Identification of Races of Colletotrichum lindemuthianum with the Aid of PCR-Based Molecular Markers.

Abstract

Inoculation of a common bean differential series is the usual method for identification of races of Colletotrichum lindemuthianum. This procedure is extremely useful for phytopathological as well as breeding purposes, but it requires strict control of the number of spores and incubation conditions. Furthermore, this method may result in misclassifications of isolates because of the subjectivity of symptom evaluation. We propose the use of DNA-based molecular markers as an auxiliary tool to aid the classification of races of C. lindemuthianum. Specific DNA bands were identified for races 73, 65, and 64 by polymerase chain reaction (PCR) amplification of bulked DNA samples from isolates of these three races with random primers. The presence of these bands was checked on four isolates previously classified by inoculation on a differential series as belonging to races 23, 72, 79, and 585. The molecular procedure showed that two of these isolates had been misclassified, confirming the high potential of the proposed procedure to aid the identification of races of C. lindemuthianum. Amplification products obtained with 44 different primers also allowed the determination of the genetic distances among isolates from races 73, 65, and 64. These data were used to cluster the isolates into three groups that coincide with the ones obtained by inoculation.