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In vitro Evaluation of Fungicides, Botanicals and Bio-agents against Colletotrichum lindemuthianum causing Anthracnose of Bean

Common bean (Phaseolus vulgaris L.) is one of the important legumes crop belongs to family leguminaceae and occupies a premier place among grain legumes in the world including India (Jan et al., 2014). It is an important constituent of people’s diets especially in developing countries. Dry bean find a unique position in the culinary items because of their high nutritional value (Padder et al., 2017). It is rich in calories, carbohydrates, protein, vitamins and minerals particularly calcium, phosphorus and iron, thus an excellent food for human consumption. Common bean suffers from many diseases caused by fungi, bacteria, viruses, nematode and also abiotic stresses.


Introduction
Common bean (Phaseolus vulgaris L.) is one of the important legumes crop belongs to family leguminaceae and occupies a premier place among grain legumes in the world including India (Jan et al., 2014). It is an important constituent of people's diets especially in developing countries. Dry bean find a unique position in the culinary items because of their high nutritional value (Padder et al., 2017). It is rich in calories, carbohydrates, protein, vitamins and minerals particularly calcium, phosphorus and iron, thus an excellent food for human consumption. Common bean suffers from many diseases caused by fungi, bacteria, viruses, nematode and also abiotic stresses. fruits since the lesions on the fruits considerably reduce the market value of the produce. In India, disease incidence has been reported to vary between 24.59 to 51.72 % Sharma and Sugha (1995). As a disease of minor important but during the last few year bean anthracnose has appeared as a potential threat to the (Sacc and Magnus) Briosi and Cavara is a major limiting factor in reduction of yield in subtropical and temperate regions. Anthracnose is mainly a seed-borne disease caused by a fungus which has a wide host range on many legume species (Goswami et al., 2011).

Isolation and pathogenicity
The present investigation on in vitro efficacy of fungicides, botanicals and bio-agents against Colletotrichum lindemuthianum was conducted at the Department of Plant Pathology, Post Graduate Institute, Dr. Panjabrao Deshmukh Krishi Vidyapeeth (Dr. PDKV), Akola. The culture of C. lindemuthianum used in this study was isolated from infected leaves of common bean plants collected from the fields of Chilli and Vegetable Research Unit, Dr. PDKV, Akola. In order to isolate pathogen infected leaf sample were cut along with healthy leaf and surface sterilized with 0.1% sodium hypochlorite solution for one minute and washing with three times by sterilized distilled water. The bits were placed in petriplates containing PDA medium. All the above operations were carried out in sterilized condition (under laminar air flow unit). Then plates were incubated at 27±2 ºC for 10 days. The fungal growth, which developed around each bit, was then transferred to PDA medium slant for sub culturing. The isolated fungi were identified as C. lindemuthianum on the basis of morphological characters and published literature. The inoculum was prepared and sprayed (1×10 6 spore/ ml) on plants of common bean within 6-12 days typical anthracnose symptoms were observed. The pathogen was reisolated on the PDA medium from the inoculated plants for confirmation of Koch's postulates.

In vitro evaluation of fungicides by poisoned food technique
Poisoned food technique was used to evaluate the efficiency of six fungicides against pathogens. Potato dextrose agar medium was prepared and distributed at the rate of 100 ml in 250 ml conical flask, autoclaved 1.05kg/cm2 for 15 min. Then before solidification of media different fungicides with desired concentration were incorporated aseptically in different flasks. These flasks shaken to facilitate uniform mixture of fungicides thoroughly and poured in Petri plate's 20 ml/plate likewise three plates for each treatment were poured. One set of three plates was poured without any fungicides to serve as a control. After solidification of medium, the plates inoculated with seven days old pathogens separately. Five mm diameter mycelial disc selected from peripheral growth of the plate by sterilized cork borer were used for inoculating the plates by keeping one disc per plate in the centre in inverted position, so as to make the mycelial growth touch the surface of medium.
The inoculated plates were incubated at room temperature for seven days. The colony diameter of the fungal pathogens on medium was recorded and percent inhibition in each treatment was calculated by using following formula (Vincent, 1927).
x 100 C Where, PI = Per cent Inhibition C = Growth of fungi in control (mm) T = Growth of fungi in treatment (mm)

In vitro evaluation of botanicals by poisoned food technique
Aqueous leaf extracts of the test botanicals were obtained by grinding the washed rhizome and leaves (100 g) in mortal and pestle with equal volume (100 ml) of sterilized distilled water. The macerate obtained was filtered through the folds of muslin cloth and the filtrate obtained formed 100% phytoextracts, which were evaluated by poisoned food method. Twenty ml of poisoned medium was poured into each stetrile petriplates. Five mm diameter mycelial disc selected from periphery of actively growing culture were cut out by sterilized cork borer were used for inoculating the plates by keeping one disc per plate in the centre in inverted position, so as to make the mycelial growth touch the surface of medium. of each agar plate. Control were also maintained by growing the pathogen on PDA plates.
The inoculated plates were incubated at room temperature for seven days. The colony diameter of the fungal pathogens on medium was recorded and percent inhibition in each treatment was calculated by using above formula.

In vitro evaluation of bio-agents by dual culture method
The lawn culture of test fungi and bio-agents viz., Trichoderma asperellum and Trichoderma reesei were prepared. Autoclaved, melted potato dextrose agar was poured in petri plates and allowed to solidify for obtaining levelled surface. The plates were inoculated with the culture of test fungi and bio-agents after solidification of media and then plates were incubated at room temperature for seven days.
Bacterial bio-agents, Bacillus subtilis and Pseudomonas fluorescens were prepared by inoculating a loopful culture in sterilized conical flask containing 100 ml of nutrient broth. Broth culture was incubated at room temperature for three days. Five mm disc of one week old test fungus and bio-agent lawn culture was cut with the help of sterilized cork borer lifted and transferred in petri plates, containing autoclaved solidified PDA medium. In each petri plates, four discs of bio-agents were inoculated at four peripheral points of the plates and the test fungi was placed in the center of petri plates. In case of Pseudomonas fluorescens and Bacillus subtilis, a three days old culture was streaked around the disc of test fungus. The test fungi grown in same condition on potato dextrose agar without bio-agents served as control. All these plates were incubated at room temperature for seven days. After an expiry of seven days incubation period the mycelial inhibition was calculated as per formula mentioned in the poisoned food method.

In vitro evaluation of bio-agents against Colletotrichum lindemuthianum
In the present investigation (Table 3 and Fig. 3) showed that two fungal and two bacterial bio-agents were tested against C. lindemuthianum. The results of dual culture technique on C. lindemuthianum reported that maximum growth inhibition was recorded with Trichoderma reesei (77.77%) followed by Bacillus subtilis (59.44%), Pseudomonas fluorescens (56.77%), and Trichoderma asperellum (48.61%). The present result in respect of antagonistic activity of bio-agents are in agreement with Fitson et al., (2014) who reported highest inhibition of the mycelial growth by Trichoderma viride (80.39%) followed by Trichoderma harzianum (75.49%) against Colletotrichum lindemuthianum. Rajesh et al., (2010) also reported that Trichoderma harzianum was most effective inhibiting the mycelial growth of Colletotrichum lindemuthianum to an extent of (73.54%) followed by Trichoderma viride (50.90%). The effective results of T. harzianum and T. viride against C. dematium was also recorded by Shovan et al., (2008) and Kothikar and Koche (2017) respectively.