Resistant Assessment And Molecular Mechanism Analysis Of Botrytis Cinerea From Strawberry In Two Regions Of Zhejiang To Iprodione And Pyimethanil

The grey mold of strawberry, caused by Botrytis cinerea Pers., is one of the most destructive diseases worldwide. It induces 20% to 30% yield loss in regular year and up to 50% loss when the conditions favor to disease prevalence. Due to the lack of the grey mold-resistant strawberry germplasm, chemical controlling is still the most important strategy in strawberry grey mold management. However, the large populations and rapid propagation capability, together with the presence of heterokaryosis and parasexuality, result in a high potential for genetically diverse populations of this fungus. Under the persistent selection pressure of a fungicide, some mutants, presented in very low rate, with fungicide-resistance would be selected, and their populations would increase rapidly if their ecological fitness is not significantly lower than fungicide-sensitive subpopulation. Consequently, the performance of the fungicides in disease controlling would be decreased, and failure of the disease control may eventually occur. To learn the frequency and the level of a fungicide-resistant subpopulation of B. cinerea population would be benefit for adjustment the strategies of fungicide application in time, thus to raise the control efficiency and to reduce the gross usage of fungicides. In addition, a system for rapid molecular detection of fungicide-resistant subpopulation in field could be established based on the understanding the molecular mechanisms of a fungicide-resistance.In this study, 98 Botrytis cinerea Pers. were collected from strawberry fields in Jiande City and Xiasha District, the main strawberry producing regions in Zhejiang Province, China, and then evaluated for the resistance to fungicide iprodione with the method of mycelial growth inhibition. Our investigations indicated that the subpopulation of B. cinerea with iprodione-resistance had been presented in Xiaya and Miaotouzhang of Jiande City. The resistant frequency was 17.5% and the resistant factor of the most resistant strain from Xiaya was 12.32. However, iprodione-resistant B. cinerea was not found in Yangcunqiao of Jiande City and Xiasha District in this study. In order to characterize the resistant molecular mechanism of B. cinerea to iprodione, the sequences of a 526 bp fregment in aa repeat domain of BcOS1 gene of 8 iprodione-resistant and 2 iprodion-sensitive strains were amplified and sequenced. Sequence alignment indicated that the resistance was associated with aa substitution at aa 365 (³⁶⁵Ile to Ser) and both 369 (³⁶⁹Gln to Pro) and 373 (³⁷³Asn to Ser). The former genotype was predominated in the resistant population.Resistance to fungicide pyrimethanil for 42 strains in B. cinerea Pers, describled as above were determined by the methods described previously. The results indicated that 19.1% isolates were of high resistance, 2.38% isolates were of middle resistance, 2.4% were of low resistance. The resistant factor for the most resistant isolate (JDxyl3) was 387.3. Among 8 high resistant isolates, 7 were from Xiaya of Jiande city. Together the factor of these strains resistant to iprodione, we suggest that a sever fungicide-resistance problem for B.cinerea is presented in Xiaya, and the application strategy of fungicides in this region should be adjusted.Anthracnose is one of the most economically important strawberry diseases According to the previous research, there are at least three species in genus Colletotrichum (C. acutatum, C. gloeosporioides and C. fragariae) are the pathogens of strawberry anthracnose. In this study, we collected 44 Colletotrichum isolates from Jiande city and Xiasha, two major regions for strawberry production in Zhejiang province. Conidia produced in acervuli were 16-20μm×4-5μm, straight, cylindrical, hyaline, aseptate, with both ends rounded. In culture, there are two distinct colonies, grey without pigment and producing a plent of conidia (type I, 38 of 44 isoaltes), and dark grey to-black and dark pigment-producing, but less conidia-producing (type II, 6 of 44 isolates) on potato dextrose agar (PDA) medium. In order to identify these isolates, two paires of species-specific primers CaInt-2/ITS4 and CgInt/ITS4, specific to C. acutatum and C. gloeosporioides/C. fragariae, were synthesed and used to amplify the sequence of internal transcribed spacer regions. A fragment of approximately 500 bp only could be amplified with primers CgInt/ITS4 from all 44 isoaltes. Together with the morphology, we suggest that all isoates in Colletotrichum we collected from strawberry are not C. acutatum, but belong to C. gloeosporioides or C. fragariae.