CHAPTER IV RESULTS AND DISCUSSION

Transcription

1 CHAPTER IV RESULTS AND DISCUSSION Coriander (Coriandrum sativum L.) belongs to family Apiaceae is one of the principal spice crop have commonly used in India since ancient times. In India, it is cultivated in about 5.52 lakh hectares and produces 4.61 lakh tones seed. Gujarat and Rajasthan both are major seed spice producing state with more than 80 per cent of the total seed spices production in India. Coriander is mainly grown in Rajasthan, Gujarat, Madhya Pradesh, Tamil Nadu and Uttar Pradesh. In Gujarat, coriander is mainly grown in Saurashtra region. During rabi 2016, coriander crop exhibited symptoms of root rot at Vegetable Research Station, JAU, Junagadh as well as in a few farmers fields nearby Junagadh. Naturally infected coriander plants with symptoms on collar region, stem and roots were observed in the field. Root rot caused by R. solani is an important disease of coriander and causes serious losses to coriander production. The loss in yield due to disease was severe in vicinity of Junagadh and hence, attempt has been made to identify the causal pathogen and to find out suitable management practices. The results obtained from the investigation were communicated hereunder. 4.1 Collection, isolation and purification of pathogen Naturally infected plants of coriander showing typical symptoms of root rot were collected from Vegetable Research Station, JAU, Junagadh has well as farmers field from Ivnagar, Shelra and Shapur of Junagadh district. Isolations were done from infected collar region, stem and root. The isolated fungus grew in the form of dirty white mycelium with black discoloration on media. The culture was purified by hyphal tip isolation technique. The purified culture was maintained by storing it at 4 C under refrigeration and transferred periodically on slants containing fresh PDA media.

2 4.2 Morphology and identification of pathogen Morphological characters form the primary basis for the identification of pathogen. Morphological characters of mycelium, sclerotia and growth patterns were observed by preparing slides of the aggressive isolates which were stained by cotton blue and examined under compound microscope. The morphological characters of mycelium and sclerotium studied are mentioned as under Mycelium The isolated fungus grew in the form of dirty white mycelium with discoloration on PDA covered the entire surface of the plate within a week. The aerial mycelium was dense and fluffy off grey white in color, while the submerged mycelium was brown to black in color (Table 4.1, Plate-1A). Branching of hyphae was at right angles and constriction observed at point of branching of the mycelium and septum also present at juncture of branching (Plate-1B). Similar type of observation for R. solani was made by Sneh et al. (1991a). The hyphae measured in the range of 3.0 to 8.2 μm in diameter. Similar mycelial diameter was also noted by Vijayan and Nair (1985). Table 4.1: Colony characters of different isolates of R. solani Sr. No. Isolates Mycelial arrangement and color Hyphal diameter (μm) Sclerotial diameter (μm) Sclerotial initiation after days 1. R. solani 1 (VRS) 2. R. solani 2 (Ivnagar) 3. R. solani 3 (Shelra) 4. R. solani 4 (Shapur) Fluffy growth with blackish colour Fluffy growth with blackish colour Fluffy growth with blackish colour Fluffy growth with blackish colour

3 4.2.2 Sclerotium Abundant sclerotial formation was observed when the culture was incubated for fifteen days. Sclerotial size was measured from slides of 15 days old pure culture of R. solani under compound microscope using ocular micrometer. The sclerotia were of irregular shapes and size measuring in the range of μm with mean diameter of 96.6 μm (Table 4.1, Plate-2A, B, C & D). 4.3 Symptomatology The symptoms of R. solani were studied on naturally infected coriander plant under field condition (Plate 3A) and artificially inoculated coriander plants (Plate-3B) under glass house condition as well as at farmer s field in severe condition (Plate-3C). The inoculated plants were successfully infected by causal fungus, whereas plants under controlled condition (i.e. without application of inoculums) remain healthy throughout the period of experiment. The aerial part of infected plant shows yellowing of older leaves first leading to formation of grayish black sunken spots on collar region. Such plants were dislodged at stem portion which resulted into wilting and drying of the plants. The root system of the affected plants exhibited decaying of secondary root and could be easily pulled out from the soil. The tap root showed symptoms of drying and the root bark shredded off easily which in due course of time wilts and died. Similar types of symptoms were also described by Kotila (1947) and Patole and Narute (2011). They observed that root rot infected soybean plant showed browning and rotting of collar region and brown discoloration of infected tissues with easy uprooting and root decay was identified as R. solani Kühn. More or less similar type of symptoms were also reported by Yang (1999) and Sonakar et al. (2014). They reported that the fungus may cause pre-emergence and post-emergence damping-off in addition to rotting of the hypocotyls and roots. Reddish brown lesions on the hypocotyls at the soil line are the typical symptoms of Rhizoctonia hypocotyls rot. 4.4 Pathogenicity test of R. solani on coriander Pathogenicity test of different four isolates of R. solani Kühn were confirmed by using artificial soil inoculation method as described in Materials and Methods. After inoculation of susceptible coriander plants, most of the plants were infected by 45

4 test pathogen and the symptoms developed were similar to that of naturally infected coriander plants. The initial symptoms exhibited by aggressive isolate at thirty days after inoculation were similar to the sign of water stress. Drooping of the leaves, petioles and finally the entire plant dried up at 50 DAS. Such plants appeared straw colored and the roots of the plants either rotted or remained undeveloped (Plate-4A). Tap root also dried and root bark shredded off easily. Shredding of tissue were the conspicuous symptoms in dried plants. The entire root system of collar rot infected plant became black and disintegrated later. The plants dried due to root rot in inoculated pots by 50 days after inoculation while in control pots the plants were free from disease (Plate-4B). According to data presented in Table 4.2 (Plate-4C), the maximum mortality was found in Rhizoctonia solani 2 (Ivnagar isolate) at 30 DAS and total plant mortality was achieved at 50 DAS. On that basis this isolate was proved aggressive one. Whereas, remaining isolates takes more time to kill the coriander plants and were found less aggressive as compared to Ivnagar isolate (Plate-5). Thus, Ivnagar isolate was used for all laboratory and pot experiment. Table 4.2: Plant mortality observed in different isolates of R. solani in pot condition Sr. No. Isolates No. of seeds sown No. of seeds germinated Days to initiation of symptoms Plant mortality at 30 DAS * Plant mortality at 50 DAS Plant mortality at 70 DAS 1. Rhizoctonia solani 1 (VRS) 2. Rhizoctonia solani 2 (Ivnagar) 3. Rhizoctonia solani 3 (Shelra) 4. Rhizoctonia solani 4 (Shapur) * DAS = Days after sowing 46

