Diversity Analysis Of Soil Fungi By PCR-RFLP Techniques And The Detection Of Oidium Heveae In Hainan Rubber Plantation

Rubber tree powdery mildew is one of the important rubber diseases. It can influence on the growth of seedling, sapling and the dry glue production, and cause considerable harm and economic loss. Through the research on the identification and pathogenic tests of the rubber powder pathogenic(Oidium heveae Steinmann) in rubber tree soil, we can better know the effect of rubber tree soil in powdery mildew infection, find more effective methods of prevention and treatment to rubber tree powdery mildew, and reduce the harm of rubber tree powdery mildew in the actual production. This research used specific primers to PCR amplification and PCR-Southern hybridization to detect whether there is Oidium heveae in the rubber tree soil. The rubber leaves inoculated by the soil dilution can detect whether there is Oidium heveae in the rubber tree soil, and the Oidium heveae in the rubber tree soil can cause rubber tree powdery mildew. PCR-RFLP analysis on soil fungi in different depths and different months of Hainan rubber plantation can be better to investigate the community structure of rubber plantation.The main topics of this thesis are as follows:Primers reported to amplification rDNA of Oidium heveae and primers designed according to genes related to Oidium heveae infect rubber tree were used to amplification the Oidium heveae and the soil samples. After comparison, the14640-S/14640-A was selected as the specific primers to detect Oidium heveae in soil.Six fungi of different genera were isolated from rubber plantation soil samples by dilution-plate method. They are Verticillium, Muco, Trichoderma, Gongronella butleri, Rhizomuco and Penicillium. Extract DNA of the fungi isolated from soil and the Colletotrichuim gloeosporioides and Corynespora cassiicola. After PCR amplification by14640-S/14640-A, the agarose gel electrophoresis has no bands. After PCR-Southern hybridization detection, there is a hybridization band different from the Oidium heveae. As a result, primers14640-S/14640-A can be used as a specific primer in detecting Oidium heveae in the soil.Using specific primers14640-S/14640-A to PCR amplification and PCR-Southern hybridization detection soil the samples, only the surface and the depth of5cm soil samples in January, the surface, depth of5cm and10cm soil samples in April,5cm depth in October, and the surface,5cm depth soil samples in November can get the same band with Oidium heveae. After sequencing comparison, we found it in agreement with the Oidium heveae sequence. This means that the DNA of Oidium heveae can be detected in rubber tree soil by the PCR amplification and PCR-Southern hybridization, Oidium heveae could survive in rubber plantation soil.Inoculated the rubber leaves by rubber soil dilution can detect whether there is Oidium heveae in the rubber tree soil, and whether the Oidium heveae in the rubber tree soil can cause rubber tree powdery mildew. The results shows that, the surface, depth of5cm and10cm soil samples in January, march, may, June, August and September, the surface, depth of5cm in February, July and November, the surface, depth of10cm in December, can cause rubber tree powdery mildew after inoculated the rubber leaves by rubber soil dilution. This means that Oidium heveae could survive in rubber plantation soil, and the Oidium heveae in the rubber tree soil can cause rubber tree powdery mildew.Verticillium, Muco, Trichoderma, Gongronella butleri, Rhizomuco and Penicillium were9.1%,9.1%,36.3%,9.1%,9.1%,27.3%in the total number of fungi isolated, respectively, means that these fungis could survive in rubber plantation soil. Trichoderma and Penicillium may be the communities of dominant fungi in rubber plantation soil.We took a PCR-RFLP analysis on soil fungi in different depths and different months of Hainan rubber plantation. The results showed that there were differences between the PCR-RFLP patterns of fungal18S rDNA and the communities of dominant fungi in rubber plantation soil have some changes along with the change of time. The agarose gel electrophoresis of fungal18S rDNA digested by restriction endonuclease is difference in same times and different depths soils. At the same time, the similarity of the PCR-RFLP patterns was increased with the increase of soil depth. This suggests that the fungal diversity in soil of Rubber Plantation was affected by the vertical distribution of soil. The communities of dominant fungi in rubber plantation soil have some changes along with the change of soil depth. To the soil samples in different months and same depth, the agarose gel electrophoresis of fungal18S rDNA digested by restriction endonuclease were also difference, but the trend of the brightest band were same. This means that the fungal diversity in soil of Rubber Plantation was affected by difference months. The communities of dominant fungi in rubber plantation soil also have some changes along with the change of months, this may related to climate.