Determining genetic diversity and regulation of sexual compatibility in Colletotrichum lentis Damm, the causal agent of anthracnose on Lens culinaris (Medik.)

Abstract

Anthracnose of lentil caused by the fungal pathogen Colletotrichum lentis is an economically important disease in Western Canada. The pathogen population is divided into two races (0 and 1) and two sexual incompatibility groups (IG-1 and IG-2). Resistance to anthracnose race 1 is found in cultivated Lens cultivars whereas for the more aggressive race 0, higher levels of resistance have been reported only from wild Lens species. Furthermore, C. lentis seems to only possess one (MAT1-2) of the two mating type idiomorphs commonly present in heterothallic ascomycete fungi with the typical bipolar mating system. The purpose of this study was to verify the phylogenetic relationship between race 0 and 1 isolates of C. lentis and to sequence and characterize the MAT1-2 of C. lentis. A morphological, multi-locus phylogenetic and host-range study was conducted with isolates of C. lentis, C. truncatum (from various host species and the epitype), C. destructivum, C. dematium, C. higginsianum, C. linicola and C. lindemuthianum. Sequence data from six conserved loci displayed 100% identity for C. lentis isolates of both races that formed a single cluster separate from other Colletotrichum species including C. destructivum, the epitype of C. truncatum and isolates from other hosts identified as C. truncatum. Conidia of C. lentis were slightly falcate with obtuse apices compared to cylindrical conidia with rounded ends of C. destructivum, and longer lunate to falcate conidia of the epitype C. truncatum. Host range tests undertaken on Lens culinaris, Pisum sativum, Cicer arietinum, Vicia faba, Glycine max, Phaseolus vulgaris, Phaseolus lunatus, Trifolium pratense, Medicago sativa, Medicago truncatula, Brassica chinensis and Arabidopsis thaliana under controlled environmental conditions revealed that the host ranges of C. linicola and C. higginsianum overlapped with that of lentil isolates. In contrast, the epitype specimen of C. truncatum was pathogenic on Pisum sativum, Phaseolus vulgaris, T. pratense and Medicago sativa, but not on L. culinaris. All Colletotrichum spp. infected Medicago truncatula and all but the lentil isolates caused disease on G. max. The mating type gene MAT1-2 of C. lentis contained two introns and three exons and an open reading frame of 726 bp coding for a putative protein of 241 amino acids including the high mobility group (HMG) domain characteristic of the MAT1-2 in fungi. The MAT1-2 nucleotide sequences of C. lentis isolates were identical irrespective of IG. An isolate from each of the two IGs, CT-21 (IG-2), CT-30 (IG-1) and a co-culture of CT-21 and CT-30 was used to study the expression levels of MAT1-2 at seven different in vitro time points (0h, 6h, 12h, 18h, 24h, 36, 48h after inoculation in glucose yeast media) and investigate for possible alternative splicing events. MAT1-2 expression for CT-21, CT-30 and the co-culture was observed at all seven time points indicating that it is constitutively expressed, and no differences in the transcript size were seen, ruling out the possibility of a splicing event.