Molecular characterization of slow-growing orange and Key lime anthracnose strains of Colletotrichum from citrus as C-acutatum

Three forms of Colletotrichum are recognized on citrus: the fast-growing gray (FGG) form, a common saprophyte and postharvest pathogen that is morphologically distinct from the other two; the slow-growing orange (SGO) form, the causal agent of postbloom fruit drop; and the Key lime anthracnose (KLA) form, the cause of lime anthracnose. Pathogenicity tests confirmed that the SGO isolates infected sweet orange flowers only, the KLA isolates infected both sweet orange flowers and Key lime foliage, and the FGG isolates did not infect either of these host tissues. Polymerase chain reaction (PCR) with the C. acutatum-specific primer CaInt2 and the internal transcribed spacer (ITS)4 primer (from a conserved sequence of ribosomal DNA [rDNA]) amplified a 490-bp fragment from the SGO and KLA isolates but not from the FGG isolates. PCR with the C, gloeosporioides-specific primer CgInt and the ITS4 primer amplified a 450-bp fragment only from the FGG isolates. The nucleotide sequence of the rDNA ITS1 of four SGO and four KLA isolates indicated either complete homology or a I-base difference from a C. acutatum reference isolate but only 80 to 81% homology to a C. gloeosporioides reference isolate. The ITS1 sequence of the FGG isolates had 97% homology to the C. gloeosporioides reference isolate but only 80% homology to the C. acutatum reference isolate. These data confirmed the classification of the SGO and KLA isolates as C. acutatum and the FGG isolates as C. gloeosporioides. Cluster analysis of rDNA and mitochondrial DNA restriction fragments divided the SGO and KLA isolates into two recognizable groups with genetic similarity of 87%. These two groups were more closely related to each other than to any of the reference isolates used (<80% similarity). It is suggested that the SGO and KLA isolates had a recent common ancestor.