Study On Anthracnose Pathogen Of Persimmon And Its Infection Mechanisms

Anthracnose of persimmon caused by Colletotrichum gloeosporioides occurred in all countries planting the persimmon. Colletotrichum gloeosporioides attacked Diospyros kaki cv. Wuheshi and caused twig blight, leaves defoliation and even whole trees death in Chongan area, Zhejiang province. It led to the vast loss of local persimmon production and already became a bottleneck of sustainable development of the industry. Species of Colletotrichum use diverse strategies for invading host tissue, providing excellent models for studying the molecular and cellular bases of fungal pathogenicity. Therefore, with the anthracnose of persimmon as the objects of research, the results of the studies concerning the relationship between morphology and molecular systematics of pathogens, the law of disease occurrence, cytology of the fungal-host interaction and host speciality are reported.A. Morphological and molecular characteristics of Colletotrichum gloeosporioides from Persimmon The morphological characters of Colletotrichum gloeosporioides isolates collected at different time fromtwigs and petioles of persimmon were investigated. No difference was found on the morphology under the condition of both natural host and culture. The setae through acervului usually were absent on lesions of twigs. Under scanning electron microscopy, apex of conidia obtuse and its base truncate; conidia were embedded within and surrounded by the mucilage. Phylogenetic tree of Collectotrichum species by UPGMA analysis of rDNA ITS1-ITS2 sequence allowed 6 isolates from persimmon to cluster into a group with C. gloeosporioides or their teleomorph Glomella cingulata from other hosts, corresponding with the morphological taxonomy. Along with the process of appressorium development, two rounds of mitosis were visualized with a DNA specific fluorochrome, DAPI. The first round took place after 3-4 h when conidia settled in the polystyrene cultures. After 7-8 h, subsequently a septum formed in the center of the conidium and divided the conidium into two cells. The second round occurred in a cell which produced the germ tube. One nucleus remained in the primary cell and another one moved to the appressorium through the germ tube.B. Various stages and amount of colltotrichum gloeosporiodes on overwintering twigs of persimmon The survival of the pathogen on the overwintering twigs of the host was investigated. On the surface ofthe twig, a few spores of Colletotrichum gloeosporioides were found, there were also a number of spores of Pestalotiopsis diospyri, Alternaria tenuissima and other fungi. The survival rate of the pathogen was 16.67% in lesions of living twigs, and 1.56% in the segments of dead diseased twigs, showing that themycelia of the pathogen in overwintering twigs could be the major source of the inoculum for the primary infection. The planting test showed that the persimmon seedlings without symptom from the diseased area got diseased in the isolated condition 150 km far from Chunan area. It is suggested that the persimmon anthracnose could be disseminated by the looking-like symptomless seedlings. The associated fungi P. diospyri and A. tenuissima hardly inhibited the hyphal growth of C. gloeosporioides on dual culture, respectively. The pathogenicity test demonstrated that both spores and mycelia of/! diospyri did not infect the host twigs, and hardly affect the pathogenicity of C. gloeosporioides on the host.C. Cytology of the infection of persimmon by colletotrichum gloeosporioides on persimmon infrastructure studies of the infection of persimmon by Colletotrichum gloeosporioides were conducted.It was found that there were primary and secondary hypha in the infected tissue, the outer of host plasma membrane was deposited by a layer of electron-opaque material , and an interfacial matrix separated the cell walls of primary hyphae from the plasma membrane deposited a compacted layer of electron-opaque material (PMDM). The previous PMDM, invaginating around primary hyphae, was degenerated gradually by primary hyphae as the primary hyphae colonized host cells further. After infecting host cell wall, the primary hyphae first formed a funnel-shaped hyphal cone in infected host cell wall and then penetrated the cell wall to reform swollen primary hyphae. In the process of penetration, mycelium produced a septum at the extreme constriction and the hyphal cone contained electron-dense cytoplasm. Necrotrophic secondary hyphae extended and propagated as well as ramified through host tissue both intercellularly and intracellularly. Secondary hyphae showed little sign of deformation as it passed through thin host cell wall and no septum and swell appeared near its top. As penetrating thick host cell walls, secondary hyphae produced a septum near the top of mycelium and its top cell swollen and caused host cell wall to splinter. The acervuli formed at 90h after inoculation. There are two stages in the process of the infection of persimmon by Colletotrichum gloeosporioides, including biotrophy and necrotrophy.D. Effects of environment factors on conidia germination, appresorium formation and pathogenicity of the persimmon anthracnose pathogens collectotrichum gloeosporioidesThe conidial germination and appressorial formation of colletotrichum gloeosporioides, the pathogen of persimmon anthracnose, was tested on concave glass slides in different environment. As the concentration of glucose went up, rates of conidia germinating increased but the percentage of appressorial formation decreased, and the length of germ tubes augmented and the diameter of appressoria was steady; with time prolonging, the percentage of conidia germinating and appressorial formation all climbed. The conidiacould germinate and form appressoria over wide pH range from 2.0 to 9.0; the optimal pH for conidial germination and appressorial formation was between pH 5.0 and pH 6.0. The pathogenicity test under different pH condition illuminated that the disease occurred on twigs at 23°C within pH 4.0-8.0, also appeared at 17°C and pH 6.0 without spore masses on lesions of twigs surface, however, it failed to take place at 17CC and pH 5.0 as well as at 15°C and pH 5.0 to pH 6.0. The optimal temperature for the mycelium growth was about 25°C, and higher temperature inhibited its growth. The scanning electron microscopy examination showed that the length of germ tube, growing longitudinally along the ridge and transversely cross the ridges and grooves, varied greatly, and the appressorial formation occurred at the bottom of groove or near.E. Cytological characteristics of the infection of different species, varieties and organs of persimmon by Colletotrichum gloeosporioidesPathogenicity and characteristics of appressorium formation and infection of Colletotrichum gloeosporioides in different species and varieties of persimmon were investigated. Pathogenicity test showed that C. gloeosporioides infected twigs and petioles of Wuheshi and twigs of Yeshi, but failed to infect leaves of Wuheshi, petioles and leaves of Yeshi and also failed to infect leaves, twigs and petioles of Dongshi and Zhejiangshi. The appressorium formation on different host surfaces was almost same and characterized geographically by that 81%~93% of appressoria formed over or near a junction of anticlinal cell walls (JACWs). Cytological studies of infection process demonstrated that 24 h after inoculation, appressorium formation on all surface of different hosts was observed. At 36 h after inoculation, filamentous infection hyphae appeared in cells of twigs and petioles of Wuheshi; 48 h after inoculation, swollen primary hyphae and narrow secondary hyphae formed in cells of twigs and petioles of Wuheshi, which also extended into adjacent cells, but infection hyphae just appeared in Yeshi; 60 h after inoculation, swollen primary hyphae and narrow secondary hyphae also were observed in cell of Yeshi twigs, but primary hyphae were limited in the initial cell, and only branched secondary hyphae extended inter- or intracelluiarlly or in adjacent cells of twigs of Yeshi and twigs and petioles of Wuheshi; Until 90 h after inoculation, no infection hyphae was found in Dongshi and Zhejiangshi. It is suggested that the formation and extension of infection hyphae of the pathogen could be the main mechanism of Colletotrichum gloeosporioides responsible for speciality to different species and varieties of persimmon.F. Ultrastructure of infection of persimmon petiole by Collectotrichum gloeosporioidesThe ultrastructure of infection of persimmon petiole by Collectotrichum gloeosporioides was examined...