Agrobacteirum-Mediated Transformation And Molecular Analysis In Colletotrichum Acutatum And C. Gloeosporioides, The Pathogens Of Colletotrichum Leaf Disease Of Rubber Tree

Anthracnose is one of the most serious foliar disease of Hevea Brasiliensis which was infected by the complex of Colletotrichum gloeosporioides and Colletotrichum acutatum and the disease was aggravate year after year. Anthracnose Pathogen was also a important mode fungi to the study about plant-pathogeny interaction.In order to study pathogenic molecular mechanism of Anthracnose Pathogen,we have succeeded in transforming Colletotrichum gloeosporioides HCGHN4and HCGYN12 by optimizing established method of Agrobacterium tumefaciens-mediated tansfomation and carrying through phenotype screening and molecular marker to transformants, based on gathering Anthracnose Pathogen of the rubber plant in main planting areas, clarifying their biological Characteristics and taxonomic status, having success with resistance evaluation of domestic major germplasm to Colletotrichum gloeosporioides and Colletotrichum acutatum.The internal transcribed spacer regions (5.8S/ITS) of the ribosomal RNA gene from 22 isolates of pathogen of rubber colletotrichum leaf disease analysis showed that only 7 isolates belonged to Colletotrichum gloeosporioides.The remaining 15 isolates were C. acutatum and the intraspecific genetic variation of Colletotrichum acutatum isolated from rubber was low, however, intraspecific genetic variation of C. gloeosporioides was relatively abundant.We analyzed the optimum AS concentration, co-culture time, induced time and concentration of Agrobacterium, the concentration of Colletotrichum spore and co-culture time etc and obtained appropriate Agrobacterium tumefaciens-mediated transformed system to Colletotrichum gloeosporioides of the rubber tree. We transformed Anthracnose Pathogen of the rubber tree and obtained 2172 T-DNA inserted transformants of Colletotrichum gloeosporioides and 2263 T-DNA inserted transformants of Colletotrichum acutatum by using duality vector pSULF.gfp containing ILV 1 gene (with a chlorimuron-ethyl resistance) and the GFP reporter geneThe PCR amplification results showed that pathogenecity transformants all had GFP gene.Twenty transformants arbitrarily selected were used to do genomic Southern blot analysis,9 transformants of C. gloeosporioides were proved to be single-copy insertion,7transformants were double-copied,3 transformants were triple-copied;8 transformants of C. gloeosporioides were proved to be single-copy insertion,7transformants were double-copied,3 transformants were triple-copied. The percent of single-copy insertion transformants are 47.4% and 44.4%We did not obtain mutant with obvious changed ability of infection, pathogenicity, sporulation, although the inserted T-DNA has influence on configuraion and growth rate of the two transformants. By means of TAIL-PCR, we get five flanking sequences. They are HCGYN12-153,HCGHN4-182,HCGHN4-56,HCGYN12-392.