In vitro evaluation of bioagents against Sclerotium rolfsii causing collar rot in brinjal

Introduction

Brinjal (Solanum melongena L.) is an important vegetable crop grown in India and other parts of the world. It can be grown in almost all parts of the country except at higher altitudes all the year around. Brinjal is said to be the native of India (Thompson and Kelly, 1957) [21] . It belongs to the economically important family Solanaceae. This solanaceous crop is India's contribution to the global palate. Regarding nutritional value per 100 g, brinjal has a very low caloric value (24 Kcal), protein (2%) and vitamin C (2.2 mg) but is a very good source of dietary fibre (3.4 g), potassium (230 mg), manganese (14 mg), calcium (9 mg), copper (0.082 mg) and thiamin (0.039 mg) (Naeem and Ozgen, 2019) [12] . The disease was first reported in India by P. Madhava Rao (1969) [9] in Vijayawada district of Andhra Pradesh and the fungus Sclerotium rolfsii sacc was first time seen in Florida on tomato plants by Peter Henry Rolfs (1892) [16] but the name was coined by Saccardo (1911) [17] . The common diseases and their pathogens are, damping off (Pythium aphanidermatum, Phytophthora spp. and Rhizoctonia spp), Leaf spots (Alternaria melongena L, Alternaria solani, Cercospora melongena, C. Solani), Wilt (Fusarium solani, Verticillium dahalae), Collar rot (Sclerotium rolfsii sacc.), Powdery mildew (Erysiphae cichoracearum), Root knot nematode (Meloidogyne javanica), Phomopsis blight and fruit rot (Phomopsis vexans) and little leaf of brinjal caused by Mycoplasma like organism which are causing In recent years, collar rot caused by Sclerotium rolfsii Sacc. has emerged as one of the serious soil-borne diseases of brinjal in western Maharashtra, causing heavy losses particularly under warm and humid conditions. The pathogen of collar rot, Sclerotium rolfsii sacc., has become one of the major threats both under nursery and field cultivated brinjal crop. It attacks the seedlings in the nursery bed as well as adult plants in the transplanted fields. The severity of the pathogen can be attributed to the ability of its sclerotia to withstand adverse conditions (Singh and Singh, 1994) [20] . S. rolfsii sacc. is a soil borne facultative pathogen, belonging to the sub division Deuteromycotina. It is especially severe on legumes, solanaceous crops and cucurbits. Collar rot disease incidence of 60-100% in brinjal has been reported (Siddique et al, 2016; Hasan and Meah, 2019) [18,6] with 16-30% yield losses (Singh and Dhancholia, 1991; Jadon, 2009) [19,7] . Infestation from seedling to flowering stages may result in 100% crop loss.

Materials and Methodology Isolation, Identification and Pathogenicity

The fungi associated collar rot of brinjal was isolated by applying tissue isolation technique (Tuite, 1969) [22] . Naturally infected brinjal plants showing typical collar rot symptoms were collected from the fields, thoroughly washed with distilled water, blot dried and cut with sharp sterilized blade into small bits (5 mm) and then surface sterilized with 1 per cent aqueous solution of Sodium hypochlorite (NaOCl) for two minutes. Subsequently, these bits were washed thoroughly by giving three sequential changes with sterile distilled water to remove traces of Sodium hypochlorite if any, blot dried and aseptically transferred on to autoclaved and cooled Potato dextrose agar PDA medium in sterile glass Petri plates (90 mm), under Laminar air-flow cabinet and incubated in BOD incubator at 27 ± 2 0C temperature. These inoculated PDA plates were observed at regular interval for growth of the pathogenic fungi. After a weak of incubation, typical fungal growths developed on PDA plates were transferred into fresh PDA plates and incubated further. By applying hyphal tip isolation technique (Tuite, 1969) [22] , purified, and subcultured the cultures and their pure cultures on Agar slant tubes were maintained in refrigerator, for further studies.

