Doctor of Philosophy in (Agriculture) PLANT PROTECTION (Plant Pathology & Nematology)

CHARACTERIZATION OF INDIGENOUS SOIL ISOLATES OF ASPERGILLUS NIGER AGGREGATE AND DEVELOPMENT OF THEIR COMMERCIAL FORMULATIONS FOR THE MANAGEMENT OF WILT DISEASE COMPLEX OF CHICKPEA AND TOMATO CAUSED BY FUSARIUM SPP. AND MELOIDOGYNE INCOGNITA

THESIS SUBMITTED FOR THE DEGREE OF Doctor of Philosophy IN (Agriculture) PLANT PROTECTION (Plant Pathology & Nematology)

BY MD. ARSHAD ANWER DEPARTMENT OF PLANT PROTECTION FACULTY OF AGRICULTURAL SCIENCES ALIGARH MUSLIM UNIVERSITY ALIGARH 202 002, INDIA

2010 ACKNOWLEDGEMENTS First and foremost, I offer my heartiest thanks to Almighty ALLAH, the most Merciful and the most Beneficent who showered upon me the power and means to complete this thesis. It is my privilege to acknowledge with deep sense of gratitude and humble submission to my research supervisor, Dr. Mujeebur Rahman Khan, Associate Professor, Department of Plant Protection, Faculty of Agricultural Sciences, Aligarh Muslim University, Aligarh, India for his able guidance, affectionate attitude and authenticate support in bringing up this work in present form. His indebted oracle, unobjectionable counseling, enduring inquisitiveness and affectionate demeanour during accomplishment of this task served as reinforcement to wrap up the present thesis. I wish to acknowledge with thanks Prof. P.Q. Rizvi, Chairman and Prof. Akhtar Haseeb, ex-Chairman, Department of Plant Protection, Faculty of Agricultural Sciences, Aligarh Muslim University, Aligarh for providing facilities to carry out the research and also to Mr. Mohd. Ali Khan, Chairman, PHET for providing space for my pot trial and some other lab facilities. I am highly grateful to Dr. M.S. Ansari, Dr. S. Ashraf for time to time support, advice and encouragement and also to other teachers of the department especially Dr. R.U. Khan, Dr. (Mrs.) M. Haseeb from Aligarh Muslim University, Aligarh. I owe a very special thanks and pious gratitude to Dr. Masood Ali (ex-Director, IIPR, Kanpur), Dr. R. Ahmad (ex-Principal Scientist), Dr. S.S. Ali (Emeritus Scientist, CSIR), Mr. Naimuddin (Senior Scientist), Dr. N.P. Singh, Dr. P.R. Choudhury and Dr. Subhojit Datta (Senior Scientist) of the Indian Institute of Pulses Research, Kanpur for providing necessary facilities and support required during molecular analysis work. I wish to extend my sincere thanks to Prof. Jawed Khan, Jamia Millia Islamia, New Delhi for supporting me to complete molecular analysis and Prof. Saleem Jawed (Jamia Hamdard, New Delhi) for help in the mycotoxin analysis. I esteemingly acknowledge the help by my endearing friends, batchmates and seniors especially Dr. F.A. Mohiddin, Mr. Haidar Ali and Dr. Tufail Ahamd, who were a constant source of enthusiasm and zeal for me and their presence tranquillized my work. I wish to

extend special thanks to Mrs. Reshu, Mr. Nazrussalam, Ms. Rumanna, Ms. Huma Naz, Dr. Wajid Hassan, Mrs. Uzma Khan, Mr. Nadeem Ahmad, Mr. Salman Ahmad and Mr. Fazil Hasan who had a yawning effect and helped me in one way or the other during my research. Very special thanks go out to Mr. Mohd. Mahmood Khan my research colleague and Mr. Ziaul Haq my junior for their extreme support. My thanks are due to the technical staff, in particular the help rendered by Mr. Amaan-ul-Rehman and Mr. Raghib Ali. I acknowledge Darakshan Apa for issuing me desired books and journals for quiet a longer time with her precious dua and encouragement. I also wish to thank Guddu Bhai, Amir Bhai, Akbar Bhai, Nahid Madam and Anjum Madam for their regular assistance and cooperation in laboratory work. My special thanks are due to Waheed bhai, Fazal Bhai, Zarar Bhai and Shaba Apa for their time to time support. I also wish to thank Bano Baji, Shanaz Baji, Anjum, Sabir, Ansar and Babu Bhai especially for the preparation of research fields. At last I am fumbling for words to express my feelings for my family. I salute my parents who always enthused my nerves and sinews with the dreams and the hopes of realizing them. I am extremely thankful to my father in-law who inspired and encouraged me for dedication in research and felt pride whatever little I achieved ever. I wish to give special thanks to my brothers for their moral support and cooperation during critical periods of the work. The love and affection for me expressed by my loving son, Huzaifa and daughter, Aamna have been invaluable assets. I am extremely thankful to my wife for patience, support and also for being serviceable towards me at every moment, whenever I looked up to her. Without her help it would not have been possible for me to finish my work and apologize for not been able to give time due to spending hours to prepare this thesis.

Md. Arshad Anwer

TABLE OF CONTENTS

Introduction 1-6 Review of literature 7-73 Materials and Methods 74-102 Isolation and identification of the wilt fungus 74 Mass culture of the wilt fungi and pathogenicity test 74 Isolation, identification and mass culture of root-knot nematode, 75 Meloidogyne incognita Isolation, identification, characterization and pure culture of 76 Aspergillus niger aggregate isolates Aspergillus niger as biocontrol agent 77-82 Screening against wilt fungi 77-79 Nematicidal effect 79-80 Compatibility with pesticides 80-81 Biosorption of toxic heavy metals by Aspergillus niger 81-82 aggregate isolates Biochemical characterization of Aspergillus niger aggregate 82-85 Molecular characterization of efficient Aspergillus niger isolates 85-86 Mass culture of efficient isolates of Aspergillus niger 87 Pot culture experiment to test effectiveness of Aspergillus niger 87-93 isolates against the wilt, root-knot and wilt disease complex Field trial 93-94 Preparation of biopesticides 94-95 Shelf life of A. niger biopesticides 95 Experiment on evaluation of performance of biopesticides of selected 95-103 isolates of Aspergillus niger aggregate under field condition Observations recorded 97-101 1. Biochemical tests of plant material 97- i. Estimation of leaf pigments 97 ii. Estimation of total phenol 97-98 iii. Estimation of salicylic acid 98 iv. Estimation of lycopene in tomato 98-99 2. Soil population of fungi 99 3. Soil population of root-knot nematode 99-100 4. Root-nodulation 100 5. Wilt incidence and severity 100 6. Root-knot severity 100-101 7. Dry weight of plants 101 8. Weight of seeds or fruit/plant 101 9. Seed index of chickpea 101 10. Seed health test 101 i. Viability test by Tetrazolium chloride 101 ii. Germination test 101 iii. Detection of pathogen associated with seeds 101

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Results 103-205 Experiment-I Collection, isolation and identification (morphological and biochemical 103-110 basis) of soil isolates of Aspergillus niger aggregate from different agricultural fields of Uttar Pradesh, India. Experiment-II RAPD profiling of effective isolates of A. niger aggregate and their 111-125 characterization for ochratoxin A production, phosphate solubilization, heavy metal bioadsorption and in vitro pathogen suppression (Fusarium oxysporum f. sp. ciceri, F. oxysporum f. sp. lycopersici and Meloidogyne incognita). Experiment-III Evaluation of Aspergillus niger isolates for antagonism against 126-162 Fusarium spp. and Meloidogyne incognita and for promotion of plant growth and yield of chickpea and tomato under pot condition. Experiment-IV Evaluation of selected isolates of Aspergillus niger for effectiveness 163-183 against Fusarium wilt, root-knot and fungus-nematode wilt disease complex of chickpea and tomato under field condition. Experiment-V Preparation of biopesticides of selected isolates of Aspergillus niger 184-205 (based on performance in Expt.-IV), and their field trial for effectiveness against the wilt, root-knot and fungus-nematode wilt disease complex of chickpea and tomato. Discussion 206-220 References 221-274 Appendices 275-285

LIST OF TABLES

Table Caption of Table Page No. No. 1 Amino acids composition (mg/100 g protein) of protein 08 from different sources. 2 Protein contents in pulses. 08 3 Area and Production of chickpea in different states of 10 India. 4 Nutritional value of chickpea and tomato per 100 gram of 12 edible portion. 5 Essential amino acid composition of seed protein fractions 13 of chickpea. 6 Nutritional composition of whole seed and Dhal 14 component of chickpea. 7 Improved varieties of chickpea recommended for general 17 cultivation in different states in India. 8 Area, production and productivity of tomato in major 19 countries of the world. 9 Area, Production and productivity of tomato in different 20 states of India. 10 Temperature requirements for different stages of tomato. 23 11 Varieties of tomato recommended for different states or all 25 over the country. 12 Estimated annual crop losses caused by pests and 27 diseases worldwide. 13 Disease of chickpea caused by various pathogens. 28 14 Disease of tomato caused by various pathogens. 29 15 Base material/carriers used for mass production of fungus 69 biocontrol agents. 16 Colony forming unit load of inocula of Fusarium spp., 89 Rhizobium and Aspergillus niger isolates at the time of application. 17 Some biochemical characteristic of Aspergillus niger 106 isolates. 18 Summary of polymorphism produced by OPA and 112 Fusarium specific synthetic primers. 19 Ochratoxin A production and phosphate solubilization of 116 efficient Aspergillus niger isolates. 20 In vitro compatibility of selected efficient Aspergillus niger 117 isolates with some common fungicides and nematicides. 21 Minimum inhibitory concentrations (MIC) of Ni+2, Cd+2, or 118 Cr+6 for some selected isolates of Aspergillus niger. 22 Biosorption of heavy metals by some selected isolates of 120 Aspergillus niger (mg/g of biomass ) in single metal system of Ni+2, Cd+2, or Cr+6. 23 Biosorption of heavy metals by efficient Aspergillus niger 121

Table Caption of Table Page No. No. isolates (mg/g of biomass ) in multi metal system of Ni+2, Cd+2, or Cr+6. 24 Inhibition in the colonization by Fusarium oxysporum f. sp. 122 ciceri and F. oxysporum f. sp. lycopersici due to volatile compounds, culture filtrates and dual culture with Aspergillus niger isolates in vitro. 25 Effect of culture filtrates of efficient Aspergillus niger 125 isolates on hatching of eggs and mortality to the juveniles of Meloidogyne incognita in vitro. 26 Effects of seed treatment and soil application of efficient 132 Aspergillus niger isolates on the dry matter production and yield of chickpea in pots inoculated with Fusarium oxysporum f. sp. ciceri, Meloidogyne incognita singly and concomitantly. 27 Effect of nursery treatment and soil application of efficient 134 Aspergillus niger isolates on the dry matter production and yield of tomato in pots inoculated with Fusarium oxysporum f. sp. ciceri, Meloidogyne incognita singly and concomitantly. 28 Effects of seed treatment with efficient Aspergillus niger 140 isolates on chlorophyll a, b and total chlorophyll of chickpea inoculated with Fusarium oxysporum f. sp. ciceri and Meloidogyne incognita, singly or concomitantly. 29 Effects of soil application of efficient Aspergillus niger 141 isolates on chlorophyll a, b and total chlorophyll of chickpea inoculated with Fusarium oxysporum f. sp. ciceri and Meloidogyne incognita, singly or concomitantly. 30 Effects of nursery treatment with efficient Aspergillus niger 142 isolates on chlorophyll a, b and total chlorophyll of tomato inoculated with Fusarium oxysporum f. sp. lycopersici and Meloidogyne incognita, singly or concomitantly. 31 Effects of soil application of efficient Aspergillus niger 143 isolates on chlorophyll a, b and total chlorophyll of tomato inoculated with Fusarium oxysporum f. sp. lycopersici and Meloidogyne incognita, singly or concomitantly. 32 Effects of seed treatment with efficient Aspergillus niger 144 isolates on total phenolic contents and salicylic acid of chickpea inoculated with Fusarium oxysporum f. sp. ciceri and Meloidogyne incognita, singly or concomitantly. 33 Effects of soil application of efficient Aspergillus niger 145 isolates on total phenolic contents and salicylic acid of chickpea inoculated with Fusarium oxysporum f. sp. vi

Table Caption of Table Page No. No. ciceri and Meloidogyne incognita, singly or concomitantly. 34 Effects of nursery treatment with efficient Aspergillus niger 146 isolates on total phenolic contents, salicylic acid and lycopene of tomato inoculated with Fusarium oxysporum f. sp. lycopersici and Meloidogyne incognita, singly or concomitantly. 35 Effects of soil application of efficient Aspergillus niger 147 isolates on total phenolic contents, salicylic acid and lycopene content of tomato inoculated with Fusarium oxysporum f. sp. lycopersici and Meloidogyne incognita, singly or concomitantly. 36 Effect of seed treatment and soil application of efficient 150 Aspergillus niger isolates on the seed index, viability and germination of seeds, and detection of associated Fusarium oxysporum f. sp. ciceri with harvested seeds of chickpea from pots inoculated with F. oxysporum f. sp. ciceri, Meloidogyne incognita singly and concomitantly. 37 Effect of nursery treatment and soil application of efficient 152 Aspergillus niger isolates on the viability and germination of seeds, and detection of associated Fusarium oxysporum f. sp. lycopersici with harvested fruits of tomato from pots inoculated with F. oxysporum f. sp. lycopersici, Meloidogyne incognita singly and concomitantly. 38 Effects of seed treatment and soil application of 171 Aspergillus niger isolates on the dry matter production and yield of chickpea in field plots infested singly or concomitantly with Fusarium oxysporum f. sp. ciceri and Meloidogyne incognita or not infested. 39 Effect of nursery treatment and soil application of efficient 172 Aspergillus niger isolates on the dry matter production and yield of tomato in field plots infested singly or concomitantly with Fusarium oxysporum f. sp. lycopersici and Meloidogyne incognita or not infested. 40 Effects of seed treatment and soil application of 193 Aspergillus niger biopesticides on the dry matter production and yield of chickpea in field plots infested singly or concomitantly with Fusarium oxysporum f. sp. ciceri, Meloidogyne incognita or uninoculated. 41 Effect of nursery treatment and soil application of 194 Aspergillus niger biopesticides on the dry matter production and yield of tomato in field plots infested singly or concomitantly with Fusarium oxysporum f. sp. lycopersici, Meloidogyne incognita or uninoculated.

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LIST OF FIGURES Figure Caption of Figure Page No. No. 1 Share of different countries in global chickpea production. 10 2 Different stages of chickpea growth: Field view (A), root 15 system showing nodules (B), single young plant (C), pods and seed of chickpea (D). 3 Share of different countries in global tomato production. 19 4 Different stages of tomato growth: Field view (A), nursery 23 beds (B), single young plant (C), fruit of tomato (D). 5 Actual crop production and annual crop losses due to plant 27 diseases, insect pests and weeds (A) and breakdown of crop losses caused by fungi, bacteria, viruses and nematodes (B). 6 World distribution map of wilt disease of chickpea. 31 7 Symptoms of chickpea (A) and tomato (B) wilt caused by 34 Fusarium oxysporum f. sp. ciceri and F. oxysporum f. sp. lycopersici, respectively. 8 Colony of Fusarium oxysporum f. sp. ciceri on PDA (A), 34 micro and macro conidia of the fungus (B). 9 Disease cycle of wilt of chickpea (Fusarium oxysporum f. 36 sp. ciceri) and tomato (F. oxysporum f. sp. lycopersici). 10 Colony of Fusarium oxysporum f. sp. lycopersici on PDA 40 (A), micro and macro conidia of the fungus (B). 11 Distribution of Meloidogyne incognita and M. javanica in 43 different states in India. 12 Roots of chickpea (A) and tomato (B) showing galls or 45 knots caused by root-knot nematode, Meloidogyne incognita. 13 Perineal patterns of females of Meloidogyne incognita (A) 47 and M. javanica (B). 14 Life cycle of Meloidogyne incognita. 47 15 Colony of Aspergillus niger on PDA (A), microscopic 54 morphology of conidial head with conidia (B). 16 Pure culture of root-knot nematode, Meloidogyne incognita 75 maintained on eggplant in earthen pots. 17 Districts of Uttar Pradesh from Aspergillus niger aggregate 76 were isolated. 18 Ingredients used in biopesticide development. 96 19 The biopesticides of Aspergillus niger isolates SkNAn5, 96 VAn4 and AnC2 packed in to different polypacks. 20 Colony characters of Aspergillus niger (A), conidial heads 104 (B) with conidia (C); colony characters of Fusarium oxysporum f. sp. ciceri (D) and its micro and macroconidia (E); colony characters of F. oxysporum f. sp. lycopersici (F) and its micro and macroconidia (G). viii

Figure Caption of Figure Page No. No. 21 Browning of infected tissue of chickpea (A) and tomato (B) 105 caused by Fusarium oxysporum f. sp. ciceri and F. oxysporum f. sp. lycopersici, respectively. 22 RAPD profile of Aspergillus niger isolates obtained with 113 primers, Primer 02 (A), Primer 08 (B), Primer 06 (C) and OPA 16 (D). 23 Dendrogram of Aspergillus niger isolates constructed using 114 UPGMA with Jaccard’s similarity Index based on 28 RAPD primers. 24 Antifungal activity of Aspergillus niger SkNAn5 against 123 Fusarium oxysporum f. sp. ciceri (A) and F. oxysporum f. sp. lycopersici (B) in dual culture test. 25 Effects of volatile compounds of Aspergillus niger SkNAn5 123 on the growth of Fusarium oxysporum f. sp. ciceri and F. oxysporum f. sp. lycopersici. 26 Chickpea plants showing symptoms of Fusarium wilt. 126 Uninoculated healthy plants (A), browning and wilting of shoot at young (B) and maturing stage (C). 27 Effects of seed treatment and soil application of Aspergillus 127 niger isolates on the wilt severity (0-5 scale) of chickpea caused by Fusarium oxysporum f. sp. ciceri , galling and egg mass production of Meloidogyne incognita alone or with F. oxysporum f. sp. ciceri. 28 Effects of nursery treatment and soil application of 128 Aspergillus niger isolates on the wilt severity (0-5 scale) of tomato caused by Fusarium oxysporum f. sp. lycopersici, galling and egg mass production of Meloidogyne incognita alone or with F. oxysporum f. sp. lycopersici. 29 Roots of chickpea (A) and tomato (B) showing severe 130 galling caused by Meloidogyne incognita. 30 An aerial view of pot experiments conducted to evaluate 131 the effects of seed/nursery treatment with Aspergillus niger isolates on wilt, root-knot and wilt disease complex of chickpea and tomato. 31 Rhizobial nodules on the roots of chickpea. 136 32 Effects of seed treatment of Aspergillus niger isolates on 137 the root nodulation of chickpea inoculated with Fusarium oxysporum f. sp. ciceri and Meloidogyne incognita or not inoculated pots. 33 Effects of soil application of Aspergillus niger isolates on 138 the root nodulation of chickpea inoculated with Fusarium oxysporum f. sp. ciceri and Meloidogyne incognita or not inoculated pots. 34 Detection of Fusarium oxysporum f. sp. ciceri and F. 154 oxysporum f. sp. lycopersici from the seeds of chickpea (A) and tomato (B), respectively. ix

Figure Caption of Figure Page No. No. 35 Effects of seed treatment and soil application of Aspergillus 156 niger isolates on the soil population of Fusarium oxysporum f. sp. ciceri in the presence and absence of Meloidogyne incognita. 36 Effects of nursery treatment and soil application of 157 Aspergillus niger isolates on the soil population of Fusarium oxysporum f. sp. lycopersici in the presence and absence of Meloidogyne incognita. 37 Effects of seed treatment and soil application of Aspergillus 158 niger isolates on the soil population of Meloidogyne incognita in the absence and presence of Fusarium oxysporum f. sp. ciceri. 38 Effects of nursery treatment and soil application of 159 Aspergillus niger isolates on the soil population of Meloidogyne incognita in the absence and presence of Fusarium oxysporum f. sp. lycopersici. 39 Rhizosphere population of Aspergillus niger isolates in 161 relation to single or concomitant inoculations with Fusarium oxysporum f. sp. ciceri and Meloidogyne incognita. 40 Rhizosphere population of Aspergillus niger isolates in 162 relation to single or concomitant inoculations with Fusarium oxysporum f. sp. lycopersici and Meloidogyne incognita. 41 An aerial view of experimental plots of chickpea (A) and 164 tomato (B). 42 Effects of seed treatment and soil application of Aspergillus 165 niger isolates on the incidence and severity of wilt of chickpea caused by Fusarium oxysporum f. sp. ciceri in the presence and absence of Meloidogyne incognita. 43 Effects of nursery treatment and soil application of 166 Aspergillus niger isolates on the incidence and severity of wilt of tomato caused by Fusarium oxysporum f. sp. lycopersici in the presence and absence of Meloidogyne incognita. 44 Effects of seed treatment and soil application of Aspergillus 168 niger isolates on the galling and egg mass production of Meloidogyne incognita alone or with Fusarium oxysporum f. sp. ciceri. 45 Effects of nursery treatment and soil application of 169 Aspergillus niger isolates on the galling and egg mass production of Meloidogyne incognita alone or with Fusarium oxysporum f. sp. lycopersici. 46 Effects of seed treatment of Aspergillus niger isolates on 174 the root nodulation of chickpea grown in plots infested with Fusarium oxysporum f. sp. ciceri and x

Figure Caption of Figure Page No. No. Meloidogyne incognita or not infested. 47 Effects of soil application of Aspergillus niger isolates on 175 the root nodulation of chickpea grown in plots infested with Fusarium oxysporum f. sp. ciceri and Meloidogyne incognita or not infested. 48 Effects of seed treatment and soil application of Aspergillus 177 niger isolates on the soil population of Fusarium oxysporum f. sp. ciceri in the presence and absence of Meloidogyne incognita. 49 Effects of nursery treatment and soil application of 178 Aspergillus niger isolates on the soil population of Fusarium oxysporum f. sp. lycopersici in the presence and absence of Meloidogyne incognita. 50 Effects of seed treatment and soil application of Aspergillus 179 niger isolates on the soil population of Meloidogyne incognita in the absence and presence of Fusarium oxysporum f. sp. ciceri. 51 Effects of nursery treatment and soil application with 180 Aspergillus niger isolates on the soil population of Meloidogyne incognita in the absence and presence of Fusarium oxysporum f. sp. lycopersici. 52 Rhizosphere population of Aspergillus niger isolates in the 182 plots infested with Fusarium oxysporum f. sp. ciceri and Meloidogyne incognita singly or concomitantly. 53 Rhizosphere population of Aspergillus niger isolates in the 183 plots infested with Fusarium oxysporum f. sp. lycopersici and Meloidogyne incognita singly or concomitantly. 54 Shelf life test of biopesticides of Aspergillus niger isolates 185 showing colony forming units/g formulation at various temperatures and durations. 55 Chickpea (A) and tomato (B) plants showing symptoms 187 caused by Fusarium oxysporum f. sp. ciceri and F. oxysporum f. sp. lycopersici. 56 An aerial view of field experiments conducted to evaluate 187 the effects of seed/nursery and soil treatment with biopesticides of Aspergillus niger on wilt, root-knot and wilt disease complex of chickpea (A) and tomato (B). 57 Effects of seed treatment and soil application of Aspergillus 188 niger biopesticides on the incidence and severity of wilt of chickpea caused by Fusarium oxysporum f. sp. ciceri in the presence or absence of Meloidogyne incognita. 58 Effects of nursery treatment and soil application of 189 Aspergillus niger biopesticides on the incidence and severity of tomato wilt caused by Fusarium oxysporum f. sp. lycopersici in the presence or absence of xi

Figure Caption of Figure Page No. No. Meloidogyne incognita. 59 Effects of seed treatment and soil application of Aspergillus 190 niger biopesticides on the galling and egg mass production of Meloidogyne incognita alone or with Fusarium oxysporum f. sp. ciceri. 60 Effects of nursery treatment and soil application of 191 Aspergillus niger biopesticides on the galling and egg mass production of Meloidogyne incognita alone or with Fusarium oxysporum f. sp. lycopersici. 61 Effects of seed treatment of Aspergillus niger biopesticides 196 on the root nodulation on chickpea grown in plots infested with Fusarium oxysporum f. sp. ciceri and Meloidogyne incognita singly or concomitantly or not infested. 62 Effects of soil application of Aspergillus niger biopesticides 197 on the root nodulation on chickpea grown in plots infested with Fusarium oxysporum f. sp. ciceri and Meloidogyne incognita singly or concomitantly or not infested. 63 Effects of seed treatment and soil application with 198 Aspergillus niger biopesticides on the soil population of Fusarium oxysporum f. sp. ciceri in the presence or absence of Meloidogyne incognita. 64 Effects of nursery treatment and soil application with 199 Aspergillus niger biopesticides on the soil population of Fusarium oxysporum f. sp. lycopersici in the presence or absence of Meloidogyne incognita. 65 Effects of seed treatment and soil application with 201 Aspergillus niger biopesticides on the soil population of Meloidogyne incognita in the presence or absence of Fusarium oxysporum f. sp. ciceri. 66 Effects of nursery treatment and soil application with 202 Aspergillus niger biopesticides on the soil population of Meloidogyne incognita in the presence or absence of Fusarium oxysporum f. sp. lycopersici. 67 Population of Aspergillus niger isolates in the rhizosphere 203 of chickpea grown in the plots infested with Fusarium oxysporum f. sp. ciceri and Meloidogyne incognita singly or concomitantly. 68 Population of Aspergillus niger isolates in the rhizosphere 204 of tomato grown in the plots infested with Fusarium oxysporum f. sp. lycopersici and Meloidogyne incognita singly or concomitantly.

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ABSTRACT

Chickpea is a major source of human food and the world’s third most important pulse crop after beans and peas. Similarly tomato is considered as ‘poor man’s orange’, a vitamin C rich vegetable and is eaten freely throughout the world. Chickpea production in India has declined considerably and tomato production has been limited due to the regular occurrence of wilt caused by Fusarium oxysporum f. sp. ciceri and F. oxysporum f. sp. lycopersici, respectively and root-knot caused by Meloidogyne spp. and the resulting fungus-nematode wilt disease complex. Annual yield losses to chickpea and tomato from the wilt vary from 10-15% in India. The disease under specific conditions may cause much greater losses or may destroy the entire crop in a field or area. Root-knot nematodes have been reported to reduce the yield of chickpea and tomato by 9-40% and 24-61% in India. The concomitant situation involving Meloidogyne spp. and Fusarium spp. leading to the development of wilt disease complex is, however, more damaging. The fungus-nematode wilt complex is one of the major constraints in the production of chickpea and tomato in India, and many growers have given up cultivation of these crops because of the disease complex. Management of wilt or root-knot is not an easy task, it becomes further difficult for wilt disease complex because of multi-pathogenic nature of the disease. When a pesticide is applied, it is targeted either against the wilt fungi or root-knot nematode, consequently the disease remains relatively unchecked. In view of lesser effectiveness of chemicals, high cost of application and adverse effects, biological control offers potential substitute for the management of wilt, root-knot and the wilt complex of chickpea and tomato. Present study was undertaken with an objective to identify and characterize indigenous efficient isolates of Aspergillus niger for effectiveness against wilt (F. oxysporum f. sp. ciceri/lycopersici), root-knot (M. incognita) and the wilt disease complex (F. oxysporum f. sp. ciceri/lycopersici + M. incognita) and to develop their low cost formulations that could be adapted by poor and small farmers in India to control the target diseases of chickpea and tomato. The study was started with the isolation of A. niger aggregate from 32 different crop fields of 40 districts of the state of Uttar Pradesh, India. Initially 16 isolates out of 236 viz., AAn1, BAn4, BuAn3, BasAn5, BudAn3, GaAn1, JaAn2, LAn3, MeAn4, SkNAn3, SkNAn5, VAn4, ANAn1, ANAn4, AnC2 and AnR3 were short listed for further study for the reason that they showed faster growth rate, had more

ammonia and siderophore production and produced hydrogen cyanide and IAA than other isolates. RAPD fingerprinting was conducted on the 16 efficient isolates of A. niger and three groups (Group I, II and III) were differentiated by RAPD markers and some amplicons were identified as isolate specific. Primer 02 (GCGACGCCTA) and Primer 06 (GATAGCCGAC) may be considered as A. niger specific. Primer OPA-16 can be treated as isolate specific for AnC2 and VAn4 (three amplicons produced by the primer as 2300 bp for AnC2 and VAn4, and 2800 bp for AnC2 only); Primer 04 (AGTGGTCGCG) can be treated as SkNAn5 specific (as it produced only 1500 bp for isolate SkNAn5); OPA-12 can be treated as VAn4 specific (as it produced 700 pb in VAn4 only). The isolates SkNAn5, VAn4, AnC2, AnR3, ANAn4 and BuAn3 which were present in the Group I by RAPD profiling, solubilized more phosphorus, had negative production of ochratoxin A, had more compatible and adsorbed more toxic heavy metals and were found to possess relatively stronger ability to suppress the wilt fungi through antibiosis (production of volatile compounds) and mycoparasitism (dual culture test). Culture filtrates of these six isolates of A. niger inhibit the hatching of eggs and induced mortality to juveniles of M. incognita. These isolates were also found more compatible with common fungicides viz., carbendazim (Bavistin 50 WP), captan (Captaf 50 WP), mancozeb (Dithane M-45 75 WP), metalaxyl (Apron 35 SD), thiram (TMTD 75 WP), and two nematicides viz., carbofuran (Furadan 3G) and nemacur (Fenamiphos) than rest of the isolates. The sixteen isolates were evaluated against target diseases on chickpea and tomato in 15 cm clay pots filled with 1 kg sterilized field soil and compost (3:1 ratio). The pathogens were inoculated in the form of 2 g sorghum seeds (SS) colonized by F. oxysporum f. sp. ciceri/lycopersici (22×108 CFUs/g) and/or nematode suspension containing 2000 freshly hatched juveniles (J2) of M. incognita/kg soil. The nematode suspension was prepared by incubating egg masses which were excised from the eggplants grown in a pure culture pots. The A. niger isolates were cultured on sorghum seeds or bagasse-soil mixture (BSM; 4:1). The A. niger isolates and pesticides were applied through seed treatment and soil application in chickpea, whereas nursery treatment and soil application were done in tomato. For seed treatment the dose was 4 g BSM or SS/kg seeds of chickpea that was applied to seeds along with the commercial Rhizobium of chickpea strain (25×108 CFUs/g formulation). In case of tomato, root-dip treatment with spore suspension of A. niger (10 g/100 seedlings) was given by grinding 10 g of colonized seeds 2

or the BSM in 100 ml distilled water. The roots of tomato seedlings were dipped in the suspension for 10 minutes. The pathogens were inoculated two days before the seed sowing or nursery transplanting. Five seeds of chickpea, Cicer arietinum cv. BGD-72 were sown in each pot. Whereas one seedling of tomato, Lycopersicon esculentum cv. Pusa Ruby was sown per pot. The pots were placed in an open space receiving uniform sunlight in a completely randomized block design. The plants were grown for four months (November to March). During this period they were regularly observed for symptom(s) attributable to a given treatment. The pots were watered uniformly with tap water whenever needed. Inoculation with F. oxysporum f. sp. ciceri/lycopersici caused wilting which appeared on 7-8 weeks old plants and gradually intensified during vegetative growth. Some seedlings in a pot succumbed to the fungus infection. Chickpea cv. BGD-72 and tomato cv. Pusa Ruby were also found susceptible to the infection by M. incognita and developed characteristic root galls and egg masses. The galls were, however, smaller in size and lesser in number in chickpea than tomato. Concomitant inoculation with both the pathogens resulted to greater wilting but lesser galling (P≤ 0.01). The infected plants exhibited significantly reduced plant growth, yield and root nodulation. Phenol and salicylic acid contents of leaf and root of chickpea and tomato were considerably greater in the plants inoculated with F. oxysporum f. sp. ciceri or F. oxysporum f. sp. lycopersici and M. incognita singly or concomitantly. Infection with wilt fungi and root-knot nematode considerably decreased the leaf pigments. Application of A. niger isolates significantly checked the suppressive effect of the pathogens resulting to corresponding increase in the plant growth and yield variables; checked the loss of chlorophyll content of leaves, lycopene content of the tomato fruit and further increased the phenol content and salicylic acid of leaves and roots of chickpea and tomato. Seeds of chickpea and tomato obtained from A. niger applied plants showed considerably greater viability, germination and lower frequency of infection of F. oxysporum f. sp. ciceri/lycopersici. Among the treatments, A. niger SkNAn5 showed greatest effectiveness as it decreased the wilt severity by 60-68% and increase the yield by 84-88%, followed by VAn4 (decreased the wilt severity by 57- 65% and increase the yield by 74-77%), AnC2 (decreased the wilt severity by 56-65% and increase the yield by 71-72%), carbendazim (decreased the wilt severity by 55-63% and increase the yield by 27-38%) / carbendazim + carbofuran (decreased the wilt severity by 56-57% and increase the yield by 38-50%). Other A. niger isolates also checked the disease and improved the yield of infected plants but less than AnC2. Soil population of pathogens 3

and A. niger isolates increased over time. In concomitantly inoculated pots, population of wilt fungi increased significantly (P≤ 0.000001) but nematode population decreased (P≤ 0.01). Application of A. niger isolates or pesticides resulted to significant decrease in the soil population of wilt fungi (P≤ 0.000001) and root-knot nematode (P≤ 0.0001) with the corresponding increase in the population of biocontrol agents. Based on the relative performance in pot experiment, SkNAn5, VAn4, AnC2, AnR3, ANAn4 and BuAn3 were found more effective against the target diseases, and hence were tested further under field condition against wilt, root-knot and wilt disease complex of chickpea and tomato during November-March 2007-08. Three microplots each of 2 × 4 m were prepared for each treatment, and were randomly distributed in the field. Inoculation of F. oxysporum f. sp. ciceri/lycopersici (sorghum colonized seeds) and M. incognita (second stage juvenile suspension) was applied in soil @ 2 g colonized sorghum seeds or 2000 juveniles/kg soil two days before seed sowing/nursery transplanting. The efficient isolates of A. niger were mass cultured on bagasse-soil mixture (4:1). The soil treatment @ 40 g/microplot were applied at the time of seed sowing/nursery planting. Seed treatment with the A. niger isolates @ 4 g/kg chickpea seed was given along with Rhizobium application. root-dip treatment with A. niger isolates was done whereas seed treatment was not done. Roots of tomato nursery were dipped in the suspension of A. niger isolates (10 g/100 seedlings) for 10 minutes. Suspension of A. niger was made by grinding 10 g of colonized seeds in 100 ml distilled water. Carbendazim and carbofuran were applied @ 2.5 kg a.i./h (2 g a.i./microplot) and @ 2 kg a.i./h (1.6 g a.i./microplot), respectively, as soil application, and 2 g a.i./kg seed as seed treatment. In root-dip treatment, two weeks old tomato seedlings were dipped in 200 ppm solution of carbendazim/carbofuran for 15 minutes before transplanting. For combined treatment of fungicide and nematicide half dose of carbendazim was mixed with the half dose of carbofuran to get the equal dose to other treatments. Thereafter seeds/nursery were sown/transplanted in four rows in a micro plot. Adequate moisture was maintained in the soil at the time of chickpea seeds sowing and irrigated just after transplanting of tomato nurseries. Two irrigations (as per requirement) in case of chickpea and bi-weekly irrigation (as per requirement) in case of tomato were given without over flooding of water from microplots to avoid contamination and plants were grown for 4 months. Soil population of the wilt fungi and A. niger isolates were estimated monthly by dilution plate method on Fusarium specific medium, and A. niger specific

medium supplemented with 50 mg/l nystatin fungicide in Petri dishes. Background population of the Fusarium spp. and A. niger were also determined. Chickpea and tomato plants grown in the wilt fungus and/or root-knot nematode infested plots exhibited the characteristic symptoms of wilt and root-knot on above ground parts and roots, respectively. Severity of wilt was greater in plots inoculated concomitantly with F. oxysporum f. sp. ciceri/lycopersici and M. incognita. Seed/nursery treatment or soil application with the A. niger isolates provided the disease control that varied with pathogen and isolate. Greatest decrease in the wilt incidence and corresponding increase in the yield of chickpea and tomato occurred with SkNAn5 (75-76% lower incidence and 59-65% greater yield) followed by VAn4 (69-70% lower incidence and 52-57% greater yield), AnC2 (65-66% lower incidence and 50-53% greater yield) and carbendazim (56-58% lower incidence and 19-20 % greater yield) compared to the control. A suppression of 47-51% was recorded in the gall formation and 50-58% in egg mass production of root-knot nematode with SkNAn5 followed by VAn4 (39-45% galls and 40-47% egg masses), AnC2 (39-45% galls and 33-43% egg masses) and carbofuran (39-44% galls and 29-43% egg masses) over respective controls. In concomitantly inoculated plots, application of SkNAn5 suppressed the wilt and root-knot disease by 55-61% and 36-43%, respectively, and increased the yield of chickpea and tomato by 41% and 44%, respectively in comparison to the control followed by VAn4, AnC2 and joint application of carbendazim and carbofuran. Joint application of carbendazim and carbofuran decreased the wilt incidence by 54-58% and increase the yield by 27-28% compared to the control. Soil population of wilt fungi increased over time in plots without a treatment. The nematode population, however, decreased in the presence of wilt fungus. Various treatments also caused substantial decrease in the soil population of pathogens, being greatest with SkNAn5 followed by VAn4 and AnC2. In such plots population of the A. niger isolates increased correspondingly. Seed/nursery treatment and soil application of the A. niger isolates against the target diseases were more or less equally effective. In view of effectiveness of A. niger SkNAn5, VAn4 and AnC2 demonstrated under field condition, their commercial formulations (biopesticides) were prepared on sawdust- soil-molasses mixture (stock culture) and fly ash-soil-molasses (immobilizing agent) in the ratio of 1:20. Shelf life test of the formulations revealed that the fly ash based carrier supported the survival as well as multiplication of the A. niger isolates during storage. At ambient temperature, the CFU count of A. niger isolates/g formulation increased 5

significantly in comparison to other temperatures. The temperature next in supporting the grater CFU count was 25°C. Greatest CFU load/g formulation (1010) was recorded during March to August. From August onwards, the CFU count gradually declined, but even in September it was greater than the control at 25°C or ambient temperature. The biocontrol fungus was, however, detected in the formulation upto 12 months. Among the three isolates of A. niger, greatest CFUs/g formulation (mean of 12 months population) was recorded for SkNAn5 (14.5 × 109), followed by VAn4 (13.2 × 109) and AnC2 (12.6 × 109). The significant differences between the CFU counts of SkNAn5 and VAn4 (P≤ 0.05); SkNAn5 and AnC2 (P≤ 0.01); and VAn4 and AnC2 (P≤ 0.05) were recorded during different storage periods. Effectiveness of the biopesticides against wilt (F. oxysporum f. sp. ciceri/lycopersici), root-knot (M. incognita) and the wilt disease complex (F. oxysporum f. sp. ciceri/lycopersici + M. incognita) of chickpea and tomato was tested in microplots (4 × 2 m2) in different fields. The biopesticides were applied in soil (40 g/microplot) or on chickpea seeds (4 g/kg seed) or on tomato nursery roots (10 g/100 seedlings; root dipped for 10 minutes in 10% A. niger biopesticide suspension). Treatments with carbendazim and carbofuran were maintained to compare effectiveness of the biopesticides applied @ 2.5 kg a.i./h (2 g a.i./microplot) and @ 2 kg a.i./h (1.6 g a.i./microplot), respectively, as soil application, and 2 g a.i./kg seed as seed treatment. In root-dip treatment, two weeks old tomato seedlings were dipped in 200 ppm solution of carbendazim/carbofuran for 15 minutes before transplanting. For combined treatment of fungicide and nematicide half dose of carbendazim was mixed with the half dose of carbofuran. Chickpea cv. BGD-72 and tomato cv. Pusa Ruby grown in the plots infested with pathogens singly or concomitantly developed characteristic wilt and root-knot symptoms, and exhibited significant yield decline being significantly greater with the concomitant infestation (P≤0.001). Application of the biopesticides checked the severity of the disease and associated yield declines. Application of biopesticide SkNAn5 decreased the wilt incidence by 75% and 79% and promoted the yield by 66% and 71% of chickpea and tomato, respectively grown in F. oxysporum f. sp. ciceri/lycopersici infested plots. Its application was also found effective against root-knot disease and suppressed the galling by 63 and 72% and promoted the yield by 27 and 28% of chickpea and tomato, respectively in comparison to the control. Application of biopesticide SkNAn5 was found highly effective against the fungus- nematode wilt disease complex of chickpea and tomato, and its seed/nursery treatment 6

substantially controlled the wilt and root-knot, and increased the yield of chickpea and tomato by 46 and 44%, respectively. The biopesticide SkNAn5 also acted as a bio-fertilizer and improved the yield of chickpea and tomato in the plots not infested with either pathogens by 39% and 41%, respectively. Soil population of the wilt fungi and root-knot nematode decreased in the plot applied with biopesticides/pesticides. Population of the A. niger isolates applied through biopesticides, however, increased in the presence as well as absence of pathogens, being greatest in the former. The present study has demonstrated that biological management of wilt, root-knot and wilt disease complex of chickpea and tomato can be satisfactorily achieved with the application of the biopesticides prepared with indigenous soil isolate of A. niger SkNAn5. Hence, it can be used in all three disease situations i.e., wilt, root-knot and fungus-nematode wilt disease complex. The isolate A. niger SkNAn5 was found ochratoxin A negative, highly tolerant to the fungicides such as carbendazim (Bavistin 50 WP), captan (Captaf 50 WP), mancozeb (Dithane M-45 75 WP), metalaxyl (Apron 35 SD), thiram (TMTD 75 WP), and two nematicides viz., carbofuran (Furadan 3G) and nemacur (Fenamiphos) commonly used by farmers. It was also found highly tolerant to toxic heavy metals such as Ni, Cr and Cd, and also showed great potential to adsorb the metals. Hence, pesticide contamination in soil will not influence effectiveness of the formulation. The pesticide tolerance ability has broadened the use of present biopesticide as this formulation in conjugation with pesticides can be applied under integrated disease management. However, a further fine tuning of the formulation and extensive field trials in different agro-climatic conditions and cropping patterns are needed before the commercial production and application.

INTRODUCTION

Agriculture productivity of India has not increased as expected in spite of continuous introduction of high yielding cultivars, and the new crop cultivars have failed to give higher yields because of several reasons. Plant diseases caused by various pathogens is one of the important constraints in obtaining the yield to the best of genetic potential of a cultivar. According to one estimate, the present global land under crop production would produce quantities of food much greater than presently required, if pest and disease free crops are grown (Khan and Jairajpuri, 2010a). Soil-borne pathogens, especially fungi are highly destructive and cause tremendous yield loss to agricultural crops. During the last several decades, numerous phytopathogenic fungi, such as Pythium, Phytophthora, Botrytis, Rhizoctonia and Fusarium have spread to new areas especially in underdeveloped and developing countries because of improper implementation of quarantine measures. The exotic pathogens have established better than the native organisms and have become rather more aggressive causing severe damage at pre-harvest as well as post harvest stages of a crop (Chet et al., 1997).

The wilt caused by Fusarium spp. and root-knot by Meloidogyne spp. are important and widely occurring diseases of a large number of vegetables, pulses and other crops, and inflict significant yield loss to them. For example, wilt of chickpea and tomato caused by F. oxysporum f. sp. ciceri and F. oxysporum f. sp. lycopersici, respectively and root-knot caused by Meloidogyne incognita or M. javanica occur almost in every growing area in India and other countries (Khan, 2005; Mandal et al., 2009; Hari and Khirbat, 2009; Singh and Mathur, 2010a). Annual yield loss to chickpea from the wilt varies from 10-15% (Trapero-Casas and Jimenez-Diaz, 1985; Jalali and Chand, 1992). Under favorable conditions the disease can cause much greater yield loss or may destroy the crop completely (Hari and Khirbat, 2009). Higher incidence of the wilt may inflict a yield decline of upto 5 q/h in chickpea (Khan, 2005). Fusarium wilt is also a major limiting factor in tomato production (Snyder and Hansen, 1940) and causes up to 15% annual loss to canning tomatoes in advanced countries like USA, but exact amount of loss in India is not known (Khan et al., 2007; Mandal et al., 2009).

Root-knot nematodes, Meloidogyne species such as M. incognita and M. javanica have been reported to cause 19-40% and 24-61% economic loss to chickpea (Ali et al., 1

2010) and tomato (Singh and Mathur, 2010a), respectively in India. Ali (1997) reported 25-60% avoidable loss in chickpea due to M. incognita. Besides direct damage, root-knot nematodes possess great capability to synergise other soil-borne pathogens leading to development of disease complex (Khan, 1993). The wilt disease complex caused by the species of Fusarium and Meloidogyne is a commonest and devastating disease and is considered an important constraint in the production of pulses and vegetables in India (Khan and Reddy, 1993; Samuthiravalli and Sivakumar, 2008). Infection of Meloidogyne spp. not only increases wilt severity but also moderates resistance of a cultivar against the fungus (Atkinson, 1892; Reed and Lateef, 1990; Castillo et al., 2003). In India, many wilt tolerant or resistant cultivars of chickpea viz., JG 74, Avrodhi, Pusa-212, ICC 12275, ICC 11322, ICC 11319, ICC 12272 and tomato viz., Florida 47 R, P48024 and P48025 become susceptible in the presence of root-knot nematodes (Dababat, 2007).

In view of enormity of the yield loss caused by various diseases to agricultural crops and the shortage of food material to feed the burgeoning population it has become essential to integrate effective and eco-friendly methods in general crop production practices. Although the age-old cultural practices like sanitation, crop rotation, mixed cropping, green manuring etc. to combat plant pathogens are environmentally safe but are slow in action and do not work in endemic situations. The pace of development of resistant varieties is slow and time taking and durability of resistance is short in spite of tremendous advancements made in field of plant genetic engineering. The chemical control too has its own limitations such as high capital investment, non-remunerative, poor-availability, selectivity, temporary effect, efficacy affected by physio-chemical and biological factors, development of resistance, contamination of food, health hazards, toxicity towards plants and animals, environmental pollution etc. To control a target organism by application of pesticides more than 100 species of non-target organisms are adversely affected (Tjamos et al., 1992). The pesticides, however, may prove less injurious, if they are applied at judicious dose. Hence to reduce the pesticide input in agriculture, it is important to utilize nonchemical options especially the biopesticides, which are highly specific to target pathogens; possess prerequisite biosafety standard and their mass production is cost effective. The microorganisms which suppress plant pathogens are commonly referred to as biocontrol agents and their commercial formulations as biopesticides. However, it is not

necessary that a biopesticide should always contain live microorganisms, the formulation may also contain microbial metabolites.

Presently awareness is growing to develop novel management practices that alone or in integration with other practices could bring about a reasonably good degree of reduction of disease potential coupled with sustainability of production, cost effectiveness and eco-friendliness. A large number of microorganisms from fungi and bacteria have shown some ability to antagonize plant diseases. As most of the soil-borne plant pathogens are fungi, biocontrol through mycoparasitism has been attempted extensively (Henis et al., 1979; Baker, 1987; Suarez et al., 2004; Sant et al., 2010). The microbial fungal can also work through other mechanisms (Khan and Anwer, 2011). Plant growth promoting organisms may also suppress plant diseases but they have not been adequately evaluated for disease management (Papavizas, 1985; Nair and Burke, 1988). These microorganisms may suppress plant pathogens through antibiosis (Vey et al., 2001), production of hormones (Osiewacz, 2002), solubilization of minerals (Harman et al., 2004; Benitez et al., 2004) and induction of host resistance (Harman et al., 2004). Among phosphate- solubilizing microorganisms (PSM), Aspergillus niger (Sen, 2000; Medina et al., 2007; Pandya and Saraf 2010; Khan and Anwer, 2009, 2011), A. awamori (Khan and Khan, 2002; Mittal et al., 2008), Penicillium digitatum (Asea et al., 1998; Mittal et al., 2008) etc. are most important microorganisms and may prove to be efficient biocontrol agents of plant pathogens if exploited properly (Khan et al., 2009).

A critical analysis of the relevant information on the biological control of wilt and root-knot diseases has revealed that majority of the research efforts have dealt with Trichoderma spp., Paecilomyces lilacinus and Pochonia (=Verticillium) chlamydosporia (Benitez et al., 2004; Federico et al., 2007; Khan and Anwer, 2011), whereas other antagonists have not been duly tested. Researchers have also tested antagonism of A. niger against plant pathogens and have found the fungus effective than Trichoderma spp. (Vassilev et al., 1996; Chet et al., 1997; Vassilev et al., 2006). However, these studies are of primitive type and lack some important biochemical (ochratoxin A negative/positive, production of HCN, NH3, H2S etc.) and molecular approaches. Moreover, due attention has not been paid towards genetic variability in A. niger aggregate in order to identify efficient strains with regard to antagonism and phosphate solubilization, and least efforts have been made to select environmentally safe strains of A. niger (ochratoxin A negative). 3

The antagonism by A. niger involves multiple mechanisms. A. niger competes with the pathogens for nutrients (Vassilev et al., 1996, 2006) and space (Mondal et al., 2000), modifies the microenvironment (Domich, et al., 1980), produces plant growth promoting substances (Mondal et al., 2000; Vaddar and Patil, 2007; Khan and Anwer, 2008) and elicit plant defense mechanisms (Bai et al., 2004; Gomathi and Gnanamanickam, 2004) and produce antibiotic (Sen, 2000; Benitez et al., 2004; El-Hasan et al., 2007), or directly parasitize the pathogenic fungi and hyphal lysis (Mondal et al., 2000). These indirect and direct mechanisms may operate together during the disease suppression (Howell, 2003). Effectiveness of A. niger against a pathogen depends on several factors such as the target fungus, plant types and environmental conditions, including soil nutrition, pH, temperature and iron concentration. Activation of each mechanism implies the production of specific compounds and metabolites such as plant growth factors (Harman et al., 2004), hydrolytic enzymes (Howell, 1998; Monte, 2001), siderophores (Eisendle et al., 2004), antibiotics (Chet et al., 1997) and carbon and nitrogen permeases (Mach and Zeilinger, 2003; Eisendle et al., 2004). There are some other most important attributes which are found in A. niger. The fungus does not produce mycotoxins and ribotoxin (Campbell, 1994). Interestingly the fungus is reported to decrease aflatoxin contamination (Wicklow et al., 1980; Horn and Wicklow, 1983). It is xerotolerant (Cooke and Whips, 1993) and grow at a wide range of temperatures (10–50 °C), pH (2.0–11.0), and osmolarity (from nearly pure water up to 34% salt) (Kis-Papo et al., 2003) and can also improve its thermostability (Zhang et al., 2007). The fungus is extremely resistant to herbicides, fungicides and pesticides at very high concentrations (Braud et al., 2006). A. niger isolates not only survive high concentration of many toxic heavy metals but also adsorb these metals (Ahmad et al., 2006).

The application of A. niger @ 8 g/kg seed, and soil with A. niger @ 30 g/pit in a field where muskmelon and watermelon crops were suffering from Fusarium wilt, R. solani and Pythium spp. resulted in 81% control of the disease. The vines were more vigorous, and even with 15% incidence of disease, yield was approximately 5% greater as compared to that in disease-free areas (Chattopadhyay and Sen, 1996). In an experiment, seed treatment with A. niger @ 8 g/kg effectively controlled the blast (Pyricularia oryzae), sheath blight (R. solani), brown spot (Helminthosporium oryzae) and false smut (Ustilaginoides virens) of rice in the field (Sehgal et al., 2001). Problems of pre and post- emergence damping-off incited by P. aphanidermatum and R. solani in fruit and vegetable 4

farms were successfully overcome by a combined treatment of seed and soil application of A. niger (Majumdar and Sen, 1998). Similarly, 93% control of charcoal rot of potato in a Macrophomina phaseolina-infested field was obtained with A. niger (Mondal, 1998). In another study, 87% control of black scurf of potato (R. solani) and a 10% increase in the yield by application of A. niger @ 8 g/kg on infected seed tubers grown in worst affected fields was reported (Sen et al., 1998). Lodhi (2004) has reported the control of soil borne diseases of potato by the application of A. niger alone or in combination with VAM fungi.

Winter sorghum can be severely damaged by Macrophomina infection; however, A. niger seed treatment brought down incidence of the disease from 30 to 7% (Das, 1998). Fusarium wilt of Hibiscus was satisfactorily controlled by the application of A. niger in the soil after uprooting the dead plants and soil population of the pathogen was found non- detectable level after a month of A. niger application (Sen, 2000). Malformation of mango caused by Fusarium moniliforme was controlled by spraying conidial suspension of A. niger over dead necrotic malformed panicles (Chand et al., 2007). Guava wilt caused by F. oxysporum f. sp. psidii and F. solani was controlled by the soil application of A. niger formulation prepared on the field wastes (Misra, 2007). Root-dip applications of A. niger resulted in significant decline in the rhizosphere population of F. oxisporum f. sp. lycopersici and increase in the tomato yield (Khan and Khan, 2001). In another study, direct soil inoculation with A. niger decreased the rhizosphere population of F. oxisporum f. sp. lycopersici by 23–49% while the tomato yield increased by 28–53% in a field experiments (Khan and Khan, 2002). Application of A. niger has also managed root-knot of tomato, significantly increased the plant growth and reduced the soil population of M. incognita (Goswami et al., 2008).

Critical analysis of the relevant researches and the evidences presented above have revealed that A. niger possesses great potential to suppress Fusarium and Meloidogyne species. In view of effectiveness of A. niger against these pathogens, it was considered desirable to study the diversity among A. niger aggregate to identify more efficient soil isolates of the fungus with multifarious activities of plant growth promotion and pathogen suppression. To exploit commercial use of the fungus, biopesticides of efficient isolates of A. niger were prepared so as to have formulations which could yield higher CFU load of A. niger and keep them viable for longer duration. The present study was undertaken with an objective to identify efficient soil isolates of A. niger aggregate (ochratoxin A negative) 5

collected from fields having different crops in 40 districts of Uttar Pradesh (North India) to control wilt (Fusarium oxysporum f. sp. ciceris and F. oxysporum f. sp. lycopersici), root- knot (Meloidogyne incognita) and the resulting fungus-nematode wilt disease complex of chickpea (F. oxysporum f. sp. ciceris + M. incognita) and tomato (F. oxysporum f. sp. lycopersici + M. incognita). To attain the objective following experiments were carried out.

1. Collection, isolation and identification (morphological and biochemical basis) of soil isolates of Aspergillus niger aggregate from different agricultural fields of Uttar Pradesh, India.

2. RAPD profiling of effective isolates of A. niger aggregate and their characterization for ochratoxin A production, phosphate solubilization, heavy metal bioadsorption and in vitro pathogen suppression (Fusarium oxysporum f. sp. ciceri, F. oxysporum f. sp. lycopersici and Meloidogyne incognita).

3. Evaluation of Aspergillus niger isolates for antagonism against Fusarium spp. and Meloidogyne incognita and for promotion of plant growth and yield of chickpea and tomato under pot condition.

4. Evaluation of selected isolates of Aspergillus niger (based on performance in Expt.-III) for effectiveness against Fusarium wilt, root-knot and fungus-nematode wilt disease complex of chickpea and tomato under field condition.

5. Preparation of biopesticides of selected isolates of Aspergillus niger (based on performance in Expt.-IV), and their field trial for effectiveness against the wilt, root-knot and fungus-nematode wilt disease complex of chickpea and tomato.

REVIEW OF LITERATURE

Pulses are widely cultivated in India especially by small holding farmers. These are low input crops because of their ability to assimilate atmospheric nitrogen. The pulse cultivation also contributes towards improvement of soil fertility and subsequently the productivity of non-leguminous crops in the rotation. Equally important is their role in the diet of the people particularly the rural people, who can not afford expensive animal protein products to meet their dietary needs of essential amino acids (Table 1). Pulses efficiently complement the cereal rich food in making a wholesome meal by balancing the amino acid and micronutrient content of the diet. No wonder they are called ‘poor man’s meat’. Presently, pulses are grown on around 67 million hectares with 61 million tonnes of production worldwide. In India, latest estimates for 2007-08 indicate that the production of pulses in the country is 15.1 million tonnes from an area of 24.45 million hectares (Ministry of Agriculture, 2009). In spite of being the largest producer in the world with 25% share in the global production (Ministry of Agriculture, 2009), India has to import over 2.0 million tonnes pulses every year to meet the domestic requirement (ASSOCHAM, 2009). The major pulses cultivated and consumed in India are chickpea, pigeonpea, mungbean, urdbean, lentil and fieldpea (Ranjekar, 2003; Table 2) contributing 39, 21, 10, 7 and 5%, respectively, to the total production of pulses in the country (Ministry of Agriculture, 2009). Though individual crops have undergone significant changes with respect to area, production and productivity, the per capita availability of pulses has declined from 64 g/capita/day to less than 35 g/capita/day as against the recommendation of 80 g/capita/day by FAO/WHO (ASSOCHAM, 2009). Besides pulses, vegetables are another important group of crops. Most of the vegetables are important cash crops in India, hence their production improves economic status of farmers, especially the smaller ones. They occupy an eminent position in the balanced diet of human beings, but a considerable proportion of the world’s population remains deprived of vegetables due to either insufficient production and supply or cost constraints (Ali and Tsou, 1997). They are important protective food and highly beneficial for the maintenance of health and prevention of disease. They contain valuable food ingredients which can be successfully utilized to build up and repair the body. They not only supply the vital protective nutrients like minerals, vitamins, important material for

roughages, but also provide energy rich food which is nutritionally far superior to the non- vegetarian food.

Table 1. Amino acids composition (mg/100 g protein) of protein from different sources. Amino acid Animal Pulse Cereal Isoleucine 46.7 45.3 39.8 Leucine 79.6 78.9 86.3 Lysine 84.3 67.1 30.5 Methionine and Cystine 37.7 25.3 41.1 Tryptophan 11.4 12.3 12.1

Table 2. Protein contents in pulses. Pulse Protein (%) Chickpea 22 Lentil 25.6 Mungbean 22.1 Pea 19.7 Pigeonpea 22.9 Urdbean 21 Source (Table 1-2): The Hindu Survey of Indian Agriculture, 2002.

Presently India is the second largest producer of vegetables in the world next to China with an annual production of 101.43 million tonnes (about 13% of the world vegetable production) from an area of 6.755 million hectare occupying 3.2% of the total cultivated area of the country (Economic survey, 2009). The per capita production of vegetables is 100 kg per annum against 250 kg/capita/year by FAO/WHO (Ministry of Agriculture, 2009). To increase the production so far 230 high yielding open pollinated varieties, 99 hybrids and 40 vegetable varieties resistant to biotic and abiotic stresses have been released by public funded research agencies in India (Ministry of Agriculture, 2009). In India, almost all kinds of vegetables are produced and consumed, among them tomato is the most commonly grown and used after potato (Ministry of Agriculture, 2009). Population in India is growing at about 1.4% annually i.e., adding 16 million people every year to the already large population base of the country. More than half of Indian children under the age of five are malnourished and about 27% of newborns are significantly underweight (WHOSIS. 2010) and more than two billion people, mostly in

developing countries, suffer from lack of sufficient micronutrients in their diets (Graham and Welch, 1994; Ali and Tsou, 1997) and 2.7 million people per year died globally due to low fruit and vegetable intake (FAO/WHO, 2004). It is estimated that India’s population will touch nearly 1.35 billion marks by 2020 AD and will require double of the pulses (30.3 million tonnes) that are produced presently in the country (Ranjekar, 2003) and about 30% more vegetables (127.2 million tonnes) produced presently other than potato and tubers (Ministry of Agriculture, 2009). Hence, the main challenge is to attain self-sufficiency in pulse and vegetable production to meet the increasing demand of protein, minerals, vitamis etc. to the poorer people. Chickpea production in India and the world Chickpea after dry beans and dry peas is the third most important grain legume in the world. Its cultivation is largely done in Asia with 90% of the global area. India is the principal chickpea producing country with 83% share in the region and 65% of acreage of world, followed by Pakistan (9%) and Turkey (6%) in production (FAOSTAT, 2009; Fig. 1). It is also grown in North and Central America, the Mediterranean region, the West Asian and North African region and Eastern Africa. It has been introduced to the Americas and gained popularity especially in Mexico. However, the crop assumed greatest significance in Indian subcontinent. The crop has expanded to new niches such as Australia and Canada. Chickpea is grown mostly as a rainfed crop under conserved moisture in the post rainy season in the semi arid tropics and in spring and winter seasons in the temperate and Mediterranean types of climate. “Kabuli types” are mostly grown in the West Asian and North African region, the America and Europe, while “Desi types” predominate in Asia, parts of Africa and Australia. The harvested area and production of chickpea in India during 2007-08 were found 7.5 million hectare and 5.75 million tonnes, which represent 30 and 38% of the national pulse acreage and tonnage, respectively (FAOSTAT, 2009). Four states viz., Madhya Pradesh, Rajasthan, Uttar Pradesh and Maharashtra together contribute 87% of the total area (Table 3). The area under chickpea has shown steady uptrend in Andhra Pradesh, Gujarat, Karnataka, Madhya Pradesh, Orissa, Maharashtra, Rajasthan and Tamil Nadu. Except Rajasthan, all other states in this category are located in Southern and Central zones. As far as the productivity rate is concerned, chickpea has observed considerable improvement from 611 kg/h in 1971-75 to 761 kg/h in 2007-08 (FAOSTAT, 2009). The productivity of chickpea in Northern states is 1.87q/h and in Southern and Central states of India along with Rajasthan is 2.02 q/h. 9

Fig. 1. Share of different countries in global chickpea production (FAOSTAT, 2009).

Table 3. Area and Production of chickpea in different states of India. State Production Area Yield (Lakh tonnes) (Lakh hectares) (Kg per h) Andhra Pradesh 96.4 142.5 679 Bihar 110.0 107.0 1028 Gujrat 41.8 81.9 512 Haryana 58.0 100.0 580 Karnataka 181.0 319.0 567 Madhya Pradesh 2493.3 2748.1 908 Maharashtra 580.0 932.0 622 Orissa 16.0 32.0 500 Punjab 6.1 6.3 968 Rajasthan 677.9 975.3 695 Tamil Nadu 5.3 8.0 663 Uttar Pradesh 779.3 822.3 948 West Bengal 30.0 27.0 1111 Source: Chickpea Research in India: Edited by Masood Ali, Shiv Kumar, N.B. Singh, IIPR, Kanpur, India.

