The Identification On Causal Agent Of Pear Black Spot And Microscopic Observation For Its Infection In Pear Leaves

In recent years, a devastating fungal disease characterized by small black spots(≤1 mm) on sandy pear(Pyrus pyrifolia Nakai) leaves has occurred in sudden outbreaks in southern China, resulting in severe defoliation and a loss of fruit quality and yield, and causes a great economic loss. However, the etiology of this disease and the production mechanism of black spot symptoms caused by the disease is not always clear, so the disease can not be controlled effectively. In this study, a large number of fungal isolates from sandy pear leaves showing typical black spots were collected from orchards of the occurrence of disease were obtained, and completed the Koch’s postulates, so the etiology of the disease was identified. The pathogen of pear leaf black spot was marked with GFP by using the agrobacterium- mediated transformation technique(ATMT). The transforma nts, which growth rate and morphological characteristics and virulence were not significant changed comparing with wild strains, were screened out. Using fluorescence microscopy, the processes of the infection and expansion of the transforma nts of pear leaf black spot in pear leaves were observed, which intended to clarify the infecting process of the strain and the producing mechanism of black spot symptom clearly. The main results were as follows:1. Identification on causal agent of pear leaf black spot. The pear leaves showing typical black spots were collected in the Fujian, Jiangxi, Guangxi and Hubei province of China, from 2013 to 2015. We collected 447 fungal isolates. Observing the colony morphology and anamorph characteristics of these isolates and ITS sequences of all isolates from Fujian and partial isolates from Jiangxi, Guangxi, Hubei, 397 fungal isolates was identified as Colletotrichum species. Twenty- five isolates from Fujian were assessed for their taxon statuses based on multilocus regions(ITS, ACT, TUB, CHS-1, GAPDH), and the Apn2/MAT(ApMat) locus, which provided molecular proof for the identification of these isolates as Colletotrichum fructicola Prihastuti. Koch’s postulates were fulfilled by inoculating conidium suspensions of the representative isolates on attached and detached leaves of sandy pear cvs. Cuiguan, Xueqing and Huali No. 2 with analogous conditions(under saturated humidity and with tiny wounds) to those that appeared in the field, and similar symptoms were elicited by the isolates, and the number of black dots induced by no injury was less than spray- inoculated. Three isolates were done pathogenicity test on the leaves of the pear, apple, Begonia and so on and analyzed its host range. And these isolates were virulent on sandy pear(Pyrus pyrifolia) and white pear(Pyrus bretschneideri), while they were not virulent on grapes and Chinese date.2. GFP-tagging of C. fructicola strain FJ-85 and the screening of the transformants. By utilizing ATMT method, C. fructicola strain FJ-85 was marked with green fluorescent protein and this study obtained 130 transformants. 23 transformants were randomly selected to determine the hygromycin B resistance and stability of the fluorescence, and detect the hph and gfp gene by RT-PCR, to verify the transformants; And through the comparation of the morphological features, growth rate and pathogenicity of transformants with wild strain FJ-85 respectively, the transformant FJ-85-114 that characterizations were stable and the growth rate and biological characteristics were not significantly changed was screened out.3. Microscopic observation for C. fructicola transformant FJ-85-114 infection in ’Cuiguan’ pear leaves. The mycelium block and the conidia suspension of the transformant FJ-85-114 were prepared, then were inoculated on the healthy ’Cuigua n’ pear leaves; After observing and comparing the infection of the strain on pear leaves by different inoculation methods. The results of observation showed that the infection varied with different inoculation methods. After the mycelium block inoculating by needling leaves, the leaves produced big dark spots. Under a fluorescence stereo microscope observed that leaf tissue had become brown when the hyphae did not reach. After the mycelium block inoculating directly and the conidia suspension inoculating by rubbing with carborundum particles or directly spraying without injury, leaves produced black spots(≤1mm). Under an inverted microscope observed preliminarily that organization between palisade tissue and spongy tissue became brown, but not observed under fluorescent microscope hyphae in the symptomatic tissue at the extension case. Thus, the black spot may be due to some kind secondary metabolites of C. fructicola.