The use of T-DNA tagging to isolate mutants of Colletotrichum gloeosporioides and Colletotrichum acutatum with reduced virulence against Hevea brasiliensis

摘要

Agrobacterium tumefaciens-mediated transformation (ATMT) is being increasingly recognized as an effective insertional mutagenesis tool in studies of filamentous fungi for gene discovery and functional analysis. We developed and optimized ATMT for 2 Colletotrichum species, Colletotrichum gloeosporioides and Colletotrichum acutatum, the causative agents of Colletotrichum leaf disease in rubber trees in Southern China. A.tumefaciens strain AGL-1 carrying an ILV1 gene and a green fluorescent protein gene were used to transform the conidia of these 2 Colletotrichum species. The transformation efficiency was correlated with the co-cultivation duration and bacterial cell concentrations, which reached 300-400 transformants per 1x10(6) conidia after optimization. Southern blot analysis indicated that about 60.0% of the C.gloeosporioides transformants and 46.2% of the C.acutatum transformants had a single copy of T-DNA in their genomes. Fungal genomic DNA segments flanking the T-DNA were identified in the transformants through thermal asymmetrical interlaced polymerase chain reaction followed by sequencing. The flanking sequences from 4 C.acutatum and 7 C.gloeosporioides transformants showed moderate or weak homology to the NCBI database entries. Some sequences matching those reported virulence-related genes. The results suggest that the T-DNA inserted mutants banks constructed are useful for the discovery of new or important genes and to elucidate their function in the 2 Colletotrichum species.