In vitro Evaluation of Fungicides, Botanicals and Bio-agents against Phomopsis vexans, the Causal Agent of Fruit Rot of Brinjal

1Department of Plant Pathology (University of Horticultural Sciences), Bagalkot-587104, Karnataka, India. 2Department of Plant Pathology (University of Horticultural Sciences)Kolar-563103, Karnataka, India. 3Department of Agronomy (University of Horticultural Sciences), Arabavi-591307, Karnataka, India. 4Horticultural Reasearch Station (University of Horticultural Sciences) Devihosur-581110, Haveri, Karnataka. India.

Brinjal or egg plant (Solanum melongena L.) is an important vegetable crop belongs to the family solanaceae.Brinjal is one of the most common, popular and principal vegetable crops grown in the tropical and sub tropical areas.It's a highly productive and usually finds its place as the poor man's crop.This crop is extensively grown in India, Pakistan, Bangladesh, China and Phillipines.South Asia accounts for almost 50 per cent of world brinjal area under cultivation (Harish et al., 2011).This sturdy crop is cultivated throughout the year, even in the hot wet monsoon season when other vegetables are in short of supply.In India, brinjal is mainly grown in the states like West Bengal, Orissa, Bihar, Gujarat, Maharastra, Andhra Pradesh, Karnataka etc. with an area of 7.22 lakh hectare with a production of 135.58 metric tonnes and productivity of 19.10 tonnes per ha (Anon., 2014).It contributes about 12.47 per cent of the total production of vegetables in India.In Karnataka, brinjal is cultivated over an area of 15,800 ha with a production of 4002.50 tonnes (Anon., 2014).It is mainly grown in the Bagalkot district.The fruit rot caused by a fungi Phomopsis vexans, is becoming severe disease in northern dry zone.The disease was first reported from the Gujarat state in 1914 and since then from many parts of India.In general, the crop loss due to this disease ranges from 15-20% (Hossain et al., 2013).It has been reported that Phomopsis vexans reduces yield and marketable value of the crop by nearly 20-30 % (Jain and Bhatnagar 1985).The fruit infection takes place during fruit formation just some days prior to the harvest of the crop.This infection becomes severe at the time of harvesting to marketing.Hence the investigation was carried out in the Department of Plant Pathology,UHS, Bagalkot, Karnataka to know the effective fungicides, botanicals and bio-agents for the effective management of fruit rot of brinjal caused by Phomopsis vexnas.

MATERIALS AND METHODS
The efficacy of 5 systemic fungicides at the concentration of 0.25, 0.5, 0.75 and 1 per cent and three non-systemic fungicides at the concentration of 0.25, 0.5, 0.75 and 1 per cent was determined.The evaluation of fungicides was based on the active ingredient consideration.Required quantity of individual fungicide was added separately into sterilized molten PDA so as to get the desired concentration of the fungicides.Later, 20 ml of the poisoned media was poured into sterilized Petri plates.Mycelium discs of five mm diameter from seven days old culture of the fungus were cut out by sterile cork borer and one such disc was placed at the centre of each plate.PDA without any fungicide served as control.Three replications were maintained for each concentration.Plates were incubated at room temperature for seven days and radial growth was measured when fungus attained maximum growth in control plates.The efficacy of the fungicides was expressed as per cent inhibition of mycelial growth over control which was calculated by using the formula of Vincent (1927).To evaluate the antifungal activity of botanicals fresh samples were washed in tap water and finally washed thrice using sterilized distilled water.They were crushed in a sterilized pestle and mortar by adding little quantity of alcohol (1:1 w/v) just enough to moisten the samples so that it was easy to crush.The extracts were strained through the two layers of muslin cloth.Finally, filtrates thus obtained from the leaves were used as stock solution (Begum and Bhuiyan, 2006).To study the antifungal mechanism of plant extracts, poisoned food technique was followed as suggested by Nene and Thapliyal (1982).For this, 5 and 10 ml of stock solutions were mixed with 95 and 90 ml of sterilized molten potato dextrose agar medium respectively so as to get 5 and 10 per cent concentration.The medium was shaken thoroughly for uniform mixing of plant extract.About 20 ml of medium was poured into each of the 90 mm sterilized Petri plates.Each plate was seeded with 5 mm mycelial discs aseptically taken from the periphery of 7 days old culture and incubated at 27±1°C till the growth of the colony reaches maximum in control plate.Three replications were maintained for each treatment.Suitable control plates were maintained.Mean colony diameter in each case was recorded.The efficacy of the botanicals was expressed as per cent inhibition of mycelial growth over control which was calculated by using the formula as given by Vincent (1927).
Bio-agents were evaluated for their efficacy through dual culture technique.Twenty ml of sterilized and cooled potato dextrose agar medium was poured into sterilized Petri plates.Fungal antagonists were evaluated by inoculating a pathogen at one side of the Petri plate and the antagonist at exactly opposite side of the same plate by leaving about 4 cm gap.For this, actively growing cultures were used.In case of bacterial antagonist evaluation, two mycelial discs of pathogen were inoculated at the periphery of the Petri plate and bacterial antagonist was streaked in the centre of the same plate.After required period of incubation i.e., when the growth in control plate recorded 90 mm in diameter, the radial growth of the pathogens was measured.Per cent inhibition over control was worked out according to the equation given by Vincent (1927)

