The experiment was conducted in the Plant Protection Laboratory of Agrotechnology Discipline, Khulna University, in the year of 2008-2009 to evaluate the effect of different botanical extracts on the growth of (C. gloeosporioides) causal agent of anthracnose of mango.
Plant extracts used Clove of garlic, rhizome of ginger, seed of mahogani, leaf of giant indian milky weed, pulp of keora fruit, keora seed in water, tobacco leaf in water were mixed (Table 1) at different concentrations (0%, 30%, 40%, 50%, 60% and 70% respectively) as botanical extracts. In this experiment, the crude extracts of the above indigenous plants were mixed with Potato Dextrose Agar (PDA) medium at different concentrations and the fungal mycelia were inoculated to grow. Data on the radial growth were recorded.
Collection of the sample Diseased mango fruit (Mangifera indica) showing typical anthracnose symptoms of Colletotrichum infection was collected from the market near the Khulna University campus. The fungus Colletotrichum gloeosporioides was isolated from the diseased mango fruit.
Isolation of the fungus: The fungus was isolated from the infected mango fruit following standard procedures (Dasgupta, 1981; Agostini and Timmer, 1992). The infected diseased samples along with healthy tissues were cut into small pieces and were surface sterilized by dipping in 0.1% sodium hypochloride (NaOCl) solution for two minutes. The treated plant tissues were washed three times with sterilized distilled water. Excess water was decanted by soaking with sterilized blotting paper. The cut pieces were then placed onto sterilized potato dextrose agar (PDA) in glass Petridish (20ml/petridish) and incubated at 28±1°C for three days for mycelium formation in petridish.
Purification and preservation To obtain a pure culture of C. gloeosporioides hyphal tip were transferred aseptically onto PDA plate by using the flame sterilized tip of an inoculation needle. The plate was incubated at room temperature for three days. Mature hyphae were collected and transferred into the test-tube slants containing PDA and incubated at room temperature for seven days. After incubation, the slants were carefully checked for contamination and then preserved at 4 °C in a refrigerator for further use.
Identification of fungus isolates upto species The fungus was identification on the basis of morphological characteristics suggested by Ellis (2009) and Agron (2009). Treatments of the experiment: The experiment was conducted in two factors completely randomized design (CRD) where factor A = plant extracts of tobacco leaves, giant Indian milky weed leaves, cloves of garlic, rhizome of ginger, and mahogani seed, keora seed, keora fruit and factor B = different level of concentrations i.e. 0%, 30%, 40%, 50%, 60% and 70%.
Preparation of botanical extracts: Botanical extracts were prepared by using a newly adapted method of standard procedure (Koul et al., 1999) Preparation of different media using botanical extracts Fresh mahogani fruit was collected directly from the mahogani tree and fruit were washed with tap water and seeds were separated from heard seed coat and taken into mortar and pestle to collect pure extract. For preparing the stock solution of mahogani seed extract, at first 150 ml pure extracts were mixed with 150 ml distilled water and then 30, 40, 50, 60 and 70 ml extracts from stock solution were mixed with 70, 60, 50, 40 and 30 ml PDA respectively in separate 250 ml conical flask to prepare 30, 40, 50, 60 and 70% concentrations of the extract. The same procedure was followed for preparing garlic, ginger and giant Indian milky weed extracts. For preparing the stock solution of tobacco leaves 250 ml of distilled water was taken into a 1000 ml beaker by using a measuring cylinder and 250 ml of tobacco crude leaves were soaked into it for 24 hours. After 24 hours, the tobacco leaves within the beaker were pressed by hand. Then the crude extract solution was filtered and collected for use. After that 30, 40, 50, 60 and 70 ml extracts from stock solution were mixed with 70, 60, 50, 40 and 30 ml PDA respectively in separate 250 ml conical flasks to prepare 30, 40, 50, 60 and 70% concentrations of the extract. The same procedure was followed for preparing keora seed extract. 150gm fresh fruits of keora collected from the market were washed with running tap water and taken into a pot and boiled for 15 minutes. Then the crude extract was filtered and collected for use. For preparing the stock solution of keora fruit extract, at first 150ml pure extract was mixed with 150 ml distilled water Then 30, 40, 50, 60 and 70 ml extracts from stock solution were mixed with 70 , 60 , 50 , 40 and 30 ml PDA respectively in separate 250 ml conical flask to prepare 30, 40, 50, 60 and 70% concentrations of extract.
Response of the identified C. gloeosporioides to seven different plant extracts Different concentrated media in conical flasks were sterilized in an autoclave at temperature of 121 0 C for 20 minutes. After autoclaving about 20 ml of the medium was poured in each 9cm sterilized petridishes. Mycelial discs were prepared using a cork borer (5mm diameter) from the tip of 5 days old cultures of the isolates. One disc of the isolate was placed at the center of a petridish after solidification of the PDA. Each treatment was replicated three times. The medium without plant extract served as a control.