Origin and domestication Chickpea (Cicer arietinum L.) is thought to have originated in Anatolia (Turkey), where three closely related wild species C. bijugum, C. echinospermum, and C. reticulatum are commonly found in nature (van der Maesen, 1984). Chickpea seeds had been occasionally recovered in pre-historic sites in the Near East (Renfrew, 1973). However, Ramanujam

(1976) reported that remnants of chickpea radiocarbon are dated at 5450 BC and there is evidence of its cultivation in the Mediterranean basin in 3000-4000 BC. The earliest record of chickpea in Northern India (Uttar Pradesh) dated at 2000 BC, and from the Southern India much later (Chowdhury et al., 1971; Vishnu-Mittre, 1974). Taxonomy of chickpea Order Fabales Family Fabaceae (Leguminosae) Subfamily Faboideae (Paplionoideae) Tribe Cicereae Genus Cicer Species arietinum

Uses of chickpea Chickpea is valued for its nutritive seeds with high protein content 12.6-30.5% (Singh et al., 1997). Although chickpea is a rich source of protein, its protein quality is limited by sulphur containing amino acids, methionine and cystine. Chickpea generally meets adult human requiremnet for all essential amino acids with trace amount of methionine and cystine and rich in fiber, minerals (phosphorus, calcium, magnesium, iron and zinc) and β-carotene (Table 4). Based on the amino acid composition, chickpea proteins were found to be of higher nutritive value as compared to other legumes (Gupta and Kapoor, 1980). The levels of different protein fractions primarily control the essential amino acid compositon of chickpea seed proteins (Table 5) Chickpea seeds are eaten fresh as green vegetables, parched, fried, roasted and boiled; as snack food, sweet and condiments; seeds are ground and the flour can be used as a soup, dhal and to make bread; prepared with pepper, salt and lemon and served as a side dish (Saxena 1990). Dhal is the splited grain without its seedcoat, dried and cooked into a thick soup or ground into flour for snacks and sweetmeats (Saxena, 1990; Hulse, 1991). The protein content of Dhal is higher than that of the whole seed due to much lower protein content of seed coat (Table 6). Sprouted seeds are eaten as a vegetable or added to salads. Young plants and green pods are eaten like spinach. A small proportion of canned chickpea is also used in Turkey and Latin America, and to produce fermented food. Animal feed is another use of chickpea in many developing countries. An adhesive may also be prepared; although not water resistant, it is suitable for plywood. Gram husks, and green or dried stems and leaves are used for livestock feed; whole seeds may be milled directly for feed. Leaves are said to yield an indigolike dye. Acid exudates from the leaves can be applied

Table 4. Nutritional value of chickpea and tomato per 100 gram of edible portion. Value per 100 grams Nutrient Unit

Chickpea Tomato Amino acids Tryptophan g 0.185 0.008 Threonine g 0.716 0.029 Isoleucine g 0.828 0.027 Leucine g 1.374 0.042 Lysine g 1.291 0.042 Methionine g 0.253 0.01 Cystine g 0.259 0.015 Phenylalanine g 1.034 0.03 Tyrosine g 0.479 0.02 Valine g 0.809 0.03 Arginine g 1.819 0.029 Histidine g 0.531 0.018 Alanine g 0.828 0.033 Aspartic acid g 2.27 0.161 Glutamic acid g 3.375 0.427 Glycine g 0.803 0.029 Proline g 0.797 0.022 Serine g 0.973 0.031 Proximates Water g 11.53 94.78 Energy kcal 364 16 Energy kj 1525 67 Protein g 19.3 1.16 Total lipid (fat) g 6.04 0.19 Ash g 2.48 0.69 Carbohydrate, by difference g 60.65 3.18 Fiber, total dietary g 17.4 0.9 Sugars, total g 10.7 - Minerals Calcium, Ca mg 105 5 Iron, Fe mg 6.24 0.47 Magnesium, Mg mg 115 8 Phosphorus, P mg 366 29 Potassium, K mg 875 212 Sodium, Na mg 24 42 Zinc, Zn mg 3.43 0.14 Copper, Cu mg 0.847 0.062 Manganese, Mn mg 2.204 0.088 Selenium, Se µg 8.2 0.4 Vitamins Vitamin C, total ascorbic acid mg 4 16 Thiamin mg 0.477 0.046 Riboflavin mg 0.212 0.034 Continued…

Table 4 Continued… Value per 100 grams Nutrient Unit

Chickpea Tomato Niacin mg 1.541 0.593 Pantothenic acid mg 1.588 0.186 Vitamin B-6 mg 0.535 0.06 Folate, total µg 557 29 Folate, food µg 557 29 Folate, DFE µg_DFE 557 29 Choline, total µg 95.2 0 Vitamin B-12 µg 0 75 Vitamin A, RAE µg_RAE 3 0 Retinol µg 0 1496 Carotene, beta µg 40 - Vitamin A, IU IU 67 0 Lycopene µg 0 8.8-42* Vitamin E (alpha-tocopherol) mg 0.82 - Vitamin K (phylloquinone) µg 9 - Lipids Fatty acids, total saturated g 0.626 0.025 14:00 g 0.009 0 16:00 g 0.501 0.019 18:00 g 0.085 0.007 Fatty acids, total monounsaturated g 1.358 0.028 16:1 undifferentiated g 0.012 0.001 18:1 undifferentiated g 1.346 0.028 Fatty acids, total polyunsaturated g 2.694 0.076 18:2 undifferentiated g 2.593 0.073 18:3 undifferentiated g 0.101 0.003 Phytosterols mg 35 4 IU: One International Unit of Vit-A = 0.3 mg of Vit A alcohol; * Rao and Rao (2007). Source: USDA National Nutrient Database for Standard Reference, Release 22 (2009)

Table 5. Essential amino acid composition of seed protein fractions of chickpea. Protein fraction (g/100 g protein) Amino acid Albumin Globulin Glutelin Prolamin Lysine 10.8 6.4 6.8 2.3 Methionine + Cystine 5.3 1.8 2.6 1.5 Phe + Try 9.3 9.0 8.1 5.7 Threonine 5.4 3.5 5.7 2.2 Valine 4.5 4.2 5.7 2.1 Isoleucine 5.1 4.4 5.4 2.3 Leucine 9.8 7.5 9.1 1.6

Table 6. Nutritional composition of whole seed and Dhal component of chickpea. Constituent (%) Whole seed Dhal Value (Kcal) 347.6 360.8 Protein 22.0 24.5 Starch 47.3 56.0 Sugar 5.8 4.9 Ash 3.2 2.8 Fat 5.3 5.7 Crude fiber 6.3 1.1 Dietary fiber 19.0 11.3 Source (Table 5-6): Chickpea Research in India: Edited by Masood Ali, Shiv Kumar, N.B. Singh, IIPR, Kanpur, India. medicinally or used as vinegar. In Chile, a cooked chickpea milk (4:1) mixture was good for feeding infants, effectively controlled diarrhea. Chickpeas yield 21% starch suitable for textile sizing, giving a light finish to silk, wool, and cotton cloth (Duke, 1981). Medicinal uses Among the food legumes, chickpea is the most hypocholesteremic agent; germinated seeds are reported to be effective in controlling cholesterol level in rats (Geervani, 1991). Glandular secretions of the leaves, stems and pods consist of malic and oxalic acids giving a sour taste. In India these acids are used to be harvested by spreading thin muslin over the crop during the night. In the morning the soaked cloth is wrung out and the acids are collected in bottles. Medicinal applications include use for aphrodisiac, bronchitis, catarrh, cutamenia, cholera, constipation, diarrhea, dyspepsia, flatulence, snakebite, sunstroke and warts. Acids are supposed to lower the blood cholesterol levels. Seeds are considered antibilious (Duke, 1981). Agronomy of chickpea Chickpea is a quantitatively long day plant (Summerfield et al., 1981; Fig. 2). It requires cool climate for its growth and development and high temperature for maturity. The optimum temperature for its growth ranges from 15-25°C. Frost at the time of flowering results failure of flowers to develop seeds or killing of the seeds inside the pod. Soil temperature of 30°C or above adversely affects the rhizobial infection in nitrogen fixation process (Dart et al., 1975). The deep rooted system makes it a preferred crop in dry tracts with an annual rainfall of 60-90 cm. Excessive rains soon after sowing or at flowering and fruiting or hailstorms at ripening cause heavy loss. Thus areas with moderate rainfall are considered suitable for chickpea production (Gaur et al., 2010).

Chickpea thrives well on a wide range of soils in India including sandy (Thar Desert), Sandy loam (Entisols of North India) and black cotton soils (Vertisols of Central India, Maharashtra and Daccan plateau). It is highly sensitive to saline and sodic soils (Chandra et al., 1988). A pH range of 6-8.5 is favourable and an acid soil with pH of 4.6 seems to increase the problem of Fusarium wilt (Kay, 1979).

Fig. 2. Different stages of chickpea growth: Field view (A), root system showing nodules (B), single young plant (C), pods and seed of chickpea (D).

Germplasm There are two types of chickpea i.e., desi (Cicer arietinum L.) and kabuli (C. kabulium). Desi type has small seeds with angular shape edge and pigmented seed coat which may vary from black brown to cream or yellow whereas kabuli type has large and round seeds with

white or pale cream seed coat. Unlike cereals, high-yielding photo-insensitive cultivars are not available in pulses and this appears to be the most important reason for low productivity of pulse crops in the country. However, recently some improved varieties of chickpea have been evolved and recommended for different agroclimatic conditions of India (Table 7). Fertilizer Management Judicious and balanced use of nutrient is required not only for quantitative and qualitative increase in yield but also to help plant growth vigorously and overcome biotic and abiotic stresses. Total uptake of nutrients by chickpea crop has been estimated at 60-200 kg N, 5-15 kg P and 60-170 kg K per hectare (Ahlawat, 2000). Macro nutrients In general, chickpea has shown response to application of nitrogen upto 15-20 kg/h. However, response to nitrogen is poor under efficient biological nitrogen fixation system when nodules develop properly (Saxena and Sheldrake 1980). Field experiments conducted in the Indo-gangetic plains of the country suggest 19-63% yield increase in chickpea due to inoculation with rhizobium (Ali et al., 2002). When chickpea is to be cultivated grown after potato, maize, sorghum, or late duration rice, a greater nitrogen dose (25-50%) is recommended due to low soil nitrogen (Ali and Mishra, 2000). Babu et al., (2000) have, however, reported that only 50% of the recommended dose of nitrogen was sufficient to realize higher yield of chickpea grown after potato. Under late sown condition, the crop responds well upto 40 kg/h. Foliar application of 2% urea at the time of pod formation is also useful (Bharud 2001; Dudhade and Patil, 2001; AICRP, Anonymous, 2003). Phosphorus is the most important nutrient for chickpea and increases protein content in the grains (Guhey et al., 2000). Conspicuous response of phosphorus application in chickpea has been observed by several workers (Dev 1987; Prabhakar and Saraf, 1991). Among various sources of phosphorus, single super phosphate (SSP) has been found more effective over DAP (Anonymous, 1994a). Phosphate solubilizing bacteria (PSB) have been found effective in increasing the efficiency of applied phosphorus. Application of PSB culture either with DAP or SSP at 40 kg P2O5/h in calcareous soil, where phosphorus fixation is a common phenomenon was found to improve chickpea yield and available phosphorus status of the soil (Saad and Sharma, 2001; Gaur et al., 2010). Foliar application of 2% DAP has been found beneficial in increasing the seed yield of chickpea under rainfed conditions in North East Plain Zone, North West Plain Zone, Central Zone and Southern Zone of the country.

Table 7. Improved varieties of chickpea recommended for general cultivation in different states in India. State Recommended varieties Andhra Pradesh ICCV 2 (Sweta), ICCV 10 (Bharati), Kranti, JG 11, ICCV 37 Assam KWR-108, BG-256, L-550, KPG-59 Bihar KWR-108, Avrodhi, BG-256, Pant G-114, Pusa-209, L-550, Pusa- 1003, Pusa 372, KPG 59, Gujarat Gram 4 Gujarat Pusa-391, Vijay, ICCV-10, ICCV-4, Pusa-240, GG-1, Pusa-1053, Pusa 372, Vishal, Gujarat Gram 1, Gujarat Gram 2 (GCP 107) Haryana GNG-469, Pusa-362, Gora Hisari, Karnal Chana, Gaurav, H-208, H-335, Pusa 1053, KPG 59 (Udai), Pusa 372, Haryana chana 1 (H 82-2), Karnal Chana 1 (CSG 8962), Samrat, Vardan, Pusa 267, KGD 1168, Chamatkar (BG 1053) Himachal Pradesh BBG-1, Haryana Chana-1, L-550 Jammu & Kashmir GNG-469, L-550, PBG-1, Haryana Chana-1 Karnataka BDN 9-3, ICCV-2, Annegiri-1 Bharati (ICCV 10), JG 11, , ICCV 4, Madhya Pradesh Vijay, Pusa 391, Pusa 256, Pusa 1053, Vishal, Phule G 5, JG 218, Gujarat Gram 1, ICCV 10, JG 322, JGG 1, JG 16, JG 315, JG 74, JG 130 Maharashtra Pusa 372, Pusa 391, Pusa 1053, PG 5, Dharwad pragati, Sweta, KAK2, Vishal, Vijay, Vikas, Phule G 5, PG 12, JG 16, ICCV 10 Orissa Radhey, ICCV 10, L 550, Pusa 372, Pusa 391, Pusa 1003, KWR 108, ICCV 10, KPG 59 Punjab Pusa 256, Pusa 261, Pusa 267, Pusa 329, Pusa 362, Pusa 372, DCP 92-3, Vardan, Samrat, Karnal Chana 1, L 551, BG 1053 , Hare Chole 1 Rajasthan Pusa 267, P 261, P 256, RSG 44, DCP 92-3, Pusa 329, Pusa 372, Pusa 1003, Vardan, Samrat, BG 1053, RSG 888 Tamil Nadu Bharati, JG 11, CO 3, CO 4 Uttar Pradesh KWR 108, Pusa 372, Vardan, Udai, DCP 92-3, Samrat, Karnal Chana 1, Pusa 1003, Gujarat, Avrodhi, Gram 4, Sadabahar, Sadbhawana Surya, BG 256 West Bengal KWR 1087, Pusa 372, KPG 59, GCP 105, B 108, B 115 NE states KPG 59 Source: Directorate of Pulses Development (DPD), Department of Agriculture & Co- operation, Ministry of Agriculture, Government of India (2009).

The response of chickpea to potassium is small and of seldom significance. The response to applied potassium has been reported in laterite, red, coastal and deltic alluvial soil of India. Generally application of 20 kg K2O/h is recommended under deficient soil conditions. Indian soil is rich in available potassium hence this element has rarely found a place in fertilizer schedule of chickpea (Subbarao and Srinivasarao, 1996). Sulphur, being a constituent of predominant amino acids (methionine, cysteine and cystine), ferrodoxin containing nitrogenase and also involved in metabolic activities of

vitamins, thiamine and coenzyme. The element is greatly required by chickpea crop. Study conducted by Srinivasarao et al. (2002) in different pulse growing regions revealed low to medium range of available sulphur in Indian soil. In sulphur deficient soil, the response to applied sulphur has been found in the range of 20-60 kg S/h (Aulakh and Pasricha, 1986). Ali and Singh (1995), however, have recorded significant increase in plant growth and yield of chickpea response upto 40 kg S/h (Ali and Singh, 1995). Micro nutrients Among the micronutrients, zinc deficiency is widely observed followed by boron and iron. Zinc is also found to reduce the growth of collar rot, wilt and root rot in chickpea (Gupta,

1999). Yield gain of 320 kg/h with the application of 25 kg ZnSO4/h has been observed (Takkar and Nayyar 1986). Boron deficiency is an emerging problem in some states like Orissa (69% samples deficient), Bihar (39%), U.P. (23%) and Gujarat (5%) (Takkar 1996). Soil application of 10 kg borax/h has given yield advantage to the tune of 306-405 kg/h in chickpea (Ali and Mishra, 1996). Seed treatment of chickpea with Mo and Fe can synergise the effect of applied phosphorus, PSB and rhizobium in terms of yield and nodulation (Sarawgi et al., 1999). Tomato production in India and the world Tomato is the world's largest vegetable crop after potato, but it tops the list of canned vegetables. Total global area under tomato is 4.62 million hectare and the global production is to the tune of 128 million tonnes with an average productivity 277 Q/h (NHB, 2010). India rank first in tomato production after China with 11.98 million tonnes and 9.4% of global production. The average productivity of tomato in our country is merely 193 Q/h verses 277 Q/h of world productivity which is 30% less, while its productivity in USA is 661 Q/h, in Spain 668 Q/h, in Brazil 566, in Greece 535 Q/h, and in Italy 517 Q/h (Table 8, Fig. 3). In India, it is now the most widely grown vegetable crop and grown throughout the country in farm gardens, small kitchen gardens and by market gardeners for fresh consumption as well as for processing purposes. In India, tomato is grown almost all over the country and ranks first with production of 11.98 million tonnes which is 8.8% of the total vegetable production of the country followed by eggplant (7.4%) (NHB, 2010). The estimated area under tomato cultivation is 0.62 million hectares which is 8.5% of the total vegetable area of the country (NHB, 2010). As far as productivity of tomato is concern national productivity is 193 Q/h (NHB, 2010), but depending on the variety, growing season, type of soil and cultural practices followed it produces 150 to 450 quintals of fruits per hectare. At present, Karnataka is the leading 18

producer of tomato contributes 13.2% of national production where as Uttar Pradesh productivity is highest in the country with 410 Q/h (Table 9).

Table 8. Area, production and productivity of tomato in major countries of the world. Country Area Production Productivity (000’ ha) (000’t) (t / ha) Brazil 61 3453 56.6 China 1305 31626 24.2 Egypt 195 7600 39.0 India 620 11980 19.3 Iran 139 4781 34.4 Italy 139 7187 51.7 Mexico 119 2800 23.5 Spain 72 4810 66.8 Turkey 260 10050 38.7 USA 167 11043 66.1 Others 1624 35281 21.7 TOTAL 4616 127993 27.73

Fig. 3. Share of different countries in global tomato production. Source (Table 10, Fig 3): National Horticulture Board (NHB) Data base (2010).

Table 9. Area, Production and productivity of tomato in different states of India. State/Union Territories Area Production Productivity % share of (000’ h) (000’t) (t/h) India production Karnataka 48.3 1580.0 32.7 13.2 Andhra Pradesh 74.1 1408.1 19.0 11.8 Orrisa 103.1 1391.9 13.5 11.6 Maharashtra 50.0 1112.5 22.3 9.3 Bihar 47.5 1062.1 22.4 8.9 West Bengal 53.5 1050.0 19.6 8.8 Gujarat 33.8 841.3 24.9 7.0 Chhatishgarh 41.3 600.6 14.5 5.0 Madhya Pradesh 35.3 529.3 15.0 4.4 Jharkhand 21.8 436.2 20.0 3.6 Haryana 22.7 384.3 17.0 3.2 Tamil nadu 27.1 353.7 13.0 3.0 Himachal Pradesh 9.6 336.3 35.2 2.8 Uttar Pradesh 6.2 254.9 40.9 2.1 Rajasthan 17.0 178.0 10.5 1.5 Punjab 6.2 150.7 24.3 1.3 Jammu &Kashmir 8.1 122.1 15.0 1.0 Uttranchal 7.6 92.5 12.1 0.8 Delhi 1.7 35.0 20.1 0.3 Tripura 1.3 24.9 19.4 0.2 Manipur 1.8 23.1 12.5 0.2 Mizoram 0.6 5.7 9.0 0.0 Nagaland 0.7 4.0 5.9 0.0 Dadra & Nagar Haveli 0.4 1.8 5.1 0.0 Pondicherry 0.1 0.7 11.8 0.0 Daman & Diu 0.0 0.0 0.9 0.0 Total 619.8 11979.7 19.3 Source: National Horticulture Board (NHB) Data base (2010)

Origin and domestication There has been long existed controversy regarding the place of domestication. The wild tomato species are native to Western South America from Ecuador South to Northern Chile and the Galapagos Islands (Thompson and Kelly, 1957; Knapp, 2002). The progenitor of the cultivated species (Lycopersicon esculentum) currently is widespread throughout warm regions of the world, but many of these are recent introductions. There are two competing hypotheses of the origin of domestication of tomato, one supporting a Peruvian origin, another is a Mexican origin. Mexico is presumed to be the most probable region of domestication with Peru as the centre of diversity for wild relatives (Larry and Joanne,

2007). Tomatoes were first recorded outside the Americas in Italy in 1544 (Sims, 1980). They were cultivated first as ornamental or curiosity plants and thought by many to be poisonous. They were first accepted as a vegetable in southern Europe during the late 16th century but in India it became popular within the last six decades (Nath et al., 1984). Taxonomy of tomato

Order Solanales Family Solanaceae Genus Lycopersicon Species esculentum

The taxonomy of tomato has been controversial since the time of Linnaeus. The controversy involves in the generic placement, Lycopersicum or Solanum, as well as interspecific relationships. Solanum lycopersicum cv. cerasiforme is thought to be an ancestor of cultivated tomato, based on its wide occurrence in the Central America and the presence of a shorter style in the flower (Cox, 2000). However, recent genetic investigations have shown that the plants known as ‘cerasiforme’ are a mixture of wild and cultivated tomatoes rather than being ‘ancestral’ to the cultivated tomatoes (Nesbitt and Tanksley, 2002). In 1753, Linnaeus placed the tomato in the genus Solanum (alongside the potato) as Solanum lycopersicum. However, in 1768, Philip Miller moved it to its own genus, naming it Lycopersicon esculentum. This name came into wide use but genetic evidence has now shown that Linnaeus nomenclature was correct to put the tomato in the genus Solanum, as Lycopersicum (Peralta et al., 2001; Peralta et al., 2006; Peralta and Spooner 2007). Hybrids of tomato and diploid Potato can be created in the lab by somatic fusion, and are partially fertile (Jacobsen et al., 1994), providing evidence of the close relationship between these species. However, the name Solanum lycopersicum has not yet been adopted by the International Code of Botanical Nomenclature (ICBN), hence to avoid confusion in the present thesis, Lycopersicon esculentum Mill. has been used as a botanical name of tomato. Use of tomato Tomatoes are now eaten freely throughout the world. It is a diet rich vegetable (Table 4) and used in diverse ways, including raw in salads, and processed into ketchup or tomato soup. Unripe green tomatoes can also be fried used to make pickled. Tomatoes are acidic, making them especially easy to preserve in home canning whole, in pieces, as tomato sauce or paste. Tomatoes are used extensively in Mediterranean cuisine, especially Italian and Middle Eastern cuisines. They are a key ingredient in pizza, and is used in pasta sauces.

Medicinal uses Tomato is one of the richest vegetables which keeps our stomach and intestine in good condition (Giovannucci, 1999). The pulp and juice are digestible, mild aperients, a promoter of gastric secretion, stimulates torpid liver and is good in chronic dyspepsia, blood purifier and also considered to be intestinal antiseptic, useful in cancer of the mouth (Miller et al., 2002; Colleen et al., 2006). Their consumption is believed to benefit the heart among other things (Sesso et al., 2003). They contain lycopene, one of the most powerful natural antioxidants (Ishida and Chapman, 2004). In some studies lycopene, especially in cooked tomatoes, has been found to help prevent prostate cancer (Boileau et al., 2003; Nkondjock et al., 2005; Canene-Adams et al., 2007). Lycopene has also been shown to improve the skin's ability to protect against harmful UV rays (Erhardt et al., 2003). Tomato consumption has been associated with decreased risk of breast cancer (Zhang et al., 2009), head and neck cancers (Freedman et al., 2008) and might be strongly protective against neurodegenerative diseases (Fall et al., 1999; Rao and Balachandran, 2002; Suganuma et al., 2002). Agronomy of tomato Tomato, Lycopersicon esculentum Mill. is a herbaceous short-lived perennials cropped as annuals (Peet, 1995; Fig. 4). It is grown throughout the country. The major tomato producing states are Karnataka, Andhra Pradesh, Orissa, Maharashtra and Bihar which together occupy about 52% of area and 55% of the production. Tomato is a moderate season crop and does not tolerate frost but different optimum temperature is required for different stages of tomato (Table 10; Shankara et al., 2005). High temperatures followed by low humidity and dry winds increase flower drop and may be no fruit set (Smith, 1932). Both high and low temperatures interfere with fruit setting. The best pollen grain germination takes place at 21 to 23°C and very poor at 10 and 38°C, but at 14°C the seeds can germinate. Temperatures below 21°C can cause fruit abortion. The fruits develop good colour and better quality when weather is warm and sunny (Singh et al., 2004). Tomato can be grown in many types of soil from light sandy to heavy clay. A well drained, fairly light fertile loam with a fair moisture holding capacity free from hard layer is ideal for growing a good crop of tomato. Good texture of the soil is of primary importance. Poor and medium quality land produce good early crop, if managed properly (Singh et al., 2004). Tomato crop prefers a soil reaction ranging from pH 6 to 7 (Choudhury, 1976). In acidic soil, liming will be beneficial (Shinjiro et al., 2009).

Fig. 4. Different stages of tomato growth: Field view (A), nursery beds (B), single young plant (C), fruit of tomato (D).

Table 10. Temperature requirements for different stages of tomato.

Stages Temperature (°C)

Min. Optimum Max. Seed germination 11 16-29 34 Seedling growth 18 21-24 32 Fruit set 18 20-24 30 Red colour development 10 20-24 30

Germplasm There are two types of tomato on the basis of growth habit, indeterminate and determinate types. In case of indeterminate type, the varieties terminate in the vegetative bud. Generally, the flowers are borne at every third internode separated by three leaves (Dhesi and Nandpuri, 1968). In case of determinate, the varieties terminate in the flower bud. They generally have a flower cluster at every internode. Such varieties do not have adequate foliage for fruiting. A large number of varieties are released for entire country and some are suitable for different agroclimatic conditions and cropping systems in different states of India (Table 11). Fertilizer Management The tomato crops responds very well to manureal and fertilizer application. The amount of plant nutrients required by the crop depends upon the variety and season of crop. A crop grown in spring-summer will require more nitrogen as compared to crop of winter season. Early maturing varieties will require less nitrogen as compared to long duration ones. Fertility status is also one of the main factors affecting the recommendation of the nutrients. However, different recommendations made for various regions of the country. Macro nutrients

Nitrogen (N) in the form of ammonium sulphate @ 80-120 kg/h, phosphorus (P2O5) in the form of super phosphate @ 20-80 kg/h and potash (K2O) in the form muriate of potash @ 35-60 kg/h required for tomato crop depending on variety (Yawalkar et al., 1962). Hybrid varieties need more nutrients than others. In addition to above doses, 15 to 20 tonnes of well decomposed farmyard manure (FYM) may also be added in the soil during field preparation. Half dose of N and full dose of P2O5 and K2O should be applied as basal while half dose of N may be applied as top dressing after 25 to 30 days of transplanting. Kamalnathan and Thambuaj (1970) recommended 100 kg of nitrogen, 80 kg phosphorus and 50 kg potash over 25 tonnes of FYM/h for the tomato plants spaced at 75×45 cm apart.

For hybrid varieties, the recommended dose per hectare is 180 kg nitrogen, 100 kg P2O5 and

60 kg K2O. 60 kg nitrogen and half of phosphorus and potassium are given at the time of transplanting. Remaining quantities of phosphorus and potassium and 60 kg nitrogen is top dressed 30 days after transplanting. A third dose of 60 kg nitrogen is applied 50 days after transplanting (Paroda and Chadha, 1996). Kamruddin et al., (1978) reported that the high doses of nitrogen produced significantly more number of flowers and fruits than the lower doses. Six sprays of 1% urea at weekly intervals, starting at the seedling stage, produced significant increase in yield of tomato (Tiwari and Chhonkar, 1967). 24

Table 11. Varieties of tomato recommended for different states or all over the country. State Recommended varieties Delhi Hissar Arun Karnataka Arka Vikas ( Sel 22 ) (heat and moisture stress resistant), Arka Saurabh (Sel - 4) , Arka Ahuti ( Sel 11 ), Arka Ashish ( IIHR - 674 ) (powdery mildew tolerant), Arka Abha ( BWR 1) (bacterial wilt resistant), Arka Alok ( BER - 5 ) (bacterial wilt resistant), Arka Vishal ( FM HYB -1), Arka Vardan ( FM hyb -2) (Nematode resistant), Arka Shreshta (bacterial wilt resistant), Arka Abhijit ( BRH 2) (bacterial wilt resistant). Hissar Arun

Madhya Sweet-72 Pradesh Maharashtra 4 F1, Hissar Arun Orissa Hissar Arun Punjab S-12, PanjabChhuhara, HS 102, Haryana HS 102, HS 110, Hissar Anmol (TLCV resistant), Hissar Arun Tamil Nadu Co-1, Co-3 Uttar HS 102, Hissar Arun, PanjabChhuhara, Pradesh For all over High yielding India Roma, Rupali, Pusa Ruby, Pusa Early Dwarf, S-152, ARTH 3, ARTH 4, HS 101 (winter season for north India), Krishna, KS 2, Matri, MTH 6, NA 601, Pant Bahar, Pusa Divya, , Pusa Gaurav, Pusa Sadabahar, Pusa Uphar; Hybrid (determinate): Pusa hybrid-1, Pusa hybrid-2, Pusa Hybrid 4, Pusa Early Dwarf, NA-501, DTH-4, NDTH-1, Phule Hybrid-1, BSS-39, ARTH- 3, NA-701, ARTH-15, Hybrid No-37, Swarna-12, NDTH-6, HOE-606, NA-701, Maitri, Rishi, HOE-616; Hybrid (indeterminate): ARTH-4, MTH-6, NA-601, Arka Vardhan, KT-4, FMH-1, BSS-20, BSS-40, BSS-90, HOE-909, Larica, Ratna, DTH-6, Sonali, ARTH-16, FM-2, NDTH-2, NDTH-4, TC-161 Disease resistant Fusarium and Verticillium wilts resistant: Vaishali, Rupali, Rashmi, Rajni, Naveen, Pant Bahar; Bacterial wilt resistant: Arka Abha ( BWR 1), Arka Alok ( BWR - 5 ), Arka Shreshta,Arka Abhijit ( BRH 2), BT 1, BT 2, BT 10, BWR 5, LE 70-5; Powdery mildew tolerant: Arka Ashish ( IIHR - 674 ); Nematode resistant: Pusa 120, Arka Vardan ( FM hyb -2), Hisar Lalit; Frost resistant: Sioux, Best of All and Marglobe; Virus resistant/tolerant: Avinash (TLCV tolerant), Pusa Sheetal, Hisar Anmole, Hisar Gaurav. Exotic varieties Processing purpose: Amish Paste, Baylor Paste, Bulgarian Triumph, Carol Chyko's Big Paste, Grandma Mary's, Bellstar, Big Red Paste, Canadian Long Red, Denali, Hungarian Italian, Oroma, Palestinian, Peasant, Polish Paste, Red Sausage, Roma, and San Marzano from USA; Debarao, Black Plum and Wonder Light from Russia; Hogheart, Italian Gold Hybrid, La Rossa VF2 and Milano from Italy; Table Purpose: Gardners delight, Chertia, Evita, Cherry Wonder from UK; Table & Processing purpose: Opalka from USA Source: Department of Agriculture & Co-operation, Ministry of Agriculture, Government of India (2009).

Micro nutrients Sometimes the crop suffers due to boron deficiency which may cause cracking in fruits. This can be corrected by spraying the crop with Borax (0.3%) at the rate of 8 to 12 kg/h at 15 days interval (Datta, 1961; Singh et al., 2004). Blossom end rot, a calcium deficiency of tomato in which leathery rot develops on or near the blossom-end of the fruit. Spray of 600 mg/l calcium nitrate 2-3 times each week at the beginning of the second fruit clusters setting may reduce the deficiency incidence (Joseph and Sikora, 2004). Magnesium (Mg) deficiency in tomato reflects with yellowing between leaf veins, which stay green, giving a marbled appearance. This begins with older leaves and spreads to younger growth. It can be confused with virus, or natural aging in the case of tomato plants. Fruits remain small and woody. Mg deficiency can be rectified in the short term by applying a foliar feed fortnightly, with Epsom salts diluted at a rate of 20 g/l of water or add dolomitic limestone or other Mg containing rocks such as Kieserite or Langbeinite, which is potassium magnesium sulfate. Reduce usage of potash fertilizers is recommended if this may be contributing to the problem (Welch, 1986).

Plant Diseases Crop plants are attacked by hundreds of diseases (Agrios, 2005). However, only a few usually a single pathogen at a given time is able to multiply to an extent to cause the disease. Diseases of crop plants are among the most important constraints in the production of adequate quantities of food. Approximately half of the world`s total agricultural production is lost due to various pests and diseases at various planting, post-planting and post-harvest stages (Agrios, 2005; Khan, 2008). The crop losses due to diseases are lesser in developed countries because of awareness by farmers towards disease management. In developing countries, however, greater yield losses occur because of unplanned agricultural practices such as use of marginal lands, low agricultural inputs and lesser concerns by farmers towards plant disease management. On average, losses inflicted by weeds, plant diseases and insect pests to agricultural crops have been estimated as 33, 26 and 22%, respectively (Khan, 2008). According to another estimate, plant diseases, weeds, and insects contribute to a 14.1, 10.2 and 12.2%, respectively, decline in crop production (FAOSTAT, 2003; Agrios, 2005; Table 12). Among different kinds of pathogens, the greatest losses are inflicted by fungi (42%) followed by bacteria (27%), viruses (18%) and nematodes (13%) (Khan and Jairajpuri, 2010a; Fig.5). Table 12. Estimated annual crop losses caused by pests and diseases worldwide. Practice Losses (US $)

Attainable cop production (2002 prices) $ 1.5 trillion Actual crop production (-36.5%)) $950 billion Production without crop protection $455 billion Losses prevented by crop protection $415 billion Actual annual losses to world crop production $550 billion Losses caused by disease only (14.1%) $220 billion Source: FAOSTAT, 2003; Agrios, 2005

Fig. 5. Actual crop production and annual crop losses due to plant diseases, insect pests and weeds (A) and breakdown of crop losses caused by fungi, bacteria, viruses and nematodes (B).

Diseases of chickpea and tomato Diseases are the most serious constraints in chickpea and tomato production causing upto 100% losses (Table 13, 14). Environmental factors and intensity of abiotic stresses may influence the incidence of the diseases, e.g. yield losses due wilt and root rot diseases increase under drought and high temperature situation in the country (Gurha, 2003). Due to nutritive nature of both the plants, a large number of plant pathogens have been found associated with chickpea and tomato. Especially tomato is more susceptible because of its succulent nature. A careful examination of relevant information has indicated that 67 fungi, 3 bacteria, 22 viruses and 80 nematodes may infect the chickpea crop and more than that infect tomato crop (Nene and Reddy 1987, Haware 1998, Singh and Sharma, 1998, Singh et al., 1999). Among these Fusarium wilt, root rots, Ascochyta blight and Botrytis gray mould are major diseases and may cause significant yield losses to chickpea plants (Gurha, 2003).

Table 13. Disease of chickpea caused by various pathogens. Disease Causal Organism(s) 27

Disease Causal Organism(s) Fungal diseases Acrophialophora wilt Acrophialophora fusispora Alternaria blight Alternaria alternata, A. tenuissima Aphanomyces root rot Aphanomyces euteiches Ascochyta blight Ascochyta rabiei (races 1, 2, 3, 4, 5, and 6) Mycosphaerella rabiei= Didymella rabiei [teleomorph] Black root rot Fusarium solani Black streak root rot Thielaviopsis basicola Botrytis gray mold Botrytis cinerea Collar rot Sclerotium rolfsii Athelia rolfsii = Corticium rolfsii [teleomorph] Colletotrichum blight Colletotrichum capsici, C. dematium Cylindrocladium root rot Cylindrocladium clavatum Damping-off Pythium debaryanum, P. irregulare, P. ultimum Downy mildew Peronospora sp. Dry root rot Macrophomina phaseolina = Rhizoctonia bataticola Foot rot Phacidiopycnis padwickii = Operculella padwickii Fusarium root rot Fusarium acuminatum, F. arthrosporioides, F. avenaceum, F. equiseti, F. solani f.sp. eumartii = Fusarium eumartii Fusarium wilt Fusarium oxysporum f.sp. ciceri Myrothecium leaf spot Myrothecium roridum Mystrosporium leaf spot Mystrosporium sp. Neocosmospora root rot Neocosmospora vasinfecta Ozonium collar rot Ozonium texanum var. parasiticum Phoma blight Phoma medicaginis Phytophthora root rot Phytophthora citrophthora, P. cryptogea, P. drechsleri, P. megasperma Pleospora leaf spot Pleospora herbarum, Stemphylium herbarum [anamorph] Powdery mildew Leveillula taurica, Oidiopsis taurica [anamorph], Erysiphe sp. Rust Uromyces ciceris-arietini, U. striatus Sclerotinia stem rot Sclerotinia sclerotiorum, S. trifoliorum Scopulariopsis leaf spot Scopulariopsis brevicaulis Seedling or seed rot Aspergillus flavus, Trichothecium roseum Stemphylium blight Stemphylium sarciniforme Trichoderma foot rot Trichoderma harzianum Verticillium wilt Verticillium albo-atrum, V. dahliae Wet root rot Rhizoctonia solani Bacterial diseases Bacterial blight Xanthomonas campestris pv. cassiae Bacterial leaf spot Burkholderia andropogonis Nematode diseases Dirty root Rotylenchulus reniformis Pearly root (cyst nematode) Heterodera ciceri, H. rosii

Root-knot (root-knot nematode) Meloidogyne incognita, M, javanica, M. arenaria, M. artiellia, Root lesion Pratylenchus brachyurus, P. thornei Viral diseases Bushy stunt Chickpea bushy stunt virus Distortion mosaic Chickpea distortion mosaic virus Filiform Chickpea filiform virus Mosaic Alfalfa mosaic virus Narrow leaf Bean yellow mosaic virus Necrosis Lettuce necrotic yellows virus Pea streak virus Proliferation Cucumber mosaic virus Stunt Bean (pea) leaf roll virus Yellowing Pea enation mosaic virus Phytoplasmic disease Phyllody Phytoplasma

Table 14. Disease of tomato caused by various pathogens. Disease Causal Organism(s) Fungal diseases Alternaria stem canker Alternaria alternata f.sp. lycopersici Anthracnose Colletotrichum coccodes, C. dematium , C. gloeosporioides, Glomerella cingulata [teleomorph] Black mold rot Alternaria alternata, Stemphylium botryosum, Pleospora tarda [teleomorph], Stemphylium herbarum, Pleospora herbarum [teleomorph] = Pleospora lycopersici, Ulocladium consortiale = Stemphylium consortiale Black root rot Thielaviopsis basicola, Chalara elegans [synanamorph] Black shoulder Alternaria alternata Buckeye fruit and root rot Phytophthora capsici, P. drechsleri, P. nicotianae var. parasitica = P. parasitica Cercospora leaf mold Pseudocercospora fuligena = Cercospora fuligena Charcoal rot Macrophomina phaseolina Corky root rot Pyrenochaeta lycopersici Didymella stem rot Didymella lycopersici Early blight Alternaria solani Fusarium crown and root rot Fusarium oxysporum f.sp. radicis-lycopersici Fusarium wilt Fusarium oxysporum f.sp. lycopersici Gray leaf spot Stemphylium botryosum f.sp. lycopersici Stemphylium lycopersici = S. floridanum, S. solani Gray mold Botrytis cinerea, B. fuckeliana [teleomorph] Late blight Phytophthora infestans Leaf mold Fulvia fulva = Cladosporium fulvum Phoma rot Phoma destructiva Powdery mildew Oidiopsis sicula, Leveillula taurica [teleomorph] Pythium damping-off and fruit rot Pythium aphanidermatum, Pythium arrhenomanes, Pythium debaryanum, Pythium myriotylum, Pythium ultimum Rhizoctonia damping-off and fruit rot Rhizoctonia solani, Thanatephorus cucumeris [teleomorph] Rhizopus rot Rhizopus stolonifer Septoria leaf spot Septoria lycopersici Sour rot Geotrichum candidum, Galactomyces geotrichum [teleomorph], Geotrichum klebahnii (= G. penicillatum) Southern blight Sclerotium rolfsii, Athelia rolfsii [teleomorph] Target spot Corynespora cassiicola Verticillium wilt Verticillium albo-atrum, Verticillium dahliae White mold Sclerotinia sclerotiorum, Sclerotinia minor Bacterial diseases Bacterial canker Clavibacter michiganensis subsp. michiganensis Bacterial speck Pseudomonas syringae pv. tomato Bacterial spot Xanthomonas campestris pv. vesicatoria Bacterial stem rot and fruit rot Erwinia carotovora subsp. carotovora Bacterial wilt Ralstonia solanacearum Pith necrosis Pseudomonas corrugata Syringae leaf spot Pseudomonas syringae pv. syringae Nematode diseases Root-knot Meloidogyne spp. Sting Belonolaimus longicaudatus Stubby-root Paratrichodorus spp., Trichodorus spp. Viral diseases Common mosaic of tomato (internalTobacco mosaic virus (TMV) browning of fruit) Curly top Curly top virus Potato virus Y Potato virus Y Pseudo curly top Pseudo curly top virus Tomato bushy stunt Tomato bushy stunt virus Tomato etch Tobacco etch virus Continued... Tomato fern leaf Cucumber mosaic virus 29

Disease Causal Organism(s) Tomato mosaic Tomato mosaic virus (ToMV) Tomato mottle Tomato mottle gemini virus Tomato necrosis Alfalfa mosaic virus Tomato spotted wilt Tomato spotted wilt virus Tomato yellow leaf curl Tomato yellow leaf curl virus Tomato yellow top Tomato yellow top virus Viroidal diseases Tomato bunchy top Tomato bunchy top viroid Tomato planto macho Tomato planto macho viroid Phytoplasmic diseases Aster yellows Mycoplasma like organism Tomato big bud Mycoplasma like organism Mescellaneous diseases Autogenous necrosis Genetic Fruit pox Genetic Gold fleck Genetic Graywall Undetermined etiology Source (Table 13-14): The American Phytopathological Society, 2010.

In case of tomato late blight, early blight, Fusarium and Verticillium wilt are the most destructive diseases causing serious losses to the crop (Madden et al., 1978; Shtienberg et al., 1989; Charoenporn et al., 2010; Sibounnavong et al., 2010 ). In addition to fungal diseases, root knot caused by Meloidogyne incognita and M. javanica is another important disease of chickpea and tomato. Root-knot nematodes are widely distributed in India (Khan, 1997) and may cause 19-40% economic loss. Ali (1997) reported 22-84% avoidable loss due to M. javanica and 25-60% due to M. incognita to chickpea. Another species of root knot nematodes, M. arenaria caused 39% loss in grain yield of chickpea in Gujarat (Patel, 1997). Information gathered from different sources has revealed that avoidable yield loss due to root knot nematodes in chickpea may range 17- 60% in Bihar, 17-56% in Gujarat, 8% in Haryana, 35-43% in Maharashtra, 22% in Punjab, 17-60% in Rajasthan and 22-23% in Uttar Pradesh (Ali et al., 2003). Tomato is more susceptible to root-knot nematodes and may exhibit 5-43% yield loss (Sasser, 1979). M. incognita causes upto 50% yield loss to tomato in many countries. In India, Nagnathan (1984) reported 61% yield loss in tomato due to M. incognita, where as 35-39% loss reported by Reddy (1985) and Jonathan et al. (2001). In addition to direct damage, root-knot nematodes synergise other pathogens to cause greater damage (Khan, 1993). The nematode infection is known to break resistance in many wilt resistant cultivars of chickpea (Ali et al., 2003) and tomato (Son et al., 2009).

Fusarium wilt of chickpea Fusarium wilt of chickpea is a serious disease in Southeast Asia including India, Bangladesh, Burma, Pakistan, Ethopia, Syria, Tunisia and Mexico (Nene et al., 1984) Chile, Iran, Sudan, USA (Haware et al., 1986), Peru (Echandi, 1970), USSR (Stepanova, 1971) and Malawi (Kannaiyan, 1981) (Fig. 6). However, it is more severe in India, Iran, Pakistan, Nepal, Burma, Spain and Tunisia (Haware and Nene, 1982a). Disease occurs in almost all chickpea types. In general, spring sown crop is more vulnerable to wilt than winter sown (Hawtin and Singh, 1984). Because of the extreme temperature shift in the northern belt of India early wilting occurs around November, and late wilting at flowering stage around Feb-March. In winter during December-February the wilt incidence remains at the lowest.

Fig. 6. World distribution map of wilt disease of chickpea

Chickpea wilt was first reported from India by Butler (1918). Annual chickpea yield losses from Fusarium wilt vary from 10-15% in India (Singh and Dahiya, 1973; Trapero-Casas and Jimenez-Diaz, 1985; Jalali and Chand, 1992) and Spain (Trapero-Casas and Jimenez-Diaz, 1985) and 40% in Tunisia (Bouslama, 1980) and reached 100% in spring-sown chickpea in some locations in Syria (Bayaa et al., 1986). Srivastava et al. (1984) observed the yield losses ranging from 10- 90%. Fusarium wilt of chickpea can often destroy the crop completely (Izquierdo and Morse, 1975; Bayaa et al., 1986; Agrawal et al., 1993; Halila and Strange, 1996). Recently, Hari and Khirbat (2009) reported that the disease can destroy the crop 100% depending on varietal susceptibility and

agroclimatic conditions. Experimentation indicated that yield loss due to chickpea wilt is strongly correlated to wilt incidence in susceptible cultivars (Bayaa et al., 1986; Erskine and Bayaa, 1996). Seeds harvested from wilted plants are lighter, wrinkled and duller than those from healthy plants (Haware and Nene, 1980). Different cultivars of chickpea show wilting at different growth stages and influence the loss in yield with different degrees of severity whereas, the disease is systemic in nature and the pathogen may infect plants at any growth stage (Haware and Nene, 1980b). Nema and Khare (1973) observed damage up to 61% at seedling stage and 43% at flowering stage. Early wilting reduced the seed number/plant and caused more yield losses than late wilting (Navas-Cortes et al., 1998). An estimated annual loss of `12 million was reported due to wilt disease in Pakistan in 1956 (Sattar et al., 1953), the disease in the country appeared in epidemic form and more than 75% crop losses were reported (Akhtar, 1956). In India the disease is estimated to cause upto 10% yield loss but figure on monetary losses is not available (Singh and Dahiya, 1973). Symptoms of Fusarium wilt on chickpea Normally, the disease occurs at most critical stages such as seedling stage, flowering stage or adult stage (Beckman, 1987). The main symptoms of the disease are; yellowing and drying of leaves from base upward, drooping of petioles and rachis, improper branching, withering of plants, browning of vascular bundles, and finally wilting of plants (Haware and Nene, 1980; Fig. 7). The fungus colonizes the xylem vessels and thus prevents the translocation of water and nutrients, resulting in wilting (Cho and Muehlbauer, 2004). The initial symptom of the disease is acropetal vein clearing of leaves (Chauhan, 1963). Symptoms appear within 3-5 weeks after sowing, the seedlings collapse and lie flat on the ground, although they retain normal green colour. Such diseased seedlings, when uprooted, generally show uneven shrinking of the stem above and below the collar region. The affected seedlings do not show any external root rot; however, when split open vertically from collar region downward, black discoloration of xylem vessels is visible. Systemic colonization of the host’s vascular system by the pathogenic Fusarium spp. ultimately reduces the conductance of water and nutrients (Mace et al., 1981; Backman, 1987). The seedlings of highly susceptible cultivars which die within 10 days after emergence may not exhibit black discoloration of internal tissues; however, internal browning from root tip upward will be visible (Nelson, 1991). In the case of infection of adult plants (6-8 weeks after sowing) are infected, the diseased plants exhibit drooping of petioles, rachis, and leaflets. Initially, drooping is observed in the upper part of the plants, but within a short time (1-2 days) it is visible on the entire plant. The lower leaves dry but do not shed at maturity.

Gupta et al. (2010) reported that in chickpea reduction in chlorophyll and increase in organic acids, polyphenols, carbohydrates and decreased in the chloroplasts and starch formation in the mesophyll cells occurred due to infection of F. oxysporum f. sp. ciceri. Fusarium oxysporum (Schlechtend. Fr.) f. sp. ciceri (Padwick) Matuo & K. Sato Taxonomic position Division : Amastigomycota Sub division : Deuteromycotina Class : Deuteromycetes Subclass : Hyphomycetidae Order : Moniliales Family : Tuberculariaceae Genus : Fusarium Species : oxysporum Forma specialis: ciceri The fungus is septate, profusely branched; growing on potato sucrose/dextrose agar at 25°C initially white turning light buff or deep brown later, fluffy or submerged (Fig. 8). The growth becomes felted or wrinkled in old cultures. Various types of pigmentations (yellow, brown, crimson) may be observed in culture. On solid medium, microconidia may be usually borne on simple and short conidiophores which arise laterally on the hyphae. They are oval to cylindrical, straight or curved and measure 2.5 – 3.5 × 5 – 11 µm. Macroconidia are borne on branched conidiophores, thin walled, 3 to 5 septate, fusoid, pointed at both ends and measure 3.5 – 4.5 × 25 – 65 µm (Fig. 8). Chlamydospores are formed in old cultures, which are smooth or rough walled, terminal and intercalary and may be forked singly or in pairs or in chains (Gupta et al., 1986). Pathotypes and pathogenic races of Fusarium oxysporum f. sp. ciceri Variation in pathotypes (symptom types) and pathogenic races have been reported in F. oxysporum f. sp. ciceri to different geographical regions. Two pathotypes, designated yellowing and wilting, have been differentiated by pathogenicity tests (Trapero-Casas and Jimenez-Diaz, 1985; Jimenez-Gascoa et al., 2002). The yellowing pathotype induces progressive foliar yellowing with vascular discoloration, followed by plant death within 40 days of inoculation. The wilting pathotype induces severe chlorosis and flaccidity combined with vascular discoloration, followed by plant death within 20 days of inoculation (Kelly et al., 1994). In addition to variation in symptom type, there are eight races of F. oxysporum f. sp. ciceri (races 0, 1A, 1B/C, 2, 3, 4, 5 and 6) which are identified by differential disease reactions on a set of chickpea cultivars (Haware and Nene, 1982b; Jimenez-Diaz et al., 1993). As with pathotypes, some of these races can be distinguished by RAPD markers (Jimenez-Gasco et al., 2001). In susceptible chickpea cultivars, races 0 and 1B/C induce the yellowing syndrome (yellowing pathotype), whereas races 1A, 2, 3, 4, 33

5 and 6 induce the wilting syndrome (wilting pathotype) (Trapero-Casas and Jimenez- Diaz, 1985; Jimenez-Diaz et al., 1993). These races also have distinct geographical distributions. Races 2, 3 and 4 have only been described from India (Haware and Nene, 1982b), whereas races 0, 1B/C, 5 and 6 are found mainly in the Mediterranean region, as well as in the California (USA) (Jimenez-Diaz et al., 1993; Halila and Strange, 1996). Race 1A has been reported in India (Haware and Nene, 1982b), California and the Mediterranean region (Jimenez-Diaz et al., 1993). However, despite variation in symptom type, race and geographical distribution, all F. oxysporum f. sp. ciceri isolates tested so far are in a single vegetative compatibility group (VCG) showing high genetic similarity and all are monophyletic (Nogales-Moncada, 1997; Jimenez-Gascoa et al., 2002).

Fig. 7. Symptoms of chickpea (A) and tomato (B) wilt caused by Fusarium oxysporum f. sp. ciceri and F. oxysporum f. sp. lycopersici, respectively.

Fig. 8. Colony of Fusarium oxysporum f. sp. ciceri on PDA (A), micro and macro conidia of the fungus (B). Disease cycle and predisposing factors of Fusarium oxysporum f. sp. ciceri

The pathogen, F. oxysporum f. sp. ciceri is a soil inhabitant, survives in infected plant debris in the soil as mycelium and in all its spore forms but, most commonly, especially in the cooler temperate regions, as chlamydospores (Fig. 9; Haware et al, 1978; Nelson et al., 1981; Satyaprasad et al., 1983; Rai and Upadhyay, 1983). Kumar et al. (1997) isolated the pathogen from seed samples obtained from five sources. The crops like lentil, pea and pigeonpea are reported to be symptomless carriers of the pathogen (Haware and Nane, 1982a). Usually, once an area becomes infested with Fusarium spp., it remains for several years (Saxena and Singh, 1987; Agrios, 2005). Details of the colonization process of F. oxysporum within and outside the vascular system have been studied by several authors (Charest et al., 1984; Brammall and Higgins, 1988; Rodriguez-Galvez and Mendgen 1995; Olivain and Alabouvette, 1997, 1999; Lagopodi et al., 2002) and the mycelial development in the vicinity of plant roots has been investigated by Steinberg et al. (1999), but little is known about the germination of F. oxysporum propagules, a key step in plant-pathogen interactions (Nelson, 1991). Root exudates stimulated microconidia germination and the level of stimulation was affected by plant age and F. oxysporum strains (Steinkellner et al., 2005). Furthermore, root exudates from non-host plants also contain compounds that stimulate microconidia germination of F. oxysporum (Steinkellner et al., 2005). Root exudates containing phenolic compounds are inhibitory to F. oxysporum microconidia germination (Steinkellner et al., 2005). When healthy plants grow in contaminated soil, the germ tube of spores or the mycelium penetrates root tips directly or enters the roots through wounds or at the point of formation of lateral roots (Navas-Cortes et al., 2000). The mycelium advances through the root cortex intercellularly, and when it reaches the xylem vessels it enters them through the pits. The mycelium then remains exclusively in the vessels and travels through them, mostly upward, toward the stem and crown of the plant. While in the vessels, the mycelium branches and produces microconidia, which are detached and carried upward in the sap stream (Navas-Cortes et al., 2000). Microconidia germinate at the point where their upward movement is stopped, the mycelium penetrates the upper wall of the vessel, and more microconidia are produced in the next vessel. The mycelium also advances laterally into the adjacent vessels, penetrating them through the pits. The fungus then invades all tissues of the plant extensively, reaches the surface of the death plant, and there sporulates profusely (Navas-Cortes et al., 2000).

Fig. 9. Disease cycle of wilt of chickpea (Fusarium oxysporum f. sp. ciceri) and tomato (F. oxysporum f. sp. lycopersici).

Effect of environmental factors on the disease The soil type, reaction, moisture and temperature are known to influence disease development. The greenhouse studies substantiate the fact that disease is more severe in light sandy soils than heavy clay ones (Chand and Khirbat, 2009). Sugha et al., (1994) attributed higher disease severity in light sandy soils to low water retention ability of these soils. Soil pH may also influence the disease intensity that increased with lowering pH, being considerably low at pH 9.2 (Chauhan, 1965). Recently, Chand and Khirbat (2009) reported that the pathogen tolerated a wide range of pH, with an optimum of pH 5.0 - 6.5. High soil temperature and deficiency of moisture appear to have a definite bearing on the incidence of the disease (Sugha et al., 1994). Soil temperature in the range of 24.8 to 28.5°C with optimum 25°C and moisture within the water holding capacity of soil were most conducive to chickpea wilt whereas temperature and soil moisture levels above and below this range delayed the incidence and slowed down the progress of wilt (Chauhan, 1963; Sugha et al., 1994a; Chand and Khirbat, 2009). Lower levels of soil moisture (10%) kept the plant mortality due to low disease, though 12% of the plants were damaged, as compared to 83% in soil with moisture at 25% level (Sattar et al., 1953). Alkanline soils favour the incidence of wilt (Chauhan, 1963). Range of inoculum densities of the pathogen did not increase with increased inoculum levels of 104 or 105 microconidia and 36

macroconidia/ml (Bhatti and Kraft,1992). Increase in phosphorus and potassium concentration did not influence the development of wilt, but it was favoured by an increase in nitrogen and organic carbon (Sugha et al., 1994b). Sugha et al. (1994a) also reported that shallow sowing of seeds and young seedlings favoured the development of the disease, whereas deeper sown seeds and older seedlings had reduced chickpea wilt. The amount of organic matter and humus content of the soil were found inversely related to wilt incidence (Chand and Khirbat, 2009). Fusarium wilt of tomato Vascular wilt caused by Fusarium oxysporum f. sp. lycopersici is found globally in almost every natural habitat and occurs throughout the world (Woltz and Jones, 1981). Fusarium wilt of tomato is the major limiting factor in the production of tomato (Snyder and Hansen, 1940). The disease causes great losses, especially on the susceptible varieties of tomato especially when soil and air temperature are rather high during the warm season (Khan and Akram, 1999; Agrios, 2005; Mandal et al., 2009). Annual losses of up to 30,000 tonnes of canning tomatoes or 10-15% of the crop damage in the USA are associated with Fusarium tomato wilt (Westcott, 1971; Benhamou et al., 1989). In India, it causes serious monitory losses but exact amount of loss is not available (Khan et al., 2007). Symptoms of Fusarium wilt on tomato Two of the earliest symptoms on young plants are clearing of veinlets and drooping of the petioles. Later, the entire plant wilts. In the field the disease appears any time if conditions are favorable but symptoms are predominant in mature plants after flowering and the beginning of fruit set. Lower leaves show yellowing and die (Ray, 2005). The chlorotic symptoms begin to appear on one side of the leaf and then all leaflets become yellow on one half of the leaf (Sherf and MacNab, 1986). Symptoms continue to appear on successive younger leaves. These symptoms appear on the entire plant or on only few branches (Fig. 7). After the disease has advanced for few weeks, browning of the vascular system can be seen in a cross section of the lower stem or by removing stem tissue near the base with a knife. The upward extent of the discoloration depends on the severity of the disease (Agrios, 2005). Young plants wilt suddenly but older plants linger on for some time. The plant as a whole is stunted. Fruit may occasionally become infected and then it rots and drops off without becoming spotted. Roots also become infected; after an initial period of stunting, the lateral roots show black rot condition. This hastens death of plants. Symptoms of wilt are more commonly observed during the warmer part of the day. In wet weather the fungal growth can be seen on dead plants as a pinkish layer (Agrios, 2005; Singh, 2008).

Fusarium oxysporum f. sp. lycopersici Snyder & Hansen Taxonomic position Division : Amastigomycota Sub division : Deuteromycotina Class : Deuteromycetes Subclass : Hyphomycetidae Order : Moniliales Family : Tuberculariaceae Genus : Fusarium Species : oxysporum Formae specialis: lycopersici The fungus has septate, hyaline mycelium which later becomes cream coloured in cultures. On the host also the mycelium is at first whitish but after formation of macroconidia it looks pinkish or somewhat purplish (Fig. 10). The fungus produces three kinds of asexual spores. Microconidia, which have one or two cells, are the most frequently and abundantly produce spores under all conditions, even inside the vessels of infected host plants. Microconidia are one celled, hyaline, ovoid or ellipsoidal, and measure 6-15 × 2.5-4 µm. Macroconidia are the typical “Fusarium” spores; they are hyaline, three to five celled, have gradually pointed and curved ends, and measure 25-33 × 3.5-5.5 µm in size (Fig. 10), and appear in sporodochia-like groups on the surface of plants killed by the pathogen. Chlamydospores are one or two celled, thick walled, round spores produced within or terminally on older mycelium or in macroconidia. All three types of spores are produced in cultures of the fungus and probably in the soil, although only chlamydospores can survive in the soil for a long (Gilman, 2001; Agrios, 2005; Singh, 2008). Pathogenic races of Fusarium oxysporum f. sp. lycopersici The fungus usually is saprophytic (nonpathogenic), some strains of this soil borne species cause agriculturally and economically damaging plant diseases. Of the phytopathogenic strains of the fungus, more than 120 formae specials (f. sp.) have been reported, each of which causes disease on a well-defined host range of plant species (Agrios 2005). Formae specials (f. sp.) lycopersici causes wilt disease only on tomato, while other f. sp. except radicis-lycopersici do not cause diseases on tomato. Within a forma specialis, races are frequently distinguished by their specific pathogenicity to different cultivars. Three races, races 1, 2, and 3 have been reported for F. oxysporum f. sp. lycopersici to date (Clayton 1923; Alexander and Tucker 1945; Grattidge and O’Brien 1982; Keigo et al., 2010). Mostly wilt of tomato is caused by Fusarium oxysporum f. sp. lycopersici race 1 (Ren et al., 2010). Disease cycle and predisposing factors of Fusarium oxysporum f. sp. lycopersici 38

The pathogen survives in soil as chlamydospores or, for some times, as saprophytically growing mycelium in infected crop debris (Fig. 9). Once it is established in soil it is difficult to eradicate in areas where high soil temperatures are uncommon. Infected seeds, wind-borne soil, surface drainage water and agricultural implements also distribute the inoculum from field to field. The pinkish superficial growth with spores on the plants and their dissemination may help secondary spread of the pathogen in the field (Katan et al., 1997). Development of the pathogenic strains of F. oxysporum f. sp. lycopersici on tomato is stimulated in the vicinity of the roots, irrespective of plant species, but here is no chemotactic response toward or away from the root. Growth stimulation is mainly related to organic nitrogen (amino acids) in the root exudates. When seedlings with broken roots are planted in infested soil the fungus grows quickly through the wounds and slowly progresses up to the stem through xylem vessels. Nematode invasion also helps the pathogen in the same manner. It begins to secrete pectolytic enzymes which work upon the pectic materials in the tracheal walls and diffuse into the xylem parenchyma walls also. As a result of production of colloidal masses by these activities the xylem vessels are plugged, checking movement of water and minerals. Various toxins (fusaric acid, lycomarasmin) are also produced by the fungus which helps in development of wilt symptoms. Roots and stems of tomato contain the saponin, tomatine, which acts as chemical barrier to infection by pathogens. In response to this barrier many pathogens of tomato, including F. oxysporum f. sp. lycopersici produce extracellular enzymes, tomatinases, which deglycosylate alpha-tomatine to yield less toxic derivatives, tomatidine and lycotetraose, thus overcoming the chemical barrier (Lairini et al., 1997; Roldan-Arjuna et al., 1999). Rodriguez-Malina et al. (2003) studied the colonization of stem tissue by the pathogen in resistant and susceptible cultivars. One day after inoculation the propagules spread discontinuously irrespective of cultivar genotype. Five days later, the pathogen is limited to stem bases. For next one week the pathogen seems to undergo incubation at the stem bases. This condition becomes permanent in resistant cultivars and no symptoms develop. In susceptible cultivars, a gradual upward colonization of the stem is seen and symptom development occurs. Effect of environmental factors on the disease Attitalla et al. (1998) reported that optimum temperature for wilt development is 28°C. The disease development was inhibited at soil temperature of 33°C and below 21°C (Katan et al., 1997). If the temperature of the soil remains at 37°C for more than a few days the fungus is killed (Katan et al., 1997). Disease incidence decline at higher temperatures and

with increased time of exposure to higher temperatures, as wilt incidence is more at 17- 20°C and at 32°C than 42°C (Katan et al., 1997). Disease is more severe at below 6.4 pH and above 7.0 pH (Attitalla et al., 1998). Other conditions which pre-dispose the plant to wilt are short day length and low light intensity (Singh, 2008). Wilt is enhanced by high doses of nitrogen (150 kg N/h) but application of potash alone (100 kg/h) or in combination with nitrogen (N 100 kg + K 100 kg/h) decreased disease incidence by more than 60% (Raj and Kapoor, 1995).

Fig. 10. Colony of Fusarium oxysporum f. sp. lycopersici on PDA (A), micro and macro conidia of the fungus (B).

Root-knot disease Root-knot nematodes, Meloidogyne spp. are threat to global crop production (Sasser, 1980). They have a very wide distribution and causes serious damage to crops especially vegetables. The importance of root-knot nematodes as major pathogens of vegetables and other crops under different climatic conditions was reviewed by Franklin (1979), Sasser (1979), Lamberti (1979) and Khan (1997). Franklin (1979) reviewed the root-knot damage in tomato, cucumber, carrots, potatoes, lettuce, celery, brassicas, legumes and several other plants in different countries of Europe and Pacific, particularly caused by the major species of root-knot nematodes. The average yield losses on worldwide basis has been estimated to be about 5% and may be more in the developing tropical and sub-tropical countries (Taylor and Sasser, 1978). In India also, root-knot nematodes, Meloidogyne spp. are considered one of the most important pests of vegetables, pulses, cereals (rice in particular), cucurbits, tobacco, sugarcane, banana, citrus, ornamentals and oil and fiber yielding plants causing considerable reduction in their yields (Gupta et al., 1986; Khan, 1997; Khan and Jairajpuri, 2010a, 2010b). Root-knot nematode disease of chickpea 40

In India major damage to chickpea is caused by three endoparasitic nematodes viz., Meloidogyne spp., Heterodera spp. and Rotylenchulus reniformis (Ali et al., 2003). The former happens to be the major nematode in all types of chickpea. Three species of the root-knot nematode, M. incognita (Kannan, 1966, Reddy, 1975, Jamal, 1976), M. javanica and M. arenaria (Mathur et al., 1969) are associated with chickpea. Of these species, M. incognita is apparently the most predominant which is closely followed by M. javanica (Sharma, 1988). The nematode is polyphagous in nature, and posses a threat to pulse cultivation in India (Ali et al., 2010). The pathogenicity of Meloidogyne spp. among pulse crops has been proved on chickpea, pigeonpea, cowpea, mungbean, pea, lentil, urdbean, rajmash and several other pulses (Gupta et al., 1986, Khan et al., 2004). Root-knot nematode disease of tomato Vegetables are highly susceptible to root-knot nematodes, four species viz., M. incognita, M. javanica, M. arenaria, and M. hapla are of particular economic importance in vegetable production (Sasser, 1979). Netscher and Sikora (1990) categorized the main species of Meloidogyne associated with major vegetable crops in subtropics and tropics and found M. incognita, M. javanica and M. arenaria are economically very important species associated with tomato. Out of these species, M. incognita is the most predominant with four races as 1, 2, 3, and 4 in attacking tomato (Khan and Esfahani, 1992). The pathogenicity of Meloidogyne spp. has been proved on many numerous varieties of tomato (Khan, 1997). Distribution and economic importance of root-knot nematode in chickpea and tomato Root-knot nematodes are widely distributed throughout the world, especially in tropical, subtropical and Mediterranean climates (Sasser, 1979; Van Gundy, 1987). Distribution of root-knot nematode summarized as occurrence of 11 species in Africa, 9 species in Central and South America, 18 species in United States, 3 species in Canada, 11 species in Europe and the Mediterranean region, 10 species in India and Sri Lanka, 4 species in Russia, 5 species in Japan and 3 species in Southeast Asia, Australia and Fiji Islands. Out of the described species of Meloidogyne, M. incognita with 4 races (R1, R2, R3 and R4), M. javanica, M. arenaria with two races (R1 and R2) and M. hapla have wide host range and distribution (Bridge, 1981; Sasser, 1979, Khan et al., 1988). On worldwide basis M. incognita is dominating over M. javanica (Taylor et al., 1982). In India 10 species viz., M. incognita, M. javanica, M. arenaria, M. hapla, M. graminicola, M. exigua, M. africana, M. bravicaudata, M. graminis and M. triticoryzae are present (Khan, 1997). Among them, four species i.e., M. incognita, M. javanica, M. arenaria and M. hapla have been observed from all the states and Union Territories of India. Occurrence of races of M. incognita and

M. arenaria has also been confirmed in India. All the four races of M. incognita (R1, R2, R3 and R4) and one race of M. arenaria (R2) are present in U.P. In other states one or two races of M. incognita have been recorded. Root-knot nematodes (M. incognita and M. javanica) have been reported to cause 19-40% economic loss to chickpea (Bridge, 1981; Upadhayay and Dwivedi, 1987; Reddy, 1985; Ali et al., 2003) and 24-61% losses to tomato in India (Nagnathan, 1984; Reddy, 1985; Jonathan et al., 2001; Walia and Bajaj, 2003; Singh and Mathur, 2010a) (Fig. 11).

