STUDIES ON ROOT ROT/ WILT OF SOYBEAN

Transcription

1 STUDIES ON ROOT ROT/ WILT OF SOYBEAN Thesis submitted to the University of Agricultural Sciences, Dharwad in partial fulfillment of the requirements for the Degree of MASTER OF SCIENCE (AGRICULTURE) IN PLANT PATHOLOGY BY SANGEETHA T. V. DEPARTMENT OF PLANT PATHOLOGY COLLEGE OF AGRICULTURE, UNIVERSITY OF AGRICULTURAL SCIENCES, DHARWAD JUNE, 2011

2 ADVISORY COMMITTEE DHARWAD JUNE, 2011 (SHAMARAO JAHAGIRDAR) CHAIRMAN Approved by : Chairman: (SHAMARAO JAHAGIRDAR) Members : 1. (M.R. RAVIKUMAR) 2. (A.R. HUNDEKAR) 3. (R.H. PATIL) 4. (S.K. DESHPANDE)

3 CONTENTS Sl. No. Chapter Particulars CERTIFICATE ACKNOWLEDGEMENT LIST OF TABLES LIST OF FIGURES LIST OF PLATES 1. INTRODUCTION 2. REVIEW OF LITERATURE 2.1 Distribution and economic importance 2.2 The pathogen 2.3 Symptomology 2.4 Cultural studies 2.5 Growth phase 2.6 Physiological studies 2.7 In vitro evaluation of chemicals 2.8 Bioagent 2.9 Botanicals 2.10 Management 2.11 Estimation of rhizosphere population 3. MATERIAL AND METHODS 3.1 General laboratory procedure 3.2 Preparation of media 3.3 Survey method and recording of incidence 3.4 Isolation of fungal pathogens 3.5 Maintenance of the cultures 3.6 Mass multiplication 3.7 Proving the pathogenecity 3.7 Morphological studies 3.8 Physiological studies 3.9 Management of wilt complex of soybean 3.10 Management of wilt of soybean under glasshouse conditions 3.11 Estimation of rhizosphere microorganisms 3.12 Screening 3.13 Statistical analysis Contd..

4 Sl. No. 4. EXPERIMENTAL RESULTS 4.1 Survey and distribution 4.2 Symptomotology 4.3 Isolation and identification 4.4 Pathogenecity studies 4.5 Growth phase Chapter Particulars 4.6 Cultural and morphological studies 4.7 Morphological characters 4.8 Physiological studies 4.9 Management of Sclerotium rolfsii Rhizoctonia sp Fusarium sp Management of wilt of soybean in glasshouse condition 4.13 Estimation of rhizosphere population 4.14 Screenings of varieties for root rot/wilt resistance 5. DISCUSSION 5.1 Survey for incidence of wilt disease in northern Karnataka 5.2 Isolation of different pathogens 5.3 Pathogenecity test 5.4 Cultural, nutritional and physiological studies of fungal pathogens 5.5 In vitro evaluation of chemicals, bioagents and botanicals 5.6 Estimation of rhizosphere population 5.7 Management of soybean wilt in glasshouse condition 5.8 Screening for resistance 6. SUMMARY AND CONCLUSIONS REFERENCES

5 LIST OF TABLES Table No. Title 1a. Survey for incidence of root rot/ wilt in soybean growing areas of Northern Karnataka 1b. Incidence of root rot/wilt disease in major soybean growing areas of northern Karnataka during Growth rates of fungi S. rolfsii, Rhizoctonia sp. and Fuarium sp. cultured in different combinations on potato dextrose agar 3. Interaction effect between S. rolfsii, Rhizoctonia sp. and Fuarium sp. on inoculated soybean seedlings 4. Growth phase of S. rolfsii, Rhizoctonia sp. and Fusarium sp. on potato dextrose broth 5. Growth of different isolates of Sclerotium rolfsii on different solid media 6. Growth of different isolates of Rhizoctoni sp.on different solid media 7. Growth of different isolates of Fusarium sp. on different media 8. Morphological characters of Sclerotium rolfsii on different solid media 9. Morphological characters of Rhizoctonia sp. on different solid media 10. Morphological characters of Fusarim sp. 11. Effect of temperature on growth of S. rolfsii 12. Effect of temperature on the germination of sclerotia 13. Effect of temperature on the growth of Rhizoctonia sp. 14. Effect of temperature on the growth of Fusarium sp. 15. In vitro evaluation of contact fungicides against S. rolfsii. 16. In vitro evaluation of systemic fungicides against S. rolfsii 17. In vitro evaluation of different combiproduct fungicides against S. rolfsii 18. In vitro evaluation of botanicals against S. rolfsii 19. In vitro evaluation of bioagents against Sclerotium rolfsii 20. In vitro evaluation of contact fungicides against Rhizoctonia sp. 21. In vitro evaluation of systemic fungicides against Rhizoctonia sp. 22. In vitro evaluation of combiproduct fungicides against Rhizoctonia sp. Contd..

6 Table No. Title 23. In vitro evaluation of botanicals against Rhizoctonia sp. 24. In vitro evaluation of bioagents against Rhizoctonia sp. 25. In vitro evaluation of contact fungicides against Fusarium sp. 26. In vitro evaluation of systemic fungicides against Fusarium sp. 27. In vitro evaluation of combiproduct fungicides against Fusarium sp. 28. In vitro evaluation of botanicals against Fusarium sp. 29. In vitro evaluation of bioagents against Fusarium sp. 30. Management of S. rolfsii with fungicides, botanicals and bioagents under glasshouse condition 31. Management of Rhizoctonia sp. with fungicides, botanicals and bioagents under glasshouse condition 32. Management of Fusarium sp. with fungicides, botanicals and bioagents under glasshouse condition 33. Estimation of rhizosphere population 34a. Reaction of advanced material against root rot / wilt complex 34b. Reaction of rust resistant material against wilt /root rot complex disease

7 LIST OF FIGURES Figure No. Title 1. Taluk wise mean severity of wilt disease in major areas of northern Karnataka during Interaction effect between S. rolfsii, Rhizoconia sp. and Fuarium sp. on inoculated soybean seedlings 3. Growth phase of S. rolfsii, Rhizoctonia sp. and Fusarium sp. on potato dextrose broth 4. Effect of temperature on growth of S. rolfsii 5. Effect of temperature on the germination of sclerotia 6. Effect of temperature on the growth of Rhizoctonia sp. 7. Effect of temperature on the growth of Fusarium sp. 8. Management of S. rolfsii with chemicals, botanicals and bioagents under glasshouse condition 9. Management of Rhizoctonia sp. with chemicals, botanicals and bioagents under glasshouse condition Management of Fusarium sp. with chemicals, botanicals and bioagents under glasshouse condition

8 LIST OF PLATES Plate No. Title 1. Disease symptoms of wilt 2. Different pathogens of wilt 3. Pathogenecity studies 4. Growth characters S. rolfsii, Rhizoctonia sp. and Fusarium sp. on different solid media 5. Effect of temperature on growth of S.rolfsii, Rhizoctonia sp. and Fusarium sp. 6. In vitro evaluation of fungicides and botanicals against S.rolfsii 7. In vitro evaluation of bioagents against S.rolfsii, Rhizoctonia sp. and Fusarium sp. 8. In vitro evaluation of fungicides and botanicals against Rhizoctonia sp. 9. In vitro evaluation of fungicides and botanicals against Fusarium sp. 10. Management of S.rolfsii under glasshouse condition with fungicides, botanicals and bioagents. 11. Management of Rhizoctonia sp. under glasshouse condition with fungicides, botanicals and bioagents 12. Management of Fusarium sp. under glasshouse condition with fungicides, botanicals and bioagents 13. Screening for wilt disease

9 1. INTRODUCTION Soybean Glycine max (L.) Merill is a protein rich oilseed crop. It is considered as a golden bean, miracle bean and wonder crop of the 20 th century because of its characters and usage. Soybean evolved from Glycine ursuriensis, a wild legume native to china, which has been used in china since eleventh century B. C. Though, soybean is a legume crop but it is widely used as oilseed due to its poor cooking ability on account of inherent presence of trypsin inhibitor that limits its usage as pulse crop. Soybean has a great potential as an exceptionally nutritive and very rich protein food. It can supply the much needed protein to human diets, because it contains more than forty per cent protein of superior quality and all the essential amino acids particularly glycine, tryptophan and lysine, similar to cow s milk and animal proteins. Soybean also contains about twenty per cent oil with an important fatty acid, lecithin and Vitamin A and D. The four percent mineral salts of soybeans are fairly rich in phosphorous and calcium. It is an economically, the most important legume in the world which contributed to agriculture economy of United States of America in the last forty years next to maize and wheat. It is also providing base for wide range of food and industrial products (Sinclair and Shurtleff, 1975).Soybean is grown throughout the world in USA, Brazil, China, Russia and India. In a country like India, people largely dependent on vegetable oil in diet and soybean plays an important role in supplementing fats and oil of vegetarian origin. Seeds are used primarily as pulse and green and dried parts are used for cattle feed (Saxena, 1976). In India, it has been grown for centuries in the low hills of Kumaun and Garhwal region of Himalaya. Cultivation of soybean in India was negligible till 1970, but production rapidly increased crossing over 6 million tonnes in At present area under soybean cultivation is 8.9 m.ha. with a production of 9.9 m.t. (Anon., 2010). The major states which cultivate soybean are Madya Pradesh, Bihar, Gujarath, Himachal Pradesh, Maharastra, Karnataka, Rajasthan and Uttar Pradesh. Soybean crop can be attacked by more than pathogens (Sinclair and Shurtleff, 1975). In India, losses due to various diseases are estimated as 12 per cent of total production. The diseases include rust, wilts, leaf spot, rots, powdery mildew, bacterial and viral diseases. Among these, soil borne diseases like root rot or collar rot or charcoal rot caused by Sclerotium rolfsii (Saccardo 1911), Rhizoctonia sp. and Fusarium sp. are gaining more importance as they reduce the plant population in the field resulting in the heavy yield losses. All the three pathogens are soil inhabitants and polyphagous facultative parasites. These are having wide hosts ranging from field crops to horticultural crops like cowpea, chick pea, ground nut, wheat, stevia, tomato, potato, gladiolus, etc. Plant pathogens exhibit variations in their morphological, biological and pathogenic characters. Soybean is being an important pulse crop is receiving wider attention in India as well as in Karnataka. Although wilt complex disease has assumed economic importance in India and Karnataka, so for there is very limited information on these pathogens, etiology, epidemiology and management strategies of wilt complex. Keeping these points in view following objectives were set as follows. 1. Survey for root rot/wilt in northern Karnataka. 2. Isolation, identification and pathogenecity studies. 3. Cultural and morphological studies. 4. In vitro evaluation of chemicals and bioagents. 5. Management of root rots /wilt. 6. To screen available sources of resistant material against root rot / wilt.

10 2. REVIEW OF LITERATURE Soybean crop can be attacked by more than pathogens (Sinclair and Shurtleff, 1975). In India losses due to various diseases are estimated as 12% of total production. The diseases include rust, wilts, leaf spot, rots, and powdery mildew, bacterial and viral diseases. Among these soil borne diseases like root rot or collar rot or charcoal rot caused by Sclerotium rolfsii, Rhizoctonia sp. and Fusarium sp. are gaining more importance as they reduces the plant population in the field resulting in the heavy yield losses. In the following pages an attempt has been made to review the work done on different pathogenic agents associated with root rot/wilt in soybean and other field crops pertaining to survey and distribution, symptomatology, cultural and physiological studies and integrated management of the disease. 2.1 Distribution and economic importance Sclerotium rolfsii Collar rot of soybean is a soil borne disease caused by S. rolfsii Sacc. It was first reported from Madya Pradesh and later on from different soybean growing areas in India (Agarwal and Kotasthane, 1971). Harlapur (1988) observed foot rot disease incidence of 9.85% and 4.66 percent in wheat under rain fed and irrigated condition respectively. Southern blight of soybean was first reported in Nigeria with yield loss of about 59 percent under severe condition (Aken and Dasgiell, 1991). Hanumanthe Gowda (1999) carried out survey on stem rot of ground nut during kharif and rabi in Dharwad, Belgaum and Haveri districts. He reported a maximum incidence of and 8.68 % in rainfed and irrigated fields, respectively Rhizoctonia sp. Muthuswamy and Mariappan (1991) observed that collar rot disease incidence was up to 77 per cent due to this pathogen. Maglekar and Raut (1997) have reported 30 per cent yield loss in soybean due to Rhizoctonia root/stem rot in Vidharbha region of Maharastra. Massod Ali and Shivakumar (2008) observed that dry root is the major constraint in the successful production of chickpea and causes per cent loss Fusarium sp. Heavy crop loss of 30 per cent in Germany and per cent in Russia was estimated due to Fusarium wilt of gladiolus (Bruhn, 1955;Protsenko, 1958; Vlasova and Shltan, 1974). Aneja et al. (1993) conducted a series of surveys throughout Haryana during to identify naturally occurring fungal pathogens of water hyacinth leaves and identified as F. chlamydosporium. 2.2 The pathogen Sclerotium rolfsii Sclerotium rolfsii Sacc. is a well known polyphagous, ubiquitous, omnivorous and most destructive soil borne fungus. This was first reported by Rolfs (1892) as cause of tomato blight in Florida. Later, Saccardo (1911) named the fungus as Sclerotium rolfsii. Higgins (1927) worked in detail on physiology and parasitism of S. rolfsii. Its perfect stage was first studied by Curzi in McClintock (1934) and Butler (1931) reported the stem rust of groundnut in USA and India respectively. Sengupta and Das (1970) studied the cross inoculation of isolates of S. rolfsii from groundnut, wheat, potato, guava and bengalgram on a most susceptible host. Although isolates were most virulent to their appropriate hosts, the specialization was not demonstrated conclusively.