RESULTS AND DISCUSSSION
The per cent inhibition of radial growth off Phomopsis vexans by different systemic and non systemic fungicides was recorded and presented in Table 1.
Table (1) revealed that there was significant difference between the fungicides, concentrations and interaction.The per cent inhibition was reduced as the concentration of fungicides increased from 0.25 per cent to 1.00 per cent.The mean inhibition per cent in carbendazim, hexaconazole and tebuconazole was 100 per cent as they inhibited 100 per cent growth of the Phomopsis vexans in all the concentrations tested.The results are depicted in the Fig. 1.At 0.25 per cent concentration carbendazim, tebuconazole and hexaconazole recorded 100 per cent inhibition followed by propiconazole (82.00%) and difenoconazole (81.67%).At 0.5 per cent concentratin it showed that carbendazim, tebuconazole and hexaconazole recorded 100 per cent inhibition followed by propiconazole (93.67%) and difenoconazole (88.00%).It is found that as the concentration increased per cent inhibition of mycelial growth also increased in all fungicides.With reference to interaction between fungicides and concentrations, carbendazim, tebuconazole and hexaconazole recorded 100.00 per cent inhibition at all concentration.The least mycelial inhibition was observed in case of captaf (63.33%).(Nene and Thapliyal, 1973) The results on the efficacy of botanicals against Phomopsis vexans (Table 2) showed that there was a significant difference between the treatments with respect to per cent inhibition of radial growth.At 5 per cent concentration garlic bulb extract and turmeric rhizome extract recorded highest inhibition (100%) followed by kokum fruit extract (65.00%) and neem seed kernel extract (45.66%).Lantana leaf extract recorded 31.33%, which was on par with drumstick leaf extract (30.33%).The lowest inhibition was recorded in drumstick leaf extract (30.33%).The results are depicted in the Fig. 2. At 10 per cent concentration maximum inhibition of radial growth was recorded in garlic and onion bulb extract, kokum fruit extract and turmeric rhizome extract all (100%).Lantana leaf extract (61.00%), clerodendron leaves extract all 52.00% and neem seed kernels extract (51.00%).The lowest inhibition of radial growth of mycelium was observed in drumstick leaf extract (43.66%).The observations of present investigation are in close conformity with Alam (2005) who tested 11 botanicals to control Phomopsis fruit rot of egg Mirelman., 1999 reported that garlic extracts also have a strong antifungal effect and inhibit the formation of mycotoxins like the aflatoxin of Aspergillus parasiticus.Allicin was assumed to be the main component responsible for the inhibition of fungal growth.A concentrated garlic extract containing 34% allicin, 44% total thiosulfinates, and 20% vinyldithiins possessed potent in vitro fungistatic and fungicidal activity against three different isolates of Cryptococcus neoformans.Pure allicin was found to have a high anticandidal activity with a minimum inhibitory concentration of 7¼g/mL.Yamada and Azuma (1977) reported that pure allicin was effective in vitro against species of Candida, Cryptococcus, Trichophyton, Epidermophyton, and Microsporum at low concentration (minimal inhibitory concentrations of allicin were between 1.57 and 6.25 ¼g/mL).Allicin inhibits both germination of spores and growth of hyphae.
The results on the efficacy of bio-agents against Phomopsis vexans (Table 3) revealed that there was significant difference between the treatments with respect to the per cent inhibition of mycelial growth of Phomopsis vexans.Highest inhibition was observed in T. harzianum-p (70.66%) which was significantly superior over all other bioagents followed by T. harzianum-29 (64.33%), and T. harzianum -28 (63.00%) and the latter two bio-agents were on par with each other and next in order.Least inhibition was observed in case of B. subtilis (6.66%) followed by P. fluorescens (9.33%).The results are depicted in the Fig. 3. Observation in present investigation are similar with those findings of Muneshwar et al. (2012) who reported the in vitro evaluation of biocontrol agents against brinjal fruit rot pathogen.Among bio control agents Trichoderma viride (Tv-1), T. virens (Ts-1), T. harzianum (Th-1) and T. viride (JMU-24) overgrew the pathogen exhibiting antagonism.The antagonism of Trichoderma spp.against many fungi is mainly due to production of acetaldehyde compound (Robinson andPark, 1966 andDennis andWebster, 1971).Though, the genus Trichoderma comprises a large number of species some of which act as biological control agents through one or more mechanisms.Sharma et al., 2012 reported that Trichoderma strains exert control against fungal phytopathogens either indirectly by competing for nutrients and space, modifying the environmental condition, promoting plant growth, plant defensive mechanisms and antibiosis, or directly by mechanisms such as mycoparasitism.

