Bioefficacy of Plant Extracts and Biocontrol Agents Against Alternaria Solani

Full Length Research Paper

Bioefficacy of plant extracts and biocontrol agents against Alternaria solani

S. A. Ganie*, M. Y. Ghani, Qazi Nissar and Shabir-u-Rehman

Division of Plant Pathology, Sher-e-Kashmir University of Agricultural Sciences and Technology, Shalimar, Srinagar-191 121, India.

Accepted 22 July, 2013

Three bioagents viz., Trichoderma harzianum Rifai, Trichoderma viride Pers. and Trichoderma virens (Miller et al) von Arx and five plant extracts viz., Artimesia absinthium L., Datura stramonium L., Urtica dioica L., Juglans regia L. and Mentha arvensis L. were evaluated in vitro against Alternaria solani (Ellis and Martin) Jones and Grout causing early blight of potato through dual culture and poisoned food technique, respectively. Among bioagents, significantly higher mycelial growth inhibition of A. solani was recorded in the case of T. harzianum (71.85%), which was followed by T. viride (65.93%) and T. virens (58.65%) proved least effective in inhibiting the mycelial growth of A. solani. Among plant extracts, D. stramonium proved superior to all other botanicals, exhibiting (61.12%) mycelial growth inhibition of A. solani. This was followed by A. absinthium (58.54%). Urtica dioica (37.34%) proved least effective in inhibiting the mycelial growth of the test fungus.

Key words: Bioagents, plant extracts, potato, Alternaria solani, dual culture, early blight.

INTRODUCTION

Potato (Solanum tuberosum L.) is one of the most of Jammu and Kashmir like Gurez, Gulmarg, Tangmarg, valuable non-cereal food crops, grown in most of the Shopian, etc. where it serves as a staple food. An overall temperature and subtropical countries of world. It production scenario of potato in Jammu and Kashmir contribute highest amount of dry matter, protein and other depicts that area, production and productivity have nutrients per unit area and time. Potato is a unique crop sharply increased in the past decade. Although, it is which can supplement the food needs of the country in a being grown in Kashmir for many years but only few substantial manner. Potato is grown in an area of 1.3 varieties are in cultivation which are either poor yielders, m/ha, with production 24 mt and productivity 18.5 t/ha. low in quality/and or susceptible to various pests and Today, India ranks fourth in the area and fifth in the diseases. production of potato in the world (Shailbala and Pathak, The intensive and extensive cultivation under most 2008). environmental conditions for potato crop production in the In 1998-1999, India produced 22.50 million tones of state failed to provide significant strides in potato yields, potato from an area of 1.28 million hectares. The year because of a number of production constraints, of which 2008 was celebrated for “International Potato Year” by frequent occurrence of many fungal diseases viz., early United States organization dated 18th October, 2007 blight (Alternaria solani), late blight (Phytophthora (Shailbala and Pathak, 2008). Potato is a highly infestans), powdery scab (Spongospora subterranean), remunerative and nutritive crop in Jammu and Kashmir Wart (Synchytrium endobioticum), leaf black (Cercospora (J&K) particularly in high altitude cold and cold arid areas concors), Fusarium Wilt (Fusarium solani f.sp. radicicola),

*Corresponding author. E-mail: [email protected]. 4398 Afr. J. Microbiol. Res.

Table 1. The ethanol extracts of the botanicals tested against leaf spot disease of potato.

Common name Local name Botanical name Plant part used Datura Datur Datura stramonium L. Leaves Artimesia Tethvan Artimesia absinthium L. Leaves Nettle Soi Urtica dioica L. Leaves Mint Podina Mentha arvensis L. Leaves Walnut Doon Juglans regia L. Bark

