灰葡萄孢对腐霉利的抗性分子机制及快速检测技术

为明确灰葡萄孢Botrytis cinerea对腐霉利的抗性现状,于2017—2018年采用单孢分离法从浙江省5个地区的草莓大棚共分离获得200个菌株.通过区分剂量法测定了其对腐霉利的抗性,对抗药性菌株的分子机制进行了分析,并根据抗药性分子机制,建立了B.cinerea腐霉利高抗基因型的环介导等温扩增(loop-med...

Molecular mechanism and rapid detection technique of the resistance to procymidone in Botrytis cinerea

To clarify the resistance of Botrytis cinerea associated with strawberry to procymidone in Zhejiang Province, a total of 200 isolates were collected from strawberry greenhouses in five regions of Zhejiang Province by the single spore isolation method from 2017 to 2018. The resistance of B. cinerea population to procymidone was determined by distinguishing dosage method and the molecular mechanism of resistance was further studied. According to the molecular mechanism of resistance, a loop mediated isothermal amplification (LAMP) technique was developed. The results showed that the total resistance frequency to procymidone was 71.5%, while most resistance isolates were of low resistance. There were three types of mutations in BcOS1 gene of procymidone-resistant B. cinerea. Type Ⅰ had the mutation at codon 365 from ATC to AGC, which lead to the amino acid change from isoleucine (Ile, I) to serine (Ser, S). The type Ⅱ had the mutation at codon 365 from ATC to AAC, which resulted in the amino acid change from isoleucine (Ile, I) to asparagine (Asn, N). These two types of mutations caused low or moderate resistance to procymidone. Type III isolates were highly resistant which contained mutations at codon 369 and 373, which mutated from CAG and AAC to CCG and AGC, respectively, and resulted in amino acids change from glutamine (Gln, Q) and asparagine (Asn, N) to proline (Pro, P) and serine (Ser, S). A LAMP detection technique was developed to detect the highly resistant phenotype (Q369P) in 50 minutes at 64 ℃. The detection limit of this assay was 10 × 10?3 ng/μL, and the sensitivity was 10 times as high as that of the conventional PCR. This study can provide theoretical basis and technical means for the further resistance management and scientific application of procymidone for the control of B. cinerea.