5 The fungus was re-isolated from the artificially inoculated diseased coriander plants and culture was produced morphological characters as identical to that of original culture in all respects that confirming the pathogenicity of all the four isolates of R. solani. Similar type of pathogenicity test were also carried out by Woodhall et al. (2007) under glass house and field condition and concluded that disease type on potato varied between different anastomosis groups (AGs) of R. solani. Similarly, Strausbaugh et al. (2011) proved pathogenicity test with 18 Rhizoctonia solani isolates (selected on the basis of genetic diversity) with uninoculated check on the silage corn under greenhouse condition. 4.5 Evaluation of agrochemicals on mycelial growth inhibition and sclerotial formation of R. solani under in vitro condition To determine the relative efficacy of various agrochemicals, they were tested against R. solani using poisoned food technique as mentioned in Materials and Methods. Various non-systemic, systemic, readymix fungicides and weedicides showed mycelial growth inhibition and sclerotial formation of the fungus in each treatments. The per cent inhibition was calculated on the basis of the difference in inhibition obtained in the treatment and check Effect of non-systemic fungicides on mycelial growth inhibition and sclerotial formation of R. solani under in vitro condition The relative efficacy of different six non-systemic fungicides were tested at the concentration of 500, 1000, 1500 and 2000 ppm using poison food technique. The data on per cent inhibition of mycelial growth and sclerotial formation were presented in Table 4.3, Plate-6 and depicted in Fig.1. Data presented in Table 4.3 revealed that mancozeb 75% WP was the most effective fungicide with per cent mean mycelial growth inhibition and significantly superior over rest of the treatments. Wettable sulphur 80% WP was the least effective fungicide with mean mycelial growth inhibition of 3.24 per cent. 47

6 Table 4.3: Effect of non-systemic fungicides on mycelial growth inhibition and sclerotial formation of R. solani under in vitro condition Fungicides Growth inhibition (%) and sclerotial formation Mean inhibition 500 ppm 1000 ppm 1500 ppm 2000 ppm (%) Chlorothalonil 75% WP (3.88) (9.62) (45.67) (50.08) (27.49) Copper hydroxide 77% WP 1.08 (0.40) 2.91 (0.44) 3.09 (0.52) (28.76) 9.87 (7.53) Copper oxychloride (3.66) (40.69) (72.47) (78.98) (48.95) 50% WP Mancozeb 75% WP (99.98) (99.98) (99.98) (99.98) (99.98) Thiram 75% WP (23.97) (25.47) (40.07) (62.98) (38.12) Wettable sulphur 80% WP 2.99 (0.48) 9.66 (2.84) (3.89) (5.76) 9.48 (3.24) Fungicide (F) Conc. (C) F x C S. Em. ± C. D. at 5% C. V. % 4.29 Sclerotial formation: ++++ = Abundant; +++ = good; ++ = moderate; + = scanty; - = no sclerotial formation. Values in parentheses are re-transformed values while outside were transformed with arcsine transformation before analysis. 48

7 Among different non-systemic fungicides, mancozeb 75% WP gave per cent mycelial growth inhibition at all concentrations (500, 1000, 1500 and 2000 ppm) which were the best treatments for inhibiting the growth of mycelium of R. solani under in vitro condition. Copper oxychloride 50% WP was found second best effective fungicide with and per cent inhibition at 1500 and 2000 ppm, respectively. Whereas, thiram 75% WP (23.97, 25.47, and per cent) and chlorothalonil 75% WP (3.88, 9.62, and per cent), respectively were found moderately effective fungicides in inhibiting the mycelial growth of the fungus at all concentrations tried. Copper hydroxide 77% WP exhibited 0.40, 0.44, 0.52 and per cent mycelial growth inhibition but it was remained at par with wettable sulphur 80% WP with 0.48, 2.84, 3.89 and 5.76 per cent mycelial growth inhibition at 500, 1000, 1500 and 2000 ppm concentration, respectively and were found least effective non-systemic fungicides. The effect of different concentrations of non-systemic fungicides on sclerotial formation was found negatively correlated with the inhibition of growth. No sclerotial formation was found in any concentration of mancozeb. Whereas, abundant sclerotial formation were found in treatment of copper hydroxide 77% WP; wettable sulphur 80% WP and chlorothalonil 75% WP at 500 and 1000 ppm. Good sclerotial formation was observed in the treatment of copper oxychloride 77% WP and thiram 75% WP at 1500 ppm concentration. While, scanty sclerotial formation was observed in copper oxychloride at 1500 and 2000 ppm concentration. Similar type of findings were also reported by Anjana and Kumar (2008) while working with various fungicides for control of R. solani. They concluded that mancozeb was superior to all others in inhibiting mycelial growth of the test fungus R. solani under in vitro condition Effect of systemic fungicides on mycelial growth inhibition and sclerotial formation of R. solani under in vitro condition The relative efficacy of different six systemic fungicides was tested at the concentration of 50, 100, 250 and 500 ppm using poison food technique. The data on per cent inhibition of mycelial growth and sclerotial formation were presented in Table 4.4, Plate-7 and depicted in Fig

8 Table 4.4: Effect of systemic fungicides on mycelial growth inhibition and sclerotial formation of R. solani under in vitro condition Fungicides Growth inhibition (%) and sclerotial formation Mean inhibition 50 ppm 100 ppm 250 ppm 500 ppm (%) Azoxystrobin 23% SC 7.29 (1.62) 9.08 (2.58) (4.85) (97.77) (26.70) Benomyl 50% WP (87.31) (87.04) (87.75) (98.88) (90.24) Carbendazim 50% WP (82.21) (83.24) (84.49) (85.41) (83.83) Fosetyl-Al 80% WP 1.08 (0.38) 4.54 (0.80) 4.71 (0.86) 4.88 (0.93) 3.80 (0.74) Tebuconazole 25.9% EC (73.29) (81.42) (83.24) (88.88) (81.70) Thiophanate-methyl 70% WP 1.08 (0.35) 4.56 (0.80) 5.00 (0.98) 5.72 (1.30) 4.09 (0.85) Fungicide (F) Conc. (C) F x C S. Em. ± C. D. at 5% C. V. % 4.92 Sclerotial formation: ++++ = Abundant; +++ = good; ++ = moderate; + = scanty; - = no sclerotial formation. Values in parentheses are re-transformed value while outside were transformed with arcsine transformation before analysis. 50

9 The data presented in Table 4.4 indicated that benomyl 50% WP was significantly superior over rest of the treatments with per cent mean mycelial growth inhibition. Whereas, carbendazim 50% WP was the next best fungicide and found equally effective with tebuconazole 25.9 % EC and showed and per cent mean mycelial growth inhibition, respectively. Azoxystrobin 23% SC found moderately effective fungicide with mean mycelial growth inhibition of per cent. The least effective fungicide was thiophanate methyl 70% WP which was found at par with fosetyl-al 80% WP with mean mycelial growth inhibition of 0.85 and 0.74 per cent, respectively. Among different systemic fungicides tested, benomyl 50% WP found the best among all treatments and gave 87.31, 87.04, and per cent inhibition of growth of the fungus at 50, 100, 250 and 500 ppm concentrations respectively, under in vitro condition. Second best effective treatment was carbendazim 50% WP (82.21, 83.24, and per cent) followed by tebuconazole 25.9% EC with 73.29, 81.42, and per cent inhibition of mycelial growth at 50, 100, 250 and 500 ppm concentrations, respectively. Azoxystrobin 23% SC gave per cent mycelial growth inhibition at 500 ppm. Whereas, same fungicide exhibited 1.62, 2.58 and 4.85 per cent mycelial growth inhibition at 50, 100 and 250 ppm, respectively. Thiophanate methyl 70% WP (0.35, 0.80, 0.98 and 1.30 per cent) and fosetyl-al 80% WP (0.38, 0.80, 0.86 and 0.93 per cent) were equally effective in mycelial growth inhibition at 50, 100, 250 and 500 ppm, respectively and were least effective systemic fungicides. The effect of different concentrations of systemic fungicides on sclerotial formation was found negatively correlated with the inhibition of growth. No sclerotial formation was found at any concentrations of benomyl 50% WP and carbendazim 50% WP. Whereas, tebuconazole 25.9% EC at 100, 250 and 500 ppm and azoxystrobin 23% SC at 500 ppm form no any sclerotia. While, abundant sclerotial formation was found in all concentration of fosetyl-al 80% WP and thiophanate methyl 70% WP as well as in azoxystrobin 23% SC at 50, 100 and 250 ppm. Scanty sclerotial formation was observed in tebuconazole 25.9% EC at 50 ppm concentration. 51