Threshold level of root-knot nematode Chickpea Threshold level of nematode under field conditions differ greatly between countries and variation is caused by differences in soil type, environmental factors prevailing during the growing season in the different climatic zones and to complex disease interrelationships (Luc et al., 2005). An increase age of chickpea plant, nematode penetration as indicated by number of galls per root system was decreased and the plants inoculated at old age were less affected and showed low degree of damage than the plant inoculated at early stage of growth (Ahmad and Naimuddin, 1988). A complete crop failure would occur in fields infected with more than 1 egg/g of soil (Nath et al., 1979; Ali, 1995). Therefore, field studies are required to estimate tolerance limits to make yield loss assessments. However, in pot experiment, the growth of chickpea was negatively affected when soil populations of

M. incognita (Nath et al., 1979) and M. javanica (Srivastava et al., 1974) exceeded 0.2 J2/g of soil. In India, threshold level in the form of nematode/g soil of M. incognita was reported 1.0-2.0 (Nath et al., 1979; Ahmad and Husain, 1988), while for M. javanica it was 0.5-1.0. Tomato Wide variation in tolerance limit in tomato plants has been observed and was found 2-100

J2/kg (Seinhorst, 1965; Barker and Olthof, 1976; Barker et al., 1985; Ferris et al., 1986). The wide variation in tolerance limits reflects the great difference in plant response to nematode infection as well as the influence of soil type and environmental conditions on disease development and severity (Ferris et al., 1986). In the San Joaquin Valley of California, U.S.A., the number of juveniles in samples taken from sandy loam soils has been used from estimating potential yield loss in processing tomato production areas (Anonymous, 1985).

Root-knot symptoms

Above ground symptoms: The nematode causes nonspecific symptoms on above ground parts which resemble to mineral deficiency. These symptoms appear in the patches of plants in a field and are characterized by stunted growth, yellowing and/or chlorosis of foliage. The leaves may dehiscent prematurely. Pods may ripe and dry prematurely and remain partially filled and undersized in case of chickpea (Reddy et al. 1990). Foliage may experience mild wilting during the periods of high rates of transpiration (Southey, 1972). Poor emergence and death of young seedlings may occur in heavily infested soil, but death of grown-up plants is rare unless some fungus or bacteria become associated and form a disease complex (Francl and Wheelar, 1993). In case of tomato where seedling infection has taken place, numerous plants die in the seedbed and seedlings do not survive transplanting. In those plants that do survive, flowering and fruit production is strongly reduced. In Thailand, wilting often occurs in non-chlorotic plants and has given rise to the term “Green wilt disease” (Netscher and Sikora, 1990).

Fig. 11. Distribution of Meloidogyne incognita and M. javanica in different states in India.

Underground symptoms: The feeding by root-knot nematode leads to swelling of infected root tissue which is called as root gall or knot (Bird, 1986). Formation of galls on roots is the most characteristic symptom of the nematode attack (Fig. 12). Second stage juvenile enter into young lateral roots, and after getting a suitable site for feeding become sedentary with their heads inserted in vascular tissue and body in the cortical region of the root. As a result of nematode pathogenesis cortical cells around the nematode (female) become hyper-plastic dividing repeatedly by mitosis resulting to enlargement of the tissue which is commonly called as gall or knot (Bird, 1972). The galls vary in number, size and shape, depending on the crop, species and population density. Causal organism Root-knot nematode, Meloidogyne spp. is a sedentary endoparasite and second stage juveniles are infective stage of the nematode. Male and female larvae penetrate host roots. The females become sedentary after getting suitable site for feeding and gradually assume obesity. Male larvae do not feed and remain or become vermiform at maturity and migrate out of root at maturity. Mature females lay hundreds of eggs, on average 200-500 in a gelatinous matrix collectively called as an egg mass. Reproduction is parthenogenetic and life cycle from egg to egg completes in around three to four weeks. The species of root- knot nematode, M. incognita and M. javanica which have been found associated with root- knot of chickpea and tomato can be differentiated on the basis of perineal pattern characters, female stylets, male heads and stylets, and second stage juveniles (Eisenback, 1985). Meloidogyne incognita Second stage juvenile: The juvenile has a dumbbelled shape labial disc and medial lips in- face view. The labial disc is small and slightly raised above the median lips. Lateral lips lie in counter with the head region. Body length, 346-463 (405) mm; tail length, 42-62 (52) mm; head end to stylet base, 14-16 (15) mm; female stylet length, 15-17 (16) mm; male stylet length, 23-25 (24) mm. Mature female: The medial lips are wider than labial disc, but both are dumbbelled shaped. Under SEM two bumps may appear on the ventral side of the labial disc. Lateral lips are large and separated from the rounded medial lips. Stylet cone is distinctly curved dorsally. The anterior portion of the cone is cylindrical, whereas the posterior part is conical. The shaft is slightly wider at posterior side. The knobs are broadly elongated and set-off from the shaft, and indented anteriorly so much that in some specimen each knob appears as two. Mature male: Head shape is very characteristic and not easily confused with any other species. The labial disc is large and round. It is ventrally

curved and raised above medial lips. The median lips are as high as the head region. There are 2-3 incomplete annulations on the head region. Stylet tip is blunt and wider than the medial portion of the cone. Lumen of the stylet opens with a projection on the ventral side, which is located one fourth distance of the cone length from the stylet tip. The shaft is cylindrical and tapers near the knobs. The knobs are set-off from the shaft. They are broadly elongate to round and are indented anteriorly.

Fig. 12. Roots of chickpea (A) and tomato (B) showing galls or knots caused by root-knot nematode, Meloidogyne incognita.

Meloidogyne javanica Second stage juvenile: The labial disc and medial lips are bow-tie shaped. The lateral lips are triangular and lie below the contour of the labial disc and medial lips. Occasionally the head region has short annulations, but generally it is smooth. Body length, 402-560 (488) mm; tail length, 51-63 (56) mm; head end to stylet base, 14-16 (15) mm; female stylet length, 14-18 (16) mm; male stylet length, 18-22 (20) mm. Mature female: The medial lips and labial disc are dumbbell shaped. The labial disc two prominent bumps ventrally. Usually lateral lips indented and they are large, elongated and set-off from the medial lips and head region. The head region may be marked by one incomplete annulation. Stylet is similar to M. incognita except that the cone is not distinctly curved dorsally and gradually increases in width posteriorly. The shaft widens only slightly posteriorly and knobs are short and wide, often anteriorly indented. Mature male: Head cap is high and almost as wide as the head region. The labial disc and median lips are large and fused together. The head region is annulated (2-3 annules) in some population, whereas in others annules are absent. Stylet cone is narrow anteriorly, but very wide posteriorly. The shaft is cylindrical and often narrows near the junction of stylet knobs. The stylet knobs are low, wide and set- off from the shaft. 45

Perineal patterns The outer cuticle of saccate females at maturity becomes smooth and transparent on most of the body except posterior part around vulva. The transverse striae and lateral lines which disappear from the main body due to obesity remain prominent and distinctive together with phasmids and appear as a finger-like structure called as perineal pattern. The pattern shows fare degree of uniformity within a species (Fig. 13). Identification through perineal pattern is more useful when a species is to be identified from the infected root samples. Preparation of pattern does not take much time compared to picking and mounting of a larvae, but sometimes it may involve some confusion. Important diagnostic characters of the two species of root-knot nematodes are as follows: Meloidogyne incognita: The pattern is characterized by the presence of high and squarish dorsal arch. The arch may contain a distinct whorl in tail terminus area. The striae are smooth to wavy and sometimes zigzagged. Distinct lateral lines are absent, but the lateral field may be marked by breaks and forks in the striae. Some striae may bend towards vulva. Meloidogyne javanica: The perineal pattern is unique because it contains lateral lines, which divides the dorsal and ventral striae. The ridges run entire width of the pattern, but gradually disappear near the tail terminus. The dorsal arch is low and rounded to high and squarish. The striae are smooth to slightly wavy, and some striae may bend towards vulval edges. Life cycle and disease development The life cycle starts from eggs. After embryonic development, larva is formed inside the egg. The first molt occurs inside the egg (Fig. 14). Under suitable environmental conditions (temperature, moisture and osmotic pressure) an egg hatches giving rise to second stage juveniles. The J2 is infective stage of the nematode. The larvae moves slowly and randomly in the soil and does not feed. The larvae, however, moves faster and in specific direction when it has received chemical stimuli through root exudates from a susceptible plant (Southey, 1972). The second stage juveniles penetrate the roots and move mostly between the undifferentiated root cells till their head reach in endodermis. After locating a suitable site for feeding in primary phloem the J2 induces formation of giant cells (2-6) around the head region (Haung, 1985; Pasha et al., 1987). After formation of giant cells, the juveniles come to rest (sedentary) with the head in the phloem and the body in cortex and feeds strictly on giant cells around the head region. The giant cells are formed due to repeated endomitosis without cytokinesis of primary phloem cells or adjacent parenchyma and pericycle cells (Hussey,1989

Fig. 13. Perineal patterns of females of Meloidogyne incognita (A) and M. javanica (B).

Fig. 14. Life cycle of Meloidogyne incognita.

Wiggers et al., 1990; Haung, 1985; Pasha et al., 1987). The juveniles feed on giant cells for several days, as a result nematode body width increases and structural changes in the body also occur. Second and third molts occur and the female becomes pyriform. The sexes can be differentiated at all stages, as the males have one gonad where females have a pair. The males at maturity migrate out of the root, wander in soil and soon die. The fourth stage female continues to grow in size and undergoes the fourth molt to become the adult female. Reproduction is parthenogenetic and fertilized eggs develop without mating. The egg laying is completed in a week. The hatched juveniles from the new eggs may infect the same root tissue to establish new infections and form secondary galls. The hatched larvae may also infect other adjacent plants whose roots have come near the site of primary infection. The length of life cycle and number of generations depends on host health and temperature. Optimum temperature for M. incognita and M. javanica is 25-30°C. The life cycle of these species completes in 21-25 days at 26-27 °C and in 50-60 days or even 80 days at 14-16°C. In general, temperatures of 25-28°C and light textured soil are best for rapid multiplication, larval movement, infection and gall formation. The second stage juveniles (infective stage) inject secretions from oesophageal glands, which lead to several physiological and morphological changes in the invaded tissue, paramount significance are giant cells (Bird, 1962). Concurrent with the establishment of giant cells, root tissue around the nematode and its feeding site undergoes hyperplasia and hypertrophy leading to development of characteristic root galls which develop within two days of penetration and are often the first discernible symptoms of nematode infection (Khan and Esfahani, 1992). Formation of galls causes impairment of absorption of water and minerals by roots, subsequently the plant show water stress symptoms (Wallace, 1987). Upward conduction also gets impaired leading to accumulation of nutrients in root tissue (N, P, K, Mg etc.).

Fungus-nematode wilt disease complex In addition to causing direct damage, nematodes particularly Meloidogyne spp. possess great potential to synergise other pathogens leading to disease complex (Khan, 1993). The nematode-fungus interaction was first recorded by Atkinson (1892), who observed that Fusarium wilt of cotton (caused by Fusarium oxysporum f. sp. vasinfectum) became more severe in the presence of root-knot nematodes (Meloidogyne spp.). The nematode fungal complex may affect the host plant by breakdown of fungal resistance, additive or synergistic pathogenic effect, and suppression of symptoms or even earlier appearance of disease symptoms. Synergistic interaction of root-knot nematodes with other pathogens 48

results to severe crop damage (Kerry, 1988). The disease complexes involving Meloidogyne spp. and Fusarium spp. have been studied and documented on several host crops, including chickpea (Kumar et al., 1998); tomato (Goode and McGuire, 1967; Suleman et al., 1997); alfalfa (Griffin 1986); beans (France and Abawi 1994); coffee (Bertrand et al., 2000); banana (Jonathan and Rajendran 1998), lentil (De et al., 2001); brinjal (Naguera and Smits, 1982), squash (Caperton et al., 1986), onion (Alice et al., 1996) and rhizome rot of ginger (Makhnotra et al., 1997). Root knot nematodes are also known to break resistance in many wilt resistant cultivars of chickpea viz., Avrodhi, JG 74, Pusa-212, ICC 12275, ICC 11322, ICC 11319 and ICC 12272 (Maheswari et al., 1997; Ali et al., 2003). Synergistic interaction between M. incognita and F. oxysporum f. sp. lycopersici have also been recently reported (Samuthiravalli and Sivakumar, 2008; Son et al., 2009). Nematode infestation predisposes the plants and changes the physiology of the host plants infavour of secondary pathogen, thus making the task easier for fungus to invade/infect the plant and cause the disease. In susceptible genotypes, nematodes advance the onset of wilt from 31 days to 16 days and increase the disease incidence from 25-50%. The presence of both organisms together in soil results in severe and sudden loss to the crop after emergence (Ramnath and Dwivedi, 1981). Reduction in growth parameters was observed when nematode was inoculated 30 days prior to fungus (Goel and Gupta, 1986). The interaction of Fusarium oxysporum f. sp. ciceri with M. incognita on chickpea cv. Dahod Yellow revealed that the organisms in combination caused significantly greater reduction in the plant height and fresh root and shoot weights. Wilt severity was higher but root galling and nematode multiplication were lower in the presence of M. incognita and F. oxysporum f. sp. ciceri (Patel et al., 2000). Similarly, wilt severity in tomato was found 1.1 (on 0-5 scale) or 11% with F. oxysporum f. sp. lycopersici but increased to 4.1 (82%) when tomato plants were simultaneously inoculated with M. incognita and the wilt fungus (Son et al., 2009). In another experiment when M. incognita and F. oxysporum f. sp. lycopersici inoculated concomitantly enhanced the suppression of plant growth parameters synergistically and reduced the plant height by 33.08 cm of tomato cv. CO3 (Samuthiravalli and Sivakumar, 2008). Root knot nematodes are not only responsible for increasing the incidence and the severity of wilt diseases, but they also break resistance to Fusarium wilt in different crops. Abawi and Barker (1984) demonstrated that Fusarium wilt resistant cultivars of tomato became susceptible in the presence of M. incognita. In another experiment Fusarium

resistant tomato cultivars viz., Florida 47 R, Florida 91 (XP 10091), Sunguard (XP 10089), Solar Set R, Sunpride, P48024 and P48025 became susceptible to the fungus in the presence of M. incognita (Dababat, 2007). Jenkins and Coursen (1957) reported that M. incognita promoted 100% wilt symptoms in Fusarium resistant tomato cv. Chesapeake while M. hapla could produce only 66% wilt symptoms in tomato. Effect of fungus and nematode infection on root nodulation Root nodules in pulses are highly specialized structures formed as a result of a sequence of interactions between the host plant and the rhizobia. Root-nodule bacteria (Rhizobium and Bradyrhizobium) and leguminous plants show a mutually beneficial symbiotic relationship presenting a unique system of biological nitrogen fixation. This symbiotic association is of great agricultural importance. After the period of nitrogen fixation, the mature nodules decays, liberating motile bacteria in the soil which normally serve as a source of inoculum for the succeeding crop of a given species of legume (Subba-Rao, 1975). Generally antagonistic interaction between wilt fungus and root nodule forming bacteria have been recorded. Infection with wilt fungus causes suppression in nodulation (Twng-Wah and Howard, 1969; Swada, 1982, 1983) but the mechanism involved is not properly determined. It appears that Fusarium infected roots due to physiological and structural modifications are rendered unsuitable for the development of root nodules. The suppression may also be due to competition between the two microorganisms at initial stage of the infection. Wilt causing Fusarium are known to cause less infection on nodulated roots than non-nodulated roots (Zambolin and Schenk, 1984). Relationship between nematodes and root nodule forming bacteria is also negative. Plant parasitic nematodes may also affect this system at various stages from its establishment to efficient functioning. Survival of root nodule bacteria in the rhizosphere and colonization in the rhizoplane are influenced by root exudates of nematode infected plants (Huang, 1987). Most investigators have reported that plant nematodes irrespective of their mode of parasitism inhibit nodulation. Nutrient depletion by the nematodes (Masefield, 1958), competition between nematode juveniles and root nodule bacteria (Epps and Chambers, 1962), devitalization of root tips (Malek and Jenkins, 1964) and suppression of lateral root formation (Oteifa and Salem, 1972) are possible causes of reduced nodulation. In soybean, reduced nodulation may result due to interference of Heterodera glycines with soybean lectin metabolism (Haung et al., 1984). The nematodes reduce the binding of rhizobia to infected roots.

Species of Meloidogyne, Heterodera and Pratylenchus etc. invade root nodules directly on legumes (Taha, 1993). Meloidogyne spp. induce histological changes in nodular tissues and giant cells develop inside the nodules (Robinson, 1961; Barker and Hussey, 1976). Nodules also develop on root galls induced by the nematode (Khan and Kounsar, 2000). Reduction in size and number of nodules and early degeneration of nodules on nematode infected leguminous plants are considered as two possible reasons for adverse effects on the nitrogen fixing capability of the roots (Khan et al., 2002). Haung et al. (1984) found a lower leghaemoglobin (Lb) content of nodules in soybean plants infected with H. glycines than uninfected plants. Chahal and Chahal (1988) also reported significant reduction in Lb, bacteroid content and nitrogenase activity in chickpea nodules on roots infected with M. incognita. Some investigations have indicated that nodulation in legume roots may be stimulated by nematode infection. Infection by M. incognita, M. hapla, Pratylenchus penetrans and Belonolaimus longicaudatus stimulated nodulation by Bradyrhizobium japonicum on soybean (Haung, 1985). M. incognita enhanced root nodulation on pea and black beans (Verdejo et al., 1988). Some reports, however, show no apparent effect of nematode infection on root nodulation. Infection of M. javanica on cowpea (Taha and Kassab, 1980) and of M. hapla, P. penetrans and B. longicausatus on peanut (Barker and Hussey, 1976) did not influence nodule formation and their development.

Biological control of plant diseases Continuous increase in global human population has put two-fold pressure on agriculture. Precious agricultural lands are being diverted from crop production to urbanization and industrialization. As a result, the net area under crop production is shrinking whereas demand for food products continues to increase at an alarming pace. According to one estimate, the present global land area under crop production would produce much greater quantities of food than present requirements if pest and disease-free crops were grown (Khan and Jairajpuri, 2010a). Hence, the primary requirement to meet food requirements of both present and future populations is to integrate plant protection techniques with crop production systems. Numerous methods of pest and disease management are available including chemical, cultural, physical, and biological, which are used depending on crop, pathogen, availability of material and demand of the situation. Consensus is developing that chemical-based farming is not ecologically sustainable and economically viable. As a result, ecological approaches are being researched more intensively. The most obvious environment-friendly alternative to 51

pesticide application for managing agriculturally important diseases is the use of biological approaches. Biological control is based on the phenomenon that every living entity has an adversary in nature to keep its population in check (Khan, 2005). Garret (1965) defined biological control as “any condition under which, or practice whereby, survival and activity of a pathogen is reduced through the agency of any other living organism (except man himself) with the result that there is a reduction in the incidence of disease caused by the pathogen”. Baker and Cook (1974) defined biological control as the “reduction of inoculum density or disease producing activities of a pathogen or parasite in its active or dormant state, by one or more organisms, accomplished naturally or through manipulation of the environment, host, or antagonists, or by mass introduction of one or more antagonists”. In 1983 Baker and Cook revised the definition to ‘the reduction of the amount of inoculum or disease producing activity of a pathogen accomplished by one or more organisms other than man’. Biological control can be achieved either by introducing bioinoculants (biocontrol agents) directly into a field or by adopting cultural practices which stimulate survival, establishment and multiplication of the bioincoulants. Hence, more scientifically, biological control of pests and diseases can be defined as: reduction in disease severity, crop damage, population or virulence of the pest or pathogen in its active or dormant state by the activity of microorganisms that occur naturally through altering cultural practices which favours survival and multiplication of the microorganisms or by introducing bioinoculants. A large number of biocontrol agents have been investigated to harness their beneficial effects on crop productivity. Biological control agents are primarily fungal and bacterial in origin. Fungal biological agents basically work through parasitism (Papavizas, 1985; Stirling, 1993) against plant pathogenic fungi and nematodes (Khan, 2005). The important genera of biocontrol fungi which have been tested against plant pathogenic fungi and nematodes include Trichoderma, Aspergillus, Chaetomium, Penicillium, Neurospora, Fusarium (saprophytic), Rhizoctonia, Dactylella, Arthrobotrys, Catenaria, Paecilomyces, Pochonia, and Glomus. All these biocontrol agents of plant pathogens have been divided into two kinds of microorganisms. Classical parasites (e.g., Trichoderma spp., Paecilomyces lilacinus, Pasteuria penetrans etc.) which have been used in the disease control since old times (Khan and Khan, 1995). In recent years, more interest has been developed in using plant growth promoting microorganisms. Of these, phosphate- solubilizing microorganisms (PSM) such as Aspergillus niger, Penicillium spp., Bacillus

subtilis, B. polymyxa, Pseudomonas fluorescens, P. stutzeri, P. striata etc. are efficient solubilizers and may prove efficient biocontrol agents of plant pathogens (Gaur, 1990, Rao, 1990; Rudresh et al., 2005; Khan et al., 2009). The PSMs may suppress rhizospheric population of pathogens by promoting host growth, inducing systemic resistance and/or producing toxic metabolites (Kirkpatric et al., 1964). Pathogen management employing phosphate-solubilizing fungi or bacteria has advantage over classical biocontrol agents as the former provide essential nutrients to plants in addition to their antagonistic ability. Among various efficient phosphate solubilizers, Aspergillus niger was selected for the present study because of three most important attributes the fungus possesses. The fungus is not included in the toxigenic species of Aspergillus responsible for mycotoxins (ochratoxin A) (Khan and Anwer, 2007). Production of ribotoxin by A. niger was negative (Campbell, 1994) and the fungus is reported to decrease aflatoxin contamination (Boller and Schroeder, 1974; Wicklow et al., 1980; Horn and Wicklow, 1983), in addition of being highly antagonistic to a variety of pests and pathogens.

Biocontrol through Aspergillus niger Van Tieghem, 1867 Systematic position Division : Ascomycota Sub division : Pezizomycotina Class : Eurotiomycetes Order : Eurotiales Family : Trichocomaceae Genus : Aspergillus Species : niger

Macroscopic morphology Colonies on potato dextrose agar at 25°C are initially white, quickly becoming black with conidial production (Fig. 15). Reverse is pale yellow and growth may produce radial fissures in the agar (Raper and Fennell, 1965; Gilman, 2001; Alexopoulos et al., 2002). Microscopic morphology Hyphae are septate and hyaline. Conidial heads are radiate initially, splitting into columns at maturity. The species is biseriate (vesicles produces sterile cells known as metulae that support the conidiogenous phialides). Conidiophores are long (400-3000 µm), smooth, and hyaline, becoming darker at the apex and terminating in a globose vesicle (30-75 µm in diameter). Metulae and phialides cover the entire vesicle. Conidia are brown to black, very rough, globose, and measure 4-5 µm in diameter (Fig. 15) (Raper and Fennell, 1965; Sutton et al., 1998; de Hoog et al., 2000).

Fig. 15. Colony of Aspergillus niger on PDA (A), microscopic morphology of conidial head with conidia (B).

Molecular characterization The introduction of molecular biology techniques has been a major force in the area of systematics, genetic diversity and population biology of microorganisms. One of the first applications of PCR in mycology was described by White et al. (1990) to establish the taxonomic and phylogenetic relationships of fungi. Internal transcribed spacers (ITS) sequences are generally constant, or show little variation within species but vary between species in a genus, and so these sequences have been widely used to develop rapid procedures for the identification of fungal species by PCR-RFLP analysis (Vigalys and Hester, 1990; Buchko and Klassen, 1990; Goodwin and Annis, 1991; Nazar et al., 1991; Anderson and Stasovski, 1992; Buscot et al., 1996). Individual strains within a particular population, some examples being toxic producing strains of Aspergillus flavus (Bayman and Cotty, 1993), and in strain authentication in species of Trichoderma (Schlick et al., 1994) can be precisely differentiated by the RAPD-PCR method. Although the black Aspergillus species, A. carbonarius and the uniseriate species (A. aculeatus, A. japanicus) can be microscopically distinguished by vesicle and conidial size and ornamentation. However, The black Aspergilli (section Nigri) form a subgroup of the genus Aspergillus. Traditionally, fungal trains are assigned to a particular species based on their morphological characteristics such as, color, shape, size, ornamentation of the conidia and the length of the conidiophore. Using these techniques, the black Aspergilli were divided into 12 species (Raper and Fennell, 1965) and later into seven species (Al-Musallam, 54

1980). The phenotype of a fungal strain can vary significantly depending on the growth conditions (Varga et al., 2000), indicating that errors may occur when assigning strains by morphological methods. The development of molecular techniques (RFLP, RAPD PCR, DNA fingerprinting and nucleotide sequencing) for the identification of fungal strains has resulted in a reclassification of black Aspergilli (Kusters-van Someren et al. 1991; Varga et al. 1993; Varga et al. 1994; Accensi et al. 1999; Parenicova´ et al. 1997; Parenicova´ et al. 2001; Abarca et al. 2004). Recently, Random Amplified Polymorphic DNA (RAPD) was used to examine the genetic variability among isolates of Trichoderma and A. niger (Gajera and Vakharia, 2010) . Mechanism of disease suppression A. niger suppresses plant pathogens by direct parasitism, lysis, competition for food, direct antibiosis or indirect antibiosis through production of’ volatile substances. Activity of biocontrol agents mainly depends on the physicochemical environmental conditions to which they are subjected. These mechanisms are complex, and what has been defined as biocontrol is the final result of varied mechanisms acting antagonistically to achieve disease control. A. niger can even exert positive effects on plants with an increase in plant growth (mineralization) and the stimulation of plant defense mechanisms. Mechanism of disease suppression may be due to competition, antibiosis or mycoparasitism. Fungistatic An effective antagonist is usually able to survive in the presence of metabolites produced by other microorganisms and plants, and multiply under extreme competitive conditions. Aspergillus spp. were found to be most resistant to herbicides, fungicides, pesticides and many toxic heavy metals at minimum inhibitory concentrations (MIC) of 125-850 µg/ml (Baytak et al., 2005; Yuh-Shan, 2005; Ahmad et al., 2006; Braud et al., 2006). Dose- response relationships of fungicide resistance in agar growth tests were examined with A. niger and A. nidulans to pentachloronitrobenzene (PCNB), 3-phenylindole, benomyl or thiabendazole, and resistance was measured at high concentrations of these chemicals (van Tuly, 1977). In an experiment adaptation of A. niger to short-term stress induced by three antifungal agents [amphotericin B (AMPH), miconazole (MCZ), and ketoconazole (KCZ)] was observed and evaluated quantitatively using individual hyphae and found that exposure to AMPH (0.075 µg/ml) stopped the growth of the hypha. After washing with PDB, the same concentration of AMPH was applied again. The growth of the test hypha was not inhibited. This phenomenon was defined as adaptation to the short-term stress of AMPH.

Similarly, adaptation was observed with MCZ (0.01 µg/ml) and KCZ (0.5 µg/ml). The time required for the A. niger hypha to restart growth after washing with PDB depended upon the concentration of MCZ or KCZ, but not upon the concentration of AMPH (Park et al., 1994). Competition for nutrients Starvation or shortage of nutrients is one of the most common causes of death of microorganisms (Chet et al., 1997). Competition resulting in limiting the nutrient supply to fungal pathogens results in their biological control (Chet et al., 1997). For instance, in most filamentous fungi iron (Fe) uptake is essential for viability (Eisendle et al., 2004) and under Fe-deficient condition most fungi excrete low molecular-weight ferric iron-specific chelators termed siderophores to mobilize environmental Fe (Eisendle et al., 2004). Siderophores play a considerable role in biocontrol of soil-borne plant pathogens (Leeman et al., 1996) and as a supplier of Fe nutrition to crop plants (Jadhav et al., 1994). Since plant pathogens may not have the cognate ferri–siderophore receptor for uptake of the Fe– siderophore complex they are prevented from proliferating in the immediate vicinity because of Fe deficiency (O’Sullivan and O’Gara, 1992). Hence, siderophore-producing bioinoculants can confer a competitive advantage in interactions in the rhizosphere (Raijmakers et al., 1995). One of the most sensitive stages for nutrient competition in the life cycle of Fusarium is chlamydospore germination (Scher and Baker, 1982). In soil the chlamydospores of F. oxysporum require adequate nutrition to maintain a germination rate of 20-30%. Germination may decrease due to sharing of nutrients with other microorganisms. Root exudates are a major source of nutrients in soil. Thus, colonization in the rhizosphere by an antagonist might reduce infection by Fusarium-like pathotypes (Cook and Baker, 1983). Aspergillus niger AN27 was found to produced both hydroxamate and catecholate groups of siderophores (Sen, 1997; Mondal et al., 2000). In another in vitro studies Vassilev et al. (1996, 2006) also demonstrated that A. niger produced siderophore on modified CAS medium. Antibiosis Antibiosis is the phenomenon of suppression of one organism by another due to release of toxic substances/metabolites into the environment. Antibiosis is important in determining the competitive saprophytic and necrotrophic ability of antagonists. The A. niger may suppress plant parasitic nematodes and pathogenic fungi through antibiosis and by stimulating host defense. Low molecular-weight compounds and antibiotics (both volatile and non-volatile) produced by Aspergillus spp. impede colonization of harmful

microorganisms such as bacteria, fungus including nematodes in the root zone (Buchi et al., 1983; Fujimoto et al., 1993; Eapen and Venugopal, 1995). Harzianic acid, alamethicins, tricholin, peptaibols, 6-penthyl-α-pyrone, y-lactone, massoilactone, viridin, gliovirin, glisoprenins, heptelidic acid, oxalic acid and enzymes are some of the chemicals possessing antibiotic properties produced by Aspergillus species (Mankau, 1969a, 1969b; Benitez et al., 2004; El-Hasan et al., 2007). A. niger, that parasitize nematode eggs, prefer eggs which are deposited in cyst or a gelatinous matrix. The oviposition nature of Heterodera spp. and Meloidogyne spp. makes them more vulnerable to attack by the fungi. As soon as the fungi identify a cyst or an egg mass they rapidly grow and colonize those eggs where larval formation is not complete. However, when larva is formed the egg becomes less vulnerable. It has been suggested that this differential vulnerability of egg and larval stage is due to chitinolytic activity of the fungi. Chitin is a major constituent of the egg shell, which is lacking in the larval cuticle. Mycoparasitism Mycoparasitism involves direct parasitism of one fungus by another and involves recognition, attack and subsequent penetration and killing of the host fungus (Harman et al., 2004). In a necrotrophic association there is direct contact between two fungi, and a nutrient exchange channel is established between them. Typical examples are the association of Arthrobotrys oligospora with R. solani (Persson et al., 1985); Trichoderma hamatum with species of Phythium, and Rhizoctonia and Sclerotium (Bruckner and Pryzybylski, 1984). A. niger also show mycoparasitism (Sen et al., 1995). Observations using scanning electron microscopy revealed that A. niger coiled around the pathogen hyphae and penetrated within. Presence of A. niger hyphae inside pathogen hyphae using fluorescent microscopy has been confirmed repeatedly in F. oxysporum f. sp. melonis, ciceri and other pathogens (Sen et al., 1997; Sharma and Sen, 1991a; 1991b). Further studies have revealed that A. niger could kill Macrophomina phaseolina, several species of Pythium, Rhizoctonia solani, Sclerotinia sclerotiorum (Sen et al., 1995) and Sclerotium rolfsii (Palakshappa et al., 1989). The dead hyphae of the pathogens were eventually invaded. These observations confirm that A. niger is a contact and invasive necrotroph (Mondal et al., 2000). Plant growth promotion Two growth promoting compounds, 2-carboxymethyl 3-n-hexyl maleic acid (compound 1) and 2-methylene-3-hexylbutanedioic acid (compound 2) isolated from A. niger are responsible for increasing root and shoot length and biomass of crop plants (Mondal et al.,

2000). Both the compounds increased germination and improved crop vigour. Compound 1 was more effective for increase in germination and shoot length, whereas compound 2 had relatively greater role in increasing the root length and biomass of cauliflower seedlings (Mondal et al., 2000). A. niger frequently enhances root growth and development, crop productivity, resistance to abiotic stresses and the uptake and use of nutrients (Sen, 2000). Plant growth production of tomato can increase upto 467% after the addition of A. niger (Vassilev et al., 1996, 2006) greater than increased by application of T. hamatum or T. koningii (300%) of filed crops (Chet et al., 1997). In an in vitro tests, A. niger PSBG-12 strain produced plant growth promoting substances such as IAA, GA and under pot culture conditions showed better effect on plant growth parameters and nutrient uptake compared to uninoculated control (Vaddar and Patil, 2007). In a field where muskmelon and watermelon crops were suffering from Fusarium wilt (sometimes R. solani and Pythium spp. were associated with the disease), treatment of seeds with A. niger (Kalisena SD) @ 8 g per kg and soil with A. niger (Kalisena SL) @ 30 g per pit resulted in 81% control of the disease. The vines were more vigorous, and even with 15% incidence of disease, yield was approximately 5% greater as compared to that in disease-free areas (Chattopadhyay and Sen, 1996). Phytohormone production Several strains of Bacillus subtilis and Pseudomonas fluorescens are well known to synthesize phytohormones such as indole acetic acid, gibberellins, cytokinins and zeatin which promote plant growth at various stages (Gracia de Salamone et al., 2001). Evidences exists which indicate that some A. niger isolate also produce IAA and other phytohormones (Mostafa and Youssef, 1962). Youssef and Mankarios (1975) reported that soil isolates of A. niger produced auxins, gibberllins and gibberellins like substances in their culture filtrates. Khan and Anwer (2008) have recorded invitro production of IAA by A. niger which resulted to significant increase in eggplant yield. Phosphorus solubilization The production of organic acid in the microenvironment around the root or in the culture media is considered the most important parameter to measure phosphate solubilization by microorganisms (Sperber, 1958; Hayman, 1975; Gaur, 1985 a, b). A. niger have been found to synthesize citric, gluconic, glycolic, oxalic and succinic acids (Sperber, 1958; Blumenthal, 2004; Ramachandran et al., 2008). The role of organic acids in solubilizing mineral phosphates and phosphorilated minerals is attributed to the lowering pH which helps in the formation of stable complex and later forms are more soluble and available

form for plants (Jennings, 1989; Li et al., 1991). It has been reported that A. niger reduce medium pH to 4.0, sufficiently low enough to solubilize phosphorus (Domich, et al., 1980; Medina et al., 2007). Different mechanisms have been suggested by some scientists for the solubilization of inorganic phosphates by A. niger. Organic acids effect: Solubilization of tricalcium phosphate in liquid medium by organic acids such as citric, gluconic, glycolic, oxalic and succinic acids depends on pH and formation of soluble Ca-complexes (Azcon et al., 1986).These organic acids lowers down soil pH and with change in soil pH, calcium phosphate [Ca3 (PO4)2] changed to calcium biphosphate [CaHPO4] or calcium triphosphate [Ca(H2PO4)2] which are more soluble and become available to plants (Jennings, 1989). The nature of the acids released is more important to the amount of acid produced because the amount of phosphate solubilized by reaction with organic acids depends primarily on the strength of the acid (Li et al., 1991). Chelating effect: Chelations of cations (bound to P) of the organic acids has been shown to be an important mechanism for P-solubilization (Bajpai and Sundra Rao, 1971a,b). The extent to which an organic acid is capable of chelating metal ions is governed by its molecular structure and vary from acid to acid. The ability of organic acids to chelate Fe- organic complex is in the order of citrate > oxalate > malonate > tartrate > aceate (Lal, 2002). Carbon dioxide effect: Carbon dioxide produced by A. niger (Ramachandran et al., 2008) in the rhizosphere increases the availability of phosphate and its uptake by crop plants

(Morean, 1959; Knight et al., 1989). Siderophores effect: In well aerated soils, ferric ion (Fe3+) is dominant form of iron which reacts with soluble phosphorus forming insoluble ferric phosphate. Nonporphyrin metabolite secreted by A. niger forms highly stable coordination compounds (chelate) with iron (Sen, 1997), a high affinity iron-binding compound called siderophore. These siderophores bind iron tightly to prohibit its reaction with soluble phosphates, and rather help release phosphate fixed as ferric phosphate. Specific tests confirmed that A. niger produced both hydroxamate and catecholate group of siderophores (Mondal et al., 2000). By this mechanism A. niger may able to increase the solubility and availability of phosphorus to plants.

Mycotoxins from Aspergillus niger Aflatoxins are toxic fungal metabolites that can inhibit human development, cause cancer, and even induce death such toxins are mainly produced by Aspergillus flavus (Hell et al., 2008; Cotty et al., 2009). The mycotoxins, ochratoxin A and diketopiperazine asperazine are currently the most problematic compounds, especially in foods and feeds such as coffee, nuts, dried fruits, and grape-based products. For chemical differentiation /identification of the less toxic species the diketopiperazine asperazine can be used as a positive marker since it is consistently produced by A. tubingensis (177 of 177 strains tested) and A. acidus (47 of 47 strains tested) but never by A. niger (140 strains tested) ( Nielsen et al., 2009). Ochratoxin A Ochratoxin A (OTA) is a toxic metabolite produced primarily by Aspergillus, but also by Penicillium and other molds. It is a white crystalline powder. Recrystallized from xylene, it forms crystals that emit green (acid solution) and blue (alkaline solution) fluorescence in ultraviolet light, the melting point of these crystals is 169ºC. The free acid of OTA is soluble in organic solvents (IARC 1983, 1993). The sodium salt is soluble in water. OTA is unstable to light and air, degrading and fading even after brief exposure to light, especially under humid conditions. Ethanol solutions are stable for longer than 1 year if kept refrigerated and in the dark. OTA is fairly stable to heat; in cereal products, up to 35% of the toxin survives autoclaving for up to 3 hours (IARC 1976). OTA is reasonably anticipated to be a human carcinogen classified 2B (for human) by IARC based on sufficient evidence of carcinogenicity in experimental animals and possible teratogenic effects on fertility (fetotoxicity, post-implantation mortality) and birth defects. It may cause cancer, (hazardous in case of skin contact, permeator or irritant), adverse reproductive effects (paternal effects and genetic material), conjunctivitis, respiratory tract irritation, and may be fatal if swallowed. Otherwise may cause digestive tract irritation with possible ulceration or bleeding from stomach. Ochratoxin A may affect the endocrine system, liver (necrosis, fatty liver degeneration), blood behavior (somnolence, altered sleep time, ataxia), respiration (acute pulmonary edema), metabolism, cardiovascular system and may damage kidney (chronic potential health effects) (NTP, 1989 and IARC, 1993).

Acute oral toxicity (LD50): 20 mg/kg bw (Rat), 46 mg/kg bw (mouse), 0.2 mg/kg bw (dog), 1.0 mg/kg bw (pig), 3.3 mg/kg bw (chicken) (Harwing et. al., 1983). Several

assays have been reported for genotoxicity with ochratoxin A (Creppy et al,. 1985; Dorrenhaus et al.,2000; Zepnik et al., 2001). Until recently, only 6-10% of members of the A. niger group isolated from corn, peanuts, raisins, onions, mango, apples, and dried meat products (Perrone et al., 2007) are known to produce OTA in least amounts (Abarca et al., 1994; Varga et al., 2000; Esteban et al., 2006). Production of ribotoxin by A. niger was negative in Eastern and Southern blot tests (Campbell, 1994). Interestingly the fungus is reported to decrease aflatoxin contamination (Wicklow et al., 1980; Horn and Wicklow, 1983). This effect of A. niger has been attributed to lowering of substrate pH and an inhibitory substance produced by the fungus for degradation of aflatoxin (Boller and Schroeder, 1974).

Effectiveness of Aspergillus niger against plant pathogenic fungi In vitro studies In an In vitro study, Muhammad and Amusa (2003) reported that A. niger inhibited 40- 60% growth of some seedling blight inducing pathogens viz., Fusarium oxysporum, Sclerotium rolfsii, Pythium aphanidermatum, Helminthosporium maydis, Macrophomina phaseolina, and Rhizoctonia solani. In another study culture filtrate of A. niger inhibited the mycelium growth of F. udum by 66.6% in dual culture test (Singh et al., 2002). A. niger also restricted the development of colonies of the Fusarium moniliforme by its over growth and finally by parasitizing over the Fusarium mycelia (Chand et al., 2007). In dual culture test, A. niger significantly suppressed all Fusarium spp. of head blight of wheat, viz. F. avenaceum, F. culmorum, F. graminearum and F. poae isolated from wheat kernels (Mullenborn et al., 2008). In another dual inoculation assay, A. niger significantly inhibited the growth of Anthracnose (Colletotrichum gloeosporioides) of Indian bean (Lablab purpureus L.) mycelia by 57% (Deshmukh et al., 2010). Pot culture Muskmelon seeds were soaked overnight in Aspergillus niger AN 27 (Kalisena SD) spore suspension and grown in sand for six days. The roots of seedlings (with fully opened cotyledonary leaves) were washed thoroughly in water to remove A. niger spores. The seeds were suspended in F. oxysporum melonis (aqueous) spore suspension. Muskmelon seedlings raised from A. niger-treated seeds showed 56% resistance to F. oxysporum melonis without physical presence of A. niger in the root zone (Radhakrishna and Sen, 1986). These seedlings were 58, 26 and 2% higher in peroxidase, polyphenol oxidase and phenylalanine ammonia lyase activity, respectively, over controls (Angappan et al., 1996). The lignin content was also higher in the tissues of treated plants and resulted in the 61

induced resistance (Kumar and Sen, 1998). When A. niger was amended in unsterilized and sterilized pot soils at the rate of 1, 2 or 3% (w/w), controlled the Fusarial wilt of pigeonpea (F. udum) by 48.3, 52.9 and 68.2% in unsterilized soil and 74.0, 83.0 and 88.4% in sterilized soil, respectively (Singh et al., 2002). In a green house conditions the damping off of chilli (Capsicum frutescens) caused by Pythium aphanidermatum was controlled by using seed coating with A. niger resulted increased germination percentage (82%), and reduced the pre (12%) and post emergence (34.2%) damping off as compared to sick soil treated as control (Chakraborty et al., 2007). Field conditions A good deal of work has been conducted in field trials with A. niger against soil-borne fungal pathogens. Dhillion (1994) suggested that biocontrol of root diseases by the soil amendment of A. niger applied with arbuscular mycorrhizal inoculum, enhanced nutrient uptake through increase solubilization of phosphate and increased stress tolerance, and yield of maize, wheat, millet, sorghum, barley and oat plants (Dhillion, 1994). In a field where muskmelon and watermelon crops were suffering from Fusarium wilt (sometimes R. solani and Pythium spp. were associated with the disease), treatment of seeds with A. niger (Kalisena SD) @ 8 g per kg and soil with A. niger (Kalisena SL) @ 30 g per pit resulted in 81% control of the disease. The vines were more vigorous, and even with 15% incidence of disease, yield was approximately 5% greater as compared to that in disease-free areas (Chattopadhyay and Sen, 1996). Seed treatment with Kalisena SD also provided 30% less sheath blight disease rice over control plants (Kumar and Sen, 1998). Problems of pre-and post-emergence damping-off incited by P. aphanidermatum and R. solani in fruit and vegetable farms was successfully overcome by a combined treatment of seed and soil application of Kalisena SD and Kalisena SL (Majumdar and Sen, 1998). Similarly, 93% control of charcoal rot of potato (Solanum tuberosum L.) in a Macrophomina phaseolina- infested field was obtained with A. niger (Kalisena SD and Kalisena SL) (Mondal, 1998). In another study 87% control of black scurf of potato (R. solani) and a 10% increase in yield by the application of A. niger @ 8g/kg on infected seed tubers and sown in worst affected fields was reported (Sen et al., 1998). In a study, Lodhi (2004) has repored the control of soil borne diseases of potato by the application of A. niger alone and in combination with VAM fungi. Winter sorghum can be strongly damaged by Macrophomina infection; however, A. niger (Kalisena SD) seed treatment brought down incidence of the disease from 30% to 7% (Das, 1998). Fusarial wilt of Hibiscus was controlled by the application of A. niger in the soil after uprooting the dead plants and soil

population of the pathogen was found non-detectable level after a month of A. niger application (Sen, 2000). In an experiment seed treatment with A. niger @ 8 g/kg seed effectively controlled the Blast (Pyricularia oryzae), Sheath blight (Rhizoctonia solani), Brown spot (Helminthosporium oryzae), False smut (Ustilaginoides virens) of rice in the field (Sehgal et al., 2001). In another study, a plant disease of international importance, malformation disease of mango caused by Fusarium moniliforme was controlled by spraying conidial suspension of A. niger over dead necrotic malformed panicles (Chand et al., 2007). Stem canker of Aonla (Emblica officinalis) caused by R. solani was controlled by the application of A. niger (Kalisena SL) in soil around the tree trunk revived the partially diseased trees and brought them to normal bearing in an orchard (Sen ,2000). Guava (Psidium guajava L.) wilt caused by Fusarium oxysporum f. sp. psidii and F. solani was controlled by the soil application of A. niger multiply on the field wastes (Misra, 2007). Wheat loose smut (Ustilago tritici) and spot blotch (Chaetomium globosum) was effectively controlled be the application of A. niger (Birthal and Sharma, 2004). In an experiment A. niger was found potent antagonist against F. udum, both in vivo and in vitro (Upadhyay and Rai, 1981). Under field conditions, 38% control of Fusarial wilt of pigeonpea was observed with A. niger application amended at the rate of 42 g/m2 (150 g/1.8x2 m2) of A. niger cultured on wheat (Singh et al., 2002). In field trials, Khan and Khan (2001, 2002) tested the effectiveness of A. niger against Fusarium wilt of tomato (Fusarium oxisporum f. sp. lycopersici). Root-dip treatment with A. awamori or A. niger resulted in a significant decline in the rhizosphere population of F. oxisporum f. sp. lycopersici. Tomato yield was greatly enhanced by A. awamori and A. niger. Direct soil-plant inoculation with A. niger and A. awamori decreased the rhizosphere population of the pathogen by 23–49% while the tomato yield increased by 28–53% in field experiments. Vassilev et al. (1996, 2006) reported an efficient biotechnological scheme for preparing a material with biocontrol and plant- growth-promoting functions. Sugar beet wastes were mineralized by an acid-producing strain of A. niger with a simultaneous solubilization of rock phosphate under conditions of solid-state fermentation. The product of this process, used as soil amendment, resulted in 347 and 467% higher (vs. unamended control) plant biomass in plant–soil experiments contaminated or not with F. oxysporum f. sp. lycopersici, respectively. Disease severity on tomato and number of F. oxysporum f. sp. lycopersici colony-forming units reached the lowest levels, particularly when plants were mycorrhized with G. deserticola.

Aspergillus nigrer against plant nematodes Paecilomyces lilacinus, Dactylaria candida and Pasteuria penetrans have been widely used in nematode control studies during last few decades (De Bach, 1964), but now a days PGPR including A. niger are receiving attention of scientists. Effects of A. niger has been evaluated against different nematodes under in vitro, pot and field conditions. In vitro studies A. niger is reputed to produce nematoxic metabolites (Dahiya and Singh, 1985). Culture filtrates of A. niger caused mortality to Radopholus similis and M. incognita after 48 h of immersion (Molina and Davide, 1986). In an in vitro trial, A. niger (isolated from egg- masses of M. incognita) was identified to be toxic, egg parasitic or opportunistic against M. incognita (Goswami et al., 2008). In another study culture filtrates of A. niger soil isolates AnC2 and AnR3 efficiently suppressed hatching of eggs and mortality of juveniles of M. incognita (Khan and Anwer, 2008). Culture filtrates of A. terreus, A. nidulans, A. niger and Acremonium strictum was very effective against the nematode in regards to egg parasitism (upto 53%), egg hatching inhibition (upto 86%) and mortality (upto 68%) compared to controls (Singh and Mathur, 2010b). Pot conditions The majority of studies exploring the potential of bioicontrol against plant nematodes have been carried out under pot conditions (Khan, 2007). In a pot experiment, chilli (Capsicum annum) seedlings were inoculated with M. javanica, A. niger and R. solani alone or in various combinations. All growth parameters were significantly greater with A. niger and lower with M. javanica or R. solani (Shah et al., 1994). Singh et al. (1991) showed that application of A. niger decreased the damage caused by M. incognita and R. solani singly or together on tomato cv. Perfection. Sharma et al. (2005) demonstrated in pot conditions that inoculation of A. niger in preinoculated juveniles of M. incognita in okra significantly decreased the galling, eggmass production and soil population and increased the growth parameters of the plant. Similarly, inoculation with A. niger, Epicoccum purphurascum, Penicillium vermiculatum and Rhizopus utgricans effectively diluted the adverse effect of R. solani and M. incognita resulting in an increase in germination of tomato cv. Pusa Ruby (Rekha and Saxena, 1999). Pant and Pandey (2001) reported maximum reduction in populations of M. incognita with T. harzianum, P. lilacinum and A. niger applied in sterilized soil in pots @ 5000 spores/pot. In an experiment second-stage larvae of root-knot nematode were incubated in microbial filtrates of many fungal and bacterial (isolated from agricultural soil) metabolites for 48 hours and then introduced to healthy tomato seedlings

in green house or controlled chamber, after two months it has been found that several bacterial and fungal species reduced the ability of second-stage larvae of root-knot nematode from penetrating, infecting, and/or developing on the tomato roots with highest A. niger isolate (Abdel-Rahman et al., 2004). In a pot experiment application of A. niger isolates (AnC2 and AnR3) significantly suppressed galling, egg mass production and soil populations of M. incognita. The isolates AnC2 and AnR3 produced the greatest quantities of siderophores, HCN, and NH3, and solubilized the greatest quantity of soil phosphorus (Khan and Anwer, 2008). Field conditions Relatively few field trials have been conducted to evaluate the effectiveness of A. niger against nematode infestations. The studies so far conducted have demonstrated nematode control to a level that can be exploited commercially (Khan, 2005). In an experiment, soil treatment by A. niger in castor beans abated the population of Rotylenchulus reniformis up to 71% (Das, 1998). A field trials was conducted by Goswami et al. (2008) to manage root- knot nematode disease infecting tomato by dual treatment of A. niger and T. harzianum both alone and together showed significant increased in health improvement of tomato plant with a remarkable reduction in M. incognita population (Goswami et al., 2008). In another field experiment application of A. niger in combination with cattle manure resulted in a significant increase (62%) in the growth of nematode-inoculated plants and also resulted in a high reduction in galling and nematode multiplication (Siddiqui and Kazuyoshi, 2009). Aspergillus niger in IPM and chemical compatibility The term integrated control describes the combined use of more than one approach to suppress losses due to pests, with greater overall effect than would result from the use of any one of the controls applied singly (Rahe and Utkhede, 1985). The integrated control of plant pathogens has been discussed by several workers, including Lewis and Papavizas (1991). A combination of broad-spectrum fungicides with biocontrol fungi is more commonly used than any other method of integrated control. The rationale for using fungi in combination with decreased amounts of pesticides stems from the need to decrease pesticide use in disease control (Lewis and Papavizas, 1991). Integrated mixture of chemicals and biological agents are used when inconsistency and low efficiency of biocontrol occur in the field. Integration of biological control and chemicals should be based on a dose of pesticide sufficiently low enough not to cause environmental contamination and other adverse effects.

Aspergillus spp. are naturally resistant to heavy metals and have been found to be quite compatible with broad-spectrum herbicides, nematicides and fungicides (Bhatnagar, 1995; Baytak et al., 2005; Yuh-Shan, 2005; Ahmad et al., 2006; Braud et al., 2006). The effect of five fungicides, benomyl (1 mg/l), dodine (50 mg/l), manzate (100 mg/l), cupric sulphate (200 mg/l) and thiabendazole (4 mg/l) was tested under in vitro conditions on the development of Aspergillus spp. and found highly resistance to tested concentrations of the fungicides (Luz et al., 2007). In another experiment when A. niger included with two fungicides, Foltaf 80W (Captafol 80%) and Blue Copper-50, for the treatment of pigeonpea wilt, the disease was more effectively controlled than the fungicides were used alone to control the disease without adversely affecting the growth of pigeon-pea (Bhatnagar, 1995). Seed treatment of chickpea with A. niger (106 spores/ml/10 g seed) in combination with carboxin (2 g/kg seed) enhanced seed germination by 10.0–13.0% and grain yields by 33-63% and reduced wilt incidence (44–60%) during a field experiment (Dubey et al., 2007). In a soil, water and air quality conservation and management program, swine wastes were treated with A. niger resulted in 90 percent reduction in Cu and Zn from the wastes (Anonymous, 2001). In adsorption efficiencies experiment, the percentage uptake of lead ion by A. niger was observed from 6.71-64.95% for dead and live biomass respectively (Awofolu et al., 2006). These characters represent broader use of A. niger biopesticides with pesticides.

Biopesticides Biopesticides are the formulations of biocontrol agents in a form which keep the organism at higher count and viable for their introduction/application in the field. Hence for large scale or commercial use of biocontrol agents, their biopesticides are necessarily produced. Among the total pesticides used in India more than 60% of the pesticides are used in the agriculture sector. The use of pesticides is highest in Andhra Pradesh (20%), followed by Punjab (10%), Tamil Nadu (9%) and Karnataka and Gujarat (6%) each. Cotton (40-50%), rice (17-18%) and vegetables (14-16%) use maximum quantity of pesticide in the country. Among the chemical pesticides, insecticides are used to a large extent of about 60% in India followed by fungicides and bactericides (20%) herbicides (17%) and other chemicals (3%). While in western countries herbicide use is the highest. The world average for herbicide use is about (45%) followed by insecticides (36%), fungicides (17%) and other chemicals (2%) (Wahab, 2003, 2005, 2009).

About 700 products of different microbials are currently available worldwide. In India about 16 commercial preparations of Bacillus thuringiensis, 38 fungal formulations based on Trichoderma, Metarhizium, Beauveria and about 45 baculovirus based formulations of Helicoverpa and Spodoptera are available. Microbials are expected to replace at least 20% of the chemical pesticides. Biotic agents are being supplied by about 128 units in the country (80 private companies). Besides, ICAR institutes (8), SAUs (10) and Central Integrated Pest Management centers (30) and 4 parasitoid producing laboratories are also supplying natural enemies (Wahab, 2003, 2004, 2009). Developing a safe, easy to use, cost-effective formulation that will keep the microorganism alive is one of the most important steps in developing a biological product. Formulation is the blending of active ingredients such as fungal spores with inert carriers such as diluents and surfactants in order to improve the physical characteristics. A final formulation must have a long shelf-life, at room temperature, be easy to handle, insensitive to abuse and must be stable over a range of -5 to 35°C. The most needed technique is drying techniques, which allows retention of maximum number of viable propagules in dried product. Most important steps of production of biopesticides are: mass production of biocontrol agents, their immobilization, shelf life test, evaluation for effectiveness, biosafety analysis, registration etc. Mass production Fermentation methods are important for mass production of microorganisms and to harvest a much better yield quantitatively as well as qualitatively. Three methods of fermentation are described by Lewis and Papavizas (1991). Liquid fermentation: This technology has been adopted to produce bacterial and fungal biomass. A suitable medium should consist of inexpensive, readily available agricultural byproducts with appropriate nutrient balance. Acceptable materials include molasses, brewer’s yeast, corn steep liquor, sulphate waste liquor, cotton seed and soya flours (Lisansky, 1985). Alegre et al. (2003) proposed liquid fermentation method consisting of molasses, wheat bran and yeast for large scale production of T. harzianum. A higher produce of Trichoderma chlamydospores was harvested through liquid fermentation technology. The preparation based on chlamydospores prevented the disease more effectively than a preparation that contained conidia only (Lewis et al., 1990; Papavizas and Lewis, 1989). Small scale fermentation in molasses-brewers yeast medium has also resulted in abundant chlamydospores production of Trichoderma (Papavizas et al., 1984).

Solid fermentation: Mass production of antagonists on solid substrates for the production of inoculum of various biocontrol fungi includes straws, wheat bran, saw dust; bagasse moistened with water or nutrient solutions through fermentation technology is referred to as solid fermentation (Papavizas, 1985a). This technology is also effective especially for those organisms, which can multiply on dry substrates. Semisolid fermentation: Semisolid fermentation is done for the fungi which do not sporulate in liquid culture. Diatomaceous earth granules impregnated with molasses (Backman and Rodriguez-kabana, 1975), wheat bran and vermiculite-wheat bran (Lewis et al., 1989) yield good produce of bioagents. This method, however, requires more area, labour intensive and the chances of contamination are high when compared to liquid fermentation. For commercial production of antagonists, different technologies have been adopted on industrial scale. Fermented Trichoderma consisted mainly of Chlamydospores and conidia with some amount of mycelial fragments. Air-dried mats have been grounded and mixed with a commercially available carrier, the formulations thus developed, contained 108 to 109 propagules/g (Table 15; Chaube et al., 2002; Chaube and Pundhir, 2010). Immobilization For field application of a biocontrol agents an efficient substrate for mass production and an inert immobilizing material are required, which could carry the maximum number of propagules of the organism with minimum volume and necessarily maintain its survival and integrity. An excellent bioinoculant is one that is introduced to an ecosystem, and subsequently survives, proliferates, becomes active and establishes itself in a new environment (Khan, 2005). For preparing a commercial formulation these attributes must be considered. In addition, the bioinoculant should be mass cultured on an inexpensive substrate in a short time. Easy application, effectiveness and consistent results under a variety of environmental conditions are other desirable features required for production of bioinoculant formulations. Different techniques of cell immobilization have been developed to devise efficient carrier systems to produce commercial formulations of bioinoculants. A number of carriers for immobilization of microorganisms have been used to develop commercial formulations of biocontrol agents viz., peat, seeds, meals, kernals, husks, bran, bagasse, farmyard manure, cow dung cake, compost, oil cakes, wood bark, vermiculite, sand, clay and liquid carriers. Three types of formulations viz., pellet, granular and liquid, are widely produced (Mukhopadhyay, 1987; Kousalya and Jeyarajan, 1990; Bhai et al., 1994; Khan and Anwer,

2011). Tiwari et al. (2004) evaluated grains of sorghum, wheat, bajra, wheat bran, rice bran and sugarcane bagasse for mass multiplication of T. viride. Grains of sorghum (cv. swanki) were found the best substrate that provided maximum spore concentration (8×109 spores)

Table 15. Base material/carriers used for mass production of fungus biocontrol agents. Antagonists Base material(s) Form of formulation Trichoderma Black gram shell, shelled maize cob, coir-pith, Powder, pellets harzianum peat, gypsum, coffee fruit skin + biogas slurry, coffee husk, coffee-cherry husk, fruit skin and berry mucilage, molasses-yeast, molasses-soy, molasses-NaNO3, mushroom-grown waste, sugarcane straw, wheat bran + biogas manure (1:1), wheat-bran + kaolin. T. viride Barley grains, black gram shell, shelled maize Pellets cob, coir-pith, peat, gypsum, coffee husk, coffee-cherry husk, fruit skin and berry mucilage, mushroom grown waste, mustard oil cake, neem cake + cow dung, poultry manure, spent tea leaf waste, sugarcane straw, talc, vermiculite + wheat bran + HCL T. virens Barley grains, coffee husk, coffee-cherry husk, Pellets fruit skin and berry mucilage, mushroom-grown waste, neem cake + cow dung, poultry manure, soil, sorghum grains, talc, wheat bran saw dust. T. longibrachiatum Talc, wheat bran + saw dust Powder Aspergillus niger Citrus pomae (waste from canning industry), Pellet, powder talc + cmc A. terreus Maize-meal + sand Powder and spore viability (92.5%) after 15 days of incubation at 27+1°C. Spores remained viable for 6 months at 5°C. Wheat bran and sawdust mixture have been used as a carrier media for the mass multiplication of T. harzianum (Elad et al., 1980; Mukhopadhyay et al., 1986). Khan et al. (2001) evaluated various agricultural and industrial wastes for mass multiplication of T. harzianum, T. virens and P. chlamydosporia, and found highest CFU, 1.2×106 on bagasse-soil-molasses for T. harzianum, 1.0×106 for T. virens and 1.1×106 on corn meal-sucrose mixture for P. chlamydosporia. Vidyasekaran et al. (1997) developed powder formulations of P. fluorescens using talc powder, peat, vermiculite, lignite and kaolinite. All freshly prepared powdered formulations were effective in controlling pigeonpea wilt, but their efficacy varied depending upon the length of storage. Talc formulation were effective even after 6 months of storage. Bhai et al. (1994) evaluated a

number of agricultural wastes which could be used as carrier and multiplication media at the same time. They reported that sterilized tea waste, coffee husk or a mixture of coffee husk and cattle manure were ideal combinations for the fast growth and multiplication of T. harzianum and T. virens. Angappan (1992) used molasses yeast medium for growing T. viride and mixed it with talc powder to develop commercial formulation. The initial population in the produce was 3 × 108 CFU/g, whereas the product should contain 2 × 107 CFU/g at the time of use. The shelf life of this product was 4 months. Seed treatment of chickpea with this product maintained the rhizosphere population of the bioagent at 11-13 × 103 CFU/g soil throughout crop. Renganathan et al. (1995) found that gypsum is a good and cheap substitute for talc. Nakkeeran and Jayrajan (1996) tested two industrial wastes, precipitated silica and calcium silicate as carrier for Trichoderma in the place of talc. The material gave a population of 0.99 and 1.04x108 CFU/g, respectively compared to 1.4 × 108 CFU/g in talc substrate after 4 months of storage. Both the substrates were much cheaper than talc. Backman and Rodriguez-Kabana (1975) used diatomaceous earth granules impregnated with 10% molasses solution for rearing T. harzianum. It was applied to peanut at 140 kg/h on 70 and 100 days after sowing to control Sclerotium rolfsii. The disease was reduced by 42% over control and yield increased by 13.5%. Several researchers have used combination of two or more agricultural materials. Elad et al. (1986) used wheat bran: saw dust : tap water mixture (3:1:4 v/v) for T. harzianum. It was applied at the time of sowing and mixed with the soil to a depth of 7-10 cm with a rotatory hoe. It increased yield of beans (15 q/h), tomato (3 q/h), cotton (5 q/h) and potato (4-6 q/h) and controlled Sclerotium rolfsii and Rhizoctonia solani. Vidhya (1995) applied the formulation of T. harzianum based on vermiculate - wheat bran (@ 250 kg/ha) to mungbean and found 41% reduction in root-rot (Macrophomina phaseolina) and 91% increase in yield. Papavizas and Lewis (1989) prepared T. virens on alginate-bran- fermenter biomass pellets and pyrax-fermenter biomass mixture. Soil application of the product checked the damping off caused by R. solani. Several other substrates such as farm yard manure (FYM), biogas plant slurry, press mud, paddy chaff, rice bran, groundnut shell (Kousalya and Jeyarajan, 1988), FYM, FYM + Sand, Saw dust, Wheat bran, pigeonpea leaves, wheat straw, and urdbean straw (Chaudhary and Prajapati, 2004) have been tested to grow T. viride and T. harzianum. The enumeration of viable CFUs revealed that pigeonpea leaves and urdbean straw were the best substrates showing 3.4 and 3.4×105 propagules at 4 months, 1.2 and 1.1 × 105 at 8 months and 1.5 and 3.0 × 104 at 12 months of storage at room temperature, while sorghum seed showed 11.4, 3.8 and 0.6 × 104

propagules at the same intervals, respectively. Next suitable substrates were wheat straw and saw dust. Cabanillas and Bakar (1989) tested some carriers like wheat grains, alginate pelletes, and diatomaceous earth granules for soil application of P. lilacinus. Kerry et al. (1984) used oat seeds to rear P. chlamydosporia for field application. Soil application of the colonized oat kernels @ 0.5 and 1.0% (w/w soil : seed) considerably reduced the population of root-knot and cyst nematodes (Godoy et al., 1983; Rodriguez-kabana et al., 1984). De Leij and Kerry (1991) did encapsulation of liquid suspension of spores and hyphae of P. chlamydosporia with sodium alginate containing 10% (w/v) kaolin or wheat bran. On soil application, the fungus proliferated in soil from only those granules which contained wheat bran as energy source. In other study, Kerry (1988) estimated approximately 9 × 104 and 4 × 104 CFUs of the fungus/g soil after 1 and 12 weeks of application of granules, respectively. Shelf life test The limiting factor in commercialization of a biocontrol agent preparation is its loss of viability of the biocontrol agents over time. Considerable efforts have been made in India itself to determine the viability of biocontrol agents in their preparations when stored at room temperature and in refrigerator. Most of the results are variable and therefore it appears that shelf life is also dependent upon species/isolate/strain. Evaluation for effectiveness In general the antagonist multiplied in an organic food base has greater shelf life than that on an inert or inorganic food base (Jeyarajan and Nakkeeran, 2000). A talc based preparation of T. virens conidia retained 82% viability at 5°C in refrigerator after 6 months while at room temperature (25-35°C) same level of viability was observed only up to 3 months. Shelf life was same, when T. virens treated chickpea or soybean seeds were stored at room temperature (Tiwari et al., 2004). Seed coating with biocontrol agents has emerged as a feasible was of delivering the antagonists, i.e. supplying the coated seeds to the farmers directly by the seed companies/agencies. The time gap between coating seeds and sowing such seeds by farmers is critical. Mukherjee (1991) quantitatively assessed the viability of T. virens on coated chickpea seeds when the seeds were stored at low temperature (5°C) and at room temperature (25-35°C), 88% of the propagules remained viable for upto 4 months. Conidia of Trichoderma in pyrophyllite survived better than fermenter biomass propagules alone at -5 to 30°C. The most suitable temperature to prolong shelf life of conidia and fermenter biomass propagules in pyrophllite were -5 to 5°C (Mukherjee, 1991). Storage at 5°C increased the shelf life of T. virens and T. hamatum

in granular formulations of pre gelatinizing starch flour upto 6 months. Chlamydospores based formulations of T. virens and T. harzianum exhibited longer shelf life (80% viability for 9 months) than conidia based formulations (80% viability after 4 months) at room temperature and a preparation of T. virens (mainly in the form of chlamydospores was from peat moss czapeks broth culture) stored at 25°C for 6 months without loss of viability (Mishra, 2002). Talc based formulations of T. harzianum can retained more than 106 viable propagules per g upto 90 days (Prasad and Rangeshwaran, 2000). Jeyarajan et al. (1994) developed talc, peat, lignite and kaolin based formulations of T. viride, which had a shelf- life of 4 months. Lewis et al. (1995) reported that among the different carriers tested; the shelf life of B. subtilis in soybean flour was increased upto three months. Ranganathan et al. (1995) also reported 4 month shelf life of T. viride in gypsum based formulations. Studies on storage temperature revealed that 20-30° C was optimum to store vermiculite fermentor biomass of Trichoderma upto 75 days without loosing the viability (Nakkeeran et al., 1997). Commercial formulations of Aspergillus niger AN27 showed an extraordinary long shelf life of more than two years at room temperature (25-35°C) when packed in polyethylene bags and stored under less than 80% relative humidity (Sen, 2000). Biosafety analysis Quality control is the most essential aspect of biopesticide production. A good quality of the preparation is necessarily required to retain the confidence of farmers on the efficacy of biocontrol formulation. Being living agents their population in a product may be influenced by storage. The other contaminating microorganisms in the product should also be within permissible limits. Registration Current legislation demands that new products are subjected to detailed study of their environment impact and toxicological effects and they are registered. As current legislation stands, there are certain categories of biocontrol agents that have an easier and quicker passage for registration. Indigenous microorganisms that are specific to a defined group of targets have a comparatively straightforward progress. Under the Section 9(3) of Pesticide Act of India (1968) information required for registration of any biopesticides includes: systematic name and common name; natural occurrence and morphological descriptions; details of manufacturing process (active and inert ingredients of formulation); Test methods (dual culture of pathogenecity); quantitative analysis (cfu on selective medium, absence of Gram negative bacteria contaminants); moisture content; shelf life; mammalian toxicity; bioefficacy; environmental toxicity and residue analysis. Because of less

awareness of growers towards biocontrol programmes, the Indian Biopesticide Industry involves more than 15-20% expenditure on marketing compared to only 1-2% marketing expenses in the case of conventional pesticides (Singhal and Sharma, 2003). So there is a need for simplification of registration requirements and government subsidies should be granted to farmers to promote biopesticide use. The registration policies may vary with the country. In USA, registration of microbial pesticide requires toxicological tests for oral, dermal, eye and other health hazards using test animals or fish. If these tests show no adverse effects and the biocontrol agent is not a pathogen, it is registered and can be sold. The cost required for research and development for biopesticide is only US$ 0.8-1.6 million as against US$ 20 millions for chemical pesticides. The toxicological tests for a biocontrol agent cost US$ 0.5 million as against US$ 10 million for chemical pesticides. The number of candidates to be tested to develop one biocontrol product will be in 100s as against 20, 000 for a chemical pesticide. It was estimated that the market size required for profit for a biocontrol agent is US$ 1.6 million per year as against US$ 4 million per year for a chemical pesticide (Cook, 1993).

