PCR-based markers for detection of Colletotrichum acutatum and C-gloeosporioides in flowering dogwood (Cornus florida)

Two fungi, Colletotrichum acutatum (= Glomerella acutata) and C. gloeosporioides (= G. cingulata) were isolated from flowering dogwood (Cornus. orida) and characterised by molecular PCR-based markers. The internal transcribed spacer (ITS) universal primer pair ITS1-F/ITS4 produced fragments of 622 bp and 613 bp in C. acutatum and C. gloeosporioides, respectively. The two sequences were 91.1% identical in a 626 bp region. Six ITS specific primer pairs were designed that produced PCR fragments only for C. acutatum. The 18S small subunit rRNA universal primer pair NS1/NS2 produced fragments of 570 bp and 560 bp for C. acutatum and C. gloeosporioides, respectively. The two fragments were 97.4% identical in a 497 bp region. Two specifc primer pairs were designed that produced PCR fragments only for the laccase or pectate lyase B gene of C. gloeosporioides. Two additional primer pairs were designed that produced specific PCR fragments only for the glutamine synthetase or a key lime pathogenicity gene of C. acutatum. These results provide useful tools to identify and distinguish C. acutatum and C. gloeosporioides in limb dieback of flowering dogwood, and this research also provides an approach for further characterisation of pathogenicity related genes.