthetic polymer-ii+carbendazim+t. harzianum Th4d (T10) was also significant in recording germination of 93.40% and vigour index-i of and vigour index-ii of All the other treatments in synthetic polymer-ii, chitosan, carbendazim and T. harzianum Th4d alone and their combination with each other were significant in increasing per cent germination and vigour index-i, vigour index-ii when compared to control. However treatment T8 i.e., carbendazim+synthetic polymer-ii and T9 (T. harzianum Th4d+synthetic polymer-ii) were not significant in increasing per cent germination, vigour index-i and vigour index-ii when compared to control. Data in Table 4.10 indicated that all the treatments were significant in increasing per cent germination and vigour index-i, vigour index-ii of castor under in vitro condition when inoculated with Fusarium oxysporum f. sp. ricini. Maximum per cent germination (95.80%) was observed in T13 when castor seed was treated with

83 chitosan+carbendazim+t. harzianum Th4d (Plate 4.18) and F. oxysporum f.sp. ricini when compared to control and also increased the vigour index-i ( ) and vigour index-ii (152.90). The per cent germination of T12 (chitosan +T. harzianum Th4d+pathogen) and T11 (chitosan+carbendazim+pathogen) were on par with each other with 93.80% and 93.20% respectively. However maximum vigour index-i ( ) and vigour index-ii (146.33) were recorded in T12 (chitosan +T. harzianum Th4d+pathogen) than the T11 (chitosan+carbendazim+pathogen) with and when compared to control. Synthetic polymer-ii in combination with carbendazim and T. harzianum Th4d (T10) was the next best treatment in recording a germination of 89.80% and vigour index-i of and vigour index-ii of under pathogen inoculated conditions. Treatment T7 (synthetic polymer-i+t. harzianum Th4d+pathogen) and T6 (synthetic polymer-i+carbendazim+pathogen) were on par with each other in increasing the germination (88.60% and 88.00%), vigour index-i ( and ). However vigour index-ii was slightly higher in T7 (127.41) than in T6 (122.14). A similar trend was observed in the rest of the treatments in T5 (carbendazim+ T. harzianum Th4d+pathogen), T4 (T. harzianum Th4d+pathogen), T3 (carbendazim+pathogen), T2 (chitosan+pathogen) and T1 (synthetic polymer- II+pathogen). But in Treatment T9 when castor seed was coated with T. harzianum Th4d first and later coated with synthetic polymer-ii and inoculated with pathogen, the per cent germination was less (58.20%) with a vigour index-i of and vigour index-ii of which was less among all the treatments when compared to control. Corlett et al. (2014) used various combinations of calcium, silicon and fungicide with commercial seed coating polymer to evaluate the vigour of barley. The results indicated that coating of polymer, fungicide, calcium and silicon resulted improvement of emergence of the barley seedlings than control and individual treatments. It was observed from the present investigation that the combination treatments synthetic polymer-ii+carbendazim (T6) and synthetic polymer-ii+t. harzianum Th4d (T7) were effective than the combinations of carbendazim+ synthetic polymer-ii (T8) and T. harzianum Th4d+ synthetic polymer-ii (T9) because the fungicide or biocontrol agent immediately available on surface, inhibits external pathogens growth before they

84 became active to infect the seed apart from the polymer coating enhances the growth internally. Whereas in carbendazim/t. harzianum Th4d + synthetic polymer-ii combination treatment the fungicide or biocontrol agent may not be effectively inhibiting the pathogen growth because it takes time to pass through the polymer layer thus the vigour is reduced due to early pathogen infection Effect of seed coating polymers in combination with fungicide and biocontrol agent against collar rot pathogen in vitro Combination of the effective fungicide 0.2% and potential biocontrol agent T. asperellum TaDOR 1% and seed coat polymers 0.25% and synthetic seed coat 0.3% were tested against groundnut collar rot pathogen Aspergillus niger under in vitro conditions and data are presented in Table 4.11 and The treatment T12 (chitosan+t. asperellum TaDOR 7316) was highly significant in increasing the per cent germination (94.60%) (Plate 4.19) and vigour index-i ( ) and vigour index-ii (347.56) when compared to control (germination %, vigour index-i and vigour index-ii ) under uninocualted conditions. Similar trend was observed with T13 (chitosan+vitavax+thiram+ T. asperellum TaDOR 7316) the per cent germination was 92.80% with a vigour index-i of and vigour index-ii of The per cent germination of treatments T10 (synthetic polymer-ii+vitavax+thiram+t. asperellum TaDOR 7316) and T11 (chitosan+vitavax+thiram) were on par with each other recording and respectively. However vigour index-i ( ) and vigour index-ii (323.17) was higher in T11 (chitosan+vitavax+thiram) than the T10 (synthetic polymer- II+vitavax+thiram+ T. asperellum TaDOR 7316) ( and ). The other treatments chitosan, vitavax+thiram alone increased the per cent germination and vigour index-i, vigour index-ii when compared to control. However treatment T9 (T. asperellum TaDOR 7316+synthetic polymer-ii) was not significant in increasing per cent germination, vigour index-i and vigour index-ii when compared to control. Data in Table 4.12 indicated that all the treatments except synthetic polymer-ii were significant in increasing per cent germination and vigour index-i, vigour index-ii of groundnut under in vitro condition when inoculated with Aspergillus niger. Maximum per cent germination (92.80%) was observed in T13 when groundnut seed

