Development Of A Pcr And Real-time Pcr Assay For Detection Of Dickeya Fangzhongdai And Catabolic Activities Comparation Of Pseudomonas Syringae Pv.Lachrymans Strains From Cucumber And Muskmelon

The Bleeding Canker is a disease caused by bacteria in 1973 in the northern region of Jiangsu Province,part of the pear tree appeared in the trunk out of the rusty color of the viscous liquid disease,the incidence of pear trees is reduced to reduce the yield caused by the whole plant is dead,To the farmers brought huge economic losses.After sampling analysis,it was found that this was a never-reported bacterial disease.Due to the incidence of pear branches will have the smell of lees with rusty color of the mucus,so named 'The Bleeding Canker',in recent years,pear rust disease in Zhejiang,Shandong,Texas also reported.But the law of the disease is not clear,up to its symptoms was similar to the quarantine disease pear dead branches,the traditional detection technology is difficult to distinguish.The pathogen of the Bleeding Canker is an untreated species of Dickeya,our laboratory named it D.fangzhongdai sp.nov.As the the Bleeding Canker is a new pathogen,the current domestic reports are still in the initial stage of research,so the molecular detection technology is still blank.Through the routine PCR and real-time quantitative PCR method,the rapid and accurate detection of pathogenic bacteria of pear water was established.The established detection system was of great significance to the disease prevention and control:10 genes with specific loci were screened out by comparing the whole genome of the Bleeding Canker with the other 13 kinds of near-endogenic bacteria by the Blast comparison method in bioinformatics.Ten pairs of specific primers were designed for homologous genes and verified by PCR amplification.Secondly,two pairs of primers with high specificity and high sensitivity were screened out for 10 pairs of designed primers.The primers were designed according to the extracellular polysaccharide gene cpsB and hrpV gene hrpV.The two pairs of primers were used to amplify the Bleeding Canker bacteria effectively,There are no amplified bands for other near and close relatives.It was indicated its specificity.The sensitivity was 10 pg/?L.A PCR method for the detection of the Bleeding Canker was established.Finally,selectingfrom the specific hrpV gene as target specific primers and TaqMan probes were designed,and a real-time quantitative PCR method for the Bleeding Canker was established.Detection sensitivity of up to 20 copies/?L,1000 times higher than the conventional PCR detection method,and only 1 hour to complete the test.The common PCR detection method established in this study is convenient and inexpensive.real-time quantitative PCR method is more sensitive and shorter,both of which can distinguish the Bleeding Canker with other Dickeya species respectively,and fill the gaps in the detection of Bleeding Canker,Which provides a new method for detecting the Bleeding Canker.Pseudomonas syringae pv.Lachrymans caused by cucumber bacterial leaf spot all over the world,resulting in varying degrees of loss,but also cause melon bacterial leaf spot.Previous studies have shown that cucumber and melon bacterial leaf spot pathogens are Pseudomonas syringae cucumber pathogens,but in the laboratory study found that Pseudomonas syringae cucumber pathogens and melon strains There are some differences in pathogenicity and molecular biology.In this study,the carbon metabolic activity of cucumber and Cucumber Bacteria was studied and compared by Biolog Bacteria Identification System.The results showed that Pseudomonas syringae cucumber and melon strains belonged to different genotypes.