Generating A T-DNA Insertional Library Of Colletotrichum Gloeosporioides From Stylo By ATMT

Stylosanthes spp originates in Latin America, and is the most important commercial forage legume in tropical areas around the world. Stylosanthes anthracnose, mainly caused by ColLetotrichum gloeosporioid.es, is a globally severe disease in stylo production. Currently, there are little progresses in anthracnose research which refer to molecular biology. In this study, an Agrobacterium tumefaciens AGL-1,containing binary vector with ILV1and GFP as report gene was used for transformation of Colletotrichum gloeosporioides. In order to gain higher transformation efficiency, the major factors affectting transformation efficiency were analyzed. The optimization of genetic transformation system of C. gloeosporioides was established, and the conditions of transformed system were optimized. As a result,the transformation efficiency was increased to300to400transformants per106conidia.To gain more insight into the molecular mechanisms of Colletotrichum gloeosporioides pathogenesis, an important pathogen of Stylosanthes, Agrobacterium tumefaciens-mediated transformation (ATMT) was used to identify mutants of C. gloeosporioides impaired in pathogenicity. An ATMT library of4,616. C. gloeosporioides transformants was generated. Thirty randomly selected transformants were detected using PCR. All the transformants tested had GFP gene comfirmed by PCR, and GFP signals were also detected. One of the nine transformants was selected for Southern blot. Southern blot analysis showed67%of the transformants had single-site T-DNA integrations. These transformants still had chlor resistance after10generations of continuous culture, whichwill help to analize function of pathogenicity gene of this pathogen.Based on the established library of Stylo Anthracnose,1230transformants were randomly selected, for screening using six inoculation methods for Stylo Anthracnose. Finally, we selected the inoculation methods of acupuncture with bacteria cakes and spraying Stylo potted seedlings, and conducted pathogenicity determination of mutant strains using the healthy young plants of susceptible varieties AFT3309and international general-purpose determination of strain Deaveur, Schofield, Cook, Graham, Oxley, Fitzroy, a total of23mutations in bacteria, which18strains were virulence weakened and five strains were completely lost in pathogenic. Genomic sequences flanking T-DNA were recovered from15mutants by thermal asymmetric interlaced PCR, had a total of five insertion site flanking sequences of the right side of the genome. Accordancewith the flanking sequences obtained ratio on the anthrax genes which had been sequenced to find the T-DNA insertion sites in the whole genome, and predicted a potential marker gene in softberry, analyzed with bioinformatics methods. The results show that the gene homology was79%between the inserted gene of t-2430mutant strains which defects in pathogenic and the gene encoding aspartate transaminase enzyme which was present in rice blast fungus. Based on the function of the enzyme, it can be suggests that its most likely associated with the pathogenicity of Stylo Anthracnose.