11 Siddaramaiah and Chandrappa (1988) proved the pathogenecity of S. rolfsii on cardamom in pot culture studies by inoculating 25 days old sclerotial culture which was grown on sand corn meal medium and observed the symptoms a week after inoculation Rhizoctonia sp. Rhizoctonia bataticola, a sclerotial fungus is an important pathogen on several crops distributed in many parts of the world. It causes complex disease syndromes like charcoal rot, collar rot, fruit rot, dry rot, stem blight, seedling blight.(reichert and Hellinger, 1947). The pathogen is soil borne causes severe losses mainly due to moisture stress. Moisture stress and nutritional deficiency helps the pathogen for developing symptoms in the host (Arya et al., 2004) Fusarium sp. The genus Fusarium was erected by Link in 1809 for the species with fusiform, nonseptate spores borne on stroma (Booth, 1971).Fusarium oxysporum f.sp. glycines (Schlect) Emend. Snyd and Hans). is a seed borne pathogen found frequently in seed lots(nasir, 2003 and Agarwal, 2006) and found reducing the germination by rotting in severely infected seeds of soybean and it also found causing Fusarium blight or wilt and root rot.(akinsanmil and Adekule, 2003). 2.3 Symptomology Sclerotium rolfsii Kulkarni et al. (1995) reported that, the pathogen affected either stem or root or tubers. The infected stem produced dark brown lesion at collar region causing wilting and ultimately plants dried up. Brownish sclerotia resembling mustard seed developed at later stages on the root and collar regions of the infected plants. Afterwards, tubers get infected and rotten in the field itself. Anahosur (2001) observed the dark brown lesion on the stem just below the soil surface followed by dropping and wilting of infected leaves and gradually wilting of entire plant. Such wilted plants showed whitish mycelial growth with sclerotial bodies resembled mustard seeds on collar region and also roots. The affected tubers showed small sunken, tan coloured spots with brownish margin. The affected tissue was tough and became soft and watery due to secondary rot causing organisms. The white mycelium of pathogen grew rapidly over the tuber surface in fan shaped outline. Basawaraj (2005) reported that, the symptom of wilt of potato caused by S. rolfsii showed characteristic yellowing of leaves, wilting of plants. White to creamy coloured round to spherical sclerotial bodies were produced on infected tubers Rhizoctonia sp. Prashanthi (1994) reported that safflower plant infected by R.bataticola was characterized by gradual yellowing and drying of leaves followed by loss of vigour and premature death. The bark of such plants could be easily peeled off. There was extensive sloughing off and shredding of affected bark. In coleus, the infection started at the collar region of plants as water soaked areas and tissues soon turned into a soft, watery mass. Later spread to the roots of the plant and caused decay, which ultimately toppled and collapsed. These infected plants can be easily pulled off from the soil and exhibited brown discoloration of roots followed by rotting of roots. In addition, the extensive sloughing off of affected bark and shredding of roots was also observed. In advanced stage, the aerial portion of the plants decayed completely. The causal organism was isolated and identified as Rhizoctonia bataticola (Sachidananda, 2005 and Ramprasad, 2005) Fusarium sp. Sunitha (1999) explained the characteristics symptoms of wilt disease of gladiolus caused by F. oxysporum f. sp. gladioli. The symptoms included interveinal leaf tip yellowing which extended down the leaf and whole leaf gradually turned brown and became narrow. As

12 infection advanced the plant suddenly wilted or turned yellow and premature death was observed. The centre of bulb turned black and rotted completely. Corms were depressed, leading to mummified corms. Laura and Allen (2000) reported that Fusarium oxysporum and Fusarium solani both cause seedling blight and root rot of soybean. The pathogen cause a rot of lateral roots, tap root and lower stem. Lesion developing on the tap root range from brown to dark purple or black. These lesins may increases in size leading to the girdling the tap root. The lower part of tap root and lateral root system rotted and destroyed. 2.4 Cultural studies Sclerotium rolfsii Sengupta and Das (1970) reported that, isolates of Sclerotium rolfsii from ground nut, wheat, potato, and bengal gram showed considerable variation in growth rate on potato dextrose agar and broth. Sullamadath et al. (1977) reported that the time taken to reach the maximum growth varied with the isolate. Pigeonpea and tobacco isolates reached maximum growth on the seventh day. Sunflower and ground nut on the ninth day while wheat and potato continued grow up to eleventh day. Manjappa (1979) found variation among the isolates of S. rolfsii isolated from different crop plants (sunflower, ground nut, wheat, redgram, tomato, niger, lucerne and tamarind). All the eight isolates showed marked difference in the rate of growth on both solid and liquid media and time taken for sclerotial initiation. The isolates also showed difference with respect to size, number and weight of sclerotia and virulence of the pathogen. Pritviraj et al. (1996) found two types of sclerotia, small and large. There was significant difference in the surface morphology consisting of thick skin, rind, cortex and medulla. The difference in size of sclerotia appears due to difference in the volume of medullary region. Ansari and Agnihotri (2000) studied the variation exists that among the forty isolates of S. rolfsii collected from different soybean growing areas in India and were categorized into groups on morphological characters of sclerotia like sclerotial arrangement, size and colour on potato dextrose agar medium Rhizoctonia sp. Vasudeva (1937) reported that R.bataticola grew equally on richard s agar, cotton root extract and synthetic agar but the sclerotial formation was favoured by cotton extract and was poor on Brown s agar. Ghosh and Sen (1973) reported that Richard s agar permitted the best growth and sclerotial formation of R.bataticola as compared to PDA, Czapek s Dox agar, Saubouraud s agar and Steinberg s agar and jute extract agar. Waseer et al. (1990) isolated R.bataticola from soybean and reported that the pathogen grew better on potato dextrose broth. Sharma et al. (2004) reported that PDA was the best medium for growth and sclerotial formation. Vanitha et al. (2009) reported that among the six culture media used to understand the cultural behaviour such as growth and sclerotial production, they found PDA was the best medium for growth and sclerotial formation of R.bataticola isolated from soybean Fusarium sp. Wardlaw (1931) described the cultural characters shown by five strains of Fusarium cubense, when grown under uniform conditions on standard media. Important differences were observed in vegetative growth, colour production, formation of chlamydospores, sclerotia and sporulation and stated that two strains were identical. Sharma and Mathur (1971) showed that monoconodial lines of the linseed wilt pathogen F. oxysporum f. sp. lini isolated from different growing regions differed in their cultural and morphological characters with marked diversity in virulence.

13 Desai (1982) reported that F. moniliformae was able to grow very well in Czapek s dox broth while least growth was observed in Elliot s broth. Richard s medium was found to be the best suited medium for the growth of F.udum the causal agent of Fusarium wilt on pigeonpea( Sataraddi et al., 2003). 2.5 Growth phase Sclerotium rolfsii Lingaraju (1977) reported that S. rolfsii reached maximum growth after 10 days of incubation on potato dextrose broth. Ramprasad (2005) reported that S. rolfsii isolated from coleus plants, reached the maximum growth after 10 days of incubation on potato dextrose broth. Ammajamma (2010) reported that S. rolfsii reached maximum growth after 10 days of incubation on potato dextrose broth Rhizoctonia sp. Israel and Ali (1964) reported that Rhizoctonia follows the curve in liquid medium having an initial period of accelerated growth followed a phase of very rapid growth and then decreased in weight. Ramamurthy (1982) reported that R. bataticola reached maximum growth after 11 days of incubation on potato dextrose broth. Ammajamma (2003) reported that R.bataticola isolated from coleus reached the maximum growth after 12 days of incubation on potato dextrose broth Fusarium sp. Sumitra (2006) reported that Fusarium oxysporum f.sp.gladioli isolated from gladiolus, reached maximum growth after 11 days of incubation on potato dextrose broth. Ammajamma (2003) reported that Fusarium chlamydospore isolated from coleus reached the maximum growth after 16 days of incubation on potato dextrose broth. 2.6 Physiological studies Effect of temperature Sclerotium rolfsii Sullamadath et al. (1977) studied variation in requirement of temperature by different isolates and found that all isolates grew well between 23 and 25ºC. The optimum temperature for groundnut isolate was 25ºC and 30º C for tobacco and potato but 35ºC for rest of the isolates. Dalvi and Raut (1986) found that optimum temperature and relative humidity required for S. rolfsii of groundnut culture were 28ºC and 77 per cent respectively. Hari et al. (1988) reported that, 26ºC was optimum temperature for growth of S. rolfsii further they observed maximum growth of S. rolfsii at 30ºC. Basamma (2008) reported that maximum growth of fungus was observed at a temperature of 30ºC and least growth was recorded at 10ºC in ground nut root rot disease Rhizoctonia sp. Jha and Sharma (2005) reported that 30-35º C as optimum temperature for the growth and sclerotial production and observed that decrease in mycelial weight above 40ºC. Bainade et al. (2006) found that temperature of 38ºC favoured the maximum growth and sclerotial formation. Vanitha et al. (2009) observed that Rhizoctonia bataticola isolates were able to grow at temperature ranging from 20 to 40ºC. maximum growth was observed at 35º C and minimum growth at 20º C. Ammajamma (2010) observed that maximum growth of R. bataticola at temperature of 30º C and the least growth was recorded at the temperature of 5º C, in coleus wilt pathogen.

14 Fusarium sp. Neal (1927) reported that Fusarium oxysporum f. sp. vasinfectum grew slowly at temperature below 10º C. The optimum temperature for development was 28 to 30ºC. Sowmya (1993) noticed maximum growth of isolates of Fusarium oxysporum f. sp cubense at 35ºC causing panama disease of banana. Sumitra (2006) found that maximum growth of Fusarium oxysporum f. sp gladioli which isolated from gladiolus crop. At the temperature of 30º C and least growth was recorded at the temperature of 5º C. 2.7 In vitro evaluation of chemicals Sclerotium rolfsii Palaiah (2002) observed sensitivity of various isolates of S. rolfsii to chemicals. Among the chemicals tested, chloropyriphos showed maximum inhibition about per cent followed by pendimethalin and thiram with an inhibition of and per cent, respectively. Prabhu and Patil(2004) reported that among the all fungicides used cent percent inhibition of S. rolfsii observed in carboxin, carbendazim 63%+ mancozeb 12% and propiconozole at 0.05, 0.10 and 0.20 per cent concentration. Tiwari and Ashok (2004) reported that carboxin, propiconozole, hexaconozole and triademefon have completely arrested the growth of Sclerotium and Rhizoctonia. Sheoraj et al. (2003) studied the efficacy of mancozeb, thiram, carboxin, dithane M- 45, sulphur dust, carbendazim, ziram, thiophanate methyl and blue copper at 2500 ppm in controlling S. rolfsii causing collar rot in lentil. Mancozeb, thiram and carboxin recorded cent per cent control of the pathogen. Banyal et al. (2008) noticed that among the all the chemicals tested they found tebuconazole and carboxin have given maximum control Rhizoctonia sp. Ilyas et al. (1975) studied the efficacy of various fungicides and observed that carbendanzim, quintozene and mancozeb reduced R. bataticola population under laboratory conditions. Kulkarni (2000) screened various chemicals against safflower root rot caused by R. bataticola and concluded that carbendanzim and propiconazole were the most effective fungicides. Lokesha (2003) evaluated four systemic and three non systemic fungicides against M. phaseolina. Benomyl significantly inhibited the growth of the fungus at 250 ppm followed by carbendanzim among systemic fungicides. Among non-systemic fungicides, mancozeb and thiram at 500ppm inhibited the fungal growth. Ammajamma (2010) screened systemic and non-systemic fungicides against R. bataticola, among systemic fungicides hexaconozole and metalaxyl have given cent per cent inhibition. Thiram was found as the most effective among the contact fungicides and combiproduct of thiram+carboxin was also found effective Fusarium sp. Dilip (1989) reported that Fusarium oxysporum f.sp.nicotianae causing wilt of tobacco was completely inhibited by carbendanzim at 0 ppm concentration which was followed by ziram and captafol. Sowmya (1993) evaluated fungicides against of Fusarium oxysporum f. sp cubense showed cent per cent inhibition with bavistin, dithane M-45 and emisan at all concentrations. Sumitra (2006) evaluating fungicides against of Fusarium oxysporum f. sp gladioli, among systemic fungicides carbendanzim and propiconozole were found more effective and chlorothalonil was most effective among the contact fungicides.

15 2.8 Bioagent Sclerotium rolfsii Bari et al. (2000) reported antagonistic activity of T. harzianum against foot rot of barley caused by S. rolfsii. T.harzianum significantly reduced the disease incidence and enhanced the seed germination and plant growth. Anahosur (2001) reported that, T.harzianum recorded least wilt incidence (10%) followed by T.viride (14%) and were the best treatments in reducing the disease of potato wilt caused by S. rolfsii. Prabhu and Patil (2004) reported that T. harzianun and T. viride were effective against S. rolfsii in soybean. Sahare et al. (2008) observed that maximum inhibition of S. rolfsii by Trichoderma harzianum and Trichoderma viride Rhizoctonia sp. Indra et al. (2003) studied the fungal antagonists like Trichoderma viride, T. harzianum, T. pseudokoningii, T. longibrachirum, and Gliocladium virens against M. phaseolina, T. harzianum and T. longibrachirum were effective in controlling the M. phaseolina. Kamalakannan et al. (2003) studied the effect of volatile and diffusible compound of two Trichoderma spp (Trichoderma viride, T. harzianum), two Pseudomonas fluorescens (Pf 6 and Pf1) isolates and Bacillus subtilis (BSC 7 and BSC 8) isolates against coleus root rot pathogens like R.solani and M. phaseolina. volatile compounds T. harzianum, and Pseudomonas fluorescens (Pf1) effectively reduced the sclerotial production and inhibited the mycelial growth of M. phaseolina Fusarium sp. Ram et al. (1997) reported that, biocontrol agents, T. harzianum and P. fluorescens when introduced to soil for control of rhizome rot of zinger caused by F. solani and Pythium myridylum inhibited the growth of pathogen. The fungal antagonists T. harzianum inhibited the growth of F. moniliforme similarly, the bacterial antagonists, Bacillus subtilis showed maximum inhibition compared to P. fluorescens in controlling F. moniliforme ( Karunakarna et al., 2003) 2.9 Botanicals Sclerotium rolfsii Mishra and Tewari (1990) reported antifungal activity of leaf extracts of Azadiractha indica and Datura stromium against S. rolfsii. Mycelial growth and sclerotial production were completely inhibited at different concentrations. Seshakiran (2002) tested the role of plant extracts in the management of S. rolfsii in groundnut and reported that among 30 plant extracts evaluated in vitro by cold aqueous method, leaf extract of Agave americana L. exhibited maximum inhibition of mycelial growth and sclerotial formation at 10 percent concentration Rhizoctonia sp. Sindhan et al. (1999) reported the efficacy of leaf extracts of Azadiracta indica, Mentha arvensis, Eucalyptus globules, Ocimum sanctum, Duranta alba, and rhizome extract of Zinger officinalis, bulb extract of Allium cepa and Allium sativum against the growth of R. solani and R. bataticola in vitro at 5, 10 and 20 % concentrations. The results showed that all the plant extracts were inhibitory to R. solani and R. bataticola even at the 5% concentration Fusarium sp. Gupta et al. (1996) reported that the leaf extract of Pongamia pinnata, Calptropis giganto L, Azadiracta indica L, were effective against F.pallidoroseum and F.oxysporum.

16 Ramprasad (2005) evaluated twelve botanicals against F. chlamydosporum. Parthenium hysterophorus was found effective in inhibiting mycelial growth and least inhibition of mycelial growth was obtained in case of Cassia occidentialis Management Sclerotium rolfsii Mukhopadhaya et al. (1992) found that seed treatment with G.virens and 0.1 per cent were very effective in controlling S. rolfsii in lentil, chickpea and ground nut. Singh and Thapliyal (1998) studied the effect of seed treatment with fungicide and bioagents on seed and seedling rots and effectively managed the disease by seed treatment with carboxin and T. harzianum and or G. virens. Mukerjee et al. (2000) noticed integration of captan (0.25 %) and G. virens (0.1%) as the best combination to manage wilt complex of french bean, caused by S. rolfsii. Patibanda et al. (2002) reported the efficiency of T. harzianum alone and in combination with fungicides agaist S. rolfsii causing wilt of ground nut. A synergistic and positive effect on disease control was observed when T. harzianum was applied to soil in integration. Prabhu and Patil (2004) reported that, seed treatment with 1g/kg + T. 6g/kg showed effective managemenmt against S. rolfsii. Khodke and Raut (2010) observed that seed treatment with thiram(3g)+ carbendazim(1g)+ Trichoderma (4g/kg ) has resulted in the least pre emergence mortality of seeds Rhizoctonia sp. Mukerjee et al. (2000) noticed integration of captan (0.25 %) and G. virens (0.1%) as the best combination to manage wilt complex of french bean, caused by S. rolfsii R. solani. Konde et al. (2008) reported that seed treatment with 3g/kg controlled the disease up to per cent in field conditions. Khodke and Raut (2010) observed that seed treatment with thiram (3g)+ carbendazim(1g) + Trichoderma (4g/kg ) has resulted in the least pre emergence mortality of seeds Fusarium sp. In field trials, soil application with methyl isothiocyanate gave excellent control against Fusarium wilt disease of gladiolus(sarbhoy and Agarwal, 1983). In field experiment, Fusarium wilt was controlled by mercuric chloride, benomyl, germison, verfazin, zineb, permidin, and thiram as a corm treatment and brassicol as soil disinfectant (Nagler, 1965; Shumilenko, 1973; Hsieh,1985, Aluerio et al., 1994) 2.11 Estimation of rhizosphere population Ghaffar et al. (1969) reported net increase in the population of fungi, bacteria and actinomycetes in amended unsterilized soil as compared to amended sterilized soil and control in pot experiments. They also reported that, suppression of Macrophomina infection of cotton seedlings were related to increase in population of antagonistic bacteria in the soil amended with alfa-alfa or barley straw. Alexander (1961) has stated that soil amended with plant residues, increase the population and activity of actinomycetes which are antagonists to S. rolfsii Palakshappa (1986) estimated the rhizosphere population of fungi, bacteria and actinomycetes by dilution plate technique in amended soil at different intervals after incorporation. He observed increase in population of fungi, bacteria and actinomycetes per gram in sterile and unsterile soils respectively with in 30 days after incorporation of organic amendments.

