| Pear is among the first three important fruit tree in China, and its growing areas and yields in China are also listed at the first worldwide. Pear ring rot and Botryosphaeria canker are two widely occurred diseases, which mainly infect stems and fruits of pear causing serious effect on growth and productivity of pear. The symptoms of these diseases on pear stems are different. Pear ring rot disease induces ring necrotic spots on pear stems, and Botryosphaeria canker usually induces large necrotic canker. However, these two diseases show the similar rot symptom on pear fruits. Previous results indicated that the pathogens of both diseases had highly similar morphological and biological characteristics. Therefore, there are still different opinions on the classification of their pathogens. In order to understand the characteristics of pathogens causing these diseases, diseased pear stem samples showing ring rot and canker were collected from main pear production provinces and their pathogens were isolated and studied. Results are as followings.1. Totally,44 and 26 pathogen stains were isolated from pear samples showing ring rot and Botryosphaeria canker. All these strains were cultured on PDA medium and their culturing features were observed. Results showed that these pathogen strains had similar colony morphological characteristics. Their colonies initially was white, then turn into grey or dark grey, and finally into black, and there were a lot of aerial hyphae in PDA medium. A few strains showed relatively slow growth speed and less aerial hyphae. Their conidia were transparent and showed spindle or narrow spindle shapes, and had thin wall and one unit cell. The sizes of conidia from these strains did not have significant difference. All those characteristics are similar to those of B. dothidea which induced apple ring rot disease.2. Some representive strains were used for pathogenicity test on in vivo and in vitro pear stems and fruits by wound inoculation of mycelium. Results showed that all tested strains had strong pathogenecity on both stems and fruits. They induced dark brown bark spots on in vivo pear stems accompanied by cankers 10 days post inoculation (dpi). On in vitro stems, necrotic spots were induced 4 dpi and later turned into dark brown spots. On pear fruits, brown rot with concentric rings was induced 2 dpi and diseased fruits quickly decay and then produce yellow-white viscous exudates.Three sets of primers targeted on the conserved sites of rDNA-ITS and genes (3-tubulin and EF1-αwere used to amplify the genomic DNA extracted from representive strains. Sequence analysis of PCR products showed that the sequences of rDNA-ITS andβ-tubulin from different strains had high similarities ranging 99.4%-100.0% and 99.5%-100.0%%, respectively. However, the sequences of gene EF1-a from those strains had some differences, and their similarities were 97.8-100%. Phylogenetic trees of rDNA-ITS and genesβ-tubulin and EF1-αwere constructed by neighbor-joining method. Results showed that all pathogen strains from both ring rot and canker diseased pear samples were highly clustered in the same group with B. dothidea in three trees and distinguished from other species in this genus. These results indicate that there is not significant difference between strains inducing ring rot and canker diseases.4. Nine polymorphic primers screened from 50 random primers were used for RAPD analysis of obtained 72 pathogen strains. An UPGMA cluster tree was constructed based on polymorphic bands. Results showed that 27 subgroups were identified at the 0.89 similarity level, which indicated a plentiful genetic diversity among these pathogen strains. However, their genetic distances were not related to their geographic and pear cultivar origins.5. The nine primers were used for amplification pooled DNAs from pathogens of pear ring rot, Botryosphaeria canker and Valsa canker. The PCR products showing specific bands in DNA pools from pear ring rot or/and Botryosphaeria canker were sequenced. Totally, eight sets of primers were designed based on obtained sequences and use further amplification of individual strain. Finally, a primer set S105-3-1/S105-3-1, which could only amplified all 72 pear ring rot and Botryosphaeria canker strains, was selected as a SCAR marker of the studied pathogens. Meanwhile, The SCAR marker products from Botryosphaeria canker strains showed two sizes. Then, some PCR products with different sizes were sequenced. Results revealed that their sizes were 795 bp and 664 bp, and their sequences shared only 65.47% similarities. Furthermore, DNAs from seven strains showing two different SCAR marker sizes were pooled, respectively, and amplified with five random primers. The similar RAPD bands were obtained from those two pools, which indicated that those strains belong to the same species, but with some molecular differences. Therefore, the obtained SCAR marker could be used as the identification of pathogen of pear ring rot and Botryosphaeria canker and also a polymorphic maker of the pathogen. |
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