Identification of the Fungal Pathogen that Causes Strawberry Anthracnose in Bangladesh and Evaluation of In Vitro Fungicide Activity

Isolation and identification of fungi

A total of 350 samples of infected tissues were collected from strawberry plants from the Rajshahi District of Bangladesh (Table 2). The fungus was isolated from infected strawberry leaves and petioles following the standard procedures of (Agostini and Timmer, 1992). Tissue was cut from the advancing margin of the lesion, surface sterilized with 0.1% sodium hypochlorite solution and washed in three three times in sterile distilled water. The sterilized tissue was dried on sterile filter paper on a clean bench, plated on potato dextrose agar (PDA) and incubated at 28℃ for 10 days. Mycelia from pure cultures were examined under dissecting and compound microscopes and identified by comparing their morphological and cultural characteristics with published descriptions (Brooks, 1931; Howard et al., 1983; Smith et al., 1990).

Pathogenecity of Colletotrichum gloeosporioides

Conidia were harvested from 7-day-old cultures by flooding Petri dishes with 10 ml of sterile distilled water and dislodging conidia by softly scraping colonies with a sterilized, narrow edged glass slide. Aqueous conidial suspensions were filtered through sterile cheese cloth to remove mycelia. Conidial suspensions were adjusted with sterilized water to a concentration of 10³ conidia/ml and sprayed onto 6 healthy, detached strawberry leaves. Six healthy, detached leaves were simultaneously sprayed with sterilized distilled water without conidia to serve as controls.

Response of Colletotrichum gloeosporioides to fungicides

Five commercial fungicides, Dithane M-45 (Manganese ethylene-bis-dithiocarbamate), Corzim 50 WP (Carbondazim), Bavistin DF (2-Benzimidazolecarbamic acid, methyl ester), Sulcox 50 WP (Copper oxychloride) and Rovral 50 WP (3-(3,5-dichlorophenyl)-N-(1-methyl ethyl)-2,4 dioxo-1-imidazolidine carboxamide), were evaluated in vitro using the poison food technique (Adams and Wong, 1991). Fungicide suspensions of 500 µg/ml, 1000 µg/ml, 1500 µg/ml, and 2000 µg/ml were prepared by dissolving requisite quantities of each fungicide in autoclaved and cooled PDA just before pouring into Petri dishes. Twenty ml of fungicide-amended media was poured into each 9 cm sterilized Petri dish. Three replicates were performed for each concentration of each fungicide. Medium without fungicide served as a control. Mycelial discs (5 mm) were cut from 7-days-old cultures using a cork borer, placed in the centre of each Petri dish after solidification of PDA and incubated at 28℃. Percent inhibition of growth of Colletotrichum gloeosporioides was recorded using the following formula:

Where, X = radius of fungal mycelia on control plate and Y = radius of fungal mycelia on fungicide-treated plate

Sporulation test

Sporulation was measured after 10 days of incubation on media amended with different concentrations of fungicide. Five mm mycelia discs were cut randomly from four different areas and were macerated in 1 ml of sterilized distilled water. A 0.1 ml aliquot of the resulting suspension was placed on a clean glass slide and the number of spores was counted under low power of a compound microscope using a haemocytometer (Islam et al., 2003).

Statistical analysis

The percent inhibition of radial growth was subjected to arcsine transformation. Analysis of variance was performed and the means were separated using Least Significance Different Test at p < 0.05 (Zaman et al., 1982). Statistical analysis was done whenever required.

Isolation and identification of fungi

Characteristic symptoms of anthracnose in infected strawberry plants are usually black leaf spots, about 1~3 mm in diameter, although the spots may also remain light gray (Fig. 1A). Leaves can become heavily spotted without dying. In cases of severe infection, dark and sunken lesions also developed on the petioles (Fig. 1B) and stolons, which resulted in leaf mortality. In ripening fruit, round, sunken and watery lesions developed, which were usually dark brown or black in color (Fig. 1C). Reddish brown streaking developed in the crowns of wilted plants.

Acervuli with setae were produced (Fig. 1H) after three weeks of incubation at 28℃ on PDA plates. Acervuli were 10 to 30 µm in diameter, with 0~2 setae, often the acervuli coalseced up to 120 µm in diameter. Setae were 24 to 80 µm in length and 4 to 6 µm in diameter, with 1~3 transverse septa.

