| Bacterial canker is a devastating disease of kiwifruit. The disease is difficult to be controlled and spreads rapidly. Therefore, it has severely hampered the development of kiwifruit industry. Due to serious harm and huge pecuniary loss, pathogen of bacterial canker in kiwifruit, Pseudomonas syringae pv. actinidiae, was classified to the rank of forest plant quarantine objects in1996. P. syringae pv. actinidiae was classified as a harmful organism by General Administration of Quality Supervision, Inspection and Quarantine of China in AQSIQ Notice [2009] No.74(Phytosanitary requirements for the export of kiwifruit from Italy to China) in2009, and then it was listed as a hazardous and harmful organism by State Forestry Administration in2013.Risk analysis of bacterial canker in Kiwifruit was carried out, and a fast and effective molecular method for the detection of kiwifruit bacterial canker was established. Bacillus which could inhibit the growth of P. syringae pv. actinidiae was screened, then biological control test was performed in pot and field scale. Results are as follows:l.On the basis of relational information collected at home and abroad, combined with China’s actual situation, and according to "national standards of Peoples Republic of China: guideline for pest risk analysis of import and export plant and plant product"(GB/T21658-2008) and "national standards of Peoples Republic of China:technical requirement of pest risk analysis for import and export plant and plant"(GB/T20879-2007), qualitative and quantitative risk assessment has been down on bacterial canker in Kiwifruit, and measures of quarantine and prevention proposed. Qualitative risk assessment showed that this disease might enter to China or spread in China with middle risk, while consequence assessment was very high. Therefore, risk grade of P. syringae pv. actinidiae was high. Quatitative risk assessment showed that the risk index value (R value) was2.06. According to the Grading standards(2.5<R<3means the highest risk,2.0<R<2.5means higher risk,1.5<R<2.0means middle risk and1.0<R<1.5means lower risk), P. syringae pv. actinidiae belong to higher harmful living beings. Suitability analysis showed that potential suitable area (suitable value>0.9) of kiwifruit canker were mainly distributed in Sichuan, Yunnan, Guizhou, Fujian, Anhui, Hunan, Hubei, Henan, Jiangxi, Shandong, Shaanxi and Tibet.2.80strains of bacteria were isolated from Kiwi tree branches which have been infectedwith bacterial canker in Deyang, Qionglai, Dujiangyan and Chongzhou. According to Koch’s postulates and the result of pathogenicity test, DY10, QL14, DJY08and CZ20were confirmed as pathogenic bacteria of canker in kiwifruit, then these strains were identified as P. syringae pv. actinidiae according to morphological characteristics, physiological and biochemical characteristics and16S rDNA sequencesinformation.3.7primers were screened from120random primers by RAPD.The selected primers could amplify discrepant, clear and stable bands from different strains. The specific random primer P7(5’-CGCAGCCGAGAT-3’) was screened from7seclected primers, which can only detect P. syringae pv. actinidiae from all the tested strains, and obtained a1300bp specific fragment of P. syringae pv. actinidiae. Based on the sequences of the specific fragment, a pair of specific primers (F7:5’-CAATCATTTCGCCAGACGC-3’; R7:5’-CTACGAGGTTAGGTTCAGAGT-3’) was designed, which amplified a band of950bp and converted the RAPD marker into SCAR marker successfully, in order to establish the rapid detection technology.4. The result of PCR, which using the specific primers amplified all the tested strains, showed that objective band (about950bp) could be only amplified from P. syringae pv. actinidiae and no bands could be amplified no matter from other strains or the control. After specificity and sensitivity of the primers were evaluated and amplification condition was optimized, the primers were used to detect P. syringae pv. actinidiae both in infected branch samples collected from orchard and the inoculated branches. Results showed that the specific primers could be used to detect P. syringae pv. actinidiae with sensitivity of100fg/μl. Therefore, under the function of the specific primers F7/R7, optimized reaction system and optimized procedure, with the help of kit to extract DNA of P. syringae pv. actinidiae, pathogenic bacteria of canker in kiwifruit could be detected by the molecular method in a short time. The rapid and effective molecular method for detection of bacterial canker in kiwifruit was established in this research, which filled the gap in molecular detection of bacterial canker in kiwifruit of our country. Compared to traditional identification methods, this method was easier, cheaper, reliable, less time-consuming, with less processes and interferences, and could detect the pathogenic bacterium in incubation period. Therefore, this method could be applied in detection of the pathogenic bacterium from plant and plant products in infected areas and non-infected areas of our country to control the spread of bacterial canker of kiwifruit.5.36strains of bacillus were obtained from rhizosphere soil of kiwi tree, among which biocontrol strain B2with the best antagonistic effect was screened according to the result of growth rate test, spreading test and oxford cup assay. Pot experiment showed that the biocontrol rate of B2’s fermentation raw liquid was90.45%. Field trail showed that the biocontrol rate was85.10%. Then B2was preliminarily identified as P. syringae pv. actinidiae by morphological characteristics, physiological and biochemical characteristics and16S rDNA sequence information. |
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