Detection and quantification of Ceratocystis fimbriata in fig plants by real-time PCR.

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  • MIYOSHI T.
    Fruit Tree Research Center, Ehime Research Institute of Agriculture, Forestry and Fisheries Ehime Prefectural Toyo Regional Office Imabari Bureau
  • SHIMIZU S.
    Fruit Tree Research Center, Ehime Research Institute of Agriculture, Forestry and Fisheries
  • SHINOZAKI T.
    Fruit Tree Research Center, Ehime Research Institute of Agriculture, Forestry and Fisheries
  • SAWADA H.
    National Institute of Agrobiological Sciences

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  • リアルタイム定量ＰＣＲ法によるイチジク株枯病菌の絶対定量および検出
  • リアルタイム テイリョウ PCRホウ ニ ヨル イチジクカブコビョウキン ノ ゼッタイ テイリョウ オヨビ ケンシュツ

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A real-time PCR assay (qPCR) based on TaqMan chemistry was developed to detect and quantify Ceratocystis fimbriata, causal agent of fig ceratocystis canker, in fig plants. Specific primers and probe were designed based on the sequences of ITS region of its rDNA. The assay was positive for 18 C. fimbriata strains isolated from fig ceratocystis canker in Japan and negative for C. fimbriata strains isolated from sweet potato black rot and other soil-borne phytopathogenic fungi tested. A standard curve was constructed by means of a previously reported recovery test: a series of 50 mg branch tissue of fig was spiked with 10-fold, serial dilutions of the pathogen’s ascospores, and each dilution was subjected to DNA extraction and qPCR. A strong linear relationship was measured between the threshold cycle value and the spore (cell) density from 101 to 107 cells per 50 mg fresh tissue. The detection limit was nearly 100 cells per 50 mg fresh tissue. When this assay was used to evaluate the density of C. fimbriata cells in tissues of both artificially and naturally infected fig plants, symptomless regions were found to contain less than 103 cells of the pathogen per 50 mg fresh tissue.<br>

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