Premium
Species‐specific real‐time PCR detection of C olletotrichum kahawae
Author(s) -
Tao G.,
Hyde K.D.,
Cai L.
Publication year - 2013
Publication title -
journal of applied microbiology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.889
H-Index - 156
eISSN - 1365-2672
pISSN - 1364-5072
DOI - 10.1111/jam.12068
Subject(s) - biology , microbiology and biotechnology , colletotrichum , ecology
Aims Colletotrichum kahawae is a strongly aggressive pathogen causing coffee berry disease and is specific to A rabica coffee ( C offea arabica ) in A frica. In this article, we developed a real‐time PCR assay for the species‐specific diagnosis of C . kahawae by designing the primers and a T aq M an probe derived from the single nucleotide polymorphism‐rich region of glyceraldehyde‐3‐phosphate dehydrogenase ( GAPDH ) gene. Methods and Results DNA markers from r DNA internal transcribed spacer, actin, β‐tubulin and GAPDH genes of the ex‐type culture of C . kahawae and 10 reference strains of C olletotrichum species were analysed for intra‐ and interspecific variations. The GAPDH gene was selected to develop a species‐specific DNA marker. A T aq M an 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.