Sensitivity analysis of Colletotrichum gloeosporioides to four fungicides

Abstract:【Objective】Colletotrichum gloeosporioides is the causal agent of grapevine anthracnose, causing a sharp decline in grape growth and yield. Infection of grape industry by Colletotrichum gloeo- sporioides has led to big yield losses in recent years. The disease will break out in warm and rainy peri- od just before and during maturing time and the conidia are transferred by wind or rain and glues itself to the grape fruit surfaces. After falling on grape fruit, it produces germ tubes and differentiates into spe- cialized appressorium structure. After infection, Colletotrichum gloeosporioides propagates into neigh- boring cells, causing lesions on grape. In practice, the grapevine anthracnose is generally controlled through fungicides. In order to investigate the sensitivity of C. gloeosporioides to mancozeb, tebucon- azole, prochloraz and difenoconazole, diseased berries were collected from different grape vineyards, and the obtained pathogen isolates were tested for their resistance to mancozeb, tebuconazole, pro- chloraz and difenoconazole. This study could offer a theoretical direction for scientific preventing of grape anthracnose effectively【. Methods】Collected diseased berries were first surface disinfected with 75% ethanol for 30 seconds and then washed with sterile water, two millimeter sized tissues from junc- tion of healthy and diseased grape anthracnose were placed on PDA including chloramphenicol and cul- tured at 25 °C for four days. For getting purified strains, disk containing mycelium picked from medium edge and inoculated to new PDA and cultured at 25 °C for eight 8 days, collected conidia were diluted into 105 per mL conidial suspensions. Thirty microlitre conidial suspensions were evenly spread on agar medium plates and incubated for twenty hours, and single conidium was selected under a microscope. The strains collected by single spore isolation were observed after culturing on PDA medium for 8 days at 25 °C under a light microscope. Furthermore, a droplet of 5 μL fresh conidial suspensions was inocu- lated on slightly wound grape berry made by toothpick prick, and then placed in a box chamber under 25 °C. The pathogenecity of grape anthracnose was examined through lesions condition after three or four days after inoculation. Each assay was repeated four times with four duplications for every time. Fi- nally, 120 Colletotrichum gloeosporioides isolates from 6 regions of Liaoning were indentified through cultivation and pathogenicity characteristic observation. Distinguishing dosages and the mycelium growth rate method were used to detect the resistance of 120 grape anthracnose isolates. Hyphae were transferred into PDA plates covering different dosages of fungicides, and the diameter of the colony was measured after eight days. The effective inhibiting medium concentrations (EC50) value was calcu- lated according to colony diameter through a software.【Results】EC50 of tebuconazole ranged from 0.677 1 to 2.691 0 mg·L-1 with an average value of 1.474 9 mg·L-1; 29.17% of the 120 isolates showed resistance to tebuconazole in sensitive assay; no strain showed high resistance and thirty strains showed low resistance to tebuconazole. The resistant frequency of grape anthracnose was from 16.67% to 45.00% in tebuconazole plate from six different regions. The lowest resistant frequency of grape an- thracnose was 16.67% from Shenyang and the highest resistance of grape anthracnose was 45.00% from Huludao area. However, the EC50 values of different strains from different regions were varied. The EC50 of mancozeb ranged from 11.156 5 to 65.934 3 mg·L-1 with a mean value of 19.834 6 mg·L-1; 28.33% of the 120 isolates showed certain resistance to mancozeb in sensitive test. The grape anthrac- nose from Huludao area was the most resistant to mancozeb-resistant strains, reaching 45%. From the analysis of EC50 distribution values, there were higher resistant strains from Dalian and Benxi areas, reaching65.9343mg·L-1 and50.0091mg·L-1,respectively;TherewasnocolonyonPDAplatescon  taining1mg·L prochlorazandresistantprochlorazstrainwasnotfound.TheEC50 valueofthestrain was less than 5 times of the sensitive baseline in difenoconazole resistant experiment and studies showed that grape anthracnose from different regions had lower resistance to difenoconazole. It was found that EC50 values of different strains from different regions were less different in prochloraz and di- fenoconazole plate. All 120 isolates were highly sensitive to prochloraz and difenoconazole with an av- erage EC50 of 0.011 5 mg·L-1 and 0.061 1 mg·L-1, respectively【. Conclusion】In this study it was showed that C. gloeosporioides had certain resistance to mancozeb and tebuconazole, and a high sensitivity to prochloraz and difenoconazole. Because mancozeb was a multi-acting site fungicide and had a long his- tory of application, it was very effortless to produce some low resistant strains. It was single site of ac- tion that may be an important reason for the development of resistant strains like tebuconazole fungi- cide. Therefore, growers need to consider the condition of the resistance situation in different regions when tebuconazole and mancozeb will be used in manufacture. Although strains with a certain resis- tance to prochloraz and difenoconazole fungicide were not found in this test, it was recommended to use fungicides with different target sites in order to avoid the emergence of resistant strains.