Species-specific real-time PCR detection of Colletotrichum kahawae

Authors

  • G. Tao,

    1. State Key Laboratory of Mycology, Institute of Microbiology, Chinese Academy of Sciences, Beijing, China
    2. Guizhou Institute of Plant Protection, Guiyang, China
    3. Guizhou Key Laboratory of Agricultural Biotechnology, Guiyang, China
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  • K.D. Hyde,

    1. Institute of Excellence in Fungal Research, Mae Fah Luang University, Chiang Rai, Thailand
    2. School of Science, Mae Fah Luang University, Chiang Rai, Thailand
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  • L. Cai

    Corresponding author
    • State Key Laboratory of Mycology, Institute of Microbiology, Chinese Academy of Sciences, Beijing, China
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Correspondence

Lei Cai, State Key Laboratory of Mycology, Institute of Microbiology, Chinese Academy of Sciences, West Bei Cheng Rd, Beijing, China. E-mail: mrcailei@gmail.com

Abstract

Aims

Colletotrichum kahawae is a strongly aggressive pathogen causing coffee berry disease and is specific to Arabica coffee (Coffea arabica) in Africa. In this article, we developed a real-time PCR assay for the species-specific diagnosis of C. kahawae by designing the primers and a TaqMan probe derived from the single nucleotide polymorphism-rich region of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene.

Methods and Results

DNA markers from rDNA internal transcribed spacer, actin, β-tubulin and GAPDH genes of the ex-type culture of C. kahawae and 10 reference strains of Colletotrichum species were analysed for intra- and interspecific variations. The GAPDH gene was selected to develop a species-specific DNA marker. A TaqMan real-time PCR assay for species-specific detection of C. kahawae was developed, and its accuracy was tested against type strains of other phylogenetically closely related species in the C. gloeosporioides species complex, with the detection sensitivity of 80 fg μl−1 of genomic DNA.

Conclusions

This real-time PCR assay is highly specific and sensitive for the diagnosis of C. kahawae and can be applied in qualitative and quantitative tests.

Significance and Impact of the Study

This protocol allows for a rapid and sensitive detection of C. kahawae and will be useful in disease management and pest detection to prevent further spread of this pathogen.

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