Study On Pathogen Identification,Molecular Detection And Control Of Xanthomonas Arboricola Pv.Pruni On Peach

In recent years,peach bacterial spot has risen to one of the most critical diseases on peaches in China,which seriously affect the healthy development of the peach industry.However,knowledge is limited about etiology,epidemiology,population structure and control measures of this disease,which brought us a lot of difficulties in preventing and managing this disease.In this study,the causal agent of peach bacterial spot was firstly isolated from samples collected from main peach growing areas in China and identified to species level.Then the genetic diversity of Xap from different geographical populations was further analyzed by rep-PCR and ISSR-PCR molecular markers.Next,the simple detection methods were developed for realizing field detection of peach bacterial spot.Finally,the bactericides for Xap were tested in the laboratory and evaluated in two orchards for two years.These results provided the theoretical foundation and certain technical guidance for disease management in peach production.The main research results are distributed as follows:（1）A total of 675 strains were isolated from 17 cities in 12 provinces of main peach growing areas in China,and were primarily identified as Xanthomonas species（14.5%）and Pantoea species（84%）by 16S r DNA sequence alignment.The two kinds of bacteria were tested through Koch’s postulate,which showed that only Xanthomonas species could cause typical perforation symptoms on healthy peach leaves,and the same bacterium was re-isolated from inoculated samples,while Pantoea species could not produce any symptoms,indicating that Xanthomonas species was the real causal agent.Based on morphological observation,Biolog microbial system phenotype identification,ftsx gene-specific PCR identification,and multilocus phylogenetic analysis,the Xanthomonas species was finally identified as Xanthomonas arboricola pv.pruni（Xap）.（2）The genetic diversity of Xap population was analyzed by using rep-PCR and ISSR-PCR molecular markers.It was shown that the percentage of polymorphic loci（PPL）was 80.99%,the genetic differentiation coefficient（Gst）was 0.5626,the gene flow（Nm）was 0.3888,indicating that there was rich genetic variation within the Xap population.In view of the results of NJ cluster analysis,STRUTURE analysis,and principal component analysis,it was shown that although relatively high similarity existed within Xap population（65%）,obvious genetic differentiation was also detected between different geographical populations（HBZY and HBJM populations）.At the same time,analysis of molecular variance and DAPC discriminant analysis showed that the obvious host specificity among walnut,peach and plum was existed,while no obvious specificity was observed for geographic distributions.（3）Xap detection method based on RPA/Cas12a system was established.The Xap-specific gene ftsx was selected as a candidate target for this detection technology.A total of 3 pairs of RPA primers were designed to conduct RPA reaction and verify the specificity,showing that RPA1F/RPA1R could detect Xap consistently and specifically.A total of 3 crRNAs were designed and evaluated for Cas12a cleavage reaction by targeting the RPA1F/RPA1R region,which showed that crRNA 2 could stably bind to the target sequence and activate the targeted ds DNA and non-specific ss DNA cleavage activity of Cas12a.Two reporter probes were designed to achieve two forms of visual detection,i.e.,FQ-reporter for fluorescence signal,and FB-reporter to show signal on lateral flow strips,which are available for users to choose freely.The nucleic acid detection method based on FQ-reporter realized the visualization by using a miniature torch,and the detection limit attained 10^-18M（8.855 fg/μL）g DNA of Xap,while the detection method based on FB-reporter had a sensitivity of up to 10^-17M（88.55 fg/μL）g DNA of Xap.In addition,the developed RPA/Cas12a system could specifically detect Xap among DNAs from other common peach pathogens and close bacteria.The detection method was further simplified by using crude DNAs extracted with Na OH solution（3minutes）.The whole operating procedure only required incubation at 37℃for 1 hour without any other equipment.The simplified method was tested for field samples from artificially inoculated diseased leaves and naturally infected perforated leaves,the expected positive results were obtained consistently.In conclusion,this study provided a flexible,effective and simple method for Xap detection,which is propagated to be applied in fields for the early diagnosis and monitoring of Xap.（4）The indoor antibacterial effect of the bactericides on Xap was tested and the field control effect of the selected bactericides was evaluated at two locations for two years.Results showed that kocide had the best control effect on peach bacterial spot.The dilution of 800×Kocide could obtain more than 90%control efficacy,while the 2000×dilution could also get more than 70%control efficacy.And when the using ratio of Kocide was diluted to 1:2000,the phytotoxicity caused by Cu²⁺accumulation could be basically eliminated.The control efficacy of antibiotic bactericides were inconsistent and unstable when they were used in the field.In general,protective bactericides had better inhibition effects than therapeutic bactericides.