Molecular Detection Of Diaporthe Citri And Establishment Of Sensitivity Baseline Of D.citri Population To Azoxystrobin

Citrus melanose caused by Diaporthe citri is a serious fungal disease in citrusproduction.In recent years,the occurrence of citrus melanose is becoming more and more serious,and the cosmetic quality and sales price of fruits are declining,which seriously affects the development of citrus industry.Early and accurate diagnosis of D.citri is of great practical significance for the effective prevention and control of citrus melanose.At present,chemical control is the main measure to control citrus disease.Azoxystrobin is a kind of highly effective methoxyacrylate fungicide（Qo I fungicides）,which is registered in the United States for prevention and control of citrus melanose.Recently,it has also been registered for prevention and control of citrus scab and anthracnose.The pesticide manufacturers have also begun to recommend it for the prevention and treatment of citrus melanose in China.Timely determination of the sensitive baseline of the D.citri population to azoxystrobin before it widely using is of a practical significance for monitoring the development of azoxystrobin resistance of the pathogen population,thus to guide the using scientifically in the future.In this paper,the rapid molecular detection of D.citri and the baseline sensitivity of D.citri population to azoxystrobin were studied,and the results were reported here:1.Molecular detection of Diaporthe citri:Specific primers for D.citri were designed based onβ-tubulin sequence,and a rapid molecular detection system was established based on conventional PCR and SYBR Green I Real-time PCR.In conventional PCR,this primer could only amplify a 244 bp specific fragment from the DNA of D.citri,but could not be amplified from the DNA of 14 other Diaporthe species which also originated from citrus.In Real-time PCR,this primer also had only one product absorption peak for D.citri,so the designed primer was highly specific.The detection sensitivity of conventional PCR was 0.78 ng/μL,and that of Real-time PCR was 0.35 ng/μL.Leaves suspected to have been infected with D.citri were detected by molecular detection system.The positive detection rate of conventional PCR was 30%,and the positive detection rate of Real-time PCR was 60%,which indicated that Real-time PCR was more sensitive than conventional PCR.2.Baseline of sensitivity of Diaporthe citri population to azoxystrobin:The sensitivity of 66 strains of D.citri from four citrus producing areas of Zhejiang,Hunan,Jiangxi and Guizhou province to azoxystrobin was determined by mycelial growth inhibition and spore germination inhibition,respectively,and the sensitivity baseline of D.citri population to azoxystrobin was established.The 66 strains of D.citri tested in this study was highly sensitive to azoxystrobin,and no resistant strains were found.The average values of EC₅₀were（0.2285±0.1138）μg/m L and（0.0256±0.0069）μg/m L,respectively.The frequency distribution curve of susceptibility of all strains of D.citri to azoxystrobin was continuous unimodal,which was consistent with normal distribution.Therefore,（0.2285±0.1138）μg/m L and（0.0256±0.0069）μg/m L could be used as the baseline of susceptibility to azoxystrobin for mycelial growth and spore germination,respectively.The established sensitivity baseline in this study can be used as a reference for monitoring the sensivity of D.citri to azoxystrobin in the field,and has a practical significance for guiding scientific fungicide use in the future.