CONCLUSION
In vitro evaluation of eight fungicides against fruit rot pathogens was taken up.Among systemic fungicides, carbendazim, propiconazole and tebuconazole showed 100 cent per cent inhibition of mycelia growth at all four concentrations and difenconazole @ 0.75 per cent.Among non systemic fungicides tested against same fungus, chlorothalonil was most effective followed by copper oxychloride and captaf found more effective respectively.In vitro evaluation of botanicals against fruit rot pathogens was studied.Highest inhibition of mycelial growth of Phomopsis vexans was observed in 5 and 10 per cent garlic extract, kokum extract and onion extract.The results of dual culture technique revealed that fungal bio agents were better than bacterial bioagents in inhibiting the growth of all fruit rot pathogens.P. vexans was effectively inhibited by T. harzianum-p.
The different antagonistic organisms used against brinjal fruit rot pathogens included, Trichoderma harzianum strains T-21, T-28, T-29, T-72, T-p, Pseudomonas fluorescens and Bacillus subtilis maintained at biocontrol unit of UHS Bagalkot.Where, I = Per cent inhibition C = Growth in control T = Growth in treatment The results found are in accords with Hossain et al. (2013) who reported that Bavistin 50 WP (0.1%) proved to be effective arresting the spore germination and mycelial growth of Phomopsis vexans.Das et al. (2014) also reported that carbendazim at 0.1% found to inhibit completely the mycelial growth of Phomopsis vexans.Muneeshwar et al. (2012) conducted the in vitro evaluation of fungicides against brinjal leaf blight and fruit rot pathogen Phomopsis vexans.Carbendazim recorded 100% inhibition of P. vexans over control at 1 ppm concentration.Suman and Sujan (2012) conducted the experiment on efficacy of various fungitoxicants alone and in combination Botanicals used for in vitro evaluation.Sl.

Fig. 3 .
Fig. 3. Evaluation of different bio-agents against Phomopsis vexans under in vitro condition

Table 1 .
In vitro evaluation of different systemic and non systemic fungicides against Phomopsis vexans *Figures presented in parenthesisare angular transformed values.

Table 2 .
In vitro evaluation of botanicals against Phomopsis vexans *Figures presented in parenthesisare angular transformed values.

Table 3 .
In vitro evaluation of bio-agents against Phomopsis vexans *Figures presented in parenthesis are angular transformed values