Collection and isolation of Colletotrichum isolates
Infected chilli fruits were collected from Ishurdi, Bogra sadar, Rangpur sadar and Gazipur sadar. Colletotrichum isolates were obtained from lesions on chilli fruit. Infected samples were then cut into small pieces of tissue taken from healthy, and the margin of diseased tissue, surface sterilized and then placed on PDA. Plates were incubated at room temperature (28-30°C) and observed periodically. The growing edges of any fungal hyphae developing from the samples were then transferred aseptically to PDA slants and were stored at 4°C.
Collected isolates of Colletotrichum sp. were characterized by the following parameters:
Morphological identification: Morphological identification was done on the basis of some important characters like hyphae (Septation, colour and width), setae (Size (length and breadth), acervuli (Colour and size) and conidia (Colour and size) etc.
Molecular identification: To confirm the morphological identification, genomic DNA was extracted from fresh mycelia grown on PDA plates using a standard fungal DNA extraction kit (Promega, USA). Internal transcribed spacer (ITS) regions of the nuclear-encoded ribosomal RNA genes (rDNA) was amplified using the ITS4-ITS5 primer pair (White et al., 1990).
Cultural variability: Cultural variability among the isolates was studied on the basis of type and colour of the colony, the growth rate of fungus, etc.
Morphological variability: The morphological variations among the various isolates of Colletotrichum sp. were studied on artificial culture with the important characters of hyphae, setae, acervuli, conidia etc.
Pathogenicity test: All four isolates were used for pathogenicity testing. Isolates were cultured on PDA at 25°C. Conidia from 7-day-old cultures were harvested by adding 5-10 mL of sterilized distilled water onto the culture, which was then gently swirled to dislodge the conidia. The conidial suspension was filtered through two layers of muslin cloth. BARI Morich-1 variety of Chilli was used in this study. Non-infected fruits (both red and green) were surface-sterilized with 1% sodium hypochlorite for 5 min and washed twice with distilled water. The fruits were blotted dry and inoculated using the wound/ drop method (Lin et al., 2002). The wound/drop method involved pin-pricking the chilli fruit wall and then placing ca. 5 μL of conidial suspension (105 conidia mL–1) over the wound. The inoculated fruits were incubated at 25°C, 98% RH. Disease reactions of the host were evaluated by measuring the length, width and area of the typical anthracnose lesion which developed on the fruits. Symptoms were evaluated 9-15 days after inoculation (DAI). Disease reaction was scored on a 0-9 point scale described by Than et al. (2008): 0 (highly resistant), no infection; 1 (resistant), 1-2% of the fruit with a necrotic lesion or a larger water-soaked lesion surrounding the infection site; 3 (moderately resistant), > 2 to 5% of the fruit with a necrotic lesion, possibly acervuli may be present, or a watery lesion covering up to 5% of the fruit surface; 5 (susceptible), > 5 to 10% of the fruit showing a necrotic lesion, possibly acervuli, or a water-soaked lesion covering up to 25% of the fruit surface; 7 (very susceptible), > 10 to 25% of the fruit covered with a necrotic lesion with acervuli; and 9 (highly susceptible), > 25% of the fruit showing necrosis, lesion often encircling the fruit, abundant acervuli.
The data were analyzed statistically for calculating the mean values and for the test of significance. The means were compared following Duncan's Multiple Range Test (DMRT).