85 was treated with chitosan+vitavax+thiram+t. asperellum TaDOR 7316 and Aspergillus niger when compared to control (Plate 4.20) and also increased the vigour index-i ( ) and vigour index-ii (200.45). The treatment T12 (chitosan + T. asperellum TaDOR 7316+pathogen) was on par with a per cent germination of 90.60% and recorded the vigour index-i and vigour index-ii of when compared to control. Chitosan in combination with vitavax+thiram (T11) was the next best treatment in recording a germination of 89.60% and vigour index-i of and vigour index-ii of under pathogen inoculated conditions. Synthetic polymer-ii+vitavax+thiram+t. asperellum TaDOR 7316+pathogen (T10) was also significant in recording germination of 87.80% and vigour index-i ( ) and vigour index-ii (173.14). A similar trend was observed in the rest of the treatments in T7 (synthetic polymer-ii+t. asperellum TaDOR 7316+pathogen), T6 (synthetic polymer- II+vitavax+thiram+pathogen), T5 (vitavax+thiram+t. asperellum TaDOR 7316+pathogen), T3 ((vitavax+thiram+pathogen) and T8 (vitavax+thiram+synthetic polymer-ii+pathogen). But among all the treatments, groundnut seed coated with synthetic polymer-ii (T1) recorded less per cent germination (36.80) with a vigour index-i of and vigour index-ii of which did not show any significant effect in restricting the pathogen under inoculated condition. Similar observations were made by Vinod kumar et al. (2014) who studied the effect of combination of polymer, fungicide and pesticide under in vitro in pigeon pea. The combination of polymer+deltamethrin+vitavax power combination resulting the highest growth than the untreated control and individual treatments. The combination treatments synthetic polymer-ii+vitavax+thiram (T6) and synthetic polymer-ii+t. asperellum TaDOR 7316 (T7) were effective than the combinations of vitavax+thiram+ synthetic polymer-ii (T8) and T. asperellum TaDOR synthetic polymer-ii (T9) because the fungicide or biocontrol agent immediately available on surface and it inhibits external pathogens growth before they became active to infect the seed apart from the polymer coating enhances the growth internally. Whereas in vitavax+thiram/t. asperellum TaDOR 7316+synthetic polymer-ii combination treatment the fungicide or biocontrol agent may not be effectively

86 inhibiting the pathogen growth because it takes time to pass through the polymer layer thus the vigour is reduced due to early pathogen infection. This was the first report on combinations of fungicide, biocontrol agent and seed coat polymers and their role in plant growth promotion and pathogen inhibition in castor and groundnut. The investigation also revealed that the compatibility between biopolymer, fungicide and biocontrol agent and their combination treatment, antagonistic to pathogens (Fusarium oxysporum f. sp. ricini and Aspergillus niger) infection was due to direct protection by stimulating the defence responses within the castor and groundnut plant during germination and simultaneously showed the synergetic effect on growth promotion beyond the pathogen effect by influencing the critical germination activating enzymes present within the seed. 4.8 EFFECT OF SEED COATING POLYMERS, EFFECTIVE FUNGICIDE AND POTENTIAL BIOCONTROL AGENT UNDER GREEN HOUSE CONDITIONS ON PLANT GROWTH The experiment was conducted to find out the effect of seed coat polymers in combination with effective fungicide and potential bioagent under green house condition. A total of 12 treatments were imposed with castor and groundnut both under inoculated (Fusarium oxysporum f. sp. ricini and Aspergillus 2 g kg -1 soil) and uninoculated condition. The data are presented in Table 4.13, 4.14, 4.15 and Effect of seed coating polymers in combination with effective fungicide and potential biocontrol agent against Fusarium wilt of castor Synthetic polymer-ii (0.3%), chitosan (0.25%), carbendazim (1 g kg -1 seed) and T. harzianum Th4d (10 g kg -1 seed) and their combinations were used for coating castor seed. The results of Table 4.13 showed that the per cent germination was maximum in T11 (chitosan+carbendazim+t. harzianum Th4d) with 98.00% followed by T10 (chitosan+t. harzianum Th4d) of 94.33% and T8 (synthetic polymer- II+carbendazim+T. harzianum Th4d) of 93.67% (Plate 4.21). Although the per cent germination was same in treatments T9 (chitosan+carbendazim), T7 (synthetic polymer- II+T. harzianum Th4d), and T2 (chitosan) there was a variation with regard to vigour index-i and vigour index-ii. Maximum vigour index-i was recorded treatment T11 (chitosan+carbendazim+t. harzianum Th4d) of followed by in T10

87 (chitosan+t. harzianum Th4d) and in T8 (synthetic polymer- II+carbendazim+T. harzianum Th4d). Among the treatments, T9 (chitosan+carbendazim) recorded a vigour index of 4677 and in T7 (synthetic polymer-ii+t. harzianum Th4d) which were on par with each other. The vigour index-ii was maximum in T11 (chitosan+carbendazim+t. harzianum Th4d) followed by T9 (chitosan+carbendazim) and T10 (chitosan+t. harzianum Th4d) and T7 (synthetic polymer-ii+t. harzianum Th4d) It was also noticed that per cent germination in T6 (synthetic polymer-ii+carbendazim+t. harzianum Th4d) was 88.67% similar to that of control. The lowest per cent germination was recorded in T11 when the castor seeds were treated with synthetic polymer-ii alone which was also not significant. All the treatments were significant in increasing the vigour index-i and vigour index-ii of castor. Treatment T4 (T. harzianum Th4d) recorded the lowest vigour index-i ( ) while T3 (carbendazim) recorded lowest vigour index-ii (113.74) when compared to other treatments. Maximum number of leaves (15.67) in castor was recorded in T11 (chitosan+carbendazim+t. harzianum Th4d) followed by T8 (synthetic polymer- II+carbendazim+T. harzianum Th4d) (14.67) and other treatments T9 (chitosan+carbendazim), T10 (chitosan+t. harzianum Th4d), T6 (synthetic polymer- II+carbendazim) and T7 (synthetic polymer-ii+t. harzianum Th4d) were on par with each other with and leaves. Treatments T3 (carbendazim), T4 (T. harzianum Th4d) and T5 (carbendazim+t. harzianum Th4d) recorded number of leaves while T2 (chitosan) and T1 ((synthetic polymer-ii) were on par with control (12.00). When Fusarium oxysporum f. sp. 2 g kg -1 soil was inoculated the maximum per cent germination (93.67%) was obtained with treatment T11 (chitosan+carbendazim+t. harzianum Th4d+pathogen) followed by T8 (synthetic polymer-ii+carbendazim+t. harzianum Th4d+pathogen) (Plate 4.22) and T9 (chitosan+carbendazim+pathogen) which were on par with 89.33% and 89.00% germination (Table 4.14). Treatments T4 (T. harzianum Th4d+pathogen), T10 (chitosan+t. harzianum Th4d+pathogen) and T3 (carbendazim+pathogen), T7 (synthetic polymer-ii +T. harzianum Th4d+pathogen) recorded the same per cent germination of 88.00% and 86.33% respectively. The maximum vigour index-i ( ) and vigour index-ii (134.89) was recorded with treatment T11 (chitosan+carbendazim+t. harzianum Th4d+pathogen)