black scurf (Rhizoctonia solani) and charcoal rot triggering systematic disease resistance mechanism, (Macrophomina phaseolina) are note worthy and have enhancing the uptake of water and nutrients especially been taking heavy toll of the produce. Early blight of nitrogen and by increasing photosynthesis of host plants. potato is one of the most important foliar diseases of The present investigation was undertaken to study the potato worldwide (Christ, 1990; Pelletier and Fry, 1990; bioefficacy of plant extracts and biocontrol agents against Shtienberg et al., 1990; Vander-Walls et al., 2001). In A. solani to devise suitable management programme of recent years, increase in A. solani disease on potato the disease. foliage has been reported in various potato growing areas (Vloutoglou and Kalogerakis, 2000). Primary damage by early blight is attributed to premature defoliation of the MATERIALS AND METHODS potato plants, resulting in tuber yield reduction. The pathogen can also attack potato tubers and produce The plant extracts were evaluated in vitro through poisoned food technique (Carpenter, 1942; Nene and Thapiyal, 1993). For shallow, dry, corky rot (Folsom and Bonde, 1925; O’Brien obtaining ethanol extracts, fresh plant material was collected, and Rich, 1976). washed with sterilized distilled water and dried at room Although, satisfactory control of the disease by using temperature, crushed and suspended in 80% ethanol and filtered various chemicals has been documented (Choulwar and after one hour through Whatman No. 1 filter paper. These where Datar, 1988; Maheshwari et al., 1991), the continuous evaporated to dryness on a water bath (40 ± 2°C), on cooling, their aqueous suspensions were prepared in the ratio of 1:1 (W/v) by use of these agrochemicals for controlling the disease adding sterilized distilled water. may pose several problems like toxicity to non-target The desired concentration, that is, 10, 20, 30, 40, 50 and 60% of organisms, development of resistance in the populations the test extracts was added to potato dextrose broth in sterilized of the pathogen and environmental pollution. Bioagents conical flasks. The medium without extract served as check. The and plant extracts are considered as new rays of hope experiment was conducted in Completely Randomised design because they are ecofriendly and can be used as an (CRD) with six treatments and three replications. Each flask was inoculated with 3 mm diameter mycelial disc taken from 10 days old effective alternative measure to control plant diseases. culture with the help of cork borer, raised on potato dextrose agar Several workers have reviewed the various medium (PDA). developments in biological control of plant pathogens The inoculated flasks were incubated at 23±1°C for 15 days. (Papavizas, 1985; Cook, 1993; Mukhopadhyay, 1994; After incubation, the medium containing the mycelial growth of the Jayaraj and Ramabadron, 1996; Dube, 2001; Ashwani et fungus was filtered through previously weighed Whatman filter al., 2004; Sendhilvel et al., 2005; Shalini and Dohroo, paper No. 41. The mycelial mat on filter paper was oven dried at 60°C and weighed. The dry mycelial weight was calculated by 2005; Harman, 2006). Several plant extracts are known subtracting weight of previously weighed filter paper from weight of to possess antimicrobial properties and are therefore, filter paper with mycelial mat. Percent inhibition of mycelial growth being exploited to achieve control over various plant was calculated using the formula of Vincent (1947): ailments (Mishra and Dixit, 1977). Skinner (1955) C – T suggested that the presence of some antibiotic Per cent inhibition = x 100 constituents or some unknown substances contribute to C the inhibitory activity of the plant extracts. Recently, plant extracts drawn from the various parts of certain plant Where, C = Weight of fungal colony in control (mg); T = weight of fungal colony in treatment (mg). The ethanol extracts of the species have been successfully tested to demonstrate botanicals were tested against leaf spot disease of potato (Table 1). their antifungal activities (Raja and Kurucheve, 1998; Antagonistic activity of various fungal antagonists viz., Datar, 1999; Ranjan et al., 1999; Singh and Navi, 2000; Trichoderma harzianum Rifai, Trichoderma viride Pers. and Bowers and Locke, 2000). The antifungal activities of Trichoderma virens (Miller et al.)von Arx were tested in vitro against different plant extracts against many plant pathogenic Alternaria solani by dual culture technique (Utkhede and Rahe, fungi have been well documented (Dubey and Dwivedi, 1983). Five millimeters discs of seven day old cultures of test pathogen as well as biocontrol agents were taken with the help of a 1991; Biswas et al., 1995). Biologicals are a natural way cork borer. Two discs, one each of pathogen and biocontrol agent, to improve agricultural production and plant yields by were placed equidistantly (60 mm) apart in each 90 mm petriplate enhancing anti-oxidative potential of the host plant, containing PDA under aseptic conditions. The petriplates containing Ganie et al. 4399

Table 2. In vitro efficacy of ethanol extracts of various botanicals in inhibiting the mycelial growth of A. solani.