10 The present investigation was in close conformity with the similar type of findings as reported by Wenham et al. (1976) during their in vitro study. They reported benomyl as the best systemic fungicides for control of R. solani and was superior to all others in inhibiting mycelial growth of the test fungus R. solani under in vitro condition. Similarly, Wasira Akhter et al. (2015) also reported bavistin 50% WP as the most effective fungicide with completely inhibiting the radial growth of R. solani, even at the lowest concentration (100 ppm) Effect of readymix fungicides on mycelial growth inhibition and sclerotial formation of R. solani under in vitro condition The relative efficacy of different six readymix fungicides were tested at the concentration of 100, 250, 500 and 1000 ppm using poison food technique. The data on per cent inhibition of mycelial growth and sclerotial formation are presented in Table 4.5, Plate-8 and depicted in Fig. 3. Data presented in Table 4.5 stated that tebuconazole 50% + tryfloxistrobin 25% WG gave significantly the maximum mean mycelial growth inhibition (76.46 per cent). The next best was metiram 55% + pyraclostrobin 5% WG with mean mycelial growth inhibition of per cent. Whereas, cymoxanil 8% + mancozeb 64% WP was the least effective readymix fungicide with 4.43 per cent mean mycelial growth inhibition. Among different readymix fungicides tested under in vitro condition, tebuconazole 50% + tryfloxistrobin 25% WG gave 86.56, and per cent mycelial growth inhibition at 250, 500 and 1000 ppm concentrations, respectively which was the best treatment for inhibiting the growth of mycelium of R. solani under laboratory conditions. The moderately effective readymix fungicides in inhibiting the mycelial growth were metiram 55% + pyraclostrobin 5% WG with per cent at 500 and 1000 ppm concentration followed by carboxin 37.5% + thiram 37.5% WP with per cent at 1000 ppm and cabendazim 12% + mancozeb 63% WP with and per cent at 500 and 1000 ppm concentration, respectively. The least effective readymix fungicides were hexaconazole 4% + zineb 68% WP (3.94, 5.40, 5.56 and 6.41 per cent) and cymoxanil 8% + mancozeb 64% WP (2.05, 2.58, 2.94 and

11 per cent) mycelial growth inhibition at 100, 250, 500 and 1000 ppm concentrations, respectively. Table 4.5: Effect of readymix fungicides on mycelial growth inhibition and Readymix fungicides Carbendazim 12% + Mancozeb 63% WP Carboxin 37.5% + Thiram 37.5% WP Cymoxanil 8% + Mancozeb 64% WP Hexaconazole 4% + Zineb 68% WP Metiram 55% + Pyraclostrobin 5% WG Tebuconazole 50% + Trifloxystrobin 25% WG sclerotial formation of R. solani under in vitro condition Growth inhibition (%) and sclerotial formation 100 ppm 250 ppm 500 ppm 1000 ppm 9.75 (2.92) (4.57) (82.89) (83.41) (13.74) (16.66) (66.45) (83.51) (2.05) 9.16 (2.58) 9.79 (2.94) (10.17) (3.94) (5.40) (5.56) (6.41) (13.74) (19.62) (99.98) (99.98) (30.87) (86.56) (88.45) (99.98) Fungicide (F) Conc. (C) F x C S. Em. ± C. D. at 5% C. V. % 3.81 Mean inhibition (%) (43.44) (45.09) (4.43) (5.32) (58.33) (76.46) Sclerotial formation: ++++ =Abundant; +++= good; ++ = moderate; + = scanty; - = no sclerotial formation. Values in parentheses are re-transformed values while outside were transformed with arcsine transformation before analysis. 53

12 The effect of different concentrations of readymix fungicides on sclerotial formation was found negatively correlated with the inhibition of growth. No sclerotial formation was found in tebuconazole 50% + trifloxystrobin 25% WG at 250, 500 and 1000 ppm concentration as well as in metiram 55% + pyraclostrobin 5% WG and carbendazim 12% + mancozeb 63% WP at 500 and 1000 ppm and carboxin 37.5% + thiram 37.5% WP at 1000 ppm. Abundant sclerotial formation were found in all concentration of cymoxanil 8% + mancozeb 64% WP and hexaconazole 4% + zineb 68% WP. While, carbendazim 12% + mancozeb 63% WP; carboxin 37.5% + thiram 37.5% WP and metiram 55% + pyraclostrobin 5% WG each at 100 and 250 ppm concentration, respectively exhibited abundant sclerotial formation. More or less similar trend was also observed by Prajapati et al. (2002). They recorded that carbendazim, carbendazim + thiram, carboxin and thiophanate methyl completely inhibited the growth of R. bataticola isolated from chickpea. Similarly, Dileep Kumar (2016) studied the broad spectrum action of a nanoform commercial fungicide trifloxystrobin 25% + tebuconazole 50% (75 WG) with improved antifungal activity against Macrophomina phaseolina Effect of weedicides on mycelial growth inhibition and sclerotial formation of R. solani under in vitro condition The relative efficacy of different six weedicides were tested at the concentrations of 100, 150, 200 and 250 ppm using poison food technique. The data on per cent inhibition of mycelial growth and sclerotial formation are presented in Table 4.6, Plate-9 and depicted in Fig. 4. The data presented in Table 4.6 revealed that quizalofop-p-ethyl 5% EC was significantly superior over rest of the treatments with per cent mean mycelial growth inhibition. This was followed by propaquizafop 10% EC with per cent mean inhibition. The least effective weedicides found were imazethapyr 10% SL and oxyfluorfen 23.5% EC with mean mycelial growth inhibition of 2.99 and 1.88 percent, respectively. 54

13 Table 4.6: Effect of weedicides on mycelial growth inhibition and sclerotial formation of R. solani under in vitro condition Weedicides Growth inhibition (%) and sclerotial formation Mean inhibition 100 ppm 150 ppm 200 ppm 250 ppm (%) Fenoxaprop-pethyle 10% EC (6.65) (8.48) (13.87) (16.62) (11.40) Imazethapyr 10% SL 7.68 (1.91) 9.20 (2.66) (3.44) (3.98) 9.73 (2.99) Oxyfluorfen 23.5% EC 5.40 (1.03) 6.63 (1.35) 9.01 (2.50) 9.33 (2.67) 7.59 (1.88) Pendimethalin 30% EC (15.13) (38.83) (71.55) (88.96) (53.61) Propaquizafop 10% EC (52.43) (62.86) (63.80) (87.68) (66.69) Quizalofop-p-ethyle 5% EC (85.53) (87.54) (88.96) (99.98) (90.50) Weedicide (W) Conc. (C) W x C S. Em. ± C. D. at 5% C. V. % 4.47 Sclerotial formation: ++++=Abundant; +++ = good; ++ = moderate; + = scanty; - = no sclerotial formation. Values in parentheses are re-transformed values while outside were transformed with arcsine transformation before analysis. 55

