Identification of pathogenicity factors of Colletotrichum gloeosporioides with forward genetic approach

• Main Authors: Chia-Chi Chang, 張家綺
• Other Authors: Miin-Huey Lee
• Format: Others
• Language: zh-TW
• Published: 2014
• Online Access: http://ndltd.ncl.edu.tw/handle/30601772802767348458

碩士 === 國立中興大學 === 植物病理學系所 === 102 === Mango (Mangifera indica L.), the king of fruits, is one of the most important fruit crops in the world. Warm and humid conditions favor the occurrence of mango anthracnose, caused by Colletotrichum gloeosporioides. Mango anthracnose causes brown spots on leaves, panicles, twigs and fruits, resulting in dramatical reduction on fruit quality and yield. To identify the pathogenicity factors of Colletotrichum gloeosporioides on mango, I used Agrobacterium tumefaciens-mediated transformation (ATMT) to generate transformants of C. gloeosporioides TYC-2 by random mutagenesis. Agrobacterium tumefaciens EHA105 carrying binary vector pBHt2 or p1300-CT74 was used for ATMT in my study. Total 326 transformants were obtained from eight ATMT experiments. After PCR or GFP screening, 181 transformants had T-DNA integrations. The transformation rate is about 0.0258%. Southern blot analysis showed that 63.6% transformants carried single T-DNA insertion by analyzing 11 randomly selected transformants. Morphological examination determined that transformant C225-1 and its single spore isolate C225-2 were albino mutants. The albino mutants displayed more abundant white-aerial hyphae and lower ability of sporulation than the wild type strain. Transformant C225-1 produced few spores, whereas transformant C225-2 almost lost the ability of sporulation on PDA and MS agar medium. I also assayed the pathogenicity of 118 transformants on young mango leaf. Transformant 48 and 76 showed reduced virulence and caused 0.7% and 21.4% of the lesion area generated by the wild type strain, respectively. Besides, transformant 76 also showed reduced virulence on wounded apple fruits. Southern blot analysis indicated that transformant C225 and 76 had only one T-DNA insertion event, whereas transformant 48 carried two copies of T-DNA. In order to find out the insertion site, I used inverse PCR to obtain the T-DNA flanking regions. DNA sequence data showed that T-DNA inserted in the gene encoding a pH-response transcription factor (pacC) in transformant 76 and in the coding region of polyketide synthase (PKS) gene in transformant C225-1 and C225-2. Further analysis of transformant C225-1 and C225-2 showed that they accumulated less melanin than the wild type strain. Interestingly, transformant C225-2 showed lower virulence on both intact immature mango leaf and wounded mature mango leaf by inoculation with mycelium plugs, which was not observed in transformant C225-1. Taken together, in this study I have setup an ATMT system for mango anthracnose pathogen C. gloeosporioides and produced 181 transformants. Two of the transformants were further characterized and were pacC and pks disrupted mutants, respectively. In addition, I have generated a binary vector for pks complementation assay. The function of the PKS in C. gloeosporioides development and pathogenicity on mango will be demonstrated in the future.