Isolation And Identification Of The Causal Agent For Kiwifruit Bacterial Canker In Wanzhou District Of Chongqing And The Detection Method Construction Of Loopmediated Isothermal Amplification

Kiwifruit bacterial canker is a devastating disease that has a serious threat to the kiwifruit industry. At the present, the disease has occurred at home and abroad. Chongqing is a main production area where the kiwifruits were planted. In recent years, the plants have suffered from bacterial canker.For control and quarantine of the local diseases, it is very necessary to confirm the causal agent for kiwifruit bacterial canker in Chongqing. The study collected diseased Actinidia chinensis cv. Hongyang twigs, leaves and so on from several orchards in Sunjia town, Xiangshui town and Tiefeng township in Wanzhou district of Chongqing. After the pathogens were isolated, they were identified by conventional methods including phenotype identification, biochemical features, physiological behavior, pathogenicity and molecular methods. Based on the avr D1 gene of Pseudomonas syringae pv. actinidiae(Psa), the primers were designed. After optimizing the parameters, a new detection method that is loop-mediated isothermal amplification(LAMP) was established and would lay the foundation for the disease diagnosis in the field. The results of main research are as follow:The identification of the the causal agent for kiwifruit bacterial canker by using conventional methods: In the study, ten isolates were obtained and named CQPsa-01, CQPsa-02, CQPsa-03, CQPsa-04, CQPsa-05, CQPsa-06, CQPsa-07, CQPsa-08, CQPsa-09, CQPsa-10, respectively. The morphological characteristics of the strains were basically identical. They were round, milk-white, margin-entire, slightlyconvex, surface-smooth, translucent on KBA medium. They were also Gram-negative. The strain M7 from Shaanxi was acted as standard strain, and the cultural, physiological and biochemical properties of ten strains were identified. The results showed that the strain M7 and the ten isolates were identical in terms of all indexes. The pathogenicity assays were performed. The ten isolates induced hypersensitive response on tobacco leaves. After culture under certain conditions, kiwifruit twigs and leaves inoculated showed the symptoms similar to typical symptoms observed in the field. Bacteria recovered from inoculated plants were consistant with the ten isolates. Koch’s postulates were fulfilled.The identification of the the causal agent for kiwifruit bacterial canker by using molecular methods: Standard strain M7 and ten isolates were amplified by the primers 27F/1492 R, that were a pair of universal primers amplifying the 16 S r DNA of the bacteria. Phylogenetic tree was performed by MEGA 4 with N-J based on 16 S r DNA sequence. The result indicated that the strain M7 and the ten isolates have the closest relatives to Psa, but were indistinguishable from P. syringae pv. theae. RG-PCR and duplex-PCR were respectively performed by the special primers Psa F1/R2, KNF/KNR and Avr Ddpx-F/R. A unique band of 280 bp and two unique DNA bands of the expected size(226bp, 492bp), respectively, were detected by RG-PCR and duplex-PCR in the strains from Chongqing, and the Psa strain M7. Duplex-PCR has the capacity to identify Psa accurately except for all Psa-related because only Psa possesses the two genes at the same time. Therefore, the ten isolates were determined to be Psa. The strain M7 and the ten isolates were also respectively amplified by the primers China F/R and F7’/R7’, the former was designed according to the Psa strain from Shaanxi and the latter was designed according to the Psa strain from Sichuan. The result showed in terms of the primers China F/R, a band of 609 bp was obtained only from three strains M7, CQPsa-08 and CQPsa-10, but no bands or two to five nonspecical bands also obtained from the other CQPsa strains. As far as the primers F7’/R7’ were concerned, several nonspecial bands obtained from all strains were almost the same. These results demonstrated that there are mutations in certain genes among the Psa strains from Shaanxi, the Psa strains from Sichuan and the Psa strains from Chongqing. And there are some differences among the Psa strains in Wanzhou district of Chongqing.The development of LAMP method aming at the the causal agent for kiwifruit bacterial canker: Using Primer Explorer V4 software program, LAMP primers were designed according to the sequence of avr D1 gene of Psa. The reaction system was initially set up and optimized. Finally, a rapid detection method aiming at Psa-LAMP was established. Amplified products were observed through white turbidity and colour change after adding SYBR Green I, then, the test results were directly determined. Typical ladder-like bands were only obtained from all Psa strains, but no bands were amplified from all strains used as negative control. The results demonstrated LAMP was specific for all Psa strains tested, but not for any of the strains of genera of plant pathogenic bacteria or endophytic bacteria from kiwifruit plants. The sensitivity threshold of LAMP was compared with that of the duplex-PCR assay and real-time PCR. The detection limits of LAMP and real-time PCR for Psa pure DNA were 5 fg, 100 fold higher than that of duplex-PCR. The detection limits of LAMP and real-time PCR for bacterial suspension were 10 CFU/PCR, 10 fold higher than that of duplex-PCR. LAMP, real-time PCR and duplex-PCR were used to detect Psa of thirty-four field samples and two artificially inoculated samples. The results showed the pathogen was not detected in all the uninfected samples which produced no amplicon with the duplex-PCR and LAMP methods. The pathogen was successfully detected in all the symptomatic disease samples which produced the two bands and typical ladder-like bands when using duplex-PCR and LAMP. For all the asymptomatic infected samples, two special bands were obtained only from one of all by duplex-PCR, but the pathogen was detected without any failure in all asymptomatic samples by LAMP. The amplification results of all actural samples using real-time PCR were the same as those of LAMP. So the results verified the high sensitivity and accuracy of LAMP basically consistant with those of real-time PCR.Comprehensive the above results, the study got the following conclusions:1) Ten isolates obtained from Wanzhou district of Chongqing were identified as Pseudomonas syringae pv. actinidiae.2) There are some mutations in certain genes among the Psa strains from Shaanxi, the Psa strains from Sichuan and the Psa strains from Chongqing. There are also some differences among the Psa strains in Wanzhou district of Chongqing.3) The study successfully developed Psa-LAMP appling to the rapid detection of the samples in the field. It offerd a new technique and alternative for an on-site diagnosis of kiwifruit bacterial canker.