Detection Of Pseudomonas Syringae Pv.actinidiae,identification Of Variety Resistance And Screening Of Bactericides

Kiwifruit（Actinidia chinensis Planch）,widely planted in China,is a kind of vine fruit tree with high economic value and rich nutrition.In recent years,with the continuous expansion of the planting area of actinidia in Fengxin County of Jiangxi Province,diseases of Kiwifruit are increasing year by year,in which,kiwifruit bacterial canker（kiwifruit canker for short）caused by Pseudomonas syringae pv.actinidiae（Psa）is one of the most difficult diseases to control in kiwifruit planting.The disease can lead to the destruction of orchards and seriously restrict the development of kiwifruit industry.At present,the detection of Psa is mostly by PCR,and the control measures are mainly chemical control combined with strengthening cultivation.However,these technologies and measures have some limitations.For this reason,this study attempted to establish a high accuracy,sensitive and simple detection system of Loop Mediated Isothermal Amplification（LAMP）,so as to realize the rapid detection of Psa.23 varieties of kiwifruit were identified for their resistance to canker.After screening out resistant and susceptible varieties,their transcriptome was sequenced by RNA-seq,and the causes of resistance and susceptibility were analyzed and compared.To obtain some effective combinations of fungicides,the antibacterial effect of fungicides on Psa was determined.The main results are as follows:（1）Specific primers for detecting Psa by LAMP were designed based on its sequence of 16S rDNA.The concentration of reagents,reaction time and temperature in the reaction system were optimized,and the optimal reaction system of LAMP was determined.It was consisted of 12.5μL of 2×reaction buffer（RM）,1.0μL of each of1μmol/L outer primers（F3 and B3）,2.0μL of each of 12μmol/L inner primers（FIP and BIP）,1.0μL of Bst DNA polymerase,2.0μL of DNA template,1.0μL of Calcein,and 2.5μL of dd H₂O.The amplification reaction conditions were 63℃for 1 h and95℃for 2 min.Meanwhile,the two pairs of primers designed in this experiment had good specificity,by which the Psa could be detected,while other pathogenic bacteria were detected negatively.（2）Resistance of 23 kiwifruit varieties to canker was determined by inoculating the detached branches of them with bacterial suspension using wound inoculation method.Results showed that there were significant differences in resistance to Psa among 23 varieties.According to the order of disease resistance from strong to weak,the varieties were Maohua 1,Ruanzao 1,Donghong,Ruanzao 2,Maohua 3,Maohua 5,Xuxiang,Maohua 2,Cuiyu,Cuixiang,Qingpihongxiang,Guichang,Zaoxian,G3,Kuimi,G9,Maohua 4,Lushanxiang,793,Wanhong,Fenghuang 1,Hongyang and Yunhai 1,of which,Yunhai 1（YH）was the most susceptible variety with 26.33 mm of average lesion length,and Maohua 1 was the most resistant variety with 2.20 mm of average lesion length.These two varieties were used as susceptible and resistant varieties respectively for subsequent transcriptome sequencing and analysis.（3）Using MH and YH as test samples,their transcriptome were sequenced by RNA-seq technology at 0 h,12 h and 48 h after Psa inoculation.Their differentially expression genes were analyzed,and the GO and KEGG enrichment analysis was performed.The results showed that upon infection of kiwifruit branches by Psa,the resistance-related genes connected with MAPK cascade reaction,flavonoid biosynthesis,signal transduction of plant hormone and carbon metabolism pathway were significantly up-regulated in resistance variety of MH,by which the defense ability of kiwifruit was improved.（4）The virulence of different combinations of pesticides to Psa was determined by filter paper method.The proportion of the combined chemicals with synergistic effect is as follows:1:1,2:1 and 1:2 for tetramycin to acetoallicin;5:1 for tetramycin to Benziothiazolinone;4:1 and 5:1 for tetramycin to Zhongshengrnycin;3:1,5:1 and1:2 for tetramycin to Bromobacterium·prochloraz;1:1 and 1:3 for tetramycin to bromothalonil.