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

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 1 × 10⁶ 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.