17 Bhagawat (1997) studied fruit rot of Sunflower caused by S. rolfsii. Population of fungi, bacteria and actinomycetes increased after incorporation of amendments. Gaikwad and Kapgaat (1990) reported that rhizosphere soil samples taken from healthy sesame plants adjacent to disease one is used for isolating fungi, bacteria and actinomycetes by soil plate technique. They isolated eight fungi and two bacteria from rhizosphere cultures found to be antagonistic to S. rolfsii when assayed individually. Meena et al. (2001) reported significant increase in the population of beneficial fungi, bacteria and actinomycetes in the soils amended with organic amendments than the population in the non amended soils. Neem cake amended soils recorded the maximum Trichoderma spp and P. fluorescens population. The inoculum levels of S. rolfsii were lower in neem cake amended soil Screening for resistant entries against major root rot/wilt causing fungi Chemicals and cultural practice have been considered as potential management practices of plant disease control. But these are associated with certain limitations. So, nowa-days host plant resistance in management of disease is an acceptable proposition. Resistant cultivars are very much essential in management of S. rolfsii. Agarwal and Kotasthane (1971) observed pre as well as post emergence killing of soybean seedlings which were infected by to S. rolfsii and further they also observed in the field that some varieties were attacked severely while other did not. They also attempted screening of 25 soybean varieties. Out of them IC-216, Taichung, EC-32, improved pelican, Monetta, Shelby, PB-1, Palmetto, Hood and Jackson showed resistance against to S. rolfsii. Out of 20 selected groundnut genotypes of interspecific hybrid derivatives, seven genotypes (326, 988, 1019, 1024, 1063, 1267 and 1364) consistently exhibited stable resistance to stem rot. Several lines with low susceptibility to S. rolfsii also possessed resistance to rust and moderate resistance to late leaf spot (Mehan et al., 1991) Uma Singh and Thapliyal (1991) reported pre and post emergence rot in five test soybean cultivars due to soil inoculation with to S. rolfsii at 10g inoculum per kg soil. Maximum pre and post emergence rot was observed in Bragg followed by PK 262 and in PK 327. Hanumananthe Gowda (1999) screened 40 groundnut genotypes against to S. rolfsii and identified AIS-I-9705, Dh-74 and INS-I-9702 as moderately resistance. None of them were found resistant to S. rolfsii.

18 3. MATERIAL AND METHODS 3.1 General laboratory procedure Glassware cleaning Corning and borosil glassware were used for all laboratory experimental studies. Before using, the glassware were kept for 24 hr in cleaning solution containing 60 ml of concentrated sulphuric acid in 0 ml of water and then cleaned using washing powder in tap water and finally rinsed with distilled water Sterilization The glassware were sterilized in an autoclave at 1.1 kg cm pressure for 20 min and then kept in hot air oven at 60º C for one hour. Both solid and liquid media were sterilized at 1.1 kg cm pressure for 20 min. Soil used for pot culture experiments was sterilized in an autoclave at 1.4 kg cm for 2 hr Surface sterilization of plants Plant materials were surface sterilized using one per cent sodium hypochlorite for one minute and then washed in sterilized water twice. 3.2 Preparation of media Name of the media used, their composition and method of preparation are briefly described below. In the laboratory studies, the standard potato dextrose agar (PDA) medium was used for culturing the Sclerotium rolfsii, Fusarium sp. and Rhizoctonia sp. The composition of PDA used is given below: Peeled potato Dextrose Agar agar Distilled water ph : 7 : 200 g : 20 g : 20 g : 0 ml Two hundred gram of peeled potatoes were cut into pieces. These pieces were boiled in water and the extract was collected by filtering through muslin cloth. Each 20 g of dextrose and agar agar were dissolved in potato extract and the final volume was made up to 0 ml by adding distilled water. A known quantity of such media was dispensed into number of conical flasks and plugged with non-absorbent cotton and finally put in sterilization cover. These were sterilized at 1.1 kg cm pressure for 20 min. 3.3 Survey method and recording of incidence Survey on incidence of collar rot of soybean was carried out during kharif 2010 in the major rainfed soybean growing districts of northern Karnataka. Survey was carried out in these areas Athani, Chikkodi, Raibag, Hukkeri, Hathargi, Belgaum, Haveri, Kundagol, Kalgatagi, Gokak and Bailhongal. In each field, in an area of 10 sq.m., the total number of plants presents and number of plants showing wilting symptoms due to collar rot in each sp.ot were counted and recorded. The per cent disease incidence was calculated by using formula. Number of plants affected Percent disease incidence = Total number of plants observed Each sample was taken in polythene bag and labelled. Samples were analysed on the day of collection or after keeping for a few days under refrigerated conditions. Root samples were used for detection of fungi associated with wilted plants.

19 3.4 Isolation of fungal pathogens Soybean plants showing wilting symptoms were collected. The infected root portion was used for isolation. The isolation was made by following standard tissue isolation procedure. The infected sp.ecimens were cut into small bits and washed in running water. These bits were surface sterilized with 1 percent of sodium hypochlorite solution for one minute, were washed thoroughly with sterile distilled water for three times to remove the traces of sodium hypochlorite and then aseptically transferred to petriplates containing the sterilized PDA medium. The plates were incubated at 27±1 ºC three days for Sclerotium and Rhizoctonia and eight days for Fusarium. The fungal growth which arose through the infected tissue was taken by inoculation loop and transferred aseptically to petriplates containing the sterilized PDA medium. The pure culture of the fungus was maintained by further growing the culture and following hyphal tip culture method under aseptic conditions (Rangaswami, 1972). 3.5 Maintenance of the cultures The fungi were sub-cultured on the PDA slants and allowed to grow at 27±1 o C temperature. The cultures so obtained were stored in refrigerator at 5 o C for further studies and were sub cultured every week for further studies. 3.6 Mass multiplication In order to get maximum growth of pathogens, for mass multiplication of inoculums, sand-corn meal medium was used. The sand corn meal medium was prepared in the proportion of 90:10 in order to get maximum inoculum of fungus. Four hundred grams of corn meal sand medium was taken in 0ml flask and watered to 20 per cent of its weight and sterilized at 1.1 kg/cm 2 pressure for 20 minutes. The pure culture of S. rolfsii, Rhizoctonia sp. and Fusarium sp. were inoculated to different flasks under aseptic conditions and incubated at 27±1 o C for 30 days. These flasks were shaken periodically to get uniform growth. The giant culture so obtained was used for further studies. 3.7 Proving the pathogenecity Detached root technique Healthy roots of susceptible cultivar JS 335 were collected and each of roots were punctured with needle. Pathogen discs of five mm were mixed in ml of water and roots were soaked in suspension for twenty minutes and roots were covered with cotton. Afterwards inoculated roots were incubated for about seven days under moist chamber. After seven days root rot index was recorded Pathogenecity studies in pots The soybean seeds were sown in pots filled with sterile soil. Twenty days old seedlings were used for inoculation with one month old giant culture. Control was maintained in which no inoculum was added. Treatments are as follows. 1. S. rolfsii. 2. Rhizoctonia sp. 3. Fusarium sp. 4. S. rolfsii + Rhizoctonia sp. 5. Rhizoctonia sp. + Fusarium sp. 6. S. rolfsii + Fusarium sp. 7. S. rolfsii. + Rhizoctonia sp. + Fusarium sp. Giant culture of S. rolfsii, Rhizoctonia sp. and Fusarium sp. were inoculated separately and in combination in set of pots. The pots were maintained at 25 per cent moisture holding capacity. Observations were made every day on the development of wilt symptoms. When the plants showed wilt symptoms, such plants were carefully up rooted and the pathogens were reisolated by standard tissue isolation method. The pathogens were compared with original culture.

20 3.7 Morphological studies Growth characters of isolates on different solid media The growth characters of different isolates of S.rolfsii, Rhizoctonia sp. and Fusarium sp. were studied on different solid media.viz., Potato dextrose agar. Czapek s -dox agar. Oat meal agar Sabouraud s agar Host extract agar All the media were sterilised at 1.1 kg/cm 2 pressure for 15 minutes. To carry out the study, 20 ml of each of the medium was poured in 90 mm petriplates. Such petriplates were inoculated with 5 mm disc cut from periphery of actively growing seven day old culture of the individual isolate grown on PDA in petriplate and incubated at 27±1 o C. Each treatment was replicated thrice. Observations were taken when the fungus covered complete petriplate in any one media. The colony diameter was recorded by averaging the radial growth of the colony in two directions. The data on radial growth were analyzed statistically. Sclerotium production measured as # Excellent sclerotial production (>60 sclerotia / plate), + + Good sclerotial production (30 to 60 sclerotia / plate) and + Poor sclerotial production (<30 sclerotia / plate Composition of different media 1. Potato dextrose agar (PDA) Peeled potato Dextrose Agar-agar Distilled water ph : 7 : 200 g : 20 g : 20 g : 0 ml (To makeup volume) Two hundred gram of cleaned, washed and peeled potato tuber was chopped into pieces. Later these pieces were boiled in distilled water and the extract was collected by filtering through muslin cloth. Dextrose and agar-agar 20 g of each were dissolved in the potato extract and the volume was made up to 0 ml by adding distilled water. 2. Czapek s dox agar Sodium nitrate (NaNO 3 ) Potassium dihydrogen phosphate (K 2 HPO 4 ) Magnesium sulphate (MgSO 4.7H 2 O) Potassium chloride (KCl) Ferrous sulphate (FeSO 4.7H 2 O) Sucrose (C 12 H 22 O 11 ) Distilled water (to makeup volume) : 3.0 g : 0.01 g ph : 7 : 0.5 g : 0.5 g : 0.5 g : 20.0 g : 0 ml Agar agar was melted in 400 ml distilled water. The other ingredients were dissolved in 400 ml of distilled water. The two solutions were mixed thoroughly and the volume was made up to one 0 ml by adding distilled water.

21 3. Oat meal agar Oat flakes Agar-agar Distilled water (to make up) ph : 7 : 30 g : 20 g :0 ml Oat flakes are boiled in 400 ml of distilled water for 20 min and the extract was filtered through a muslin cloth. Agar agar was melted separately in 400 ml of water. Both the solutions were mixed thoroughly. The volume was made up to 0 ml distilled and sterilized at 1.04 kg cm -2 for 15 min. 4. Sabouraud s agar Dextrose Peptone Agar-agar Distilled ph : 20g : 10 g : 20 g : 0 ml : 7 Agar agar was melted in 400 ml of distilled water. All other ingredients were dissolved in 400 ml of distilled water. The two solutions were mixed mixed thoroughly and the volume was made up to 0 ml by adding distilled water. This was sterilized at 1.04 kg cm -2 for 15 min. 5.Host extract agar Soybean roots Dextrose Agar- agar Distilled water : 0 ml ph : 200 g : 20 g : 20 g : 7 Soybean roots were boiled in 400 ml of water for one hour at º C and then the extract was filtered through muslin cloth and mixed with dextrose. Agar- agar was melted in 400 ml of water. Both the solutions were mixed thoroughly and the volume was made up to 0 ml by adding distilled water. This was sterilized at 1.04 kg cm -2 for 15 min Growth phase The growth study was conducted on potato dextrose broth.twenty ml of the potato dextrose broth was dispensed into each of 150 ml flasks. These flasks were sterilized at 1.1 kg/cm 2 pressure for 15 minutes. Each of the flasks was inoculated aseptically with five mm culture disc obtained from the growing periphery of seven day old culture of the fungus grown on PDA in petriplate. The flasks were incubated at 27±1 o C. Each treatment was replicated thrice. The culture was filtered through Whatman no.42 filter paper which was previously dried at 60 0 C in an electrical oven for three days to obtain constant weight. The mycelia mat on the filter paper was washed thoroughly with distilled water to remove any salts likely to be associated with it. The filter paper along with the fungal mat was then dried in oven at 60 0 C for three days to obtain constant weight and weight of the fungal mat was recorded using analytical balance. The above said procedure of harvesting and assessing the weight of the growth of the fungus was started from second day of incubation and continued with two days interval till 20 th day of incubation since the weight of the fungal growth fall down after attaining the maximum growth on tenth day. Growth is expressed as the dry mycelia weight of the fungus.

