Application of amplified restriction fragment length polymorphism for genetic characterization of Colletotrichum pathogens of alfalfa

Amplified restriction fragment length polymorphism (AFLP) was used to assess the levels of genomic variations among species and isolates of the genus Colletotrichum. Our objective was to characterize at the molecular level two alfalfa pathogens, isolates Arl-NW and 57RR, which are unusually aggressive to anthracnose-resistant alfalfa cultivars and whose taxa has been uncertain based on morphological criteria. The fingerprint patterns obtained were complex but did enable us to place these two isolates within the species C. trifolii and C. gloeosporioides, respectively. The diversity detected with AFLP among and within Colletotrichum species from alfalfa and other crops corroborated their published taxonomy based on morphology, ribosomal DNA sequence, and random amplified polymorphic DNA analyses. Similarity matrices generated with three primer pairs were highly correlated and, thus, were combined to determine the similarity among the fungal species and isolates that were analyzed. Analysis of the data generated with each of the primer pairs individually and application of either distance or parsimony methods supported the placement of these two isolates. The parsimony method of data analysis was more confirmatory in the placement of Phoma medicaginis as an outgroup than the distance method, using either simple matching or Jaccard's coefficients to generate the similarity matrices. Our conclusion is that the AFLP technique will be useful for identification of individual isolates within complex genera such as Colletotrichum because of its ability to generate large numbers of polymorphisms and the consistency of polymerase chain reaction amplification.