14 Among various weedicides tested in laboratory condition, quizalofop-p-ethyl 5% EC gave 85.53, 87.54, and per cent inhibition of growth of the fungus at 100, 150, 200 and 250 ppm concentrations which was the best treatment for inhibiting the growth of mycelium of R. solani under in vitro condition. Second best treatment was propaquizafop 10% EC with 52.43, 62.86, 63.8 and per cent mycelial growth inhibition at 100, 150, 200 and 250 ppm. Pendimethalin 30% EC gave and per cent mycelial growth inhibition at 200 and 250 ppm concentrations, respectively. Whereas, fenoxaprop-p-ethyle 5% EC (6.65, 8.48, and per cent); imazethapyr 10% SL (1.91, 2.66, 3.44 and 3.98 per cent) and oxyfluorfen 23.5% EC (1.03, 1.35, 2.50 and 2.67 per cent) found least effective in inhibiting the mycelial growth at 100, 150, 200 and 250 ppm concentrations, respectively. The effect of different concentrations of weedicides on sclerotial formation was found negatively correlated with the inhibition of growth. No sclerotial formation was found in any concentration of quizalofop-p-ethyle 5% EC as well as in propaquizafop 10% EC and pendimethalin 30% EC at 250 ppm. Abundant sclerotial formation was found in all concentration of fenoxaprop-p-ethyle 10% EC, imazethapyr 10% SL and oxyfluorfen 23.5% EC as well as in pendimethalin 30% EC at 100 ppm. The present investigation was in close conformity with the similar type of findings as reported by Bauske and Kirby (1992). They noted that pendimethalin herbicide did not cause an increase in disease caused by R. solani on field location and soybean cultivar. On contrary to this, soybean treated with imazethapyr had increased Rhizoctonia disease severity levels both in green house and field experiments were reported by Bradley et al. (2002). However, inhibitory effect of quizalofop-p-ethyle against R. solani has also been reported by De et al (2007). 56

15 4.6 Effect of phytoextracts on mycelial growth inhibition and sclerotial formation of R. solani under in vitro condition The relative efficacy of different ten phytoextracts was tested at 5, 10 and 15 per cent concentrations using poison food technique. The data on per cent inhibition of mycelial growth and sclerotial formation were presented in Table 4.7, Plate-10 and depicted in Fig. 5. The data presented in Table 4.7 showed that Curcuma longa was the best among all phytoextracts and significantly inhibited the mycelial growth with mean inhibition of per cent. Whereas, Zinziber officinale was the next best followed by Parthenium hysterophorus with and per cent mean inhibition of mycelial growth of fungus. While, Azadirachta indica and Ocimum sanctum were the least effective phytoextracts with mean inhibition of 1.28 and 0.75 per cent, respectively. Among different phytoextracts tested under laboratory condition, the maximum inhibition of per cent was found in Curcuma longa at 15% concentration. Whereas, Parthenium hysterophorus (2.43, 3.13 and per cent), Allium sativum (7.33, 9.25 and per cent) and Zinziber officinale (15.37, and per cent) were the next best phytoextracts in inhibiting the mycelial growth of the fungus. While, Allium cepa (1.45, 1.69 and per cent), Pongamia pinnata (1.57, 1.78 and per cent), Tagetes erecta (1.76, 2.71 and per cent) and Adhatoda vasica (1.48, 7.1 and per cent) extracts were found moderately and equally effective in inhibiting the growth of the fungus at all concentration. It was observed that, Azadirachta indica (0.62, 1.57 and 1.67 per cent) and Ocimum sanctum (0.55, 0.79 and 0.92 per cent) extract remain less effective in inhibiting the mycelial growth of the fungus at all concentration tried. The abundant sclerotial formation was found in all treatment of phytoextracts at all concentration except Curcurma longa, which shows scanty sclerotial formation at 15 per cent concentration. The effectiveness of phytoextracts against R. solani was reported earlier by Patole and Narute (2011) in soybean. Similarly, Sonakar et al. (2014) also found inhibitory effect of Allium sativum on mycelium growth of R. solani in soybean. It is evident from the reports of same author in same year that out of different plant extracts tested in laboratory, Garlic and Madar exhibited and per cent inhibition, respectively against Rhizoctonia solani. 57

16 Table 4.7: Effect of phytoextracts on mycelial growth inhibition and sclerotial Phytoextracts formation of R. solani under in vitro condition Growth inhibition (%) and sclerotial formation 5% 10% 15% Mean inhibition (%) Adhatoda vasica Ness. (Ardusi) 6.92 (1.48) (7.10) (10.46) (6.34) Allium cepa L. (Onion) 6.88 (1.45) 7.47 (1.69) (18.07) (7.07) Allium sativum L. (Garlic) (7.33) (9.25) (30.45) (15.67) Azadirachta indica L. (Neem) 4.48 (0.62) 7.17 (1.57) 7.41 (1.67) 6.35 (1.28) Curcuma longa L. (Turmeric) (19.27) (19.49) (70.45) (36.40) Ocimum sanctum L. (Tulsi) 4.19 (0.55) 5.07 (0.79) 5.49 (0.92) 4.92 (0.75) Parthenium hysterophorus L. (Carrot grass) 8.73 (2.43) (3.13) (47.24) (17.60) Pongamia pinnata L. (Karanj) 7.19 (1.57) 7.65 (1.78) (16.47) (6.60) Tagetes erecta L. (Marigold) 7.57 (1.76) 9.37 (2.71) (15.46) (6.64) Zingiber officinale Rosc. (Ginger) (15.37) (19.24) (20.80) (18.47) Phytoextract (P) Conc. (C) P x C S. Em. ± C. D. at 5% C. V. % 6.80 Sclerotial formation: ++++=Abundant; +++ = good; ++ = moderate; + = scanty; - = no sclerotial formation. Values in parentheses are re-transformed values while outside were transformed with arcsine transformation before analysis. 58

17 4.7 Effect of biocontrol agents on mycelial growth inhibition and sclerotial formation of R. solani under in vitro condition The hazardous effect of chemicals used in plant disease management has diverted attention to find out the alternative methods having little or no adverse effect on environment. Notable success of disease control through the use of antagonistic microorganism in the laboratory, glass house and field have been achieved during past several years and based on this information, there is a possibility of developing biological control of plant disease under field conditions as well as in glass house. However, inadequate information on the performance of the antagonists under varying condition is a major constraints in the large scale adoption of this technology. In present study, different fungal and bacterial biological control agents are manipulated and results so obtained under laboratory conditions are communicated hereunder Effect of fungal biocontrol agents on mycelial growth inhibition and sclerotial formation of R. solani under in vitro condition An experiment was conducted to determine the antagonistic action of five fungal biocontrol agents viz, Trichoderma harzianum, T. virens, T. koningii, T. viride and T. hamatum against the test fungus by dual culture technique. Based on observations on radial growth of antagonist and test fungus per cent inhibition was calculated. The results presented in Table 4.8, Plate-11 and depicted in Fig. 6 revealed that all the biocontrol agents inhibited the growth of test fungus over the control. Out of five fungal biocontrol agents tested, Trichoderma harzianum showed significantly the maximum inhibition (66.10%) and remained statically at par with T. viride (64.36%) and T. koningii (63.03%). While, T. hamatum (53.91%) and T. virens (50.36%) found comparatively poor in inhibiting the mycelial growth of the fungus. Scanty sclerotial formation was found only in Trichoderma harzianum while moderate sclerotial formation was found in rest of the fungal biocontrol agents. The successfull suppression of R. solani by T. harzianum was earlier reported by Gveroska and Ziberoski (2011). The antagonistic effect of T. harzianum and T. viride against R. solani was also reported by Taha et al. (2012). Our result also in 59