Conclusion Perusal of the vast information on wilt, root-knot and fungus-nematode wilt disease complex of chickpea and tomato and critical analysis of the researches presented above have shown that the two crops which are nutritionally very important for human diet especially in the Indian dietary system, are regularly attacked by the wilt and root-knot disease and exhibit significant yield loss. Various management options are available and are applied by the growers but fail to offer satisfactory disease control. In view of adverse effects of chemicals, biocontrol agents may prove an effective substitute if applied at right time and in a proper way. Among various biocontrol agents, A. niger has demonstrated great potential for being multifarious in disease suppression and plant growth promotion. However, application of A. niger biopesticides does provide disease control and yield enhancement of varied degree. This has necessitated for search of efficient indigenous isolates of A. niger with regard to biosafety, pathogen antagonism, phosphate solubilization, compatibility with pesticides and ability to establish in soil. The present study was taken up to consider these parameters so as to efficient multifarious A. niger isolates and to develop their biopesticides to achieve ecologically sustainable and economically viable management of wilt, root-knot and wilt disease complex of chickpea and tomato.

MATERIALS AND METHODS

Isolation and identification of the wilt fungus The root and stem samples of chickpea and tomato plants showing characteristic wilt symptoms were collected from naturally infected farmer’s fields in Aligarh to isolate wilt fungus, Fusarium oxysporum (Schlechtend. Fr.) f. sp. ciceri (Padwick) Matuo & K. Sato and Fusarium oxysporum f. sp. lycopersici Snyder & Hansen from the root and stem tissue of chickpea and tomato, respectively. The samples were packed in sterilized poly bags during transport to the laboratory. The samples were cut into small pieces (2-5mm) and were surface sterilized in 2.5% NaOCl (vol/vol) for 15 sec and rinsed 2-3 times in sterile distilled water and blotted dry on a sterile paper towel. The pieces were placed aseptically to a Petri plate containing solidified potato dextrose agar (PDA) (Appendix 1) amended with 5% lactic acid (vol/vol) to inhibit bacterial growth. The inoculated plates were incubated in a BOD incubator at 25±2°C. The fungal colonies developed in the plates were examined microscopically to identify F. oxysporum f. sp. ciceri and F. oxysporum f. sp. lycopersici (Updhayay and Rai, 1992; Gillman, 2001). The fungi were subcultured on PDA slants in culture tubes. The fungi were also compared with the standard cultures procured from the Indian Agricultural Research Institute (IARI), New Delhi and Institute of Microbial Technology (IMTECH), Chandigarh. The isolates got identified from the National Bureau of Agriculturally Important Microorganisms (NBAIM), Mau, India where they have been deposited with accession no. FPFS-2213, 2214. Mass culture of the wilt fungi and pathogenicity test Pure cultures were established for pathogenicity testing by transferring stored cultures to plated PDA and incubated at 25+2°C for 5 days. The local isolates of F. oxysporum f. sp. ciceri and F. oxysporum f. sp. lycopersici were multiplied separately on sorghum seeds, which were soaked overnight in 5% sucrose and 0.0003% chloramphenicol solution (Whitehead, 1957). The seeds were transferred to conical flasks of 500 ml capacity. The flasks were autoclaved twice at 15 kg/cm2 pressure at 121°C for 15-20 minutes. Thereafter, the flasks were inoculated with the pure culture of F. oxysporum f. sp. ciceri and F. oxysporum f. sp. lycopersici incubated for 8-10 days in an incubator at 25+2°C. During incubation, the flasks were shaken manually for a few minutes daily for uniform colonization of seeds. The inoculum so prepared was incorporated in 25 cm earthen pots (diameter and height) containing autoclaved 2 kg soil (loam soil and farm yard manure, 3:1) and mixed thoroughly. Thereafter, 5-6 surface sterilized seeds of chickpea cv. BGD-72 and

74 one seedling (2 week old) of tomato cv. Pusa Ruby/pot were sown in the pots separately and five pots were maintained for each crop. The pots were irrigated with tap water regularly to maintain adequate moisture. Symptoms developed were observed 30 days after sowing. To verify Koch’s postulates, the pathogen was re-isolated from roots and/or stem of infected plants. Inoculation and pathogenicity tests were done as described above. Isolation, identification and mass culture of root-knot nematode, Meloidogyne incognita Root samples of eggplant, Solanum melongena L. showing root knots were collected from fields. The samples were brought to lab and association of Meloidogyne incognita (Kofoid and White) Chitwood was confirmed using perineal pattern technique (Barker et al., 1985). Pure culture of M. incognita was prepared by singly egg mass inoculation technique (Khan and Khan, 1991). A female along with the attached egg mass was excised from a gall. The species, M. incognita was identified on perineal pattern characters (Barker et al., 1985). Thereafter, the egg mass was placed near the roots of a seedling of eggplant cv. Pusa Purple Long grown in sterilized soil in an earthen pot. The nematode culture from this plant was raised in sterilized soil on eggplant in numerous pots. For field inoculation, the nematode culture was prepared from the egg masses excised from the eggplants maintained in pots (Fig. 16). The egg masses were placed on wire gauze in a Bergmann funnel and incubated at 25-30°C for 6-10 days. The hatched juveniles were collected from the funnel.

Fig. 16. Pure culture of root-knot nematode, Meloidogyne incognita maintained on eggplant in earthen pots.

Isolation, identification, characterization and pure culture of Aspergillus niger aggrigate isolates Extensive surveys were conducted in crop fields in forty districts of Uttar Pradesh, India in a way that the entire state was covered (Fig. 17). Soil was collected in sterilized polythene bags from different crop fields in an area. The samples were brought to the laboratory and stored inside the room away from the direct sunlight. The soil samples were processed within a couple of days using a standard serial dilution technique (Wakman, 1927) to isolate Aspergillus niger isolates. Ten gram soil was taken in 100 ml distilled water and stirred for 15 min. The suspension was left for 15 min and 1 ml was transferred to a test tube containing 9 ml distilled water. The procedure was repeated three times to obtain a dilution of 1: 10,000, which was pipetted over PDA in a Petri plate (0.3 ml / plate) under a laminar flow. Three plates were maintained for each treatment. Inoculated plates were incubated at

Fig. 17. Districts of Uttar Pradesh from Aspergillus niger aggrigate were isolated. 76

25±2°C for 5 days in a BOD incubator. After incubation, the plates were examined and isolates belonging to A. niger aggrigate were identified on the basis of cultural and morphological characters as described by Raper and Fennel (1965) and Gilman (2001). The pure culture of the isolates was prepared by inoculating culture tubes containing PDA slants with spores from an identified colony of A. niger. The tubes were incubated at 25±2°C in a BOD incubator for 5 days. The isolates were also got identified from the NBAIM, Mau, India, where they have been deposited. The details of the isolates, coding and accession number are presented in Appendix 2.

Aspergillus niger as biocontrol agent Screening against wilt fungi The A. niger aggrigate isolates were screened against the wilt fungi, F. oxysporum f. sp. ciceri and F. oxysporum f. sp. lycopersici in vitro for the following tests. Monoculture growth rates of Aspergillus niger aggrigate isolates and wilt fungi PDA discs (6 mm diameter) were cut from five days old cultures of A. niger aggrigate isolates, F. oxysporum f. sp. ciceri and F. oxysporum f. sp. lycopersici in the plates with the help of a sterilized cork borer. The discs were placed individually at the center in 9 cm Petri plates containing sterilized and solidified PDA. The plates were incubated at 25±2°C for 7 days and radial growths were measured. Dual culture test A dual culture test was conducted in vitro to assay the antagonistic ability of the isolates of A. niger aggrigate against F. oxysporum f. sp. ciceri and F. oxysporum f. sp. lycopersici (Morton and Stroube, 1955; Broadbent et al., 1971). The PDA discs (6 mm diameter) of F. oxysporum f. sp. ciceri or F. oxysporum f. sp. lycopersici and A. niger were cut from the five days old culture plates with sterilized cork borer and placed on solidified PDA 5 cm apart in the central area in Petri plates. Each plate was inoculated either with F. oxysporum f. sp. ciceri or F. oxysporum f. sp. lycopersici and A. niger. The plates were incubated at 25±2°C. The process of inoculation of fresh plates was continued for five consecutive days. The observations on the growth and ability of A. niger aggrigate to restrict and colonize F. oxysporum f. sp. ciceri and f. sp. lycopersici were recorded on 5 days after inoculation. Mycoparasitism From the zone of contact of hyphal growth of two fungi in dual culture plates, the mycelia were picked gently by a sterilized needle and put in a drop of cotton blue stain on a glass slide. The mycelial mats were then transferred to a clean slide in a drop of lactophenol. The

77 mycelium was teased by a needle, covered with a cover slip and observed under a compound microscope at 10x, 40x and 100x magnifications to determine penetration and lyses of the pathogenic mycelium by A. niger isolates. Antibiosis Antibiosis is one of the most important attribute in deciding the competitive saprophytic ability of antagonists. Antibiosis occurs when toxic metabolites or antibiotics either volatile or nonvolatile produced by one organism have a direct effect on another organism. Effect of volatile and non-volatile compounds produced by the A. niger aggrigate on F. oxysporum f. sp. ciceri and F. oxysporum f. sp. lycopersici was studied in Petri plates. Volatile compounds PDA discs (6 mm) of five days old culture of A. niger isolates were placed aseptically on solidified PDA at the center in Petri plates (Dennis and Webster, 1971a). The plates were incubated at 25±2°C for 2, 4, 8 and 10 days followed by immediate replacement of lid with a solidified freshly inoculated PDA plate with F. oxysporum f. sp. ciceri or F. oxysporum f. sp. lycopersici. A set of plates with PDA medium without A. niger isolate at the lower side and F. oxysporum f. sp. ciceri or F. oxysporum f. sp. lycopersici inoculated plates at the upper side served as control. The plates in pair were sealed together with cellophane adhesive tape and incubated for 10 days at 28±1°C. Three plates were maintained for each treatment. After incubation, the colony diameter of F. oxysporum f. sp. ciceri and F. oxysporum f. sp. lycopersici in different plates was measured and growth inhibition was calculated according the formula: I = (C – T)/C × 100 Where, I = Percent growth inhibition; C = Radial growth in control (mm); T = Radial growth in treated plates (mm). Non-volatile compounds Conical flasks containing 150 ml potato dextrose broth were inoculated with 6 mm discs of 5 days old culture of A. niger isolates separately, and were placed on an electric shaker on 150 rpm inside an incubator at 27°C for 15 days. Thereafter the broth was filtered through Whatman No. 42 filter paper under aseptic conditions and the filtrate was collected in sterilized Erlenmeyer flasks. The culture filtrates thus obtained, was centrifuged at 6000 rpm for 15 minutes to make it cell free. Fungicidal effect The cell free filtrate of A. niger aggrigate isolates was added to double strength PDA (40°C) to obtain final concentration of 10%, 25% and 50% (vol/vol). To obtain these 78 concentrations, 10, 25 and 50 ml cell free culture filtrates (CFCF) of the isolates were mixed with 90, 75 and 50 ml of double strength PDA, respectively. Twenty ml of this medium was poured into sterilized Petri plates. After solidification, separate plates were centrally seeded with 6 mm discs of the F. oxysporum f. sp. ciceri and F. oxysporum f. sp. lycopersici from 5 days old PDA culture and were incubated in an incubator at 25±2°C for 10 days. A set of plates inoculated with the test pathogen not amended with the CFCF served as a check. Three replicates for each treatment were maintained. Observations on radial growth of mycelium were recorded periodically (Dennis and Webster, 1971b) and percent inhibition of colonization by F. oxysporum f. sp. ciceri and F. oxysporum f. sp. lycopersici was determined by the formula described above. Nematicidal effect The effect of cell free culture filtrates (CFCF) of the A. niger isolates were also tested for nematicidal effect on hatching and mortality of Meloidogyne incognita. Five ml CFCF was transferred to a glass cavity block to which 10 surface sterilized egg masses of relatively same size were added. The egg masses were surface sterilized by immersing in 0.5% NaOCl for 2-3 minutes. Thereafter, the masses were rinsed several times with distilled water on a sterilized 60-mesh sieve (250 µm mesh diameter) to remove the residues of NaOCl. Two sets of blocks in which egg masses were immersed in distilled water and broth alone (uninoculated) served as control. The cavity blocks were incubated at 25-27°C for 5 days in an incubator. To avoid evaporation during incubation, the blocks were placed inside Petri plates containing 10 ml distilled water and covered with their lids. Three cavity blocks were maintained for each treatment and the experiment was conducted three times. After incubation, larvae present in the suspension were counted. To examine the effect on mortality of juveniles, four concentrations of CFCF viz., 25, 50, 75 and 100% were tested. Four ml CFCF was placed in a glass cavity slide to which 1 ml suspension containing approximately 100 freshly hatched and surface sterilized juveniles (J2) of M. incognita were added. The juveniles were surface sterilized by immersing in 0.5% NaOCl for 2-3 minutes and were immediately rinsed several times with distilled water on a sterilized 500 mesh sieve (24 µm mesh diameter) to remove the residues of NaOCl. Juveniles kept in the broth alone (without A. niger) and in distilled water served as control. Each treatment was replicated three times. The slides were placed on glass supports in Petri plates containing distilled water and covered with a lid having water soaked blotter paper and incubated at 25-27°C for 2 days. After incubation, numbers of

79 dead juveniles characterized by the relaxed and immobile body posture were counted. The experiment was repeated three times. Compatibility of the biocontrol agents with pesticides Seven pesticides viz., carbendazim (Bavistin 50 WP), captan (Captaf 50 WP), mancozeb (Dithane M-45 75 WP), metalaxyl (Apron 35 SD), thiram (TMTD 75 WP), carbofuran (Furadan 3G) and nemacur (Fenamiphos) were tested against the A. niger isolates using poisoned food technique (Grover and Moore, 1961). Double strength PDA (50 ml) was taken in an Erlenmeyer flasks of 250 ml capacity and sterilized in an autoclave at 15 kg/m2 for 15-20 min. Different concentrations of pesticides viz., 10, 25, 50, 125, 250,500,1000, 2000, 3000 and 5000 µg/ml were prepared in distilled water. Fifty ml of a concentration was aseptically transferred to the Erlenmeyer flask containing 50 ml liquefied PDA. Three Petri plates (90 mm diameter) for each concentration of the fungicides were prepared by pouring 20 ml PDA aliquots in each plate and allowed to solidify. Thereafter the plates were seeded centrally with a 3 mm disc of 5 days old culture of A. niger isolates. PDA plates without a fungicide but inoculated with the fungi served as control. The inoculated plates were incubated at 25±2°C for 5 days. The radial growth of the colony in each treatment was measured and the percent inhibition of growth was calculated by the formula, and ED90 (maximum inhibition concentration) and ED50 (safe tolerance concentration) were determined. I = (C – T)/C × 100

Biosorption of toxic heavy metals by Aspergillus niger aggrigate isolates To determine the biosorption of toxic heavy metals in single and multiple metal systems by the A. niger isolates, following tests were conducted. Determination of minimum inhibitory concentration (MIC) Minimum inhibitory concentration (MIC) is defined as the minimum concentration of a substance that inhibits the visible growth of a microorganism. Sensitivity of A. niger isolates against three heavy metals viz., Ni2+, Cr6+ and Cd2+ was determined by using spot plate method (Volesky, 1990). Sabouraud dextrose agar (SDA) medium (Hi-Media Lab Ltd. Mumbai) containing Ni2+, Cr6+ and Cd2+ at different concentrations of 25, 50, 75, 100, 125, 150, 175, 200, 225, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475 and 500 µg/ml was prepared and poured in Petri plates. Plates were inoculated with 1 ml inoculum of A. niger (106 CFU/ml). Plates without metal served as control and three plates were maintained for

80 each treatment. The plates were incubated at 25±2°C for 5 days to observe the growth of fungi on the spotted area. Metal biosorption assay To determine the heavy metals biosorption by A. niger isolates following standard procedure as described by Yan and Viraraghvan (2000) was adapted. 1. Preparation of Aspergillus niger biomass: Culture of A. niger isolates (6-7 days old) maintained on PDA slant at 25±2°C was to prepare spores of suspension (spores suspension 1×105 CFU/ml) which was used to inoculate in yeast peptone glucose broth (YPGB) (Appendix 3). To get the required concentration (1×105 CFU/ml) of A. niger, spores from the PDA slant were taken in distilled water and counted in Haemocytometer. The fungus was grown at 23°C in the YPGB in a conical flask kept on a rotary shaker agitated at 125 rpm. The mycelial mass was harvested after 3-4 days of growth by filtering through sieve (150 µm mesh diameter). The harvested mycelial biomass was washed with generous amount of de-ionized water. The live fungal biomass so obtained was stored at -20°C or used immediately. 2. Preparation of Aspergillus niger biomass powder: Live harvested mycelial biomass (50 g) was treated with 0.5N NaOH for 30 min, followed by washing with adequate amount of distilled water until the pH of the solution reached to neutral range (pH 6.8-7.2) and autoclaved at 15 kg/cm2 for 20 min. The pretreated biomass was dried at 60°C for 24 hr in hot-air oven and converted into powder form by grinding in mortar and pestle. 3. Bioadsorption and analysis: Bioadsorption experiment was conducted for two metal systems, single and multiple. For single metal system separate solutions of four concentrations viz. 2, 4 and 6 mM concentration were prepared containing Ni2+, Cd2+ and 6+ Cr in the form of NiCl2, CdCl2.H2O and K2Cr2O7, respectively. For multiple metal system a single solution of all the metals with the same concentration as in single metal system was prepared. The solutions were maintained at pH 5. The dried and powdered dead biomass of A. niger isolates (100 mg) was added to 250 ml capacity flask containing 100 ml of each single and multi metals solution. The flasks were kept on a rotary shaker for 18 h at 30°C and at 125 rpm. Thereafter, the solution along with powdered biomass of A. niger was centrifuged at 10,000 rpm for 15 min and supernatant was collected. Content of the supernatant (1 g) was dissolved in aquaregia (2 ml of 65% HNO3 + 6 ml of HCL) and digested on a hot plate until dense fume evolved and the liquid was largely volatilized and a clear solution was obtained. The clear solution was filtered through Whatman filter paper (No. 42). The filtrate was analyzed for the various metals after proper dilution in atomic 81 absorption spectrophotometer (AAS: M/S GBC Scientific Equipment Pvt. Ltd., Dandenong Victoria, Australia). Bioadsorption experiments were carried out in three replicates and average values were used in the analysis. Bioadsorption capacity i.e., amount of metal ions (mg) bioadsorbed/g of dried biomass was calculated using the following equation: Q = (Ci – Cf)/m × V Where, Q = mg of metal ion bioadsorbed/g of biomass; Ci = initial metal ion concentration mg/l; Cf = final metal ion concentration mg/l; m = mass (g) of biomass in the reaction mixture; V = volume (l) of the reaction mixture.

Biochemical characterization of Aspergillus niger aggrigate Soil isolates of A. niger aggrigate were characterized in-vitro with the following tests to identify isolates with greater effectiveness for mycotoxin production, fungus suppression and phosphate solubilization. i. Ochratoxin A analysis and production ii. Ammonification iii. Hydrogen sulphide production iv. HCN production v. Siderophore production vi. IAA production vii. Phosphate solubilization i. Ochratoxin A analysis and production Ochratoxin A (OTA) was detected in the samples by high-performance liquid chromatography (HPLC), following the method of Scudamore and MacDonald (1998) with some modifications. Fifty grams of a finely grounded A. niger isolate sample was added to a 250 ml Erlenmeyer flask containing a 100 ml mixture of methanol: water (9:1). The mixture was shaken for 30 min and filtered to remove any particulate matter. A 10 ml aliquot of the above extract was mixed with 40 ml of distilled water and filtered through a micro fiber filter. Ten ml of the filtered sample was taken and added to an immunoaffinity column (Ochra Test TM, Vicam, Digen Ltd. Oxford, UK). The column was washed with 10 ml phosphate buffer solution (PBS) containing 0.01% Tween 20, and then with 10 ml double distilled water. Ochratoxin A was eluted from the column with methanol (HPLC grade), again at a flow rate of 1–2 drops per second.

The HPLC apparatus (Hewlett Packard, Palo Alto, CA, USA) used to determine OTA and was equipped with a spectrofluorescence detector (excitation, 330 nm; emission, 460 nm) and a C18 column (Supelcosil LC-ABZ, Supelco; 150×4.6 mm, 5 µm particle size), connected to a precolumn (Supelguard LC-ABZ, Supelco; 20×4.6 mm, 5 µm particle size). The mobile phase was pumped at 1.0 ml/min and consisted of an isocratic system as follows: 57% acetonitrile, 41% water and 2% acetic acid. Ochratoxin A was quantified on the basis of the HPLC fluorometric response compared with the OTA standard (Sigma Aldrich Co., St. Louis, MO, USA, purity > 99%). The lowest limit of detection was 1 ng/g . Each sample was analyzed three times. Ochratoxin A production was tested on all the 16 selected isolates of A. niger aggrigate. Ochratoxin A was determined using the method of Teren et al. (1996) with some modifications as follows: the isolates were grown in stationary cultures in 25 ml quantities of yeast extract sucrose medium (2% yeast extract, 15% sucrose) at 30°C for 10 days in the dark. After incubation, a portion (1 ml) of each culture medium was mixed with 1 ml of chloroform and centrifuged at 4000 g for 10 min. The chloroform phase was transferred to a clean tube, evaporated to dryness and re-dissolved in 0.5 ml of methanol. The OTA was quantified as described previously. ii. Ammonification Organic nitrogen compounds are subject to dissimilation by a wide variety of heterotrophic micro-organisms to yield ammonia and other end-products. The test for production of ammonia was detected by using the standard method of Deshmukh (1997). A peptone broth containing an organic nitrogen substrate was used to test the ability of A. niger aggrigate isolates to degrade proteins, causing ammonia to form. After incubation, the presence of ammonia, indicative of ammonification, was detected by the yellow color when Nesler’s reagent was added to samples of test cultures. Peptone broth (4%) (Appendix 4) was taken in culture tubes and autoclaved at 15 kg/cm2 pressure at 121°C for 15 to 20 min. The tubes were inoculated with a loopful of culture of A. niger isolates and incubated at 25±2°C for 7 days. Thereafter 1ml Nessler’s reagent (Hi-Media India) was added in each tube, change of color to yellow was indicative for positive for presence of ammonia. Deep yellow to brownish color was indicative of maximum production of ammonia. iii. Hydrogen sulphide production Test for the production of hydrogen sulphide was performed using the standard method (Deshmukh, 1997). Isolates of A. niger were inoculated in the culture tubes containing peptone water. Whatman filter paper no.1 impregnated in saturated solution of lead acetate 83 was kept hanging in the inoculated tube of peptone water with the support of cotton plugs and the tubes were incubated at 25±2oC in a BOD incubator for one week. A change in color of the filter paper from white to black was indicative of positive test for production of

H2S. iv. HCN production The test for production of hydrogen cyanide (HCN) was performed using the method of Bakker and Schipper (1987). Isolates of A. niger were inoculated in Petri plates containing sabarose dextrose agar (SDA medium with 4.4 g/l glycine; Appendix 5). A Whatman filter paper no. 2 soaked in alkaline picric acid solution was placed in the lid of each plate. The plates were incubated in an incubator at 25±2°C for 7 days and change in color of the filter paper from yellow to orange brown was recorded as positive for HCN. v. Siderophore production Siderophore production was detected using the chrome azurol assay (CAS) (Schwyn and Neilands, 1987). The medium contains iron-CAS-HDTMA (Hexadecyltrimethylammonium bromide) complex which is blue coloured. The presence of iron chelator (siderophore) is indicated by the decolorization of the blue-coloured ferric-dye complex, resulting in a yellow halo around the microbial colony. Contamination of iron is avoided by treating the chemicals and glassware for removal of iron (deferration). For removal of iron from the glass surfaces all glassware was soaked in 2N HCl for at least 24 hours and held with double distilled water to remove the acid. Removal of contaminating iron from media components like sucrose, casamino acids, MgSO4.7H2O, CaCl2. 2H2O was achieved by extraction with 3% (w/w) 8-Hydroxyl quinoline in chloroform twice. Fresh culture of A. niger isolates were inoculated on CAS medium and incubated at 25±2oC for 5 days. Occurrence of yellow halo around the fungus colony was indicative of positive test otherwise negative for siderophore production. vi. Indole acetic acid (IAA) production The IAA test was done using the standard method (Gordon and Weber, 1951); Brick et al., 1991). A loopful of test organism was inoculated in 10 ml of Luria Bertani broth (LBB; Appendix 6) amended with 35 mg tryptophan/100 ml, and was incubated at 25±2oC for 4 days on a rotary shaker. The broth was centrifuged at 10,000 rpm for 15 min, 2 ml of the supernatant was taken to which 2-3 drops of O-phosphoric acid was added. Four ml of

Ferric chloride-perchloric acid (FeCl3-HClO4) reagent was added to the aliquot. The samples were incubated for 25 min at room temperature to observe the change in color to pink was indicative of positive test. 84 vii. Phosphate solubilization A loopful of A. niger aggrigate was aseptically inoculated on solidified Pikovskaya’s agar medium (Pikovskaya, 1948) (Appendix 7) in the centre of the plates. The plates were incubated at 25±2°C for 5–7 days. Colonies of the isolates of A. niger aggrigate showing a clearing or solubilization zone were considered phosphate solubilizers. Quantitative estimation of phosphate solubilization in liquid medium Test organisms were cultured in 100 ml Pikovskaya’s liquid medium for 5 days at 25±2°C. The culture broth was centrifuged at 15,000 rpm for 30 minutes. Two ml supernatant was taken in a culture tube to which 10 ml chloromolybydic acid (Appendix 8) was added. The mixture was shaken thoroughly and diluted with distilled water to 45 ml. Thereafter 0.3 ml chlorostannous acid (Appendix 9) was added and the final volume was made to 50 ml. After 10 minutes, the % transmittance of resultant blue colour was measured at 430 nm in a spectrophotometer (SHIMADZU, 2450 PC, Japan). Quantification values were recorded from a standard curve prepared with various phosphorus concentrations (Appendix 10). Molecular characterization of efficient Aspergillus niger isolates An additional PCR-RAPD test has been performed for the identification of efficient A. niger isolates. To identify A. niger isolate through specific primer and to distinguish them amont the isolates, Rapid Amplified Polymorphic DNA (RAPD) profiles were prepared (Hardys et al., 1992) through following protocol. . Isolation of genomic DNA Genomic DNA from each soil isolate of A. niger was isolated following modified protocol of George et al. (1993). The vacuum dried fungus mycelium (3 g fresh or frozen) was crushed in 15 ml of Grinding Buffer (Appendix 15) using mortar and pestle. The resultant paste was transferred to sterile centrifuge tubes using sterile spatula. The tube was incubated in water bath at 60-65°C for one hour with gentle inversion at about every 15 min. Thereafter, 3 ml of 10 M ammonium acetate was added in to the tube and kept for another 30 min at 65°C followed by centrifugation at 10,000 rpm for 10 min. The supernatant was transferred to another tube to which an equal volume of chilled isopropanol was added and kept at -20°C for 60 min or 0°C over night. DNA was pelleted out by centrifugation (6,000 rpm for 25 min or 10,000 rpm for 15 min at 4°C), washed twice with 70% ethanol and dissolved in T10E1 buffer (Appendix 16). Dissolved DNA solution was extracted with chloroform: iso-amyl alcohol (24:1) and RNA was removed by RNAse (enzyme) treatment (@ 4 µl/ml of supernatant from stock of 10 mg/ml of RNAse) at 37°C for one hour. RNAse treated DNA was further extracted twice with chloroform: iso-amyl alcohol (24:1) for 85 further purification. DNA was re-precipitated in chilled ethanol (100%) and dissolved in

T10E1 buffer. Purified DNA was checked for its quality and quantity by 0.8% agarose gel electrophoresis using uncut lambda (λ) DNA as standard (300 ηg/µl). Dilution of the DNA solution was done using T10E1 buffer to a concentration of approximately 25 ηg/2µl for use in PCR analysis. DNA amplification by PCR PCR amplification was carried out in 0.2 ml thin-wall PCR tubes using a Whatman Biometra (model T1 Thermocycler, Germany) thermal cycler. A total of 46 RAPD primers were screened in the study. Twenty primers were Operon series A (Operon Technologies, Almeda, CA, USA) and twenty six primers were Fusarium specific (Appendix 17) custom synthesized from Genetix Biotech Asia Pvt. Ltd., India. Polymerase chain reaction (PCR) mixture of 25 µl contained 25 ηg of genomic DNA templates, 0.6 U of Taq DNA polymerase (Bangalore Genei, Bangalore, India), 0.3 µM of decamer primer, 2.5 µl of 10X

PCR assay buffer (50 mM KCl, 10 mM Tris-Cl, 1.5 mM MgCl2) and 0.25 µl of pooled dNTPs (100 mM each of dATP, dCTP, dGTP and dTTP from Fermentas Life Sciences, USA). PCR cycle conditions were as follows: initial denaturating step at 94˚C for 3 min followed by 44 cycles of 92°C for 1 min, 37°C for 1 min and 72°C for 2 min. In the last cycle, primer extension at 72°C for 7 min was provided. Documentation of gel and data analysis PCR products were electrophoretically separated on a 1.5% agarose gel containing ethidium bromide [@ 1 µl (10 mg/ml) per 250 ml agarose solution] using 1X TBE buffer (pH 8.0) and 60 V current was run for 3 h. The amplified product were visualized and photographed under UV light source. O’ Gene RullerTM 100 bp DNA Ladder Plus (Fermentas Life Sciences, USA) was used as molecular weight marker. DNA bands were scored ‘1’ for its presence and ‘0’ for its absence for each primer- genotype combination. The binary data matrix was then utilized to generate genetic similarity data among genotypes. Only unambiguous bands were scored for the estimation of genetic similarity between the isolates using Jaccard’s similarity coefficient. Based on the data dendrograms were generated by using the SAHN clustering programme selecting the unweighted pair group method with arithmetic mean (UPGMA) algorithm (Nei and Li, 1979) in NTSYS-pc (Rohlf, 1992). Support for clusters was evaluated by bootstrap analysis with Win Boot software (Yap and Nelson, 1995). One thousand (1000) samples were generated by re-sampling with replacement of characters with in the combined 1/0 data matrix. 86

Mass culture of efficient isolates of Aspergillus niger Bagasse-soil mixture (BSM) was used to prepare mass culture of efficient isolates of A. niger. Coarse powder of bagasse and field soil (loam) were mixed in the ratio of 4:1 and filled in conical flasks of 500 ml capacity. The flasks were sealed with cotton plugs and butter paper and autoclaved twice at 15 kg/cm2 pressure at 121°C for 15-20 minutes. The flasks were inoculated with efficient isolates of A. niger separately and incubated in a BOD incubator at 25±2°C for 8-10 days. For large-scale production, BSM medium was filled in heat resistant plastic bags and autoclaved twice at 15 kg/cm2 pressure at 121°C for 15-20 minutes. The medium in polybags was inoculated with the liquid culture of A. niger isolates prepared on the potato dextrose broth in conical flasks with the help of sterilized pipette. After inoculation, the bags were sealed, shaken and kept in an incubator at 27±2°C for 8-10 days and shaken periodically. During this period the BSM was fully colonized by the A. niger isolates and ready for field application. Before application, colony forming unit (CFU) of the fungus in the BSM was determined using dilution plate method.

Pot culture experiment to test effectiveness of Aspergillus niger isolates against the wilt, root-knot and wilt disease complex Effectiveness of 16 isolates of A. niger selected on the basis of in vitro tests was tested against wilt (F. oxysporum f. sp ciceri and F. oxysporum f. sp. lycopersici), root-knot (M. incognita) and fungus-nematode wilt disease complex (F. oxysporum f. sp ciceri + M. incognita / F. oxysporum f. sp. lycopersici + M. incognita) of chickpea and tomato was evaluated in pot culture experiment. Crop culture Chickpea: Certified seeds of chickpea, Cicer arietinum (L) cv. BGD-72 were procured from an authorized dealer. The BGD-72 matures in 114-124 days. The mycoflora examination of seeds (external and internal) through blotter paper test (Tempe, 1970) revealed absence of F. oxysporum f. sp. ciceri or other potential pathogenic fungi on the seeds. The seeds were sown in pots (5 seeds/pot) or in rows in microplots (57 seeds/row, 4 rows/microplot) where antagonists had already been applied. One week after sowing irrigation was done, and the plants were allowed to grow for four months. In pots, one week old seedlings were thinned to one/pot. During this period they were regularly observed for any symptom. Tomato: Certified seeds of tomato, Lycopersicon esculentum Mill. cv. Pusa Ruby were procured from an authorized dealer. The mycoflora examination of seeds through above

87 method (Tempe, 1970) revealed absence of F. oxysporum f. sp. lycopersici or other potential pathogenic fungi on the seeds. The seeds were sown in raised bed and watered daily or after one day as per requirement. Five leaves stage seedlings of tomato (two weeks old) were transplanted at the center of the pots where antagonists had already been applied. Seedlings were irrigated with tap water immediately after transplanting and it continued as per requirement till harvest. In field, seedlings were transplanted in rows in microplots (12 seedlings/row, 4 rows/microplot) where antagonists had already been applied. Just after transplanting irrigation was done to establish the seedlings and allowed to grow for four months. During the experimental period, plants were regularly observed for any visible symptom. Inoculum and dose of pathogens Sorghum seeds colonized by F. oxysporum f. sp. ciceri and F. oxysporum f. sp. lycopersici were grinded separately with known volume of distilled water in an electric grinder. The suspension containing 2 g fungus colonized seeds (Table 16) was mixed in 1 kg soil filled in a pot. For field trials, the fungus was applied @ 2 g/kg soil by mixing in 10 cm × 10 cm top layer of soil. Weight of the top soil to 10 cm depth in a microplot of 2 m × 4 m was estimated as 355 kg. Hence, the fungus suspension containing 710 g colonized seeds grinded in 10 liter tap water was sprinkled and mixed in the top soil in a microplot to achieve uniform distribution of the pathogen. The inoculation was done two days prior to seed sowing or nursery transplanting. The colony forming unit load of F. oxysporum f. sp. ciceri and F. oxysporum f. sp. lycopersici/g seed was estimated using the dilution plate method and is given in Table 16. Root-knot nematode, Meloidogyne incognita: Nematode inoculation was done @ 2,000 second stage juveniles/kg soil in both pots and field trials. Ten liters water containing 710000 second stage juveniles of M. incognita was added to the top soil in a microplot The amount of inoculum level was determined considering 355 kg soil up to 10 cm depth per microplot (2 × 4 m2). The inoculation was done two days before the F. oxysporum f. sp. ciceri or F. oxysporum f. sp. lycopersici inoculation. Dose of Aspergillus niger isolates In pot study, 1 g of A. niger cultured on sorghum seeds (Table 16) was applied to a pot filled with 1 kg sterilized soil. Whereas, in field trial, soil application of the A. niger isolates or its biopesticides was done @ 40 g material/microplot at the time of seed sowing or nursery plantation. The application was done in rows where the seeds/nurseries were to be sown. For seed treatment, the dose was 4 g BSM or biopesticide/kg seeds of chickpea that 88

Table 16. Colony forming unit load of inocula of Fusarium spp., Rhizobium and Aspergillus niger isolates at the time of application. Pot Expt. Field Expt. I Field Expt. II Chickpea Soil application: F. oxy f. sp. ciceri 22×108 CFUs/g SS 20×108 CFUs/g SS 21×108 CFUs/g SS Rhizobium 25×108 CFUs/g F 25×108 CFUs/g F 26×108 CFUs/g F AAn1 2×109 CFUs/g SS - - ANAn1 1×109 CFUs/g SS - - ANAn4 11×109 CFUs/g SS 12×109 CFUs/g BSM - AnC2 12×109 CFUs/g SS 15×109 CFUs/g BSM 21×109 CFUs/g BP AnR3 12×109 CFUs/g SS 13×109 CFUs/g BSM - BAn4 4×109 CFUs/g SS - - BasAn5 6×109 CFUs/g SS - - BuAn3 10×109 CFUs/g SS 11×109 CFUs/g BSM - BudAn3 6×109 CFUs/g SS - - GaAn1 9×109 CFUs/g SS - - JaAn2 8×109 CFUs/g SS - - LAn3 2×109 CFUs/g SS - - MeAn4 4×109 CFUs/g SS - - SkNAn3 9×109 CFUs/g SS - - SkNAn5 20×109 CFUs/g SS 21×109 CFUs/g BSM 25×109 CFUs/ BP VAn4 16×109 CFUs/g SS 18×109 CFUs/g BSM 22×109 CFUs/ BP Seed treatment: AAn1 2×106 CFUs/ seed - - ANAn1 1×106 CFUs/ seed - - ANAn4 11×106 CFUs/ seed 13×106 CFUs/ seed - AnC2 13×106 CFUs/ seed 16×106 CFUs/ seed 22×106 CFUs/ seed AnR3 13×106 CFUs/ seed 14×106 CFUs/ seed - BAn4 4×106 CFUs/ seed - - BasAn5 6×106 CFUs/ seed - - BuAn3 11×106 CFUs/ seed 12×106 CFUs/ seed - BudAn3 6×106 CFUs/ seed - - GaAn1 10×106 CFUs/ seed - - JaAn2 9×106 CFUs/ seed - - LAn3 2×106 CFUs/ seed - - MeAn4 4×106 CFUs/ seed - - SkNAn3 10×106 CFUs/ seed - - SkNAn5 22×106 CFUs/ seed 23×106 CFUs/ seed 27×106 CFUs/ seed VAn4 17×106 CFUs/ seed 19×106 CFUs/ seed 24×106 CFUs/ seed Tomato Soil application: F. oxy f. sp. 22×108 CFUs/g SS 21×108 CFUs/g SS 22×108 CFUs/g SS lycopersici AAn1 2×109 CFUs/g SS - - ANAn1 1×109 CFUs/g SS - - ANAn4 11×109 CFUs/g SS 12×109 CFUs/g BSM - AnC2 12×109 CFUs/g SS 15×109 CFUs/g BSM 21×109 CFUs/g BP AnR3 12×109 CFUs/g SS 13×109 CFUs/g BSM - BAn4 4×109 CFUs/g SS - -

Pot Expt. Field Expt. I Field Expt. II BasAn5 6×109 CFUs/g SS - - BuAn3 10×109 CFUs/g SS 11×109 CFUs/g BSM - BudAn3 6×109 CFUs/g SS - - GaAn1 9×109 CFUs/g SS - - JaAn2 8×109 CFUs/g SS - - LAn3 2×109 CFUs/g SS - - MeAn4 4×109 CFUs/g SS - - SkNAn3 9×109 CFUs/g SS - - SkNAn5 20×109 CFUs/g SS 21×109 CFUs/g BSM 25×109 CFUs/ nursery VAn4 16×109 CFUs/g SS 18×109 CFUs/g BSM 22×109 CFUs/g BP Nursery application: AAn1 1×109 CFUs/ nursery - - ANAn1 5×108 CFUs/ nursery - - ANAn4 5×109 CFUs/ nursery 6×109 CFUs/ nursery - AnC2 6×109 CFUs/ nursery 8×109 CFUs/ nursery 10×109 CFUs/ nursery AnR3 6×109 CFUs/ nursery 7×109 CFUs/ nursery - BAn4 2×109 CFUs/ nursery - - BasAn5 3×109 CFUs/ nursery - - BuAn3 5×109 CFUs/ nursery 6×109 CFUs/ nursery - BudAn3 3×109 CFUs/ nursery - - GaAn1 5×109 CFUs/ nursery - - JaAn2 4×109 CFUs/ nursery - - LAn3 1×109 CFUs/ nursery - - MeAn4 2×109 CFUs/ nursery - - SkNAn3 5×109 CFUs/ nursery - - SkNAn5 10×109 CFUs/ 11×109 CFUs/ 13×109 CFUs/ nursery nursery nursery VAn4 8×109 CFUs/ nursery 9×109 CFUs/ nursery 11×109 CFUs/ nursery SS, fungus colonized sorghum seeds; F, formulation; BSM, bagasse-soil mixture (4:1); BP, biopesticide. was applied to seeds along with the commercial Rhizobium of chickpea strain (25×108 CFUs/g formulation). In case of tomato, root-dip treatment with A. niger isolates (10 g/100 seedlings) was done whereas seed treatment was not done. Spore suspension of A. niger was made by grinding 10 g of colonized seeds or the biopesticides in 100 ml distilled water. The roots of tomato seedlings were dipped in the suspension for 10 minutes (Table 16). Dose of pesticides Carbendazim (Bavistin 50 WP) as a fungicide and carbofuran (Furadan 3G) as nematicide were applied in the experiments to compare efficacy of A. niger isolates.

Fungicide: Carbendazim (Bavistin 50 WP) was applied in pots @ 0.25 g a.i./pot whereas in microplots, the dose was @ 2.5 kg a.i./h (2 g a.i./microplot) which was applied in a broadcast manner a week after the seed sowing or nursery transplanting. Broadcast of the fungicide was done after mixing the chemical with small amount of fly ash (to increase the volume) and for seed treatment the fungicide was applied @ 2g a.i./kg seeds. For root- dip treatment seedlings were immersed in 100 ppm solution of the fungicide for 15 minutes before transplanting. Nematicide: Soil application of carbofuran (Furadan 3G) in pots was done at 0.1 g a.i./pot (1 kg soil). Application of the nematicide in microplots was done @ 2 kg a.i./h (1.6 g a.i./microplot), and for seed treatment the doze was 2 g a.i./kg seed. To prepare a combined treatment of fungicide and nematicide half dose of carbendazim was mixed with the half dose of carbofuran to get the equal dose to other treatments. In root-dip treatment, seedlings were dipped in 200 ppm solution of carbofuran for 15 minutes before transplanting. Treatment and plant culture Clay pots (15 cm diameter and height) were filled with steam-sterilized soil amended with compost (3:1), and following 73 treatments each for chickpea and tomato for soil application and seed treatment were incorporated. Three replicates were maintained for all treatments. Additional 9 pots per treatment were taken to estimate monthly soil population of root-knot nematode and fungi and three pots per treatment for salicylic acid (SA) estimation of shoots and roots. The pots were arranged in a completely randomized design in an open space receiving uniform sunlight and irrigated with tap water on alternate day (250 ml/pot). 01. Plant (Control) 02. Plant + AnC2 03. Plant + AnR3 04. Plant + AAn1 05. Plant + BAn4 06. Plant + BasAn5 07. Plant + BudAn3 08. Plant + GaAn1 09. Plant + JaAn2 10. Plant + LAn3 11. Plant + MeAn4 12. Plant + SkNAn3 13. Plant + SkNAn5 14. Plant + VAn4 15. Plant + BuAn3 16. Plant + ANAn1 91

17. Plant + ANAn4 18. Plant + Carbendazim 19. Plant + Carbofuran 20. Plant + Fusarium oxysporum f. sp. ciceri / lycopersici (Control) 21. Plant + F. oxysporum f. sp. ciceri / lycopersici + AnC2 22. Plant + F. oxysporum f. sp. ciceri / lycopersici + AnR3 23. Plant + F. oxysporum f. sp. ciceri / lycopersici + AAn1 24. Plant + F. oxysporum f. sp. ciceri / lycopersici + BAn4 25. Plant + F. oxysporum f. sp. ciceri / lycopersici + BasAn5 26. Plant + F. oxysporum f. sp. ciceri / lycopersici + BudAn3 27. Plant + F. oxysporum f. sp. ciceri / lycopersici + GaAn1 28. Plant + F. oxysporum f. sp. ciceri / lycopersici + JaAn2 29. Plant + F. oxysporum f. sp. ciceri / lycopersici + LAn3 30. Plant + F. oxysporum f. sp. ciceri / lycopersici + MeAn4 31. Plant + F. oxysporum f. sp. ciceri / lycopersici + SkNAn3 32. Plant + F. oxysporum f. sp. ciceri / lycopersici + SkNAn5 33. Plant + F. oxysporum f. sp. ciceri / lycopersici + VAn4 34. Plant + F. oxysporum f. sp. ciceri / lycopersici + BuAn3 35. Plant + F. oxysporum f. sp. ciceri / lycopersici + ANAn1 36. Plant + F. oxysporum f. sp. ciceri / lycopersici + ANAn4 37. Plant + F. oxysporum f. sp. ciceri / lycopersici + Carbendazim 38. Plant + Meloidogyne incognita (Control) 39. Plant + M. incognita + AnC2 40. Plant + M. incognita + AnR3 41. Plant + M. incognita + AAn1 42. Plant + M. incognita + BAn4 43. Plant + M. incognita + BasAn5 44. Plant + M. incognita + BudAn3 45. Plant + M. incognita + GaAn1 46. Plant + M. incognita + JaAn2 47. Plant + M. incognita + LAn3 48. Plant + M. incognita + MeAn4 49. Plant + M. incognita + SkNAn3 50. Plant + M. incognita + SkNAn5 51. Plant + M. incognita + VAn4 52. Plant + M. incognita + BuAn3 53. Plant + M. incognita + ANAn1 54. Plant + M. incognita + ANAn4 55. Plant + M. incognita + Carbofuran 56. Plant + M. incognita + F. oxysporum f. sp. ciceri / lycopersici (Control) 57. Plant + M. incognita + F. oxysporum f. sp. ciceri / lycopersici + AnC2 58. Plant + M. incognita + F. oxysporum f. sp. ciceri / lycopersici + AnR3 59. Plant + M. incognita + F. oxysporum f. sp. ciceri / lycopersici + AAn1 60. Plant + M. incognita + F. oxysporum f. sp. ciceri / lycopersici + BAn4 61. Plant + M. incognita + F. oxysporum f. sp. ciceri / lycopersici + BasAn5 62. Plant + M. incognita + F. oxysporum f. sp. ciceri / lycopersici + BudAn3 63. Plant + M. incognita + F. oxysporum f. sp. ciceri / lycopersici + GaAn1 64. Plant + M. incognita + F. oxysporum f. sp. ciceri / lycopersici + JaAn2 65. Plant + M. incognita + F. oxysporum f. sp. ciceri / lycopersici + LAn3 66. Plant + M. incognita + F. oxysporum f. sp. ciceri / lycopersici + MeAn4

67. Plant + M. incognita + F. oxysporum f. sp. ciceri / lycopersici + SkNAn3 68. Plant + M. incognita + F. oxysporum f. sp. ciceri / lycopersici + SkNAn5 69. Plant + M. incognita + F. oxysporum f. sp. ciceri / lycopersici + VAn4 70. Plant + M. incognita + F. oxysporum f. sp. ciceri / lycopersici + BuAn3 71. Plant + M. incognita + F. oxysporum f. sp. ciceri / lycopersici + ANAn1 72. Plant + M. incognita + F. oxysporum f. sp. ciceri / lycopersici + ANAn4 73. Plant + M. incognita + F. oxysporum f. sp. ciceri / lycopersici + Carbendazim +Carbofuran

Field trial Based on the relative performance of 16 isolates of A. niger against the wilt and root-knot tested in pots, six efficient isolates viz., AnC2, AnR3, SkNAn5, Van4, BuAn3 and ANAn4 were selected for evaluation against the target diseases under field. The experiments were conducted during November, 2007 - March, 2008 in a field of 60 m × 50 m in the Faculty Farm, Aligarh Muslim University. The soil was sandy clay loam (66.7% sand, 19% silt, clay 14.3%), with 43% water holding capacity, 7.9 pH, 0.016% inorganic carbon, 1.9% organic carbon and 15.2 kg/h available phosphorus. The land was prepared by adding 10 tonnes farm yard manure during ploughing. The major climatic conditions viz., temperature, humidity and rainfall from November to March were 18.5°C (3-37°C), 68% (38-93%) and 0.09 mm (0-6.0 mm), respectively during the experiment (November, 2007 - March, 2008; Appendix 11). In the field 395 microplots each of 2 × 4 m dimension were prepared with 0.5 m wide and 0.25 m high bunding (margins) to permit flood irrigation to individual plots. The width of the bunding was considered adequate to minimize possible lateral movement of nematodes or microorganisms and over flooding of water. The following 33 treatments were maintained for each chickpea and tomato, for seed and soil application of A. niger isolates. For each treatment of a crop, three microplots (replicates) were maintained which were randomly distributed in the field. 01. Plant (Control) 02. Plant + AnC2 03. Plant + AnR3 04. Plant + SkNAn5 05. Plant + VAn4 06. Plant + BuAn3 07. Plant + ANAn4 08. Plant + Carbendazim 09. Plant + Carbofuran 10. Plant + Fusarium oxysporum f. sp. ciceri / lycopersici (Control) 11. Plant + F. oxysporum f. sp. ciceri / lycopersici + AnC2 12. Plant + F. oxysporum f. sp. ciceri / lycopersici + AnR3 13. Plant + F. oxysporum f. sp. ciceri / lycopersici + SkNAn5 14. Plant + F. oxysporum f. sp. ciceri / lycopersici + VAn4 93

15. Plant + F. oxysporum f. sp. ciceri / lycopersici + BuAn3 16. Plant + F. oxysporum f. sp. ciceri / lycopersici + ANAn4 17. Plant + F. oxysporum f. sp. ciceri / lycopersici + Carbendazim 18. Plant + Meloidogyne incognita (Control) 19. Plant + M. incognita + AnC2 20. Plant + M. incognita + AnR3 21. Plant + M. incognita + SkNAn5 22. Plant + M. incognita + VAn4 23. Plant + M. incognita + BuAn3 24. Plant + M. incognita + ANAn4 25. Plant + M. incognita + Carbofuran 26. Plant + M. incognita + F. oxysporum f. sp. ciceri / lycopersici (Control) 27. Plant + M. incognita + F. oxysporum f. sp. ciceri / lycopersici + AnC2 28. Plant + M. incognita + F. oxysporum f. sp. ciceri / lycopersici + AnR3 29. Plant + M. incognita + F. oxysporum f. sp. ciceri / lycopersici + SkNAn5 30. Plant + M. incognita + F. oxysporum f. sp. ciceri / lycopersici + VAn4 31. Plant + M. incognita + F. oxysporum f. sp. ciceri / lycopersici + BuAn3 32. Plant + M. incognita + F. oxysporum f. sp. ciceri / lycopersici + ANAn4 33. Plant + M. incognita + F. oxysporum f. sp. ciceri / lycopersici + Carbendazim +Carbofuran

Preparation of biopesticides Three isolates of A. niger aggrigate viz., SkNAn5, VAn4 and AnC2 found highly effective against target diseases evaluated under field condition, hence there commercial formulations (biopesticide) were prepared on sawdust, fly ash, soil and molasses (Khan et al., 2010; US Patent no. US 7,815,903 B2). The mass culture or stock culture of A. niger isolates was prepared on sawdust, soil and molasses mixture, whereas immobilization of the fungus was done on a fly ash based carrier. Production of mass/stock culture of Aspergillus niger isolate A mixture of sawdust-soil-5% molasses (15:5:1) was used to prepare mass (stock) culture of A. niger isolates. The mixture amended with 10 mg chloramphenicol/kg was filled in heat resistant polybags, sealed and steam sterilized at 15 kg/cm2 pressure at 121°C for 15 minutes. Liquid pure cultures of A. niger for inoculation of the material was prepared in potato dextrose broth supplemented with 10 mg chloramphenicol/liter. Thereafter the bags containing 500 g autoclaved sawdust-soil-molasses mixture were inoculated with homogenized pure culture of the A. niger isolates (5 ml/bag) by sterilized needle and syringe. The puncture made in the polybag to insert the needle was resealed with cello tape. The bags were incubated at room temperature (25-30°C) or at 25±2°C in an incubator for 10-12 days in an incubator. During incubation the bags were shaken daily for a few minutes to achieve uniform colonization by the A. niger isolate on the material. Luxuriant and uniform colonization by the A. niger isolates occurred within the incubation duration. 94

Immobilization of Aspergillus niger isolates A mixture of fly ash, soil (loam) and 5% molasses in the ratio of 15:3:1 plus chloramphenicol 10 mg/kg formulation was used as a carrier to immobilize A. niger isolates (Fig. 18). The fly ash was collected from a coal fired thermal power station, Kasimpur, Aligarh; where bituminous coal is burnt. Some of the important physico-chemical characteristics of the ash were: pH 8.9, conductivity 7.6 m mhos/cm, cation exchange capacity 9.3 m mhos/cm, sulphate 9.72%, carbonate 1.07%, bicarbonate 2.6%, chloride 1.85%, nitrogen 0.0%, phosphorus 0.093%, potassium 0.82%, calcium 1.06%, magnesium 0.90%, manganese 64.5 mg/g, copper 117.8 mg/g, zinc 85.1 mg/g and boron 198.5 mg/g. The ash-soil mixture was solarized under thin and transparent polythene sheet for 3-4 weeks (38°C ambient temperature) or filled in heat resistant polybags and autoclaved at 15 kg/cm2 pressure at 121°C for 15 minutes. Thereafter, stock culture and carrier were mixed in the ratio of 1: 15 and filled in polybags. The bags were sealed and incubated for 10-15 days at room temperature (25-30°C) or inside an incubator at 25+2°C. After incubation, the number of colony forming units/g formulation was determined using dilution plate method. The CFUs of contaminant(s) in the formulation was also counted. The formulations were packed in airtight polypacks of different size viz., 200, 500 and 1000 g (Fig. 19). Shelf life Shelf life of A. niger biopesticides was tested at five temperature regimes i.e., 5°C, 10°C, 15°C, 25°C and ambient for 12 months (January to December). The viability and CFU load of A. niger and contaminants was determined fortnightly. Experiment on evaluation of performance of biopesticides of selected isolates of Aspergillus niger aggrigate under field condition A field experiment was conducted during November, 2008 - March, 2009 to test efficacy of the biopesticides containing A. niger isolates viz., AnC2, SkNAn5 and Van4. The major climatic conditions viz., temperature, humidity and rainfall from November, 2008 - March, 2009 were 19.5°C (5-35°C), 61% (23-97%) and 0.07 mm (0-6.6 mm) during the experiment (Appendix 12). The following 21 treatments were maintained for each chickpea and tomato, for seed and soil application of A. niger isolates. For each treatment of a crop, three microplots (replicates) were maintained which were randomly distributed in the field. 01. Plant (Control) 02. Plant + AnC2 03. Plant + SkNAn5 04. Plant + VAn4 05. Plant + Carbendazim

Fig. 18. Ingredients used in biopesticide development.

Fig. 19. The biopesticides of Aspergillus niger isolates SkNAn5, VAn4 and AnC2 packed in to different polypacks.

06. Plant + Carbofuran 07. Plant + Fusarium oxysporum f. sp. ciceri / lycopersici (Control) 08. Plant + F. oxysporum f. sp. ciceri / lycopersici + AnC2 09. Plant + F. oxysporum f. sp. ciceri / lycopersici + SkNAn5 10. Plant + F. oxysporum f. sp. ciceri / lycopersici + VAn4 11. Plant + F. oxysporum f. sp. ciceri / lycopersici + Carbendazim 12. Plant + Meloidogyne incognita (Control) 13. Plant + M. incognita + AnC2 14. Plant + M. incognita + SkNAn5 15. Plant + M. incognita + VAn4 16. Plant + M. incognita + Carbofuran 17. Plant + M. incognita + F. oxysporum f. sp. ciceri / lycopersici (Control) 18. Plant + M. incognita + F. oxysporum f. sp. ciceri / lycopersici + AnC2 19. Plant + M. incognita + F. oxysporum f. sp. ciceri / lycopersici + SkNAn5 20. Plant + M. incognita + F. oxysporum f. sp. ciceri / lycopersici + VAn4 21. Plant + M. incognita + F. oxysporum f. sp. ciceri / lycopersici + Carbendazim + Carbofuran Observations recorded During the course of growth or at harvest of different experiments conducted in the pots or field following observations were recorded. 1. Biochemical tests of plant material i. Estimation of leaf pigments Chlorophyll a, chlorophyll b and total chlorophyll were estimated by grinding 1 g of fresh leaves from interveinal areas of one month old tomato and chickpea plants in 40 ml 80% acetone with the help of mortar and pestle. The suspension was decanted in a Buchner funnel having two Whatman filter paper No.1. The filtration was done with the help of suction pump. The residue was ground thrice by adding acetone. The suspension was decanted in Buchner funnel and filtered in vacuum. At last, mortar and pestle were rinsed with acetone and solution was transferred in Buchner funnel and filtered. The filtrate was transferred to 100 ml volumetric flask and the volume was made upto the capacity by adding acetone. Using spectrophotometer (Spectronic 20, USA), the optical density (OD) of the filtrate was read at 645 and 663 nm for chlorophyll. The chlorophyll contents were calculated by using the Arnon (1949) formulae. Chlorophyll a (µg/ml) = 12.7 (A663) - 2.69 (A645) Chlorophyll b (µg/ml) = 22.9 (A645) - 4.68 (A663) Total chl (µg/ml) = 17.76 (A645) + 7.34 (A663) Where, A663 is the OD at 663 ηm; A645 is the OD at 645 ηm. ii. Estimation of total phenol Leaf samples (1g) from tomato and chickpea plants 2 weeks after inoculation were homogenized in 10 ml 80% methanol and agitated for 15 minutes at 70°C (Zieslin and Ben

Zaken, 1993). The leaf samples from the three plants replicates of a treatment were processed separately. One milliliter of the methanol extract was added to 5 ml of distilled water and 250 µl of Folin-Ciocalteau reagent (1N) and the solution was kept at 25°C. The absorbance of the developed blue color was measured using a Spectrophotometer (Spectronic 20, USA) at 725ηm. Catechol was used as the standard. The amounts of phenolics were expressed as µg catechol /gm fresh weight of leaf sample (Sharma et al., 2005). iii. Estimation of salicylic acid The salicylic acid (SA) of the leaves and roots of the chickpea and tomato was estimated separately 2 week after inoculation. The leaves and roots (5 g) were cut into small pieces of size 0.5-1.0 cm, soaked in water for overnight, than filtered through the Whatman filter paper no.1 and extracted in ethyl acetate. The ethyl acetate fraction was taken and sodium sulphate added to remove the moisture and filtrate was evaporated to dryness in water bath. The stock solution was prepared by the addition of 10 ml methanol. The stock solution was used for recording the absorbance in a spectrophotometer (SHIMADZU, 2450 PC, Japan) at 306 ηm. The absorbance was fixed at 306 ηm and the readings were recorded at different ppm of SA and standard curve was prepared for the estimation of SA concentration in the leaf sample (Pankaj et al., 2005). From the standard curve the concentration of SA in the sample was calculated according to the formula y = mx ± c (Lowery et al., 1951). iv. Estimation of lycopene in tomato Extraction of lycopene was performed according to Fish et al. (2002). Fully ripe tomato fruits were first chopped and homogenized in a laboratory homogenizer. 0.5 g samples were weighed and 5 ml of 0.05% (w/v) butylated hydroxytoluene (BHT) in acetone, 5 ml ethanol and 10 ml hexane were added. The recipient was introduced in ice and stirred on a magnetic stirring plate for 15 min. After shaking, 3 ml of deionized water were added to each vial and the samples were shaken for 5 min on ice. Samples were then left at room temperature for 5 min to allow the separation of both phases. The absorbance of the hexane layer (upper layer) was measured in a spectrophotometer (SHIMADZU, 2450 PC, Japan) at 503 ηm blanked with hexane. A standard solution of lycopene in hexane (0.04 mg/ml) was also prepared, wrapped in foil, and stored in the dark at 4ºC. Working mixtures of relevant concentrations were made by appropriate combination and dilution with hexane. The absorbance was fixed at 503 ηm and the readings were recorded at different ppm of standard solution of lycopene. The standard curve was plotted and concentration of lycopene in the sample was calculated 98 according to the formula y = mx ± c (Lowery et al., 1951) or the lycopene concentration in the sample was determined using the following formula (Vosburgh and Cooper, 1941):

3.1206 × A 503 × Volume made up × Dilution × 100 Lycopene (mg/100g sample) = ------1 × Wt of sample × 1000

Where, A 503 is the OD at 503 ηm.

2. Soil population of fungi Soil population of F. oxysporum f. sp. ciceri, F. oxysporum f. sp. lycopersici and A. niger isolates was estimated monthly using dilution plate method (Wakman, 1927). Soil was collected from the rhizosphere of the plants from pots or microplots and was mixed together to make a composite sample. Ten gram of the soil was taken in a conical flask to which 90 ml sterile water was added. The soil-water mixture was stirred over a magnetic stirrer for 5 minutes. One ml of this suspension was transferred to 9 ml sterile water in a test tube. One ml sample was then transferred to another tube containing 9 ml sterile water. The process was repeated till the desired dilution of 10-6 achieved. Each suspension was shaken over magnetic stirrer for few seconds and was in motion while being drawn into the micropipette. From the final dilution 0.1 ml suspension was aseptically spread under Laminar flow over solidified Fusarium specific medium (Appendix 13) and A. niger specific medium supplemented with 50 mg/l nystatin fungicide (Appendix 14) in Petri plates and three plates were maintained for each dilution. The agar plates were prepared four days previously to ensure that the medium in the plate was free from contamination. After inoculation the plates were then incubated at 27+2°C for 5 days to get the colonies. After incubation, the plates were examined under a colony counter to determine soil population of the target microorganism on the basis of morphological test characters. 3. Soil population of root-knot nematode Soil population of root-knot nematode, M. incognita was determined by Cobb’s decanting and sieving method (modified) followed by the Baermann funnel technique (Southey, 1986). A composite soil sample was made by collecting the soil from three randomly selected pots or rhizosphere of five plants in each microplot in case of field trials. The soil was sifted through a coarse sieve. The soil sample (1 kg) was mixed in 5 liters of water in a plastic bucket. The soil-water mixture was stirred and then allowed to stand for 1-2 minutes.

The suspension was decanted over a combination of 3 sieves (60, 200 and 500 mesh), the catch from the final sieve was carefully washed and transferred to a beaker. A small coarse sieve with two layers of wet paper towels was kept in a Baermann funnel filled with water. The nematode suspension from the beaker was gently poured onto the sieve and allowed to stand overnight. The nematode juveniles because of the random and continuous wringling movement migrate through the paper pores into the water and gradually settled down in the bottom of rubber tubing of the funnel. The nematode suspension recovered from the Baermann funnel was taken into a beaker and counted in a counting plate under a stereomicroscope. 4. Root-nodulation Two month old chickpea plants were randomly selected from three pots and were uprooted carefully to observe nodulation. From each microplot, 5 plants (2 months old) were randomly uprooted to count nodules on per root system. Pink and healthy nodules were recognized as functional nodules, where as dark brown and degenerated ones as nonfunctional nodules. 5. Wilt incidence and severity Two months old plants of chickpea and tomato were visually observed to determine wilt severity on 0-5 scale and incidence (%) according to the following formulae: Number of wilted plants in microplot Wilt incidence (%) = ------× 100 Total number of plants in a microplot

Number of branches / twigs showing wilt symptom Wilt severity (%) = ------× 100 Total number of branches / twigs of a plant

The severity was converted into wilt index on 0-5 scale. 0= no wilting, 1= 1-19%, 2= 20- 39%, 3= 40-59%, 4= 60-79%, 5= 80-100%.