Conc. (%) Percent inhibition of mycelial growth over control Botanical 10 20 30 40 50 60 Mean Artimesia (Artimesia absinthium) 44.77* (6.69) 52.12 (7.21) 59.31 (7.70) 61.44 (7.83) 65.19 (8.07) 68.41 (8.27) 58.54 (7.65) Datura (Datura strmonium) 46.40 (6.81) 56.16 (7.49) 61.81 (7.86) 63.87 (7.99) 66.95 (8.18) 71.57 (8.45) 61.12 (7.81) Nettle (Urtica dioica) 27.65 (5.25) 30.50 (5.52) 33.15 (5.75) 40.07 (6.33) 44.50 (6.67) 48.22 (6.94) 37.34 (6.11) Walnut (Juglans regia) 28.49 (5.33) 33.87 (5.81) 38.35 (6.19) 40.50 (6.36) 43.01 (6.55) 45.69 (6.75) 38.31 (6.18) Mint (Mentha arvensis) 21.72 (4.66) 30.84 (5.55) 35.01 (5.91) 41.11 (6.41) 46.49 (6.81) 52.96 (7.27) 38.02 (6.16) Mean 33.68 (5.80) 40.69 (6.37) 45.52 (6.74) 49.39 (7.02) 53.22 (7.29) 57.37 (7.57)

CD(p=0.05); Treatment: 0.17; concentration: 0.19; treatment x concentration: 0.43. *Mean of three replications; *figures in parentheses are arc sine transformed values.

PDA without biocontrol agent served as control. Each Plant Pathology SKUAST-K, Shalimar. These biocontrol a minimum of inhibition of 33.68% at 10% and treatment was replicated four times. The experiment was agents were maintained on PDA by periodic sub-culture at increased gradually to 57.37% at 60% laid out in a completely randomized design with three monthly intervals. Mass culture of fungal biocontrol agents concentration. Data further revealed a significant treatments and three replications. The petriplates were was made on wheat bran in 500 ml Erlenmeyer flasks. The incubated at 23 ± 2°C. Observations on the growth of data was subjected to statistical analysis as per the interaction between botanical and concentration. biocontrol agent and pathogen were recorded after 10 days methods described by Panse and Sukhatame (1978). The All the botanicals showed fifty percent mycelial of incubation and percent mycelial inhibition was calculated software used for analysis was Minitab. growth inhibition at 60% concentration or beyond according to Vincent (1947). Based on the growth and except Urtica dioica and Juglans regia which mycoparasitic nature, biocontrol agents were grouped into exhibited only 48.22 and 45.69% inhibition, various categories as per the scale given by Bell et al. RESULTS (1982) with slight modifications (Munshi and Dar, 2004): respectively. Among the botanicals tested at 50 and 60% 1. Strong antagonist: Growth of biocontrol agent was very The efficacy of different botanicals against A. concentration, D. stramonim proved superior with fast, it covered the entire medium surface and completely solani was evaluated in vitro by poisoned food mycelial growth inhibition of 66.95 and 71.57% overgrew the pathogen. technique. The data on in vitro efficacy of test over check. This was closely followed by A. 2. Antagonistic: Growth of biocontrol agent was very fast, it covered at least 2/3rd medium surface but without showing botanicals in inhibiting the mycelial growth of absinthium 50 and 60% concentration with mycoparasitic action. Alternaria solani is presented in Table 2. An mycelial growth inhibition of 65.19 and 68.41%, 3. Moderate antagonist: Growth of biocontrol agent was insight into the data reveals that all the botanicals respectively over the control. At lowest very slow but rapidly it overgrew the pathogenic one in significantly inhibited mycelial growth of A. solani concentration of 10 and 20%, D. stramonium and contact. at all concentrations tested. Datura stramonium A. absinthium were superior to other test 4. Slow antagonist: Growth of biocontrol agent and pathogen was similar in magnitude, none appeared to be proved significantly superior to all other botanicals botanicals. Similarly, at 30% concentration, D. dominant to the other. exhibiting 61.12% of mycelial growth inhibition of stramonium and A. absinthium proved effective 5. Poor antagonist: Growth of the pathogen was fast, it the test fungus. This was followed by Artimesia over the control. Urtica dioica, Mentha arvensis colonized at least 2/3rd of the surface and appeared to absinthium (58.54%). The other botanicals in and Juglans regia exhibited relatively least withstand encroachment by the biocontrol agent. order of their efficacy were Juglans regia inhibition over the control at all test concen- 6. Non antagonist: Growth of the pathogen was very fast, it (38.31%)>Mentha arvensis (38.02%)>Urtica trations. completely overgrew the biocontrol agent and covered the entire medium surface. dioica (37.34%). On an overall basis, the extent of Three biocontrol agents viz., T. harzianum, T. Pure culture of biocontrol agents viz., T. viride, T. mycelial growth inhibition by the test botanicals viride and T. virens were screened in vitro against harzianum and T. virens were obtained from Division of increased with increase in their concentration with A. solani by dual culture technique on potato 4400 Afr. J. Microbiol. Res.