18 harmony with Khan et al. (2014). They have studied the effects of T. harzianum, T. hamatum and T. viride against R. solani. Table 4.8: Effect of fungal biocontrol agents on mycelial growth inhibition and sclerotial formation of R. solani under in vitro condition Sr. No. Fungal biocontrol agents Per cent inhibition over control* 1. Trichoderma harzianum (66.10) 2. Trichoderma virens (50.36) 3. Trichoderma koningii (63.03) 4. Trichoderma viride (64.36) 5. Trichoderma hamatum (53.91) S. Em. ± 0.65 C. D. at 5% 1.95 C. V. % 2.57 Sclerotial formation * Average of four repetition Sclerotial formation: ++++=Abundant; +++ = good; ++ = moderate; + = scanty; - = no sclerotial formation. Values in parentheses are re-transformed value while outside were transformed with arcsine transformation before analysis Effect of bacterial biocontrol agents on mycelial growth inhibition and sclerotial formation of R. solani under in vitro condition An experiment was conducted to determine the antagonistic action of six bacterial biocontrol agents viz., Pseudomonas fluorescens 2, P. fluorescens 3, P. fluorescens 4, Bacillus subtilis 1, B. subtilis 2 and Bacillus cereus against the test fungus by dual culture technique. Based on observations on radial growth of test fungus on antagonist amended plates and control plates with test fungus, per cent inhibition was calculated. The results presented in Table 4.9, Plate-12 and depicted in Fig. 7 revealed that out of six bacterial biocontrol agents tested under in vitro condition, Pseudomonas fluorescence 4 showed significantly the maximum mycelial growth 60

19 inhibition of per cent. The next best treatment was P. fluorescens 2 with per cent mycelial growth inhibition. Whereas, P. fluorescens 3 recorded per cent mycelial growth inhibition, but it was remained at par with B. subtilis 2 with per cent mycelial growth inhibition. While, Bacillus cereus (11.14%) was least effective and remained statistically at par with Bacillus subtilis 1 (10.92 %) exhibiting poor mycelial growth inhibition. agents. There were abundant sclerotial formation was found in all bacterial biocontrol Table 4.9: Effect of bacterial biocontrol agents on mycelial growth inhibition and sclerotial formation of R. solani under in vitro condition Sr. No. Bacterial biocontrol agents Per cent inhibition over control* 1. Pseudomonas fluorescens (23.73) 2. Pseudomonas fluorescens (14.26) 3. Pseudomonas fluorescens (32.30) 4. Bacillus subtilis (10.92) 5. Bacillus subtilis (12.91) 6. Bacillus cereus (11.14) S. Em. ± 0.56 C. D. at 5% 1.67 C. V. % 4.64 * Average of four repetition Sclerotial formation: Sclerotial formation =Abundant; +++ = good; ++ = moderate; + = scanty; - = no sclerotial formation. Values in parentheses are re-transformed values while outside were transformed with arcsine transformation before analysis. 61

20 The present findings are corroborated with the results of Prasanna et al. (2010) who reported the antagonistic effect of P. fluorescens against R. solani. On the other hand Nihad and Mutlag (2015) studied that Pseudomonas and Bacillus genera were the most feasible biocontrol microorganism which suppress several pathogens like R. solani and reduced the incidence of tomato root rot and damping-off diseases caused by R. solani. 4.8 Isolation, purification and identification of rhizosphere microflora Antagonistic microflora from rhizosphere soil of coriander and fenugreek plants were isolated by serial dilution as per procedure given in section The fungal and bacterial rhizosphere microflora were isolated on Potato Dextrose Agar (PDA) and Nutrient Agar (NA) medium, respectively. A total of five fungal and six bacterial rhizosphere microflora were isolated from rhizosphere soil as given bellow. Name of isolates Name of plant Blast results Fungal isolate-1 Coriander plant Trichoderma viride-i Fungal isolate-2 Coriander plant Trichoderma viride-ii Fungal isolate-3 Fenugreek plant Trichoderma viride-iii Fungal isolate-4 Coriander plant Trichoderma longibrachiatum Fungal isolate-5 Fenugreek plant Trichoderma viride-iv Bacterial isolate-1 Coriander plant Psedomonas putida Bacterial isolate-2 Fenugreek plant Pseudomonas fluorescens Bacterial isolate-3 Coriander plant Bacillus subtilis Bacterial isolate-4 Coriander plant Enterobacter cloacae Bacterial isolate-5 Fenugreek plant Azotobacter vinelandi Bacterial isolate-6 Coriander plant Bacillus cereus The fungal antagonists were purified by single spore method and were maintained on PDA medium, while bacteria were purified by streak plate method and maintained on Nutrient agar medium. Fungi isolated from rhizosphere soil were designated as F1 to F5 and six bacteria were designated as B1 to B6. Based on colony, morphological and molecular characters, fungal and bacterial isolates were identified as mention in Table

21 Kumar et al. (2011) isolated five bacterial strains (TR1 to TR5) from root nodules of fenugreek (Trigonella foenum-graecum) were tested for their plant growth promotory traits and biocontrol potential against Fusarium oxysporum. On the basis of morphological, physiological, biochemical and molecular characteristics, strains TR1, TR3 and TR5 were identified as Ensifer meliloti and TR2 as Rhizobium legumeinosarum Molecular identification of microflora Fungal and bacterial biocontrol agents isolated from rhizosphere of coriander and fenugreek plants were identified by proper molecular tools i.e. 18S for fungi and 16S for bacteria. Agents which showed antagonistic effect against Rhizoctonia solani were identified and their sequences with identical per cent presented in Table Bhadania (2015) isolated 17 endophytic bacteria from wild malvaceae family plants among them isolates AIK35, AIB49, AIB52 and WC68 showed good antagonism towards growth of Fusarium oxysporum and Rhizoctonia solani. Based on 16s rrna analysis isolates, AIK35, AIB49, AIB52 and WC68 were identified as a Providencia rettgeri strain NCTC 11801, Microvirga zambiensis strain WSM3693, Providencia vermicola strain OP1 and Alcaligenes faecalis strain NBRC 13111, respectively. Cornelia et al. (2000) recognized about 35 Trichoderma species on the basis of morphological and molecular characters. Besides the role of a few of these species in biotechnology, several seem to play prominent roles in soil ecosystems. With a goal of investigating global biodiversity in Trichoderma isolates, there were no report on the occurrence of Trichoderma spp. in Russia (Moscow and Ural areas), Siberia (Krasnoyarsk area) and the Himalayan Mountains. The ITS1 and 2 sequence of the rdna cluster of the 75 isolates obtained was compared with that of ex-type strains and taxonomically established isolates of Trichoderma. Thirty-nine isolates were positively identified as T. atroviride, T. virens, T. hamatum, T. asperellum, T. koningii and T. oblongisporum. A further 26 isolates yielded six closely related ITS1 and 2 sequence types, which are highly similar yet different from the ex-(neo) type strains of T. harzianum and T. hamatum. 63

