Development Of Padlock And LAMP Based Methods For Detection Of Xanthomonas Axonopodis Pv.Glycines And Pseudomonas Syringae Pv.Glycinea

Bacterial pustule and bacterial blight are two severe seed-borne diseases,which dramatically reduce soybean yield.Xanthomonas axonopodis pv.glycine and Pseudomonas syringae pv.glycinea are pathogenic bacterias of these two diseases respectively.The diseases spread to more areas and cause more serious economic losses to soybean industry in domestic these years.Since they are both seed-borne diseases,one of the most effective measures is to use seeds free from the diseases.In this study,we estabilished rapid,sensitive and reliable detecting systems to these two pathogenic bacterias based on Padlock Probe and LAMP separately,the results we achieved are as follows:First of all,we developed Xanthomonas axonopodis pv.glycine and Pseudomonas syringae pv.glycinea detecting systems separately based on padlock probe combined with dot-blot hybridization.After comparing lots of house-keeping genes,we finally selected recF from Xanthomonas axonopodis pv.glycine and rpoN from Pseudomonas syringae pv.glycinea as target genes and designed specific padlock probes named as PLP-Xag and PLP-Psg respectively.The results indicated that PLP-Xag and PLP-Psg could detect the pathogens of the two diseases specifically and their sensitivity could be up to 100 fg/μL,which is more sensitive than general PCR.Besides,seedlots with 0.1%infestation could be detected with this system.Finally,we detected 45 samples of commercial soybean seeds,and found 4 samples positive with the bacterial pustule pathogen and 2 samples with bacterial blight pathogen.Based on above results,the detecting systems based on padlock probe could be applied to health inspecting for soybean seeds.Secondly,we developed Xanthomonas axonopodis pv.glycine and Pseudomonas syringae pv.glycinea detecting systems separately based on LAMP.After consulting related references and comparing large numbers of house-keeping genes,we eventually selected glyR from Xanthomonas axonopodis pv.glycine and rpoN from Pseudomonas syringae pv.glycinea as target genes separately and designed specific primers for these two microorganisms named as LAMP-Xag and LAMP-Psg.After reacting under 65 ℃for 1 h,the results could be visible directly with SYBR Green Ⅰ.The results indicated that the detecting system based on LAMP showed good specificity and with sensitivity up to 10 fg/μL and 1 pg/μL respectively,which is more sensitive than general PCR.The detecting results of infected seedlots and commercial soybean seeds with LAMP system are consistent with the padlock system.Based on above results,the detecting systems based on LAMP could also be applied to health inspecting for soybean seeds.Our study indicated that the detecting systems based on padlock probe combined with dot-blot hybridization and LAMP could meet the purpose for bacterial pustule and bacterial blight pathogens detection,and provied new available data for the development ofdetecting systems for other soybean pathogenic bacterias.