Table 3. In vitro effect of various antagonists on mycelial growth of A. solani in dual culture.

Antagonist Growth inhibition (%) Zone of inhibition* Trichoderma harzianum 71.85 (30.19) - Trichoderma viride 65.93 (33.62) + Trichoderma virens 58.65 (37.85) - CD (p = 0.05) 2.88

*+ Inhibition zone present; - inhibition zone absent; figures in parenthesis are arc sine transformed values.

dextrose agar medium. Data on radial mycelial growth, of mycelial growth with increase in their concentration. D. zone of inhibition and degree of antagonism recorded 10 stramonium (66.95 and 71.57%) and A. absinthium days after incubation at 25 ± 2C is presented in Table 3. (65.19 and 68.41 %) proved significantly superior at 50 All the test isolates significantly inhibited the mycelial and 60% concentration. At lower concentration of 10 and growth of test pathogen when compared with the control 20% also, D. stramonium (46.40 and 56.16%) and A. (Table 3). T. harzianum was superior to all other test absinthium (44.77 and 52.12%) were significantly isolates by exhibiting the maximum mycelial growth superior to the rest of the test botanicals, respectively. inhibition of 71.85%. T. harzianum did not show any zone The presence of antibiotic constituent in the form of of inhibition but completely covered the mycelial growth phenolic, resinous, gummy and non-volatile substances of test pathogen in 10 days and thus was highly of unknown nature in different botanicals is reported by antagonistic in nature. The next best biocontrol agent T. Skinner (1955). Amonkar and Banergi (1971) have viride exhibited radial mycelial growth inhibition of attributed such antimicrobial properties to the presence of 65.93%. It was observed that T. viride mycoparasitized A. diallyl-disulphide and diallyl-tri-sulphide in Allium sativum solani in dual culture within 11 days of incubation. It also and fixed oils in D. stramonium. Similar results on the showed zone of inhibition. Among the biocontrol agents, efficacy of different plant extracts against Alternaria spp. T. virens was least effective and inhibited 58.65% have been reported by Roopa (2012). Several workers mycelial growth of A. solani. T. virens neither colonized while studying the in vitro effect of different plant extracts the fungal mycelium of the pathogen nor mycoparasitized on Fusarium solani and other Fusarium spp. have it; which was accordingly categorized as moderate reported almost similar findings (Vimala et al., 1993; Arya antagonist. et al., 1995; Lolpuri, 2002). The presence of essential oils in garlic (A. sativum) and tulsi (Ocimum sanctum) fixed oils in datura (D. alba) and canabinol in cannabis DISCUSSION (Canabis sativa) are also considered responsible for such inhibitions (Anonymous, 1972). Vanitha (2010) reported Currently, efforts are being made to manage plant that EC formulation of winter green oil exhibited 100% diseases through the use of different plant extracts or inhibition of mycelial growth of Alternaria chlamydospora. their products. A number of plants have been reported to Similar results on the efficacy of different plant extracts possess antifungal activity (Shekhawat and Prasada, against A. alternata have also been reported by Feng and 1971; Dixit and Tripathi, 1975; Neeraj and Verma, 2010; Zheng (2007), Raghavendra et al. (2009) and Zaker and Derbalah et al., 2011). These include bulb extracts of Mosallanejad (2010). Suleiman (2010) reported that garlic and and leaf extracts of datura and mentha methnol extracts of leaves of pawpaw showed highest (Shivpuri et al., 1997; Shivpuri and Gupta, 2001; mycelial growth inhibition against A. solani. Ravikumar Chattopadhyay et al., 2002; Singh et al., 2003). and Rajkumar (2013) reported that out of the 39 plants In the present study, efforts were made to explore the selected, 13 plant extracts significantly reduced the possibility of using extracts of locally available plants for mycelial growth of A. solani. the management of Alternaria leaf spot of potato. In vitro The in vitro bioassay of biocontrol agents revealed that evaluation revealed that all the five test extracts at all the three biocontrol agents tested significantly various concentrations were significantly effective in inhibited the radial mycelial growth of A. solani. Among inhibiting the mycelial growth of A. solani. It was the antagonists, T. harzianum caused maximum mycelial observed that ethanol extract of D. stramonium growth inhibition of 71.85% in dual culture, followed by T. irrespective of concentrations, exhibited maximum viride (65.93%) in A. solani. T. harzianum and T. virens average mycelial growth inhibition of 61.12%. This was showed strong mycoparasitic activity and completely followed by A. absinthium (58.54%). The least mycelial overgrew the host mycelium once in contact with it. inhibition was exhibited by U. dioica (37.34%). It was Amongst the above biocontrol agents only T. viride further noticed that test botanicals increased the inhibition developed zone of inhibition against the pathogen. These Ganie et al. 4401