Conidial sizes of the 22 isolates were compared and the mean for each isolate ranged from 12.9 to 16.5 µm long and 4.4 to 6.5 µm wide (Table 1). The size and shape of conidia from these isolates fit the description for conidia of C. gloeosporioides (anamorph = G. cingulata) (Smith and Black, 1990). (Smith and Black, 1990) reported that the principal pathogen responsible for anthracnose disease of strawberry are C. acutatum, C. fragariae and C. gloeosporioides but perithecia are produced only by C. gloesporioides (Table 1). Our fungi formed typical perithecia in culture and differed from the other two species of Colletotrichum (Smith and Black, 1990). We concluded that our fungi were C. gloeosporioides because the length, width and other morphological characteristics of the conidia match published descriptions.

The conidia that developed on the acervuli of all the 22 isolates were hyaline, cylindrical and single celled. Isolates SR001, SC225, SC217, SC228, SI001, SF001, SS001 and SS002 produced setae in culture, while the remaining isolates did not (Table 2). These results deviated from the results of (Smith and Black, 1990). They reported that C. gloeosporidies does not produce setae in culture but that C. fragariae does. The acervuli of C. gloesporioides is setae or glabrous (Mordue, 1971). All the 22 isolates were produced cylindrical conidia and white to dark brown colonies, which fits the result of (Smith and Black 1990).

The growth of the fungi after 7 days of incubation at 28℃ on PDA plates was 7.5 to 9 cm. According to (Smith and Black 1990), C. gloeosporioides can be easily differentiated from C. acutatum and C. fragariae by their growth on PDA plates. The greatest difference in growth occurred at 32℃, where the average diameter of 5-day-old C. acutatum was 13 mm, compared to 69 mm for C. fragariae and 63 mm for C. gloeosporioides.

Colony characteristics on PDA plates were also observed. The texture was loose, the top surface was smooth, acervuli were setose or glabrous in culture and colony color was white in early stages but turned grayish in later stages. These observations support the results of (Azad et al., 2005). Based on the mode of conidiogenesis, morphology, cultural characteristics and growth rates, we identified the fungi as C. gloeosporioides.

Pathogenecity of Colletotrichum gloeosporioides

Watery, irregular blotches and pale brown spots 1 to 3 mm in diameter appeared on leaves 3 to 5 days after inoculation. Tissues with lesions began to rot and/or blight when lesions enlarged and coalesced 6 to 8 days after inoculation (Fig. 1D and 1E). Control leaves had no symptoms. C. gloeosporidies was consistently re-isolated from diseased leaves. Our results demonstrate that isolates were pathogenic to strawberry

Response of Colletotrichum gloeosporioides to fungicides

A progressive increase in percent inhibition of radial growth of the pathogen was observed with the increase in concentration of the fungicides (Table 3). Bavistin DF was the most effective fungicide in this experiment. Growth of the fungus was totally checked using all four concentrations of Bavistin DF. (Meah and Khan 1986) used Bavistin DF, Rovral WP, Dithane M-45 and Topsin M to test the in vitro control of C. gloesporioides, the cause of fruit rot of guava. They observed that Bavistin DF (0.2%), Roval WP (0.2%) and Topsin M (0.2%) were effective in inhibiting mycelial growth. (Alam et al., 2000) found that Bavistin DF, Dithane M-45 and Tilt 250 EC were the most effective fungicides of Bipolaris sorokiniana at 500 µg/ml to 2,500 µg/ml and 1/10 to 1/1000 ml (Ahmed et al., 1991) evaluated eight fungicides and observed that Dithane M-45 gives the best control of anthracnose (Colletotrichum gloeosporioides), followed by Bordeaux mixture. Our results agree with these earlier findings. We found that Bavistin DF was highly effective and that Dithane M-45. Rovral WP, Corzim WP and Sulcox WP were less effective fungicides against C. gloesporioides.

Sporulation test

No spores were formed when the fungus was grown on media containing Bavistin. However, the fungus produced spores on PDA medium amended with the other fungicides. (Alam et al., 2000) reported that 100% germination inhibition occurred with the application of Bavistin and Thiovit after 15 minutes and 25 minutes of immersion in 0.25% Cupravit. With the increase in the fungicide concentration and the immersion period, the inhibition of conidial germination of this fungus also increased.