6. Root-knot severity: Root-knot severity in terms of galls, egg masses per root system was determined on four month old plants of chickpea and tomato at harvest. Roots were rinsed with water and observed for presence of galls. The galls were gently teared to excise creamy females, which were transferred to water in cavity blocks. Perineal pattern of the females was prepared and temporarily mounted in glycerin (Khan, 2008). The species was identified on the basis of perineal pattern (Sasser, 1954). To count egg mass, roots were treated with phloxine B solution (0.159 g/l) which gave stain to the egg mass (Hartman, 1983). The galls 100 and egg masses were indexed on 0-5 scale (Taylor and Sasser, 1978), 0 = 0 galls or egg masses/root system, 1 = 1-2 galls or egg masses/root system, 2 = 3-10 galls or egg masses/root system, 3 = 11-30 galls or egg masses/root system, 4 = 31-100 galls or egg masses/root system, and 5 = >100 galls or egg masses/root system. Some egg masses were also found embedded in the galled tissue, hence were observed after gently tearing the galled tissue. 7. Dry weight of plants: The chickpea and tomato plants after collecting pods and fruits were wrapped with paper and kept in an oven at 60°C for 2 days to determine dry weight of root and shoot. 8. Weight of seeds or fruit/plant: Weight of chickpea seeds was determined at harvest and tomato fruit weight per plant was calculated by adding the multiple harvested production. 9. Seed index of chickpea: Weight of 100 seeds in gram is called seed index. Weight of 100 seeds per treatment in gram was recorded. 10. Seed health test: Seed health test of chickpea and tomato was carried out to determine the sanitary condition of the seed with special reference to seed-borne disease. To know the seed health following three tests were performed i. Viability test by Tetrazolium chloride: Seeds were soaked in 2% solution of tetrazolium chloride (2, 3, 5- triphenyl Tetrazolium chloride) for 5-10 minutes (Lakon, 1939; Singh, 2000). The viable or alive seeds took bright red colouration which became more intense in the embryo while the dead seeds remained in their original colour. ii. Germination test: Petri plate method for germination test was performed (Singh, 2000). PDA (20ml/Petri plate) or two blotter or filter papers soaked in distilled water were kept in the bottom of the Petri plate and 10 were placed on the surface of the paper with equal spacing in the Petri plate and germination counts were made between 5-8 days. iii. Detection of pathogen associated with seeds: It is not necessary that all the germinated seedlings may grow into healthy plants. However, to detect pathogen(s) associated with seeds, they were divided into two sets, one set was surface sterilized with 0.5% NaOCl solution for 2 minutes (to observe the pathogen associated with inside seeds) and washed the seeds in distilled water and other set was not surface sterilized. Ten seeds from each set were kept in the Petri plates separately on wet blotter paper treated with chloramphenicol (0.0003%) at 2 cm apart and incubated at 20°C for not more than 15 days in BOD incubator. During incubation observation and identification of fungus if present was done daily.

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Statistical analysis Three replicates were maintained for each treatment and the observations taken from five plants from a microplot were averaged and considered as one replicate. For pot experiments, 3 replicates were maintained for each treatment with additional 9 pots/treatment for the monthly soil population of nematode and fungi, and 3 pots/treatment for estimation of salicylic acid and leaf pigments from plants. The data on plant growth, yield etc. were subjected to a two-factor analysis of variance. Pathogens were considered as one factor, whereas isolates of A. niger as second factor. The data on wilt incidence, root knot, soil population etc. were analyzed for single factor ANOVA. The data on wilt incidence was angularly transformed before the analysis. Least significance difference (LSD) was calculated at P<0.05, P<0.01 and P<0.001 for all variables to compare individual treatments. Duncan’s multiple range test was applied to identify efficient isolates of A. niger. The data has been presented in tabulated form. Regression analysis among plant growth parameters and disease severity, disease incidence was performed and coefficient of regression was calculated and presented in graphical forms. For biosorption of heavy metals, the data was statistically analyzed for test of significance for mean values of three replicates and LSD was calculated at P<0.05. Entire statistical analysis was done using SPSS-10 for Windows or Minitab-15.

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RESULTS Experiment-I

IN-VITRO EXPERIMENTS

Characteristics of Aspergillus niger aggregate isolates The Aspergillus niger isolates were identified on the basis of cultural and morphological characters. The fungus grew upto 90 mm in 4-7 days on potato dextrose agar (PDA) and produced initially white mycelium which quickly became black due to production of conidia (Fig. 20). The mycelium was hyaline, septate and produced conidia on biseriate metulae of conidial heads. Conidiophores were long measured 600-3000 µm, smooth, hyaline, darker at the apex and terminated in a globose vesicle of 40-65 µm diameters. Conidia were black, globose, and measured 4-5 µm in diameter (Fig. 20). Characteristics of wilt fungus Fusarium oxysporum f. sp. ciceri The wilt inducing fungus was identified based on cultural and morphological characteristics of the fungal structures grown on PDA. The fungus grew upto 90 mm in 5-6 days on potato dextrose agar (PDA) and produced and hyaline cottony mycelium which became pale yellow (Fig. 20). The mycelium was septate and produced three types of spores. Microconidia were small elliptical or with 1-2 septa, whereas the macroconidia were long or curved (fusaroid; Fig. 20). Chlamydospores were oval or spherical and formed in older cultures from any cell of the hyphae. Fusarium oxysporum f. sp. lycopersici The fungus was identified on cultural and morphological characteristics of the fungal structures grown on PDA. The fungus grew upto 90 mm in 6-7 days on potato dextrose agar (PDA) and produced septate, hyaline mycelium which later became pinkish or purplish (Fig. 20). The fungus produced three kinds of asexual spores, microconidia were small elliptical with 1 septa, whereas the macroconidia were long, hyaline, three to five celled, had gradually pointed and curved ends, and measured 25-30 × 4-5.5 µm (Fig. 20). Chlamydospores were one or two celled, thick walled, round spores produced within or terminally on older mycelium. To fulfill Koch’s postulates the inoculum of F. oxysporum f. sp. ciceri and F. oxysporum f. sp. lycopersici were prepared on sorghum seeds and inoculated in different

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Fig. 20. Colony characters of Aspergillus niger (A), conidial heads (B) with conidia (C); colony characters of Fusarium oxysporum f. sp. ciceri (D) and its micro and macroconidia (E); colony characters of F. oxysporum f. sp. lycopersici (F) and its micro and macrocondia (G).

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pots containing autoclaved soil. Healthy and surface sterilized seeds of chickpea cv. BGD- 72 and nursery of tomato cv. Pusa Ruby were sown in F. oxysporum f. sp. ciceri and F. oxysporum f. sp. lycopersici inoculated pots, respectively. Symptoms of the diseases were observed 30 days after sowing. The symptoms were identical to those recorded in naturally infested plants. Browning was visible through the bark as streaks or bands (Fig 21). Relative colonization of Aspergillus niger aggregate isolates Growth of monoculture A. niger varied with isolates and covered the entire PDA in Petri plates (90 mm) between 4-7 days of inoculation. The radial growth of 7 days mean varied from 52-70 mm with maximum 70 mm of isolate VAn4 (Table 17). Biochemical characters of Aspergillus niger aggregate isolates None of the isolates produced hydrogen sulphide. Eight isolates of A. niger viz., BuAn3, LAn3, SkNAn3, SkNAn5, VAn4, ANAn4, AnC2 and AnR3 produced indole acetic acid (IAA). All isolates produced ammonia with varied amount. Isolates also produced siderophores being greater with SkNAn3, SkNAn5, VAn4, ANAn1, ANAn4, AnC2 and AnR3. The 33 isolates out of 236 produced hydrogen cyanide (Table 17). Conclusion Based on in vitro monoculture growth and some above biochemical tests, 16 isolates of A. niger viz., AAn1, BAn4, BuAn3, BasAn5, BudAn3, GaAn1, JaAn2, LAn3, MeAn4, SkNAn3, SkNAn5, VAn4, ANAn1, ANAn4, AnC2 and AnR3 were selected for further study for the reason that they showed faster growth rate, had more ammonia and siderophore production and produced hydrogen cyanide and IAA than other isolates.

Fig. 21. Browning of infected tissue of chickpea (A) and tomato (B) caused by Fusarium oxysporum f. sp. ciceri and F. oxysporum f. sp. lycopersici, respectively.

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Table 17. Some biochemical characteristics of Aspergillus niger isolates. S. No A. niger Production of biochemical compounds Radial isolate growth X Y H2S Ammonia HCN Siderophore IAA mean of 7 days (mm)Z 1. AAn1 - ++ + **** - 66* 2. AAn2 - ++ + *** - 62 3. AAn3 - + - ** - 60 4. AAn4 - + - *** - 66* 5. AAn5 - + - ** - 54 6. AgAn2 - + - ** - 54 7. AgAn3 - + - ** - 54 8. AgAn4 - + - ** - 58 9. AgAn5 - + - ** - 56 10. AzAn1 - ++ - *** - 58 11. AzAn3 - + - ** - 52 12. AzAn4 - + - ** - 54 13. AzAn5 - + - ** - 58 14. BahAn1 - + - ** - 56 15. BahAn3 - + - ** - 62 16. BahAn4 - + - ** - 56 17. BahAn5 - + - ** - 58 18. BaAn1 - + - ** - 60 19. BaAn2 - ++ + ** - 62 20. BaAn4 - + - ** - 60 21. BaAn5 - + - ** - 58 22. BAn1 - + - ** - 56 23. BAn2 - ++ + ** - 58 24. BAn3 - + - *** - 58 25. BAn4 - ++ + **** - 68* 26. BAn5 - + + *** - 68* 27. BuAn2 - + - ** - 62 28. BuAn3 - ++ + **** + 66* 29. BuAn4 - ++ - **** - 58 30. BuAn5 - ++ + **** - 58 31. BarAn1 - + - ** - 56 32. BarAn2 - + - ** - 62 33. BarAn3 - + - ** - 64 34. BarAn4 - ++ - *** - 68* 35. BasAn1 - + - ** - 64 36. BasAn2 - + - *** - 64 37. BasAn4 - + - ** - 64 38. BasAn5 - ++ + **** - 66* 39. BiAn1 - + - *** - 64 40. BiAn2 - + - ** - 64 41. BiAn3 - + - ** - 64 42. BiAn4 - + - ** - 58 43. BiAn5 - + - ** - 60 44. BudAn1 - + - ** - 62 45. BudAn2 - + - ** - 62 46. BudAn3 - ++ + **** - 68* 47. BudAn4 - + - ** - 58 48. BudAn5 - + - ** - 68* Continued…

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Table 17 Continued… S. No A. niger Production of biochemical compounds Radial isolate growth X Y H2S Ammonia HCN Siderophore IAA mean of 7 days (mm)Z 49. ChAn1 - ++ + *** - 60 50. ChAn2 - + - ** - 60 51. ChAn3 - + - ** - 56 52. ChAn5 - + - ** - 60 53. EtAn1 - ++ - *** - 60 54. EtAn2 - + - ** - 68* 55. EtAn3 - ++ + *** - 58 56. EtAn4 - + - ** - 56 57. EtAn5 - + - ** - 56 58. FaAn1 - + - ** - 58 59. FaAn3 - + - ** - 62 60. FaAn4 - + - ** - 60 61. FaAn5 - + - ** - 58 62. FazAn1 - + - ** - 62 63. FazAn2 - + - ** - 58 64. FazAn3 - + - ** - 66 65. FazAn4 - ++ - *** - 60 66. FazAn5 - ++ - *** - 62 67. GoAn1 - + - ** - 60 68. GoAn2 - + - ** - 60 69. GoAn3 - + - ** - 62 70. GoAn4 - + - ** - 58 71. GoAn5 - + - ** - 58 72. GaAn1 - ++ + **** - 68* 73. GaAn2 - + - ** - 58 74. GaAn3 - + - ** - 56 75. GaAn5 - + - ** - 56 76. GorAn1 - ++ + *** - 58 77. GorAn2 - + - ** - 56 78. GorAn3 - + - ** - 58 79. GorAn4 - ++ - *** - 62 80. GorAn5 - + - ** - 58 81. JhAn3 - + - ** - 56 82. JhAn4 - + - ** - 56 83. JhAn5 - + - ** - 60 84. JaAn2 - ++ + **** - 66* 85. JaAn3 - + - ** - 62 86. JaAn4 - + - ** - 58 87. JaAn5 - + - ** - 62 88. KaAn1 - ++ - *** - 60 89. KaAn2 - + - ** - 56 90. KaAn3 - + - ** - 62 91. KaAn4 - + - ** - 58 92. KaAn5 - + - ** - 58 93. LkAn1 - + - ** - 60 94. LkAn2 - + - ** - 58 95. LkAn3 - + - ** - 58 96. LkAn4 - + - ** - 58 97. LkAn5 - + - ** - 62 98. LaAn2 - ++ + *** - 64 Continued… 107

Table 17 Continued… S. No A. niger Production of biochemical compounds Radial isolate growth X Y H2S Ammonia HCN Siderophore IAA mean of 7 days (mm)Z 99. LaAn3 - ++ - **** - 62 100. LaAn4 - + - ** - 62 101. LaAn5 - + - ** - 64 102. LAn1 - + - ** - 58 103. LAn2 - + - ** - 56 104. LAn3 - ++ + **** + 68* 105. LAn4 - + - ** - 60 106. LAn5 - + - ** - 56 107. MaAn1 - + - ** - 60 108. MaAn2 - + - ** - 56 109. MaAn3 - + - ** - 62 110. MaAn4 - + - ** - 62 111. MaAn5 - + - ** - 58 112. ManAn1 - + - ** - 56 113. ManAn3 - + - ** - 56 114. ManAn4 - + - ** - 60 115. ManAn5 - + - ** - 58 116. MNAn1 - + - ** - 60 117. MNAn2 - + - ** - 58 118. MNAn3 - + - ** - 60 119. MNAn4 - + - ** - 60 120. MNAn5 - ++ - *** - 66* 121. MeAn1 - + - ** - 54 122. MeAn2 - + - ** - 56 123. MeAn3 - + - ** - 56 124. MeAn4 - ++ + **** - 66* 125. MeAn5 - + - ** - 60 126. PAn2 - + - ** - 60 127. PAn3 - + - ** - 58 128. PAn4 - + - ** - 62 129. RaAn1 - + - ** - 56 130. RaAn2 - ++ - *** - 58 131. RaAn3 - + - ** - 58 132. RaAn4 - + - ** - 58 133. RaAn5 - ++ + *** - 64 134. SaAn2 - + - ** - 58 135. SaAn3 - + - ** - 58 136. SaAn4 - + - ** - 60 137. SaAn5 - + - ** - 62 138. SoAn1 - + - ** - 62 139. SoAn2 - + - ** - 60 140. SoAn3 - + - ** - 56 141. SoAn4 - + - ** - 62 142. SoAn5 - + - ** - 58 143. SuAn1 - + - ** - 60 144. SuAn2 - + - ** - 46 145. SuAn3 - + - ** - 56 146. SuAn4 - + - ** - 56 147. SuAn5 - + - ** - 58 148. SkNAn1 - + - ** - 60 Continued… 108

Table 17 Continued… S. No A. niger Production of biochemical compounds Radial isolate growth X Y H2S Ammonia HCN Siderophore IAA mean of 7 days (mm)Z 149. SkNAn2 - + - ** - 62 150. SkNAn3 - ++ + ***** + 66* 151. SkNAn4 - ++ + **** - 58 152. SkNAn5 - ++ + ***** + 68* 153. SiNAn1 - ++ - *** - 58 154. SiNAn3 - + - ** - 60 155. SiNAn4 - + - ** - 60 156. SiNAn5 - + - ** - 64 157. SiAn1 - + - ** - 62 158. SiAn2 - + - ** - 58 159. SiAn3 - + - ** - 58 160. SiAn4 - + - ** - 56 161. SiAn5 - + - ** - 56 162. UAn1 - ++ - *** - 58 163. UAn2 - + - ** - 58 164. UAn3 - ++ - *** - 62 165. UAn4 - + - ** - 58 166. UAn5 - + - ** - 50 167. VAn1 - + - ** - 54 168. VAn2 - + - ** - 56 169. VAn3 - + - ** - 56 170. VAn4 - ++ + ***** + 70* 171. VAn5 - + - ** - 64 172. ANAn1 - ++ + ***** - 66* 173. ANAn2 - ++ + *** - 64 174. ANAn3 - + - ** - 62 175. ANAn4 - ++ + ***** + 68* 176. ANAn5 - + - ** - 54 177. AnPM1 - + - ** - 60 178. AnPM2 - + - ** - 52 179. AnPM3 - + - ** - 54 180. AnPM4 - + - ** - 58 181. AnPM5 - + - *** - 58 182. AnPP1 - ++ + *** - 56 183. AnPP2 - + - ** - 66* 184. AnPP3 - + - ** - 62 185. AnPP4 - + - ** - 60 186. AnPP5 - + - ** - 60 187. AnC1 - + - ** - 56 188. AnC2 - ++ + ***** + 66* 189. AnC3 - + - ** - 56 190. AnC4 - + - *** - 56 191. AnC5 - + - ** - 58 192. AnS1 - + - ** - 58 193. AnS2 - + - ** - 64 194. AnS3 - + - ** - 62 195. AnS4 - + - ** - 58 196. AnS5 - + - ** - 60 197. AnPa1 - + - ** - 56 198. AnPa2 - + - ** - 62 Continued… 109

Table 17 Continued… S. No A. niger Production of biochemical compounds Radial isolate growth X Y H2S Ammonia HCN Siderophore IAA mean of 7 days (mm)Z 199. AnPa3 - ++ - *** - 58 200. AnPa4 - + - ** - 56 201. AnPa5 - + - *** - 58 202. AnMa1 - + - *** - 52 203. AnMa2 - + - ** - 54 204. AnMa4 - + - ** - 58 205. AnMa5 - + - ** - 60 206. AnR1 - + - ** - 56 207. AnR2 - + - ** - 58 208. AnR3 - ++ + ***** + 66* 209. AnR4 - ++ - **** - 54 210. AnR5 - + - ** - 56 211. AnL1 - + - ** - 56 212. AnL2 - + + ** - 58 213. AnL3 - + - *** - 62 214. AnL4 - + - *** - 60 215. AnL5 - + - ** - 60 216. AnT1 - + - ** - 60 217. AnT2 - + - ** - 56 218. AnT3 - + + *** - 65 219. AnT4 - + - ** - 56 220. AnPo1 - + - ** - 54 221. AnPo2 - + - ** - 56 222. AnPo3 - ++ - **** - 58 223. AnPo4 - + - ** - 58 224. AnPo5 - + - ** - 56 225. AnRa1 - + - ** - 56 226. AnRa2 - + - *** - 60 227. AnRa4 - + - *** - 56 228. AnM1 - + + ** - 58 229. AnM2 - + - ** - 56 230. AnM3 - + - ** - 66* 231. AnM4 - + + ** - 62 232. AnM5 - + - ** - 60 233. AnCp2 - ++ - *** - 60 234. AnCp3 - + - *** - 58 235. AnCp4 - + - ** - 60 236. AnCp5 - + - ** - 56 LSD (P≤0.01) 4.3 Each value is the mean of three replicates, + production, - no production; X + less ammonia production, ++ high ammonia production; Y width of yellow halo around the colony: * very narrow, ** narrow, *** wide, **** wider, *****widest; Z Means followed by asterisk (*) in the column are significantly different from others at P≤0.01. H2S, Hydrogen sulphide; HCN, Hydrogen cyanide; IAA, Indole acetic acid.

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Experiment-II

RAPD PROFILING OF EFFECTIVE ISOLATES OF ASPERGILLUS NIGER AGGREGATE AND THEIR CHARACTERIZATION FOR OCHRATOXIN A PRODUCTION, PHOSPHATE SOLUBILIZATION, HEAVY METAL BIOADSORPTION AND IN VITRO PATHOGEN SUPPRESSION (FUSARIUM OXYSPORUM F. SP. CICERI, F. OXYSPORUM F. SP. LYCOPERSICI AND MELOIDOGYNE INCOGNITA)

Molecular characterization of selected Aspergillus niger isolates RAPD fingerprinting (polymorphism) Among the 28 primers tested in the PCR amplification, 22 primers showed clear and unambiguous amplification while the rest 6 primers did not give amplification of several reactions tried or produced faint or fizzy lanes (Table 18). Scorable 22 RAPD primers led to amplification of 727 fragments ranging from about 3,500 bp (OPA-11) to 200 bp (Primer-06), out of 169 bands 167 bands were found to be polymorphic and two of them were monomorphic (Primer-02 and Primer-06). Most of the A. niger isolates had common bands of 0.5, 0.8 and 1.3 kb size. A maximum number of 13 amplified products were obtained by Primer-01. Moreover, Primer-03 produced 12 bands followed by OPA-17 (11 bands), OPA-02 and OPA-16 with 10 bands each. The primer OPA 14 amplified a minimum of one (1) band. On an average, 7.23 bands per primer were obtained and 8 primers out of 22 primers (36.4%) used in the study produced DNA bands greater than the average value of 7.2. Two RAPD products (monomorphic band) produced by Primer 02 (650 bp) and Primer 06 (850 bp), which may be considered as Aspergillus specific as they were present in all isolates of A. niger tested. Whereas the three amplicons produced by primer OPA-16 can be treated as isolate specific as 2300 bp for AnC2 and VAn4, and 2800 bp for AnC2 only. Primer 04 produced only 1500 bp for isolate SkNAn5, Primer 01 amplified 700 bp in ANAn1 and OPA-12 produced 700 pb in VAn4 only (Table 18). DNA amplification patterns as detected by some of the RAPD primers in the A. niger isolates have been provided in Figure 22.

Highest similarity (0.891) was measured between isolate AnC2 and VAn4 (Fig. 23).

High degree of similarity (> 80%) was also observed between ANAn4 and VAn4; BuAn3 and SkNAn5; AnC2 and AnR3, SkNAn5; VAn4 and AnR3, SkNAn5; AnR3 and SkNAn5;

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Table 18. Summary of polymorphism produced by OPA and Fusarium specific synthetic primers. Primers Total no. of bands No. of Polymorphic No(s)Y and Mol wt of unique profile in base Percent uniqueness AmplifiedX bands pairs (bp) with isolate (Y/X)×100 OPA-01 - - - - OPA-02 10 10 4 (300, BuAn3; 800, BudAn3; 1400, MeAn4; 2000, BuAn3) 40 OPA-03 7 7 - 0 OPA-04 5 5 3 (500, ANAn1; 1600, ANAn1; 2100, GaAn1) 60 OPA-05 - - - - OPA-06 - - - - OPA-07 - - - - OPA-08 7 7 4 (600, SkNAn3; 750, MeAn4; 800, ANAn1; 1300, BuAn3) 42.85 OPA-09 6 6 2 (800, AnR3; 900, MeAn4) 33.33 OPA-10 7 7 2 (1700, ANAn4; 2700, BuAn3) 28.57 OPA-11 5 5 2 (600, LAn3; 3500, MeAn4) 40 OPA-12 4 4 1 (700, VAn4) 25 OPA-13 5 5 2 (1300, BudAn3; 1700, ANAn1) 20 OPA-14 1 1 - 0 OPA-15 8 8 2 (500, MeAn4; 2700, BuAn3) 25 OPA-16 10 10 2 (1800, BudAn3; 2800, AnC2) 20 OPA-17 11 11 2 (600, MeAn4; 2500, AnR3) 18.18 OPA-18 9 9 2 (300, MeAn4; 3400, BuAn3) 22.22 OPA-19 - - - - OPA-20 - - - - Primer 01 13 13 1 (700, ANAn1) 7.69 Primer 02 9 8 - - Primer 03 12 12 2 (300, ANAn1; 1650, BAn4) 16.66 Primer 04 6 6 1 (1500, SkNAn5) 16.66 Primer 05 7 7 4 (200, ANAn1; 650 MeAn4; 800, MeAn4; 1500,MeAn4) 57.14 Primer 06 5 4 2 (400, MeAn4; 500, JaAn2) 40 Primer 07 5 5 - - Primer 08 7 7 - -

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Fig. 22. RAPD profile of Aspergillus niger isolates obtained with primers, Primer 02 (A), Primer 08 (B), Primer 06 (C) and OPA 16 (D). Lane M-100 bp marker, Lane 1-16 different isolates of Aspergillus niger as lane 1-ANAn4, 2-AnPM3, 3-AAn1, 4-ANAn1, 5-GaAn1, 6- AnC2, 7-BasAn5, 8-VAn4, 9-AnR3, 10-Ban4, 11-BudAn3, 12-JaAn2, 13-LAn3, 14-MeAn4, 15-SkNAn3 and 16-SkNAn5. 113

Fig. 23. Dendrogram of Aspergillus niger isolates, constructed using UPGMA with Jaccard’s similarity Index based on 28 RAPD primers. Number at branch points indicate support for isolates clustered to the right of the number, values are percent of bootstrap sample that exhibit the cluster (no number at branch indicates support less than 10%). The major clusters are indicated on right margin.

LAn3 and SkNAn3; and SkNAn3 and SkNAn5. Whereas the least similarity (0.273) was recorded between AnC2 and Ban4. The isolates BasAn5 and MeAn4; AnR1 and BAn4; BAn4 and JaAn2; BudAn3 and MeAn4, SkNAn5 also showed considerable diversity (32%). Multivariate (cluster) analysis of genetic similarity data grouped the isolates in to three major clusters (Group I, Group II and Group III) and five major subgroups (IAa, IAb, IB, IIIA and IIIB) (Fig. 23). The Group I consisted of 10 isolates sharing 63.5 to 89.1% genetic similarity and divided into three main groups viz. IAa, IAb, IB (Fig. 23). Two isolates, AnC2 and VAn4 positioned themselves separately in sub-cluster of IAb of Group I. Isolates AAn1and GaAn1 formed separate sub group IIIA of III Group, BAn4 and BudAn3 formed another sub group IIIB of Group III with genetic similarity 68.0% and 70.0%, respectively (Fig. 23). Two isolates ANAn1and MeAn4 formed Group II sharing 47.7% similarity (Fig. 23). Bootstrap analysis used to evaluate the degree of support for clusters within the dendrogram has revealed that out of the groups and subgroups classified, coefficient of probability for reproducibility ranged 80-90% for I and III Groups, 60-29% for Group II, all subgroups of Group III and all subgroups of Group I except three sub clusters (IAa; AnR3 and VAn4, AnC2; SkNAn5 and VAn4, AnC2). These three sub clusters of Group I showed 20-30% probability (Fig. 23). 114

Ochratoxin A production None of the isolates produced ochratoxin A (< 1ηg/g) or produced 0.2 ηg/g (by BudAn3), or 0.3 ηg/g (by AAn1, GaAn1, MeAn4), or 0.4 ηg/g (by BAn4) (Table 19). In rest of the isolates (BuAn3, BasAn5, JaAn2, LAn3, SkNAn3, SkNAn5, VAn4, ANAn1, ANAn4, AnC2 and AnR3) production of ochratoxin A was not detected. Phosphate solubilization In Petri dishes all A. niger isolates solubilized phosphate from 20-27 mm. The quantitative estimation of phosphorus solubilization in liquid medium showed solubilization from 5.1- 11.2 (µg P/ml). Very high significant differences in solubilization of phosphate at P≤ 0.0001 level were seen among the isolates. The A. niger isolate SkNAn5, VAn4, AnC2 were the most efficient (P≤ 0.0001) isolates followed by AnR3, ANAn4 and BuAn3 with regard to phosphate solubilization (Table 19). Compatibility of Aspergillus niger isolates with pesticides The compatibility test of the A. niger with pesticides varied with isolates and the maximum inhibition in the growth of the fungus (ED90) was recorded at 530-660 µg carbendazim/ml, 945-1210 captan/ml, 705-820 µg mancozeb/ml, 2325-2650 µg metalaxyl/ml, 160-182 µg thiram/ml, 39-49 µg carbofuran/ml and 1180-1320 µg nemacur/ml (Table 20). The pesticide concentrations at 60-83 µg carbendazim/ml, 151-178 µg captan/ml , 220-265 µg mancozeb/ml, 700-1150 µg metalaxyl/ml, 26-35 µg thiram/ml, 20-29 µg carbofuran/ml and

960-1050 µg nemacur/ml medium appeared to be safe tolerance limits (ED50) for A. niger isolates (Table 20). Tolerance for metalaxyl was about 5 times greater than carbendazim as

2325-2650 µg of metalaxyl and 530-660 µg carbendazim/ml inhibited 90% (ED90) growth of A. niger isolates. The isolates also showed 33-36 times more tolerance for nemacur than carbofuran (Table 20). Compatibility with the pesticides of these isolates significantly (P≤ 0.0001) varied and the order was SkNAn5, VAn4 > AnC2, AnR3 > ANAn4, BuAn3 > rest of the A. niger isolates (Table 20).

Compatibility of Aspergillus niger isolates with toxic heavy metals and biosorption The tolerance of toxic metals in the form of maximum inhibitory concentration (MIC) by A. niger isolates depicted 350-400 µg Ni+2/ml, 150-175 µg Cd+2/ml and 350-400 µg Cr+6/ml medium (Table 21). The MIC of heavy metals significantly (P≤ 0.0001) varied with the isolates and order was SkNAn5, VAn4, AnC2 > AnR3, ANAn4, ANAn1 > BuAn3 > rest of the A. niger isolates (Table 21).

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Table 19. Ochratoxin A production and phosphate solubilization of efficient Aspergillus niger isolates. A. niger Ochratoxin A Phosphate solubilization isolate (ηg/g) In Petri plate (diameter in mm) In liquid medium Dia. of halo Dia. of colony Difference (µg P/ml) AAn1 0.3 86 64 22b 7.3b ANAn1 0 85 64 21c 6.5c ANAn4 0 86 64 22b 8.9b AnC2 0 90 65 25a 10.0a AnR3 0 82 59 23b 8.6b BAn4 0.4 75 54 21c 5.4c BasAn5 0 82 61 21c 6.0c BuAn3 0 69 47 22b 7.5b BudAn3 0.2 84 64 20c 5.2c GaAn1 0.3 80 59 21c 5.8c JaAn2 0 86 66 20c 5.3c LAn3 0 72 52 20c 5.1c MeAn4 0.3 79 59 20c 5.3c SkNAn3 0 82 61 21c 5.9c SkNAn5 0 90 63 27a 11.2a VAn4 0 86 60 26a 10.1a LSD (P≤0.0001) 2.0 2.0 Each value is the mean of three replicates. Means followed by different alphabets in column are significantly different from each other at P≤0.0001.

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Table 20. In vitro compatibility of selected efficient Aspergillus niger isolates with some common fungicides and nematicides.

A. niger Carbendazim Captan Mancozeb Metalaxyl Thiram Carbofuran Nemacur isolate MTC* MIC MTC MIC MTC MIC MTC MIC MTC MIC MTC MIC MTC MIC AAn1 62e 560e 157c 1040d 235f 740d 735e 2425e 26e 161h 21f 40h 970g 1210c ANAn1 60f 540f 155c 1025e 220i 715f 720f 2375f 26e 160h 20g 39i 970g 1200d ANAn4 65d 590c 155c 1040d 245d 760c 1080c 2525c 28c 172c 24c 43e 1010c 1230c AnC2 70c 600c 165b 1100b 250c 790b 1100b 2500c 30b 175b 25b 45c 1020b 1260b AnR3 69c 590c 158c 1050c 245d 770c 1090b 2525c 30b 173c 24c 44d 1010c 1240b BAn4 63e 560e 158c 1045c 240e 755d 750d 2475d 27d 163g 22e 41g 990e 1210c BasAn5 60f 550f 156c 1035e 225h 720f 710f 2350f 26e 165f 21f 41g 980f 1200d BuAn3 65d 580d 159c 1055c 240e 760c 1070c 2500c 28c 171d 23d 42f 1000d 1230c BudAn3 61f 550f 155c 1030e 230g 720f 720f 2400e 27d 168e 20g 40h 970g 1200d GaAn1 55g 530g 154d 980f 220i 710f 710f 2350f 27d 165f 20g 39i 970g 1190d JaAn2 62e 565e 156c 1040d 235f 750d 740e 2450d 26e 163g 21f 42f 1000d 1225c LAn3 55g 540f 151e 945g 220i 705g 700g 2325g 27d 169e 20g 40h 970g 1200d MeAn4 60f 555e 155c 1025e 225h 715f 715f 2370f 27d 169e 20g 39i 960h 1180d SkNAn3 63e 560e 157c 1050c 240e 735e 755d 2475d 26e 162g 22e 40h 1000d 1225c SkNAn5 83a 660a 178a 1210a 265a 820a 1150a 2650a 35a 182a 29a 49a 1050a 1320a VAn4 81b 640b 175a 1200a 260b 810a 1140a 2600b 35a 181a 28a 48b 1050a 1300a LSD (P≤0.001) 1.9 12.5 3.3 11.6 4.4 10.4 10.5 26.9 0.8 1.2 1.0 0.9 8.1 20.8 Each value is the mean of three replicates. Means followed by different alphabets in column are significantly different from each other at P≤0.0001. MTC – Maximum tolerance concentration, MIC – Maximum inhibition concentration; * Concentrations are in (µg/ml).

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Table 21. Minimum inhibitory concentrations (MIC) of Ni+2, Cd+2, or Cr+6 for some selected isolates of Aspergillus niger. A. niger Minimum inhibitory concentrations (µg/ml) isolate Ni+2 Cd+2 Cr+6 AAn1 375b 175a 350c ANAn1 400a 150b 400a ANAn4 400a 175a 375b AnC2 400a 175a 400a AnR3 400a 175a 375b BAn4 375b 150b 350c BasAn5 350c 150b 350c BuAn3 375b 175a 375b BudAn3 350c 150b 350c GaAn1 375b 150b 350c JaAn2 350c 150b 350c LaAn3 375b 150b 350c MeAn4 350c 150b 350c SkNAn3 400a 175a 400a SkNAn5 400a 175a 400a VAn4 400a 175a 400a LSD (P≤0.0001) 11.3 9.5 11.1 Each value is the mean of three replicates. Means followed by different alphabets in column are significantly different from each other at P≤0.0001.

Varying levels of metal biosorption by A. niger isolates were found in single and multi-metal systems. In single or multi-metal system biosorption of Ni+2, Cd+2, and Cr+6 ions by the A. niger isolates varied with initial metal concentration of the medium and was found more at 4 mM concentration than 2 or 6 mM (Table 22-23). In single-metal system A. niger isolates adsorbed Ni+2 more than Cd+2 than Cr+6 (Table 22). In multi-metal system +6 +2 +2 adsorption of Cr was more than Cd whereas, Ni was adsorbed maximum as adsorbed in single-metal system (Table 23). In single-metal system A. niger isolates adsorbed Ni+2 6.3-6.7, 25.5-29.6 and 17.3-20.2 mg/g biomass at 2, 4 and 6 mM metal concentration, respectively; Cd+2 7.2-8.6, 19.4-21.4 and 16.8-18.1 mg/g biomass at 2, 4 and 6 mM metal concentration, respectively; and Cr+6 7.4-8.5, 18.2-19.5 and 16.0-16.6 mg/g biomass at 2, 4 and 6 mM metal concentration, respectively (Table 22). In multi-metal system, A. niger isolates adsorbed Ni+2 4.6-5.9, 13.7-16.3 and 8.6- 10.0 mg/g biomass at 2, 4 and 6 mM metal concentration, respectively; Cd+2 3.7-4.5, 10.2-12.3 and 7.4-9.9 mg/g biomass at 2, 4 and 6 mM metal concentration, respectively; and Cr+6 3.3-4.4, 11.4-13.2 and 8.1-9.2 mg/g

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biomass at 2, 4 and 6 mM metal concentration, respectively (Table 23). The biosorption of heavy metals by the A. niger isolates varied at P≤ 0.05 (2 mM concentration) and P≤ 0.01 (4, 6 mM concentration) for single and multi-metal systems and order was SkNAn5 > VAn4 > AnC2 > AnR3 > ANAn4 > BuAn3 > Ban4 > ANAn1 > rest of the A. niger isolates (Table 22, 23). Mycoparasitism A. niger isolates interacted readily with the mycelium of F. oxysporum f. sp. ciceri and F. oxysporum f. sp. lycopersici as was evident by the overgrowth and lysis of mycelium of the pathogenic fungi by A. niger isolates. Microscopic examination of mycelium from the zone of interaction revealed parallel running and coiling of the Fusarium mycelium by the mycelium of A. niger isolates. All the A. niger isolates completely utilized the Fusarium mycelium in 6-10 days on PDA plates (Fig. 24). Dual culture test In dual culture test, no isolates overgrew F. oxysporum f. sp. ciceri or F. oxysporum f. sp. lycopersici in 5 or 6 days after inoculation. F. oxysporum f. sp. ciceri and F. oxysporum f. sp. lycopersici were overgrown by SkNAn5, VAn4, AnC2 in 7 days, by AnR3, ANAn4, BuAn3 and Ban4 in 8 days and by rest of the isolates in 9 or 10 days of inoculation. Mean of 6, 7, 8, 9 and 10 days percentage inhibition of the F. oxysporum f. sp. ciceri (70-78%) or F. oxysporum f. sp. lycopersici (67-76%) mycelium by the A. niger isolates revealed wide variation among the isolates even at P≤ 0.0001 level of probability. The order of effectiveness of inhibition by isolates was SkNAn5, VAn4, AnC2, AnR3 > ANAn4, BuAn3 > rest of the A. niger isolates (Table 24). Effect of volatile compounds Effects of 5 days old cultures of A. niger isolates for 2, 4, 8 and 10 days were tested against F. oxysporum f. sp. ciceri and F. oxysporum f. sp. lycopersici. Volatile compounds produced by 5 days old isolates of A. niger isoaltes significantly (P≤ 0.0001) reduced the radial mycelial growth of F. oxysporum f. sp. ciceri (41-58%) or F. oxysporum f. sp. lycopersici (34-53%). Among the A. niger isolates, suppression of Fusarium growth through volatile compounds varied greatly (P≤ 0.0001), which depicted the variation in the amount of production of volatile compounds by the isolates. The order of A. niger isolates which produced efficiently more amount of volatile compounds are SkNAn5 > VAn4 > AnC2, AnR3 > ANAn4 > BuAn3 > Ban4 > ANAn4 > rest of the A. niger isolates (Table 24, Fig. 25 ).

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Table 22. Bioadsorption of heavy metals by some selected isolates of Aspergillus niger (mg/g of biomass ) in single metal system of Ni+2, Cd+2, or Cr+6. Bioadsorption of heavy metals (mg/g of biomass of A. niger)

Ni+2 (mM) Cd+2 (mM) Cr+6 (mM) A. niger isolate 2* 4 6 2* 4 6 2* 4 6 AAn1 6.5a 27.9c 18.0c 7.8b 19.4d 17.4b 7.9c 18.8b 16.0b ANAn1 6.4b 28.3b 18.8b 8.1b 20.3b 17.5b 8.4a 19.2a 16.2a ANAn4 6.5a 28.7b 18.9b 8.1b 20.3c 17.5b 8.1b 19.0a 16.1a AnC2 6.5a 28.9b 19.2b 8.3a 20.9b 17.8a 8.3a 19.0a 16.1a AnR3 6.5a 28.9b 19.2b 8.2b 20.8b 17.6a 8.2b 19.0a 16.1a BAn4 6.3b 27.9c 18.2c 7.9b 20.5b 17.4b 8.0c 18.9b 16.0 BasAn5 6.3b 28.0c 18.2c 7.9b 20.3c 17.4b 8.1b 18.9b 16.0 BuAn3 6.5a 27.9c 18.0c 7.8b 20.6b 17.6a 8.2b 19.0a 16.1a BudAn3 6.3b 27.9c 18.1c 7.8b 20.3c 17.4b 8.1b 18.9b 16.0b GaAn1 6.3b 27.8c 18.1c 7.9b 20.3c 17.4b 8.0c 18.9b 16.0b JaAn2 6.3b 28.0c 18.5b 8.0b 19.5d 17.5b 8.1b 18.9b 16.0b LaAn3 6.3b 27.9c 18.0c 7.8b 19.4d 17.5b 8.0c 18.9b 16.0b MeAn4 6.4b 25.5d 17.3d 7.2c 19.4d 16.8c 7.4d 18.2c 15.6b SkNAn3 6.4b 28.3b 18.8b 8.2b 20.8b 17.6a 8.3a 19.1a 16.2a SkNAn5 6.7a 29.6a 20.2a 8.6a 21.4a 18.1a 8.5a 19.5a 16.6a VAn4 6.5a 28.5b 19.1b 8.3a 20.9b 17.8a 8.4a 19.2a 16.3a LSD (P≤0.05) 0.29 0.41 0.64 0.44 0.33 0.37 0.19 0.35 0.38 LSD (P≤0.01) 0.41 0.57 0.82 0.59 0.45 0.50 0.26 0.47 0.50 Each value is the mean of three replicates. * Means followed by different alphabets in the column are significantly different from each other at P≤0.05 and in other column at P≤0.01.

120

Table 23. Bioadsorption of heavy metals by efficient Aspergillus niger isolates (mg/g of biomass ) in multi metal system of Ni+2, Cd+2, or Cr+6. Bioadsorption of heavy metals (mg/g of biomass of A. niger)

A. niger isolate Ni+2 (mM) Cd+2 (mM) Cr+6 (mM)

2* 4 6 2* 4 6 2* 4 6 AAn1 5.3c 15.8b 9.5c 3.8b 10.2d 7.4d 3.3c 11.4c 8.1c ANAn1 5.4c 15.8b 9.7b 4.0b 10.8c 9.1b 4.0b 12.5b 8.6b ANAn4 5.5b 15.9a 9.6b 3.9b 11.4b 8.9c 4.0b 12.6a 8.6b AnC2 5.5b 15.9a 9.6b 4.1a 11.5b 9.0b 4.1a 12.8a 8.9a AnR3 5.5b 15.9a 9.6b 4.0b 11.3b 8.9c 4.1a 12.7a 8.8b BAn4 5.3c 15.7b 9.5c 3.9b 10.8c 8.9c 4.0b 12.5b 8.7b BasAn5 5.3c 15.7b 9.1d 3.8b 10.6c 7.7d 3.8b 12.0b 8.5b BuAn3 5.5b 15.9a 9.7b 3.9b 11.1b 8.6c 4.1a 12.6a 8.7b BudAn3 5.3c 15.7b 9.4c 3.8b 10.5c 7.6d 3.5c 11.9c 8.4c GaAn1 5.3c 15.6b 9.4c 3.8b 10.6c 7.6d 3.5c 11.9c 8.4c JaAn2 5.3c 15.7b 9.5c 3.9b 10.6c 7.7d 3.7b 11.9c 8.5b LaAn3 5.3c 15.7b 9.5c 3.9b 10.8c 8.9c 4.0b 12.7a 8.4c MeAn4 4.6d 13.7c 8.6e 3.7b 10.4d 7.6d 3.5c 11.9c 8.4c SkNAn3 5.4c 15.9a 9.7b 4.0b 10.8c 9.0b 4.0b 12.6a 8.7b SkNAn5 5.9a 16.3a 10.0a 4.5a 12.3a 9.9a 4.4a 13.2a 9.2a VAn4 5.6b 16.0a 9.8a 4.2a 11.6b 9.4b 4.2a 12.9a 8.9a LSD (P≤0.05) 0.12 0.27 0.15 0.43 0.44 0.33 0.31 0.45 0.21 LSD (P≤0.01) 0.16 0.37 0.20 0.53 0.60 0.44 0.42 0.61 0.28 Each value is the mean of three replicates. * Means followed by different alphabets in the column are significantly different from each other at P≤0.05 and in other column at P≤0.01.

121

Table 24. Inhibition in the colonization by Fusarium oxysporum f. sp. ciceri and F. oxysporum f. sp. lycopersici due to volatile compounds, culture filtrates and dual culture with Aspergillus niger isolates in vitro. A. niger Fusarium oxysporum f. sp. Fusarium oxysporum f. sp. ciceri lycopersici

isolate % inhibition % inhibition VC DCT Culture filtrates VC DCT Culture filtrates 10% 25% 50% 10% 25% 50% AAn1 50d 72c 19d 57d 83c 43d 72b 18c 53e 81c ANAn1 49d 72c 17e 55e 80c 42d 71b 18c 50f 80d ANAn4 53c 75b 22b 63c 86b 47c 73b 20b 54e 83c AnC2 55b 76a 23b 65b 90a 50b 74a 22a 58c 86b AnR3 54b 76a 22b 63c 87b 48b 74a 21b 56d 83c BAn4 51c 73b 19d 58d 85b 44d 73b 19c 41i 82c BasAn5 43e 70d 16e 51g 68e 36g 69c 14e 50f 65f BuAn3 52c 74b 21c 62c 86b 46c 73b 20b 55d 81c BudAn3 47d 71c 17e 54e 73d 41e 70c 17d 49f 78d GaAn1 47d 71c 18d 54e 70e 40e 70c 16d 47g 77e JaAn2 44e 70d 17e 51g 69e 37f 68c 15e 41i 75e LAn3 44e 71c 16e 53f 71e 38f 68c 15e 44h 77e MeAn4 41f 70d 16e 50g 64f 34g 67d 14e 39j 58g SkNAn3 47d 72c 17e 55e 75d 41e 70c 17d 50f 78d SkNAn5 58a 78a 25a 68a 93a 53a 76a 23a 63a 89a VAn4 56b 76a 23b 66b 90a 51a 75a 22a 60b 87a LSD (P≤0.0001) 1.9 1.9 1.1 1.4 3.3 2.1 2.2 1.9 1.9 2.0 Each value is the mean of three replicates. VC - volatile compounds, CF - culture filtrates, DCT - dual culture test. Values of VC are the mean of 2, 4, 8 and 10 days of inhibition; values of DCT are the mean of 6, 7, 8, 9 and 10 days of inoculation. Means followed by different alphabets in column are significantly different from each other at P≤0.0001.

122

Fig. 24. Antifungal activity of Aspergillus niger SkNAn5 against Fusarium oxysporum f. sp. ciceri (A) and F. oxysporum f. sp. lycopersici (B) in dual culture test.

Effect of non-volatile compounds (culture filtrates) When F. oxysporum f. sp. ciceri or F. oxysporum f. sp. lycopersici were grown on the PDA amended with 10, 25 and 50% concentrations of culture filtrates of A. niger isolates, radial growth of the pathogen was inhibited (P≤ 0.0001) compared to PDA without culture filtrates. The maximum decrease of 64-93% in the radial growth of F. oxysporum f. sp. ciceri and 58-89% of F. oxysporum f. sp. lycopersici was recorded with 50% filtrate of A. niger isolates (Table 24).

Fig. 25. Effects of volatile compounds of Aspergillus niger SkNAn5 on the growth of Fusarium oxysporum f. sp. ciceri and F. oxysporum f. sp. lycopersici. 123

Nematicidal effects of Aspergillus niger isolate in-vitro Nematode hatching and mortality The hatching of juveniles from egg masses in potato dextrose broth (not inoculated with A. niger) and distilled water was statistically identical (P≤ 0.05). The hatching, however, was inhibited in culture filtrates (P≤ 0.001). The hatching decreased by 30-55%, 46-68%, 66- 79% and 77-85% in 25, 50, 75 and 100% culture filtrates of A. niger compared with the control (broth alone) after 5 days of incubation, respectively (Table 25). The culture filtrates of A. niger isolates also induced mortality (P≤ 0.001) to the hatched juveniles of M. incognita (Table 25). The juvenile mortality was 25-32% and 100% with the 25 and 50% (onwards) culture filtrate of A. niger isolates after 24 hrs of incubation, respectively (Table 25). Among A. niger isolates a significant difference (P≤ 0.001) in decrease in egg hatching and increase in juveniles mortality was seen and order was SkNAn5 > VAn4 > AnC2 > AnR3 > ANAn4 > BuAn3 > Ban4 > ANAn1 > rest of the A. niger isolates (Table 25). Conclusion On the basis of preliminary evaluation of A. niger isolates done for some important in vitro tests viz., monoculture growth rate, ammonia production, siderophore production, hydrogen cyanide and indole acetic acid production, phosphate solubilization and Ochratoxin A production, compatibility with pesticides and toxic heavy metals, in vitro mycoparasitism and nematicidal effect, sixteen isolates viz., AAn1, BAn4, BuAn3, BasAn5, BudAn3, GaAn1, JaAn2, LAn3, MeAn4, SkNAn3, SkNAn5, VAn4, ANAn1, ANAn4, AnC2 and AnR3 performed more efficiently and qualified the parameters tested for being a good biocontrol agents.

124

Table 25. Effect of culture filtrates of efficient Aspergillus niger isolates on hatching of eggs and mortality to the juveniles of Meloidogyne incognita in vitro. Concentration of culture filtrate of Aspergillus niger in distilled water

A. niger 25% 50% 75% 100% isolates Hatching (%) Mortality (%) Hatching (%) Mortality (%) Hatching (%) Mortality (%) Hatching (%) Mortality (%) Control(PDB) 100g (90) 0 (0) 100g (90) 0 (0) 100c (90) 0 (0) 100c (90c) 0 (0) AAn1 39c (54.2) 28c (26c) 56d (40.0) 100 (90) 71c (25.7) 100 (90) 80b (17.8b) 100 (90) ANAn1 38d (55.4) 32b (29b) 55d (40.7) 100 (90) 71c (25.9) 100 (90) 80b (17.8b) 100 (90) ANAn4 44b (50.6) 30c (27c) 54e (41.2) 100 (90) 69d (28.0) 100 (90) 81b (17.2b) 100 (90) AnC2 50a (42.2) 36a (32a) 55d (40.7) 100 (90) 77b (21.1) 100 (90) 84a (14.8a) 100 (90) AnR3 53a (42.4) 34a (31a) 66b (30.6) 100 (90) 77b (20.5) 100 (90) 84a (14.4a) 100 (90) BAn4 40c (53.8) 29b (29b) 55d (40.7) 100 (90) 71d (26.4) 100 (90) 81b (17.2b) 100 (90) BasAn5 30f (62.8) 30c (27c) 49f (46.0) 100 (90) 67e (29.5) 100 (90) 80b (17.8b) 100 (90) BuAn3 44b (50.6) 29c (26c) 60c (36.3) 100 (90) 74b (23.2) 100 (90) 80b (18.2b) 100 (90) BudAn3 33e (59.9) 31b (28b) 49f (46.3) 100 (90) 70d (26.9) 100 (90) 80b (17.8b) 100 (90) GaAn1 34e (59.5) 32b (29b) 51e (43.9) 100 (90) 69d (27.8) 100 (90) 80b (17.6b) 100 (90) JaAn2 31f (61.6) 33a (30a) 54e (41.3) 100 (90) 69d (27.4) 100 (90) 80b (17.8b) 100 (90) LAn3 31f (61.8) 28c (25c) 46f (48.6) 100 (90) 66e (31.0) 100 (90) 77b (21.1b) 100 (90) MeAn4 30f (62.9) 30c (27c) 49f (46.0) 100 (90) 67e (29.5) 100 (90) 80b (17.8b) 100 (90) SkNAn3 35e (57.9) 31b (28b) 53e (42.2) 100 (90) 69d (28.0) 100 (90) 80b (17.6b) 100 (90) SkNAn5 55a (40.7) 36a (32a) 68a (28.9) 100 (90) 79a (18.8) 100 (90) 84a (14.4a) 100 (90) VAn4 54a (41.3) 34a (31a) 64b (32.0) 100 (90) 78a (20.0) 100 (90) 85a (13.9a) 100 (90) LSD (P≤0.001) 2.8 2.7 2.8 2.8 4.0 Each value is the mean of three replicates; Figures in parenthesis are angular transformed values; PDB - Potato dextrose broth; Means followed by different alphabets in column are significantly different from each other at P≤0.001.

125

Experiment-III EVALUATION OF ASPERGILLUS NIGER ISOLATES FOR ANTAGONISM AGAINST FUSARIUM SPP. AND MELOIDOGYNE INCOGNITA AND FOR PROMOTION OF PLANT GROWTH AND YIELD OF CHICKPEA AND TOMATO UNDER POT CONDITION Symptoms Fusarial wilt Inoculation with the wilt fungi, Fusarium oxysporum f. sp. ciceri or F. oxysporum f. sp. lycopersici caused characteristic symptoms of wilt disease of chickpea and tomato, respectively. The first sign of the disease was stunted growth and mild chlorosis that appeared at seedling stage. Some plants at the seedling stage exhibited drooping that led to their mortality. The seedlings which escaped early infection showed chlorosis and stunted growth at one month of age. At the advanced stage of plant growth, the whole plant, branches or twigs become brown and subsequently dried and died (Fig. 26). The severity of chickpea and tomato wilt on an average was 3.5 and 3.8 on 0-5 scale, respectively (Fig 27- 28). Treatments of Aspergillus niger decreased the severity of wilt symptoms. Decrease in the disease was 15-68% (seed treatment) and 13-59% (soil application) in chickpea; 12-60% (nursery treatment) and 9-51% (soil application) in tomato over respective controls. Application of carbendazim controlled the wilt severity in chickpea by 62 and 53% with seed and soil application, respectively, whereas in tomato 65-55% decrease in the disease was recorded with nursery and soil application, respectively (Fig. 27-28). Among the A. niger isolates, SkNAn5 isolate was found the most effective against the disease and suppressed the wilt severity by 60-68%, followed by VAn4 (57-65% decrease), AnC2 (56- 65%), AnR3 (54,62%), ANAn4 (54-61%) and BuAn3 (53-60%) (Fig. 27-28). The overall performances of the treatments was SkNAn5 > VAn4 > AnC2 > carbendazim > AnR3 > ANAn4 > BuAn3.

Fig 26. Chickpea plants showing symptoms of Fusarium wilt. Uninoculated healthy plants (A), browning and wilting of shoot at young (B) and maturing stage (C). 126

Mi Foc+Mi Mi Foc+Mi

SEED TREATMENT SOIL APPLICAITON

Mi Foc+Mi Mi Foc+Mi

Fig. 27. Effects of seed treatment and soil application of Aspergillus niger isolates on the wilt severity (0-5 scale) of chickpea caused by Fusarium oxysporum f. sp. ciceri , galling and egg mass production of Meloidogyne incognita alone or with F. oxysporum f. sp. ciceri. Vertical bars indicate the standard error of three replicates. Mi – M. incognita, Foc – F. oxysporum f. sp. ciceri, Czm – carbendazim, Cbf - carbofuran.

127

Fig. 28. Effects of nursery treatment and soil application of Aspergillus niger isolates on the wilt severity (0-5 scale) of tomato caused by Fusarium oxysporum f. sp. lycopersici, galling and egg mass production of Meloidogyne incognita alone or with F. oxysporum f. sp. lycopersici. Vertical bars indicate the standard error of three replicates. Mi – M. incognita, Fol – F. oxysporum f. sp. lycopersici, Czm – carbendazim, Cbf - carbofuran. 128

Root-knot symptoms

Chickpea or tomato plants inoculated with 2000 J2 of Meloidogyne incognita/pot, exhibited stunted growth and mild chlorosis. On an average 62 galls and 58 egg masses/root system were recorded on chickpea and 89 galls and 82 egg masses/root system on tomato (Fig. 27- 29). The applied treatments suppressed the gall formation and egg mass production but to a varied extent. Seed treatment with A. niger isolates resulted to greater suppression in the number of galls (23-54%) and egg masses (22-61%) than soil application (22-43% galls; 20-46% egg masses) on chickpea. In the same manners nursery treatment of tomato caused greater suppression in the number of galls (18-61%) and egg masses (18-67%) than soil application (decreased 14-44% galls; 14-48% egg masses). Application of carbofuran caused 34-47% and 31-46% decrease in the number of galls and egg masses, respectively in both the crops. The isolate SkNAn5 caused greatest suppression in the galling (51-54%) and egg mass production (53-61%), followed by VAn4 (41-48%; 43-49%), AnC2 (40-48%; 35- 46%), AnR3 (36-42%; 33-43%), ANAn4 (31-42%; 30-42%) and BuAn3 (29-39%; 29-37%) (Fig. 27-28). Effectiveness of carbofuran to suppress root-knot disease was less than AnC2 (P≤ 0.0001) and better than AnR3 isolate (P≤ 0.001). Wilt disease complex Severity of wilt caused by F. oxysporum f. sp. ciceri or F. oxysporum f. sp. lycopersici in chickpea and tomato increased significantly (P≤ 0.01) in the pots also infested with M. incognita but gall formation and egg mass production were lower (P≤ 0.01) in comparison to the pots with either pathogen (Fig. 27-28). Application of A. niger isolates resulted to considerable decrease in the severity of wilt and root-knot. Seed treatment of A. niger isolates checked wilting in chickpea (20-62%) relatively better than soil application (19- 56%) over concomitantly inoculated control. In the same way nursery treatment of A. niger isolates resulted to tomato wilting control (21-64%) better than soil application (19-55%) over concomitant inoculated control. The combined treatment of carbendazim and carbofuran however, checked the wilt symptoms by 39-56% and 39-57% in chickpea and tomato, respectively. Gall formation and egg mass production of concomitantly inoculated plants were reduced by 20-80% and 19-75% due to seed and soil application of A. niger isolates in chickpea, respectively in both the crops. In tomato, nursery and soil treatment of A. niger led to 9-38% decrease in gall formation and 24-52% in egg mass production in concomitantly inoculated plants. Soil application or seed/nursery treatment with carbendazim+carbofuran suppressed the gall formation (24-35%) and egg mass production (33-67%) (Fig. 27-28). Among the A. niger isolates the order of effectiveness (P≤ 0.01) 129

against the wilt and root-knot on chickpea and tomato was SkNAn5 > VAn4 > AnC2 > AnR3 > ANAn4 > BuAn3 rest of the A. niger isolates (Fig. 27-28). Effect of carbendazim+carbofuran in controlling the wilt in concomitantly inoculated plants was less than AnC2 and better than AnR3 isolate.

Fig 29. Roots of chickpea (A) and tomato (B) showing severe galling caused by Meloidogyne incognita .

Dry matter production and yield Chickpea plants applied with A. niger isolates produced dry matter 17-58% and yield 21- 52% greater than those not applied with the isolates (Table 26). With same treatments dry matter of tomato increased by 17-60% and the yield by 21-54% (Table 27). This growth promoting effect was greater with seed/nursery treatment (Fig. 30). Infection by the wilt fungus in chickpea and tomato suppressed the dry matter production and yield by 20 and 18%, and 26 and 29% over control, respectively (Table 26- 27). Application of A. niger treatments compensated the yield loss but to a varying extent. Seed treatment with A. niger isolates promoted the dry matter production and yield of chickpea by 25-89% and 31-84%, respectively. Corresponding values for soil application were 22-88% and 31-82%, respectively (Table 26). In tomato, A. niger isolates promoted the dry matter (25-94%) and yield (30-88%) with nursery treatment, and 24-91% and 21- 85% with soil application, respectively (Table 27). The plants also gave significantly greater yield with carbendazim treatment. Inoculation of chickpea and tomato plants with root-knot nematode, M. incognita resulted to dry matter (13 and 19%) and yield (11 and 18%) less than the control, respectively (Table 26-27). Application of A. niger isolates checked the suppressive effect of the nematode and significantly promoted the dry matter and yield of nematode infected plants in comparison to the nematode inoculated control, the effect was 2-5% greater with 130

Fig. 30. An aerial view of pot experiments conducted to evaluate the effects of seed/nursery treatment with Aspergillus niger isolates on wilt, root-knot and wilt disease complex of chickpea and tomato. the seed or nursery treatment than soil application. Seed or nursery treatment with A. niger isolates increased the yield by 14-37 in chickpea and 14-38% in tomato. Whereas soil application with A. niger increase the yield by 15-34% in chickpea and 14- 36% in tomato (Table 26-27). The yield of chickpea and tomato were also significantly improved due to application with carbofuran. Concomitant inoculations with the wilt fungus and root-knot nematode greatly suppressed the dry matter production (43 and 52%) and yield (39 and 53%) of chickpea and tomato over uninoculated control, respectively (Table 26-27). Application of A. niger isolates significantly improved the considered variables of infected plants over concomitantly inoculated control. Enhancements in the yield of infected chickpea plants recorded with seed/nursery and soil treatment were 22-58% and 21-56% greater than concomitantly inoculated control, respectively. The yield of infected tomato plants increased by 23-60% with nursery treatment and 22-56% with soil application of A. niger isolates compared to the concomitantly inoculated control. Enhancement in the dry matter with A. niger isolates was 2-5% greater than yield enhancement. Seed/nursery or soil treatment with carbendazim+carbofuran significantly (P≤ 0.0001) increased the dry matter

131

Table 26. Effects of seed treatment and soil application of efficient Aspergillus niger isolates on the dry matter production and yield of chickpea in pots inoculated with Fusarium oxysporum f. sp. ciceri, Meloidogyne incognita singly and concomitantly. Dry shoot weight (g) Yield/plant (g)

Treatment Seed treatment Soil application Seed treatment Soil application Control 18.4 18.4 6.6 6.6 AAn1 25.5 (38.7) 25.2 (37) 8.5 (28.8) 8.3 (26) ANAn1 24.9 (35.3) 24.7 (34.4) 8.4 (28) 8.4 (27.2) ANAn4 27.3 (48.3) 28.3 (53.9) 9 (36.8) 9.2 (39.9) AnC2 28.2 (53.2) 27.8 (51) 9.6 (44.9) 9.5 (43.8) AnR3 28.2 (53.5) 28.1 (52.8) 9.3 (41.3) 9.5 (44.2) BAn4 25.7 (39.9) 24.8 (35) 8.5 (28.7) 8.5 (28.3) BasAn5 23 (25.1) 23.7 (28.9) 7.9 (19.8) 8 (20.5) BuAn3 27.1 (47.5) 27.5 (49.5) 9.1 (37.6) 9 (36.9) BudAn3 24.9 (35.4) 25 (35.7) 8.2 (24.9) 8.1 (23.3) GaAn1 24.4 (32.7) 24 (30.4) 8.2 (23.7) 8.2 (24.6) JaAn2 23.9 (30.1) 24.2 (31.4) 7.9 (20.4) 8 (21.6) LAn3 25 (35.8) 24.3 (32.1) 8.2 (24.4) 8 (20.9) MeAn4 21.5 (17) 21.5 (16.6) 8 (21.1) 8 (21.1) SkNAn3 24.8 (35) 24 (30.4) 8.3 (25.1) 8.2 (24.6) SkNAn5 29.1 (58. 4) 29 (57.3) 10.1 (52.4) 10 (51.7) VAn4 28.3 (54) 27.8 (51.2) 9.8 (48.7) 9.7 (46.3) Carbendazim (Czm) 20.1 (9.1) 19.8 (7.5) 7.1 (6.9) 7 (5.4) Carbofuran (Cbf) 19.8 (7.9) 19.6 (6.6) 7 (6.1) 6.9 (4.6) Control (F) 14.8 (-19. 6) 14.8 (-19.6) 5.4 (-18.4) 5.4 (-18.4) F + AAn1 22.9 (54.9) 22.8 (53.8) 7.7 (42.6) 7.5 (39.4) F + ANAn1 22.4 (51.1) 22.8 (54.1) 7.7 (42.3) 7.5 (39.5) F + ANAn4 25.8 (74.2) 26.7 (80.3) 8.6 (59) 8.7 (61.7) F + AnC2 26.5 (79.4) 26.3 (77.6) 9.2 (71) 9.1 (68.8) F + AnR3 25.9 (75.3) 26.5 (79.2) 8.9 (64.6) 9 (66.2) F + BAn4 23.5 (58.5) 23.3 (57.1) 7.7 (43.3) 7.8 (44.8) F + BasAn5 21 (41.8) 21.2 (42.9) 7.1 (31.3) 6.9 (28.1) F + BuAn3 25.3 (71.3) 25.7 (73.8) 8.5 (56.9) 8.4 (55.1) F + BudAn3 22.1 (49.5) 22.8 (54) 7.3 (35.7) 7.1 (31.5) F + GaAn1 21.7 (46.9) 22.3 (50.6) 7.4 (37.2) 7.3 (36) F + JaAn2 21.7 (46.9) 21.3 (44) 7.1 (30.6) 7.1 (31.2) F + LAn3 22.4 (51.1) 21.8 (47.1) 7.2 (33.5) 7.1 (31.6) F + MeAn4 18.4 (24.5) 18.1 (22.4) 7 (30.1) 7.1 (30.9) F + SkNAn3 22.3 (51) 22.2 (49.9) 7.5 (39) 7.4 (37.1) F + SkNAn5 28 (89.1) 27.8 (87.8) 9.9 (83.9) 9.8 (82) F + VAn4 27.1 (83.3) 27.1 (82.8) 9.4 (74.3) 9.3 (72.6) F + Carbendazim (Czm) 20.2 (36.2) 19.8 (33.8) 8.6 (27.4) 7.5 (39.3)

Continued...