findings are in conformity with Rudresh et al. (2005) who Fusarium wilt in the green house. Plant disease 3:300-305. noticed 72.1 and 77.0% growth inhibition of R. solani and Carpenter JB (1942). A toximetric study of some eradicant fungicides. Phytopathology 32:845. F. oxysporum, respectively by T. harzianum. Earlier rapid Chattopadhyay C, Meena PD, Kuamr S (2002). Management of growth of T. harzianum covering the entire colonies of Sclerotinia rot of Indian mustard using ecofriendly strategies. J. Sclerotinia sclerotiorum and strong antagonism of Mycol. Plant Pathol. 32:194-200. Trichoderma spp. against S. sclerotiorum have been Choulwar AB, Datar VV (1988). Cost linked spray scheduling for the management of tomato early blight. Indian Phytopathol. 41(4):603- reported by many workers (Lee and Wu, 1979; Singh, 606. 1998). The formation of inhibition zone by T. viride Christ BJ (1990). Influence of potato cultivars on the effectiveness of against A. solani in the present study suggests the fungicide control of early blight. Am. Potato J. 67:3-11. involvement of strong antibiosis, possibly due to Cook RJ (1993). Making greater use of introduced micro-organisms for biological control of palnt pathogens. Ann. Rev. Phytopathol. 31:53- production of some diffusible substances. 80. Various volatile metabolites viz., derivatives of lactones, Datar VV (1999). Bioefficacy of palnt extracts against Macrophomona alcohols and terpens etc., produced by T. viride (Zeppa phasealina (Tassi) Goid, the incitant of charcoal- rot of sorghum. J. et al., 1990), and chemicals like gliotoxin and gliovirin etc. Mycol. Plant Pathol. 29:251-253. Derbalah AS, El-Mahrouk MS, El-Sayed AB (2011). Efficacy and safety produced by Gliocladium sp. are reported to be of some plant extracts against tomato early blight disease caused by responsible for the formation of inhibition zone (Wilhite et Alternaria solani. Plant Pathol. J. 10(3):115-121. al., 1994; Howell and Stipanovic, 1995). Similar observa- Dixit SN, Tripathi SC (1975). Fungistatic properties of some seedling tions with regard to Trichoderma spp. and T. virens have extracts. Curr. Sci. 44:279-280. Dube HC (2001). Rhizobacteria in biological control and plant growth been made by Elad et al. (1980), Tu (1980) and Tu and promotion. J. Mycol. Plant Pathol. 31:9-21. Vartaja (1981). Dubey RC, Dwivedi RC (1991). Fungitoxic properties of some plant The present findings are also in conformity with that of extracts against vegetative growth and sclerotic viability of Munshi (1998) and Munshi and Dar (2004) who noticed Macrophomina Phaseolina. Indian Phytopathol. 44: 411-413. Elad Y, Chet I, Katan J (1980). Trichoderma harzianum, a biocontrol the formation of inhibition zone by Gliocladium sp. agent effective against Sclerotium rolfsii and Rhizoctonia solani. against Fusarium pallidoroseum. T. viride and T. Phytopathology 70: 119-121. harzianum have also been reported to be effective fungal Folsom D, Bonde R (1925). Alternaria solani as a cause of tuber rot in antagonists against Sclerotium rolfsii (Alice et al., 1998) potatoes. Phytopathology 15: 282-286. Gokulapalan C, Nair MC (1984). Antagonism of a few fungi and bacteria and Rhizoctonia solani (Roy, 1977; Elad et al., 1980; against Rhizoctonia solani. Indian Journal of Microbiology 24 : 57-58. Gokulapalan and Nair, 1984). Harman GE (2006). Overview of mechanisms and uses of Trichoderma spp. Phytopathology. 