Molecular Detection of Phyllosticta citricarpa on Citrus Black Spot

doi:10.7668/hbnxb.2017.05.020

Abstract

Abstract: In order to distinguish P.citricarpa, P.citriasiana, P.capitalensis and P.citrichinaensis,upstream primers for P.citricarpa, P.capitalensis and P.citrichinaensis were designed in ITS1 and 18S regions,and these primers were used together with the ITS4 primer.Then,the specificity and annealing temperature of primers were screened,and the sensitivity of the specific primers were verified.Screened primer was used to detect P.citricarpa when DNA was extracted from black/tan spot lesions on fruits or leaves.The results showed that 593 bp specific fragments were only obtained from P.citricarpa by specific primer Pc1/ITS4,551 bp specific fragments were only obtained from P.capitalensis by specific primer Pct4/ITS4,and 706 bp specific fragments were only obtained from P.citrichinaensis by specific primer Pcc1/ITS4,when annealing temperature at 60℃.These specific primers were never amplified any fragment from the common Citrus pathogens.The sensitivity test results showed that the sensitivity of specific primers Pc1/ITS4 and Pct4/ITS4 was 200 pg,and the sensitivity of specific primer Pcc1/ITS4 was 20 pg.The screened specific primer Pc1/ITS4 could detect P.citricarpa from DNA of black spot lesions on fruit.Therefore,using the specific primers,combined with the simple method of DNA extraction,the molecular detection for the pathogen of Citrus black spot could be completed in a short time.

Key words: Citrus black spot, Phyllosticta citricarpa, Phyllosticta citrichinaensis, Phyllosticta capitalensis, Specific primers

CLC Number:

S436.66

WANG Xinghong. Molecular Detection of Phyllosticta citricarpa on Citrus Black Spot[J]. ACTA AGRICULTURAE BOREALI-SINICA, 2017, 32(5): 130-135. doi: 10.7668/hbnxb.2017.05.020.

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