22 Table 4.10: Molecular identification of rhizosphere microflora of coriander and fenugreek Isolates Sequence (Request ID, BLAST) Identification (Percentage match) F1 F2 F3 F4 CCGAGTTTACAACTCCCAAACCCAATGTGAACCATACCAAACTGTTGCCTCGGCGGGGTCACGCCCCGGGTGCGTCGC AGCCCCGGAACCAGGCGCCCGCCGGAGGGACCAACCAAACTCTTTCTGTAGTCCCCTCGCGGACGTTATTTTTACAGC TCTGAGCAAAAATTCAAAATGAATCAAAACTTTCAACAACGGATCTCTTGGTTCTGGCATCGATGAAGAACGCAGCG AAATGCGATAAGTAATGTGAATTGCAGAATTCAGTGAATCATCGAATCTTTGAACGCACATTGCGCCCGCCAGTATTC TGGCGGGCATGCCTGTCCGAGCGTCATTTCAACCCTCGAACCCCTCCGGGGGGTCGGCGTTGGGGACTTCGGGAACCC CTAAGACGGGATCCCGGCCCCGAAATACAGTGGCGGTCTCGCCGCAGCCTCTCATGCGCAGTAGTTTGCACAACTCGC ACCGGGAGCGCGGCGCGTCCACGTCCGTAAAACACCCAACTTCTGAAATG (MA4E21SB013) CCGAGTTTACAACTCCCAAACCCAATGTGAACCATACCAAACTGTTGCCTCGGCGGGGTCACGCCCCGGGTGCGTCGC AGCCCCGGAACCAGGCGCCCGCCGGAGGGACCAACCAAACTCTTTCTGTAGTCCCCTCGCGGACGTTATTTTTACAGC TCTGAGCAAAAATTCAAAATGAATCAAAACTTTCAACAACGGATCTCTTGGTTCTGGCATCGATGAAGAACGCAGCG AAATGCGATAAGTAATGTGAATTGCAGAATTCAGTGAATCATCGAATCTTTGAACGCACATTGCGCCCGCCAGTATTC TGGCGGGCATGCCTGTCCGAGCGTCATTTCAACCCTCGAACCCCTCCGGGGGGTCGGCGTTGGGGACTTCGGGAACCC CTAAGACGGGATCCCGGCCCCGAAATACAGTGGCGGTCTCGCCGCAGCCTCTCATGCGCAGTAGTTTGCACAACTCGC ACCGGGAGCGCGGCGCGTCCACGTCCGTAAAACACCCAACTTCTGAAATG (MA4J3KVV016) CCGAGTTTACAACTCCCAAACCCAATGTGAACCATACCAAACTGTTGCCTCGGCGGGGTCACGCCCCGGGTGCGTCGC AGCCCCGGAACCAGGCGCCCGCCGGAGGGACCAACCAAACTCTTTCTGTAGTCCCCTCGCGGACGTTATTTTTACAGC TCTGAGCAAAAATTCAAAATGAATCAAAACTTTCAACAACGGATCTCTTGGTTCTGGCATCGATGAAGAACGCAGCG AAATGCGATAAGTAATGTGAATTGCAGAATTCAGTGAATCATCGAATCTTTGAACGCACATTGCGCCCGCCAGTATTC TGGCGGGCATGCCTGTCCGAGCGTCATTTCAACCCTCGAACCCCTCCGGGGGGTCGGCGTTGGGGACTTCGGGAACCC CTAAGACGGGATCCCGGCCCCGAAATACAGTGGCGGTCTCGCCGCAGCCTCTCATGCGCAGTAGTTTGCACAACTCGC ACCGGGAGCGCGGCGCGTCCACGTCCGTAAAACACCCAACTTCTGAAATG (MA4KKWSR013) TGTGAACGTTACCAATCTGTTGCCTCGGCGGGATTCTCTTGCCCCGGGCGCGTCGCAGCCCCGGATCCCATGGCGCCC GCCGGAGGACCAACTCCAAACTCTTTTTTTCTCTCCCGTCGCGGCTCTGTTTTATTTTTGCTCTGAGCCTTTCTCGGCGA CCCTAGCGGGCGTCTCGAAAATGAATCAAAACTTTCAACAACGGATCTCTTGGTTCTGGCATCGATGAAGAACGCAGC GAAATGCGATAAGTAATGTGAATTGCAGAATTCAGTGAATCATCGAATCTTTGAACGCACATTGCGCCCGCCAGTATT CTGGCGGGCATGCCTGTCCGAGCGTCATTTCAACCCTCGAACCCCTCCGGGGGGTCGGCGTTGGGGATCGGCCCTCAC CGGGCCGCCCCCGAAATACAGTGGCGGTCTCGCCGCAGCCTCTCCTGCGAGTAGTTTGCACACTCGCACCGGGAGCGC GGCGCGGCCA (MA4NAK1H013) Trichoderma viride-i strain (100 % identity) Trichoderma viride-ii strain (100 % identity) Trichoderma viride-iii strain (100 % identity) Trichoderma longibrachiatum strain (100 % identity) 64