96: 190-194. Howell CR, Stipanovic RD (1995). Mechanism in the biocontrol of Conclusion Rhizoctonia solani induced cotton seedling disease by Gliocladium virens : antibiosis. Phytopathology 85: 469-471. Jayaraj J, Ramabadran R (1996). Evaluation of certain organic Early blight of potato is an economically important substrates and adjuvants for mass multiplication of T. richoderma disease in Kashmir valley caused by A. solani (Ellis and harzianum Rifai. J. Biol. Control. 10:129-131. Martin) Jones and Grout. Under in vitro conditions, the Lee YA, Wu WS (1979). Memories of the College of Agriculture, disease was well controlled by plant extracts and National Taiwan University 19(1): 96-107. Lolpuri ZA (2002). Management of fungal wilt complex of brinjal biocontrol agents. These need to be further evaluated (Solanum melongena L.). M.Sc. Thesis Submitted to S.K. University under in vivo conditions so as to make minimum use of of Agricultural Sciences and Technology of Kashmir, Shalimar, pp. 1- chemicals. 63. Maheshwari SK, Gupta PC, Gandhi SK (1991). Evaluation of different fungitoxicants against early blight of tomato (Lycopersicon REFERENCES esculentum Mill). Agricultural Science Digest Karnal 11(4): 201-202. Mishra SB, Dixit SN (1997). Antifungal spectrum of the leaf extracts of Alice D, Ramamorthy V, Muthusamy M, Seetharaman K (1998). Allium sativum Linn. Geobias 4: 176. Biological control of Jasmine wilt incited by Sclerotium rolfsii. Indian Mukhopadhyay AN (1994). Biocontrol of soil borne fungal plant J. Plant Protect. 26:91-95. pathogens. Current status, future prospects and potential limitations. Amonkar SV, Banerji A (1971). Antifungal activity of certain plant Indian Phytopathology33: 1-14. extracts against Macrophomina phaseolina. Science 74:1343-1344. Munshi NA (1998). Studies on the Fusarial blight of mulberry. Ph.D. Anonymous (1972). The wealth of India. Council of Scientific and thesis, SKUAST, J&K, Srinagar, pp. 1-198. Industrial Research (CSIR), New Delhi, India. pp. 237. Munshi NA, Dar GH (2004). In vitro evaluation of some locally isolated Arya A, Chauhan R, Arya C (1995). Effect of allicin and extracts of garlic microfungi for antagonism against mulberry Fusarial blight pathogen and bignonia on two fungi. Indian J. Mycol. Plant Pathol. 25:316-318. (Fusarium pallidoroseum (Cook.) Saac.). Sercologia (In press). Ashwani T, Sharma YP, Lakhanpal TN (2004). Effect of volatile Neeraj, Verma S (2010). Alternaria diseases of vegetable crops and compounds released by Gliocladium virens and Trichoderma spp. on new approaches for its control. Asian J. Biol. Sci. 1(3): 681-692. growth of Dematophora necatrix. J. Mycol. Plant Pathol. 34:37-40. Nene YL, Thapliyal PN (1993). Fungicides in Plant Disease Control. III Bell DK, Wells HD, Markham CR (1982). In vitro antagonism of edition. Oxford and IBH Publishing Company, New Delhi, India pp trichoderma species against six fungal plant pathogens. 531-532. Phytopathology 72:379-382. O’Brien MJ, Rich AE (1976). Potato diseases. USDA Agricultural Hand Biswas S, Das NK Qadri SMH, Chandra BS (1995). Evaluating different Book, pp. 474. plant extracts against three major diseases of mulberry. Indian Panse VG, Sukhatame PV (1985). Statistical Methods for Agricultural Phytopathol. 48:342-346. Workers. Indian Council of Agricultural Research, New Delhi, p. 347. Bowers JH, Locke JC (2000). Effect of botanical extracts on the Papavizas GC (1985). Trichoderma and Gliocladium: Biology, Ecology population density of Fusarium oxysporum in soil and control of and their potential for biological control. Ann. Rev. Phytopathol. 51: 4402 Afr. J. Microbiol. Res.

