Molecular and morphological determination of Colletotrichum trifolii isolates originating from alfalfa

Colletotrichum trifolii is a fungal pathogen responsible for anthracnose disease in alfalfa. The isolates of C. trifolii were obtained from diseased alfalfa stems collected from field in Serbia. It was determined by pathogenicity examination that four isolates (Luc-7, Luc-17, Luc-27, Luc-33) can cause stem lesions on inoculated alfalfa plant. Our isolates were compared using reference isolates of C. trifolii (CBS 158.83). Isolates on MA and CDA developed olive green to grey colonies with white margin, while the substrate got dark olive green color. Conidiophores were hyaline, varied in length and produced a succession of conidia apically. Conidia were hyaline, straight, rounded at the ends, and non-septated with average size 7.85x3.87 μm. Average sizes of appressoria were 7.5-16.5 x 5.5 x 8.9 μm. The polymerase chain reaction (PCR) with one set of specific primers was used for the detection of examined isolates of Colletotrichum trifolii. Amplification of desired DNA fragment (590 bp) was determined using specific primer pair TB3-F/TB3-R. Achieved results of amplification indicated that the isolates Luc-7, Luc-17, Luc-27, as well as CBS 158.83, had traits of C. trifolli. Amplification and band on about 430 bp appeared in isolate Luc-33, which also belonged to C. trifolli species