132

Table 26 Continued… Dry shoot weight (g) Yield/plant (g)

Treatment Seed treatment Soil application Seed treatment Soil application Control (N) 15.9 (-13.4) 15.9 (-13.4) 5.9 (-11.3) 5.9 (-11.3) N + AAn1 19.9 (25.2) 20.1 (26.5) 7.1 (20.5) 7 (18.2) N + ANAn1 19.5 (22.6) 19.9 (24.9) 7.1 (20.7) 6.9 (17.8) N + ANAn4 21.6 (35.6) 22.2 (39.5) 7.6 (28.7) 7.7 (31.1) N + AnC2 21.5 (35.2) 22.1 (38.9) 7.8 (32.7) 7.7 (30.9) N + AnR3 21.4 (34.8) 22 (38.4) 7.6 (28.6) 7.8 (32.2) N + BAn4 19.9 (25.3) 20.2 (26.8) 7.2 (21.8) 7.2 (21.4) N + BasAn5 19.1 (19.8) 19.5 (22.7) 6.7 (12.9) 6.7 (14.4) N + BuAn3 21.6 (35.6) 21.4 (34.7) 7.6 (28) 7.4 (25.9) N + BudAn3 19.9 (25) 19.9 (25.1) 6.8 (15.3) 6.8 (15.6) N + GaAn1 19.3 (21.6) 19.8 (24.5) 7 (18.7) 6.9 (17.1) N + JaAn2 19 (19.6) 19 (19.4) 6.7 (14) 6.8 (15.3) N + LAn3 19.6 (23.4) 19.8 (24.5) 6.8 (15.8) 6.8 (15.1) N + MeAn4 18.5 (16.2) 18.3 (14.9) 6.7 (13.9) 6.7 (14.2) N + SkNAn3 19.8 (24.3) 19.5 (22.4) 7.1 (19.6) 7 (18.9) N + SkNAn5 22.5 (41.2) 22.8 (43.6) 8.1 (36.6) 7.9 (33.8) N + VAn4 22 (38.1) 22.4 (40.6) 7.9 (33.9) 7.8 (32) N + Carbofuran (Cbf) 21 (32.2) 20.7 (30.1 8 (36.4) 7.5 (28.1) Control (F+N) 10.5 (-42.7) 10.5 (-42.7) 4.1 (-38.6) 4.1 (-38.6) F + N + AAn1 14.9 (42.3) 14.9 (41.8) 5.2 (27.8) 5.3 (30.3) F + N + ANAn1 14.3 (36.4) 14.3 (36.5) 5.4 (31.5) 5.3 (29) F + N + ANAn4 16.2 (54) 16.7 (58.7) 5.8 (41.4) 5.9 (43.5) F + N + AnC2 16.6 (58.4) 16.2 (54.2) 6.2 (50.1) 6.1 (49.5) F + N + AnR3 16.2 (54.6) 16.5 (57.6) 5.9 (44.7) 6.1 (48.2) F + N + BAn4 15.1 (43.4) 14.8 (40.6) 5.4 (32.5) 5.4 (32.1) F + N + BasAn5 14 (33.3) 13.6 (29.6) 5 (22) 5 (21.1) F + N + BuAn3 15.7 (49.7) 15.8 (50.1) 5.8 (42.1) 5.8 (41.9) F + N + BudAn3 14.5 (38.1) 14.6 (39.4) 5.1 (24.1) 5 (22.5) F + N + GaAn1 14 (33.4) 14.2 (34.8) 5.2 (26.3) 5.2 (27.3) F + N + JaAn2 14.2 (35.1) 13.7 (30.6) 5.1 (24.7) 5 (21.9) F + N + LAn3 14.4 (37.4) 14 (32.9) 5.1 (23.2) 5.1 (23.2) F + N + MeAn4 12.4 (18) 12.6 (19.8) 5 (22) 5 (22.6) F + N + SkNAn3 14.4 (37.6) 14.4 (36.7) 5.2 (26.2) 5.1 (25.5) F + N + SkNAn5 17 (61.6) 17 (61.6) 6.5 (58.3) 6.4 (55.5) F + N + VAn4 16.7 (59.3) 16.7 (59.1) 6.3 (53.4) 6.2 (50.8) F + N + Czm + Cbf 15.1 (44.1) 14.7 (40.3) 5.7 (38.1) 5.4 (32.7) LSD (P≤0.0001) 2.4 2.3 0.33 0.3 F-value (P≤0.0001) Control agents (df=18) 50.8* 41.3* 410* 230* Pathogens (df=2) 41.9* 27.4* 327* 326* Interaction (df=17) 18.7* 72.3* 139* 91* Values in parenthesis are percent increase (+ve) or decrease (-ve) over respective control [plants not inoculated with either pathogen but applied with Aspergillus niger isolates were compared with uninoculated Control; wilt fungus inoculated plants applied with A. niger isolates were compared with fungus inoculated Control (F); nematode inoculated plants applied with A. niger isolates were compared with nematode inoculated Control (N); concomitantly inoculated plants applied with A. niger isolates were compared with concomitantly inoculated Contorl (F+N)]. *Significantly different from the control at P≤0.0001.

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Table 27. Effect of nursery treatment and soil application of efficient Aspergillus niger isolates on the dry matter production and yield of tomato in pots inoculated with Fusarium oxysporum f. sp. ciceri, Meloidogyne incognita singly and concomitantly. Treatment Dry shoot weight (g) Yield/plant (g)

Nursery Soil Nursery Soil treatment application treatment application Control 36.3 36.3 360 360 AAn1 50.6 (39.3) 50.2 (38.2) 468 (30) 458 (27.3) ANAn1 49.5 (36.4) 49.3 (35.9) 462 (28.4) 460 (27.8) ANAn4 54.7 (50.6) 56.9 (56.8) 493 (36.9) 510 (41.8) AnC2 55.7 (53.4) 55.4 (52.6) 527 (46.3) 523 (45.4) AnR3 56.3 (55.2) 56 (54.2) 513 (42.4) 524 (45.5) BAn4 51.3 (41.4) 49.2 (35.5) 464 (28.9) 466 (29.4) BasAn5 45.9 (26.5) 47.1 (29.8) 434 (20.5) 437 (21.5) BuAn3 53.9 (48.6) 55.2 (52.2) 503 (39.6) 494 (37.2) BudAn3 49.7 (37) 49.6 (36.6) 451 (25.4) 448 (24.4) GaAn1 48.8 (34.5) 48 (32.2) 446 (24) 451 (25.2) JaAn2 47.4 (30.6) 48 (32.2) 438 (21.6) 441 (22.6) LAn3 49.7 (36.9) 48 (32.3) 452 (25.5) 436 (21.1) MeAn4 42.6 (17.4) 42.5 (17.1) 440 (22.3) 440 (22.1) SkNAn3 49.6 (36.5) 47.8 (31.7) 453 (25.9) 454 (26) SkNAn5 58 (59.8) 57.3 (57.8) 554 (54) 547 (52) VAn4 56 (54.3) 56 (54.2) 544 (51.1) 533 (47.9) Carbendazim (Czm) 40 (10.3) 38.7 (6.7) 405 (12.4) 381 (5.8) Carbofuran (Cbf) 39.8 (9.7) 38.9 (7.1) 402 (11.8) 374 (3.9) Control (F) 26.8 (-26.3) 26.8 (-26.3) 257 (-28.5) 257 (-28.5) F + AAn1 42 (56.8) 42 (56.9) 367 (42.8) 360 (40.1) F + ANAn1 40.9 (52.6) 41.7 (55.7) 370 (44.1) 363 (41.1) F + ANAn4 47.6 (77.4) 48.5 (80.9) 413 (60.7) 417 (62.1) F + AnC2 48.8 (82.1) 48.8 (82) 442 (72.1) 438 (70.5) F + AnR3 47.6 (77.6) 48.2 (79.7) 423 (64.7) 433 (68.7) F + BAn4 43.2 (61.1) 42.9 (60) 371 (44.4) 379 (47.4) F + BasAn5 38.6 (44) 38.5 (43.7) 341 (32.7) 332 (29) F + BuAn3 46.5 (73.4) 47.5 (77.2) 406 (58.1) 403 (56.8) F + BudAn3 40.3 (50.4) 41.6 (55.3) 351 (36.5) 342 (33.1) F + GaAn1 39.9 (48.7) 41 (53.1) 354 (37.9) 355 (38.1) F + JaAn2 39.5 (47.4) 39.2 (46.3) 338 (31.4) 339 (32.1) F + LAn3 40.8 (52.1) 39.6 (47.9) 344 (33.8) 341 (32.7) F + MeAn4 33.4 (24.5) 33.1 (23.6) 334 (30.1) 336 (21.0) F + SkNAn3 40.8 (52.1) 40.7 (51.9) 361 (40.6) 354 (37.7) F + SkNAn5 52 (94) 51.1 (90.8) 482 (87.5) 476 (85.3) F + VAn4 50 (86.6) 50.1 (86.8) 456 (77.4) 452 (75.9) F + Carbendazim 35.8 (33.6) 34.3 (28.1) 355 (38.2) 315 (22.7) (Czm)

Continued…

134

Table 27 Continued… Treatment Dry shoot weight (g) Yield/plant (g)

Nursery Soil Nursery Soil treatment application treatment application Control (N) 29.3 (-19.2) 29.3 (29.3) 296 (-17.8) 296 (-17.8) N + AAn1 36.8 (25.5) 37.3 (27.3) 358 (20.9) 353 (19.1) N + ANAn1 36.1 (23.1) 36.9 (25.8) 359 (21.3) 351 (18.7) N + ANAn4 39.8 (35.9) 41.2 (40.6) 382 (28.9) 389 (31.5) N + AnC2 39.7 (35.6) 41.1 (40.4) 395 (33.3) 393 (32.7) N + AnR3 39.9 (36.1) 40.8 (39.3) 381 (28.8) 395 (33.5) N + BAn4 36.9 (25.9) 37.5 (27.9) 364 (23) 362 (22.3) N + BasAn5 35.4 (20.7) 36.3 (23.9) 336 (13.5) 339 (14.6) N + BuAn3 40.1 (36.9) 39.8 (35.7) 382 (29) 375 (26.7) N + BudAn3 36.7 (25.4) 36.7 (25.2) 343 (15.8) 345 (16.4) N + GaAn1 35.7 (22) 36.6 (25) 354 (19.5) 348 (17.6) N + JaAn2 35.2 (20.3) 35 (19.6) 339 (14.4) 343 (16) N + LAn3 36.2 (23.5) 36.5 (24.6) 343 (15.9) 341 (15.2) N + MeAn4 34.1 (16.5) 33.7 (14.9) 339 (14.4) 340 (15) N + SkNAn3 36.6 (25) 36.2 (23.4) 356 (20.3) 353 (19.1) N + SkNAn5 41.5 (41.7) 42.8 (46) 407 (37.6) 401 (35.6) N + VAn4 41 (39.8) 41.2 (40.8) 401 (35.3) 394 (33) N + Carbofuran (Cbf) 38.5 (31.5) 36.5 (24.6) 415 (40.2) 387 (30.8) Control (F+N) 17.6 (-51.6) 17.6 (-51.6) 168 (-53.2) 168 (-53.2) F + N + AAn1 25.5 (44.6) 25.3 (43.5) 217 (28.9) 220 (30.9) F + N + ANAn1 24.1 (37.2) 24.1 (37.1) 222 (32.1) 220 (30.7) F + N + ANAn4 27.5 (56) 28.4 (61.6) 240 (42.6) 243 (44.9) F + N + AnC2 28 (59.3) 27.6 (56.9) 255 (52) 256 (52.4) F + N + AnR3 27.7 (57.2) 28.1 (59.7) 245 (46) 253 (50.8) F + N + BAn4 25.4 (44.2) 25 (42.2) 224 (33.2) 225 (33.9) F + N + BasAn5 23.5 (33.5) 22.9 (30) 206 (22.5) 205 (22) F + N + BuAn3 26.4 (50) 26.6 (50.9) 239 (42.1) 242 (44) F + N + BudAn3 24.5 (39.2) 24.6 (39.7) 210 (25.1) 208 (23.7) F + N + GaAn1 23.6 (34.3) 24.1 (36.7) 213 (26.9) 214 (27.5) F + N + JaAn2 24.1 (37) 23.1 (31) 210 (25.2) 207 (23.1) F + N + LAn3 24.5 (39.5) 23.7 (34.7) 207 (23.4) 207 (23.4) F + N + MeAn4 20.8 (18.3) 21.1 (20.1) 206 (22.5) 207 (23.4) F + N + SkNAn3 24.4 (38.4) 24.1 (37.2) 212 (26.2) 212 (26) F + N + SkNAn5 28.5 (62.2) 28.8 (63.4) 268 (59.5) 262 (55.7) F + N + VAn4 28.1 (59.8) 28.3 (61) 261 (55.3) 254 (51.2) F + N + Czm + Cbf 25.1 (42.6) 24.1 (36.8) 253 (50.3) 225 (34.2) LSD (P≤0.0001) 2.4 2.9 7.3 7.3 F-value (P≤0.0001) Control agents 184* 187* 2001* 1554* (df=18) Pathogens (df=2) 111* 225* 1330* 1622* Interaction (df=17) 33.5* 31.3* 347* 463* Values in parenthesis are percent increase (+ve) or decrease (-ve) over respective control [plants not inoculated with either pathogen but applied with Aspergillus niger isolates were compared with uninoculated Control; wilt fungus inoculated plants applied with A. niger isolates were compared with fungus inoculated Contorl (F); nematode inoculated plants applied with A. niger isolates were compared with nematode inoculated Contorl (N); concomitantly inoculated plants applied with A. niger isolates were compared with concomitantly inoculated Contorl (F+N)]. *Significantly different from the control at P≤0.0001.

135

production and yield of chickpea and tomato. The A. niger isolates SkNAn5, VAn4, AnC2, AnR3, ANAn4 and BuAn3 performed significantly better (P≤ 0.0001) than other isolates in enhancing the yield of chickpea as well as tomato plants inoculated with the pathogens or not inoculated over respective controls. Root nodulation Chickpea roots developed luxuriant nodules (49 nodules/root system) (Fig. 31) and it further increased (58%) in the presence of A. niger isolates. Infection by F. oxysporum f. sp. ciceri and M. incognita singly or concomitantly resulted to decrease in the number of functional and total nodules per root system in comparison to the control (Fig. 32-33). Decrease in the nodulation by concomitant inoculation of the pathogens was significantly greater than their individual effects (P≤ 0.05). Application of A. niger isolates checked the suppressive effect of pathogens on root nodulation. All the isolates through seed or soil treatment promoted functional nodules of wilt fungus inoculated plants by 18-89% and 23- 82%, respectively, in comparison to the control (Fig. 32-33). Root nodulation in nematode infected plants also improved significantly due to A. niger application. Number of functional nodules of nematode infected plants increased by 11-41% due to the soil or seed application of A. niger isolates over control (Fig. 32-33). Almost similar effect of A. niger isolates was recorded on the nodulation of concomitantly inoculated plants and the order of nodule promotion with A. niger isolates was SkNAn5 > VAn4 > AnC2 > AnR3 > ANAn4 > BuAn3 > Ban4 > rest of the A. niger isolates (Fig. 32- 33). The number of non-functional nodules was, however, decreased due to A. niger treatments (Fig. 32-33). Carbofuran application increased the functional nodules of nematode infected plants by 11-15% (Fig. 32-33) in comparison to the control. Combined application of carbendazim and carbofuran resulted to 17% increase in the functional nodules over control (Fig. 32-33).

Fig 31. Rhizobial nodules on the roots of chickpea. 136

FUNCTIONAL NODULES

NON FUNCTIONAL NODULES

TOTAL NODULES

Control Foc Mi Foc+Mi Fig. 32. Effects of seed treatment of Aspergillus niger isolates on the root nodulation of chickpea inoculated with Fusarium oxysporum f. sp. ciceri and Meloidogyne incognita or not inoculated pots. Vertical bars indicate the standard error of three replicates. Foc- F. oxysporum f. sp. ciceri, Mi- M. incognita, Czm – carbendazim, Cbf - carbofuran. 137

Control Foc Mi Foc+Mi Fig. 33. Effects of soil application of Aspergillus niger isolates on the root nodulation of chickpea inoculated with Fusarium oxysporum f. sp. ciceri and Meloidogyne incognita or not inoculated pots. Vertical bars indicate the standard error of three replicates. Foc- F. oxysporum f. sp. ciceri; Mi- M. incognita; Czm - carbendazim; Cbf - carbofuran. 138

Leaf chlorophyll Chlorophyll a, b and total chlorophyll content of leaves of chickpea and tomato decreased considerably in response of inoculation with the wilt fungi (P≤ 0.05) and root-knot nematode (P≤ 0.05) singly or concomitantly (P≤ 0.01) in comparison to uninoculated control (Table 28-31). Treatments with A. niger isolates improved the leaf chlorophyll a, b and total chlorophyll of chickpea and tomato plants inoculated singly with wilt fungus (P≤ 0.001) and the nematode (P≤ 0.001) or concomitantly with both pathogens (P≤ 0.0001) (Table 28-31). Relative performance of A. niger isolates in improving leaf pigments varied significantly and the order was SkNAn5 > VAn4 > AnC2 > AnR3 > ANAn4 > BuAn3 > rest of the A. niger isolates. Application of pesticides also significantly improved the contents of leaf pigment, and overall effects were more or less same as A. niger isolates. Increase in the chlorophyll production with A. niger treatments in tomato plants was significantly (P≤ 0.05) greater than in chickpea. Leaf phenol Total phenol content of chickpea and tomato leaves of plants applied with A. niger isolates increased (P≤ 0.05) over control (Table 32-35). The phenol contents also increased considerably in response of inoculation with wilt fungi (P≤ 0.01) and root-knot nematode (P≤ 0.01) singly and concomitantly (P≤ 0.001) in comparison to uninoculated control (Table 32-35). The phenol increased further in the inoculated plants applied with A. niger isolates (Table 32-35). Seed/nursery treatment with A. niger isolates produced phenol significantly (P≤ 0.05) greater than soil treatment. Significant variation in the performance of A. niger isolates was seen on the plants inoculated with wilt fungus (P≤ 0.001) and root- knot nematode (P≤ 0.001), singly and concomitantly (P≤ 0.0001) (Table 32-35). Greatest increase in the phenol content was recorded with SkNAn5 followed by AnC2, AnR3, ANAn4 and BuAn3. Application of pesticides also increased leaf phenol as increased by A. niger isolates in all treatments, but the degree of increase was less than BuAn3 and greater than other isolates of A. niger. Production of phenol in the plants applied with A. niger isolates was significantly (P≤ 0.05) greater in tomato than chickpea.

139

Table 28. Effects of seed treatment with efficient Aspergillus niger isolates on chlorophyll a, b and total chlorophyll of chickpea inoculated with Fusarium oxysporum f. sp. ciceri and Meloidogyne incognita, singly or concomitantly. Chlorophyll (mg/g fresh weight of leaf tissue)

Control Mi Foc Foc+Mi Treatment Chl. a Chl. b Total Chl. Chl. a Chl. b Total Chl. Chl. a Chl. b Total Chl. Chl. a Chl. b Total Chl. Control 0.704 0.695 1.399 0.628 0.590 1.218 0.597 0.528 1.126 0.520 0.493 1.013 AAn1 0.781 0.782 1.5630.685 0.651 1.336 0.675 0.6081.283 0.643 0.633 1.276 ANAn1 0.779 0.780 1.558 0.683 0.634 1.317 0.673 0.605 1.279 0.639 0.629 1.268 ANAn4 0.789 0.791 1.580 0.690 0.657 1.348 0.683 0.616 1.299 0.655 0.647 1.302 AnC2 0.792 0.795 1.588 0.693 0.660 1.353 0.687 0.620 1.307 0.662 0.654 1.315 AnR3 0.789 0.792 1.581 0.691 0.658 1.349 0.684 0.617 1.301 0.657 0.648 1.305 BAn4 0.783 0.785 1.568 0.686 0.653 1.339 0.678 0.610 1.288 0.647 0.637 1.284 BasAn5 0.785 0.787 1.571 0.687 0.654 1.342 0.679 0.612 1.291 0.649 0.640 1.289 BuAn3 0.787 0.789 1.576 0.689 0.656 1.345 0.682 0.614 1.296 0.653 0.644 1.297 BudAn3 0.777 0.778 1.555 0.682 0.648 1.330 0.672 0.604 1.275 0.637 0.626 1.263 GaAn1 0.776 0.776 1.552 0.681 0.647 1.328 0.670 0.602 1.272 0.634 0.623 1.258 JaAn2 0.774 0.774 1.548 0.680 0.646 1.325 0.668 0.601 1.269 0.632 0.620 1.252 LAn3 0.775 0.775 1.550 0.680 0.646 1.326 0.669 0.601 1.271 0.633 0.622 1.255 MeAn4 0.770 0.770 1.539 0.676 0.642 1.319 0.664 0.596 1.260 0.625 0.612 1.238 SkNAn3 0.776 0.777 1.553 0.681 0.648 1.329 0.671 0.603 1.274 0.636 0.625 1.260 SkNAn5 0.798 0.802 1.599 0.697 0.665 1.362 0.693 0.625 1.318 0.670 0.664 1.334 VAn4 0.794 0.797 1.591 0.694 0.662 1.356 0.689 0.621 1.310 0.664 0.657 1.321 Carbendazim (Czm) 0.786 0.788 1.573 - - - 0.680 0.613 1.293 - - - Carbofuran (Cbf) 0.783 0.785 1.568 0.686 0.653 1.339 ------Czm+Cbf 0.786 0.789 0.000 - - 0.000 - - 0.000 0.650 0.640 0.000 LSD (P≤0.05) 0.004 0.006 0.004 0.041 0.004 0.005 0.005 0.005 0.004 0.005 0.004 0.005 (P≤0.01) 0.005 0.007 0.006 0.054 0.005 0.007 0.007 0.006 0.006 0.007 0.006 0.007 (P≤0.001) 0.007 0.009 0.007 0.068 0.006 0.008 0.008 0.008 0.007 0.009 0.007 0.009 (P≤0.0001) 0.008 0.012 0.009 0.087 0.008 0.011 0.011 0.010 0.009 0.011 0.009 0.012 Each value is the mean of three replicates. Chl – chlorophyll; Czm - carbendazim, Cbf - carbofuran; Mi - Meloidogyne incognita, Foc - Fusarium oxysporum f. sp. ciceri. 140

Table 29. Effects of soil application of efficient Aspergillus niger isolates on chlorophyll a, b and total chlorophyll of chickpea inoculated with Fusarium oxysporum f. sp. ciceri and Meloidogyne incognita, singly or concomitantly. Chlorophyll (mg/g fresh weight of leaf tissue)

Control Mi Foc Foc+Mi Treatment Chl. a Chl. b Total Chl. Chl. a Chl. b Total Chl. Chl. a Chl. b Total Chl. Chl. a Chl. b Total Chl. Control 0.704 0.695 1.399 0.628 0.590 1.218 0.597 0.528 1.126 0.520 0.493 1.013 AAn1 0.734 0.735 1.469 0.644 0.612 1.256 0.635 0.5711.206 0.604 0.595 1.199 ANAn1 0.732 0.733 1.465 0.642 0.596 1.238 0.633 0.569 1.202 0.601 0.591 1.192 ANAn4 0.741 0.744 1.485 0.649 0.618 1.267 0.642 0.579 1.221 0.616 0.608 1.224 AnC2 0.745 0.748 1.493 0.652 0.621 1.272 0.646 0.583 1.229 0.622 0.615 1.237 AnR3 0.742 0.744 1.486 0.649 0.618 1.268 0.643 0.580 1.223 0.617 0.609 1.227 BAn4 0.736 0.738 1.474 0.645 0.614 1.259 0.637 0.574 1.211 0.608 0.599 1.207 BasAn5 0.738 0.740 1.477 0.646 0.615 1.261 0.639 0.575 1.214 0.610 0.602 1.212 BuAn3 0.740 0.742 1.482 0.648 0.617 1.265 0.641 0.577 1.218 0.614 0.606 1.219 BudAn3 0.730 0.731 1.462 0.641 0.609 1.250 0.631 0.568 1.199 0.599 0.588 1.187 GaAn1 0.729 0.730 1.459 0.640 0.608 1.248 0.630 0.566 1.196 0.596 0.586 1.182 JaAn2 0.728 0.728 1.456 0.639 0.607 1.246 0.628 0.565 1.193 0.594 0.583 1.177 LAn3 0.728 0.729 1.457 0.639 0.608 1.247 0.629 0.565 1.194 0.595 0.584 1.180 MeAn4 0.723 0.723 1.447 0.636 0.604 1.239 0.624 0.560 1.185 0.588 0.576 1.163 SkNAn3 0.730 0.730 1.460 0.640 0.609 1.249 0.631 0.567 1.197 0.598 0.587 1.185 SkNAn5 0.750 0.753 1.503 0.655 0.625 1.280 0.651 0.588 1.239 0.630 0.624 1.254 VAn4 0.746 0.749 1.496 0.653 0.622 1.274 0.647 0.584 1.232 0.624 0.617 1.242 Carbendazim (Czm) 0.738 0.740 0.000 - - - 0.639 0.576 0.000 - - - Carbofuran (Cbf) 0.736 0.738 0.000 0.645 0.614 0.000 ------Czm+Cbf 0.738 0.740 0.000 ------0.611 0.630 0.000 LSD (P≤0.05) 0.003 0.005 0.004 0.038 0.004 0.005 0.005 0.004 0.004 0.005 0.004 0.005 (P≤0.01) 0.005 0.007 0.006 0.051 0.005 0.006 0.006 0.006 0.006 0.007 0.006 0.007 (P≤0.001) 0.006 0.009 0.007 0.064 0.006 0.008 0.008 0.007 0.007 0.008 0.007 0.009 (P≤0.0001) 0.008 0.011 0.009 0.082 0.008 0.010 0.010 0.010 0.009 0.011 0.009 0.011 Each value is the mean of three replicates. Chl – chlorophyll; Czm - carbendazim, Cbf - carbofuran; Mi - Meloidogyne incognita, Foc - Fusarium oxysporum f. sp. ciceri. 141

Table 30. Effects of nursery treatment with efficient Aspergillus niger isolates on chlorophyll a, b and total chlorophyll of tomato inoculated with Fusarium oxysporum f. sp. lycopersici and Meloidogyne incognita, singly or concomitantly. Chlorophyll (mg/g fresh weight of leaf tissue)

Control Mi Fol Fol+Mi Treatment Chl. a Chl. b Total Chl. Chl. a Chl. b Total Chl. Chl. a Chl. b Total Chl. Chl. a Chl. b Total Chl. Control 0.830 0.790 1.620 0.732 0.659 1.391 0.692 0.582 1.273 0.591 0.538 1.129 AAn1 0.932 0.947 1.878 0.845 0.828 1.673 0.837 0.789 1.626 0.808 0.812 1.620 ANAn1 0.929 0.943 1.872 0.843 0.938 1.780 0.834 0.833 1.667 0.804 0.791 1.595 ANAn4 0.942 0.959 1.900 0.854 0.855 1.709 0.847 0.848 1.696 0.822 0.813 1.635 AnC2 0.947 0.964 1.911 0.858 0.860 1.718 0.853 0.854 1.706 0.829 0.821 1.650 AnR3 0.943 0.960 1.902 0.854 0.856 1.711 0.848 0.849 1.698 0.824 0.814 1.638 BAn4 0.935 0.950 1.885 0.848 0.849 1.697 0.840 0.840 1.680 0.812 0.801 1.614 BasAn5 0.937 0.953 1.889 0.850 0.851 1.700 0.842 0.842 1.685 0.815 0.805 1.620 BuAn3 0.940 0.956 1.896 0.852 0.854 1.706 0.845 0.846 1.691 0.820 0.809 1.629 BudAn3 0.927 0.941 1.867 0.841 0.841 1.683 0.832 0.831 1.663 0.801 0.788 1.589 GaAn1 0.924 0.939 1.863 0.840 0.840 1.679 0.830 0.828 1.659 0.798 0.785 1.583 JaAn2 0.922 0.936 1.859 0.838 0.838 1.676 0.828 0.826 1.654 0.795 0.782 1.577 LAn3 0.923 0.937 1.861 0.839 0.839 1.677 0.829 0.827 1.656 0.797 0.783 1.580 MeAn4 0.917 0.930 1.846 0.833 0.832 1.666 0.822 0.820 1.642 0.788 0.772 1.560 SkNAn3 0.925 0.940 1.865 0.840 0.841 1.681 0.831 0.830 1.661 0.800 0.787 1.586 SkNAn5 0.954 0.973 1.926 0.864 0.867 1.730 0.860 0.862 1.722 0.839 0.832 1.672 VAn4 0.949 0.967 1.916 0.859 0.862 1.721 0.855 0.856 1.711 0.832 0.824 1.656 Carbendazim (Czm) 0.938 0.954 0.000 - - - 0.843 0.843 0.000 - - - Carbofuran (Cbf) 0.935 0.950 0.000 0.848 0.950 0.000 ------Czm+Cbf 0.940 0.950 0.000 ------0.820 0.820 0.000 LSD (P≤0.05) 0.004 0.006 0.005 0.045 0.004 0.006 0.006 0.005 0.005 0.006 0.005 0.006 (P≤0.01) 0.005 0.008 0.007 0.061 0.006 0.007 0.007 0.007 0.007 0.008 0.007 0.008 (P≤0.001) 0.007 0.010 0.008 0.076 0.007 0.009 0.009 0.009 0.008 0.010 0.008 0.010 (P≤0.0001) 0.009 0.013 0.011 0.098 0.009 0.012 0.012 0.012 0.011 0.013 0.011 0.013 Each value is the mean of three replicates. Chl – chlorophyll; Czm - carbendazim, Cbf - carbofuran; Mi - Meloidogyne incognita, Fol - Fusarium oxysporum f. sp. lycopersici. 142

Table 31. Effects of soil application of efficient Aspergillus niger isolates on chlorophyll a, b and total chlorophyll of tomato inoculated with Fusarium oxysporum f. sp. lycopersici and Meloidogyne incognita, singly or concomitantly. Chlorophyll (mg/g fresh weight of leaf tissue)

Treatment Control Mi Fol Fol+Mi

Chl. a Chl. b Total Chl. Chl. a Chl. b Total Chl. Chl. a Chl. b Total Chl. Chl. a Chl. b Total Chl. Control 0.830 0.790 1.620 0.732 0.659 1.391 0.692 0.582 1.273 0.591 0.538 1.129 AAn1 0.857 0.871 1.728 0.778 0.762 1.540 0.770 0.726 1.496 0.744 0.747 1.490 ANAn1 0.854 0.868 1.722 0.776 0.863 1.638 0.767 0.766 1.534 0.740 0.728 1.468 ANAn4 0.866 0.882 1.748 0.785 0.787 1.572 0.780 0.780 1.560 0.757 0.748 1.504 AnC2 0.871 0.887 1.758 0.789 0.791 1.580 0.784 0.786 1.570 0.763 0.755 1.518 AnR3 0.867 0.883 1.750 0.786 0.788 1.574 0.781 0.781 1.562 0.758 0.749 1.507 BAn4 0.860 0.874 1.734 0.780 0.781 1.561 0.773 0.773 1.546 0.747 0.737 1.485 BasAn5 0.862 0.876 1.738 0.782 0.783 1.564 0.775 0.775 1.550 0.750 0.740 1.490 BuAn3 0.865 0.880 1.744 0.784 0.785 1.569 0.778 0.778 1.556 0.754 0.745 1.499 BudAn3 0.852 0.866 1.718 0.774 0.774 1.548 0.766 0.764 1.530 0.737 0.725 1.462 GaAn1 0.851 0.864 1.714 0.772 0.772 1.545 0.764 0.762 1.526 0.734 0.722 1.457 JaAn2 0.849 0.861 1.710 0.771 0.771 1.542 0.762 0.760 1.522 0.732 0.719 1.451 LAn3 0.850 0.862 1.712 0.772 0.772 1.543 0.763 0.761 1.524 0.733 0.721 1.454 MeAn4 0.843 0.855 1.699 0.767 0.766 1.533 0.757 0.754 1.511 0.725 0.711 1.435 SkNAn3 0.851 0.865 1.716 0.773 0.773 1.547 0.765 0.763 1.528 0.736 0.724 1.459 SkNAn5 0.878 0.895 1.772 0.794 0.797 1.592 0.791 0.793 1.584 0.772 0.766 1.538 VAn4 0.873 0.889 1.762 0.791 0.793 1.584 0.786 0.788 1.574 0.766 0.758 1.524 Carbendazim (Czm) 0.863 0.878 1.740 - - - 0.776 0.776 1.552 - - - Carbofuran (Cbf) 0.860 0.874 1.734 0.780 0.874 1.654 ------Czm+Cbf 0.864 0.877 1.741 ------0.752 0.742 1.493 LSD (P≤0.05) 0.004 0.005 0.004 0.039 0.004 0.005 0.005 0.005 0.004 0.005 0.004 0.005 (P≤0.01) 0.005 0.007 0.006 0.052 0.005 0.006 0.006 0.006 0.006 0.007 0.006 0.007 (P≤0.001) 0.006 0.009 0.007 0.065 0.006 0.008 0.008 0.008 0.007 0.008 0.007 0.009 (P≤0.0001) 0.008 0.011 0.009 0.084 0.008 0.010 0.010 0.010 0.009 0.011 0.009 0.011 Each value is the mean of three replicates. Chl – chlorophyll; Czm - carbendazim, Cbf - carbofuran; Mi - Meloidogyne incognita, Fol - Fusarium oxysporum f. sp. lycopersici. 143

Table 32. Effects of seed treatment with efficient Aspergillus niger isolates on total phenolic contents and salicylic acid of chickpea inoculated with Fusarium oxysporum f. sp. ciceri and Meloidogyne incognita, singly or concomitantly. Salicylic acid (ppm/g fresh weight of tissue) Total Phenol content (mg/g catechol fresh weight of leaf tissue) Treatment Leaf Root

Control Mi Foc Foc+Mi Control Mi Foc Foc+Mi Control Mi Foc Foc+Mi Control 110.0 133.1 141.9 168.3 14.5 17.2 18.1 20.4 18.1 22.1 23.2 26.9 AAn1 140.1 150.2 168.5 196.0 18.9 19.6 21.8 24.1 24.4 25.7 28.7 32.5 ANAn1 139.2 149.7 167.7 195.2 18.7 19.6 21.7 24.0 24.2 25.6 28.5 32.3 ANAn4 143.1 152.0 171.2 198.8 19.3 19.9 22.2 24.5 25.0 26.1 29.3 33.1 AnC2 144.6 152.8 172.5 200.2 19.5 20.0 22.4 24.6 25.3 26.2 29.5 33.3 AnR3 143.4 152.1 171.5 199.0 19.3 19.9 22.3 24.5 25.0 26.1 29.3 33.1 BAn4 141.0 150.8 169.3 196.8 19.0 19.7 22.0 24.2 24.5 25.8 28.9 32.7 BasAn5 141.6 151.1 169.9 197.4 19.1 19.8 22.0 24.3 24.7 25.9 29.0 32.8 BuAn3 142.5 151.6 170.7 198.2 19.2 19.8 22.1 24.4 24.9 26.0 29.2 32.9 BudAn3 138.5 149.4 167.2 194.6 18.6 19.5 21.7 23.9 24.0 25.5 28.4 32.2 GaAn1 137.9 149.0 166.7 194.1 18.6 19.5 21.6 23.8 23.9 25.4 28.3 32.1 JaAn2 137.3 148.7 166.2 193.5 18.5 19.4 21.5 23.8 23.8 25.4 28.2 32.0 LAn3 137.6 148.9 166.4 193.8 18.5 19.4 21.5 23.8 23.9 25.4 28.3 32.1 MeAn4 135.7 147.7 164.6 191.9 18.2 19.3 21.3 23.6 23.4 25.2 27.9 31.7 SkNAn3 138.2 149.2 166.9 194.3 18.6 19.5 21.6 23.9 24.0 25.5 28.4 32.2 SkNAn5 146.7 154.0 174.4 202.1 19.8 20.2 22.7 24.9 25.7 26.5 29.9 33.7 VAn4 145.2 153.2 173.1 200.7 19.6 20.1 22.5 24.7 25.4 26.3 29.6 33.5 Carbendazim (Czm) 141.9 - 170.1 - 19.1 - 22.1 - 24.7 - 29.0 - Carbofuran (Cbf) 141.0 150.8 - - 19.0 19.7 - - 24.5 25.8 - - Czm+Cbf 141.7 - - 197.7 19.0 - - 24.3 24.6 - - 32.8 LSD (P≤0.05) 2.3 2.2 2.3 0.5 0.2 0.2 0.2 0.2 0.2 0.2 0.2 0.2 (P≤0.01) 3.1 3.0 3.1 0.7 0.2 0.2 0.2 0.2 0.3 0.2 0.2 0.3 (P≤0.001) 3.9 3.9 3.9 0.9 0.3 0.3 0.3 0.3 0.4 0.3 0.3 0.4 (P≤0.0001) 5.1 5.0 5.1 1.1 0.4 0.3 0.3 0.4 0.5 0.4 0.4 0.5 Each value is the mean of three replicates. Czm - carbendazim, Cbf - carbofuran; Mi - Meloidogyne incognita, Foc - Fusarium oxysporum f. sp. ciceri.

144

Table 33. Effects of soil application of efficient Aspergillus niger isolates on total phenolic contents and salicylic acid of chickpea inoculated with Fusarium oxysporum f. sp. ciceri and Meloidogyne incognita, singly or concomitantly. Total Phenol content (mg/g catechol Salicylic acid (ppm/g fresh weight of tissue) fresh weight of leaf tissue) Leaf Root Treatment Control Mi Foc Foc+Mi Control Mi Foc Foc+Mi Control Mi Foc Foc+Mi Control 110.0 133.1 141.9 168.3 14.5 17.2 18.1 20.4 18.1 22.1 23.2 26.9 AAn1 131.6 141.2 158.4184.2 17.7 18.5 20.5 22.6 22.9 24.2 27.0 30.6 ANAn1 130.8 140.7 157.7 183.5 17.6 18.4 20.4 22.5 22.7 24.1 26.8 30.4 ANAn4 134.5 142.8 160.9 186.8 18.1 18.7 20.9 23.0 23.5 24.5 27.5 31.1 AnC2 135.9 143.6 162.2 188.1 18.3 18.8 21.1 23.2 23.8 24.7 27.8 31.3 AnR3 134.8 143.0 161.2 187.1 18.2 18.7 20.9 23.0 23.5 24.5 27.6 31.1 BAn4 132.5 141.7 159.2 185.0 17.8 18.5 20.6 22.8 23.1 24.3 27.1 30.7 BasAn5 133.1 142.0 159.7 185.5 17.9 18.6 20.7 22.8 23.2 24.3 27.2 30.8 BuAn3 133.9 142.5 160.4 186.3 18.1 18.6 20.8 22.9 23.4 24.4 27.4 31.0 BudAn3 130.2 140.4 157.2 182.9 17.5 18.3 20.4 22.5 22.6 24.0 26.7 30.3 GaAn1 129.7 140.1 156.7 182.4 17.4 18.3 20.3 22.4 22.5 23.9 26.6 30.2 JaAn2 129.1 139.8 156.2 181.9 17.4 18.3 20.2 22.3 22.4 23.9 26.5 30.1 LAn3 129.4 139.9 156.4 182.2 17.4 18.3 20.2 22.4 22.4 23.9 26.6 30.1 MeAn4 127.5 138.9 154.8 180.4 17.1 18.1 20.0 22.1 22.0 23.7 26.2 29.8 SkNAn3 130.0 140.3 156.9 182.7 17.5 18.3 20.3 22.4 22.5 24.0 26.7 30.2 SkNAn5 137.9 144.8 163.9 190.0 18.6 19.0 21.3 23.4 24.2 24.9 28.1 31.7 VAn4 136.5 144.0 162.7 188.7 18.4 18.8 21.1 23.2 23.9 24.7 27.9 31.4 Carbendazim (Czm) 133.3 - 159.9 - 18.0 - 20.7 - 23.2 - 27.3 - Carbofuran (Cbf) 132.5 141.7 - - 17.8 18.5 - - 23.1 24.3 - - Czm+Cbf 133.3 - - 185.7 17.9 - - 22.9 23.1 - - 30.9 LSD (P≤0.05) 2.2 2.1 2.2 0.5 0.2 0.1 0.1 0.1 0.2 0.2 0.2 0.2 (P≤0.01) 3.0 2.8 3.0 0.7 0.2 0.2 0.2 0.2 0.3 0.2 0.2 0.3 (P≤0.001) 3.7 3.7 3.7 0.8 0.3 0.2 0.2 0.3 0.3 0.3 0.3 0.4 (P≤0.0001) 4.7 4.7 4.7 1.1 0.3 0.3 0.3 0.3 0.4 0.4 0.4 0.5 Each value is the mean of three replicates. Chl – chlorophyll; Czm - carbendazim, Cbf - carbofuran; Mi - Meloidogyne incognita, Foc - Fusarium oxysporum f. sp. ciceri 145

Table 34. Effects of nursery treatment with efficient Aspergillus niger isolates on total phenolic contents, salicylic acid and lycopene of tomato inoculated with Fusarium oxysporum f. sp. lycopersici and Meloidogyne incognita, singly or concomitantly. Salicylic acid (ppm/g fresh weight of tissue) Total Phenol content (mg/g Lycopene (mg/100 g fresh catechol fresh weight of leaf tissue weight of fruit) Treatment Leaf Root

Control Mi Fol Fol+Mi Control Mi Fol Fol+Mi Control Mi Fol Fol+Mi Control Mi Fol Fol+Mi Control 90.4 114.4 119.5 144.5 19.3 23.1 24.4 28.9 26.3 32.8 34.0 41.1 8.3 7.3 6.8 5.8 AAn1 117.1 130.3 143.7 170.2 26.0 26.9 30.2 34.9 36.7 38.9 43.3 50.9 9.5 8.4 7.7 7.2 ANAn1 116.3 129.8 143.0 169.4 25.8 26.8 30.0 34.7 36.4 38.8 43.0 50.6 9.5 8.3 7.7 7.2 ANAn4 119.7 131.9 146.2 172.8 26.6 27.2 30.8 35.5 37.8 39.6 44.2 51.9 9.6 8.5 7.8 7.4 AnC2 121.1 132.7 147.4 174.0 27.0 27.4 31.1 35.8 38.3 39.9 44.7 52.4 9.7 8.6 7.9 7.5 AnR3 120.0 132.1 146.4 173.0 26.7 27.3 30.8 35.6 37.9 39.6 44.3 52.0 9.7 8.5 7.9 7.5 BAn4 117.9 130.8 144.5 171.0 26.2 27.0 30.4 35.1 37.0 39.1 43.6 51.2 9.6 8.4 7.8 7.3 BasAn5 118.4 131.1 144.9 171.5 26.3 27.1 30.5 35.2 37.3 39.3 43.8 51.4 9.6 8.5 7.8 7.3 BuAn3 119.2 131.6 145.7 172.2 26.5 27.2 30.7 35.4 37.6 39.4 44.0 51.7 9.6 8.5 7.8 7.4 BudAn3 115.7 129.5 142.5 168.9 25.6 26.7 29.9 34.6 36.2 38.6 42.8 50.4 9.5 8.4 7.7 7.1 GaAn1 115.2 129.2 142.0 168.4 25.5 26.6 29.8 34.5 36.0 38.5 42.6 50.3 9.4 8.3 7.6 7.1 JaAn2 114.7 128.9 141.6 167.9 25.4 26.5 29.7 34.4 35.8 38.4 42.5 50.1 9.4 8.3 7.6 7.1 LAn3 114.9 129.0 141.8 168.1 25.4 26.6 29.7 34.4 35.9 38.5 42.5 50.2 9.4 8.3 7.6 7.1 MeAn4 113.2 128.0 140.2 166.4 25.0 26.3 29.3 34.0 35.2 38.0 41.9 49.5 9.3 8.3 7.6 7.0 SkNAn3 115.5 129.4 142.3 168.6 25.6 26.6 29.8 34.6 36.1 38.6 42.7 50.4 9.4 8.4 7.7 7.1 SkNAn5 122.9 135.9 148.2 186.1 27.4 27.7 31.5 36.2 39.0 40.3 45.3 53.1 9.8 8.6 8.0 7.7 VAn4 121.6 134.4 146.1 174.6 27.1 27.5 31.2 35.9 38.5 40.0 44.9 52.6 9.7 8.6 7.9 7.6 Carbendazim (Czm) 118.7 - 145.2 - 26.4 - 30.5 - 37.4 - 43.8 - 9.6 - 7.8 - Carbofuran (Cbf) 117.9 130.8 - - 26.2 27.0 - - 37.0 39.1 - - 9.6 8.4 - - Czm+Cbf 118.8 - - 171.7 26.4 - - 35.3 37.3 - - 51.5 9.5 - - 7.4 LSD (P≤0.05) 2.62 2.46 2.62 0.59 0.19 0.18 0.18 0.18 0.24 0.20 0.20 0.26 0.12 0.10 0.13 0.12 (P≤0.01) 3.52 3.36 3.52 0.79 0.25 0.24 0.24 0.25 0.32 0.27 0.27 0.35 0.16 0.13 0.17 0.16 (P≤0.001) 4.37 4.37 4.37 0.98 0.31 0.28 0.29 0.30 0.40 0.34 0.34 0.44 0.21 0.17 0.22 0.20 (P≤0.0001) 5.66 5.60 5.66 1.26 0.40 0.37 0.38 0.40 0.52 0.44 0.44 0.57 0.25 0.20 0.27 0.25 Each value is the mean of three replicates. Czm - carbendazim, Cbf - carbofuran; Mi - Meloidogyne incognita, Fol - Fusarium oxysporum f. sp. lycopersici. 146

Table 35. Effects of soil application of efficient Aspergillus niger isolates on total phenolic contents, salicylic acid and lycopene content of tomato inoculated with Fusarium oxysporum f. sp. lycopersici and Meloidogyne incognita, singly or concomitantly. Salicylic acid (ppm/g fresh weight of tissue) Total Phenol content (mg/g Lycopene (mg/100 g fresh catechol fresh weight of leaf tissue weight of fruit) Treatment Leaf Root

Control Mi Fol Fol+Mi Control Mi Fol Fol+Mi Control Mi Fol Fol+Mi Control Mi Fol Fol+Mi Control 90.4 114.4 119.5 144.5 19.3 23.1 24.4 28.9 26.3 32.8 34.0 41.1 8.3 7.3 6.8 5.8 AAn1 107.7 119.9 132.2 156.6 23.9 24.7 27.8 32.1 33.8 35.8 39.8 46.9 8.8 7.7 7.1 6.7 ANAn1 107.0 119.4 131.6 155.9 23.7 24.6 27.6 32.0 33.5 35.7 39.6 46.6 8.7 7.6 7.1 6.6 ANAn4 110.2 121.4 134.5 158.9 24.5 25.1 28.3 32.7 34.8 36.4 40.7 47.8 8.9 7.8 7.2 6.8 AnC2 111.4 122.1 135.6 160.1 24.8 25.2 28.6 33.0 35.2 36.7 41.1 48.2 8.9 7.9 7.3 6.9 AnR3 110.4 121.5 134.7 159.2 24.6 25.1 28.4 32.7 34.9 36.5 40.8 47.9 8.9 7.8 7.2 6.9 BAn4 108.4 120.3 132.9 157.3 24.1 24.8 27.9 32.3 34.1 36.0 40.1 47.1 8.8 7.8 7.1 6.7 BasAn5 108.9 120.6 133.4157.8 24.2 24.9 28.0 32.4 34.3 36.1 40.3 47.3 8.8 7.8 7.2 6.8 BuAn3 109.7 121.1 134.0 158.5 24.4 25.0 28.2 32.6 34.6 36.3 40.5 47.6 8.8 7.8 7.2 6.8 BudAn3 106.5 119.2 131.1155.4 23.6 24.5 27.5 31.8 33.3 35.5 39.4 46.4 8.7 7.7 7.1 6.6 GaAn1 106.0 118.9 130.7 154.9 23.5 24.5 27.4 31.7 33.1 35.4 39.2 46.2 8.7 7.7 7.0 6.5 JaAn2 105.5 118.6 130.2 154.4 23.3 24.4 27.3 31.6 32.9 35.3 39.1 46.1 8.7 7.7 7.0 6.5 LAn3 105.7 118.7 130.5 154.7 23.4 24.4 27.4 31.7 33.0 35.4 39.1 46.1 8.7 7.7 7.0 6.5 MeAn4 104.1 117.7 129.0 153.1 23.0 24.2 27.0 31.3 32.4 35.0 38.6 45.5 8.6 7.6 7.0 6.4 SkNAn3 106.2 119.0 130.9 155.2 23.5 24.5 27.5 31.8 33.2 35.5 39.3 46.3 8.7 7.7 7.0 6.6 SkNAn5 113.1 125.0 136.3 171.2 25.2 25.5 29.0 33.3 35.9 37.1 41.7 48.9 9.0 7.9 7.3 7.1 VAn4 111.9 123.7 134.5 160.6 24.9 25.3 28.7 33.1 35.4 36.8 41.3 48.4 8.9 7.9 7.3 7.0 Carbendazim (Czm) 109.2 - 133.6 - 24.3 - 28.1 - 34.4 - 40.3 - 8.8 - 7.2 - Carbofuran (Cbf) 108.4 120.3 - - 24.1 24.8 - - 34.1 36.0 - - 8.7 7.8 - - Czm+Cbf 108.9 - - 158.0 24.2 - - 32.5 34.2 - - 47.4 8.8 - - 6.8 LSD (P≤0.05) 2.24 2.11 2.24 0.50 0.16 0.15 0.15 0.15 0.21 0.17 0.17 0.22 0.10 0.08 0.11 0.10 (P≤0.01) 3.01 2.87 3.01 0.67 0.21 0.20 0.20 0.21 0.28 0.23 0.23 0.30 0.13 0.11 0.14 0.13 (P≤0.001) 3.74 3.74 3.74 0.84 0.26 0.24 0.25 0.26 0.35 0.29 0.29 0.37 0.18 0.14 0.19 0.18 (P≤0.0001) 4.84 4.79 4.84 1.08 0.34 0.32 0.32 0.34 0.45 0.37 0.37 0.48 0.22 0.17 0.23 0.21 Each value is the mean of three replicates. Czm - carbendazim, Cbf - carbofuran; Mi - Meloidogyne incognita, Fol - Fusarium oxysporum f. sp. lycopersici. 147

Salicylic acid Salicylic acid (SA) concentration in chickpea and tomato leaves increased significantly due to application of A. niger isolates (P≤ 0.01) or pesticides (P≤ 0.01) or inoculation with wilt fungus (P≤ 0.01) and root-knot nematode (P≤ 0.05), singly or concomitantly with both the pathogens (P≤ 0.001) (Table 32-35). Leaves produced more SA due to application of A. niger isolates, and the increase further varied due to inoculation with wilt fungus (P≤ 0.001) and nematode (P≤ 0.01), singly or concomitantly (P≤ 0.001) compared to respective inoculated control (Table 32-35). Seed/nursery treatment with A. niger isolates accumulated SA in leaves significantly (P≤ 0.01) greater than soil treatment. Effect of A. niger isolates varied the inoculation with wilt fungus (P≤ 0.001), root-knot nematode (P≤ 0.01) and fungus+nematode (P≤ 0.01) (Table 32-35). Overall increase in SA due to A. niger treatments was significantly (P≤ 0.05) greater in tomato than chickpea over respective controls. Among the A. niger isolates tested, greatest effectiveness with regard to increase in SA contents of chickpea and tomato leaves irrespective of inoculation was recorded with SkNAn5 followed by AnC2, AnR3, ANAn4 and BuAn3. Increase in leaf SA with pesticides was same as increased by A. niger isolates in all the treatments and the amount of increase was almost equal to BuAn3. Salicylic acid accumulation in roots was 25-32% greater than the leave in chickpea and 35-42% in tomato. Effect of pathogens and A. niger isolates singly or jointly on SA contents of root was similar to leaf. Similarly SA contents in tomato roots was greater (P≤ 0.05) than the chickpea with various treatments. Lycopene content of tomato fruit Lycopene content of ripe fruits of tomato (fully red) from the plants treated with the A. niger isolates increased (P≤ 0.001) over control, however, effect of the isolates varied to a great extent (P≤ 0.001) (Table 34-35). Inoculation with wilt fungus (P≤ 0.01) and root-knot nematode (P≤ 0.05), singly and concomitantly (P≤ 0.001) decreased the tomato lycopene in comparison to uninoculated control (Table 34-35). However, treatment with A. niger isolates improved the lycopene content of tomato fruit inoculated wilt fungus (P≤ 0.001) and nematode (P≤ 0.001) singly or concomitantly (P≤ 0.0001) (Table 34-35). Nursery treatment with A. niger isolates produced significantly (P≤ 0.05) more lycopene than soil treatment. Effect of pesticides in all treatments on lycopene content of tomato fruits was same as with A. niger isolates. Over all order of A. niger isolates and pesticides in increasing lycopene contents was SkNAn5 > VAn4 > AnC2 > AnR3 > ANAn4 > pesticides > BuAn3 > rest of the A. niger isolates.

148

Seed index of chickpea Seed index (SI, weight of 100 harvested seeds in gram) of chickpea plant decreased considerably due to inoculation with wilt fungus (SI = 19 g; P≤ 0.01) and root-knot nematode (SI = 20 g; P≤ 0.05) singly or concomitantly (SI = 16.4 g; P≤ 0.001) in comparison to uninoculated control (SI = 21.3 g) (Table 36). Treatment with A. niger isolates improved the seed index of plants inoculated with wilt fungus (P≤ 0.01) and nematode (P≤ 0.01) singly or concomitantly over respective controls (P≤ 0.01) (Table 36). There was no difference (P≤ 0.05) in seed or soil application of the treatments on seed index. Effect of pesticides on seed index in all treatments was same as A. niger isolates. In general the order of A. niger and pesticides in increasing seed index was SkNAn5 > VAn4 > AnC2 > AnR3 > ANAn4 > BuAn3 > pesticides > rest of the A. niger isolates. Seed health Viability and germination Viability and germination of the harvested chickpea seeds decreased significantly in response of inoculation with wilt fungus (11 and 17%), nematode (6 and 6.5%) singly or concomitantly with both pathogens (23 and 25%) in comparison to uninoculated control, respectively (Table 36). In the same was viability and germination of tomato seeds decreased significantly in response of inoculation with wilt fungus (14 and 19%), nematode (8 and 12%) singly or concomitantly with both pathogens (30 and 36%) in comparison to uninoculated control, respectively (Table 37). Application of A. niger isolates improved the viability and germination of both the seeds from the plants inoculated with wilt fungus (P≤ 0.001) and root-knot nematode (P≤ 0.05) singly or concomitantly (P≤ 0.001) (Table 36-37). Significant variation (P≤ 0.01) in the performance of A. niger isolates was recorded with the inoculation of pathogens. The isolate found most effective in improving the viability and germination was SkNAn5 followed by VAn4, AnC2, AnR3, ANAn4 and BuAn3. Germination percentage was found less than viability percentage, but insignificantly (P≤ 0.05). Seed or soil treatment showed no significant difference (P≤ 0.05). Pesticide application also improved viability and germination in all treatments as increased by A. niger isolates, but the degree of effectiveness was less than BuAn3 and greater than others.

149

Table 36. Effect of seed treatment and soil application of efficient Aspergillus niger isolates on the seed index, viability and germination of seeds, and detection of associated Fusarium oxysporum f. sp. ciceri with harvested seeds of chickpea from pots inoculated with F. oxysporum f. sp. ciceri, Meloidogyne incognita singly and concomitantly. Out of 10 seeds Detection of Foc with 10 seeds

Seed index Viable Germinated Sterilized Unsterilized

Treatment Seed Soil Seed Soil Seed Soil Seed Soil Seed Soil tr. app. tr. app. tr. app. tr. app. tr. app. Control 21.3 21.3 9.3 9.3 8.7 8.7 0.3 0.3 0.7 0.7 AAn1 25.2 24.6 10.0 9.7 9.0 8.7 0 0 0.3 0.3 ANAn1 25.2 24.6 10.0 10.0 9.0 8.7 0 0 0.3 0.3 ANAn4 25.5 24.9 10.0 9.7 10.0 9.7 0 0 0 0 AnC2 26.4 25.6 10.0 10.0 10.0 10.0 0 0 0 0 AnR3 25.5 24.9 10.0 10.0 10.0 9.7 0 0 0 0 BAn4 25.3 24.7 10.0 10.1 9.7 9.3 0.3 0.3 0.7 0.7 BasAn5 21.6 21.5 9.7 9.7 8.7 8.7 0 0 0 0.3 BuAn3 25.5 24.9 10.0 10.0 9.7 8.7 0 0 0 0 BudAn3 22.7 22.5 9.3 9.0 8.7 8.7 0 0 0.3 0.3 GaAn1 21.6 21.6 9.3 9.3 8.7 8.7 0 0 0.3 0.3 JaAn2 21.6 21.6 9.7 9.3 8.7 8.7 0 0 0.3 0.3 LAn3 21.6 21.5 9.3 9.0 8.7 9.3 0 0 0.3 0.3 MeAn4 21.5 21.5 9.3 9.0 8.7 9.3 0.3 0.3 0.7 0.7 SkNAn3 23.8 23.4 9.7 9.3 8.7 9.3 0 0 0 0.3 SkNAn5 26.8 26.0 10.0 10.0 10.0 10.0 0 0 0 0 VAn4 26.7 25.9 10.0 10.0 10.0 9.7 0 0 0 0 Carbendazim (Czm) 25.4 24.8 10.0 9.7 8.7 8.7 0 0 0 0 Carbofuran (Cbf) 21.7 21.4 10.0 10.0 8.7 8.7 0.3 0.3 0.7 0.7 Control (F) 19.0 19.0 7.7 7.7 7.0 7.0 4.3 4.3 7 7 F + AAn1 24.8 23.9 8.7 8.7 7.3 7.0 3.3 3.3 4.3 4.3 F + ANAn1 24.7 23.8 8.7 8.3 7.3 7.3 3.3 3.3 4.3 4.3 F + ANAn4 25.2 24.3 8.7 8.7 8.0 8.0 3.3 3.3 4.0 3.7 F + AnC2 26.5 25.4 9.0 8.7 8.3 8.0 2.3 2.3 3.3 3.3 F + AnR3 25.2 24.3 8.3 8.3 7.7 7.7 2.3 2.3 3.3 3.3 F + BAn4 24.9 24.0 8.7 8.3 7.3 7.3 3.3 3.3 4.3 4.7 F + BasAn5 19.4 19.3 8.0 8.0 7.0 7.0 3.3 3.3 4.7 4.7 F + BuAn3 25.2 24.3 8.7 8.7 7.7 7.7 2.3 2.3 4.3 4.3 F + BudAn3 21.1 20.8 7.7 7.7 7.3 7.3 3.3 3.0 4.7 4.7 F + GaAn1 19.5 19.4 7.7 7.7 7.3 7.3 3.3 3.3 4.7 4.7 F + JaAn2 19.5 19.4 8.0 7.7 7.3 7.0 3.3 3.7 4.7 5.0 F + LAn3 19.4 19.3 7.7 8.0 7.3 7.3 3.3 3.7 5.0 5.0 F + MeAn4 19.3 19.3 7.7 7.7 7.0 7.0 3.7 4.0 6.3 6.7 F + SkNAn3 22.7 22.1 8.3 8.0 7.3 7.3 3.3 3.3 4.3 4.3 F + SkNAn5 27.1 25.9 9.7 9.3 9.3 9.0 1.0 1.3 2.7 3.0 F + VAn4 27.0 25.8 9.3 9.3 8.7 8.3 1.3 1.7 3.3 3.3 F+Carbendazim(Czm) 25.1 24.2 8.7 8.0 8.0 7.3 2.3 2.3 3.7 3.3 Continued ...

150

Table 36 Continued… Control (N) 20.0 20.0 8.7 8.7 7.7 7.7 0.7 0.7 1.3 1.3 N + AAn1 23.4 22.9 9.3 9.0 8.0 7.7 0.7 0.7 1.0 1.0 N + ANAn1 23.3 22.8 9.3 9.0 8.0 7.7 0.7 0.7 1.0 1.0 N + ANAn4 23.6 23.1 9.3 9.3 8.0 8.0 0.7 0.7 0.7 1.0 N + AnC2 24.3 23.7 10.0 9.3 8.7 8.3 0.3 0.3 0.7 1.0 N + AnR3 23.6 23.1 9.7 9.3 8.3 8.3 0.3 0.3 0.7 1.0 N + BAn4 23.4 22.9 9.0 9.3 8.0 8.0 0.7 0.7 1.0 1.0 N + BasAn5 20.2 20.2 9.0 9.0 8.0 8.0 0.7 0.7 1.0 1.3 N + BuAn3 23.6 23.1 9.7 9.7 8.3 8.3 0.3 0.3 1.0 1.0 N + BudAn3 21.2 21.0 8.7 9.0 8.0 7.7 0.7 0.7 1.0 1.3 N + GaAn1 20.3 20.2 8.7 8.7 8.0 7.7 0.7 0.7 1.0 1.3 N + JaAn2 20.3 20.2 8.7 8.7 8.0 7.7 0.7 0.7 1.0 1.3 N + LAn3 20.2 20.2 8.7 8.7 8.0 7.7 0.7 0.7 1.0 1.3 N + MeAn4 20.2 20.1 8.7 8.7 8.0 7.7 0.7 0.7 1.0 1.3 N + SkNAn3 22.1 21.8 9.0 9.0 8.0 8.0 0.7 0.7 1.0 1.3 N + SkNAn5 24.7 24.0 10.0 9.7 9.3 9.0 0.3 0.3 0.7 1.0 N + VAn4 24.6 23.9 10.0 9.7 9.3 8.7 0.3 0.3 0.7 1.0 N+Carbofuran(Cbf) 23.5 23.0 9.7 9.3 8.3 8.3 0.3 0.3 0.7 1.0 Control (F+N) 16.4 16.4 7.0 7.0 5.3 5.3 7.3 7.3 8.0 8.0 F + N + AAn1 21.9 21.1 7.7 7.7 6.3 6.0 4.7 5.0 5.7 6.3 F + N + ANAn1 21.8 21.0 7.7 7.3 6.3 6.0 4.7 5.0 5.7 5.7 F + N + ANAn4 22.3 22.0 8.3 8.3 7.3 7.3 4.7 5.0 5.7 5.7 F + N + AnC2 23.5 22.5 8.3 8.3 7.7 7.3 2.7 3.0 3.7 4.0 F + N + AnR3 22.3 22.1 8.0 8.0 7.3 7.0 3.7 3.7 4.3 4.7 F + N + BAn4 22.0 21.2 7.7 7.7 7.0 7.0 4.7 5.3 6.3 6.7 F + N + BasAn5 16.8 16.7 7.7 7.3 6.3 6.0 4.7 5.3 6.7 7.0 F + N + BuAn3 22.3 21.4 8.0 8.0 7.3 6.0 4.7 5.0 4.3 4.7 F + N + BudAn3 18.4 18.1 7.7 7.3 6.3 6.0 4.7 5.3 5.3 6.0 F + N + GaAn1 16.9 16.8 7.3 7.3 7.3 6.0 4.7 5.3 6.0 6.7 F + N + JaAn2 16.9 16.8 7.3 7.3 6.3 6.0 4.7 5.7 6.3 6.7 F + N + LAn3 16.8 16.7 7.3 7.3 6.3 6.0 4.7 4.7 6.0 6.7 F + N + MeAn4 16.7 16.6 7.0 7.3 6.3 5.7 5.3 5.7 6.7 7.3 F + N + SkNAn3 19.9 19.4 7.3 7.7 6.3 6.0 4.7 4.7 5.3 6.0 F + N + SkNAn5 24.1 22.9 8.7 8.3 8.7 8.3 2.7 3.0 3.7 4.0 F + N + VAn4 24.0 22.9 8.3 8.0 7.7 7.3 2.7 3.3 3.7 4.3 F + N + Czm + Cbf 22.2 21.3 8.0 7.7 7.3 7.0 3.7 3.7 4.3 4.7 LSD (P≤0.01) 0.98 0.91 0.66 0.67 0.57 0.57 0.20 0.20 0.18 0.21 F-value (P≤0.01) Control agents (df=18) 16.8* 10.5* NS 5.7* 9.0* 11.4* 6.8* 4.71* 30.2* 16.6* Pathogens (df=2) 38.6* 7.9* 29* 28* 34.6* 9.81* 4368* 4360* 3627* 11000* Interaction (df=17) 46.2* 32.0* 6.61* 6.65* 23.7* 22.6* 343* 522* 462* 508* Each value is the mean of three replicates. Plants not inoculated with either pathogen but applied with Aspergillus niger isolates were compared with uninoculated Control; wilt fungus inoculated plants applied with A. niger isolates were compared with fungus inoculated Contorl (F); nematode inoculated plants applied with A. niger isolates were compared with nematode inoculated Contorl (N); concomitantly inoculated plants applied with A. niger isolates were compared with concomitantly inoculated Contorl (F+N). *Significantly different from the control at P≤0.01; NS- Not significant at P≤0.01. Foc - Fusarium oxysporum f. sp. ciceri.