693-699. Singh Y (1998). Biological control of Sclerotinia rot of rapseed and Pelletier JR, Fry WE (1990). Characterization of resistance to early mustard caused by Sclerotinia sclerotiorum. Plant Disease Res. blight in three potato cultivars, receptivity. Phytopathology 80(4): 361- 13:144-146. 366. Skinner FA (1955). Modern Methods of Plant Analysis (Eds. Peach SK, Raghavendra MP, Satish S, Raveesha KA (2009). Akaloid extracts of Tracey MV). Springer- Verlog. Viertev Brand III: 626-725 [cf: Indian Prosopis juliflora (Sw.). DC. (Mimosaceae) against Alternaria Phytopathol. 54:271-274. alternata. J, Biopest. 2(1): 56-59. Suleiman MN (2010). Fungitoxic activity of neem and pawpaw leaves Raja J, Kurucheve V (1998). Influence of pant extracts and buffalo urine extracts on Alternaria solani, causal organism of yam roots. Adv. on the growth and sclerotial germination of Macrophomina Environ. Biol. 4(2):159-161. Phaseolina. Indian Phytopathol. 51: 102-103. Tu JC (1980). Gliocladium virens: A destructive mycoparasite of Ranjan S, Phookan AK, Bhagabat KN (1999). Efficacy of some plant Sclerotinia sclerotiorum. Phytopathology 70:670-674. extracts in the management of sheath blight disease of rice. J. Mycol. Tu JC, Vartaja V (1981). The effect of hypoparasite (Gliocladium virens) Plant Pathol. 29:336-339. on Rhizoctonia solani on Rhizoctonia root rot of white beans. Canad. Ravikumar MC, Rajkumar HG (2013). Antifungal activity of plant J. Bot. 59:22-27. extracts against Alternaria solani, the causal agent of early blight of Utkhede RS, Rahe JE (1983). Interaction of antagonist and pathogens tomato. Arch. Phytopathol. Plant Protect. 4(1):166-172. in biological control of onion white rot. Phytopathology 73: 890-893. Roopa VS (2012). Antagonistic effect of some plant root extract on seed Vander-Walls JE, Korsen L, Aveling TAS (2001). A review of early blight borne fungi. Int. J. Res. Pharm. Biomed. Sci. 3(4): 1716-1718. of potato. Afr. Plant Protect. 70:91-102. Roy AK (1977). Parasitic activity of Trichoderma viride on sheath blight Vanitha S (2010). Developing new botanical formulation using plant oils fungus of rice. J. Plant Disease Protect. 84: 675-683. and testing their physical stability and antifungal activity against Rudresh DL, Shivaprakash MK, Prasad RD (2005). Potential of Alternaria chlomydospora causing leaf blight in Solanum nigrum. Trichoderma spp. as bio control agents of pathogens involved in wilt Res. J. Agric. Sci. 1(4):385-390. complex of chickpea. J. Biol. Control 19:157-166. Vimala R, Sivaprakasam K, Setharaman K (1993). Effect