22 3.8 Physiological studies Temperature requirement The spores of pathogenic agent were placed in cavity slides containing 2% sucrose solution and three replications were maintained. The different temperature regimes like 15, 20, 25, 30 and 40 ºĊ for 12 hours were used for inoculation. Further, sclerotia germinated per cavity slide were observed and percent germination of sclerotia was calculated. For this experiment potato dextrose broth was used. The different temperatures tried for the growth of the fungi were 15, 20, 25, 30 and 40ºC. For each treatment level, three replications were maintained. The flasks were inoculated at respective temperatures. The dry mycelial weight was recorded and results were analysed statistically. 3.9 Management of wilt complex of soybean In vitro evaluation of chemicals against S. rolfsii, Rhizoctonia sp. and Fusarium sp. The efficacy of four non-systemic fungicides (at the concentration of 0.15, 0.2, 0.25 and 0.3%), four systemic fungicides (at the concentration of 0.05, 0.1 and 0.15 %) and four combiproducts (at the concentration of 0.05, 0.1 and 0.15%) were assayed in vitro against S. rolfsii, Rhizoctonia sp. and Fusarium sp. The fungicides used are given hereunder. Contact fungicides Common name Chemical name Trade name Captan N-trichloromethyl mercapta 4-cyclohexene-1,2- dis carboximide N-trichloromethyl thiotetra hydro othalamide Captaf 50 WP Mancozeb Manganese zinc ethylene bis dithiocarbomate + zinc Indofil M 45 Zineb Zinc ethylene bis dithiocarbomate Dithane Z-78 Systemic fungicides Common name Chemical name Trade name Hexaconazole (RS)-2-(2,4-dichloro phenyl)-1-(14-1, 2, 4- triazole-1 yl) hexane-2-1 Contaf 5%EC Propiconazole 1-(2,4 di chlorophenyl) -4-ropyl-1, 3-dioxolan- 2-methyl) H-1,4-triozole Tilt 25EC Thiophanate methyl Dimethyl 4,4(o-phenlene) bis (3- thioalphanate) Roko 50 WP Carbendazim 2-(methoxy-carbomyl)-benzimidazole Bavistin 50 WP

23 Combiproducts Common name Chemical name Trade name Carbendazim 12% + Mancozeb 63% Methyl benzimidazole carbonate + Manganese zinc ethylene bis dithiocarbomate + zinc Saff 75% WP Zineb 64% + Hexaconazole 4%WP Zinc ethylene bis dithiocarbomate + (RS)-2-(2,4- dichloro phenyl)-1-(14-1,2,4-triazole-1 yl) hexane-2-1 Avthar 68%WP Tricyclozole 18% + Mancozeb 62% Manganese zinc ethylene bis dithiocarbomate + zinc Merger 80%WP Carboxin 37.5% + Thiram 37.5% 3-(3,5-diclorophenyl )-N-(1-methylethyl )-2,4- dioxo-1-lemadazolidine carboximide + Tetramethyl thiuram disulphide Vitavax 75% WP power Cymoxanil 8%+ Mancozeb Manganese zinc ethylene bis dithiocarbomate + zinc Curzate Captan 70% + Hexaconozole 5% N-trichloromethyl mercapta 4-cyclohexene-1,2- dis carboximide N-trichloromethyl thiotetra hydro othalamide + RS)-2-(2,4-dichloro phenyl)-1-(14-1,2,4-triazole-1 yl) hexane-2- Taquat 75 WP Required quantity of individual fungicide was added separately into sterilized molten and cooled potato dextrose agar so as to get the desired concentration of the fungicides. Later, 20 ml of the poisoned medium was poured into sterilized petriplate. Mycelial disc of five mm size from actively growing zone of seven days old culture was cut by a sterile cork borer and one such disc was placed at the centre of each agar plate. Control treatment was maintained without adding any fungicide to the medium. Three replications were maintained for each concentration. Then such plates were incubated at room temperature and radial growth was measured when fungus attained maximum growth in control plates. Per cent inhibition of mycelial growth over control was calculated by using the formula given by Vincent (1947). C T I = C Where, I = Percent inhibition C = Growth in control T = Growth in treatment Media composition King s B medium For maintenance of P. fluorescens fresh culture, the King s B medium was used. The composition of King s B medium was as follows (Sands and Rovira, 1970).

24 Peptone 20 g Agar agar Glycerol K 2 HPO 4 MgSO 4.7H 2 O 1.5 g 12 g 8 ml 1.5 g ph 7.0 Distilled water 0 ml (To makeup volume) All the components excluding the agar were mixed together as dry powder before distilled water was added. After adjusting of ph to 7.0, the agar was added and medium was autoclaved at 1.1 kg/cm 2 pressure for 15 minutes and cooled to 45 0 C and then 10 ml of freshly prepared antibiotic mixture was added per liter of medium. Cooling was essential to maintain the activity of the antibiotics. Nutrient agar (NA) For maintenance of Bacillus subtilis fresh culture, nutrient agar medium was used. The composition of nutrient agar was as follows Glucose (C 6 H 12 O 6 ) Peptone Beef extract Agar Distilled water 5.0 g 5.0 g 3.0 g ph g 0 ml (To makeup volume) All the components should be mixed together as dry powder before distilled water was added. Medium was autoclaved 1.1 kg/cm 2 pressure for 15 minutes In vitro evaluation of bioagents against S. rolfsii, Rhizoctonia sp. and Fusarium sp. The efficacy of six Bioagents was tested against S. rolfsii, Rhizoctonia sp. and Fusarium sp.for radial growth inhibition on the potato dextrose agar media using dual culture technique under in vitro condition. List of Bioagents used against S. rolfsii, Rhizoctonia sp. and Fusarium sp. 1. Trichoderma viride Pers. 2. Trichoderma harzianum Rifai, T. 3. Trichoderma koningii 4. Trichoderma virens Miller. 5. Pseudomonas fluorescens Migula. 6. Bacillus subtilis Cohn. Dual culture test Bioagents were evaluated for their efficacy through dual culture technique. Both bio control agents and test fungus were cultured on potato dextrose agar in order to get fresh and active growth of fungus. The cultures of antagonistic micro organisms used in the present study were obtained from the Project Directorate of Biological Control (PDBC) Bengaluru and IABT Dharwad, Karnataka state. Twenty ml of sterilized and cooled potato dextrose agar was poured into sterile petriplate and allowed to solidify. For evaluation of fungal bio control agents, mycelial disc of test fungus was inoculated at one end of the petriplate and antagonistic fungus was placed opposite to it on the other end. In case of evaluation of bacterial antagonist the bacterium was

25 streaked at the middle of the petriplates and mycelial disc of the test fungus was placed on either side at the centre of each half of the plate. The plates were incubated at 27±1 o C and zone of inhibition was recorded by measuring the clear distance between the margin of the test fungus and antagonistic organism. The colony diameter of the pathogen in control plate was also recorded. The per cent inhibition of the growth of the pathogen was calculated by using the formula given by Vincent (1947) In vitro evaluation of botanicals against S. rolfsii, Rhizoctonia sp. and Fusarium sp. The present investigation was carried out to evaluate the available botanicals. The efficacy of six botanicals viz. Cristol, Crude neem oil, Crude pongamia oil, Achook ( Azadirechtin 1500 ppm), Nimbicidine (Azadirechtin 300 ppm) and Neem seed kernel extract (NSKE) at the concentration of 5, 7.5, and 10 %. The plates were incubated at 27±1 o C and zone of inhibition was recorded by measuring the clear distance between the margin of the test fungus and antagonistic organism. The colony diameter of the pathogen in control plate was also recorded. The per cent inhibition of the growth of the pathogen was calculated by using the formula given by Vincent (1947) Management of wilt of soybean under glasshouse conditions Evaluation of chemicals, botanicals and bioagents against S. rolfsii, Rhizoctonia sp. and Fusarium sp. Pot culture experiment was carried out in the glasshouse to study the effect of various treatments including agrochemicals, bioagents and botanicals. A pot experiment was conducted in the glasshouse of AICRP on soybean, MARS University of Agricultural Sciences, Dharwad during 2011 to find out best treatment for control of wilt of soybean. The soybean seeds of Variety JS-335 were sown in pots. The effective chemicals evaluated in vitro studies were further evaluated in pot culture. Each treatment was replicated thrice. The giant culture was inoculated to each pot at the rate of 4 g per pot. The giant cultures along with treatments listed below were drenched individually to respective sets of pots. Pot without drenching with chemical serves as control. The treatments for S. rolfsii, Rhizoctonia sp. and Fusarium sp. are as follows. S. rolfsii, Rhizoctonia sp. Fusarium sp. Mancozeb 0.3 % Mancozeb 0.25% Mancozeb 0.15 % Carbendazim 0.2% Propiconazole 0.05% Captan 0.25% Propiconazole 0.05% Carbendazim 0.05% Propiconazole 0.05% Hexaconazole 0.05% Hexaconazole 0.05% Carbendazim 0.05% Thiophanate methyl 0.1 % Thiophanate methyl 0.05% Hexaconazole 0.05% Curzate 0.15 % Curzate 0.05% Thiophanate methyl0.05 % Saaf 0.15% Saaf 0.05% Saaf 0.05% Taquat 0.05% Taquat 0.05% Taquat 0.05% Merger 0.2 % Merger 0.2 % Merger 0.2 % Avathar 0.1 % Avathar 0.05 % Vitavax power 0.1% Vitavax power 0.1% Vitavax power 0.05% Achook 5% Achook 5% Achook 5% Nimbicidine 5% Nimbicidine 5% Nimbicidine 5% Neem oil 5% Neem oil 5% Neem oil 5% Trichoderma viride (2 g) Trichoderma viride (2 g) Trichoderma viride (2 g) Trichoderma harzianum (2 g) Trichoderma harzianum (2 g) Trichoderma harzianum (2 g) - Control Control Control Observations were recorded on growth parameters and disease incidence 20 and 45 days after imposing treatment and the percent disease incidence was calculated by following formula.

26 % Disease incidence = Number of plants affected Total number of plants observed 3.11 Estimation of rhizosphere microorganisms Soil sample from different treatments were collected at the time of sowing and 30 days after planting from rhizosphere and population of microorganisms count was done by adopting serial dilution method. After mixing, 10 g of soil from each sample was transferred to aseptically to 250 ml conical flask contending ml sterile water separately and mixed thoroughly by shaking for 10 mins. One ml sample was drawn from suspension and transferred to 9 ml of sterile water blank. Fifteen ml of appropriate media was transferred to petriplates and one ml suspension from each of the treatment was transferred to sterile petriplates containing media sp.read thoroughly to get the uniform growth. The plates were incubated at room temperature. Microbial counts were assessed on fifth day for fungi. The dilution and medium used for experiment Dilution Medium 1. Fungi: 10-4 Martin s rose Bengal agar. Composition of media Martin s rose bengal agar. Peptone Potassium dihydrogen phosphate (KH 2 PO 4 ) Magnesium sulphate (MgSo 4. 7H 2 O) Dextrose Rose bengal Agar-agar Distilled water : 5 g : 1 g : 0.05 g : 10g : g : 15 g : 0 ml ph : Screening A field experiment was conducted during kharif 2010 at sick plot of Ugar khurd Research Station, Nippani. 40 IVT trial material and rust differentials were screened against the wilt disease; each line was sown in a row of four meter in a non replicated trial. The lines used were Bragg, RU 18, MAUS 61, Ps 104, S 1525, H2P5, H2P19, DSb 16, RKS 54, MAUS 1281, MAUSS 1039, NRC 77, SL 688, IS-02-2-Sel-3, MAUS 1440, L 129, JS 9305, JS 335, VisSoya 59, RAU S5, AMS1, NRC 80, MAUS 1259, MAUS 1188, MAUS 1259, SL 799, DS 12-13, PI F, EC , PI , JS 33J EC (7), JS 9305 EC (20), JS 335 EC , JS 335 x EC (7), DSb 1 EC (14), JS 9305 EC (16), JS 335 EC (34), JS 335, PI , PI B, PI , JS Observation was recorded on per cent disease incidence and lines were grouped based on scale given by Shenai et al. (1994).

27 Scale for wilt/ root rot Scale Grade 0 No infection % % % >75% 3.13 Statistical analysis Statistical analysis was carried out as per the procedure given by Panse and Sukhatme (1967). Data in percentage were transformed to arc sine and square root values and analysis was done by using M-Stat C.

28 4.1 Survey and distribution 4. EXPERIMENTAL RESULTS The root trot/ wilt complex has become major production constraint in Karnataka. The various aspects on association of causative agents, their physiological, morphological, cultural aspects in vitro evaluation of chemicals, bioagents and botanicals were taken up. The results on these various aspects are presented in this chapter. Roving survey was conducted during kharif 2010 in major soybean growing areas of northern Karnataka to assess the distribution and incidence of root rot/wilt. The data is presented in the Table 1a, 1b and Fig. 1. The results revealed that per cent disease ranged from 3.36 to per cent in different locations. Irrespective of the location maximum disease incidence was observed in Ugar khurd area (Ugar sugar 36.30%) followed by Badal Ankalagi (22.68%), Ugarkhurd (15.30 %) and Kavalageri (12.19%). In Dharwad maximum disease was recorded at Kavalgiri(12.19 %) followed by Murakatti (10.56%), less disease was observed in Narendra (2.27%).While in Khalghatagi maximum incidence was recorded in Devikoppa (9.64%) followed by Dummawad (4.89%) and Mishrilkoli(3.17%). In Haveri maximum incidence was recorded Devagiri(7.90%) followed by Motebennur (4.60 %) and Haveri(3.90%). In Baihongal taluk in maximum incidence was recorded in Badal Ankalagi (22.687%) followed by Budarakatti (12.87%). Where as in Hukkeri taluk maximum disease incidence was recorded in Yamakanmaradi (5.48 %) followed by Shankeshwar (4.55%). In Chikkodi taluk maximum disease was recorded in Yebaratti (6.30%) followed by Chikkodi (4.78%). In Athani taluk maximum disease was recorded in Ugar Khurd area (Ugar sugar 36.30%) followed by Ugarkhurd (15.30 %). Less incidence of disease was recorded in Kudachi (2.17%). Where as in taluk wise maximum incidence of disease was reported in Athani taluk (19.08%) followed by Baihongal (17.77%) and Dharwad (8.34%) while, less incidence of disease was reported from Raibag (3.36 %). 4.2 Symptomotology First external symptoms noticed was necrosis or yellowing of older leaves followed by discolouration. The infected plants at the early stage showed seedling blight symptom (Plate 1) while the infected plant at the later stage showed general yellowing followed by defoliation of leaves and general wilting(plate 1). When the infected plants uprooted the cross section of the infected roots revealed brownish black discolouration of vascular bundles. 4.3 Isolation and identification The repeated isolation of fungi associated with root rot complex samples collected from different soybean growing areas which yielded three major species of Fusarium, Sclerotium and Rhizoctonia. (Plate 2). Description of all these fungi agreed with description of Fusarium sp. by Booth (1971), Sclerotium rolfsii by Mundkur (1934) and Rhizoctonia bataticola by (Taub) Butler (Pycnidial stage of Macrophomina phaseolina by Ashby (1927). (Plate 2) 4.4 Pathogenecity studies Pathogenecity studies were conducted at two stages in order to ascertain the degree of association of all the three fungi in root rot complex Interaction of the pathogen on culture media. The observations on interaction of three major pathogens are presented in Table 2 and Fig. 2. No significant interaction was observed between the three fungi on media. However, after 4 days of incubation, 30 mm and mm colony diameter was recorded in simultaneous inoculation of S. rolfsii + Rhizoctonia sp. respectively.

