Resistance to QoIs fungicides in Botrytis cinerea populations for strawberries in hilly area of Jiangsu

Abstract: 【Objective】Gray mold caused by Botrytis cinerea is a major fungal disease for strawberries in Jiangsu province, which is considered as one of the most important strawberry-producing areas in China.Recently, effective management of this disease has become an urgent need for the farmers as a result of the failure of many fungicides in controlling this pathogen.In order to investigate the resistance distribution and molecular mechanisms of methoxy-acrylates fungicides (Qo Is) in Botrytis cinerea isolates, samples were collected from the strawberry fields in Jiangsu province and were used to provide measures of the resistance management.【Methods】Employing the methods of discriminative dose (a concentration that fully inhibits mycelial growth of the sensitive strains) and effective concentration (inhibits mycelia growth by 50%relative to the control, EC₅₀) values were identified to distinguish sensitivity to azosxystrobin and pyraclostrobin, fungicides resistance distribution and resistance phenotypes of B.cinerea isolates to azosxystrobin and pyraclostrobin.According to previous studies, the discriminatory concentration of azosxystrobin and pyraclostrobin was 10 mg·L^-1, and the plates were amended with 100 mg·L^-1salicylhydroxamic acid (SHAM) to inhibit the alternative respiratory pathways.Then the effective concentration that inhibits mycelia growth by 50%relative to the control (EC50) values of azoxystrobin was determined for 19 sensitive isolates and 77 resistant isolates.Azoxystrobin or pyraclostrobin solution was added to PDA to produce final concentrations of 0, 0.01, 0.03, 0.1, 0.3 and 1 mg·L^-1for sensitive isolates and 0, 3, 10, 30, 100 and 300 mg·L^-1for resistant isolates.The plates were amended by using 100 mg·L^-1SHAM to further inhibit the alternative respiratory pathway.For each isolate, three replicates per concentration were used.Disease severity on detached strawberry fruit for different resistance phenotypes of B.cinerea isolates to azoxystrobin and pyraclostrobin were tested.Furthermore, the molecular mechanisms of Qo Is fungicides were determined by the sequence analysis of target genes.First, DNA from fungal mycelia was extracted using a DNA kit.Two primer pairs, cytb129-F (5’-GCATAAAGC ATTGGGGCTAA-3’) +cytb129-R (5’-CCGTCTGGCGTCACTATAAAT-3’) , Qo13ext (5’-GGTATAACCCGACGGG GT-TATAGAATAG-3’) +Qo14ext (5’-AACCATCTCCATCCACCATACCTAC A AA-3’) were used.PCR products were examined by electrophoresis in a 1.2%agarose gel in 1×TAE buffer【.Results】236 isolates were collected from the Jiangning district of Nanjing city and the Jurong district of Zhenjing city in Jiangsu province.Among the 236 isolates, 192 (81.4%) showed resistance and 44 (18.6%) showed sensitivity to Qo Is fungicides by using the methods of the discriminative dose.A positive cross resistance existed between azoxystrobin and pyraclostrobin.There were different frequencies of resistance in different sampling areas.The frequency of resistance from high to low were Huayang town (100%) , Baitu town (88.9%) , Shishi town (83.3%) , Chunhua town (83.3) , Maoshan town (80%) , Tuqiao town (70.8%) , Tianwang town (70.0%) and Houbai town (52.0%) .The 19 sensitive isolates were divided into sensitive isolates (EC50<1mg·L^-1) and decreased sensitivity isolates (1≤EC50<5 mg·L^-1) .The average EC50 values of the 15 sensitive isolates and 4 decreased sensitivity isolates to azoxystrobin were 0.269 8 mg·L^-1and 4.159 6 mg·L^-1, respectively.The EC50 values of the 18 sensitive isolates were 0.025 7-0.112 7 mg·L^-1to pyraclostrobin.The average values were 0.055 9 mg·L^-1and the 1 decreased sensitivity isolates were 1.598 8 mg·L^-1.The EC50values of the whole 77 azoxystrobin-resistance isolates were more than 100 mg·L^-1.The 77 pyraclostrobin-resistance isolates were divided into moderate resistance isolates (10≤EC50<100 mg·L^-1, PyrMR) and highly resistance isolates (EC50≥100 mg·L^-1, PyrHR) .The average EC50 values of the 64 PyrMR (83.12%) were 53.070 9 mg·L^-1.The detached strawberry fruit assay inoculated resistant isolates indicated that there was no or less control efficiency when using the recommended doses (a.i 166.67 mg·L^-1) of azoxystrobin and pyraclostrobin.All of the resistant isolates harbored the G143A point mutation.B.cinerea populations were divided into two types according to the structure of the cyt b gene, with or without the third intron (Bcbi-143/144) .All the resistance isolates and some sensitive isolates (47.4%) were without Bcbi-143/144, however, the 52.6%sensitive isolates had Bcbi-143/144.【Conclusion】The examination of the population of high level resistance isolates from the strawberry fields of the Jiangsu province hilly area showed that the Qo Is fungicides had become the dominant population (81.4%) .Detached strawberry fruit assay inoculated resistant isolates indicated, that the control efficiencies were 3.02%and 18.91%, respectively, when using the recommended doses (a.i 166.67 mg·L^-1) of azoxystrobin and pyraclostrobin.We recommend that Qo Is fungicides should not be used in the protection of strawberry gray mold.As tested, all the resistant isolates harbored the G143A point mutation.B.cinerea populations were divided into two types according to the structure of the cyt b gene, with or without the third intron Bcbi-143/144.