88 followed by T9 (chitosan+carbendazim+pathogen) which recorded the vigour index-i of and vigour index-ii of The treatments T10 (chitosan+t. harzianum Th4d+pathogen), T7 (synthetic polymer-ii+t. harzianum Th4d+pathogen), T5 (carbendazim+t. harzianum Th4d+pathogen), T4 (T. harzianum Th4d+pathogen) treatments were on par with each other with regard to vigour index-i. The vigour index- II recorded similar values in the treatments T10 (chitosan+t. harzianum Th4d+pathogen), T7 (synthetic polymer-ii+t. harzianum Th4d+pathogen), T4 (T. harzianum Th4d+pathogen), T8 (synthetic polymer-ii+carbendazim+t. harzianum Th4d+pathogen) and T5 (carbendazim+t. harzianum Th4d+pathogen). Maximum number of leaves (13.33) were recorded with treatment T11 (chitosan+carbendazim+t. harzianum Th4d+pathogen) followed by T9 (chitosan+carbendazim+pathogen) of leaves. While the treatments T6 (synthetic polymer-ii+carbendazim+pathogen), T7 (synthetic polymer-ii+t. harzianum Th4d+pathogen), T8 (synthetic polymer-ii+carbendazim+t. harzianum Th4d+pathogen), T10 (chitosan+t. harzianum Th4d+pathogen) and T3 (carbendazim+pathogen), T4 (T. harzianum Th4d+pathogen) recorded the same number of leaves with and respectively. The per cent disease incidence was also recorded in all the treatments and the results are presented in Table All the treatments were effective in reducing the disease incidence. The lower disease incidence was recorded in treatment T11 (chitosan+carbendazim+t. harzianum Th4d+pathogen) with 8.67% followed by T10 (chitosan+t. harzianum Th4d+pathogen) (14.00%) and T9 (chitosan+carbendazim+pathogen) (17.33%) while the maximum disease incidence was recorded in treatments T1 (synthetic polymer-ii+pathogen) with 65.67% and T2 (chitosan+pathogen) of 39.33%. The treatments T8 (synthetic polymer- II+carbendazim+T. harzianum Th4d+pathogen), T7 (synthetic polymer-ii+t. harzianum Th4d+pathogen), T6 (synthetic polymer-ii+carbendazim+pathogen), T5 (carbendazim+t. harzianum Th4d+pathogen), T4 (T. harzianum Th4d+pathogen) and T3 (carbendazim+pathogen) recorded a disease incidence of 20.67%, 25.00%, 26.33%, 27.67%, 32.00% and 31.00% respectively when compared to control. Among the treatments, the seeds coated with polymer in combination with fungicide and biocontrol agent treatment exhibited superiority in maintaining the seed quality and pathogen inhibition. The application of polymer first on seed allowed it to penetrate into the seed through the seed coat and enhanced the germination process. The

89 application of fungicide and biocontrol agent over polymer treated seed reduced the external pathogen infection to the seed and simultaneously passed through the polymer film coating and protects their vigour besides reduction of disease incidence. So, coating the seeds with polymer followed by fungicide and biocontrol agent combination sequence resulted in higher plant growth and lower disease incidence Evaluation of seed coating polymers in combination with effective fungicide and potential biocontrol agent against collar rot of groundnut For coating the groundnut seed, synthetic polymer-ii (0.3%), chitosan (0.25%), vitavax+thiram (2 g kg -1 seed) and T. asperellum TaDOR 7316 (10 g kg -1 seed) and their combinations were used. The results of Table 4.15 showed that the per cent germination was maximum in T10 (chitosan+t. asperellum TaDOR 7316) with 98.33% followed by T11 (chitosan+vitavax+thiram+t. asperellum TaDOR 7316) 96.33% and T9 (chitosan+vitavax+thiram) of 95.00% (Plate 4.23). The treatments T8 (synthetic polymer-ii+vitavax+thiram+t. asperellum TaDOR 7316), T7 (synthetic polymer-ii+t. asperellum TaDOR 7316), T6 (synthetic polymer-ii+vitavax+thiram) and T2 (chitosan) were on par with each other with regard to per cent germination of 93.67%, 93.00%, 92.00%, 92.00% respectively. Whereas the treatment T3 (vitavax+thiram) recorded the less (83.00%) per cent germination than control (84.33%). There was much variation among treatments with regard to vigour index-i and vigour index-ii. Maximum vigour index-i was recorded treatment T10 (chitosan+t. asperellum TaDOR 7316) of followed by in T11 (chitosan+vitavax+thiram+t. asperellum TaDOR 7316) and T9 (chitosan+vitavax+thiram) recorded Among the treatments T7 (synthetic polymer-ii+t. asperellum TaDOR 7316) recorded a vigour index of and in T6 (synthetic polymer-ii+vitavax+thiram) which were on par with each other. The treatment T3 (vitavax+thiram) recorded the lowest vigour index-i ( ) which was less than control ( ). The vigour index-ii was maximum in T10 (chitosan+t. asperellum TaDOR 7316) of followed by T11 (chitosan+vitavax+thiram+t. asperellum TaDOR 7316) of The treatments T3 (vitavax+thiram) and T4 (T. asperellum TaDOR 7316) recorded the lowest vigour index-ii ( and ) which were lower than the control (113.28).