29 Table 1a: Survey for incidence of root rot/ wilt in soybean growing areas of Northern Karnataka Taluk Location Cropping situation % wilt/root rot incidence Average Dharwad Kalghatagi Haveri Bailhongal Hukkeri Chikkodi Athani Raibag Gokak Narendra Rainfed 2.27 Murakatti Rainfed Kavalgeri Rainfed Dummawad Rainfed 4.89 Devikoppa Rainfed 9.64 Mishrikoli Rainfed 3.17 Devagiri Rainfed 7.90 Motebennur Rainfed 4.60 Haveri Rainfed 3.90 Budarakatti Rainfed Badal Ankalagi Rainfed Yamakanmaradi Rainfed 5.84 Hebbal Rainfed 2.97 Hathargi Rainfed 3.60 Shankeshwar Rainfed 4.55 Yebaratti Rainfed 6.30 Chikkodi Rainfed 4.78 Manjari Irrigated 5.64 Ugarkhurd Irrigated 15.3 Ugarkhurd (Ugarsugar) Rainfed 36.3 Chinchali Irrigated 4.56 Kudachi Irrigated 2.17 Musguppi Rainfed 3.56 Vaderhatti Rainfed Kundagol Kundgol Rainfed

30 Table 1b: Incidence of root rot/wilt disease in major soybean growing areas of northern Karnataka during Taluk Per cent disease incidence (%) Dharwad 8.34 Kalghatagi 5.90 Haveri 5.46 Bailhongal Hukkeri 4.24 Chikkodi 5.54 Athani Raibag 3.36 Gokak 4.10 Kundagol Per cent disease incidence (PDI) Dharwad Kalghatagi Haveri Bailhongal Hukkeri Chikkodi Athani Raibag Gokak Kundagol Isolates Fig. 1 : Taluk wise mean severity of wilt disease in major areas of northern Karnataka during Fig. 1 : Taluk wise mean severity of wilt disease in major areas of northern Karnataka during

31 A colony growth of mm and mm diameter was recorded when inoculated in combination of Fusarium sp. + Rhizoctonia sp. respectively. While in Fusarium sp. + Rhizoctonia sp. colony growth of mm and mm was recorded respectively and 38.66, 26.33, mm diameter in simultaneous inoculation of Rhizoctonia sp. + S. rolfsii+ Fusarium sp. recorded respectively (Plate 3), when compared to 81.66, 86, mm in S. rolfsii, Rhizoctonia sp and Fusarium sp. alone respectively. While after 6 days of incubation, 36mm and mm colony diameter was recorded in simultaneous inoculation of S. rolfsii + Rhizoctonia sp. respectively. A colony growth of mm and 55.55mm diameter was recorded in when inoculated in combination of Fusarium sp. + Rhizoctonia sp. respectively. While in Fusarium sp. + Rhizoctonia sp. colony growth of 31 mm and mm was recorded respectively and42.33, 30, 18.66mm diameter in simultaneous inoculation of Rhizoctonia sp.+ S. rolfsii+ Fusarium sp. recorded respectively, when compared to 90, 90, mm in S. rolfsii, Rhizoctonia sp. and Fusarium sp. alone respectively. Rhizoctonia sp. overgrew S. rolfsii and Fusarium sp. to some extent Inoculation of pathogenic fungi on soybean seedlings The pathogenecity studies were conducted in glasshouse on susceptible cultivar JS The data on percentage of diseased plants inoculated with the all the pathogens is presented in the Table 3a and Plate 3. After 20 days of inoculation of the giant culture of the different pathogens in isolation and also in combination, revealed that maximum per cent disease incidence in case of dual inoculation of S. rolfsii + Rhizoctonia sp. (83.33%) and mixed inoculation of S. rolfsii + Rhizoctonia sp.+ Fusarium sp. (83.33%) followed by dual inoculation of S. rolfsii + Fusarium sp and Fusarium sp.+ Rhizoctonia sp. (75 %). The pathogens alone also recorded more than 50 per cent disease incidence. Maximum per cent incidence in single inoculation of S. rolfsii and Fusarium sp. with per cent. Least disease incidence was recorded in Rhizoctonia sp. (58.33 per cent). Root length (root rot index) was maximum (5 cm) in treatment S. rolfsii+ Rhizoctonia sp.and dual inoculation of S. rolfsii + Rhizoctonia sp. and triple inoculation of S. rolfsii + Rhizoctonia sp. + Fusarium sp. followed by S. rolfsii+ Fusarium sp. (4.33 cm) and Fusarium sp. (4.08 cm). The root rot index was minimum Rhizoctonia sp. (2.37 cm) Detached root technique The pathogenecity studies were conducted in lab on susceptible cultivar JS-335.The data on percentage of diseased plants inoculated with the all the pathogens is presented in the Table 3b and Plate 3. After 7 days of inoculation of the culture of the different pathogens in isolation and also in combination, revealed that maximum per cent disease incidence in case of dual inoculation of S. rolfsii + Rhizoctonia sp. (%) and mixed inoculation of S. rolfsii + Rhizoctonia sp.+ Fusarium sp. (%) followed by dual inoculation of S. rolfsii + Fusarium sp. (75%), Fusarium sp.+ Rhizoctonia sp. (75 %). The pathogens alone also recorded more than 50 per cent disease incidence. Maximum per cent incidence in single inoculation of Fusarium sp. with 75 per cent. Least disease incidence was recorded in Rhizoctonia sp.and S. rolfsii (50%). Root length (root rot index) was maximum (8.66 cm) in mixed inoculation of S. rolfsii + Rhizoctonia sp.+ Fusarium sp. followed by Fusarium sp. (7.50 cm)root rot index). The root rot index was minimum Rhizoctonia sp. (4.50). 4.5 Growth phase The growth phase studies of all three fungi of root rot complex was studied on PDA broth. The results are presented in the Table 4 and Fig Growth phase of S. rolfsii The maximum dry mycelial weight (287.3 mg) of S. rolfsii was recorded after 10 days of inoculation. The growth of S. rolfsii decreased subsequently from days ( mg). There was no significant difference with respect to dry mycelial weight up to 6 days of inoculation. There was significant difference in dry mycelial weight after ten days of inoculation when compared to all the other incubation periods. As the maximum growth was observed on 10 th day after inoculation, this period was used as maximum growth period for further studies.

32 Table 2: Growth rates of fungi S. rolfsii, Rhizoctonia sp. and Fuarium sp. cultured in different combinations on potato dextrose agar Colony diameter (mm) Incubation period S. rolfsii + Rhizoctonia sp. Rhizoctonia sp. + Fuarium sp. S. rolfsii + Fuarium sp S. rolfsii + Rhizoctonia sp. + Fuarium sp S. rolfsii Rhizoctonia sp. Fuarium sp. 4 days days

33 Table 3a: Interaction effect between S. rolfsii, Rhizoctonia sp. and Fusarium sp. on inoculated soybean seedlings Treatment Per cent disease incidence Root length rotted (cm) S. rolfsii Fusarium sp Rhizoctonia sp S. rolfsii + Fusarium sp Fusarium sp.+ Rhizoctonia sp S. rolfsii + Rhizoctonia sp S. rolfsii + Fuarium sp. + Rhizoctonia sp Control Table 3b: Interaction effect between S. rolfsii, Rhizoctonia sp. and Fusarium sp. on inoculated soybean roots Treatment Per cent disease incidence Root length rotted (cm) S. rolfsii Fusarium sp Rhizoctonia sp S. rolfsii + Fusarium sp Fusarium sp.+ Rhizoctonia sp S. rolfsii + Rhizoctonia sp S. rolfsii + Fuarium sp. + Rhizoctonia sp Control 0 0

34 Field affected by wilt disease in Baihongal Field affected by wilt disease in Ugar Khurd Soybean plant affected by Sclerotium rolfsii Soybean plant showing collar rot symptom Plate 1. Disease symptoms of wilt

35 Sclerotium rolfsii Rhizoctonia sp. Fusarium sp. Palte 2: Different pathogens of wilt disease

36 Interaction of three different fungi Mycelial interaction of Sclerotium, Rhizoctonia and Fusarium Pathogenicity studies Plate 3. Pathogenicity studies

37 90 Per cent disease incidence Root length rotted (cm) Per cent disease incidence Root length rotted (cm) T1 - S. rolfsii T2 - Fusarium sp. T3 - Rhizoctonia sp. T1 + T2 T2 + T3 T1 + T3 T1 + T2 + T3 Control 0 Treatments Fig. 2: Interaction effect between S. rolfsii, Rhizoconia sp.and Fuarium sp. on inoculated soybean seedlings Fig. 2: Interaction effect between S. rolfsii, Rhizoconia sp.and Fuarium sp. on inoculated soybean seedlings

38 Table 4: Growth phase of S. rolfsii, Rhizoctonia sp. and Fusarium sp. on potato dextrose broth Days after inoculation Mean dry mycelial weight (mg) S. rolfsii Rhizoctonia sp. Fusarium sp SEm± C.D. (0.01)

39 350 S. rolfsii Rhizoctonia sp. Fusarium sp. 300 Mean dry mycelial weight (mg) Days after inoculation Fig. 3: Growth phase of S. rolfsii, Rhizoctonia sp. and Fusarium sp. on potato dextrose broth Fig. 3: Growth phase of S. rolfsii, Rhizoctonia sp. and Fusarium sp. on potato dextrose broth

40 4.5.2 Growth phase of Rhizoctonia sp. In case of Rhizoctonia sp. the maximum dry mycelial weight of (233 mg) was recorded after 12 days of inoculation followed by (219 mg) after 14 days. The maximum dry mycelial weight differed significantly with rest of days of inoculation. After 14 days, dry mycelial weight reduced from days ( mg). As the maximum growth was observed on 12 th day after inoculation, this period was used as maximum growth period for further studies Growth phase of Fusarium sp. After two days of inoculation,the maximum growth of mg and gradually increased up to 6 th day of inoculation. After 6 th day there was significant increase in growth and attained maximum dry mycelial weight (206 mg) after 16 days of inoculation. There was no significant difference in dry mycelial weight between 16 and 18 days after inoculation. As the maximum growth was observed on 16 th day after inoculation, this period was used as maximum growth period for further studies. 4.6 Cultural and morphological studies Five different media were screened for the study of growth characters of different isolates of S. rolfsii, Rhizoctonia sp. and Fusarium sp. (Plate 4) and data are presented in the Table 5, 6 and Growth characters of S. rolfsii on different solid media Among the different isolates mean maximum growth of (89.60 mm) was recorded SrUKD followed by SrKGI (87.98 mm), however the mean minimum growth (77.26 mm) was recorded in SrHTG. Among the different media screened irrespective of the isolates the maximum colony diameter (87.36 mm) was recorded in Czapek s agar medium followed by (85.18 mm) in PDA. The mean minimum (81.98 mm) colony diameter was recorded in Sabouraud s agar. The colony growth was recorded in SrHTG ranging from mm while maximum colony growth ranging from mm recorded in SrGKK isolate. For SrUKD isolate the maximum colony diameter (90 mm) was recorded in Oat meal agar and Host extract agar followed by mm in Czapek s agar. While, SrGKK isolate all the media except the Sabouraud s agar supported maximum growth (88.60 mm.) Czapek s agar (89.60 mm) followed by PDA (87.50 mm). In Sr KGI, the maximum colony diameter of (90 mm) was recorded in Sabouraud s agar followed by (89.60 mm) Czapek s agar. The SrHTG recorded maximum colony diameter (81.60mm) in Host extract agar followed by (79.00 mm) in Czapek s agar Growth of different isolates of Rhizoctonia sp. on different solid media Among the different isolates of Rhizoctonia sp. mean maximum growth of (89.80 mm) was recorded in RbRBG isolate followed by mm in RbHVR and mm in RbCKI. However, mean minimum growth (86.26 mm) was recorded in RbUKD. Among the different media screened, irrespective of the isolates the maximum colony growth (90.00 mm) was recorded in Host extract and Sabouraud s agar followed by mm in Czapek s agar. The mean minimum colony diameter (87.07 mm) was recorded in PDA. For RbUKD isolate maximum colony diameter (90.00 mm) was recorded in Host extract agar, Sabouraud s agar and Czapek s agar followed by mm in Oat meal agar. Where as in RbCKI except Czapek s agar (80.00mm) all other media supported maximum (90.00mm) growth. For RHVR isolate except Oat meal agar (87.60 mm) all other media supported the maximum (90.00 mm) growth. While in RbRBG isolate except Oat meal agar (89.00mm) all other media supported the maximum (90.00 mm) growth Growth of different isolates of Fusarium sp.on different media Among the different isolates, the mean maximum growth (71.14 mm) was recorded in FsUKD followed by 66.50mm in FsKDL and minimum colony diameter (64.78 mm) was recorded FsBGL. Irrespective of the isolates mean maximum growth (90 mm) was recorded in PDA followed by mm in Czapek s agar.

41 Table 5: Growth of different isolates of Sclerotium rolfsii on different solid media Growth in colony diameter (mm) Isolate Potato dextrose agar Oat meal agar Host extract agar Czapek s agar Saboraud s agar Mean SrUKD SrGKK SrDWR SrKGI SrHTG Mean A (Isolate) B (Media) A B SEm± C.D. (0.01) (UKD - Ugarkhurd, GKK - Gokak, DWR - Dharwad, KGI - Khalgatgi, HTG - Hathargi)

42 Plate 4: Growth characters of Sclerotium, Rhizoctonia and Fusarium on different solid media

43 Table 6: Growth of different isolates of Rhizoctoni sp.on different solid media Growth in colony diameter (mm) Isolate Potato dextrose agar Oat meal agar Host extract agar Czapek s agar Saboraud s agar Mean RbUKD RbCKI RbHVR RbRBG Mean A (Isolate) B (Media) A B SEm± C.D. (0.01) (UKD - Ugarkhurd, CKI - Chikkodi, HVR - Haveri, RBG - Raibag)

44 Table 7: Growth of different isolates of Fusarium sp. on different media Growth in colony diameter (mm) Isolate Potato dextrose agar Oat meal agar Host extract agar Czapek s agar Saboraud s agar Mean FsKDL FsBGL FsUKD Mean A (Isolate) B (Media) A B SEm± C.D. (0.01) KDL Kundagol, BGL Bailhongal, UKD - Ugarkhurd

45 The mean minimum growth (50.26 mm) was recorded in Sabouraud s agar. For FsKDL maximum colony growth (90.00mm) was recorded in PDA and minimum was recorded in Host extract agar and Sabouraud s agar (54.00 mm). While for FsBGL maximum growth (90.00mm) mm) was recorded in PDA followed by mm in Host extract agar and minimum colony growth (47.33 mm) was recorded in Czapek s agar. While in FsUKD maximum growth (90.00 mm) was recorded in PDA followed by mm in Czapek s agar and minimum colony growth (50.20 mm) was recorded in Sabouraud s agar. 4.7 Morphological characters The most virulent isolate of Ugarkhurd was selected for further studies such as morphological, in vitro evaluation of chemicals. Various morphological parameters were studied for Sclerotium, Rhizoctonia and Fusarium including mycelial colour, sclerotial shape, spore formation etc Morphological characters of Sclerotium rolfsii The data on morphological characters are presented I the Table 8, Plate 4. In case of S. rolfsii PDA supported good growth of colony, raised colony with smooth margin and helped in initiation of sclerotial bodies after 7 days of incubation. While in Oat meal agar and Sabouraud s agar helped in initiation of sclerotial bodies after 9 days of incubation. Colony character in Oat meal agar was characterized by smooth margin, flat colony while in Sabouraud s agar light colony, good mycelial growth was observed and the sclerotial bodies were observed in group. In case of Host extract agar there was good growth of colony, smooth margin, cottony mycelia, helping the sclerotial production on 11 th day of incubation. In Czapek s agar colony was characterised by wavy margin, flat colony sclerotial production at 11 th day of incubation. The extent of sclerotial body production were more than 60 per plate in case of PDA and Sabouraud s agar while there was good production of sclerotial bodies in Host extract agar and Czapek s agar(30-60 sclerotia per plate). However, there was poor sclerotial production (less than 30 per plate) was observed in Oat meal agar. The colour of sclerotial bodies varied from light brown to dark brown. Light brown in Host extract agar, dark brown in Sabouraud s and Czapek s agar, brown in PDA and Oat meal agar. Shape of sclerotial body was spherical except for the Czapek s in which sclerotial bodies were sub spherical Morphological characters of Rhizoctonia sp. The data on morphological characters are presented I the Table 9, Plate 4. In Oat meal agar, good growth of Rhizoctonia sp. with sparse mycelium. However no micro sclerotial production was observed. Host extract agar supported good growth of mycelium and formation of micro sclerotia on 6 th day after incubation. In Sabouraud s agar sparse mycelium growth was observed with production of sclerotial bodies on 6 th day of incubation. The growth of Rhizoctonia sp. was good with dense mycelium, smooth margin, raised colonies in PDA. Sclerotial formation was observed on 6 th day of incubation. In case of Czapek s agar good dense growth of mycelia with raised colonies was observed. Sclerotial formation was observed on 6 th day of incubation. There was excellent production of sclerotial bodies in PDA and Czapek s agar while good in Sabouraud s and Host extract agar and there was no sclerotial production in Oat meal agar Morphological characters of Fusarim sp. The data on morphological characters are presented I the Table 10, Plate 4. In Czapek s agar, good mycelial growth was observed with smooth margin having a dirty white colour and showed a good sporulation. Where as in PDA, good dense growth of mycelia was observed with smooth margin having pink cottony growth and sporulation was excellent. In case of Host extract agar, moderate growth of the mycelium was observed with smooth margin having a light pink centre with white mycelium and sporulation was good while in Oat meal agar moderate growth of mycelium was observed with smooth margin having a cream colour colony and sporulation was poor.