23 F5 B1 B2 CCGAGTTTACAACTCCCAAACCCAATGTGAACCATACCAAACTGTTGCCTCGGCGGGGTCACGCCCCGGGTGCGTCGC AGCCCCGGAACCAGGCGCCCGCCGGAGGGACCAACCAAACTCTTTCTGTAGTCCCCTCGCGGACGTTATTTTTACAGC TCTGAGCAAAAATTCAAAATGAATCAAAACTTTCAACAACGGATCTCTTGGTTCTGGCATCGATGAAGAACGCAGCG AAATGCGATAAGTAATGTGAATTGCAGAATTCAGTGAATCATCGAATCTTTGAACGCACATTGCGCCCGCCAGTATTC TGGCGGGCATGCCTGTCCGAGCGTCATTTCAACCCTCGAACCCCTCCGGGGGGTCGGCGTTGGGGACTTCGGGAACCC CTAAGACGGGATCCCGGCCCCGAAATACAGTGGCGGTCTCGCCGCAGCCTCTCATGCGCAGTAGTTTGCACAACTCGC ACCGGGAGCGCGGCGCGTCCACGTCCGTAAAACACCCAACTTCTGAAATG (MA4PXH0T013) AGGCGACGATCCGTAACTGGTCTGAGAGGATGATCAGTCACACTGGAACTGAGACACGGTCCAGACTCCTACGGGAG GCAGCAGTGGGGAATATTGGACAATGGGCGAAAGCCTGATCCAGCCATGCCGCGTGTGTGAAGAAGGTCTTCGGATT GTAAAGCACTTTAAGTTTTTTTGGGGAGGGCAGTAAGCTAATACCTTGCTGTTTTGACGTTACCGACAGAATAAGCAC CGGCTAACTCTGTGCCAGCAGCCGCGGTAATACAGAGGGTGCAAGCGTTAATCGGAATTACTGGGCGTAAAGCGCGC GTAGGTGGTTCGTTAAGTTGGATGTGAAAGCCCCGGGCTCAACCTGGGAACTGCATCCAAAACTGGCGAGCTAGAGT ACGGTAGAGGGTGGTGGAATTTCCTGTGTAGCGGTGAAATGCGTAGATATAGGAAGGAACACCAGTGGCGAAGGCG ATGGACTGATACTGACACTGAGGTGTTGCCCGAAAGCGTGGGGAGCAAACAGGATTAGATACCCTGGTAGTCCACGC CGTAAACGATGTCAACTAGCCGTTGGAATCCTTGAGATTTTAGTGGCGCAGCTAACGCATTAAGTTGACCGCCTGGGG AGTACGGCCGCAAGGTTAAAACTCAAATGAATTGACGGGGGCCCGCACAAGCGGTGGAGCATGTGGTTTAATTCGAA (MA4SSGVH013) GGGTACTTGTACCTGGTGGCGAGCGGCGGACGGGTGAGTAATGCCTAGGAATCTGCCTAGTAGTGGGGGATAACGTC CGGAAACGGGCGCTAATACCGCATACGTCCTACGGGAGAAAGTGGGGGATCTTCGGACCTCACGCTATTAGATGAGC CTAGGTCGGATTAGCTAGTTGGTGAGGTAATCAAGGCGACGATCCGTAAGAATTCACCTGGTCTGAGAGGATGATCA GTCACACTGGAACTGAGACACGGTCCAGACTCCTACGGGAGGCAGCAGTGGGGAATATTGGACAATGGGCGAAAGC CTGATCCAGCCATGCCGCGTGTGTGAAGAAGGTCTTCGGATTGTAAAGCACTTTAAGTTGGGAGGAAGGGCAGTTAC CTAATACGTGATTGTTTTGACGTTACCGACAGAATAAGCACCGGCTAACTCTGTGCCAGCAGCCGCGGTTGCAAGCGT TAATCGGAATTACTGGGCCCCAAATTTTTTGTAAAGCGCGCGTAGGTGGTTTGTTAAGTTGGATGTGAAAGCCCCGGG CTCAACCTGGGAACTGCATCCAAAACTGGCAAGCTAGAGTATGGTAGAGGGTGGTGGAATTTCCTGTGTAGCGGTGA AATGCGTAGATATAGGAAGGAACACCAGTGGCGAAGGCGACCACCTGGACTGATACTGACACTGAGGTGCGAAAGC GTGGGGAGCAAACAGGATTAGATACCCTGGGCCGTAAACGATGTCAACTAGCCGTTGGGAGCCTTGAGCTCTTTTTTG CCTTAGTGGCGCAGCTAACGCATTAAGTTGACCGCCTGGGGAGTACGGCCGCAAGGTTAAAACTCAAATGAATTGAC GGGGGCCCGCACAAGCGGTGGAGCATGTGGTTTAATTCGAAGCAACGCGAAGAACCTTACCAGGCCTTGACATCCAA TGAACTTTCTAGAGATAGATTGGTGCCTTCGGGAACATTGAGACAGGTGCT (MA52JNKV016) Trichoderma viride-iv strain (100 % identity) Psedomonas putida strain (100 % identity) Pseudomonas fluorescens strain (95 % identity) 65 61

24 B3 B4 GCAAGTCGAGCGGACAGATGGGAGCTTGCTCCCTGATGTTAGCGGCGGACGGGTGAGTAACACGTGGGTAACCTGCC TGTAAGACTGGGATAACTCCGGGAAACCGGGGCTAATACCGGATGGTTGTTTGAACCGCATGGTTCAGACATAAAAG GTGGCTTCGGCTACCACTTACAGATGGACCCGCGGCGCATTAGCTAGTTGGTGAGGTAACGGCTCACCAAGGCGACG ATGCGTAGCCGACCTGAGAGGGTGATCGGCCACACTGGGACTGAGACACGGCCCAGACTCCTACGGGAGGCAGCAGT AGGGAATCTTCCGCAATGGACGAAAGTCTGACGGAGCAACGCCGCGTGAGTGATGAAGGTTTTCGGATCGTAAAGCT CTGTTGTTAGGGATAACGGAGTGCCGTTCAAATAGGGCGGCACCTTGACGGTACCTAACCAGAAAGCCACGGCTAAC TACGTGCCAGCAGCCGCGGTAATACGTAGGTGGCAAGCGTTGTCCGGAATTATTGGGCGTAAAGGGCTCGCAGGCGG TTTCTTAAGTCTGATGTGAAAGCCCCCGGCTCAACCGGGGAGAAAACATTGGAAACTGGGGAACTTGAGTGCAGAAG AGGAGAGTGGAATTCCACGTGTAGCGGTGAAATGCGTAGAGATGTGGAGGAACACCAGTGGCGAAGGCGACTCTCTG GTCTGTAACTGACGCTGAGGAGCGAAAGCGTGGGGAGCGAACAGGATTAGATACCCTTTGGATCCACGCCGTAAACG ATGAGTGCTAAGTGTTAGGGGGTTTCCGCCCCTTAGTGCTGCAGCTAACGCATTAAGCACTCCGCCTGGGGGAGTACG GTCGCAAGACTGAAACTCAAAGGAATTGACGGGGGCCCGCACAAGCGGTGGAGCATGTGGTTTAATTCGAAGCAACG CGAAGAA (MA53YM7D01R) GAACGGTAGCACAGAGAGCTTGCTCTCGGGTGACGAGTGGCGGACGGGTGAGTAATGTCTGGGAAACTGCCTGATGG AGGGGGATAACTACTGGAAACGGTAGCTAATACCGCATAACGTCGCAAGACCAAAGAGGGGGACCTTCGGGCCTCTT GCCATCAGATGTGCCCAGATGGGATTAGCTAGTAGGTGGGGTAACGGCTCACCTAGGCGACGATCCCTAGCTGGTCT GAGAGGATGAGGCCTTTAACACTGGAACTGAGACACGGTCCAGACTCCTACGGGAGGCAGCAGTGGGGAATATTGCA CAATGGGCGCAAGCCTGATGCAGCCATGCCGCGTGTATGAAGAAGGCCTTCGGGTTGTAAAGTACTTTCAGCGGGGA GGAAGGTGTTGTGGTTAATAACCACAGCAATTGACGTTACCCGCAGAAGAAGCACCGGCTAACTCCGTGCCAGCAGC CGCGGTAATACGGAGGGTGCAAGCGTTAATCGGAATTACTGGGCGTAAAGCGCACGCAGGCGGTCTGTCAAGTCGGA TGTGAAATCCCCGGGCTCAACCTGGGAACTGCATTCGAAACTGGCAGGCTGGAGTCTTGTAGAGGGGGGTAGAATTC CAGGTGTAGCGGTGAAATGCGTAGAGATCTGGAGGAATACCGGTGGCGAAGGCGCTGGACAAAGACTGACGCTCAG GTGCGTTTTTTAAAGCGTGGGGAGCAAACAGGATTAGATACCCTGGTAGTCCACGCCGTAAACGATGTCGATTTGGAG GTTGTGCCCTTGAGGCGTGGCTTCCGGAGCTAACGCGTTAAATCGACCGCCTGGGGAGTACGGCCGCAAGGTTAAAA CTCAAATGAATTGACGGGGGCCCGCACAAGCGGTGGAGCATGTGGTTTAATTCGATGCAACGCGAAGAACCTTACCT GGTCTTGACATCCACAGAACTTTCCAGAGATGGATTGGTGCCTTCGGGAACTGTGAGACAGGTGCTGCATGGCTGTCG TCAGCTCGTGTTGTGAAATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCTTATCCTTTGTTGCCAGCGGTTAGGCCG GGAACTCAAAGGAGACTGCCAGTGATAAACTGGAGGAAGGTGGGGATGACGTCAAGTCATCATGGCCCTTACGACCA GGGCTACACACG (MA5AUTDT013) Bacillus subtilis strain (99 % identity) Enterobacter cloacae strain (99 % identity) 66 62