Pathogenicity

The pathogenicity of pathogens was tested by sick soil inoculation technique in the earthen pot under greenhouse condition by using susceptible brinjal variety Manjari Gota. To prove the pathogenicity of the fungus by soil inoculation techniques, the inoculum of the fungus was prepared as per the method given by Biswas Sen (2000) [3] . Sterilized sand maize meal medium was prepared in 250 ml conical flask (10 g maize meal + 90 g washed sand + 15 ml distilled water) was inoculated with two discs of 5 mm diameter mycelial plug of three days old culture grown on PDA plates. Inoculated flasks were incubated at room temperature (27± 2 ºC). After 7th days of incubation, the inoculum was mixed with sterilized soil in 1:1 ratio. Each earthen pot was filled with 3 kg of sterilized soil and the inoculum mixture was applied @ 60 g/kg soil. The next steps of soil inoculation were carried out according to the procedure described by Agrwal and Kotasthane (1971) [1] . Surface sterilized seeds were sown in each pot on 6th days of soil inoculation. Three replications of each treatment were maintained. Uninoculated soil in earthen pot served as control. Observation on pre emergence mortality was taken on 7th day of sowing while post mergence mortality on 21st days of sowing. The infected seeds and seedling were subjected to re-isolation to confirm Koch´s postulates

In vitro evaluation of the bioagents

Most potential fungal and bacterial biocontrol agents were evaluated in vitro against the test fungi S. rolfsii, by applying Dual culture technique (Dennis and Webster, 1971) [4] and using PDA as basal culture medium. Seven days old cultures of the test bioagents and test fungi grown on respective culture media were used for the study. One each 5 mm culture disc of the test fungus and the test fungal bio agents cut out with sterilized cork-borer was placed at equidistance and exactly opposite to each other on autoclaved and solidified PDA medium in Petri plates. For bacterial biocontrol agents, a culture disc (5 mm) for the test fungus was placed along periphery of the PDA plate and exactly opposite to it pure culture suspension of the test bacterial biocontrol agent was streaked with inoculation needle loop. The PDA plates separately inoculated (in the centre) alone with pure culture disc of the test fungus were maintained as untreated control Observations on radial mycelial growth or diameter of colony of the test pathogen were taken after seven days of inoculation. Per cent mycelial growth inhibition of the pathogen with the bioagents, over the untreated control, was calculated by using the formula given by Vincent (1947) [23] .

Where, I = Per cent inhibition (mm), C = Growth of fungus in control plate (mm) T = Growth of fungus in treatment plate (mm)

Result: A total of four fungal bioagents and two bacterial bioagents were evaluated for their antagonistic activity in vitro by using the dual culture technique against Sclerotium rolfsii which exhibited a wide range of mycelial growth and inhibition of the test pathogen. The results revealed that all the bioagents exhibited antifungal activity against S. rolfsii and significantly inhibited mycelial growth as compared to the untreated control. The results revealed that among the different treatments, Trichoderma harzianum was found to be the most effective bioagent in suppressing the growth of S. rolfsii, which recorded the minimum mean colony diameter of 29.43 mm with highest inhibition (67.30 followed by Trichoderma asperellum which recorded a mean colony diameter of 31.00 mm with 65.55 per cent inhibition. The next effective treatment was Pseudomonas fluorescens which showed 32.22 mm colony diameter with 64.20 per cent inhibition over control. Similar results were recorded by Kumar and Sen (2000) [8] , Pan and Mukhopadhyay (2001) [13] , Rakholiya and Jadeja (2010) [15] , Bhuiyan et al. (2012) [2] and Manu et al. (2012) [11] they reported that T. harzianum showed strong antagonistic activity toward S. rolfsii under in vitro condition. Dutta and Das (2002) [5] , Pan et al. (2009) [14] and Madhavi and Bhattiprolu (2011) [10] found that T. harzianum was more growth inhibitory followed by T. asperellum against S. rolfsii.

Conclusion

All six bioagents evaluated in vitro significantly inhibited mycelial growth over untreated control, of the test pathogenic fungi S. rolfsii causing collar rot in brinjal. However, in S. rolfsii, significantly highest mycelial growth inhibition resulted with T. harzianum 67.30%) followed by T. asperellum (65.55%), Pseudomonas fluorescens (64.20%), Bacillus subtilis (61.58%), T. hamatum (52.73%), T. virens (52.00%).

Acknowledgement

International Journal of Agriculture and Food Science https://www.agriculturaljournals.com

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