46 Table 8: Morphological characters of Sclerotium rolfsii on different solid media Media Colony character * Sclerotial production # Colour Shape Potato dextrose agar Good growth rate, smooth margin, raised colony, sclerotial initiation on 7 th day Brown Spherical Oat meal agar Good growth, smooth margin, flat colony, sclerotial initiation on 9 th day + Brown Spherical Saboraud s agar Good growth, light cottony mycelia, sclerotial initiation on 9 th day. Sclerotial bodies in group Dark brown Spherical Host extract agar Good growth, smooth margin cottony mycelia, sclerotial initiation on 11 th day. + + Light brown Spherical Czapek s agar Good growth, wavy margin, flat colony, sclerotial initiation on 11 th day. + + Dark brown Subspherical *Good growth: mean colony diameter >70 mm Moderate growth : mean colony diameter 50 to 70 mm Slow growth: <50 mm # Excellent sclerotial production (>60 sclerotia / plate) + + Good sclerotial production (30 to 60 sclerotia / plate) + Poor sclerotial production (<30 sclerotia / plate

47 Table 9: Morphological characters of Rhizoctonia sp. on different solid media Media Colony character Sclerotial production Oat meal agar Good growth, sparse mycelium, no micro sclerotial production - Host extract agar Good growth, dense mycelium, sclerotial formation on 6 th day. + + Saboraud s agar Good growth, sparse mycelium, sclerotial formation on 6 th day. + + Potato dextrose agar Good growth, dense mycelium, smooth margin, raised colony, sclerotial formation on 5 th day Czapek s agar Good growth, dense mycelium, raised colony, sclerotial formation on 6 th day *Good growth: mean colony diameter >70 mm Moderate growth : mean colony diameter 50 to 70 mm Slow growth: <50 mm # Excellent sclerotial production (>60 sclerotia / plate) + + Good sclerotial production (30 to 60 sclerotia / plate) + Poor sclerotial production (<30 sclerotia / plate) - No sclerotial production

48 4.8 Physiological studies The temperature requirement on spore germination and mycelial dry weight for Sclerotium, Rhizoctonia and Fusarium. The data on temperature requirement are presented in Table 11 to 14, Fig. 4, 5, 6 and Plate Effect of temperature on growth of S. rolfsii The maximum dry mycelial weight (347.6 mg) was recorded in case of S. rolfsii SrKGI isolate followed by SrUKD isolate at 30 ºC. At 15 ºC the dry mycelial weight of different isolates ranged from 90 to 176.3, while at the 20º C the dry mycelial weight ranged from to 230 mg. At 40ºC temperature there was no growth of pathogen. Irrespective of temperature requirement the dry mycelial weight was maximum ( mg) in SrKGI isolate followed by mg in SrUKD isolate Effect of temperature on germination of sclerotial body At 15º, 25º and 30ºC the germination of sclerotial bodies was per cent where as in 20º C 75 per cent of sclerotial germination was observed. Maximum mycelial growth (2.25 mm) was recorded at 30º C followed by 25ºC (1.62 mm) and 20ºC (0.75 mm).no sclerotial germination at 40º C Effect of temperature on growth of Rhizoctonia sp. The maximum dry mycelial weight ( mg) was recorded in case of Rhizoctonia sp. at 30ºC in RbHVR isolate followed by RbCKI ( mg). While at 15 ºC the maximum dry mycelial weight was in RbHVR (106.6 mg) followed by RbRBG (103.3 mg) and least dry mycelial weight (71.33 mg) was recorded in RbUKD. While at 20º C dry mycelial weight ranged from mg to mg. At 25º C, maximum dry mycelial weight ( mg) was recorded in RbHVR isolate followed by RbUKD ( mg). Where as at 40º C, there was no growth of pathogen. Irrespective of the temperature requirement maximum growth ( mg) was recorded in RbHVR followed by RbCKI ( mg). Among the temperature regime 30º C was found to be more congenial temperature for the growth of fungus with mg of dry mycelial weight Effect of temperature on germination of spores The maximum per cent (75.15) germination of spore was recorded at 30ºC followed by 29.7 per cent at 25ºC and per cent at 20ºC while at the 40ºC there was no germination of spores. Among the different temperature regime 30ºC was found to be more congenial temperature for the growth of pathogen. 4.9 Management of Sclerotium rolfsii In vitro evaluation of contact fungicides against S. rolfsii. The data on bioassay of three contact fungicides at the concentration ranging from 0.15 to 0.3 per cent are presented in the Table 15 and Plate 6. The maximum per cent inhibition of mycelial growth per cent was observed in Mancozeb followed by per cent in case of Captan at 0.15 per cent concentration. While at 0.2 per cent concentration, maximum percent inhibition of mycelial growth (72.90 %) was observed in Mancozeb followed by per cent in Captan and least mycelial inhibition was observed in Zineb (37.90%). At 0.25 and 0.3 concentration there was marginal increase in the per cent inhibition in mycelial growth in case of Captan and Mancozeb. While there was significant increase in per cent inhibition of mycelial growth in Zineb at 0.3 concentration, the maximum per cent inhibition was observed (81.80 per cent) in case of Mancozeb followed by per cent in case of Zineb which was statistically on par with the Captan (68.88 %). Irrespective of the concentrations maximum (75.50%) per cent inhibition of mycelial growth was observed in Mancozeb, which differed significantly when compared to Captan (65.55 per cent), Zineb (53.50 %).

49 Table 10: Morphological characters of Fusarim sp. Media Colony character Sporulation Czapek s dox agar Good growth, dirty white mycelium, smooth margin + + Potato dextrose agar Good growth, Light pink cottony growth, smooth margin, dense mycelium Host extract agar Moderate growth, uniform mycelium, smooth margin, light pink centre with white mycelium. + + Oat meal agar Moderate growth, smooth margin, cream colour colony + Saboraud s agar Moderate growth, light pink colony, indistinct margin. + + *Good growth: mean colony diameter >70 mm Moderate growth: mean colony diameter 50 to 70 mm Slow growth: <50 mm # Excellent spores production (>60 spores/microscopic field) + + Good spores production (30 to 60 spores/microscopic field) + Poor spores production (<30 spores/microscopic field)

50 Table 11: Effect of temperature on growth of S. rolfsii Dry mycelial weight (mg) Isolate 15º C 20 ºC 25º C 30ºC 40ºC Mean SrUKD SrGKK SrDWR SrKGI SrKTG Mean A (Isolate) B (Media) A x B SEm± C.D. (0.01) (UKD - Ugarkhurd, GKK - Gokak, DWR - Dharwad, KGI - Khalgatgi, HTG - Hathargi) Table 12: Effect of temperature on the germination of sclerotia Temperature % germination of sclerotial bodies Mycelial growth on cavity slide (mm) 15º C º C º C º C º 0 0

51 Plate 5: Effect of temperature on growth of S.rolfsii, Rhizoctonia sp. and Fusarium sp.

52 350 15º C 20 ºC 25º C 30ºC 40ºC 300 Mean dry mycelial weight (mg) SrBGM SrGKK SrDWR SrKGI SrKTG Isolates Fig. 4: Effect of temperature on growth of S. rolfsii Fig. 4: Effect of temperature on growth of S. rolfsii

53 120 % germination of sclerotial bodies Mycelial growth on cavity slide (mm) % germination of sclerotial bodies Mycelial growth on cavity slide (mm) º C 20º C 25º C 30º C 40º Temperature Fig. 5: Effect of temperature on the germination of sclerotia 0 Fig. 5: Effect of temperature on the germination of sclerotia

54 Table 13: Effect of temperature on the growth of Rhizoctonia sp. Dry mycelial weight (mg) Isolate 15º C 20 ºC 25º C 30ºC 40ºC Mean RbUKD RbCKI RbHVR RbRBG Mean A (Isolate) B (Media) A x B SEm± C.D. (0.01) (UKD - Ugarkhurd, CKI - Chikkodi, HVR - Haveri, RBG - Raibag) Table 14: Effect of temperature on the growth of Fusarium sp. Temperature No of spore germinated/ per microscopic field No of spore ungerminated/ per microscopic field % germination of spores 15ºC ºC ºC ºC ºC

55 250 15º C 20 ºC 25º C 30ºC 40ºC 200 Dry mycelial weight (mg) RbATI RbCKI RbHVR RbRBG Isolates Fig. 6: Effect of temperature on the growth of Rhizoctonia sp. Fig. 6: Effect of temperature on the growth of Rhizoctonia sp.

56 90 No of spore germinated/ per microscopic field 80 No of spore ungerminated/ per microscopic field No of spore germinated/ per microscopic field and No of spore ungerminated/ per microscopic field % germination of spores % germination of spores 0 15ºC 20 ºC 25 ºC 30 ºC 40 ºC 0 Temperature Fig. 7: Effect of temperature on the growth of Fusarium sp. Fig. 7: Effect of temperature on the growth of Fusarium sp.

57 4.9.2 In vitro evaluation of systemic fungicides against S. rolfsii The data on bioassay of four systematic fungicides at the concentration ranging from 0.05 to 0.2 per cent are presented in the Table 16 and Plate 6. Among the treatments, cent per cent inhibition of mycelial growth was observed in Hexaconazole and Propiconazole in all the concentration. At 0.5 per cent concentration maximum inhibition (59.86 %) was observed in Thiophanate methyl followed by per cent in Carbendazim. While at 0.1 per cent concentration maximum inhibition of mycelial growth was observed in in Thiophanate methyl which is significantly superior over the per cent inhibition in case of Carbendazim. There was marginal increase in the mycelial growth at 0.2 per cent in Thiophanate methyl (85.40 %) which is statistically superior over the per cent in Carbendazim. Irrespective of concentration maximum per cent of inhibition of mycelial growth (75.22 %) was observed in Thiophanate methyl and least inhibition of mycelial growth was observed in Carbendazim (56.20%) In vitro evaluation of different combiproduct fungicides against S. rolfsii Bioassay of six combiproduct fungicide (Plate 6) ranging from 0.05 to 0.2 per cent concentrations are presented in the Table 17. Among the different fungicides maximum inhibition of mycelial growth was observed in Captan + Hexaconazole, Carboxin + Thiram and Zineb + Hexaconazole in all the concentrations. In the remaining fungicides, the maximum per cent of inhibition of mycelial growth was observed in Tricyclozole + Mancozeb (58.11%) followed by per cent in Carbendazim + Mancozeb and per cent in Cymoxanil +Mancozeb at 0.05 per cent concentration. While at 0.1 per cent, the maximum per cent of inhibition was observed in Tricyclozole + Mancozeb (66.20 %) followed by Cymoxanil + Mancozeb (64.70 %) and least inhibition was observed in Carbendazim + Mancozeb (60.50%). Where as at 0.2 per cent, there was marginal increase in the inhibition of mycelial growth. The maximum per cent of mycelial inhibition (85.10%) was observed in Tricyclozole + Mancozeb followed by Carbendazim + Mancozeb (74 %) and per cent in Cymoxanil +Mancozeb. Irrespective of the concentrations Tricyclozole + Mancozeb has given maximum per cent inhibition (64.8 %) which is statistically superior over the in Cymoxanil + Mancozeb and per cent in Carbendazim +Mancozeb In vitro evaluation of botanicals against S. rolfsii. The data on bioassay of six botanicals (Plate 6) at the concentration ranging from 5 to 10 per cent are presented in the Table 18. The cent per cent inhibition of mycelial growth was observed in Azadirachtin 1500 ppm and Neem oil irrespective of concentration of the botanicals. Where as in other botanicals maximum per cent inhibition of mycelial growth (80.70 %) was observed in Nimbicidine followed by per cent in Cristol and least inhibition of mycelial growth was recorded in Pongamia oil (22.5 %) at 5 per cent concentration. While at 7.5 per cent concentration, maximum inhibition of mycelial growth was recorded in the Nimbicidine (89.60%) followed by Cristal per cent and least inhibition was observed in Pongamia oil (30.7 %). There was increase in the per cent inhibition at 10 per cent as recorded in Nimbicidine (92.50%) followed by per cent in Cristol and least inhibition was recorded in Pongamia oil (45.4 %).While NSKE has shown no inhibition in all the concentrations. Irrespective of the concentrations Azadirachtin 1500 ppm and Neem oil have recorded the cent per cent inhibition of mycelial growth followed by per cent in Nimbicidine and per cent in Cristol. Least inhibition of mycelial growth was recorded in Pongamia oil (32.80%) In vitro evaluation of bioagents against Sclerotium rolfsii The competitive ability of antagonists against Sclerotium rolfsii was studied by dual culture method (Plate 7). The per cent inhibition of mycelial growth was worked out. The data is presented in the Table 19. The results revealed that fungal bioagents retarded the growth of Sclerotium rolfsii significantly. The highest per cent inhibition of mycelial growth was observed in T.viride (55.80%) followed by T.harzianum (55.70%) but they did not differ significantly.

