DNA Barcoding Detection Technology On Plant Pathogenic Bacteria Of Pseudomonas

Pseudomonas is one of the bacteria with wide distribution and numerous varieties.SomePseudomonas species resides in phyllosphere bacterial communities as a commensal bacterium and some causes diseases in common crops as a plant pathogen.The classification system of this genus is complicated and it might changes while the identification technology develops,these all bring much difficulty for identification and detection for Pseudomonas.Meanwhile,six Chinese quarantine Pseudomonas were all distinguished on a pathovars level,and therefore it is of great significant to study how to identify and detect Pseudomonas on the infraspeices level quickly and efficiently.DNA barcoding technology as one of the detection method is a combination of molecular biology and bioinformatics.This technology provides an idea of rapid and accurate detection and identification for Pseudomonas.In order to select the proper DNA barcoding gene for Pseudomonas,we choose eight reported genes frequently used in Pseudomonas studies and acquired 107 Pseudomonas strains to evaluate which is the most effective gene for distinguishing Pseudomonas pathovars.After calculating amplification rate and sequencing success rate,NJ phylogenetic trees analysis,genetic distance analysis,Wilcoxon's rank-sum test and Barcoding gap calculation,we concluded that rpoD and gapA were most powerful genes to discriminate most Pseudomonas pathovars and initially established a nonspecific detection method for Pseudomonas using DNA barcoding technology.Some plant pathogenic Pseudomonas were not listed on Chinese quarantine list but could also cause severe losses such as P.syringae pv.lachrymans.This pathovar infects cucumbers(Cucumic sativus)and induces angular leaf spot which occurs in many areas in China.We can apply DNA barcoding method to identify this pathovar and specific detection method can also be utilized when P.syringae pv.lachrymans is set as the target strain.Droplet digital PCR(ddPCR)is an emerging quantitative PCR method which is widely used in medical testing and genetic detecting and it has great potential in specific detection of plant pathogenic bacteria.In our study,we applied specific primers and probe combining ddPCR to test and verify the feasibility and applicability of this method.Through specificity test and sensitivity test we could conclude that ddPCR was effective for specific detection of P.syringae pv.lachrymans while the DNA concentration is above 2.4×10-4 ng/?l.