Species-specific real-time PCR detection of Colletotrichum kahawae
Article first published online: 21 DEC 2012
© 2012 The Society for Applied Microbiology
Journal of Applied Microbiology
Volume 114, Issue 3, pages 828–835, March 2013
How to Cite
Tao, G., Hyde, K.D. and Cai, L. (2013), Species-specific real-time PCR detection of Colletotrichum kahawae. Journal of Applied Microbiology, 114: 828–835. doi: 10.1111/jam.12068
- Issue published online: 18 FEB 2013
- Article first published online: 21 DEC 2012
- Accepted manuscript online: 16 NOV 2012 09:26PM EST
- Manuscript Accepted: 10 NOV 2012
- Manuscript Revised: 8 NOV 2012
- Manuscript Received: 2 AUG 2012
- National Natural Science Foundation of China. Grant Number: 31070020
- Knowledge Innovation Program of the CAS. Grant Number: KSCX2-EW-J-6/KSCX2-YW-Z-1026
- China Postdoctoral Science Foundation. Grant Number: 2011M500421
- King Abdulaziz City of Science and Technology. Grant Number: 10-Bio-965-02
- Coffea arabica ;
- coffee berry disease;
- Colletotrichum gloeosporioides ;
- molecular diagnosis;
- plant disease;
- the glyceraldehyde-3-phosphate dehydrogenase gene
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.
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.