Mechanisms, Detection Techniques,and Management Of Fungicide Resistance In Colletotrichum Gloeosporioides Complex Isolates From Strawberry And Grape

Anthrax, caused by Colletotrichum gloeosporioides complex, is an important disease of strawberry and grape plants characterized by the wide incidence and difficulty of management in productions.The main fungicides to control Anthrax are benzimidazoles, Qo center inhibitors(Qo Is) and the sterol demethylation inhibitors(DMIs). All the above fungicides belong to site-specific chemicals with a high degree of specificity choice which lead the serious and widespread of resistance through continuously high-dose selection pressures.A total of 68 isolates of C.gloeosporioides were collected form strawberry fields in different regions in Zhejiang province for evaluating their resistance to thiophanate-methyl by method of distinguishing dosages, assess the temperature effect on sensitivity and compare biological characteristics between different phenotypes of benzimidazole-resistant isolates(Ben S, Ben R1 and Ben R2). Results indicated that the frequency of resistance to thiophanate-methyl is as much as98.53%. and all resistant isoaltes were Ben R1(benzimidazole-resistant and diethofencarb-sensitive). Isolates of different phenotypes could express their resistance. at different temperatures and there were no significant differences in biological characteristics between them.Further study showed that molecular mechanisms of benzimidazole-resistance were E198 A and F200 Y single mutation of β-tubulin gene respectively for Ben R1 and Ben R2(benzimidazole-resistant and diethofencarb-resistant) isolates.The sensitivity of C. gloeosporioides from strawberry to pyraclostrobin, a Qo inhibitor(Qo I)fungicide, was assessed through the mycelial growth rate method and the relationship between alternative respiration and sensitivity to pyraclostrobin during mycelial growth in C.gloeosporioides was analyzed. The results showed that the medium effective concentrations(EC50Q) of the 65 isolates collected in strawberry fields from Linan, Jiandei and Zhuji of Zhejiang province ranged from 0.09 to 24.26 μg/m L, with an average value of 6.65 μg/m L in the absence of SHAM, a specific inhibitor of alternative oxidase. In the presence of 50 μg/m L SHAM, the EC50(Q+S) ranged from 0.06 to 48.98 μg/m L, with an average value of 10.58 μg/m L. The factor value F = EC50 Q / EC50(Q + S) for the impact of alternative respiration on sensitivity to pyraclostrobin ranged from 0.02 to 11.17, with an average value of 0.96.And, the F value for 78.46% isolates was less than 1. The transcript levels of alternative oxidase encoding gene, AOX, in mycelia ofstrawberry and grape C. gloeosporioides isolates with different sensitivity to pyraclostrobin were studied through real-time quantitative PCR. Results indicated that treatment with pyraclostrobin significantly increased the AOX transcript levels after 6 to 12 h in both strawberry and grape islates.However, no significant correlation between the AOX transcript levels and the sensitivity to pyraclostrobin was observed. This research suggested that the alternative respiration pathway in different fungal pathogens is very complex. Interestingly, all except one wild sensitive strawberry isolates showed mutation at codon 143 of cytb gene, and no alteration was found in the grape isolates.In this study, we developed a loop-mediated isothermal amplification(LAMP) assay targeting the β-tubulin sequence for visual detection of C. gloeosporioides. The LAMP assay was positive only for C. gloeosporioides,and could specifly detect the benzimidazole-resistant isolates.To manage resistance, best ratio of prochloraz and pyraclostrobin we screened out is 1:3 and another scheme is prochloraz and difenoconazole mixed in a 1:5 ratio through the mycelial growth rate method. in vitro.