90 Maximum number of leaves (43.33) in groundnut was recorded in treatment T11 (chitosan+vitavax+thiram+t. asperellum TaDOR 7316) followed by T10 (chitosan+t. asperellum TaDOR 7316) of and T9 (chitosan+ vitavax+thiram) of leaves. The treatments T7 (synthetic polymer-ii+t. asperellum TaDOR 7316), T8 (synthetic polymer-ii+vitavax+thiram+t. asperellum TaDOR 7316) and T5 (vitavax+thiram+ T. asperellum TaDOR 7316), T6 (synthetic polymer-ii+vitavax+thiram) were on par with each other regarding number of leaves. Treatments T1 (synthetic polymer-ii) recorded number of leaves which was lower than control (29.33). When Aspergillus 2 g kg -1 soil was inoculated there was much significant variation among the treatments regard for per cent germination. The maximum per cent germination (93.67%) was obtained with treatment T11 (chitosan+vitavax+thiram+t. asperellum TaDOR 7316+pathogen) (Plate 4.24) followed by T10 (chitosan+t. asperellum TaDOR 7316+pathogen) of 91.00% (Table 4.16). The treatments T9 (chitosan+vitavax+thiram+pathogen), T7 (synthetic polymer-ii+t. asperellum TaDOR 7316+pathogen) were recorded the 87.00% and 84.33% germination. Whereas T1 (synthetic polymer-ii+pathogen) and untreated check showed total seed rotting (100%) and inhibited germination. The maximum vigour index-i ( ) and vigour index-ii (136.44) was recorded with treatment T11 (chitosan+vitavax+thiram+t. asperellum TaDOR 7316+pathogen) followed by T10 (chitosan+t. asperellum TaDOR 7316+pathogen) which recorded the vigour index-i of and vigour index-ii of The treatments T9 (chitosan+vitavax+thiram+pathogen) and T7 (synthetic polymer-ii+t. asperellum TaDOR 7316+pathogen) recorded vigour index-i of and respectively. Whereas the treatments T5 (vitavax+thiram+ T. asperellum TaDOR 7316+pathogen) and T3 (vitavax+thiram+pathogen) recorded similar values of and with regard to vigour index-i. The treatments T5 (vitavax+thiram+t. asperellum TaDOR 7316+pathogen), T3 (vitavax+thiram+pathogen) recorded the vigour index-ii of 89.86, which were on par with each other. Maximum number of leaves (27.33) were recorded with treatment T11 (chitosan+vitavax+thiram+t. asperellum TaDOR 7316+pathogen) followed by T10 (chitosan+t. asperellum TaDOR 7316+pathogen) with leaves. Whereas the treatments T7 (synthetic polymer-ii+t. asperellum TaDOR 7316+pathogen), T6 (synthetic polymer-ii+vitavax+thiram+pathogen), T8 (synthetic polymer-ii+ vitavax+thiram+ T. asperellum TaDOR 7316+pathogen) recorded 19.33, and

91 17.33 leaves respectively. The treatments T3 (vitavax+thiram+pathogen) and T5 (vitavax+thiram+ T. asperellum TaDOR 7316+pathogen) recorded same number of leaves which were on par with treatment T4 (T. asperellum TaDOR 7316+pathogen) had leaves. The per cent disease incidence was also recorded in all the treatments and the results are presented in Table All the treatments were effective in reducing the disease incidence. The lower disease incidence was recorded in treatment T11 (chitosan+vitavax+thiram+t. asperellum TaDOR 7316+pathogen) with 23.00% followed by T10 (chitosan+t. asperellum TaDOR 7316+pathogen) of 29.33% while the maximum was recorded in treatments T1 (synthetic polymer-ii+pathogen) of 100% and T2 (chitosan+pathogen) of 58.00% and T4 (T. asperellum TaDOR 7316+pathogen) of 55.00%. The treatments T9 (chitosan+vitavax+thiram+pathogen), T7 (synthetic polymer-ii+t. asperellum TaDOR 7316+pathogen), T6 (synthetic polymer- II+vitavax+thiram+pathogen), T5 (vitavax+thiram+ T. asperellum TaDOR 7316+pathogen) and T8 (synthetic polymer-ii+ vitavax+thiram+ T. asperellum TaDOR 7316+pathogen) recorded a disease incidence of 36.33%, 47.67%, 50.33%, 51.33% and 53.67% respectively when compared to control. Similar observations were made by Vinod kumar et al. (2014) who studied the effect of combination of polymer, fungicide and pesticide under in vivo on pigeon pea. The combination of polymer+deltamethrin+vitavax+thiram resulted the highest plant height, no. of primary branches, no. of secondary branches, no. of pods per plant, pod weight per plant, seed yield per plant and seed yield than the untreated control and individual treatments. This was the first report on role of different combinations of fungicide, biocontrol agent and seed coat polymers on plant growth and pathogen inhibition under in vivo condition. The investigation also revealed that the compatibility between biopolymer, fungicide and biocontrol agent and their combination treatment which was antagonistic to pathogen (Fusarium oxysporum f. sp. ricini and Aspergillus niger) infection and gave direct protection by stimulating the defence responses within the castor and groundnut plant during germination. The castor seed coated with chitosan+carbendazim+t. harzianum Th4d and groundnut seed coated with chitosan+vitavax+thiram+t. asperellum TaDOR 7316 showed a synergetic effect on growth promotion beyond the pathogen effect.

92 SUMMARY AND CONCLUSIONS

93 Chapter V SUMMARY AND CONCLUSIONS Quality seed is the basis for profitable production. Loss of viability and vigour under high temperature and humid conditions is a common phenomenon in many crop seeds. Seed coating materials like polymers provides protection from the stress, increases the seedling and field emergence of seeds, improves the germination, plant stand and plant growth promotion and reduces fungal invasion which makes the treated seed both useful and environment friendly. The present investigation focuses on the effect of seed coat polymers, biocontrol agents and fungicides and their combinations against two major soil borne diseases castor wilt caused by Fusarium oxysporum f. sp. ricini and groundnut collar rot caused by Aspergillus niger. The pathogenicity of castor wilt pathogen (F. oxysporum f. sp. ricini) was proved by preparing conidial suspension (10 6 ml -1 conidia) of the test fungus was inoculated (0.1 ml) on each castor seed (GCH-4). The pathogen was re-isolated from the infected seeds and roots onto potato dextrose agar medium. The same procedure was followed for the proving of pathogenicity of groundnut collar rot pathogen (A. niger). A total of three different synthetic polymers (synthetic polymer-i, synthetic polymer-ii, synthetic polymer-iii), one commercial biopolymer with different concentrations (0.1, 0.2, 0.3, 0.4, and 0.5%) and natural biopolymer chitosan (0.2% and 0.25%) were used as seed coat polymers to evaluate their effect on different growth parameters on castor and groundnut seed. When synthetic polymers and chitosan were coated on castor and groundnut and tested for their effect on germination and growth parameters, it was found that the polymers were significant in increasing the per cent germination, vigour index-i and vigour index II. Among the treatments chitosan was more effective than synthetic polymers and among synthetic polymers, synthetic polymer-ii was good in increasing the per cent germination and vigour index.