58 Table 15: In vitro evaluation of contact fungicides against S. rolfsii. Percent inhibition of mycelial growth Fungicides Concentration (%) Mean Captan (53.35)* (52.47) (54.45) (56.05) (54.08) Mancozeb (57.27) (58.61) (61.94) (64.82) (60.66) Zineb (42.59) (46.66) (51.59) (56.30) (49.29) A (Fungicide) B (Conc.) A x B (F x C) SEm± C.D. (0.01) * Figures in the parenthesis are angular transformed values Table 16: In vitro evaluation of systemic fungicides against S. rolfsii Percent inhibition of mycelial growth Fungicide Concentration (%) Mean Carbendazim (37.52)* (51.19) (56.99) (48.57) Hexaconozole (89.96) (89.96) (89.96) (89.96) Propiconozole (89.96) (89.96) (89.96) (89.96) Thiophanate methyl (50.55) (63.84) (67.51) (60.63) A (Fungicide) B (Conc.) A B (F C) SEm± C.D. (0.01) * Figures in the parenthesis are angular transformations

59 Table 17: In vitro evaluation of different combiproduct fungicides against S. rolfsii Percent inhibition of mycelial growth Fungicide Concentration (%) Mean Captan + Hexaconazole (89.96)* (89.96) (89.96) () Carbendazim + Mancozeb (48.24) (51.02) (59.42) (63.40) Carboxin + Thiram (89.96) (89.96) (89.96) () Cymoxanil + Mancozeb (48.0) (53.32) (56.82) (63.50) Tricyclozole + Mancozeb (49.75) (54.41) (67.32) (64.80) Zineb + Hexaconazole (89.96) (89.96) (89.96) () A (Fungicide) B (Conc.) A B (F C) SEm± C.D. (0.01) * Figures in the parenthesis are angular transformations

60 Plate 6: In vitro evaluation of fungicides and botanicals against Sclerotium rolfsii

61 Sclerotium rolfsii Rhizoctonia sp. Fusarium sp. Plate 7: In vitro evaluation of bioagents against S.rolfsii, Rhizoctonia sp. and Fusarium sp. (Bioagents : 1-T.virens, 2-T. harzianum, 3-T. koningii, 4-T. viride, 5-B. subtilis, 6-P. Fluorescens)

62 Table 18: In vitro evaluation of botanicals against S. rolfsii Botanicals Azadirachtin 1500 ppm Cristol Neem oil Nimbicidine NSKE Pongamia oil Percent inhibition of mycelial growth Concentration (%) Mean (10)* (8.46) (10) (9.03) 0.00 (1) (4.84) A (Fungicide) (10) (8.59) (10) (9.51) 0.00 (1) (5.62) B (Conc.) (10) (8.66) (10) (9.67) 0.00 (1) (6.80) A B (F C) SEm± C.D. (0.01) *Values in the parenthesis are x + 1 transformations (10) (8.57) (10) 87.6 (9.41) 0.00 (1) (5.75) Table 19: In vitro evaluation of bioagents against Sclerotium rolfsii Bioagent Per cent inhibition of mycelial growth Trichoderma harzianum (7.53)* Trichoderma viride (7.54) Trchoderma virens (7.11) Trichoderma koningii (5.22) Bacillus subtilis 0.00 (1) Psedomonas flourescens 0.00 (1) SEm± 0.57 C.D. (0.01) 2.34 * Figures in the parenthesis are x + 1transformations

63 The next treatment was T.virens which inhibited the mycelial growth upto per cent. Least inhibition of mycelial growth was recorded in Trichoderma koningii (43.00%).In bacterial bioagents, Bacillus subtilis and Pseudomonas fluorescens there was no inhibition of pathogen Rhizoctonia sp In vitro evaluation of contact fungicides against Rhizoctonia sp. The data on bioassay of three contact fungicides at the concentration ranging from 0.15 to 0.3 per cent are presented in the Table 20. Three contact fungicides are assessed against Rhizoctonia sp. (Plate 8). The maximum per cent inhibition of mycelial growth (68.80%) in Captan followed by per cent in Mancozeb and least inhibition of mycelial growth (13.60 %) was recorded at 0.15 per cent concentration. While at 0.2 per cent concentration, the maximum inhibition of mycelial growth (77.70 %) was recorded in Captan followed by per cent in Mancozeb and least inhibition of mycelial growth (27.33 %) was observed in Zineb. There was marginal increase in the inhibition of mycelial growth at 0.25 and 0.3 per cent inhibition. At 0.25 per cent concentration, maximum inhibition of mycelial growth (81.00 %) was observed in Mancozeb followed by per cent in Captan and per cent in Zineb. Where as at 0.3 per cent concentration maximum inhibition of mycelial growth (92.50 %) was observed in Mancozeb followed by per cent in Captan. Least inhibition of mycelial growth (51.00 %) was observed in Zineb. Irrespective of the concentrations maximum inhibition of mycelial growth (78.42 %) was observed by Mancozeb followed by per cent in Captan which are significantly superior over the per cent of inhibition in Zineb In vitro evaluation of systemic fungicides against Rhizoctonia sp. The data on bioassay of three non systematic fungicides at the concentration ranging from 0.15 to 0.3 per cent are presented in the Table 21 and Plate 8. Experimental results revealed that systemic fungicides were effective in controlling the growth of pathogen. Cent per cent inhibition was observed in Thiophanate methyl and Carbendazim at all the concentrations and they were significantly superior over the other treatments. In remaining fungicides the maximum inhibition of mycelial growth (90.73 %) was recorded in Propiconazole followed by per cent in Hexaconazole at 0.05 per cent concentration.while at 0.1 per cent, maximum inhibition of mycelial growth (94.03 %) was recorded in Propiconazole followed by per cent in Hexaconazole.Where at 0.2 per cent concentration Propiconazole also shown cent per cent inhibition of mycelial growth. Hexaconazole recorded per cent of inhibition of mycelial growth at 0.2 per cent. Irrespective of the concentrations Carbendazim and Thiophanate methyl have shown cent per cent inhibition which were statistically superior over other two treatments. Next best treatment was Propiconazole with per cent of inhibition of mycelial growth followed by per cent inhibition in Hexaconazole In vitro evaluation of combiproduct fungicides against Rhizoctonia sp. The data on bioassay of six combiproduct fungicides at the concentration ranging from 0.05 to 0.2 per cent are presented in the Table 22. The maximum per cent inhibition of mycelial growth (91.83%) was observed in Carbendazim + Mancozeb followed by per cent in Carboxin + Thiram, per cent in Captan +Hexaconazole, per cent in Cymoxanil + Mancozeb, per cent in Zineb + Hexaconazole and least inhibition of mycelial growth (65.90 %) was recorded in Tricyclozole + Mancozeb at 0.05 per cent concentration. While at 0.15 per cent concentration, maximum inhibition of mycelial growth (97.40%) was recorded in Carbendazim + Mancozeb which was significantly superior over the per cent inhibition in Carboxin + Thiram. Next best treatment was Captan +Hexaconazole with mycelial inhibition of per cent which was statistically on par with per cent inhibition of mycelial growth in Cymoxanil + Mancozeb. Least inhibition (78.10 %) was recorded in Tricyclozole + Mancozeb.

64 Plate 8: In vitro evaluation of fungicides and botanicals against Rhizoctonia sp.

65 Table 20: In vitro evaluation of contact fungicides against Rhizoctonia sp. Per cent inhibition of mycelial growth Fungicide Concentration (%) Mean Captan (55.57)* (61.54) (62.96) (67.06) 77.4 (61.78) Mancozeb (53.94) (60.13) (64.23) (74.08) (63.09) Zineb (21.52) (31.22) (42.06) (45.26) (35.07) A (Fungicide) B (Conc.) A x B (F x C) SEm± C.D. (0.01) * Figures in the parenthesis indicate angular transformations Table 21: In vitro evaluation of systemic fungicides against Rhizoctonia sp. Per cent inhibition of mycelial growth Fungicide Concentration (%) mean Carbendazim (89.96)* (89.96) (89.96) (89.96) Hexaconozole (71.15) (72.26) (79.15) (74.19) Propiconozole (72.27) ( (89.96) (78.32) Thiophanate methyl (89.96) (89.96) (89.96) (89.96) A (Fungicide) B (Conc.) A x B (F x C) SEm± C.D. (0.01) * Figures in the parenthesis indicate angular transformations.

66 Table 22: In vitro evaluation of combiproduct fungicides against Rhizoctonia sp. Per cent inhibition of mycelial growth Fungicide Concentration (%) Mean Carbendazim + Mancozeb (73.37)* 97.4 (80.46) (89.96) (81.26) Cymoxanil + Mancozeb (66.15) (70.24) (80.46) (72.28) Captan + Hexaconazole (67.05) (70.76) (71.15) (69.65) Zineb + Hexaconazole (65.00) (66.43) (70.82) (67.41) Tricyclozole + Mancozeb (53.94) (62.26) (89.96) (68.72) Carboxin + Thiram (70.69) (77.76) (89.96) (79.27) A (Fungicide) B (Conc.) A x B (F x C) SEm± C.D. (0.01) * Figures in the parenthesis indicate angular transformations

67 As the concentration increased to 0.2 per cent there was marginal increase in the inhibition of mycelial growth. In Carbendazim + Mancozeb, Tricyclozole + Mancozeb and Carboxin + Thiram cent per cent inhibition was observed. Next best treatment at 0.2 per cent concentration were per cent in Cymoxanil + Mancozeb followed by per cent in Zineb + Hexaconazole and least inhibition of mycelial growth(89.56 %) was recorded in Captan +Hexaconazole. Irrespective of the concentration of the fungicides maximum inhibition of mycelial growth(96.41 %) was observed in Carbendazim + Mancozeb which is significantly on par with the Carboxin + Thiram with mycelial inhibition of per cent. Next best treatment was Cymoxanil + Mancozeb with per cent which is significantly superior over the Captan + Hexaconazole (87.57) and Zineb + Hexaconazole (86.50%) of inhibition. Least inhibition of mycelial growth was observed in Tricyclozole + Mancozeb (82.66 %) In vitro evaluation of botanicals against Rhizoctonia sp. The data on bioassay of six botanicals at the concentration ranging from 5 to 10 per cent are presented in the Table 23 and Plate 8. Among the different botanicals assessed 1500ppm was more effective and significant over the all the other treatments. In other botanicals, the maximum per cent inhibition of mycelial growth (83.30 %) was recorded in Nimbicidine followed by percent in Neem oil, per cent in Cristol and least inhibition of mycelial growth was recorded in Pongamia oil at 5 per cent concentration. While at 7.5 per cent, cent per cent inhibition of mycelial growth was observed as the concentration increased. At the same concentration maximum per cent of inhibition of mycelial growth (89.93 per cent) was recorded in Nimbicidine followed by per cent in Cristol and least inhibition of mycelial growth (36.60 %) was recorded in Pongamia oil. There was a significant increase in the inhibition of mycelial growth as the concentration increased. At 10 per cent concentration. cent per cent of mycelial growth was recorded in Azadirachtin 1500 ppm, Neem oil and in Nimbicidine followed by per cent in Cristol and least inhibition of mycelial growth (47.90 %) was recorded in Pongamia oil. While in NSKE there was no inhibition of mycelial growth of pathogen in all the concentrations. Irrespective of the concentration maximum inhibition of mycelial growth (92.85 %) was recorded in Neem oil which is significantly superior over per cent in Nimbicidine and per cent in Cristol and least inhibition of mycelial growth was observed in Pongamia oil (37.41%) In vitro evaluation of bioagents against Rhizoctonia sp. The competitive ability of antagonists against pathogen was studied by dual culture method (Plate 7). The per cent inhibition of mycelia growth was worked out. The data are presented in the Table 24. The competitive ability of antagonists against pathogen was studied by dual culture method. The per cent inhibition of mycelia growth was worked out. The results revealed that fungal bioagents retarded the mycelial growth significantly. The highest per cent inhibition of mycelial growth was observed in Trichoderma koningii (25.90%). The next best treatments were Trichoderma viride and Trichoderma harzianum with the inhibition of and per cent respectively but they did not differ significantly. Least inhibition of mycelial growth (20.10 %) was recorded in Trichoderma virens. In bacterial bioagents, Bacillus subtilis and Pseudomonas fluorescens there was no inhibition of pathogen Fusarium sp In vitro evaluation of contact fungicides against Fusarium sp. The data on bioassay of three contact fungicides (Plate 9) at the concentration ranging from 0.15 to 0.3 per cent are presented in the Table 25. The maximum per cent inhibition of mycelial growth (84.33%) was recorded in Mancozeb followed by per cent in Captan and least inhibition of mycelial growth (20.30 %) was observed in Zineb at 0.15 per cent concentration.

68 Table 23: In vitro evaluation of botanicals against Rhizoctonia sp. Botanicals Azadirachtin1 500 ppm Cristol Neem oil Nimbicidine Per cent inhibition of mycelial growth Concentration (%) Mean (10.05) (6.78) (8.91) (9.18) (10.05) (7.15) (10.05) (9.53) (10.05) (7.41) (10.05) (10.05) (10.05) (7.11) (9.67) (9.59) NSKE 0 (1) 0 (1) 0 (1) 0 (1) Pongamia oil (5.33) A (Fungicide) 36.6 (6.13) B (Conc.) (6.97) A x B (F x C) SEm± C.D. (0.01) Values in the parenthesis are x + 1 transferred values (6.14) Table 24: In vitro evaluation of bioagents against Rhizoctonia sp. Bioagent Per cent inhibition of mycelial growth Trichoderma harzianum (4.94)* Trichoderma viride (4.92) Trchoderma virens (4.56) Trichoderma koningii (5.16) Bacillus subtilis 0.00 (1) Psedomonas flourescens 0.00 (1) SEm± C.D. (0.01) * Values in the parenthesis are x + 1 transformations.

69 Plate 9: In vitro evaluation of fungicides and botanicals against Fusarium sp.

70 Table 25: In vitro evaluation of contact fungicides against Fusarium sp. Fungicide Captan Mancozeb Zineb Per cent inhibition of mycelial growth Concentration (%) Mean (53.89)* (66.66) (26.62) (55.01) (68.75) (30.20) A (Fungicide) B (Conc.) (60.17) (78.72) 33.2 (35.37) A x B (F x C) SEm± C.D. (0.01) *Figures in the parenthesis angular transformation (60.55) (78.72) 64.4 (53.38) (58.16) (73.21) (36.4) Table 26: In vitro evaluation of systemic fungicides against Fusarium sp. Carbendazim Hexaconozole Propiconozole Fungicide Thiophanate methyl Per cent inhibition of mycelial growth Concentration (%) Mean (69.41)* (64.87) (70.12) (66.55) A (Fungicide) (71.58) (66.55) (74.08) (70.15) B (Conc.) (78.73) (70.42) (89.96) (89.96) A x B (F x C) SEm± C.D. (0.01) * Figures in the parenthesis are angular transformations (73.24) (67.28) 93.4 (78.05) 90.7 (75.33)

71 While at the 0.2 per cent concentration, maximum inhibition of mycelial growth (86.90 %) was recorded in Mancozeb followed by Captan (67.16 %) and least inhibition of mycelial growth (25.06%) was observed in Zineb. There was significant increase in the inhibition of mycelial growth in Captan and Mancozeb at 0.25 and 0.3 per cent concentration. At 0.25 per cent the maximum inhibition of mycelial growth (96.20 %) was recorded in Mancozeb followed by per cent in Captan. While at the 0.3 per cent concentration, maximum inhibition of mycelial growth was recorded in Mancozeb followed by per cent in Captan and least inhibition of mycelial growth (64.40%) was recorded in Zineb. Irrespective of the concentrations maximum per cent inhibition of mycelial growth (90.90 %) was recorded in Mancozeb which differed significantly when compare to Captan (71.85%) and Zineb (36.40 %) In vitro evaluation of systemic fungicides against Fusarium sp. The data on bioassay of four systemic fungicides at the concentration ranging from 0.05 to 0.2 per cent are presented in the Table 26 and Plate 9. The maximum per cent inhibition of mycelial growth (87.70 %) was recorded in Propiconazole which is on par with Carbendazim (87.66 %).followed by per cent inhibition in Thiophanate methyl at 0.05 per cent concentration. While at 0.1 per cent concentration, maximum per cent inhibition of mycelial growth (92.50 %) was recorded in Propiconazole followed by per cent in Carbendazim, per cent in Thiophanate methyl and per cent in Hexaconazole. As the concentration increased there is marginal increase in inhibition of mycelial growth (84.50 %) in Hexaconozole at 0.2 per cent concentration while there was cent per cent in inhibition of mycelial growth in Propiconazole and Thiophanate methyl followed by per cent in Carbendazim. Irrespective of the concentrations maximum inhibition of mycelial growth (93.40 %) was recorded in Propiconazole which was statistically superior over the percent in Carbendazim, percent in Thiophanate methyl and comparatively less inhibition of mycelial growth was recorded in Hexaconazole (83.62 %) In vitro evaluation of combiproduct fungicides against Fusarium sp. The data on bioassay of six combiproduct fungicides at the concentration ranging from 0.05 to 0.2 per cent are presented in the Table 27 and Plate 9. The maximum per cent inhibition of mycelial growth (91.60 %) was recorded in Carbendazim + Mancozeb followed by percent in Captan + Hexaconazole and percent in Carboxin + Thiram. Where as less than fifty percent inhibition of mycelial growth was recorded in Zineb + Hexaconazole (46.26 %), Tricyclozole + Mancozeb(33.26%) and Cymoxanil + Mancozeb(23.20 %) at 0.05 percent concentration. While at the 0.1 percent concentration cent percent inhibition of mycelial growth was recorded in Carbendazim + Mancozeb followed by Captan + Hexaconazole (76.43 %), Carboxin + Thiram( ), Tricyclozole + Mancozeb (62.90 %) and per cent in Zineb + Hexaconazole and least inhibition of mycelial growth(31.80 %) was recorded in Cymoxanil + Mancozeb. There was significant increase in the inhibition of mycelial growth at 0.2 per cent concentration. Maximum (%) inhibition of mycelial growth was recorded in Carbendazim + Mancozeb followed by Carboxin + Thiram(87.33%), Captan + Hexaconazole(80.30 %), Tricyclozole + Mancozeb(74.20 %), Zineb + Hexaconazole (66.96 %) and least inhibition of mycelial growth was recorded in Cymoxanil + Mancozeb(50.13%). Irrespective of the concentrations maximum inhibition of mycelial growth (97.20 %) was recorded in Carbendazim + Mancozeb which is statistically superior over the other treatments. Next best treatments are Captan + Hexaconazole (75.83 %) followed by Carboxin + Thiram (73.63 %). Zineb + Hexaconazole (56.98 %t) and Tricyclozole + Mancozeb (56.78 %) are on par with each other in inhibiting the mycelial growth and least inhibition of mycelial growth (35.04 %) was recorded in Cymoxanil + Mancozeb In vitro evaluation of botanicals against Fusarium sp. The data on bioassay of six botanicals (Plate 9) at the concentration ranging from 5 to 10 per cent are presented in the Table 28.