of plant Sendhilvel V, Buvaneshwari S, Kanimozhi S, Raguchamder T (2005). extracts on the incidence of brinjal wilt. Madras Agric. J. 80: 409-412. Management of cowpea root-rot caused by Macrophomina Vincent JM (1947). Distribution of fungal hyphae in the presence of phaseolina (Tassi) Goid. Using plant growth promoting rhizobacteria. certain inhibitors. Nature 15:850. J. Biol. Control 19:41-46. Vloutoglou I, Kalogerakis SN (2000). Effects of inoculum concentration, Shailbala, Pathak C (2008). Harnessing the potential of potato to meet wetness duration and plant age on development of early blight increasing food demand. Kurukshetra 56(3):45-48. (Alternaria solani) and on shedding of leaves in tomato plants. Plant Shalini V, Dohroo NP (2005). Novel approach for screening different Pathol. 49:339-345. antagonists against Fursrium oxysporum f.sp. pisi causing Furarium Wilhite SE, Lumesden RD, Strayney DC (1994). Mutational analysis of wilt of autumn pea Plant disease Research. 20:58-61. gliotoxin production by the biocontrol fungus Gliocladium virens in Shekhawat, Prasada (1971). Antifungal properties of some plant relation to suppression of Pythium damping off. Phytopathology extracts: Inhibition of spore germination. Neeraj and Shilpi Verma, 84:816-821. 2010; Derbalah et.al., 2011. Indian Phytopathol. 800-802. Wu F, Zheng Z (2007). Essential oils to control Alternaria alternata in Shivpuri A, Gupta RBL (2001). Evaluation of different fungicides and vitro and in vivo. Food Control 18:1126-1130. plant extracts against Sclerotinia sclerotiorum causing stem rot of Zaker M, Mosallanejad H (2010). Antifungal activity of some plant mustard. Phytopathology 52:272-274. extracts on Alternaria alternata causing alternaria leaf spot of potato. Shivpuri A, Sharma OP, Jhamaria SL (1997). Fungitoxic properties of Pak. J. Biol. Sci. 13:1023-1029. plant extracts against pathogenic fungi. J. Mycol. Plant Pathol. 27:29- Zeppa G, Allegrone G, Barbeni M, Gaurda PA (1990). Variability in the 31. production of metabolities by Trichoderma viride. Annali di Shtienberg D, Bergeron SN, Nicholson AG, Fry WE, Ewing EE (1990). Microbiologia ed. Enzymologia. Rev. Plant Pathol. 70: 4735; 90:171- Development and evaluation of a general model for yield loss 176. assessment in potatoes. Phytopathology. 80:466-472. Singh R, Singh SB, Palat R (2003). Management of sclerotinia stem rot of ajowan through fungicides and biopesticides. Ann. Plant Prot. Sci. 11:165. Singh SD, Navi SS (2000). Garlic as a biocontrol agent for sorghum ergot. J. Mycol. Plant Pathol. 30:350-354.