Study On Cytology Of Infection Of Persimmon By Colletotrichum Gloeosporioides And Cloning Its CGTA1 Gene

Anthracnose of persimmons often occurred in almost all countries planting persimmon in the world. Recent years, Anthracnose of persimmon caused by Colletotrichum gloesporioides took place severely on Diospyros kaki cv. Wuheshi in the Chunan area, Zhejiang province, leading to severe economic losses. It was also become a bottleneck of local sustainable development in persimmon industry. Fungal pathogens exhibited two modes of both biotrophy and necrotrophy in the process of infecting the perssimon and its primary hyphae penetrate the cell wall from one to the other, which can produce an interfacial matrix between the pathogen and the host, being similar to the cytological characters that C. lindemuthianum infected its host to form. Species of Colletotrichum use diverse strategies for invading host tissues , providing excellent models for studying the molecular and cytological bases of fungal pathogenicity. In this thesis, it is illustrated the effect of different development process of the twig and petiole of the persimmon on the occurrence and development of anthracnose disease and the mechanisms of resistance of the mature organs to pathogen on the cytological levels. Meanwhile, the cloning of CGTA1 (C. gloeosporioides transcriptional activator 1) was conducted for stating the mechanisms involved in the switch between biotrophic and necrotrophic phases in the infection process.1,Effect of development of the persimmon organs on occurrence and development of anthracnose diseaseThe artificial inoculation tests demonstrated that the resistance of twigs and petioles of Diospyros kaki cv. Wuheshi to Colletotrichum gloeosporioides was increased gradually with organs (twigs and petioles) development. The resistance was charactered by extending the latent period of the disease. On the petioles, the latent period for the disease was prolonged for 52 h in the later inoculation than in the early inoculation. Similarly, on the twigs, the latent period was prolonged for 40 h. Cytological studies on the infection process showed that the large primary hypha became thiner when they were passing through a cell wall and then penetrated the cell wall to form a swollen primary hyphae, which produced the smaller secondary hyphae. Compared with our previous results, it was considered that the petiole development affected the fungal biotrophic stage to switch to necrotrophic stage, leading to extend the biotrophic stage and make the latent period of the disease be postponed as well.2,Cloning of the CGTA1Based on the conserved amino acid sequence of CLTA1 (C. lindemuthianum transcriptional activator 1) and other homologous genes from several filamentous fungus, a pair of degenerate primers was designed and synthesized. A 687bp fragment was amplified from the genomic DNA with the degenerate primers. Compared with the CLTA1, this sequence is 67.24% identical to the corresponding one of CLTA1 and there is only the difference of 2 bp size between their corresponding sequences. The results indicated that this sequence obtained may be the fragment of the targeted gene.Based on the known fragment obtained, three nested specific primers were designed TAIL-PCR strategy was performed to amplify the unknown 5' and 3' flanking sequences of the known fragment from genomic DNA of C. gloesporioidesis and a 2844 bp sequence was obtained. Sequence analysis showed that this sequence is the targeted CGTA1, containing the 2514 bp complete open reading frame, 130 bp 5'-noncoding region and and 200 bp 3'-noncoding region and being 65.1% identical to the CLTA1. The results of BLAST search showed that the cloned CGTA1 is homologous to fungal zinc cluster (Zn[II]₂Cys₆) genes.