72 Table 27: In vitro evaluation of combiproduct fungicides against Fusarium sp. Per cent inhibition of mycelial growth Fungicide Concentration (%) Mean Captan + Hexaconazole (57.20)* (60.93) (63.63) (60.59) Carbendazim + Mancozeb (73.12) (89.96) (89.96) 97.2 (84.35) Carboxin + Thiram (50.37) (69.27) (69.27) (59.65) Cymoxanil + Mancozeb (28.78) (34.10) (45.15) (36.01) Tricyclozole + Mancozeb (35.35) (52.27) (59.47) (49.03) Zineb + Hexaconazole (42.80) (49.35) (54.89) (49.01) A (Fungicide) B (Conc.) A x B (F x C) SEm± C.D. (0.01) * Figures in the parenthesis are angular transformations.

73 Table 28: In vitro evaluation of botanicals against Fusarium sp. Botanicals Azadirachtin1 500 ppm Cristol Neem oil Nimbicidine NSKE Pongamia oil Per cent inhibition of mycelial growth Concentration (%) Mean (89.96)* (39.62) (56.95) (47.27) (37.45) (45.34) A (Fungicide) (89.96) (42.19) (89.96) (52.47) (44.16) (48.73) B (Conc.) (89.96) (46.98) (89.96) (58.77) (46.85) (54.63) A x B (F x C) SEm± C.D. (0.01) * Figures in the parenthesis are angular transformations (89.96) (42.93) (78.96) (52.84) (42.82) (49.62) Table 29: In vitro evaluation of bioagents against Fusarium sp. Bioagent Per cent inhibition of mycelial growth Trichoderma harzianum (67.20)* Trichoderma viride (56.03) Trchoderma virens (51.63) Trichoderma koningii (54.01) Bacillus subtilis (42.48) Psedomonas flourescens (44.98) SEm± 0.94 C.D. (0.01) 3.81 * Values in the parenthesis angular transformations.

74 Among the different treatments, cent percent inhibition of mycelial growth was recorded at all the concentrations. In other botanicals the maximum per cent inhibition of mycelial growth (70.30 %) was recorded in Neem oil followed by Nimbicidine (54.00 %), Pongamia oil (50.90 %) and less than fifty per cent inhibition of mycelial growth was recorded in Cristol (40.80 %) and NSKE(37.00 %) at 5 per cent concentration. While at the 7.5 per cent concentration, cent percent inhibition of mycelial growth was recorded in Neem oil followed by Nimbicidine (62.93 %), Pongamia oil(56.80 %) and least inhibition of mycelial growth(45.50 %) was recorded in Cristol. There was significant increase in the inhibition of mycelial growth at 10 per cent concentration, cent percent inhibition of mycelial growth was recorded in Neem oil followed by Nimbicidine( %), Pongamia oil(66.20 %), Cristol (53.50 %) and least inhibition of mycelial growth (53.26 %) was recorded in NSKE. Irrespective of the concentration maximum per cent inhibition of mycelial growth (90.10%) was recorded in Neem oil which is statistically superior over the Nimbicidine (63.46 %). Next best treatments was Pongamia oil(57.96 %). Least inhibition of mycelial growth (46.27 %) was recorded in NSKE which is on par with Cristol (40.80 %) In vitro evaluation of bioagents against Fusarium sp. The competitive ability of antagonists against pathogen was studied by dual culture method (Plate 7). The data is presented in the Table 29. The results revealed that fungal bioagents retarded the growth of Fusarium sp. significantly. The highest per cent inhibition of mycelial growth (85.10 %) was observed in T.harzianum followed by T.viride (69.40%). The next best treatment was T.koningii which inhibited the mycelial growth up to per cent. In fungal bioagents, least inhibition of mycelial growth (61.90 %) was recorded in Trichoderma virens, where as in bacterial bioagents the maximum inhibition of mycelial growth (50.37 %) was recorded in Pseudomonas fluorescens followed by Bacillus subtilis with the inhibition of per cent Management of wilt of soybean in glasshouse condition Management of S. rolfsii in glasshouse condition. The studies of glass house management revealed that most of the chemicals which were found effective in vitro were not effective in glass house condition. The results are presented in the Table 30 and Plate 10. Among the different chemicals, botanicals and bioagents, cent per cent incidence of disease was observed in Tricyclozole + Mancozeb, Thiophanate methyl, Mancozeb, Cymoxanil + Mancozeb, Captan, Azadirachtin 1500 ppm and Nimibicidine after 20 days and 40 days after sowing, pre emergence death of seedlings was more. While in Carbendazim + Mancozeb (88.30%) disease incidence was observed after 20 days followed by per cent wilting after 40 days of sowing. Zineb + Hexaconazole, Hexaconazole, Propiconazole, Captan + Hexaconazole, T. viride and T. harzianum were found effective in controlling the disease, no wilting was observed in these treatments. Maximum plant height (52.20 cm) was observed in Zineb + Hexaconazole after 40 days after sowing followed by T. harzianum (42 cm), Captan + Hexaconazole (37 cm), T. viride(32.40 cm). Least plant height was observed in neem oil (1.70 cm) Management of Rhizoctonia sp. in glasshouse condition. The results are presented in the Table 31 and Plate 11.Among the different chemicals, botanicals and bioagents, cent percent incidence of disease was observed in Zineb + Hexaconazole, Tricyclozole + Mancozeb, Mancozeb, Carbendazim + Mancozeb, Cymoxanil + Mancozeb and Azadirachtin 1500 ppm. While in Thiophanate methyl, Hexaconazole, Propiconazole, Captan + Hexaconazole, T. viride and T. harzianum, Neem oil and Nimbicidine were found effective in controlling the disease. Carbendazim + Mancozeb has shown 50 per cent wilting after 20 days after sowing and after 40 days it has reached to cent per cent. After 20 days of sowing maximum height of plant was recorded in T. viride(32.0 cm) followed by Thiophanate methyl (28.50 cm) while at the 40 days after sowing maximum plant height was recorded in T. viride(46.8 cm) followed by T. harzianum (36.70 cm), Thiophanate methyl (35.80 cm).pre emergence deaths of plants was more as compared to post emergence death of plants.

75 Table 30: Management of S. rolfsii with fungicides, botanicals and bioagents under glasshouse condition Treatment (%) Per cent disease incidence (20 DAS) Per cent disease incidence (40 DAS) Plant height (cm) (20 DAS) Plant height (cm) (40 DAS) Control (10) (10) Zineb + Hexaconazole (0.1) 0.00 (1) 0.00 (1) Tricyclozole + Mancozeb (0.2) (0) (0) Thiophanate methyl (0.1) (10) (0) Mancozeb (0.3) (10) (0) Hexaconazole (0.05) 0.00 (1) 0.00 (1) Propiconazole (0.05) 0.00 (1) 0.00 (1) Captan + Hexaconazole (0.05) 0.00 (1) 0.00 (1) Carbendazim + Mancozeb (0.15) (9.47) (1) Carboxin + Thiram (0.15) 66.6 ( Cymoxanil + Mancozeb (0.15) (10) (0) Captan (0.3) (10) (0) Azadirachtin 1500 ppm (5) (10) (0) Neem oil (5) (10) (0) Nimbicidine (5) (10) (0) T. viride (2) 0.00 (1) 0.00 (1) T. harzianum (2) 0.00 (1) 0.00 (1) SEm± C.D. (0.01) * Values in the parenthesis are x + 1 transformations

76 Palte 10: Management of S.rolfsii under glasshouse condition with fungicides, botanicals and bioagents.

77 Per cent disease incidence (20 DAS) Plant height (cm) (20 DAS) Per cent disease incidence (40 DAS) Plant height (cm) (40 DAS) Treatment Fig. 8: Management of S. rolfsii with chemicals, botanicals and bioagents under glasshouse condotion Fig. 8: Management of S. rolfsii with chemicals, botanicals and bioagents under glasshouse condition

78 Management of Fusarium sp. in glasshouse condition The results are presented in the Table 32 and Plate 12. Among the different chemicals, botanicals and bioagents, cent per cent incidence of disease was observed in Tricyclozole + Mancozeb, Mancozeb, Carboxin + Thiram, Captan, Propiconazole and Azadirachtin 1500 ppm. Hexaconazole has shown per cent wilt incidence after 20 days later it reached to cent per cent after 40 days. In case of Carbendazim + Mancozeb (58.33%) wilt incidence was observed after 20 days and per cent disease in 40 days after sowing. While Thiophanate methyl, Carbendazim, T.viride and T. harzianum, Neem oil and Nimbicidine were effective in controlling the disease. After 20 days after sowing maximum plant height was observed in T. harzianum(34.20 cm) followed by T. viride (32 cm), Thiophanate methyl (30.80 cm) and least plant height was observed in Captan + Hexaconazole (4 cm). While after 40 days of sowing maximum plant height was recorded in T. harzianum(49.30) followed by T. viride(46 cm) where as plant height has increased from 29 cm to 42 cm in Neem oil.pre emergence deaths of plants was more as compared to post emergence death Estimation of rhizosphere population Rhizosphere microbial population in management of wilt of soybean was estimated by serial dilution plate technique as described in material and methods. Data on microbial population at 30 DAS and 45 DAS after sowing are presented in the Table 33. In S. rolfsii, the population of fungi at 30 DAS sowing ranged from 2.5 x 10 4 to 65 x 10 4 cfu/g soil. Highest population was noticed in Neem oil (65 x 10 4 cfu/g soil ) followed by Nimbicidine (45 x10 4+ cfu/g soil). More population was observed in botanicals as compared to chemicals and bioagents. After 45 DAS fungi population increased in botanicals and bioagents and decreased in the chemical treatments. Highest population was observed in Nimbicidine after 45 DAS (84.2 x 10 4 cfu/g soil). In Rhizoctonia sp., the population of fungi at 30 DAS sowing ranged from 3.5 x 10 4 to 53 x 10 4 cfu/g soil. After 45DAS population of fungi ranged from 2.2 x 10 4 to x 10 4 cfu/g soil, highest population was observed in Neem oil (93.20 x 10 4 cfu/g soil). In Fusarium sp. the population of fungi ranged from 6.4 x 10 4 to 220x 10 4 cfu/g soil. Highest population was recorded in Neem oil (220 x 10 4 cfu/g soil) followed by Nimbicidine (170.4 x10 4 cfu/g soil). Where as after 45 DAS fungi population ranged from 5.20 x10 4 to x10 4+ cfu/g soil. Highest population was recorded in Neem oil (235.3 x10 4 cfu/g soil) there is an increasing trend in the population in botanicals and bioagents and decrease in population in chemical treatments Screenings of varieties for root rot/wilt resistance. The results on screening of advanced lines and rust differentials against wilt of soybean are presented in the Table 35 a and 35b Screening of advanced material against root rot/ wilt during 2010 The results of screening under sick plot at Ugarkhurd revealed that among the advanced material, seven lines showed reaction of 1 grade (1-10 % wilt incidence) with resistant reaction followed by another seven lines in scale 2 with moderately resistant reaction. The line MACS 1140 recorded under scale 3, 13 lines showed highly susceptible reaction Screening of rust resistant lines against root rot/ wilt during 2010 The screening of rust resistant lines against root rot / wilt complex revealed that two lines PI and EC showed resistant reaction followed by 4 lines moderately resistant reaction. Rest of other recorded susceptible to highly susceptible reaction.

79 Table 31: Management of Rhizoctonia sp. with fungicides, botanicals and bioagents under glasshouse condition Treatment (%) Per cent disease incidence (20 DAS) Per cent disease incidence (40 DAS) Plant height (cm) (20 DAS) Plant height (cm) (40 DAS) Control (10) (10) Zineb + Hexaconazole (0.05) (10) (10) Tricyclozole + Mancozeb (0.2) (10) (10) Thiophanate methyl (0.05) 0.00 (1) 0.00 (1) Mancozeb (0.25) (10) (10) Hexaconazole (0.05) 0.00 (1) 0.00 (1) Propiconazole (0.05) 0.00 (1) 0.00 (1) Captan + Hexaconazole (0.05) 88.3 (9.43) (10) Carbendazim + Mancozeb (0.05) 50 (4.92) (7.16) Carboxin + Thiram (0.05) (0) (10) Cymoxanil + Mancozeb (0.05) (10) (10) Carbendazim (0.05) 0.00 (1) 0.00 (1) Azadirachtin 1500 ppm (5) (10) (10) Neem oil (5) 0.00 (1) 0.00 (1) Nimbicidine (5) 0.00 (1) 0.00 (1) T. viride (2) 0.00 (1) (1) T. harzianum (2) 0.00 (1) (1) SEm± C.D. (0.01) * Values in the parenthesis are x + 1 transformations

80 Plate 11: Management of Rhizoctonia sp. under glasshouse condition with fungicides, botanicals and bioagents

81 Per cent disease incidence (20 DAS) Plant height (cm) (20 DAS) Per cent disease incidence (40 DAS) Plant height (cm) (40 DAS) Treatment Fig. 9: Management of Rhizoctonia sp. with chemicals, botanicals and bioagents under glasshouse condotion Fig. 9: Management of Rhizoctonia sp. with chemicals, botanicals and bioagents under glasshouse condition

82 Plate 12: Management of Fusarium sp. under glasshouse condition with fungicides, botanicals and bioagents Plate 13: Screening for wilt disease