25 B5 B6 TTGAGTTTGATCCCTGGCTCAGATTGAACGCTGGCGGCAGGCCTAACACATGCAAGTCGAGCGGCAGCGGGACCTTC GGGTTGCCGGCGAGCGGCGGACGGGTGAGTAATGCCTAGGAATCTGCCTGTTAGTGGGGGATAACGCGGGGAAACTC GCGCTAATACCGCATACGTCCTACGGGAGAAAGCGGGGGCTCTTCGGACCTCGCGCTAACAGATGAGCCTAGGTCGG ATTAGCTAGTTGGTGGGGTAATGGCCCACCAAGGCGACGATCCGTAACTGGTCTGAGAGGATGATCAGTCACACTGG AACTGAGACACGGTCCAGACTCCTACGGGAGGCAGCAGTGGGGAATATTGGACAATGGGCGAAAGCCTGATCCAGCC ATGCCGCGTGTGTGAAGAAGGTCTTCGGATTGTAAAGCACTTTAAGTCGGGAGGAAGGGCTGTAGGCTAATACCTTG CAGTTTTGACGTTACCGACAGAATAAGCACCGGCTAACTTCGTGCCAGCAGCCGCCGATTCCTACGAAGGGTGCAAG CGTTAATCGGAATTACTGGGCGTAAAGCGCGCGTAGGTGGTTCAGCAAGTTGGATGTGAAAGCCCCGGGCTCAACCT GGGAACTGCATCCAAAACTACTGGGCTAGAGTACGGTAGAGGGTGGTGGAATTTCCTGTGTAGCGGTGAAATGCGTA GATATAGGAAGGAACACCAGTGGCGAAGGCGACCACCTGGACTGATACTGATGCGAAAGCGTGGGGAGCAAACAGG ATTAGATACCCTGGTAGTCCACGCCGTAAACGATGTCGACTAGCCGTTGGGCTCCTTGAGAGCTTAGTGGCGCAGCTA ACGCATTAAGTCGACCGCCTGGGGAGTACGGCCGCAAGGTTAAAACTCAAATGAATTGACGGGGGCCCGCACAAGCG GTGGAGCATGTGGTTTAATTCGAAGCAACGCGAAGAACCTTACCTGGCCTTGACATCCTGCGAACTTGGTAGAGATAC CTTTCGGGAGCGCAGAGACAGGTGCTGCATGGCTGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGTAACG AGCGCAACCCTTGTCCTTAGTTACCAGCACCTCGGGTGGGCACTCTAAGGAGACTGCCGGTGACAAACCGGAGGAAG GTGGGGATGACGTCAAGTCATCATGGCCCTTACGGCCAGGGCTACACACGTGCTACAATGGTCGGTACAGAGGGTTG CCAAGCCGCGAGG (MA5DUX07013) CAGGCTCAGGATGAACGCTGGCGGCGTGCCTAATACATGCAAGTCGAGCGAACGGATTAAGAGCTTGCTCTTAAGAA GTTAGCGGCGGACGGGTGAGTAACACGTGGGTAACCTGCCCATAAGACTGGGATAACTCCGGGAAACCGGGGCTAAT ACCGGATAACATCTAGCACCGCATGGTGCAAGATTGAAAGGCGGCTTCGGCTGTCACTTATGGATGGACCCGCGTCG CATTAGCTAGTTGGTGAGGTAACGGCTCACCAAGGCGACGATGCGTAGCCGACCTGAGAGGGTGATCGGCCACACTG GGACTGAGACACGGCCCAGACTCCTACGGGAGGCAGCAGTAGGGAATCTTCCGCAATGGACGAAAGTCTGACGGAG CAACGCCGCGTGAGCGATGAAGGCCTTCGGGTCGTAAAGCTCTGTTGTTAGGGAAGAACAAGTGTGAGTTGAATAAG CTCATGCCTTGACGGTACCTAACCAGAAAGCCACGGCTAACTACGTGCCAGCAGCCGCGGTAATACCAAGCGTTATCC GGAATTATTGGGCGTAAAGCGCGCGCAGGCGGTTTCTTAAGTCTGATGTGAAAGCCCACGGCTCAACCGTGGAGGGT CATTGGAAACTGGGAGACTTGAGTGCAGAAGAGGAGAGTGGAATTCCATGTGTAGCGGTGAAATGCGTAGAGATATG GAGGAACACCAGTGGCGAAGGCGACTCTCTGGTCTGTAACTGTGAGGCGCGAAAGCGTGGGGAGCAAACAGGATTA GATACCCTGGTAGTCCACGCCGTAAACGATGAGTGCTAAGTGTTAGAGGGTTTCCGCCCTTTAGTGCTGAAGTTAACG CATTAAGCACCCTGGGGAGTACGGCCGCAAGGCTGAAACTCAAAGGAATTGACGGGGGCCCGCACAAGCGGTGGAG CATGTGGTTTAATTCGAAGCAACGCGAAGAACCTTACCAGGTCTTGACATCCTCTGACAACCCTAGAGATAGGGCTTC CCCTTCGGGGGCAGAGTGACAGGTGGTGCATGGTTGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGCAAC GAGCGCAACCCTTGATCTTAGTTGCCATCATTCAGTTGGGCACTCTAAGGTGACTGCCGGTGACAAACCGGAGGAAG GTGGGGATGACGTCAAAT (MA5DUPDP013) Azotobacter vinelandi strain (98 % identity) Bacillus cereus strain (99 % identity) 63 67

26 4.8.2 Effect of rhizosphere fungal antagonists on mycelial growth inhibition and sclerotial formation of R. solani under in vitro condition An experiment was conducted to determine the antagonistic action of fungal antagonists from coriander and fenugreek rhizosphere. Five fungal antagonists of Trichoderma were tested against the test fungus by dual culture technique. Based on observations on radial growth of antagonist and test fungus, per cent inhibition was calculated. The results presented in Table 4.11, Plate-13 and depicted in Fig. 8 revealed that all the fungal antagonists tested against R. solani were effective in checking the growth of the pathogen. Out of five antagonists tested Trichoderma longibrachiatum showed significantly the maximum mycelial growth inhibition (65.62%) which was statistically remained at par with T. viride 3 (63.24%) and T. viride 2 (62.63%). While, Trichoderma viride 4 (55.22%) was statistically at par with T. viride 1 (54.36%) and inhibited minimum fungal growth. Table 4.11: Effect of rhizosphere fungal antagonists on mycelial growth inhibition and sclerotial formation of R. solani under in vitro condition Sr. No. Rhizosphere fungal antagonists Per cent inhibition over control* (%) 1. Trichoderma viride (54.36) 2. Trichoderma viride (62.63) 3. Trichoderma viride (63.24) 4. Trichoderma longibrachiatum (65.62) 5. Trichoderma viride (55.22) S. Em.± 0.85 C. D. at 5% 2.57 C. V. % 3.35 Sclerotial formation *Average of four repetitions Sclerotial formation: ++++ = Abundant; +++ = good; ++ = moderate; + = scanty; - = no sclerotial formation. Values in parentheses are re-transformed values while outside were transformed with arcsine transformation before analysis. 68