94 In castor among all the treatments, seed treated with 0.25% recorded highest seed germination (97.40%), root length (18.76 cm), shoot length (14.76 cm), vigour index-i ( ), fresh weight (19.24 g), dry weight (1.76 g) and vigour index-ii (171.74) when compared to synthetic polymer I, II, III, commercial biopolymer and control. Among the synthetic polymers higher germination was obtained in synthetic polymer-ii at 0.3% (92.80%) and was on par with biopolymer at 0.1% (92.40%) followed by synthetic polymer-i at 0.1% (91.80%). Maximum vigour index-i was obtained in synthetic polymer-ii at 0.3% ( ) followed by biopolymer at 0.1% ( ) and 0.2% ( ). Seed coated with synthetic polymer-ii at 0.3% recorded maximum vigour index-ii (496.16) followed by 0.2% concentration (442.78) and 0.1% concentration (398.05). In case of groundnut though synthetic 0.3% showed higher germination and vigour than untreated control seed, the highest germination (91.80%), root length (15.94 cm), shoot length (12.60 cm), vigour index-i ( ), fresh weight (39.76 g), dry weight (6.12 g) and vigour index-ii (562.24) was recorded with chitosan (0.25%) than control and other polymer treatments. Based on the above studies it was found that synthetic polymer-ii at a concentration of 0.3% and 0.25% was best among all treatments and were used for further studies. Further a study was carried out to find the effect of seed coat polymers on castor and groundnut when stored at different storage intervals. Among the two seed coat polymers 0.25% was significant in maintaining the quality of castor and groundnut seed when compared to control. The per cent germination of castor decreased from first month onwards when treated with 0.25% but the rate of reduction in germination percentage from the initial month till the end of 6 th month of storage was observed lesser in seeds treated with chitosan compared to synthetic polymer-ii treated and untreated seeds. The per cent germination, root length, shoot length, vigour index-i, vigour index-ii of castor seed coated with 0.25% at the beginning were 96%, cm, cm, and , respectively and after 6 months of storage observed values are 84.20%, cm, cm, and , respectively.

95 Similarly castor seed coated with synthetic 0.3% recorded the per cent germination (93.20%), root length (16.06 cm), shoot length (14.06 cm), vigour index-i ( ), vigour index-ii (155.44) at the beginning and after 6 months of storage observed values are 73.80%, cm, cm, and respectively. With regard to groundnut there was a gradual decrease in per cent germination in chitosan treated seed. From initial month onward to 4 th month there was a decrease in germination with no change in the 5 th month and further in the 6 th month the germination decreased. However when treated with synthetic 0.3% there was a gradual decrease in germination with a significant variation from month to month. Initially the synthetic polymer-ii coated seed showed good per cent germination but later on there was a rapid decrease in germination and deterioration than the untreated seed from the 1 st month of storage to 6 th month of storage. The per cent germination, root length, shoot length, vigour index-i, vigour index-ii of groundnut seed coated with 0.25% at the beginning were 91.60%, cm, cm, and respectively and after the 6 months of storage observed values are 80.60%, cm, cm, and respectively. Similarly groundnut seed coated with synthetic 0.3% recorded the per cent germination (85.80%), root length (14.80 cm), shoot length (9.24 cm), vigour index-i ( ), vigour index-ii (480.63) at the beginning and after 6 months of storage observed values are 66.60%, cm, 6.98 cm, and respectively. Further the castor and groundnut seeds coated with polymers were tested for their effect on castor wilt pathogen (F. oxysporum f. sp. ricini) and groundnut collar rot pathogen (A. niger) in pot culture. Castor and groundnut seed were coated separately with synthetic 0.3% and 0.25% and sown in inoculated soil containing the pathogen. Uninoculated bags for each treatment served as control. Data on per cent germination, vigour index-i, II and growth parameters were recorded at 30 DAS. In castor under uninoculated conditions there was a significant increase in per cent germination (95.20%), vigour index-i ( ), vigour index-ii (197.96) and growth parameters when the castor seeds were treated with chitosan alone when compared to control. But there was no significant difference in per cent germination (88.20%), vigour

96 index-i ( ), vigour index-ii (99.10) and growth parameters when treated with synthetic polymer-ii compared to control. However when chitosan and synthetic polymer-ii coated castor seeds were grown in F. oxysporum f. sp. ricini inoculated soil there was significant difference in per cent germination when compared to control. The per cent germination was 84.60% and 70.60% in chitosan coated and synthetic polymer-ii coated seed respectively with a minimum per cent disease incidence of 34.60% when compared to control (90.20) and synthetic polymer-ii coated seed (63.60%). Similarly the vigour index-i and vigour index-ii of castor seed coated with chitosan was and respectively while with synthetic polymer-ii it was and respectively. In case of groundnut a significant difference was observed in per cent germination (89.40), vigour index-i ( ), vigour index-ii (166.87) when coated with 0.25% but significant difference was not observed in per cent germination (84.60%) when coated with synthetic polymer-ii. However the vigour index-i and vigour index-ii were significant when compared to control under uninoculated condition. But when groundnut seeds coated with 0.25% were grown in A. niger inoculated soil a significant difference in per cent germination (75.80), vigour index-i ( ) and vigour index-ii (75.01) was recorded, whereas total seed rotting (100%) was observed inhibiting the germination in both synthetic polymer-ii coated and untreated seeds of groundnut. Studies on the efficacy of six fungicides viz., carbendazim, mancozeb, thiram, tebuconazole, carbendazim+mancozeb, vitavax+thiram against F. oxysporum f. sp. ricini and A. niger at recommended, half the recommended and above the recommended doses showed that carbendazim against F. oxysporum f. sp. ricini and vitavax+thiram against A. niger recorded cent per cent inhibition at all concentrations tested. These fungicides were used for further studies. Similarly a potential bioagent was selected by screening, Trichoderma harzianum Th4d and Trichoderma asperellum TaDOR 7316 against F. oxysporum f. sp. ricini and A. niger in dual culture method. Trichoderma harzianum Th4d against F. oxysporum f. sp.

