Molecular Detection of Verticillium albo-atrum by PCR Based on Its Sequences

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We developed one species-specific PCR assays for rapid and accurate detection of the pathogenic fungi Verticilliumalbo-atrum in diseased plant tissues and soil. Based on differences in internal transcribed spacer (ITS) sequences ofVerticillium spp., a pair of species-specific primers, Vaa1/Vaa2, was synthesized. After screening 17 isolates of V. albo-atrum, 121 isolates from the Ascomycota, Basidiomycota, Deuteromycota, and Oomycota, the Vaa1/Vaa2 primers amplifiedonly a single PCR band of approximately 330 bp from V. albo-atrum. The detection sensitivity with primers Vaa1/Vaa2 was10 fg of genomic DNA. Using ITS1/ITS4 as the first-round primers, combined with Vaa1/Vaa2, the nested PCR procedureswere developed, and the detection sensitivity increased 1 000-fold to 10 ag. The detection sensitivity for the soil pathogenswas 100-conidia g-1 soil. The PCR-based methods developed here could simplify both plant disease diagnosis and pathogenmonitoring as well as guide plant disease management. Based on differences in internal transcribed spacer (ITS) sequences of Vetiverillium spp., A pair of species-specific primers, Vaa1 / Vaa2, was synthesized. After screening 17 isolates of V. albo-atrum, 121 isolates from the Ascomycota, Basidiomycota, Deuteromycota, and Oomycota, the Vaa1 / Vaa2 primers amplifiedonly a single PCR band of approximately 330 bp from V. albo- atrum. The detection sensitivity with primers Vaa1 / Vaa2 was10 fg of genomic DNA. Using ITS1 / ITS4 as the first-round primers, combined with Vaa1 / Vaa2, the nested PCR procedures developed, and the detection sensitivity increased 1000-fold to 10 ag. The detection sensitivity for the soil pathogens was 100-conidia g-1 soil. The PCR-based methods developed here can simplify both plant disease diagnosis and pathogen monitoring as well as guide plant disease management .
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