151

Table 37. Effect of nursery treatment and soil application of efficient Aspergillus niger isolates on the viability and germination of seeds, and detection of associated Fusarium oxysporum f. sp. lycopersici with harvested fruits of tomato from pots inoculated with F. oxysporum f. sp. lycopersici, Meloidogyne incognita singly and concomitantly. Out of 10 seeds Detection of Fol with 15 seeds

Viable Germinated Sterilized Unsterilized Treatment

Nursery Soil Nursery Soil Nursery Soil Nursery Soil tr. app. tr. app. tr. app. tr. app. Control 9 9 8.3 8.3 0.7 0.7 1 1 AAn1 10 9.3 9 8.7 0.3 0.3 0.7 0.7 ANAn1 10 9.7 9 8.7 0.3 0.3 0.7 0.7 ANAn4 10 9.7 10 9.7 0.3 0.3 0.3 0.3 AnC2 10 9.7 10 9.7 0.3 0.3 0.3 0.3 AnR3 10 9.7 10 9.7 0.3 0.3 0.3 0.3 BAn4 10 9.3 9.7 9.3 0.7 0.7 1 1 BasAn5 9.7 9.3 8.7 8.7 0.3 0.3 0 0.7 BuAn3 10 9.7 9.7 8.7 0.3 0.3 0 0 BudAn3 9.3 9.3 8.7 8.7 0.3 0.3 0.7 0.7 GaAn1 9.3 9.3 8.7 8.7 0.3 0.3 0.7 0.7 JaAn2 9.7 9.3 8.7 8.7 0.3 0.3 0.7 0.7 LAn3 9.3 9 8.7 9.3 0.3 0.3 0.7 0.7 MeAn4 9.3 9 8.7 9.3 0.7 0.7 1 1 SkNAn3 9.7 9.3 8.7 9.3 0 0 0 0.7 SkNAn5 10 10 10 10 0 0 0 0 VAn4 10 10 10 9.7 0 0 0 0 Carbendazim 10 9.7 8.7 8.7 0 0.3 0 0.7 (Czm) Carbofuran (Cbf) 10 9.7 8.7 8.7 0.7 0.7 0.7 1 Control (F) 7.7 7.7 6.7 6.7 5.7 5.7 9 9 F + AAn1 8.7 8.7 7.3 7 4.3 4.3 5.7 5.7 F + ANAn1 8.7 8.3 7.3 7.3 4.3 4.3 5.7 5.7 F + ANAn4 8.7 8.7 8 8 4.3 4.3 5.3 5.7 F + AnC2 9 8.7 8.3 8 3 3 4.3 4.3 F + AnR3 8.3 8.3 7.7 7.7 3 3 4.3 4.3 F + BAn4 8.7 8.3 7.3 7.3 4.3 4.3 5.7 6 F + BasAn5 8 8 7 7 4.3 4.3 6 6 F + BuAn3 8.7 8.7 7.7 7.7 3 3 5.7 6 F + BudAn3 7.7 7.7 7.3 7.3 4.3 4 6 6 F + GaAn1 7.7 7.7 7.3 7.3 4.3 4.3 6 6 F + JaAn2 8 7.7 7.3 7 4.3 4.7 6 6.7 F + LAn3 7.7 8 7.3 7.3 4.3 4.7 6.7 6.7 F + MeAn4 7.7 7.7 7 7 4.7 5.3 8 8.7 F + SkNAn3 8.3 8 7.3 7.3 4.3 4.3 5.7 6 F + SkNAn5 9.7 9.3 9.3 9 1.3 1.7 3.7 4 F + VAn4 9.3 9.3 8.7 8.3 1.7 2.3 4.3 4.3 F+Carbendazim 8.7 8 8 7.3 3 3 4.7 4.3 (Czm)

Continued…

152

Table 37 Continued …

Control (N) 8.3 8.3 7.3 7.3 1 1 1.7 1.7 N + AAn1 9 9 8 7.7 0.7 0.7 1.3 1.3 N + ANAn1 9 9 8 7.7 0.7 0.7 1.3 1.3 N + ANAn4 9.3 9.3 8 8 0.7 0.7 1 1.3 N + AnC2 9.7 9.3 8.7 8.3 0.3 0.3 1 1.3 N + AnR3 9.7 9.3 8.3 8.3 0.3 0.3 1 1.3 N + BAn4 9 9.3 8 8 0.7 0.7 1.3 1.3 N + BasAn5 9 9 8 8 0.7 0.7 1.3 1.7 N + BuAn3 9.7 9.7 8.3 8.3 0.3 0.3 1.3 1.3 N + BudAn3 8.7 9 8 7.7 0.7 0.7 1.3 1.7 N + GaAn1 8.7 8.7 8 7.7 0.7 0.7 1.3 1.7 N + JaAn2 8.7 8.7 8 7.7 0.7 0.7 1.3 1.7 N + LAn3 8.7 8.7 8 7.7 0.7 0.7 1.3 1.7 N + MeAn4 8.7 8.7 8 7.7 0.7 1 1.3 1.7 N + SkNAn3 9 9 8 8 0.7 0.7 1.3 1.7 N + SkNAn5 10 9.7 9.3 9 0.3 0.3 1 1.3 N + VAn4 10 9.7 9.3 8.7 0.3 0.3 1 1.3 N+Carbofuran(Cbf) 9.7 9.3 8.3 8.3 0.3 0.7 1 1.3 Control (F+N) 6.3 6.3 5.3 5.3 9 9 13 13 F + N + AAn1 7.7 7.7 6.3 6 6.3 6.7 7.3 8.3 F + N + ANAn1 7.7 7.3 6.3 6 6.3 6.7 7.3 8 F + N + ANAn4 8.3 8.3 7.3 7.3 6 6.3 7.3 8 F + N + AnC2 8.3 8.3 7.7 7.3 3.7 3.7 4.7 5 F + N + AnR3 8 8 7.3 7 4.7 4.7 5.7 6 F + N + BAn4 7.7 7.7 7 7 6.3 6.7 8 8.7 F + N + BasAn5 7.7 7.3 6.3 6 6.3 6.7 8.7 9 F + N + BuAn3 8 8 7.3 6 6 6.7 5.7 6 F + N + BudAn3 7.7 7.3 6.3 6 6.3 7 7 7.7 F + N + GaAn1 7.3 7.3 7.3 6 6.3 7 7.7 8.7 F + N + JaAn2 7.3 7.3 6.3 6 6.3 7.3 8 8.7 F + N + LAn3 7.3 7.3 6.3 6 6.3 6.7 7.7 8.7 F + N + MeAn4 7 7.3 6.3 5.7 7 7.7 8.7 9.3 F + N + SkNAn3 7.3 7.7 6.3 6 6.3 6.3 6.7 7.7 F + N + SkNAn5 9 8.3 8.7 8.3 3.3 3.7 4.7 5 F + N + VAn4 8.3 8 7.7 7.3 3.7 3.7 5 5.7 F + N+Czm+Cbf 8 7.7 7.7 7 4.7 5 5.7 6.3 LSD (P≤0.01) 0.69 0.65 0.6 0.58 0.22 0.24 0.2 0.23 F-value (P≤0.01) Control agents 6.3* 6.1* 19.4* 22.3* 7.2* 4.93* 68.4* 29.7* (df=18) Pathogens (df=2) 33* 25* 54.5* 12.2* 5500* 4590* 6680* 12200* Interaction (df=17) 12.71* 10.2* 43.6* 31.1* 563* 520* 678* 915* Each value is the mean of three replicates. Plants not inoculated with either pathogen but applied with Aspergillus niger isolates were compared with uninoculated Control; wilt fungus inoculated plants applied with A. niger isolates were compared with fungus inoculated Contorl (F); nematode inoculated plants applied with A. niger isolates were compared with nematode inoculated Contorl (N); concomitantly inoculated plants applied with A. niger isolates were compared with concomitantly inoculated Contorl (F+N). *Significantly different from the control at P≤0.01; Fol - Fusarium oxysporum f. sp. lycopersici.

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Seed infestation with the wilt fungus Wilt fungus was mainly detected from the harvested seeds of Fusarium inoculated plants (Fig. 34). The fungus infestation was significantly greater (P≤ 0.0001) on the seeds surface sterilized or unsterilized obtained from Fusarium inoculated plants compared to uninoculated plants. The infestation was further greater (P≤ 0.00001) on the seeds of plants concomitantly inoculated with the wilt fungus and root-knot nematode compared to only Fusarium inoculated plants (Table 36-37). Application of A. niger isolates considerably decreased the infestation of wilt fungus in seeds from plants inoculated with Fusarium singly (P≤ 0.01) or concomitantly (P≤ 0.001) (Table 36-37). Application of pesticides also decreased the Fusarium infection of seeds. Among the A. niger isolates and pesticides, variation in the performance was seen and the order of effectiveness was SkNAn5 > VAn4 > AnC2 > AnR3 > pesticides > ANAn4 > BuAn3. In Fusarium inoculated plants, decrease in the seed infestation of wilt fungus with A. niger isolates was significantly (P≤ 0.01) greater in unsterilized seeds than sterilized, but in concomitantly inoculated plants, the decrease was greater (P≤ 0.001) from sterilized seeds than unsterilized (Table 36-37).

Fig. 34. Detection of Fusarium oxysporum f. sp. ciceri and F. oxysporum f. sp. lycopersici from the seeds of chickpea (A) and tomato (B), respectively.

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Soil Population Wilt fungi Soil population of the wilt fungus, F. oxysporum f. sp. ciceri gradually increased during the four months of crop period reaching to a peak of 5.8 × 107 (log 7.76) CFUs/g soil in February in comparison to the planting population of 2.4 × 107 (log 7.38) CFUs/g soil (Fig. 35). Seed or soil treatment of A. niger isolates, however, decreased the pathogen population to a varying extent and the February population (peak) decreased to almost undetectable level of log 0.69 CFUs/g soil with SkNAn5 isolate followed by VAn4 (log 1.84 CFUs/g soil), AnC2 (log 2.0 CFUs/g soil), AnR3 (log 3.10 CFUs/g soil), ANAn4 (log 4.11 CFUs/g soil) and BuAn2 (log 4.01 CFUs/g soil) (Fig. 35). Seed treatment with carbendazim decreased the pathogen population to log 5.27 CFUs/g (Fig. 35). In the presence of root- knot nematode, M. incognita, soil population of the wilt fungus increased significantly (P≤ 0.000001) with an increase of 500-700% in comparison to absence of nematode (Fig. 35). Population of the fungus in concomitantly inoculated plants drastically decreased (P≤ 0.000001) due to application of A. niger isolates in comparison to the respective month control (Fig. 35). Variation in the soil population of tomato wilt fungus, F. oxysporum f. sp. lycopersici in the presence or absence of root-knot nematode and with the nursery or soil treatment of tomato plants with A. niger isolates was almost same as of F. oxysporum f. sp. ciceri in chickpea rhizosphere (Fig. 36). Significant difference (P≤ 0.000001) among the A. niger isolates was recorded for soil population of wilt fungi in both the crops. Carbendazim+carbofuran application in concomitantly inoculated plants decreased (P≤ 0.000001) the soil population of the wilt fungi. Relative effectiveness of A. niger isolates and pesticides in decreasing the population of wilt fungi was SkNAn5 > VAn4 > AnC2 > AnR3 > ANAn4 > BuAn3 > pesticides > Ban4 > ANAn4 > rest of the A. niger isolates (Fig. 35-36). Root-knot nematode Soil population of root-knot nematode, M. incognita gradually increased with the progress of time from December onwards and reached its peak at harvest that was 105% greater in chickpea and 190% greater in tomato than planting population of 2000 J2/kg soil (Fig. 37- 38). In the presence of wilt fungus, nematode population was, however, significantly less than the respective month controls (P≤ 0.01) in both the crops. Among the A. niger isolates, only six isolates suppressed the nematode population (43-65%) over the initial population in

155

With Root-Knot Nematode Without Root-Knot Nematode With Root-Knot Nematode Without Root-Knot Nematode

Fig. 35. Effects of seed treatment and soil application of Aspergillus niger isolates on the soil population of Fusarium oxysporum f. sp. ciceri in the presence and absence of Meloidogyne incognita. Vertical bars indicate the standard error of three replicates; Czm – carbendazim, Cbf - carbofuran.

156

With Root-KnotAbsence Nematode Without Root-KnotPresence Nematode With Root-KnotAbsence Nematode Without Root-Knot Presence Nematode

Fig. 36. Effects of nursery treatment and soil application of Aspergillus niger isolates on the soil population of Fusarium oxysporum f. sp. lycopersici in the presence and absence of Meloidogyne incognita. Vertical bars indicate the standard error of three replicates; Czm – carbendazim, Cbf - carbofuran.

157

)/kg soil

Juveniles (J Juveniles 3

×10

With Wilt Fungus Without Wilt Fungus With Wilt Fungus Without Wilt Fungus

Fig. 37. Effects of seed treatment and soil application of Aspergillus niger isolates on the soil population of Meloidogyne incognita in the absence and presence of Fusarium oxysporum f. sp. ciceri. Vertical bars indicate the standard error of three replicates; Czm – carbendazim, Cbf - carbofuran.

158

)/kg soil 2 Juveniles (J Juveniles 3 ×10 With Wilt Fungus Without Wilt Fungus With Wilt Fungus Without Wilt Fungus

Fig. 38. Effects of nursery treatment and soil application of Aspergillus niger isolates on the soil population of Meloidogyne incognita in the absence and presence of Fusarium oxysporum f. sp. lycopersici. Vertical bars indicate the standard error of three replicates; Czm – carbendazim, Cbf - carbofuran.

159

both the crops. Greatest decrease in the nematode population in chickpea root-zone occurred with A. niger isolates viz., SkNAn5 (57, 65%), VAn4 (53, 61%), AnC2 (49, 57%), AnR3 (52, 56%), ANAn4 (51, 55%) and BuAn3 (46, 55%) over initial nematode population (Fig. 37). Similarly, the nematode population in tomato root-zone with these isolates was decreased by 43-61% (P≤ 0.0001) (Fig. 38). In the presence of wilt fungus, nematode population decreased over time, the decrease was, however, insignificant (P≤ 0.05) (Fig. 37- 38). Seed or nursery treatment with the A. niger isolates or pesticides decreased the nematode population and order of decrease was more or less similar to the treatments where nematode was applied alone (Fig. 37-38). Aspergillus niger isolates Soil population of A. niger isolates in chickpea and tomato significantly increased during the experimental period (Fig. 39-40). In the soils infested with wilt fungus alone or together with nematode, population of A. niger isolates increased during all months of sampling being greatest in March in comparison to planting population with 21-75% and 75-310% increase, respectively (Fig. 39-40). As far as increase in soil population with respect to respective month control is concerned, A. niger isolates were significant at P≤ 0.000001 level. In nematode inoculated pots, increase in A. niger isolates population was significantly greater (P≤ 0.000001) in comparison to planting population only, but soil population of some isolates viz., SkNAn5, VAn4, AnC2, AnR3, ANAn4 also increased with respect to month control (P≤ 0.0001) (Fig. 39-40). Conclusion Six isolates of A. niger isolates viz., ANAn4, AnC2, AnR3, BuAn3, SkNAn5 and VAn4 performed better and qualified almost all the parameters tested in these experiments against the wilt fungi and root-knot nematode on chickpea and tomato. These isolates tested negative for ochratoxin A production, and showed more phosphate solubilization activity (P≤ 0.0001), greater compatibility with pesticides and heavy metals (P≤ 0.0001), and more fungicidal (P≤ 0.0001) and nematicidal effects (P≤ 0.001). Application of these isolates decreased wilt and root-knot symptoms (P≤ 0.0001) and improved dry matter production and yield (P≤ 0.0001), leaf chlorophyll (P≤ 0.001), leaf phenol (P≤ 0.001), salicylic acid of leaf (P≤ 0.01) and root (P≤ 0.001), lycopene content (P≤ 0.001) of tomato fruits etc., with greater increase in seed index (P≤ 0.01) of chickpea, viability and germination (P≤ 0.01) of harvested seeds, and lower seed infestation with wilt fungus (P≤ 0.001). The isolates also suppressed the rhizosphere population of wilt fungi (P≤ 0.000001) and root-knot (P≤ 0.0001) nematode, while their own populations increased greatly (P≤ 0.0001).

160

CFU/g soil CFU/g 6

×10

Fig. 39. Rhizosphere population of Aspergillus niger isolates in relation to single or concomitant inoculations with Fusarium oxysporum f. sp. ciceri and Meloidogyne incognita. Vertical bars indicate the standard error of three replicates; Foc - F. oxysporum f. sp. ciceri, Mi - M. incognita.

161

CFU/g soil 6

×10

Fig. 40. Rhizosphere population of Aspergillus niger isolates in relation to single or concomitant inoculations with Fusarium oxysporum f. sp. lycopersici and Meloidogyne incognita. Vertical bars indicate the standard error of three replicates; Fol - F. oxysporum f. sp. lycopersici, Mi - M. incognita. 162

Experiment-IV EVALUATION OF SELECTED ISOLATES OF ASPPERGILLUS NIGER FOR EFFECTIVENESS AGAINST FUSARIUM WILT, ROOT-KNOT AND FUNGUS-NEMATODE WILT DISEASE COMPLEX OF CHICKPEA AND TOMATO UNDER FIELD CONDITION

Based on preliminary evaluation of Aspergillus niger isolates done in pot culture (Expt. III) for effectiveness against wilt, root-knot and fungus-nematode disease complex and for promotion of morphological (plant growth, yield etc.) and biochemical parameters of chickpea and tomato (chlorophyll, phenol contents, salicylic acid of leaves and roots, lycopene content), six isolate viz., ANAn4, AnC2, AnR3, BuAn3, SkNAn5 and VAn4 performed better than rest and qualified most of the parameters for being a good biocontrol agents. A trial was conducted in microplots of 4 × 2 m2 size (Fig. 41) to ascertain the performance of these isolates under field condition. Effects of the A. niger isolates were compared with efficacious pesticides namely carbendazim and carbofuran. The isolates were cultured on baggase-soil mixture (BSM) and were applied to seeds/ (4 g/kg seed) and soil (40 g/microplot). In case of tomato, root-dip treatment with A. niger isolates (10 g/100 seedlings) was done. For this treatment spore suspension of A. niger was made by grinding 10 g of colonized sorghum seeds in 100 ml distilled water in which roots of tomato seedlings were dipped for 10 minutes.

Symptoms

Fusarial wilt In the plots infested with Fusarium oxysporum f. sp. ciceri and F. oxysporum f. sp. lycopersici the wilt incidence was 65 and 66% on chickpea cv. BGD-72 and on tomato cv. Pusa Ruby, respectively (Fig. 42-43). Application of A. niger isolates or pesticides influenced the disease incidence. Seed/nursery treatment with A. niger isolates brought down the wilt incidence to 15-26% in chickpea and 16-28% in tomato (Fig. 42-43). In the plots where carbendazim was applied through seed/nursery treatment, the wilt incidence was 26 and 29% in chickpea and tomato, respectively (Fig. 42-43). The severity of wilt due to the pathogen was 68 and 74% in chickpea and tomato, respectively. Application of A. niger isolates decreased the severity of wilt to a varied extent. Lowest severity of 22 and 28% was recorded due to seed treatment with SkNAn5 followed by 24 and 32% with VAn4 and AnC2 in

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chickpea and tomato, respectively (Fig. 42-43). Seed treatment with carbendazim reduced severity to 26 and 34% in chickpea and tomato respectively. Response of wilt disease to soil application of A. niger isolates was more or less similar to that recorded due to seed treatment.

Fig. 41. An aerial view of experimental plots of chickpea (A) and tomato (B).

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SEED TREATMENT SOIL APPLICATION

Foc Foc+Mi Foc Foc+Mi

Fig. 42. Effects of seed treatment and soil application of Aspergillus niger isolates on the incidence and severity of wilt of chickpea caused by Fusarium oxysporum f. sp. ciceri in the presence and absence of Meloidogyne incognita. Vertical bars indicate the standard error of three replicates. Foc – F. oxysporum f. sp. ciceri, Mi – M. incognita; Czm – carbendazim, Cbf - carbofuran.

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NURSERY TREATMENT SOIL APPLICATOIN

Fig. 43. Effects of nursery treatment and soil application of Aspergillus niger isolates on the incidence and severity of wilt of tomato caused by Fusarium oxysporum f. sp. lycopersici in the presence and absence of Meloidogyne incognita. Vertical bars indicate the standard error of three replicates; Fol – F. oxysporum f. sp. lycopersici, Mi – M. incognita; Czm – carbendazim, Cbf - carbofuran.

166

Root knot Disease Characteristic galls developed on the roots of chickpea and tomato plants grown in the plots infested with the juveniles of Meloidogyne incognita (2000 J2/kg soil) (Fig. 44-45). On average, 58 and 83 galls and 51 and 75 egg masses/root system were formed on chickpea and tomato, respectively. Application of A. niger isolates suppressed the gall formation to a varied extent (Fig. 44-45). Seed treatment with A. niger isolate SkNAn5 resulted to 51 and 48% decrease in the galling, followed by 45 and 40% with VAn4 and AnC2 isolates in comparison to the control of chickpea and tomato, respectively. The gall formation was decreased by 44 and 43% due to application of carbofuran in chickpea and tomato, respectively. Wilt disease complex Chickpea and tomato plants grown in the plots infested concomitantly with F. oxysporum f. sp. ciceri or F. oxysporum f. sp. lycopersici and M. incognita exhibited severe stunted growth with chlorotic, wilted and dried leaves. Wilt symptoms in terms of incidence and severity were significantly greater compared to the wilting recorded in the plots inoculated with wilt fungus alone. In the plots infested with root-knot nematode, M. incognita a considerable increase in wilt incidence (P≤ 0.001) and severity (P≤ 0.01) was recorded in chickpea and tomato as compared to the absence of nematode (Fig. 42-43). The wilt incidence and severity in concomitantly infested plots considerably decreased due to seed/nursery application of A. niger isolates. Greatest decrease was recorded with seed/nursery treatment with SkNAn5 i.e., 75 and 68% in chickpea and 76 and 62% in tomato followed by VAn4 (69 and 65% in chickpea, and 70 and 57% in tomato) and AnC2 (65 and 65% in chickpea, and 66 and 57% in tomato). Joint treatment with carbendazim and carbofuran decreased the wilt incidence and severity by 58 and 62% in chickpea and 56 and 54% in tomato, respectively and the galling by 27 and 31%. Application of A. niger isolates caused a significant (P≤ 0.01) decrease in the gall formation with SkNAn5 by 36-43% and followed by VAn4 (25-35%) and AnC2 (27- 33%) (Fig. 44-45). The decrease in gall formation with A. niger isolates was significantly (P≤ 0.05) greater in tomato than chickpea. The egg mass production of the nematode showed almost identical response to various treatments. Overall effect of soil application was similar to seed/nursery treatments but it was 2-5% less effective (Fig. 42-45).

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SEED TREATMENT SOIL APPLICATION

Mi Foc+Mi Mi Foc+Mi Fig. 44. Effects of seed treatment and soil application of Aspergillus niger isolates on the galling and egg mass production of Meloidogyne incognita alone or with Fusarium oxysporum f. sp. ciceri. Vertical bars indicate the standard error of three replicates. Mi – M. incognita, Foc – F. oxysporum f. sp ciceri; Czm – carbendazim, Cbf - carbofuran.

168

NURSERY TREATMENT SOIL APPLICATOIN

Mi Fol+Mi Mi Fol+Mi

Fig. 45. Effects of nursery treatment and soil application of Aspergillus niger isolates on the galling and egg mass production of Meloidogyne incognita alone or with Fusarium oxysporum f. sp. lycopersici. Vertical bars indicate the standard error of three replicates. Mi – M. incognita, Fol – F. oxysporum f. sp lycopersici; Czm – carbendazim, Cbf - carbofuran.

169

Plant growth and yield Seed treatment with A. niger isolates resulted to significant increase in the dry weight of chickpea (P≤ 0.01) and tomato (P≤ 0.001) plants (Table 38-39). Greatest increase in the plant dry weight with A. niger isolates was recorded with SkNAn5 (41 and 45%) followed by VAn4 (38 and 41%) and AnC2 (37 and 40%) in chickpea and tomato, respectively in comparison to uninoculated control. Infestation with F. oxysporum f. sp. ciceri or F. oxysporum f. sp. lycopersici resulted to 27 and 31% decrease in the plant dry weight and 24 and 33% in yield of chickpea and tomato, respectively (Table 38-39). Application of A. niger isolates decreased the suppressive effect of the wilt fungi leading to significant increase in the dry weight of chickpea and tomato plants. Isolates SkNAn5, VAn4 and AnC2 promoted the dry matter production by 62, 58 and 56% in chickpea and 70, 64 and 61% in tomato compared to wilt fungus inoculated control. Seed treatment with carbendazim resulted to 25 and 24% increase in the dry weight of fungus inoculated plants of chickpea and tomato, respectively. Infection by root-knot nematode, M. incognita caused significant decrease (15 and 23%) in the dry weight of chickpea and tomato, respectively over uninoculated control (Table 38-39). Treatments with the A. niger SkNAn5, VAn4 and AnC2 increased the dry weight of chickpea and tomato by 29, 31%; 27, 30% and 25, 26% over infected controls, respectively. Carbofuran application increased the dry weight of nematode inoculated plants by 23 and 21% in chickpea and tomato, respectively. Concomitant inoculations with M. incognita and F. oxysporum f. sp. ciceri or F. oxysporum f. sp. lycopersici decreased the dry weight of chickpea by 45% and tomato by 54% compared to uninoculated control (Table 38-39). This decrease was significantly greater than the sum of reductions caused by the fungus and nematode separately. Treatments with A. niger isolates considerably decreased the suppressive effect of the pathogens. Applications of A. niger isolate SkNAn5, VAn4 and AnC2 improved the dry weight of chickpea by 43, 42 and 41% and tomato by 46, 44 and 44%, respectively whereas combined application of carbendazim and carbofuran by 31 and 30%. Yield in terms of weight of grains or fruits per plant was significantly promoted in chickpea (P≤ 0.01) and tomato (P≤ 0.001) due to application of the used A. niger isolates in comparison to the control (Table 38-39). Highest increase in the yield was recorded with isolate SkNAn5, VAn4 and AnC2 by 37, 34 and 31% in chickpea and 41, 38 35% in tomato, respectively. The wilt fungi decreased the yield of chickpea and tomato by 24 and 33%, respectively compared to the controls (Table 38-39). Seed treatment with A. niger isolates SkNAn5, VAn4, AnC2 and carbendazim enhanced the yield of chickpea by 59, 52, 50 and

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Table 38. Effects of seed treatment and soil application of Aspergillus niger isolates on the dry matter production and yield of chickpea in field plots infested singly or concomitantly with Fusarium oxysporum f. sp. ciceri and Meloidogyne incognita or not infested. Dry shoot weight (g) Yield/plant (g)

Treatment Seed treatment Soil application Seed treatment Soil application Control 21.2 21.2 7.1 7.1 ANAn4 28.4 (33.8) 29.2 (37.7) 8.9 (25.8) 9.1 (27.9) AnC2 29.1 (37.2) 28.8 (35.7) 9.3 (31.4) 9.3 (30.7) AnR3 29.1 (37.4) 29.0 (37.0) 9.2 (28.9) 9.3 (30.9) BuAn3 28.2 (33.3) 28.5 (34.6) 9.0 (26.3) 8.9 (25.8) SkNAn5 29.9 (40.9) 29.7 (40.1) 9.7 (36.7) 9.7 (36.2) VAn4 29.2 (37.8) 28.8 (35.8) 9.5 (34.1) 9.4 (32.4) Carbendazim (Czm) 22.6 (6.4) 22.3 (5.2) 7.4 (4.8) 7.4 (3.7) Carbofuran (Cbf) 22.4 (5.5) 22.2 (4.7) 7.4 (4.3) 7.3 (3.2) Control (F) 15.4 (-27.4) 15.4 (-27.4) 5.4 (-24.4) 5.4 (-24.4) F + ANAn4 20.0 (29.7) 20.3 (32.1) 6.7 (23.6) 6.7 (24.7) F + AnC2 24.0 (55.6) 23.8 (54.3) 8.1 (49.7) 8.0 (48.2) F + AnR3 20.0 (30.1) 20.3 (31.7) 6.8 (25.8) 6.8 (26.5) F + BuAn3 19.8 (28.5) 19.9 (29.5) 6.6 (22.8) 6.6 (22.0) F + SkNAn5 25.0 (62.4) 24.9 (61.4) 8.6 (58.7) 8.5 (57.4) F + VAn4 24.4 (58.3) 24.3 (58.0) 8.2 (52.0) 8.1 (50.8) F+Carbendazim(Czm) 19.3 (25.3) 19.0 (23.7) 6.4 (19.2) 6.4 (18.1) Control (N) 18.1 (-14.8) 18.1 (-14.8) 6.0 (-15.3) 6.0 (-15.3) N + ANAn4 20.7 (14.2) 21.0 (15.8) 6.7 (11.5) 6.7 (12.4) N + AnC2 22.6 (24.7) 23.0 (27.3) 7.4 (22.9) 7.3 (21.6) N + AnR3 20.6 (13.9) 20.9 (15.4) 6.7 (11.4) 6.8 (12.9) N + BuAn3 20.7 (14.3) 20.6 (13.9) 6.7 (11.2) 6.6 (10.4) N + SkNAn5 23.3 (28.9) 23.6 (30.5) 7.5 (25.6) 7.4 (23.7) N + VAn4 22.9 (26.7) 23.2 (28.4) 7.4 (23.7) 7.3 (22.4) N + Carbofuran (Cbf) 22.2 (22.5) 21.9 (21.1) 7.5 (25.5) 7.4 (23.6) Control (F+N) 11.7 (-44.7) 11.7 (-44.7) 3.6 (-49.3) 3.6 (-49.3) F + N + ANAn4 14.2 (21.6) 14.4 (23.5) 4.2 (16.5) 4.2 (17.4) F + N + AnC2 16.5 (40.9) 16.1 (38.0) 4.9 (35.1) 4.8 (34.7) F + N + AnR3 14.3 (21.9) 14.4 (23.0) 4.2 (17.9) 4.3 (19.3) F + N + BuAn3 14.0 (19.9) 14.0 (20.0) 4.2 (16.9) 4.2 (16.8) F + N + SkNAn5 16.7 (43.1) 16.9 (44.0) 5.1 (40.8) 5.0 (38.8) F + N + VAn4 16.6 (41.5) 16.5 (41.4) 4.9 (37.4) 4.9 (35.5) F + N + Czm + Cbf 15.3 (30.9) 15.0 (28.2) 4.6 (26.7) 4.4 (22.9) LSD (P≤0.01) 1.1 1.3 0.24 0.21 F-value (P≤0.01) Control agents (df=8) 99.4* 148.8* 278* 315* Pathogens (df=2) NS NS 34.3* 40.3* Interaction (df=7) 25.2* 25.8* 83.9* 63.5* Values in parenthesis are percent increase (+ve) or decrease (-ve) over respective controls [plants not inoculated with either pathogen but applied with Aspergillus niger isolates were compared with uninoculated Control; wilt fungus inoculated plants applied with A. niger isolates were compared with fungus inoculated Contorl (F); nematode inoculated plants applied with A. niger isolates were compared with nematode inoculated Contorl (N); concomitantly inoculated plants applied with A. niger isolates were compared with concomitantly inoculated Contorl (F+N)]. *Significantly different from the control at P≤0.01. NS- Not significant at P≤0.01, but significant at P≤0.05.

171

Table 39. Effect of nursery treatment and soil application of efficient Aspergillus niger isolates on the dry matter production and yield of tomato in field plots infested singly or concomitantly with Fusarium oxysporum f. sp. lycopersici and Meloidogyne incognita or not infested. Dry shoot weight (g) Yield/plant (g)

Treatment Seed treatment Soil application Seed treatment Soil application Control 42.7 42.7 470 470 ANAn4 58.9 (37.9) 60.9 (42.6) 600 (27.6) 617 (31.4) AnC2 59.8 (40.1) 59.5 (39.4) 633 (34.7) 630 (34.0) AnR3 60.4 (41.4) 60.1 (40.7) 620 (31.8) 630 (34.1) BuAn3 58.3 (36.4) 59.4 (39.1) 610 (29.7) 601 (27.9) SkNAn5 61.9 (44.9) 61.2 (43.3) 660 (40.5) 653 (39.0) VAn4 60.1 (40.7) 60.0 (40.6) 650 (38.3) 639 (35.9) Carbendazim (Czm) 46.0 (7.7) 44.8 (5.0) 514 (9.3) 490 (4.4) Carbofuran (Cbf) 45.8 (7.3) 45.0 (5.3) 512 (8.9) 484 (2.9) Control (F) 29.3 (-31.4) 29.3 (-31.4) 314 (-33.2) 314 (-33.2) F + ANAn4 37.9 (29.4) 38.3 (30.7) 386 (23.1) 388 (23.6) F + AnC2 47.1 (60.8) 47.1 (60.7) 481 (53.3) 478 (52.2) F + AnR3 37.9 (29.5) 38.2 (30.3) 391 (24.6) 396 (26.1) F + BuAn3 37.5 (27.9) 37.9 (29.3) 383 (22.1) 382 (21.6) F + SkNAn5 49.7 (69.5) 49.0 (67.2) 517 (64.7) 512 (63.1) F + VAn4 48.1 (64.1) 48.1 (64.2) 494 (57.3) 490 (56.2) F+Carbendazim(Czm) 36.2 (23.5) 35.1 (19.7) 381 (20.2) 364 (15.9) Control (N) 33.0 (-22.6) 33.0 (-22.6) 356 (-24.3) 356 (-24.3) N + ANAn4 37.5 (13.6) 38.1 (15.4) 395 (11.0) 398 (12.0) N + AnC2 41.7 (26.3) 42.9 (29.9) 443 (24.6) 442 (24.2) N + AnR3 37.5 (13.7) 37.9 (14.9) 395 (11.0) 401 (12.7) N + BuAn3 37.6 (14.0) 37.5 (13.6) 395 (11.0) 392 (10.2) N + SkNAn5 43.2 (30.8) 44.2 (34.1) 455 (27.8) 450 (26.4) N + VAn4 42.7 (29.5) 43.0 (30.2) 449 (26.1) 443 (24.4) N + Carbofuran (Cbf) 40.3 (22.1) 38.7 (17.2) 456 (28.1) 433 (21.6) Control (F+N) 19.7 (-54.2) 19.7 (-54.2) 199 (-57.7) 199 (-57.7) F + N + ANAn4 23.9 (21.3) 24.3 (23.4) 231 (16.2) 233 (17.1) F + N + AnC2 28.3 (43.9) 28.0 (42.1) 275 (38.5) 276 (38.8) F + N + AnR3 24.0 (21.8) 24.2 (22.7) 234 (17.5) 237 (19.3) F + N + BuAn3 23.4 (19.0) 23.5 (19.4) 231 (16.0) 232 (16.7) F + N + SkNAn5 28.8 (46.0) 28.9 (46.9) 286 (44.0) 281 (41.2) F + N + VAn4 28.4 (44.2) 28.6 (45.1) 280 (40.9) 274 (37.9) F + N + Czm + Cbf 25.6 (29.8) 24.8 (25.8) 254 (27.8) 246 (23.9) LSD (P≤0.001) 2.6 2.5 6.9 6.3 F-value (P≤0.001) Control agents (df=8) 465* 428* 3992* 3980* Pathogens (df=2) 630* 345* 1768* 3436* Interaction (df=7) 38.8* 54.2* 509* 645* Values in parenthesis are percent increase (+ve) or decrease (-ve) over respective controls [plants not inoculated with either pathogen but applied with Aspergillus niger isolates were compared with uninoculated Control; wilt fungus inoculated plants applied with A. niger isolates were compared with fungus inoculated Contorl (F); nematode inoculated plants applied with A. niger isolates were compared with nematode inoculated Contorl (N); concomitantly inoculated plants applied with A. niger isolates were compared with concomitantly inoculated Contorl (F+N)]. *Significantly different from the control at P≤0.001.

172

19%, and tomato by 65, 57, 53 and 20%, respectively, compared to the inoculated control. Root-knot infection caused 15 and 23% decline in the yield of chickpea and tomato over control (Table 38-39). Application of all six isolates of A. niger induced significant increase in the yield of chickpea and tomato, being greatest with SkNAn5 (26, 28%) followed VAn4 (27, 26%) and AnC2 (23, 25%). Application of carbofuran nematicide resulted to a 26 and 28% increase in the yield of chickpea and tomato, respectively in comparison to nematode inoculated control. Concomitant inoculation with F. oxysporum f. sp. ciceri or F. oxysporum f. sp. lycopersici and M. incognita drastically suppressed the yield of chickpea and tomato (Table 38-39). The decrease was significantly (P≤ 0.01) greater than the sum of the reductions caused by the fungus and nematode separately. A. niger isolates promoted the yield of infected plants by 17-41% in chickpea and 16-44% in tomato. Application of SkNAn5, VAn4 and AnC2 induced a remarkable enhancement i.e., 41, 37 and 35% in chickpea and 44, 41 and 39% in tomato compared to concomitantly inoculated control. Effect of soil application of the A. niger isolates on the yield of chickpea and tomato plants infected with wilt fungus and/or root-knot nematode was more or less similar to the seed/nursery treatments. Root nodulation Formation of rhizobial nodules on the roots of chickpea was quite good (Fig. 46-47) and on average 35 nodules were formed out of which 73% were functional and 27% were non- functional (Fig. 46-47). The nodulation was synergized with the treatments of A. niger isolates. The number of functional nodules increased by 44-58% over control due to seed treatment with A. niger isolates (Fig. 46-47). Application of carbendazim or carbofuran caused significant decline in the number of functional nodules (14-17%). Infection by F. oxysporum f. sp. ciceri and M. incognita singly or concomitantly suppressed the nodulation (P≤ 0.05). Inhibition in the nodulation due to pathogens was relatively greater with concomitant inoculations. Seed treatment with A. niger isolates suppressed the negative effect of pathogens on functional nodules. Number of non-functional nodules decreased invariably on those treatments where functional nodules were promoted. Carbendazim, carbofuran and carbendazim+carbofuran increased the number of functional nodules but it was much less than the effect of A. niger isolates. Effects of soil application of A. niger isolates was more or less similar to the seed treatments.

173

Fig. 46. Effects of seed treatment of Aspergillus niger isolates on the root nodulation of chickpea grown in plots infested with Fusarium oxysporum f. sp. ciceri and Meloidogyne incognita or not infested. Vertical bars indicate the standard error of three replicates; Foc- F. oxysporum f. sp. ciceri, Mi- M. incognita, Czm – carbendazim, Cbf - carbofuran. 174

Fig. 47. Effects of soil application of Aspergillus niger isolates on the root nodulation of chickpea grown in plots infested with Fusarium oxysporum f. sp. ciceri and Meloidogyne incognita or not infested. Vertical bars indicate the standard error of three replicates; Foc- F. oxysporum f. sp. ciceri, Mi- M. incognita, Czm – carbendazim, Cbf - carbofuran. 175

Rhizosphere population

Wilt fungi Rhizosphere population of wilt fungi decreased significantly during December (14.3%) but increased with the progress of season and reached its peak in February to log 7.79 CFUs/g soil in chickpea in comparison to planting population of November (log 7.44 CFUs/g soil) (Fig. 48-49). Seed treatment with the A. niger isolates or fungicide considerably (P≤ 0.000001) decreased the population of F. oxysporum f. sp. ciceri in comparison to control. The decrease was highest by the isolates to undetectable level in the peak month by SkNAn5 (log 0.75 CFUs/g soil) followed by VAn4 (log 1.98 CFUs/g soil) and AnC2 (log 3.13 CFUs/g soil), the decrease being significantly (P≤ 0.000001) greater than the other isolates. Presence of root-knot nematode however increased the population of the pathogenic fungus, significantly greater than the fungus alone (670%, P≤ 0.000001). Application of all A. niger isolates significantly (P≤ 0.000001) decreased the rhizosphere population in comparison to concomitantly inoculated control (log 7.44 CFUs/g soil), being greatest with SkNAn5 (log 3.3 CFUs/g soil), followed by VAn4 (log 4.32 CFUs/g soil), AnC2 (log 5.32 CFUs/g soil) and AnR3 (log 5.39 CFUs/g soil). Effects of pesticides on the rhizosphere population of Fusarium spp. was almost similar to the isolate AnR3 or AnC2. Effect of soil application of A. niger isolates on the rhizosphere population was more or less similar to seed treatment and population of F. oxysporum f. sp. lycopersici in the rhizosphere of tomato was almost similar to chickpea (Fig. 48-49). Root-knot nematode Soil population of root-knot nematode, Meloidogyne incognita monitored monthly increased gradually and periodically in the root zone of chickpea and tomato during December to March in comparison to planting population (Fig. 50-51). The increase was significant (P≤ 0.01) in all the months except December. The A. niger isolates or carbofuran effectively (P≤ 0.001) decreased the root-knot nematode population. Seed/nursery treatment with SkNAn5, VAn4 and AnC2 suppressed the nematode population during all months of sampling by 32- 75%, 27-65% and 19-60% (chickpea) and 30-75%, 25-65% and 17-59% (tomato), respectively. Carbofuran treatment decreased the population by 17-59% in chickpea and 15- 58% in tomato. In the plots, where root-knot nematode and wilt fungus was inoculated concomitantly, the nematode population was decreased 10-44% in chickpea and 8-43% in tomato compared to nematode alone (Fig. 50-51). Application of all A. niger isolates significantly suppressed the nematode population, being greater in the plots infested with the

176

Fig. 48. Effects of seed treatment and soil application of Aspergillus niger isolates on the soil population of Fusarium oxysporum f. sp. ciceri in the presence and absence of Meloidogyne incognita. Vertical bars indicate the standard error of three replicates; Czm – carbendazim, Cbf – carbofuran.

177

Fig. 49. Effects of nursery treatment and soil application of Aspergillus niger isolates on the soil population of Fusarium oxysporum f. sp. lycopersici in the presence and absence of Meloidogyne incognita. Vertical bars indicate the standard error of three replicates; Czm - carbendazim; Cbf – carbofuran.

178

SEED TREATMENT SOIL APPLICATION

)/kg soil )/kg 2

Juveniles (J Juveniles

×10 With Wilt Fungus Without Wilt Fungus With Wilt Fungus Without Wilt Fungus

Fig. 50. Effects of seed treatment and soil application of Aspergillus niger isolates on the soil population of Meloidogyne incognita in the absence and presence of Fusarium oxysporum f. sp. ciceri. Vertical bars indicate the standard error of three replicates; Czm - carbendazim; Cbf – carbofuran.

179

NURSERY TREATMENT SOIL APPLICATION

)/kg soil 2

Juveniles (J Juveniles

×10

With Wilt Fungus Without Wilt Fungus With Wilt Fungus Without Wilt Fungus

Fig. 51. Effects of nursery treatment and soil application with Aspergillus niger isolates on the soil population of Meloidogyne incognita in the absence and presence of Fusarium oxysporum f. sp. lycopersici. Vertical bars indicate the standard error of three replicates; Czm – carbendazim, Cbf – carbofuran.

180

nematode alone. Combined application of carbendazim and carbofuran decreased the nematode population by 8-53% in chickpea and 7-53% in tomato over planting population. Effect of soil application of A. niger isolates on nematode population was more or less similar to seed/nursery treatment (Fig. 50-51). Aspergillus niger isolates Population of A. niger isolates was increased 28-90% in chickpea and 32-99% in tomato during the course of experiment in comparison to planting population (Fig. 52-53). In the plots, infested with F. oxysporum f. sp. ciceri or F. oxysporum f. sp. lycopersici, the populations of A. niger isolates were increased greater than non infested plots (P≤ 0.0001). Similar trend was found in the plots infested with root-knot nematode alone or concomitantly with the wilt fungi. Greatest increase in the population of A. niger isolates was recorded for SkNAn5, followed by VAn4, AnC2, AnR3, ANAn4 and BuAn3. Effect of soil application was more or less identical in both the crops (Fig. 52-53). Conclusion The study has shown that the wilt fungi and root-knot nematode singly suppressed the yield of chickpea and tomato. Presence of root-knot nematode exacerbate the pathogenic effect of wilt fungi resulting to 22-25% greater yield decline. Application of selected isolates of A. niger checked the suppressive effect of pathogens and promoted the yield of chickpea (10- 59%) and tomato (11-65%). Among the six isolates tested, the isolate SkNAn5, VAn4 and AnC2 provided significantly greater control of the disease and yield enhancement of chickpea and tomato. These isolates also caused greater suppression of soil population of the target pathogens, while their own populations increased correspondingly.

181

SEED TREATMENT

CONTROL F. oxysporum f. sp. ciceri M. incognita Foc+Mi

SOIL APPLICATION

Foc+Mi

CFU/g soil CONTROL F. oxysporum f. sp. ciceri M. incognita

×10

Fig. 52. Rhizosphere population of Aspergillus niger isolates in the plots infested with Fusarium oxysporum f. sp. ciceri and Meloidogyne incognita singly or concomitantly. Vertical bars indicate the standard error of three replicates; Foc - F. oxysporum f. sp. ciceri, Mi - M. incognita.

182

NURSERY TREATMENT CONTROL F. oxysporum f. sp. lycopersici M. incognita Fol+Mi

SOIL APPLICATION

CFU/g soil 6 CONTROL F. oxysporum f. sp. lycopersici M. incognita Fol+Mi

×10

Fig. 53. Rhizosphere population of Aspergillus niger isolates in the plots infested with Fusarium oxysporum f. sp. lycopersici and Meloidogyne incognita singly or concomitantly. Vertical bars indicate the standard error of three replicates; Fol - F. oxysporum f. sp. lycopersici, Mi - M. incognita.

183

Experiment-V PREPARATION OF BIOPESTICIDES OF THE MOST EFFICIENT ISOLATES OF ASPERGILLUS NIGER, AND THEIR FIELD TRIAL FOR EFFECTIVENESS AGAINST THE WILT, ROOT-KNOT AND FUNGUS- NEMATODE WILT DISEASE COMPLEX OF CHICKPEA AND TOMATO

In view of greater effectiveness of three isolates of A. niger viz., SkNAn5, VAn4 and AnC2 against wilt, root-knot and the wilt disease complex of chickpea and tomato tested under field condition (Expt. IV), their commercial formulations (biopesticides) were prepared on sawdust-fly ash based material. The shelf life test of the biopesticides was tested at five temperature regimes i.e., 5, 10, 15, 25°C and ambient (January to December) for 12 months. Effectiveness of the biopesticides of A. niger SkNAn5, VAn4 and AnC2 was evaluated against wilt, root-knot and the fungus-nematode wilt disease complex of chickpea and tomato under field condition. The biopesticides were applied to seeds (4 g/kg seed) or soil (40 g/microplot). In case of tomato, root-dip treatment with A. niger isolates (10 g/100 seedlings) was done in place of seed treatment. Spore suspension of A. niger was made by mixing 10 g of biopesticides in 100 ml distilled water. The roots of tomato seedlings were dipped in the suspension for 10 minutes. Shelf-life of the formulations The shelf-life test revealed that the formulation maintained a high count of viable propagules of A. niger isolates during storage, evidenced by a much greater CFU load during the period (Fig. 54). At ambient temperature, the CFU count of A. niger isolates/g formulation increased significantly in comparison to other temperatures. The temperature next in supporting the grater CFU count was 25°C. Greatest CFU load/g formulation (1010) was recorded during March to August (Fig. 54). From August onwards, the CFU count gradually declined, but even in September it was greater than the control at 25°C or ambient temperature. The biocontrol fungus was, however, detected in the formulation upto 12 months. Among the three isolates of A. niger, greatest CFUs/g formulation (mean of 12 months population) was recorded for SkNAn5 (14.5 × 109), followed by VAn4 (13.2 × 109) and AnC2 (12.6 × 109). The significant differences between the CFU counts of SkNAn5 and VAn4 (P≤ 0.05); SkNAn5 and AnC2 (P≤ 0.01); and VAn4 and AnC2 (P≤ 0.05) were recorded during different storage periods.

184

Fig. 54. Shelf life test of biopesticides of Aspergillus niger isolates showing colony forming units/g formulation at various temperatures and durations. Vertical bars indicate the standard error of three replicates.

185

SYMPTOMS Fusarial wilt Characteristic symptoms of wilt developed on the chickpea and tomato plants grown in the plots infested with Fusarium oxysporum f. sp. ciceri or F. oxysporum f. sp. lycopersici, respectively. Incidence of the wilt was 58% with 66% severity in chickpea and 62% incidence with 75% severity in tomato (Fig. 55-58). The wilting was, however, checked due to application of biopesticides of A. niger isolates and fungicide, carbendazim but to a varying extent. Seed/nursery treatment and soil application of A. niger SkNAn5 decreased the wilt incidence by around 79% and severity by 65%, followed by VAn4 isolate (incidence 70% and severity 60%) and AnC2 (incidence 65% and severity 57%) in both the crops over respective control (Fig. 56-58). Application of carbendazim resulted to a decrease of 53% incidence and 58% severity, and 52% incidence and 63% severity in the wilt symptoms on chickpea and tomato, respectively. Root-knot Root-knot nematode, M. incognita caused 60 and 86 galls and 53 and 77 egg masses/root system of chickpea and tomato, respectively (Fig. 59-60). Gall formation and egg mass production decreased significantly in chickpea (P≤ 0.001) and tomato (P≤ 0.0001) due to application of A. niger isolates with marginal difference in seed and soil treatments in both the crops. Seed/nursery treatment of A. niger SkNAn5 suppressed the galling and egg mass production by 60% and 70% in chickpea and 70% and 76% in tomato, respectively. Application of carbofuran decreased the galls and egg masses by 38 and 19% (soil application), and 46 and 33% (seed treatment) in chickpea and 33 and 17% (soil application), and 40 and 28% in tomato (nursery treatment), respectively (Fig. 59-60). Fungus-nematode wilt disease complex Concomitant inoculation with F. oxysporum f. sp. ciceri or F. oxysporum f. sp. lycopersici and M. incognita in chickpea and tomato, respectively resulted to greater wilting of plants compared to Fusarium alone (Fig. 57-58). The incidence of wilt in tomato was almost similar as in chickpea, but the wilting on tomato was 4-5% greater than chickpea in both the pathogens infested plots. Seed/nursery treatment with A. niger SkNAn5 biopesticide decreased the wilt incidence by 59 and 62%, and severity by 61 and 67% in chickpea and tomato, respectively over control. The isolate VAn4 decreased the wilt incidence by 57-60% and severity by 60-62% and AnC2 decreased wilt incidence by 55-57% and severity by 57- 60% (Fig. 57-58). Seed/nursery treatment of combined application of carbendazim and

186

Fig. 55. Chickpea (A) and tomato (B) plants showing symptoms caused by Fusarium oxysporum f. sp. ciceri and F. oxysporum f. sp. lycopersici.

Fig. 56. An aerial view of field experiments conducted to evaluate the effects of seed/nursery and soil treatment with biopesticides of Aspergillus niger on wilt, root-knot and wilt disease complex of chickpea (A) and tomato (B). 187

SEED TREATMENT SOIL APPLICATION

incidenceWilt (%)

SEED TREATMENT SOIL APPLICATION

(0-5 scale) Wilt severity

Foc Foc+Mi Foc Foc+Mi

Fig. 57. Effects of seed treatment and soil application of Aspergillus niger biopesticides on the incidence and severity of wilt of chickpea caused by Fusarium oxysporum f. sp. ciceri in the presence or absence of Meloidogyne incognita. Vertical bars indicate the standard error of three replicates; Foc – F. oxysporum f. sp. ciceri, Mi – M. incognita, Czm – carbendazim, Cbf – carbofuran.

188

NURSERY TREATMENT SOIL APPLICATION

(%) incidence Wilt

NURSERY TREATMENT SOIL APPLICATION

Wilt severity (0-5 scale) severity (0-5 scale) Wilt

Fol Fol+Mi Fol Fol+Mi

Fig. 58. Effects of nursery treatment and soil application of Aspergillus niger biopesticides on the incidence and severity of tomato wilt caused by Fusarium oxysporum f. sp. lycopersici in the presence or absence of Meloidogyne incognita. Vertical bars indicate the standard error of three replicates; Fol – F. oxysporum f. sp. lycopersici, Mi – M. incognita, Czm - carbendazim; Cbf – carbofuran.

189

SEED TREATMENT SOIL APPLICATION

root system / Galls

SEED TREATMENT SOIL APPLICATION

Egg/ root masses system

Mi Foc+Mi Mi Foc+Mi

Fig. 59. Effects of seed treatment and soil application of Aspergillus niger biopesticides on the galling and egg mass production of Meloidogyne incognita alone or with Fusarium oxysporum f. sp. ciceri. Vertical bars indicate the standard error of three replicates; Mi – M. incognita, Foc – F. oxysporum f. sp. ciceri.

190

NURSERY TREATMENT SOIL APPLICATION

/ rootGalls system

NURSERY TREATMENT SOIL APPLICATION

/ root system Egg masses

Mi Fol+Mi Mi Fol+Mi

Fig. 60. Effects of nursery treatment and soil application of Aspergillus niger biopesticides on the galling and egg mass production of Meloidogyne incognita alone or with Fusarium oxysporum f. sp. lycopersici. Vertical bars indicate the standard error of three replicates; Mi – M. incognita, Fol – F. oxysporum f. sp. lycopersici.

191

carbofuran suppressed the wilt by 57-60%. Soil application was 2-6% less effective than the seed/nursery treatment (Fig. 57-58). Chickpea and tomato plants grown in the plots infested concomitantly with the wilt fungus and root-knot nematode developed significantly less number of galls (25-29%) and egg masses (30-32%) than those grown in the plots infested with nematode alone. Application of A. niger SkNAn5 suppressed the galling and egg mass production by 43-45% and 58-63%, respectively over control followed by the isolate VAn4 (32-36% and 50-55%) and AnC2 (31-35% and 49-53%) in both the crops (Fig. 59-60). Seed/nursery treatment with carbendazim+carbofuran reduced the galling by 28-32% and egg mass production by 32- 45%. Soil application was 2-5% less effective than the seed/nursery treatment against the root-knot nematode. Plant growth and yield Dry matter and yield of chickpea and tomato increased significantly (P≤ 0.001) due to application of A. niger isolates on seeds/nursery or in soil. Seed/nursery treatment showed greater yield enhancement than soil application (Table 40-41). Plants in the plots infested with F. oxysporum f. sp. ciceri or F. oxysporum f. sp. lycopersici, produced 23 and 19% less dry matter and yield (chickpea) and 29 and 31% less dry matter and yield (tomato), respectively over control (Table 40-41). Application of biopesticides or pesticides checked the suppressive effect of pathogens resulting to promotion in the growth and yield of chickpea and tomato. Seed/nursery treatment with SkNAn5, VAn4 and AnC2 enhanced the dry matter production and yield by 70 and 66%, 63 and 56%, 59 and 53%, respectively in chickpea (Table 40) 78 and 71%, 69 and 57%, 63 and 53% in tomato, respectively (Table 41). Carbendazim application resulted to 20 and 27% (seed/nursery treatment) and 19 and 16% (soil application) increase in the yield in chickpea and tomato grown in the wilt infested plots, respectively over control. Plants grown in nematode infested plots produced 20 and 30% and 21 and 26% dry matter production and yield less than control, respectively (chickpea and tomato; Table 40- 41). Seed/nursery treatment with the three biopesticides suppressed the pathogenic effect of the nematode and increased (P≤ 0.001) the dry matter production and yield in comparison to the control. Greatest yield enhancement occurred with A. niger SkNAn5 followed by VAn4 and AnC2. Seed/nursery treatment with carbofuran enhanced the yield and dry matter production by 23-26% in chickpea and 23-28% in tomato over control (Table 40-41).

192

Table 40. Effects of seed treatment and soil application of Aspergillus niger biopesticides on the dry matter production and yield of chickpea in field plots infested singly or concomitantly with Fusarium oxysporum f. sp. ciceri, Meloidogyne incognita or uninoculated. Dry shoot weight (g) Yield (g grains/plant)

Treatment Seed treatment Soil application Seed treatment Soil application Control 22.3 22.3 7.5 7.5 AnC2 31.0 (39.1) 30.7 (37.5) 10.0 (33.0) 9.9 (32.2) SkNAn5 32.3 (45.0) 32.1 (44.2) 10.5 (40.4) 10.5 (39.8) VAn4 31.3 (40.4) 30.8 (38.3) 10.2 (36.5) 10.1 (34.7) Carbendazim (Czm) 23.8 (6.5) 23.5 (5.3) 7.9 (4.9) 7.8 (3.8) Carbofuran (Cbf) 23.6 (5.6) 23.4 (4.7) 7.8 (4.4) 7.7 (3.3) Control (F) 17.1 (-23.3) 17.1 (-23.3) 6.1 (-19.1) 6.1 (-19.1) F + AnC2 27.2 (58.9) 26.9 (57.6) 9.3 (52.7) 9.2 (51.1) F + SkNAn5 29.1 (69.9) 28.9 (68.8) 10.1 (65.8) 10.0 (64.3) F + VAn4 27.9 (63.0) 27.8 (62.6) 9.5 (56.2) 9.4 (54.9) F + Carbendazim (Czm) 21.5 (25.8) 21.2 (24.1) 7.3 (19.6) 7.2 (18.5) Control (N) 17.8 (-20.2) 17.8 (-20.2) 5.9 (-21.3) 5.9 (-21.3) N + AnC2 22.4 (25.9) 22.9 (28.6) 7.3 (24.1) 7.2 (22.7) N + SkNAn5 23.5 (32.0) 23.8 (33.9) 7.6 (28.4) 7.5 (26.3) N + VAn4 22.9 (28.5) 23.2 (30.4) 7.4 (25.4) 7.3 (23.9) N + Carbofuran (Cbf) 21.9 (22.8) 21.6 (21.3) 7.4 (25.7) 7.3 (23.8) Control (F+N) 11.3 (-49.3) 11.3 (-49.3) 3.5 (-53.3) 3.5 (-53.3) F + N + AnC2 16.2 (43.3) 15.8 (40.2) 4.8 (37.2) 4.8 (36.7) F + N + SkNAn5 16.8 (48.7) 16.9 (49.8) 5.1 (46.1) 5.0 (43.9) F + N + VAn4 16.4 (45.2) 16.4 (45.1) 4.9 (40.8) 4.9 (38.7) F + N + Czm + Cbf 14.9 (31.5) 14.6 (28.8) 4.5 (27.2) 4.3 (23.3) LSD (P≤0.001) 1.7 1.8 0.29 0.32 F-value (P≤0.001) Control agents (df=5) 282* 197* 480* 872* Pathogens (df=2) 116* 76* 192* 768* Interaction (df=4) 62* 117* 146* 111* Each value is the mean of three replicates plots. All values are significantly different from their respective controls at P≤0.001 except carbendazim and carbofuran (without pathogen) at P≤0.05. Values in parenthesis are percent increase (+ve) or decrease (-ve) over respective control [plants not inoculated with either pathogen but applied with biopesticides of Aspergillus niger isolates were compared with uninoculated Control; wilt fungus inoculated plants applied with A. niger isolates were compared with fungus inoculated Contorl (F); nematode inoculated plants applied with A. niger isolates were compared with nematode inoculated Contorl (N); concomitantly inoculated plants applied with A. niger isolates were compared with concomitantly inoculated Contorl (F+N)]. *Significantly different from the control at P≤0.001.

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Table 41. Effect of nursery treatment and soil application of Aspergillus niger biopesticides on the dry matter production and yield of tomato in field plots infested singly or concomitantly with Fusarium oxysporum f. sp. lycopersici, Meloidogyne incognita or uninoculated. Dry shoot weight (g) Yield (g fruits/plant)

Treatment Nursery treatment Soil Nursery Soil application treatment application Control 48.3 48.3 480 480 AnC2 68.0 (40.9) 67.3 (39.4) 647 (34.7) 643 (34.0) SkNAn5 71.9 (48.9) 69.2 (43.3) 674 (40.5) 667 (39.0) VAn4 69.0 (42.8) 67.9 (40.6) 664 (38.3) 653 (35.9) Carbendazim (Czm) 52.1 (7.9) 50.7 (5.0) 516 (7.3) 501 (4.4) Carbofuran (Cbf) 51.8 (7.3) 50.9 (5.3) 513 (6.9) 494 (2.9) Control (F) 34.3 (-29.0) 34.3 (-29.0) 330 (-31) 330 (-31.3) F + AnC2 55.8 (62.6) 55.1 (60.7) 506 (53) 502 (52.2) F + SkNAn5 61.0 (77.9) 57.3 (67.2) 566 (71.5) 561 (70.0) F + VAn4 57.8 (68.6) 56.3 (64.2) 519 (57) 515 (56.2) F + Carbendazim 42.4 (23.8) 41.0 (19.7) 418 (27) 382 (15.9) (Czm) Control (N) 34.1 (-29.5) 34.1 (-29.5) 355 (-26.0) 355 (-26.0) N + AnC2 43.3 (27.1) 44.3 (29.9) 443 (24.6) 441 (24.2) N + SkNAn5 45.7 (33.9) 45.7 (34.1) 454 (27.8) 449 (26.4) N + VAn4 44.7 (31.2) 44.4 (30.2) 448 (26.1) 442 (24.4) N + Carbofuran 41.8 (22.5) 40.0 (17.2) 455 (28.1) 432 (21.6) (Cbf) Control (F+N) 18.3 (-62.1) 18.3 (-43.8) 191 (-60.3) 191 (-60.3) F + N + AnC2 26.7 (45.6) 26.0 (42.1) 264 (38.5) 265 (38.8) F + N + SkNAn5 28.0 (52.9) 26.9 (46.9) 275 (44.0) 269 (41.2) F + N + VAn4 27.0 (47.8) 26.6 (45.1) 269 (40.9) 263 (37.9) F + N + Czm + Cbf 23.9 (30.7) 23.0 (25.8) 258 (35.2) 236 (23.9) LSD (P≤0.0001) 2.7 3.0 8.6 7.9 F-value (P≤0.0001) Control agents 276* 301* 3541* 3608* (df=5) Pathogens (df=2) 386* NS 7472* 5189* Interaction (df=4) 101* 135* 1238* 367* Each value is the mean of three replicates plots. All values are significantly different from their respective controls at P≤0.001 except carbendazim and carbofuran (without pathogen) at P≤0.05. Values in parenthesis are percent increase (+ve) or decrease (-ve) over respective control [plants not inoculated with either pathogen but applied with biopesticides of Aspergillus niger isolates were compared with uninoculated Control; wilt fungus inoculated plants applied with A. niger isolates were compared with fungus inoculated Contorl (F); nematode inoculated plants applied with A. niger isolates were compared with nematode inoculated Contorl (N); concomitantly inoculated plants applied with A. niger isolates were compared with concomitantly inoculated Contorl (F+N)]. *Significantly different from the control at P≤0.0001. NS- Not significant at P≤0.0001, but significant at P≤0.001.

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Concomitant infestation with F. oxysporum f. sp. ciceri or F. oxysporum f. sp. lycopersici and M. incognita greatly suppressed the dry matter production (49-53%) and yield (60-62%) of chickpea and tomato (Table 40-41). Application of biopesticides considerably suppressed the pathogenic effects of the wilt fungus and nematode leading to significant promotion of yield. Greatest increase in the yield in concomitantly infested plots was obtained due to seed/nursery treatment of SkNAn5 i.e., 46% in chickpea and 44% in tomato. A joint application of carbendazim and carbofuran improved the yield by 27% (chickpea) and 35% (tomato) in comparison to the respective control (Table 40-41). Soil application of biopesticides was more or less similar in effectiveness as seed/nursery treatment. Root nodulation Nodule formation on the roots of chickpea was quite good (Fig. 61-62) and on average 36 nodules were formed out of which 79% were functional and 21% were non-functional (Fig. 61-62). The nodulation was synergized due to the treatments with A. niger isolates. The number of functional nodules increased by 60-65% over control due to seed treatment with A. niger isolates (Fig. 61-62). Application of carbendazim or carbofuran caused significant decline in the number of functional nodules (11-16%). Infection by F. oxysporum f. sp. ciceri and M. incognita singly or concomitantly significantly also suppressed the nodulation (P≤ 0.05). Inhibition in the nodulation due to pathogens was greater with concomitant inoculations. Seed treatment with A. niger isolates suppressed the negative effect of pathogens on nodulation. Number of non-functional nodules decreased invariably with those treatments where functional nodules were increased. Application of carbendazim, carbofuran or carbendazim+carbofuran increased the number of functional nodules of infected plants over control, but it was much less than the effect of A. niger isolates. Effects of soil application of A. niger isolates was more or less similar to the seed treatments.

Rhizosphere population

Wilt fungi In the plots infested with F. oxysporum f. sp. ciceri or F. oxysporum f. sp. lycopersici without any treatment, soil population of the wilt fungi decreased from November to December, but increased greatly from January onwards and reached to the peak in the month of February to log 7.8 CFUs/g soil (121%) in comparison to planting population of log 7.45 CFUs/g soil (Fig. 63-64). Seed treatment with the A. niger isolates or fungicide resulted to considerable

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Fig. 61. Effects of seed treatment of Aspergillus niger biopesticides on the root nodulation on chickpea grown in plots infested with Fusarium oxysporum f. sp. ciceri and Meloidogyne incognita singly or concomitantly or not infested. Vertical bars indicate the standard error of three replicates; Foc- F. oxysporum f. sp. ciceri, Mi- M. incognita, Czm – carbendazim, Cbf - carbofuran.

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Fig. 62. Effects of soil application of Aspergillus niger biopesticides on the root nodulation on chickpea grown in plots infested with Fusarium oxysporum f. sp. ciceri and Meloidogyne incognita singly or concomitantly or not infested. Vertical bars indicate the standard error of three replicates; Foc- F. oxysporum f. sp. ciceri, Mi- M. incognita, Czm – carbendazim, Cbf - carbofuran.

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With Root-Knot Nematode Without Root-Knot Nematode With Root-Knot Nematode Without Root-Knot Nematode

Fig. 63. Effects of seed treatment and soil application with Aspergillus niger biopesticides on the soil population of Fusarium oxysporum f. sp. ciceri in the presence or absence of Meloidogyne incognita. Vertical bars indicate the standard error of three replicates; Czm – carbendazim, Cbf – carbofuran.