97 ricini and Trichoderma asperellum TaDOR 7316 against A. niger showed maximum inhibition of the pathogen which were used for further studies. Studies were conducted to find out the effect of synthetic polymer, biopolymer chitosan, effective fungicide and potential biocontrol agent individually and their combinations for germination, growth promotion and pathogen inhibition. Combination of the effective fungicide 0.1% and potential biocontrol agent T. harzianum 1% and seed coat polymers 0.25% and synthetic seed coat 0.3% were tested against castor wilt pathogen Fusarium oxysporum f. sp. ricini under in vitro conditions. The treatment T13 (chitosan+carbendazim+t. harzianum Th4d) was highly significant in increasing the per cent germination (98.00%) and vigour index-i ( ) and vigour index-ii (210.32) when compared to control (germination %, vigour index-i , vigour index-ii ) under uninoculated conditions. Similar trend was observed with T12 (chitosan+t. harzianum Th4d) the per cent germination was 94.60% with a vigour index-i of and vigour index-ii of Synthetic polymer-ii+carbendazim+t. harzianum Th4d (T10) was also significant in recording germination of 93.40% and vigour index-i ( ) and vigour index-ii (174.47). However the treatment carbendazim+synthetic polymer-ii (T8) and T. harzianum Th4d+synthetic polymer-ii (T9) were not significant in increasing per cent germination, vigour index-i and vigour index-ii when compared to control. Maximum per cent germination (95.80) was also observed when inoculated with F. oxysporum f. sp. ricini in T13 when castor seeds were treated with chitosan+carbendazim+t. harzianum Th4d and F. oxysporum f. sp. ricini with an increase in vigour index-i ( ) and vigour index-ii (152.90) when compared to control. Synthetic polymer-ii in combination with carbendazim and T. harzianum Th4d (T10) was the next best treatment in recording a germination of 89.80% and vigour index-i of and vigour index-ii of under inoculated conditions. But in Treatment T9 when castor seed was coated with T. harzianum Th4d first and later coated with synthetic polymer-ii and inoculated with pathogen the per cent germination was less (58.20%) with

98 a vigour index-i of and vigour index-ii of which was less among all the treatments when compared to control. In groundnut also the combination of the effective fungicide 0.2% and potential biocontrol agent T. asperellum TaDOR 1% and seed coat polymers 0.25% and synthetic seed coat 0.3% were tested against groundnut collar rot pathogen Aspergillus niger under in vitro conditions. The treatment T12 (chitosan+t. asperellum TaDOR 7316) was highly significant in increasing the per cent germination (94.60%) and vigour index-i ( ) and vigour index-ii (347.56) when compared to control (germination %, vigour index-i and vigour index-ii ) under uninocualted conditions. Whereas in pathogen (Aspergillus niger) inoculated condition all the treatments except synthetic polymer-ii were significant in increasing per cent germination and vigour index-i, vigour index-ii of groundnut under in vitro condition. Maximum per cent germination (92.80%) was observed in T13 when groundnut seed were treated with chitosan+vitavax+thiram+t. asperellum TaDOR 7316 and Aspergillus niger when compared to control and also increased the vigour index-i ( ) and vigour index-ii (200.45). Synthetic polymer-ii+vitavax+thiram+t. asperellum TaDOR 7316+pathogen (T10) was also significant in recording germination of 87.80% and vigour index-i ( ) and vigour index-ii (173.14). But among all treatments, the treatment T1 when groundnut seed was coated with synthetic polymer-ii recorded less per cent germination (36.80%) with a vigour index-i of and vigour index-ii of which did not show any significant effect in restricting the pathogen under inoculated condition. In light of these results, the next logical step was to determine whether or not a combination of chitosan with another compatible biocontrol strategy could lead to enhanced growth and possibly to better control of castor wilt and collar rot of groundnut. Taking the cue from the in vitro studies except two treatments i.e., carbendazim+ synthetic polymer-ii and T. harzianum Th4d+ synthetic polymer-ii for Fusarium oxysporum f. sp. ricini, vitavax+thiram+synthetic polymer-ii and T. asperellum TaDOR 7316+synthetic polymer-ii for Aspergillus niger further studies were carried out in green house using the rest of treatments both under inoculated and uninoculated condition.

99 Synthetic polymer-ii (0.3%), chitosan (0.25%), carbendazim (1 g kg -1 seed) and T. harzianum Th4d (10 g kg -1 seed) and their combinations were used for coating castor seed. The treated seeds were sown in polythene bags containing inoculum of Fusarium oxysporum f. sp. ricini (@ 2 g kg -1 soil mixed with sterilized soil. A similar set of treatments were maintained in uninoculated soil. All the treatments were significant in increasing the per cent germination, vigour index-i and vigour index-ii of castor grown in F. oxysporum f. sp. ricini inoculated and uninoculated soil when compared to control. Maximum per cent germination of 98.00% was recorded in T11 (chitosan+carbendazim+t. harzianum Th4d) followed by T10 (chitosan+t. harzianum Th4d) with 94.33% and T8 (synthetic polymer-ii+carbendazim+t. harzianum Th4d) with 93.67%. Maximum number of leaves (15.67) in castor was recorded in T11 (chitosan+carbendazim+t. harzianum Th4d) followed by T8 (synthetic polymer- II+carbendazim+T. harzianum Th4d) (14.67). Whereas in pathogen (F. oxysporum f. sp. 2 g kg -1 soil) inoculated soil maximum per cent germination (93.67%) was obtained with treatment T11 (chitosan+carbendazim+t. harzianum Th4d+pathogen) followed by T8 (synthetic polymer-ii+carbendazim+t. harzianum Th4d+pathogen). The maximum vigour index-i ( ) and vigour index-ii (134.89) were recorded with treatment T11 (chitosan+carbendazim+t. harzianum Th4d+pathogen) followed by T9 (chitosan+carbendazim+pathogen) which recorded the vigour index-i of and vigour index-ii of Maximum number of leaves (13.33) were recorded with treatment T11 (chitosan+carbendazim+t. harzianum Th4d+pathogen) followed by T9 (chitosan+carbendazim+pathogen) of leaves. The per cent disease incidence was also recorded in all the treatments. All the treatments were effective in reducing the disease incidence. The lowest disease incidence was recorded in treatment T11 (chitosan+carbendazim+t. harzianum Th4d+pathogen) with 8.67% followed by T10 (chitosan+t. harzianum Th4d+pathogen) of 14.00% and T9 (chitosan+carbendazim+pathogen) of 17.33% while the maximum per cent incidence was recorded in treatments T1 (synthetic polymer-ii+pathogen) of 65.67% and T2 (chitosan+pathogen) of 39.33%.

100 In groundnut synthetic polymer-ii (0.3%), chitosan (0.25%), vitavax+thiram (2 g kg -1 seed) and T. asperellum TaDOR 7316 (10 g kg -1 seed) treatments along with combinations were imposed both in inoculated (Aspergillus niger) and uninoculated condition. The treated seeds were sown in polythene bags in normal sterilized soil and as well as in pathogen infested (Aspergillus 2 g kg -1 soil) soil. The per cent germination was maximum in T10 (chitosan+t. asperellum TaDOR 7316) with followed by T11 (chitosan+vitavax+thiram+t. asperellum TaDOR 7316) 96.33% and T9 (chitosan+vitavax+thiram) of 95.00%. The treatment T3 (vitavax+thiram) recorded lowest (83.00) germination per cent than control (84.33%). Much variation was observed among treatments with regard to vigour index-i and vigour index-ii. Maximum vigour index-i was recorded in treatment T10 (chitosan+t. asperellum TaDOR 7316) of followed by in T11 (chitosan+vitavax+thiram+t. asperellum TaDOR 7316) and T9 (chitosan+vitavax+thiram) of The treatment T3 (vitavax+thiram) recorded the lowest vigour index-i ( ) which was lower than the control ( ). The vigour index-ii was maximum in T10 (chitosan+t. asperellum TaDOR 7316) of followed by T11 (chitosan+vitavax+thiram+t. asperellum TaDOR 7316) of The treatments T3 (vitavax+thiram) and T4 (T. asperellum TaDOR 7316) recorded lowest vigour index-ii ( and ) which was lower than control (113.28). Maximum number of leaves (43.33) in groundnut was recorded in T11 (chitosan+vitavax+thiram+t. asperellum TaDOR 7316) followed by T10 (chitosan+t. asperellum TaDOR 7316) of and T9 (chitosan+vitavax+thiram) of leaves. Treatments T1 (synthetic polymer-ii) recorded number of leaves which were lower than control (29.33). While under inoculated condition a significant variation among treatments was observed. The per cent germination (93.67), number of leaves (27.33) vigour index-i ( ), vigour index-ii (136.44) was maximum in treatment T11 (chitosan+vitavax+thiram+t. asperellum TaDOR 7316+pathogen) followed by T10 (chitosan+t. asperellum TaDOR 7316+pathogen) which was just reverse when compared to uninoculated condition. The per cent disease incidence was also recorded and found that all the treatments were effective in reducing the disease incidence and also promoting good growth of

101 groundnut. Lowest disease incidence of 23.00% was recorded in treatment T11 (chitosan+vitavax+thiram+t. asperellum TaDOR 7316+pathogen) followed by T10 (chitosan+t. asperellum TaDOR 7316+pathogen) with 29.33% while the maximum was recorded in treatments T1 (synthetic polymer-ii+pathogen) of 100% disease incidence followed by T2 (chitosan+pathogen) with 58.00% and T4 (T. asperellum TaDOR 7316+pathogen) with 55.00%. It can be concluded from the present investigation that 0.25% was more effective than synthetic polymer II in increasing the germination and vigour index I and vigour index II of both castor and groundnut. Seed coating with chitosan could also protect the seed from deterioration and increased the viability upto six months of storage. Further, it was also found that the seed coat polymers reduced the disease incidence of wilt of castor and collar rot of groundnut and promoted good growth. Studies on the combination of the effective fungicide 0.1% and potential biocontrol agent T. harzianum 1% and seed coat polymers 0.25% and synthetic seed coat 0.3% against castor wilt pathogen Fusarium oxysporum f. sp. ricini both under in vitro and in vivo conditions indicated that chitosan+carbendazim+t. harzianum Th4d combination was effective in reducing the wilt disease incidence in castor. Similarly studies on the combination of the effective fungicide 0.2% and potential biocontrol agent T. asperellum TaDOR 1% and seed coat polymers 0.25% and synthetic seed coat 0.3% against groundnut collar rot pathogen Aspergillus niger both under in vitro and in vivo conditions indicated that chitosan+vitavax+thiram+t. asperellum TaDOR 7316 combination was effective in reducing the collar rot disease incidence in groundnut. It was the first report under both under in vitro and in vivo condition on role of different combinations of fungicide, biocontrol agent and polymers on plant growth and pathogen inhibition. In both castor and groundnut, seed was treated with chitosan+fungicide+biocontrol agent showed the synergetic effect on growth promotion beyond the pathogen effect by influencing the critical germination activating enzymes present within the seed.

102 FUTURE Research Work Examination of better ways like nano particles, seed primimg and pelleting to incorporate these combinations into Integrated Disease Management strategies remains to be pursued in many major crops especially against soil borne diseases. Results on antifungal activity of those seed treatment combinations provides information for further research under in vivo conditions in large scale which will provide the decreasing of indiscriminate use of fungicides and which will be useful to the farmers and nature. The combination of polymer, fungicide and biocontrol agent effect on spermosphere beneficial microflora, PGPR and the compatibility between them will be useful to promote the plant growth as well as in reduction of soil borne plant pathogens in many crops.