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WithRoot-Knot Nematode Without Root-Knot Nematode With Root-Knot Nematode Without Root-Knot Nematode

Fig. 64. Effects of nursery treatment and soil application with Aspergillus niger biopesticides on the soil population of Fusarium oxysporum f. sp. lycopersici in the presence or absence of Meloidogyne incognita. Vertical bars indicate the standard error of three replicates; Czm – carbendazim, Cbf - carbofuran

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(P≤ 0.000001) decrease in the population of wilt fungi over control. The population of Fusarium spp. reached to undetectable level in the month of February with A. niger SkNAn5 (log 0.75 CFUs/g soil) followed by the isolate VAn4 (log 1.98 CFUs/g soil) and AnC2 (log 3.13 CFUs/g soil). Presence of root-knot nematode, however, resulted to increase in the population of the pathogenic fungi in comparison to the fungus alone (660%, P≤ 0.000001). Application of A. niger isolates significantly (P≤ 0.000001) decreased the rhizosphere population of Fusarium spp. in comparison to concomitantly inoculated control, being greatest with SkNAn5 (log 2.5 CFUs/g soil) followed by VAn4 (log 4.32 CFUs/g soil), AnC2 (log 5.35 CFUs/g soil) compared to the control (log 7.45 CFUs/g soil). Pesticide treatments also decreased the rhizosphere population of wilt fungi to log 5.43 CFUs/g soil. Effect of soil application of A. niger isolates on the rhizosphere population was more or less similar to seed/nursery treatment (Fig. 63-64). Root-knot nematode Soil population of root-knot nematode, M. incognita gradually increased over the course of experiment in chickpea and tomato in comparison to planting population (Fig. 65-66). The increase was significant (P≤ 0.01) in all the months except December. In tomato the increase in the population of root-knot nematode was much higher (48.8%) than chickpea. The A. niger isolates and carbofuran efficiently (P≤ 0.001) decreased the root-knot nematode population. Seed/nursery treatment with SkNAn5, VAn4 and AnC2 significantly decreased the nematode population during all months of sampling by 32-66%, 26-52% and 17-35% in chickpea and 25-62%, 20-40% and 11-34% in tomato, respectively. Carbofuran treatment resulted to 12-28% decrease in the population in chickpea and 8-20% in tomato root-zone. In the plots, infested with root-knot nematode and wilt fungi concomitantly, the nematode population decreased by 7-44% (chickpea) and 9-46% (tomato) compared to nematode alone (Fig. 65-66). Application of all A. niger isolates significantly decreased (P≤ 0.0001) the nematode population; decrease in the population was greater (P≤ 0.01) in concomitant infection than the nematode alone. Carbendazim+carbofuran treatment decreased the nematode population by 5-50% in chickpea and 2-56% in tomato as compared to the planting population. Effect of soil application of A. niger isolates on nematode population was more or less similar to seed/nursery treatment (Fig. 65-66). Aspergillus niger isolates Population of A. niger isolates increased by 60-109% in chickpea and 66-120% in tomato during the course of experiment in comparison to planting population (Fig. 67-68). In the plots, infested with F. oxysporum f. sp. ciceri or F. oxysporum f. sp. lycopersici, population

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SEED TREATMENT SOIL APPLICATION

)/kg soil 2

Juveniles (J Juveniles

×10 With Wilt Fungus Without Wilt Fungus With Wilt Fungus Without Wilt Fungus

Fig. 65. Effects of seed treatment and soil application with Aspergillus niger biopesticides on the soil population of Meloidogyne incognita in the presence or absence of Fusarium oxysporum f. sp. ciceri. Vertical bars indicate the standard error of three replicates; Czm – carbendazim, Cbf – carbofuran.

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NURSERY TREATMENT SOIL APPLICATION

)/kg soil 2

Juveniles (J Juveniles

×10 With Wilt Fungus Without Wilt Fungus With Wilt Fungus Without Wilt Fungus

Fig. 66. Effects of nursery treatment and soil application with Aspergillus niger biopesticides on the soil population of Meloidogyne incognita in the presence or absence of Fusarium oxysporum f. sp. lycopersici. Vertical bars indicate the standard error of three replicates; Czm – carbendazim, Cbf – carbofuran.

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SEED TREATMENT

Control F. oxysporum f. sp. ciceri M. incognita Foc+Mi

CFU/g soil 6 SOIL APPLICATION

×10

Control F. oxysporum f. sp. ciceri M. incognita Foc+Mi

Fig. 67. Population of Aspergillus niger isolates in the rhizosphere of chickpea grown in the plots infested with Fusarium oxysporum f. sp. ciceri and Meloidogyne incognita singly or concomitantly. Vertical bars indicate the standard error of three replicates; Foc - F. oxysporum f. sp. ciceri, Mi - M. incognita.

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NURSERY TREATMENT

M. incognita Fol+Mi Control F. oxysporum f. sp. lycopersici

CFU/g soil 6 SOIL APPLICATION

×10

Control F. oxysporum f. sp. lycopersici M. incognita Fol+Mi

Fig. 68. Population of Aspergillus niger isolates in the rhizosphere of tomato grown in the plots infested with Fusarium oxysporum f. sp. lycopersici and Meloidogyne incognita singly or concomitantly. Vertical bars indicate the standard error of three replicates; Fol - F. oxysporum f. sp. lycopersici, Mi - M. incognita.

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of A. niger isolates increased greatly (P≤ 0.000001) as compared to planting population and also to their respective control (P≤ 0.0001). Similar trend was found in the plots infested with wilt fungus and/or root-knot nematode. Effect of soil application was more or less identical in both the crops (Fig. 67-68). Conclusion The study has shown that biopesticides of A. niger can be successfully prepared on the sawdust-fly ash based formulation. The formulation effectively maintained a higher CFU load of A. niger (108-10 CFU/g formulation) during 2-8 months. Application of the biopesticides effectively controlled the wilt, root-knot and fungus-nematode wilt disease complex of chickpea and tomato, and significantly improved the yield of both crops. The biocontrol agents also established in soil, whereas soil population of the pathogens decreased gradually and drastically. Overall seed/nursery treatment with the biopesticides based on A. niger isolates suppressed the wilt (61-65%), root-knot (70-76%) and wilt disease complex (60-64%) and improved the yield of chickpea (29-66%) and tomato (27-71%). The A. niger isolates also synergized with Rhizobium resulting to 60-65% greater nodulation. Based on the in vitro and in vivo performance of 236 isolates of A. niger collected from diversified locations, seed/nursery treatment with the biopesticide of A. niger SkNAn5 was found highly effective and is recommended for chickpea and tomato cultivation in Fusarium and/or Meloidogyne infested, prone fields or regions. In view of low cost of the formulation and its handy application, the seed/nursery treatment may also be integrated with general cultural practice of the crops, as A. niger SkNAn5 proved growth promoting and enhanced 39-41% yield in non infested soils.

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DISCUSSION

The fungus, Aspergillus niger is wide spread in distribution and is found in almost all kind of soils (Domich et al., 1980; Nigam and Robinson, 2004). The fungus also possesses capability to tolerate a wide range of pH, salinity, alkalinity (Kis-Papo et al., 2003), and even soil pollutants including heavy metals (Baytak et al., 2005; Yuh-Shan, 2005; Ahmad et al., 2006), and other toxicants (Anon., 2001; Awofolu et al., 2006; Braud et al., 2006). Because of this ability A. niger was found in all 236 soil samples collected from different localities in the state of U.P. of India. The fungus was identified on the basis of morphological characters especially hyaline and septate mycelium and conidia 4-5 µm in diameter formed on biseriate metulae of conidial heads. Conidiophores were long and the length ranged 600-3000 µm. Roper and Funnel (1965) suggested that all black Aspergilli having conidia of 4-5 µm diameter on biseriate metulae of 400-3000 µm long conidiophores are A. niger and these characters have also been confirmed by other researchers (Al-Musallam, 1980; Kozakiewicz, 1989; Sutton et al., 1998 and de Hoog et al., 2000). Antagonism by A. niger isolates against plant pathogenic fungi and nematodes has been observed by various researchers (Molina and Davide, 1986; Dhillion, 1994; Chattopadhyay and Sen, 1996; Kumar and Sen, 1998; Lodhi, 2004; Khan, 2005; Chand et al., 2007; Misra, 2007; Goswami et al., 2008; Khan and Anwer, 2007, 2008; Goswami et al., 2008; Siddiqui and Kazuyoshi, 2009). A. niger may suppress plant pathogenic fungi by direct parasitism (Mondal et al., 2000), lysis (Mondal et al., 2000; Benitez et al. 2004; El-Hasan et al., 2007), food competition (Sen, 1997; Mondal et al., 2000; Vassilev et al., 1996, 2006), direct and indirect antibiosis (Buchi et al., 1983; Fujimoto et al., 1993; Eapen and Venugopal, 1995; Mankau, 1969a, 1969b; Benitez et al., 2004; El-Hasan et al., 2007) and/or through production of volatile compounds etc. (Buchi et al., 1983; Fujimoto et al., 1993; Eapen and Venugopal, 1995). All 236 A. niger isolates tested produced ammonia and siderophore with varied amount. However, only a few of them produced indole acetic acid (8 isolates) and hydrogen cyanide (33 isolates). Production of IAA, gibberallic acid and other phytohormones by some isolates of A. niger has been reported earlier (Mostafa and Youssef, 1962; Vaddar and Patil, 2007). Siderophore production was recorded in all isolates, but the amount of production varied among the isolates. Siderophore production by A. niger on modified CAS medium was reported (Vessilev et al., 2005a; Sen, 1997; Mondal et al., 2000). Hydrogen cyanide is an important volatile compound and highly suppressive against plant

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pathogenic fungi, bacteria and nematodes (Fujimoto et al., 1993; Eapen and Venugopal, 1995). The HCN is known to be produced by A. niger (Fujimoto et al., 1993). In vitro studies were also carried out to test the Ochratoxin A production and phosphate solubilization by the isolates of A. niger. All isolates solubilized phosphorus evidenced by the zone of solubilization on agar plates, but varied with isolates; diameter of the zone was wider, and also in liquid medium more solubilization was seen with SkNAn5, followed byVAn4, AnC2, AnR3, ANAn4 and BuAn3. A. niger is well known for phosphate solubilization (Domich, et al., 1980; Medina et al., 2007). Domich, et al. (1980) and Medina et al. (2007) have reported similar variations in the behavior of A. niger isolates for phosphate solubilization. Production of organic acids (Sperber,1958; Blumenthal, 2004; Ramachandran et al., 2008), reducing the medium pH (Domich, et al., 1980; Medina et al., 2007), production of siderophores (Azcon et al., 1986; Jennings, 1989; Li et al., 1991) and Carbon dioxide producion (Ramachandran et al., 2008) by A. niger are some important determinants of phosphorus solubilization. None of the tested isolates of A. niger produced ochratoxin A (< 1ηg/g) or produced up to 0.4 ηg/g (by five isolates) in in vitro studies. Only a 6-10% of members of the A. niger aggregate isolated from corn, peanuts, raisins, onions, mango, apples, and dried meat products are known to produce OTA in least amounts of less than 1ηg/g (Abarca et al., 1994; Varga et al., 2000; Esteban et al., 2006; Perrone et al., 2007). Even production of ribotoxin by A. niger was found negative in Eastern and Southern blot tests (Campbell, 1994). Interestingly it was found that A. niger decreased the aflatoxin contamination (Wicklow et al., 1980 ; Horn and Wicklow, 1983). The sixteen isolates of A. niger which were found more effective and efficient for various parameters tested also showed considerable genetic variation using 28 RAPD decamer primers. The variation ranged from 11% (between isolates VAn4 and AnC2) to 73% (between AnC2 and BAn4). Pekarek et al. (2006) have recorded 12-78% molecular variation among 89 isolates of A. niger using 31 RAPD markers. A. niger is classified as an asexual deuteromycetes (Raper and Fennel, 1965; Fennell, 1977). Evidences suggest that many asexual fungi have higher than expected levels of genetic variation and are undergoing some means of genetic exchange (Pekarek et al., 2006). The rate of mitotic recombination (crossing over and haploidization) in A. niger were calculated to be 100X higher than meiotic crossing over in the sexual species of A. nidulans (Lhoas, 1967). The parasexual behavior of A. niger may have been responsible for the genetic variation in the isolates (Debets, 1998). Some studies have experimentally proved that parasexuality in A. niger is occurring under controlled field conditions (Zeigler et al., 1997; Souza-Paccola et al., 2003). Genetic

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variability through parasexualism may equal or greater than those occurring the sexual reproduction (Agrios, 2005). On the basis of genetic variability recorded among 16 isolates three groups were differentiated by RAPD markers and some amplicons were identified isolate specific. Primer 02 and Primer 06 may be considered as A. niger specific. Primer OPA-16 can be treated as isolate specific for AnC2 and VAn4 (three amplicons produced by the primer as 2300 bp for AnC2 and VAn4, and 2800 bp for AnC2 only); Primer 04 can be treated as SkNAn5 specific (as it produced only 1500 bp for isolate SkNAn5); Primer 01 can be treated as ANAn1 specific (as it amplified 700 bp in ANAn1 only) and OPA-12 can be VAn4 specific (as it produced 700 pb in VAn4 only). This indicates that RAPD test can distinguish the isolates of A. niger as the technique has been successfully used for genetic variability and relatedness in the complex group of black Aspergilli (Megnegneau et al., 1993) and genetic variability and in identifying human pathogenic isolate of A. fumigatus (Aufauvre-Brown et al., 1992) or plant pathogenic isolate of Fusarium solani f. sp. cucurbitae (Crowhurst et al., 1991). Biochemical tests especially the production of ochratoxin A, IAA and phosphate solubilization also showed variation among the isolates of A. niger tested. The non producing ochratoxin A isolates viz., ANAn4, BuAn3, AnC2, VAn4, SkNAn5, AnR3, LAn3, SkNAn3, JaAn2 and BasAn5; IAA producing isolates viz. ANAn4, BuAn3, AnC2, VAn4, SkNAn5, AnR3, LAn3 and SkNAn3, belonged to the Group I differentiated by RAPD test. These isolates also produced siderophore, ammonia and solubilized phosphorus greater than other isolates. Ochratoxin A producing (very small amount) isolates viz., AAn1, GaAn1, BAn4 and BudAn3, belonged to the Group III differentiated by RAPD test. These isolates also produced siderophores, ammonia and solubilized phosphorus, but less than other isolates. This indicates that the genetic variability revealed through RAPD test was authentic. This is in accordance with several researchers who used RAPD fingerprinting to differentiate isolates of A. niger (Kusters-van Someren et al. 1991; Varga et al. 1993; Varga et al. 1994; Accensi et al. 1999; Parenicova´ et al. 1997; Parenicova´ et al. 2001; Abarca et al. 2004; Gajera and Vakharia, 2010). However, researches have indicated than RAPD fingerprinting is not a reliable test to identify a species or to differentiate isolates of a species. However, within the limited infrastructure available with us, other reliable tests such as RFLP and 18s- nucleotide sequencing could not be done. On the basis of in vitro tests 16 isolates viz., AAn1, BAn4, BuAn3, BasAn5, BudAn3, GaAn1, JaAn2, LAn3, MeAn4, SkNAn3, SkNAn5, VAn4, ANAn1, ANAn4, AnC2 and AnR3 found to be more efficient in production of biochemicals and monoculture

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colonization, and hence were selected for further study. Numerous evidences exist which have shown remarkable variability for antibiosis and antagonism against pathogens among isolates of biocontrol fungi including A. niger (Sen, 2000). The isolates A. niger SkNAn5, VAn4, AnC2, AnR3, ANAn4, and BuAn3 were also found more compatible with common fungicides viz., carbendazim (Bavistin 50 WP), captan (Captaf 50 WP), mancozeb (Dithane M-45 75 WP), metalaxyl (Apron 35 SD), thiram (TMTD 75 WP), and two nematicides viz., carbofuran (Furadan 3G) and nemacur

(Fenamiphos) than rest of the isolates. The LD50 and LD90 concentrations were much greater than the recommended doses of the pesticides for soil or seed treatment. However, the tolerant levels varied with the isolates and pesticides. A. niger isolates also expressed tolerance to Ni, Cd and Cr and their considerable biosorption; however, the efficiency varied with the isolate in both single and multi-metal system and the order was SkNAn5 > VAn4 > AnC2 > AnR3 > ANAn4 > BuAn3 > rest of the A. niger isolates. Biosorption of Ni+2, Cd+2 and Cr+6 by A. niger have been reported by other researchers (Kapoor and Viraraghavan, 1995; Gupta et al., 2000; Ahmad et al., 2006; Tripathi et al., 2007). Sixteen out of 236 isolates of A. niger suppressed the wilt fungi, F. oxysporum f. sp. ciceri and F. oxysporum f. sp. lycopersici in in vitro tests, the degree of antagonism, however, varied with the isolate. Diversity in virulence and growth patterns within A. niger species are well known to occur (Buchi et al., 1983; Fujimoto et al., 1993; Eapen and Venugopal, 1995). In dual culture, hyphae of some A. niger isolates viz., SkNAn5, VAn4, AnC2, AnR3, ANAn4 and BuAn3 coiled around the hyphae of Fusarium as soon as the two colonies came in contact, whereas in other soil isolates, coiling did not occur until the A. niger hyphae had penetrated far into the host fungus colony. These six most efficient isolates overgrew the Fusarium in 7-8 days, where as rest of the isolates in 10 days of inoculation on PDA plates. Similar pattern of varied aggressiveness among isolates of A. niger against various pathogenic fungi has been reported (Chand et al., 2007; Mullenborn et al., 2008; Deshmukh et al., 2010). The tested isolates also produced certain volatile compounds and suppressed the colonization by F. oxysporum f. sp. ciceri and F. oxysporum f. sp. lycopersici. The isolates SkNAn5, VAn4, AnC2, AnR3, ANAn4 and BuAn3 caused greater reduction in the radial growth of Fusarium than other isolates. The effectiveness of the volatile compounds produced by A. niger isolates varied with the age of culture and duration of exposure. Five days old culture of A. niger isolate caused greatest reduction in the radial growth of the Fusarium when exposed for 10 days than 6, 7, 8 or 9 days. In a classical study Buchi et al. (1983) investigated the production of volatile compounds by different

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Aspergillus spp. isolates and their suppressive effects on plant pathogenic fungi, Fusarium, Rhizoctonia, Pythium, Sclerotium etc. The volatile compounds play a proactive role in the suppression of soil borne plant pathogens (Lim and The, 1990; Angappan, 1992; Gao et al., 2001, 2002; Dubey et al., 2007). Suppressive effects of nonvolatile compounds (culture filtrates) of A. niger isolates on the radial growth of F. oxysporum f. sp. ciceri and F. oxysporum f. sp. lycopersici were found to be concentration dependent. Higher concentrations of the culture filtrates (25% and 50%) were more inhibitory than the lower concentration (10%). Sen et al. (1995) reported the production of nonvolatile compounds by A. niger which proved inhibitory to various pathogens. Harzianic acid, alamethicins, tricholin, peptaibols, 6-penthyl-α-pyrone, y- lactone, massoilactone, viridin, gliovirin, glisoprenins, heptelidic acid, oxalic acid were detected in the culture filtrates of A. niger and suspected to be involved in the inhibition of radial growth of pathogens (Benitez et al., 2004; El-Hasan et al., 2007). Singh et al. (2002) reported that culture filtrate of A. niger inhibited the mycelium growth of F. udum. In another study, Muhammad and Amusa (2003) reported that A. niger culture filtrates inhibited F. oxysporum, Sclerotium rolfsii, Pythium aphanidermatum, Helminthosporium maydis, Macrophomina phaseolina, and Rhizoctonia solani causing seedling blight of various crops. The culture filtrates of A. niger isolates were also found nematicidal as they suppressed egg hatching and induced mortality to the juveniles of Meloidogyne incognita. The nematicidal effects of the culture filtrates were found to be concentration as well as isolate dependent. Higher concentrations of the culture filtrates (50%, 75% and 100%) were more inhibitory than lower concentration (25%). Isolates SkNAn5, VAn4, AnC2, AnR3, ANAn4 and BuAn3 had more nematicidal effects than other isolates. A. niger is reported to produce nematoxic metabolites that may have been involved in the nematode suppression (Dahiya and Singh, 1985; Gokte and Swarup, 1988; Khan and Akram, 2000; Siddiqui and Eteshamul Haque, 2001; Khan and Anwer, 2008). Culture filtrates of A. niger also caused mortality to Radopholus similis and M. incognita (Molina and Davide, 1986; Goswami et al., 2008; Khan and Anwer, 2008). Recently Singh and Mathur (2010b) confirmed the nematicidal effects of culture filtrates of A. niger and reported inhibition in egg hatching and mortality to the juveniles of M. incognita. Chickpea cultivation is affected by a number of diseases. Among them, wilt disease caused by Fusarium oxysporum f. sp. ciceri is a serious disease in major chickpea-growing areas in India (Nene et al., 1984). The annual yield loss to the crop from Fusarium wilt vary

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from 10-15% in India (Jalali and Chand, 1992) and can destroy the crop up to 100% (Hari and Khirbat, 2009). Similarly, Fusarium wilt of tomato is the major limiting factor in the chickpea production (Snyder and Hansen, 1940) and causes considerable loss to the crop (Khan et al., 2007; Mandal et al., 2009). Majid and Khan (2010) have reported 54% disease incidence in tomato grown in the wilt fungus infested plots. In the present study, infection with F. oxysporum f. sp. ciceri and F. oxysporum f. sp. lycopersici resulted to the wilt incidence of 58-65% and 62-66% in chickpea and tomato, respectively. The wilt symptoms on chickpea may appear 3-5 weeks after sowing (Backman, 1987). The initial visible symptom of the disease is loss of turgidity in leaves and acropetal vein clearing (Chauhan, 1963). The disease occurs at any stages such as seedling stage, flowering stage or adult stage (Beckman, 1987) with symptoms of yellowing and drying of leaves, drooping of petioles, withering of plants, browning of vascular bundles (Haware and Nene, 1980b). In the present study, chickpea responded similarly to the inoculation with the fungus and developed typical symptoms of the disease. The first sign of the disease was mild chlorosis and stunted growth that appeared at seedling stage. Some of the stunted seedlings succumbed to the infection. The percent wilting in chickpea seedlings and their mortality was greater in pots than microplots. Under field conditions, recognizable wilt symptoms developed when plants were 6-8 weeks old. Gurha et al. (2003) reported six week as critical age of chickpea plant for appearance of symptoms. The seedlings, which escaped early infection exhibited stunted growth and leaf chlorosis at one month age. At a later stage, leaves/branches wilted, drooped or dried. The earliest symptoms of wilting on young tomato plants are clearing of veinlets and drooping of the petioles. In the field, the disease may appear at any time in mature plants after flowering or at the beginning of fruit set (Ray, 2005). The chlorotic symptoms begin to appear on one side of the leaf and then all leaflets become yellow on one half of the leaf (Sherf and MacNab, 1986). After the disease having advanced for a few weeks, browning of the vascular system can be seen (Agrios, 2005; Singh, 2008). Young plants wilt suddenly but older plants hang on for some time (Agrios, 2005; Singh, 2008). Fruit may occasionally become infected, but they drops off without becoming spotted (Agrios, 2005; Singh, 2008). Roots also become infected; after an initial period of stunting, the lateral roots show black rot condition. In the present study, tomato cv. Pusa Ruby grown in the soil infested with F. oxysporum f. sp. lycopersici in pot or plot developed typical symptoms of the disease. The first sign of the disease was chlorosis, stunted growth and sudden death that appeared at seedling stage. The percent wilting in tomato seedlings and their mortality was greater in pots

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than microplots. Under field conditions, recognizable wilt symptoms developed on 6-9 weeks old plants. Ray (2005) and Ren et al. (2010) reported 6-7 weeks as critical age of tomato for appearance of symptoms. The mycelium of the Fusarium grows profusely in vascular tissue especially in xylem tissue and causes clogging and browning of vessels from the root to the stem (Cho and Muehlbauer, 2004; Agrios, 2005). As a result black streaks gradually develop in xylem tissue whereas brown to black bands appear on the stem surface of partially wilted plants and extend upward from the base. When the bark of such bands is peeled off, browning or blackening of the wood can be seen (Dubey and Singh, 2004; Agrios, 2005). In the present study, transverse sections of roots and stems of chickpea and tomato revealed the presence of the fungus in xylem tissue. The fungus also colonized on PDA when the surface sterilized root and stem pieces of infected chickpea and tomato plants were inoculated on the medium in Petri plates. The isolated fungus was then re-inoculated in the respective plants to establish its pathogenecity. Nene et al. (1980) have reported that wilt fungus can be isolated from any part of the infected chickpea plant from lateral fine roots to pedicel and pod hull. The used chickpea cv. BGD-72 and tomato cv. Pusa Ruby are found to be highly susceptible to wilt and exhibited 18-24% and 29-33% yield loss, respectively at 2 g/kg soil of Fusarium spp. Application of A. niger isolates as seed/nursery treatment or soil application proved considerably effective against the wilt fungi infecting chickpea and tomato in pots. As a result plant growth and yield of both the crops increased significantly. The most effective isolates emerged from the pot trial were SkNAn5, VAn4, AnC2, AnR3, ANAn4 and BuAn3. In the pot culture test, the six isolates controlled the wilt of chickpea and tomato with seed/nursery treatment and soil application greater than other isolates being highest with isolate SkNAn5 (60-68% decrease), followed by VAn4 (57-65%), AnC2 (56-65%), AnR3 (54,62%), ANAn4 (54-61%) and BuAn3 (53-60%). Singh et al. (2002) reported 48-88% decrease in wilt severity of pigeonpea with A. niger in a pot experiment. A. niger is an established antagonist of soil borne fungal pathogens in particular against Fusarium spp. (Sen, 2000). The suppression in Fusarium wilt due to application of A. niger isolates may have resulted through competition for nutrients that has been found to be involved in the antagonism exerted by the fungus against F. oxysporum (Cook and Baker, 1983; Chet et al., 1997; Mondal et al., 2000; Eisendle et al., 2004; Vassilev et al., 1996, 2006). A. niger produces antibiotics and other metabolites which are potentially capable of inhibiting the colonization of Fusarium spp. (Sen, 2000).

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The metabolic changes that occur in diseased plants leads to accumulation of phenolic compounds (Farkas and Kiraly, 1962; Bashan et al., 1985; Sindhu et al., 1995; Raju et al., 2008). Activation of systemic acquired resistance (SAR) in plants triggered by salicylic acid (SA) contributes to restrict invasion and infection of the pathogen (O'Connell and Panstruga, 2006; Firdous et al., 2007; Doehlemann et al., 2008; Esmailzadeh et al., 2008). In chickpea and tomato, activation of SAR results in a significant reduction of disease symptoms caused by F. oxysporum (Houssien et al., 2010) and M. incognita (JataKumar et al., 2006). However, SA contents usually get increased in response of inoculation/infection with pathogens (Singh et al., 2003; Saikia et al., 2006; Raju et al., 2008; Garcia-Limones et al., 2008; Gupta et al., 2010). In the present study, phenol and salicylic acid contents of leaf and root of chickpea and tomato were considerably greater in the plants inoculated with F. oxysporum f. sp. ciceri or F. oxysporum f. sp. lycopersici and M. incognita singly or concomitantly. Infection with wilt fungi (Achore et al., 1993) and root-knot nematodes (Khan and Khan, 1987) resulted to considerable decrease in the leaf pigments, as recorded in the present study. However, A. niger isolates significantly checked the loss of chlorophyll content of leaves, lycopene content of the tomato fruit and further increased the phenol content and salicylic acid of leaves and roots of chickpea and tomato caused by F. oxysporum f. sp. ciceri or F. oxysporum f. sp. lycopersici and M. incognita singly or concomitantly as already reported by Sen (2000). Among the treatments, A. niger SkNAn5 was the most efficient isolate followed by VAn4, AnC2, carbendazim / carbofuran / carbendazim + carbofuran, and other isolates. It has been reported that systemic fungicides may first activate some defensive responses in the host (Guest, 1984; Jones et al., 1991; Molina et al., 1998). Garcia et al. (2001) determined that carbendazim (2.6 mM) increased SA as well as the accumulation of phenolics. Based on relative effectiveness of six isolates of A. niger against the wilt, root-knot and fungus-nematode wilt diseases complex evaluated under field condition, biopesticides of three isolates viz., SkNAn5, VAn4 and AnC2 were prepared on sawdust-fly ash based material. The formulation contained 109-10 CFU/g formulation. The shelf life tests revealed highest CFU load during 2-8 months of inoculation. Increase in CFU count during storage indicates that the A. niger isolates utilized the nutrients present in fly ash (Fakoussa and Hofrichter, 1999; Schmidt and Noack, 2000) and the sugars of molasses (Maneerat, 2005). Molasses contains 48-56% total sugar (mainly sucrose), 9-12% non-sugar organic matter, 2- 4% protein, 1.5-5% potassium, 0.4-0.8% calcium, 0.06% magnesium, 0.6-2.0% phosphorus,

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1.0-3.0 mg biotin/kg, 15-55 mg pantothenic acid/kg, 2500-600 mg inositol/kg and 1.8 mg thiamine/kg (Curtin, 1983; Makkar and Cameotra, 1997). These nutrients can act as a good source of carbohydrates for sustenance and multiplication of the biocontrol fungi (Patel and Desai, 1997; Maneerat, 2005). The shelf life test of the formulations has revealed that the present biopesticides contained CFU load of A. niger isolates on or above the standard level of 108-9/g formulation (Tilak, 1993). The highest CFU counts were recorded at room temperature (22-37°C) or 25°C during 2-8 months of storage. These temperatures and durations well suit Indian condition as ambient temperature except during December- February. A. niger is xerotolerant (Cooke and Whips, 1993) and can grow at a wide range of temperatures (10–50 °C), pH (2.0–11.0), and salt (34%) (Kis-Papo et al., 2003; Zhang et al., 2007). The 1-2 months period is sufficient for transportation and distribution of the biopesticides to local pesticide dealers and another 1-2 months for the procurement of the biopesticides by farmers and its application in the field. Hence, the total duration from manufacturing to application of biopesticides (2-4 months) lies within the period of higher CFU load as determined by the shelf life test. It is, therefore, well expected that when the biopesticides will be applied in the field the formulation shall contain the standard CFU load of the biocontrol agent. Effectiveness of the newly prepared biopesticides was evaluated against the target disease on chickpea and tomato in small plots under field condition. Inoculation with F. oxysporum f. sp. ciceri and F. oxysporum f. sp. lycopersici resulted to 58 and 62% wilt incidence with an average severity of 3.3 and 3.5 on 0-5 scale on chickpea and tomato, respectively. Application of the biopesticides checked the wilt incidence by 65-79% and severity by 57-65% being highest with SkNAn5 (74-79% incidence and 59-65% severity) in both the crops. Effect of carbendazim (decreased 53-58% wilt incidence and 52-63% wilt severity) was less than SkNAn5, VAn4 and AnC2. Other researchers have also shown that if biofungicides contain standard CFUs of efficient strains of the antagonists they can provide disease control equal to fungicides (Khan, 2005). In field trials, Khan and Khan (2001, 2002) tested the effectiveness of A. niger against Fusarium wilt of tomato and found the BCA effective against the disease. In another field experiment with seeds treatment of A. niger checked the Fusarium wilt of muskmelon and watermelon by 81% (Chattopadhyay and Sen, 1996). Other researchers have also confirmed effectiveness of A. niger against different diseases of crops (Upadhyay and Rai 1984; Kumar and Sen, 1998; Mondal, 1998; Sen et al., 1998; Sen, 2000; Sehgal et al., 2001; Singh et al., 2002; Birthal and Sharma, 2004; Misra, 2007; Khan and Anwer, 2011).

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Root-knot nematodes are sedentary endoparasites and they induce certain structural, physiological and biochemical changes in the host plant (Haung, 1985; Hussy, 1989). Second stage juvenile enter into young lateral roots, and after getting a suitable site for feeding become sedentary with their heads inserted in vascular tissue and body in the cortical region of the root. As a result of nematode pathogenesis cortical cells around the nematode (female) become hyperplastic dividing repeatedly by mitosis resulting to enlargement of the tissue which is commonly called as gall or knot (Bird, 1972). Galled roots become short, thick and deshaped. Gall formation and suppression of normal root growth impair absorption capacity of the root system resulting to the appearance of water stress symptoms in foliage, especially during periods of moisture stress and/or high transpiration rate (Wilcox-Lee and Loria, 1987). Photosynthesis decreases but respiration increases (Wilcox-Lee and Loria, 1987) accompanied by greater allocation of photosynthates to roots particularly infected tissue and the giant cells (Wallace, 1987). Cumulatively the infection leads to poor growth and reduced yield of the host. Infected plants show nutrient deficiency symptoms as stunted growth with pale green foliage. In the present study the chickpea cv. BGD-72 and tomato cv. Pusa Ruby inoculated with 2000 J2/kg soil showed poor shoot growth and mild leaf chlorosis, and visible galls formed on the roots. The galls were, however, smaller in size and lesser in number in chickpea than tomato. Solanaceous vegetables are more suitable for gall formation than leguminous crops (Sasser, 1989) probably due to nodulation (Taha, 1993). Application of A. niger isolates or their commercial formulations (biopesticides) greatly suppressed the gall formation and egg mass production. Application of SkNAn5 and its biopesticide caused the highest decrease in the number of galls (51-70%) and egg masses (48-76%) per root system followed by VAn4, AnC2 and carbofuran in pots and fields. The fungus, A. niger produces nematoxic metabolites (Dahiya and Singh, 1985; Khan et al., 2009) which may cause mortality to nematodes (Molina and Davide, 1986). Some strains of A. niger have also been identified as egg parasite or opportunistic parasite of M. incognita (Khan, 2007; Goswami et al., 2008). A. niger is an active colonizer and soil invader and preferably grows on organic matter (Domich et al., 1980; Nigam and Robinson, 2004). The fungus may have colonized larvae and subsequently adults, eggs or egg masses, and resulted to significant decline in galls and soil population of M. incognita in the pots and plots. A. niger produces certain enzyme/metabolites such as harzianic acid, alamethicins, tricholin, peptaibols, 6-penthyl-α-pyrone, y-lactone, massoilactone, viridin, gliovirin, glisoprenins, heptelidic acid, oxalic acid and hydrolytic enzymes (Benitez et al., 2004; El-Hasan et al.,

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2007), which may also have been involved in the antagonism against root-knot nematodes. Siddiqui and Kazuyoshi (2009) have reported a significant decrease in galling with the application of A. niger. The rhizosphere population of the wilt fungi were significantly lower in the month of January, but increased to its peak in February. Khan et al. (2004) have reported higher population of F. oxysporum f. sp. ciceri during Feb-March over January. Population of the A. niger isolates, however, increased gradually in the wilt fungus infested soil. The A. niger isolates apparently would have colonized better in the rhizosphere or roots of chickpea and tomato plants growing in wilt fungus infested soil. For effective management of soil borne diseases the introduced antagonist should colonize in the rhizosphere and/or roots (Weller, 1988). A. niger is reported to colonize roots of several crops (Sen, 2000; Khan and Anwer, 2008), possibly due to an improved ability to compete for root exudates (Gamliel and Katan, 1992). Greater increase in the A. niger population in Fusarium indicates that a rhizosphere rich in spores/propagules of the wilt fungus serves as a better substrate for the multiplication of the A. niger (Khan et al., 2004). Soil population of the pathogens significantly decreased due to application of A. niger isolates. Greatest decrease in the soil population of Fusarium and M. incognita occurred with the application of SkNAn5, followed by VAn4, AnC2 isolate and carbendazim/carbofuran. The A. niger isolates may have suppressed the nematode population with the actions as explained for decrease in galls and egg masses. An inverse relationship was recorded between the soil populations of a pathogen and the A. niger isolates. Soil population of the A. niger isolates increased proportionately with the decrease in disease severity and pathogen population. The antagonist, A. niger may parasitize F. oxysporum f. sp. ciceri (Khan, 2005) and F. oxysporum f. sp. lycopersici (Khan and Khan, 2001, 2002) and draw nutrition from mycelium to grow and sporulate (Mondal et al., 2000). Likewise, A. niger also possesses ability to parasitize root-knot nematodes (Buchi et al., 1983; Fujimoto et al., 1993; Eapen and Venugopal, 1995) and its population increased in the pots/plots infested with M. incognita. Greatest increase in the population was recorded for SkNAn5 in the pots/plots that had nematode and Fusarium together than alone. The fungus multiplies efficiently on eggs and egg masses of Meloidogyne spp. (Khan et al., 2005a). In addition nematode population was much lower in the presence of Fusarium spp. irrespective of other treatments. Fusarium spp. are well documented to preferably invade feeding sites of root-knot nematodes (Francl and Wheeler, 1993), as a result the nematode development and egg mass production are suppressed greatly, but Fusarium population correspondingly increases. However, A. niger

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isolates showed affinity to both, wilt fungus and root-knot nematode, as a result in concomitantly infested plots greater increase in the population of A. niger occurred. Synergistic interaction between wilt fungi (Fusarium spp.) and root-knot nematodes (Meloidogyne spp.) is well established on a number of crops including legumes like, alfalfa (Griffin, 1986), cowpea (Thomason et al., 1959), pea (Davis and Jenkins, 1963), beans (Ribero and Ferraz, 1984), mungbean (Khan et al., 2002), pigeonpea and chickpea (Khan, 2005) and vegetables like tomato (Khan and Akram, 2000), eggplant (Khan and Anwer, 2008) and other vegetable crops (Khan and Husain, 1991). The interaction between these two pathogens has been found to be generally synergistic (Francl and Wheeler, 1993). In such interactions the Fusarium wilt has become severe in the presence of nematodes resulting to significantly greater crop damage. In the present study also, M. incognita and F. oxysporum f. sp. ciceri/lycopersici interacted synergistically and caused grater suppression in plant growth and yield parameters of chickpea cv. BGD-72 and tomato cv. Pusa Ruby. Severity of wilt symptoms was enhanced when both the pathogens were present together in comparison to F. oxysporum f. sp. ciceri/f. sp. lycopersici alone. Root-knot nematode infection leads to alteration in root exudation of the infected plants. Root exudates of nematode infected plants contain greater concentration of Ca, Mg, Na, K, Cu, and Fe, and during first fourteen days of infection, carbohydrates are the major organic constituents of root exudates, and nitrogenous compounds predominate afterwards (Van Gundy et al., 1977). According to Powell (1971) and Webster (1985), root-knot nematode infection predisposes the host plant to wilt fungi. During development of giant cells, infected roots of host plant exhibit decreased concentration of cellulose and lignin, and considerable increase in the amino acids, hemicelluloses, lipids, minerals, nucleotides, organic acids, proteins, DNA and RNA (Khan, 1993). These biochemical changes enrich the medium, which is the cause for rapid growth and colonization of the wilt fungi on the galled tissue (Francl and Wheeler, 1993). Giant cells being rich nutritionally, serve a good site for the colonization of the fungi. The giant cells, located in the vascular tissue act as a launching pad for the fungus for spread in xylem tissue and also to transport toxins. Fusarium spp. produce fusaric acid and other toxins that contribute in wilting of plants (Bell and Mace, 1981; Glick, 1995). The concomitant inoculation with M. incognita and F. oxysporum f. sp. ciceri/f. sp. lycopersici in the present study, exacerbated the wilt symptoms but severity of the nematode symptoms (galling) significantly decreased compared to nematode alone (control). Fusarium spp. have shown strong affinity to feeding sites of sedentary endoparasites. The giant cells formed by M. incognita are rapidly invaded by the wilt fungi utilizing its contents (Webster,

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1985); as a result developing nematode females starve to death. In addition, the metabolites produced by F. oxysporum f. spp. may suppress hatching of eggs and induce mortality to larvae (Ciancio et al., 1988). The sedentary endoparasites such as Meloidogyne spp. show relatively decreased populations in soils infested with Fusarium or Verticillium spp. (Morgan-Jones et al., 1983; Hasan, 1989; Fazal et al., 1994). Nodule formation on chickpea in F. oxysporum f. sp. ciceri infected soil in pot or field decreased considerably. Suppressive effects of Fusarium spp. on root nodules has been observed in a number of studies (Twng-Wah and Howard, 1969; Sawada, 1982, 1983), but the mechanism involved is not properly understood. Infected roots with altered physiology and morphology (rotting, decaying etc.) may become less suitable for the infection by the Rhizobium and development of root nodules. The suppression may also be due to competition between the two microorganisms at initial stage of the infection. Fusaric acid produced by the Fusarium spp. (Toyoda and Utsumi, 1991) may also be involved in the inhibition of Rhizobium. Root-knot nematode also adversely affected the nodulation. The suppression may have occurred due to nutritional interference, particularly carbohydrates or physiological changes brought about by the nematode infection and/or competition for infection site (Taha, 1993). Number of functional nodules were decreased but non functional nodules increased significantly as a result of infection of M. incognita. This may be due to invasion of nodules by the nematode and causing histological changes in the nodular tissue (Taha and Raski, 1969; Barker and Hussey, 1976; Khan et al., 2002; Khan, 2005) thereby rendering them nonfunctional. In the present study, seed or soil application of carbendazim and carbofuran caused some suppressive effect on root nodulation. Inhibitory effects of the fungicides were reported by Graham et al. (1980). Researches have suggested that a field factor such as dilution of the fungicide in the soil or migration of the bacteria away from the toxic chemical may be involved in determining fungicidal toxicity (Castro et al. 1997). Rhizobia exposed to the chemicals were viable, but their fatty acid composition and substrate utilization profile were significantly altered. Resulting in changes in cellular metabolism (Rose, 1989). In addition, cellular fatty acids are involved in nodulation, but their role remains unclear (Pueppke 1996). Thus, the alteration of fatty acid by the chemicals might suggest a decreased capacity of Rhizobium to nodulate. Lesser nodules were formed in the field compared to pots. This may be due to competition with ineffective indigenous strains of Rhizobium and other rhizobia (McLoughlin et al., 1988).

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In the present study some isolates of A. niger were found to be efficient plant growth promoters as their application increased chickpea and tomato yield from 26-41% after their seed/nursery or soil application. Among the isolates, greater increase in chickpea and tomato yield was recorded with SkNAn5 (36-41%). Plant growth promotion activity of A. niger is well established (Chattopadhyay and Sen, 1996; Mondal et al., 2000; Ramachandran et al., 2008), and researchers have reported significant yield enhancement (Dhillion, 1994; Misra, 2007; Siddiqui and Kazuyoshi, 2009; Khan and Anwer, 2011). Root colonization by A. niger frequently enhances root growth, development, crop productivity, resistance to abiotic stresses and the uptake and use of nutrients (Mondal et al., 2000). Plant growth promoting effect of A. niger may also contribute towards promotion of plant growth of infected plants in addition to exerting antagonism against pathogenic microorganisms in rhizosphere (Mostafa and Youssef, 1962; Youssef and Mankarios, 1975). A. niger may also solubilize minerals such as phosphorus (Domich, et al., 1980; Medina et al., 2007), synthesize phytohormones (Youssef and Mankarios, 1975; Khan and Anwer, 2007) and produces siderophores that can solubilize and sequester iron from the soil and provide it to plants cells (Mondal et al., 2000; Khan and Anwer, 2007). It has been evidenced that the plants have an ability to incorporate Fe+3 of siderophores into their biomass (Reid et al., 1984; Barker et al., 1985). Application of A. niger to plants results in improved seed germination, increased plant size and augmentation of leaf area and weight (Chakraborty et al., 2007). Overall seed/nursery treatment with A. niger biopesticides was relatively more efficient than soil application. The amount of A. niger isolates added to the a microplot through soil application (40 g/microplot) was much greater than applied through seed/nursery (4 g/kg seed or 10 g/100 nurseries). But with soil application, the CFUs dispersed in greater area giving rise to a much smaller CFU count/unit area, whereas with seed/nursery treatment the antagonist remained concentrated on or around the seed or root zone. In case of chickpea, the germinating seeds attract rhizobacteria (Scher et al., 1985) and are rapidly colonized due to profuse exudation of a wide range of amino acids, carbohydrates, organic acids (Hayman, 1969; Lynch, 1978). The A. niger propagules applied through seed/nursery treatment thus received greater nutrients through exudates of germinating seeds to full root growth and also had faced less competition and exerted more because of their preoccupation as well as aggregation in the root zone, as a result the applied isolates multiplied with a greater pace evidenced by the higher CFUs/g soil compared to soil application. Apparently the greater population of A. niger would have resulted to greater phosphate solubilization, hormone

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production and/or pathogen suppression that reflected into a greater disease control, better plant growth and improved yield of chickpea and tomato. It can be concluded that a biopesticide of A. niger SkNAn5 prepared on sawdust-fly ash based formulation may carry 109-10 CFU of the biocontrol agent which can be maintained as viable propagules for 6-8 months. The seed/nursery treatment of the biofungicides @ 4 g/kg seed or 10 g/100 seedlings may satisfactorily control the wilt (60-66%) and root-knot (63-72%), and improve the yield of chickpea (59-66%) and tomato (65-71%). The biofungicide can be incorporated into general cultivation practice as the A. niger SkNAn5 also acted as a growth promoter and improved the yield of two crops by 39-41% grown in non-infested soil. In view of low cost of the formulation and its handy application, the seed/nursery treatment with A. niger SkNAn5 biopesticide can also be adopted by the small farmers in India and other developing countries.

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APPENDICES

Appendix-1 Potato Dextrose Agar (PDA) Potato 200 g Dextrose 20 g Agar 15 g Water 1 l

Appendix-2 Isolation details with coding of Aspergillus niger aggregate collected from 40 different districts of Uttar Pradesh, India S.No. District Area/Block Crop field Accession A. niger No. isolate (FBCAN) code 1. Allahabad Naini Pigeonpea 1887 AAn1 2. Allahabad Karchana Mustard 1888 AAn2 3. Allahabad Phulpur Potato 1889 AAn3 4. Allahabad Meja Cauliflower 1890 AAn4 5. Allahabad Sirsa Cabbage 1891 AAn5 6. Agra Malpura Brinjal 1892 AgAn2 7. Agra Agra city Tomato 1893 AgAn3 8. Agra Fetehabad Chilli 1894 AgAn4 9. Agra Fatahpur Mustard 1895 AgAn5 10. Azamgarh Lalganj Mustard 1896 AzAn1 11. Azamgarh Phariha Rice 1897 AzAn3 12. Azamgarh Muhammadabad Potato 1898 AzAn4 13. Azamgarh Azamgarh city Cauliflower 1899 AzAn5 14. Bahraich Kaisavganj Wheat 1900 BahAn1 15. Bahraich Baundi Mustard 1901 BahAn3 16. Bahraich Sisai Tomato 1902 BahAn4 17. Bahraich Nawabganj Chickpea 1903 BahAn5 18. Ballia Rasra Sugarcane 1904 BaAn1 19. Ballia Ratsanar Tomato 1905 BaAn2 20. Ballia Nawangar Pigeonpea 1906 BaAn4 21. Ballia Chitthara Mustard 1907 BaAn5 22. Banda Naraini Spinach 1908 BAn1 23. Banda Atarra Onion 1909 BAn2 Continued…

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Appendix 2 Continued… S.No. District Area/Block Crop field Accession A. niger No. isolate (FBCAN) code 24. Banda Bisenda Chickpea 1910 BAn3 25. Banda Baveru Garlic 1911 BAn4 26. Banda Tindpara Marigold 1912 BAn5 27. Bulandshahar Anupshahar Wheat 1913 BuAn2 28. Bulandshahar Ramghat Chilli 1914 BuAn3 29. Bulandshahar Dabai Lentil 1915 BuAn4 30. Bulandshahar Khurja Sugarcane 1916 BuAn5 31. Bareilly Nawabganj Kidneybean 1917 BarAn1 32. Bareilly Faridpur Wheat 1918 BarAn2 33. Bareilly Collectorganj Doobhgrass 1919 BarAn3 34. Bareilly Sirauli Potato 1920 BarAn4 35. Basti Basti city Tomato 1921 BasAn1 36. Basti Kapatanganj Brinjal 1922 BasAn2 37. Basti Abha Papaya 1923 BasAn4 38. Basti Gopalpur Sugarcane 1924 BasAn5 39. Bijnor Najibabad Guava 1925 BiAn1 40. Bijnor Barhapur Wheat 1926 BiAn2 41. Bijnor Afzalgarh Mustard 1927 BiAn3 42. Bijnor Bijnor city No crop 1928 BiAn4 43. Bijnor Chandpur Mango 1929 BiAn5 44. Baudaun Babrala Sugarcane 1930 BudAn1 45. Baudaun Bisauli Mentha 1931 BudAn2 46. Baudaun Baudaun city Sorghum 1932 BudAn3 47. Baudaun Alapur Brinjal 1933 BudAn4 48. Baudaun Usaihat Okra 1934 BudAn5 49. Chitrakoot Chitrakoot city Mustard 1935 ChAn1 50. Chitrakoot Karwi Pigeonpea 1936 ChAn2 51. Chitrakoot Manikpur Chickpea 1937 ChAn3 52. Chitrakoot Raipura Tomato 1938 ChAn5 53. Etawah Chakarnagar Wheat 1939 EtAn1 54. Etawah Bukedar Potato 1940 EtAn2 55. Etawah Bharthana Sugarcane 1941 EtAn3 56. Etawah Etawah city Onion 1942 EtAn4 57. Etawah Kalan Chickpea 1943 EtAn5 58. Farrukhabad Muhammadabad Tomato 1944 FaAn1 59. Farrukhabad Farrukhabad city Sugarcane 1945 FaAn3 60. Farrukhabad Shamshabad Chickpea 1946 FaAn4 61. Farrukhabad Kaimganj Marigold 1947 FaAn5 62. Faizabad Faizabad city Brinjal 1948 FazAn1 Continued…

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Appendix 2 Continued… S.No. District Area/Block Crop field Accession A. niger No. isolate (FBCAN) code 63. Faizabad Amanigang Mentha 1949 FazAn2 64. Faizabad Bikapur No crop 1950 FazAn3 65. Faizabad Khajurhat Okra 1951 FazAn4 66. Faizabad Gosainganj Sugarcane 1952 FazAn5 67. Gonda Karnalganj Pigeonpea 1953 GoAn1 68. Gonda Paraspur Wheat 1954 GoAn2 69. Gonda Begamganj Sugarcane 1955 GoAn3 70. Gonda Gonda city Mustard 1956 GoAn4 71. Gonda Srinagar Potato 1957 GoAn5 72. Ghaziabad Ghaziabad city Chilli 1958 GaAn1 73. Ghaziabad Hapur Chickpea 1959 GaAn2 74. Ghaziabad Faridnagar Mustard 1960 GaAn3 75. Ghaziabad Muradnagar Tomato 1961 GaAn5 76. Gorakhpur Rudrapur Cucumber 1962 GorAn1 77. Gorakhpur Beghat khas Wheat 1963 GorAn2 78. Gorakhpur Mundera Chickpea 1964 GorAn3 79. Gorakhpur Dumri Sugarcane 1965 GorAn4 80. Gorakhpur Gorakhpur city Mustard 1966 GorAn5 81. Jhansi Gursarai Pigeonpea 1967 JhAn3 82. Jhansi Garantha Chickpea 1968 JhAn4 83. Jhansi Kakarbai Tomato 1969 JhAn5 84. Jalaun Orai Tomato 1970 JaAn2 85. Jalaun Khanwa Brinjal 1971 JaAn3 86. Jalaun Bagra Wheat 1972 JaAn4 87. Jalaun Rampura Chickpea 1973 JaAn5 88. Kanpur Kalyanpur Pigeonpea 1974 KaAn1 89. Kanpur Mangalpur Moongbean 1975 KaAn2 90. Kanpur Akbarpur Lentil 1976 KaAn3 91. Kanpur Gausganj Wheat 1977 KaAn4 92. Kanpur Panki Tomato 1978 KaAn5 93. Lakhimpur Kheri Kheri Tomato 1979 LkAn1 94. Lakhimpur Kheri Tatarpur Potato 1980 LkAn2 95. Lakhimpur Kheri Kothia Cauliflower 1981 LkAn3 96. Lakhimpur Kheri Palia Cabbage 1982 LkAn4 97. Lakhimpur Kheri Dudwa Brinjal 1983 LkAn5 98. Lalitpur Bijrautha Brinjal 1984 LaAn2 99. Lalitpur Gona Mustard 1985 LaAn3 100. Lalitpur Mahrauni Wheat 1986 LaAn4 101. Lalitpur Kalgawan Chickpea 1987 LaAn5 Continued…

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Appendix 2 Continued… S.No. District Area/Block Crop field Accession A. niger No. isolate (FBCAN) code 102. Lucknow Amousi Okra 1988 LAn1 103. Lucknow Chinhat Sugarcane 1989 LAn2 104. Lucknow Gosaiganj Wheat 1990 LAn3 105. Lucknow Amethi Guava 1991 LAn4 106. Lucknow Dilwarnagar No crop 1992 LAn5 107. Mahoba Kabrai Potato 1993 MaAn1 108. Mahoba Srinagar Cauliflower 1994 MaAn2 109. Mahoba Jaitpur Wheat 1995 MaAn3 110. Mahoba Kulpahad Tomato 1996 MaAn4 111. Mahoba Kohnia Pigeonpea 1997 MaAn5 112. Mainpuri Karhat Sugarcane 1998 ManAn1 113. Mainpuri Mainpuri city Chickpea 1999 ManAn3 114. Mainpuri Bewar Potato 2000 ManAn4 115. Mainpuri Bhongaon Tomato 2001 ManAn5 116. Mahamaya Nagar Iglas Chickpea 2002 MNAn1 117. Mahamaya Nagar Hathras Pigeonpea 2003 MNAn2 118. Mahamaya Nagar Sasni Wheat 2004 MNAn3 119. Mahamaya Nagar Sikndrarau Cabbage 2005 MNAn4 120. Mahamaya Nagar Akbarabad Tomato 2006 MNAn5 121. Merrut Merrut city No crop 2007 MeAn1 122. Merrut Shahjahanpur Sugarcane 2008 MeAn2 123. Merrut Rajpura Wheat 2009 MeAn3 124. Merrut Mawana Brinjal 2010 MeAn4 125. Merrut Sardhana Tomato 2011 MeAn5 126. Pilibhit Amaria Potato 2012 PAn2 127. Pilibhit Deoria Wheat 2013 PAn3 128. Pilibhit Bilsanda Sugarcane 2014 PAn4 129. Rampur Shahabad Wheat 2015 RaAn1 130. Rampur Milak Pigeonpea 2016 RaAn2 131. Rampur Champrana Sugarcane 2017 RaAn3 132. Rampur Bilaspur Tomato 2018 RaAn4 133. Rampur Nepatnagar Potato 2019 RaAn5 134. Saharanpur Muzaffarabad Pigeonpea 2020 SaAn2 135. Saharanpur Chilkana Mustard 2021 SaAn3 136. Saharanpur Balia Kheri Brinjal 2022 SaAn4 137. Saharanpur Deoban Cauliflower 2023 SaAn5 138. Sonbhadra Aikdehi Onion 2024 SoAn1 139. Sonbhadra Muirpur Tomato 2025 SoAn2 140. Sonbhadra Dudhinagar Pigeonpea 2026 SoAn3 Continued…

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Appendix 2 Continued… S.No. District Area/Block Crop field Accession A. niger No. isolate (FBCAN) code 141. Sonbhadra Ranital Chickpea 2027 SoAn4 142. Sonbhadra Renukut Mustard 2028 SoAn5 143. Sultanpur Lampura Mustard 2029 SuAn1 144. Sultanpur Sultanpur city Chickpea 2030 SuAn2 145. Sultanpur Musafirkhana Sugarcane 2031 SuAn3 146. Sultanpur Jagdishpur Pigeonpea 2032 SuAn4 147. Sultanpur Piparpur Tomato 2033 SuAn5 148. Santkabir Nagar Khalilabad Wheat 2034 SkNAn1 149. Santkabir Nagar Alinagar Mustard 2035 SkNAn2 150. Santkabir Nagar Santkabir Nagar Chickpea 2036 SkNAn3 city 151. Santkabir Nagar Dudhara Papaya 2037 SkNAn4 152. Santkabir Nagar Mehdawal Amla 2038 SkNAn5 153. Siddharth Nagar Siddharth Nagar Wheat 2039 SiNAn1 main 154. Siddharth Nagar Khalilabad Tomato 2040 SiNAn3 155. Siddharth Nagar Asanhara Potato 2041 SiNAn4 156. Siddharth Nagar Naugarh Cauliflower 2042 SiNAn5 157. Sitapur Mahmudabad Wheat 2043 SiAn1 158. Sitapur Kherabad Sugarcane 2044 SiAn2 159. Sitapur Kamlaapur Wheat 2045 SiAn3 160. Sitapur Sitapur city Mustard 2046 SiAn4 161. Sitapur Hargaon Chickpea 2047 SiAn5 162. Unnao Mahan Wheat 2048 UAn1 163. Unnao Hasanganj Mustard 2049 UAn2 164. Unnao Rasulabad Cauliflower 2050 UAn3 165. Unnao Purwa Cabbage 2051 UAn4 166. Unnao Maurava Papaya 2052 UAn5 167. Varanasi Varanasi Chickpea 2053 VAn1 168. Varanasi Sarnath Pigeonpea 2054 VAn2 169. Varanasi Aunrihar Mustard 2055 VAn3 170. Varanasi Varanasi city Sugarcane 2056 VAn4 171. Varanasi Mughalsari cauliflower 2057 VAn5 172. Ambedkar Nagar Akbarpur Brinjal 2058 ANAn1 173. Ambedkar Nagar Belana Okra 2059 ANAn2 174. Ambedkar Nagar Jalalpur Chilli 2060 ANAn3 175. Ambedkar Nagar Asapur Papaya 2061 ANAn4 176. Ambedkar Nagar Birhar Mango 2062 ANAn5 177. Aligarh Bijauli Pearl millet 2063 AnPM1 178. Aligarh Atrauli Pearl millet 2064 AnPM2 Continued…

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Appendix 2 Continued… S.No. District Area/Block Crop field Accession A. niger No. isolate (FBCAN) code 179. Aligarh Jawan Pearl millet 2065 AnPM3 180. Aligarh Chandous Pearl millet 2066 AnPM4 181. Aligarh Buroily Pearl millet 2067 AnPM5 182. Aligarh Atrauli Pigeonpea 2068 AnPP1 183. Aligarh Khair Pigeonpea 2069 AnPP2 184. Aligarh Fatahpur Pigeonpea 2070 AnPP3 185. Aligarh Aligarh city Pigeonpea 2071 AnPP4 186. Aligarh Pisawa Pigeonpea 2072 AnPP5 187. Aligarh Akrabad Cauliflower 2073 AnC1 188. Aligarh Mehrawal Cauliflower 2074 AnC2 189. Aligarh Hathras Cauliflower 2075 AnC3 190. Aligarh Atrauli Cauliflower 2076 AnC4 191. Aligarh Fatahpur Cauliflower 2077 AnC5 192. Aligarh Pisawa Sunflower 2078 AnS1 193. Aligarh Khair Sunflower 2079 AnS2 194. Aligarh Chharra Sunflower 2080 AnS3 195. Aligarh Tappal Sunflower 2081 AnS4 196. Aligarh Atrauli Sunflower 2082 AnS5 197. Aligarh Pisawa Papaya 2083 AnPa1 198. Aligarh Buroily Papaya 2084 AnPa2 199. Aligarh Sikandarpur Papaya 2085 AnPa3 200. Aligarh Fatahpur Papaya 2086 AnPa4 201. Aligarh Chharra Papaya 2087 AnPa5 202. Aligarh Atrauli Mango 2088 AnMa1 203. Aligarh Aligarh city Mango 2089 AnMa2 204. Aligarh Buroily Mango 2090 AnMa4 205. Aligarh Fatahpur Mango 2091 AnMa5 206. Aligarh Polwal Rice 2092 AnR1 207. Aligarh Akrabad Rice 2093 AnR2 208. Aligarh Dhanipur Rice 2094 AnR3 209. Aligarh Lodha Rice 2095 AnR4 210. Aligarh Pisawa Rice 2096 AnR5 211. Aligarh Gonda Lentil 2097 AnL1 212. Aligarh Dhanipur Lentil 2098 AnL2 213. Aligarh Iglas Lentil 2099 AnL3 214. Aligarh Chharra Lentil 2100 AnL4 215. Aligarh Aligarh city Lentil 2101 AnL5 216. Aligarh Pravan Tomato 2102 AnT1 217. Aligarh Tappal Tomato 2103 AnT2 Continued…

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Appendix 2 Continued… S.No. District Area/Block Crop field Accession A. niger No. isolate (FBCAN) code 218. Aligarh Fatahpur Tomato 2104 AnT3 219. Aligarh Chharra Tomato 2105 AnT4 220. Aligarh Sikandarpur Potato 2106 AnPo1 221. Aligarh Khair Potato 2107 AnPo2 222. Aligarh Aligarh city Potato 2108 AnPo3 223. Aligarh Pravan Potato 2109 AnPo4 224. Aligarh Buroily Potato 2110 AnPo5 225. Aligarh Pisawa Radish 2111 AnRa1 226. Aligarh Sikandarpur Radish 2112 AnRa2 227. Aligarh Fatahpur Radish 2113 AnRa4 228. Aligarh A.M.University Mustard 2114 AnM1 229. Aligarh Khair Mustard 2115 AnM2 230. Aligarh Buroily Mustard 2116 AnM3 231. Aligarh Gangiri Mustard 2117 AnM4 232. Aligarh Pravan Mustard 2118 AnM5 233. Aligarh Aligarh city Chickpea 2119 AnCp2 234. Aligarh Buroily Chickpea 2120 AnCp3 235. Aligarh Pravan Chickpea 2121 AnCp4 236. Aligarh Sikandarpur Chickpea 2122 AnCp5

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Appendix-3 Yeast Peptone Glucose Broth (YPGB) Yeast 3 g Peptone 10 g Glucose 20 g Water 1 l

Appendix-4 Peptone Broth Peptone 10 g NaCl 5 g Water 1 l pH 7

Appendix-5 Sabouraud Dextrose Agar (SDA) Glucose 40 g Peptone 10 g Agar 20 g Glycine 4.4 g Water 1 l

Appendix-6 Luria Bertani Broth (LBB) Bacto tryptone Glucose 10 g NaCl 5 g Yeast extract 5 g Water 1 l pH 7

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Appendix-7 Pikovskaya’s Medium Glucose 10 g Ca3(PO4)2 5 g (NH4)2SO4 0.5 g NaCl 0.2 g MgSO4.7H2O 0.1 g KCl 0.2 g MnSO4 Trace FeSO4. 7H2O Trace Yeast extract 0.5 g Agar 15 g Water 1l pH 7.2

Appendix-8 Chloromolybdic acid reagent 15 g of ammonium molybdate was dissolved in about 400 ml of warm distilled water to which 342 ml of 12 N HCl was added showly and finally the volume was made one liter with distilled water.

Appendix-9 Chlorostannous acid reagent

The reagent was prepared by dissolving 2.5 g of SnCl2. 2H2O crystals in 10 ml concentrated HCl and finally the volume was made 100 ml with distilled water.

Appendix-10 Standard phosphorus solution (100 ppm) 0.4390 g of dried KH2PO4 was dissolved in 400 ml distilled water. 0.25 ml of 7N H2SO4 was added to it and the volume was made upto one liter. Standard phosphorus solution (2 ppm) 2 ml of 100 ppm standard solution was diluted to 100 ml distilled water.

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Appendix-11 Climatic conditions that prevailed during cropping season 2007- 08 Months Temperature (°C) Relative humidity (%) Rainfall (mm)

Min. Max. Mean Min. Max. Mean Nov 11.0 32.0 21.5 53.0 89.0 71.0 - Dec 5.0 26.5 15.8 47.0 93.0 70.0 - Jan 3.0 26.5 14.8 38.0 89.0 63.5 - Feb 4.0 28.5 16.3 49.0 92.0 70.5 0.39 March 11.5 37.0 24.3 49.0 85.0 67.0 0.02 Source: Metrological Data Collection Unit, Department of Physics, Aligarh Muslim University, Aligarh, India.

Appendix-12 Climatic conditions that prevailed during cropping season 2008- 09 Months Temperature (°C) Relative humidity (%) Rainfall (mm)

Min. Max. Mean Min. Max. Mean Nov 10.0 34.0 22.0 27.0 93.0 60.0 0.22 Dec 7.5 27.5 17.5 33.0 95.0 64.0 - Jan 5.0 25.5 15.3 41.0 97.0 69.0 - Feb 8.5 30.0 19.3 23.0 95.0 59.0 - March 11.5 35.0 23.3 25.0 82.0 53.5 0.12 Source: Metrological Data Collection Unit, Department of Physics, Aligarh Muslim University, Aligarh, India.

Appendix-13 Fusarium specific medium / Nash and Snyder medium / PCNB Agar Peptone 5 g MgSO47H2O 0.5 g KH2PO4 1 g Agar 20 g Water 1 l After autoclaving, the medium was cooled to 45 °C and supplemented with 300 mg streptomycin and 1 g PCNB (75% WP).

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Appendix-14 Aspergillus niger specific medium / Sabouraud Dextrose Agar (SDA) Glucose 40 g Peptone 10 g Agar 20 g Water 1 l

Appendix-15 Grinding Buffer 3M sodium acetate 10 ml 4M NaCl 37.5 ml 0.5M Etylenediamine tetracetic acid (EDTA) 30 ml 1 M Tris Cl 15 ml Poly vinyl prrolidone (PVP) 6 g Sodium lauryl sulphate (SDS) 4.2 g pH 8.0 Water Add to make total volume 300 ml

Appendix-16

T10E1 Buffer 1 M Tris 1 ml 0.5M Etylenediamine tetracetic acid (EDTA) 200 µl pH 8.0 Water Add to make total volume 100 ml

Appendix-17 Fusarium specific synthetic decamer primers Primer Sequence Primer 01 GTCACCCGGA Primer 02 GCGACGCCTA Primer 03 GCGGCATTGT Primer 04 AGTGGTCGCG Primer 05 CCAGACAAGC Primer 06 GATAGCCGAC Primer 07 GCTGGACATC Primer 08 GATCTCAGCG

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