103 LITERATURE CITED

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117 Plate 3.1 Pure culture of Fusarium oxysporum f. sp. ricini Plate 3.2 Pure culture of Aspergillus niger

118 Plate 3.3 Mass multiplication of Fusarium oxysporum f. sp. ricini on sorghum grains Plate 3.4 Mass multiplication of Aspergillus niger on sorghum grains

119 Plate 3.5 Pure culture of Trichoderma harzianum Th4d Plate 3.6 Pure culture of Trichoderma asperellum TaDOR 7316

120 Plate 4.1 Castor seed inoculated with Fusarium oxysporum f. sp. ricini Plate 4.2 Groundnut seed inoculated with Aspergillus niger

121 Synthetic 0.3 % 0.25 % Untreated Control Plate 4.3 Effect of synthetic polymer and biopolymer chitosan on growth of castor Synthetic 0.3 % 0.25 % Untreated Control Plate 4.4 Effect of synthetic polymer and biopolymer chitosan on growth of groundnut

122 Initial Synthetic 0.3 % 0.25 % Untreated Control After 6 Months Synthetic 0.3 % 0.25 % Untreated Control Plate 4.5 Effect of synthetic polymer and biopolymer chitosan on growth of castor at different storage intervals

123 Initial Synthetic 0.3 % 0.25 % Untreated Control After 6 Months Synthetic 0.3 % 0.25 % Untreated Control Plate 4.6 Effect of synthetic polymer and biopolymer chitosan on growth of groundnut at different storage intervals

124 Synthetic Polymer-II Synthetic Polymer-II + Pathogen Chitosan Chitosan + Pathogen Untreated Control Untreated + Pathogen Plate 4.7 Effect of synthetic polymer and biopolymer chitosan treated seed on growth parameters and antifungal activity against wilt of castor in pot culture

125 Synthetic Polymer-II Synthetic Polymer-II + Pathogen Chitosan Chitosan + Pathogen Untreated Control Untreated + Pathogen Plate 4.8 Effect of synthetic polymer and biopolymer chitosan treated seed on growth parameters and antifungal activity against collar rot disease of groundnut in pot culture.

126 Plate 4.9 Effect of fungicides on the radial growth Fusarium oxysporum f. sp. ricini at recommended concentration in vitro 1) Carbendazim 2) Mancozeb 3) Thiram 4) Tebuconazole 5) Carbendazim + Mancozeb 6) Vitavax Power (vitavax + thiram)

127 Plate 4.10 Effect of fungicides on the radial growth Fusarium oxysporum f. sp. ricini at half the recommended concentration in vitro 1) Carbendazim 2) Mancozeb 3) Thiram 4) Tebuconazole 5) Carbendazim + Mancozeb 6) Vitavax Power (vitavax + thiram)

128 Plate 4.11 Effect of fungicides on the radial growth Fusarium oxysporum f. sp. ricini at above the recommended concentration in vitro 1) Carbendazim 2) Mancozeb 3) Thiram 4) Tebuconazole 5) Carbendazim + Mancozeb 6) Vitavax Power (vitavax + thiram)

129 Plate 4.12 Effect of fungicides on the radial growth Aspergillus niger at recommended concentration in vitro 1) Carbendazim 2) Mancozeb 3) Thiram 4) Tebuconazole 5) Carbendazim + Mancozeb 6) Vitavax Power (vitavax + thiram)

130 Plate 4.13 Effect of fungicides on the radial growth Aspergillus niger at half the recommended concentration in vitro 1) Carbendazim 2) Mancozeb 3) Thiram 4) Tebuconazole 5) Carbendazim + Mancozeb 6) Vitavax Power (vitavax + thiram)

131 Plate 4.14 Effect of fungicides on the radial growth Aspergillus niger at above the recommended concentration in vitro 1) Carbendazim 2) Mancozeb 3) Thiram 4) Tebuconazole 5) Carbendazim + Mancozeb 6) Vitavax Power (vitavax + thiram)

132 1 2 Plate 4.15 Effect of biocontrol agents on the radial growth Fusarium oxysporum f. sp. ricini in vitro 1) Trichoderma harzianum Th4d 2) Trichoderma asperellum TaDOR 7306

133 1 2 Plate 4.16 Effect of biocontrol agents on the radial growth Aspergillus niger in vitro 1) Trichoderma harzianum Th4d 2) Trichoderma asperellum TaDOR 7316

134 Synthetic Polymer-II + Carbendazim + T. harzianum Th4d Chitosan + Carbendazim + T. harzianum Th4d Untreated Control Plate 4.17 Effect of synthetic polymer and biopolymer chitosan in combinations with fungicide and biocontrol agent on growth of castor in vitro Synthetic Polymer-II + Carbendazim + T. harzianum Th4d + Pathogen Chitosan + Carbendazim + T. harzianum Th4d + Pathogen Untreated Seed + Pathogen Plate 4.18 Effect of synthetic polymer and biopolymer chitosan in combinations with fungicide and biocontrol agent on growth of castor against Fusarium oxysporum f. sp. ricini inoculation in vitro

135 Synthetic Polymer-II + Vitavax Power + T. asperellum Ta DOR 7316 Chitosan + T. asperellum Ta DOR 7316 Untreated Control Plate 4.19 Effect of synthetic polymer and biopolymer chitosan in combinations with fungicide and biocontrol agent on growth of groundnut in vitro Synthetic Polymer-II + Vitavax Power + T. asperellum Ta DOR 7316 Chitosan + Vitavax Power + T. asperellum Ta DOR 7316 Untreated Seed + Pathogen Plate 4.20 Effect of synthetic polymer and biopolymer chitosan in combinations with fungicide and biocontrol agent on growth of groundnut against Aspergillus niger inoculation in vitro

136 Synthetic Polymer-II + Carbendazim + T. harzianum Th4d Chitosan + Carbendazim + T. harzianum Th4d Untreated Control Plate 4.21 Effect of synthetic polymer and biopolymer chitosan in combinations with fungicide and biocontrol agent on growth of castor under green house conditions

137 Synthetic Polymer-II + T. harzianum Th4d + Pathogen Chitosan + Carbendazim + T. harzianum Th4d + Pathogen Untreated Seed + Pathogen Plate 4.22 Effect of synthetic polymer and biopolymer chitosan combinations with fungicide and biocontrol agent on growth of castor against Fusarium oxysporum f. sp. ricini infected soil under green house condition

138 Synthetic Polymer-II + Vitavax Power + T. asperellum Ta DOR 7316 Chitosan + T. asperellum Ta DOR 7316 Untreated Control Plate 4.23 Effect of synthetic polymer and biopolymer chitosan combinations with fungicide and biocontrol agent on growth of groundnut under green house conditions

139 Synthetic Polymer-II + T. asperellum Ta DOR Pathogen Chitosan + Vitavax Power + T. asperellum Ta DOR Pathogen Untreated Seed + Pathogen Plate 4.24 Effect of synthetic polymer and biopolymer chitosan in combinations with fungicide and biocontrol agent on growth of groundnut against Aspergillus niger infested soil under green house conditions