Development Of Detection Methods For Four Important Plant Pathogens On The Base Of Isothermal Amplification

Pseudomonas syringae pv.lachrymans and Cucumber green mottle masaic virus(CGMMV)are common pathogens that infect Cucurbitaceae crops.Prunus necrotic ringspot virus(PNRSV)and Plum pox virus(PPV)are two important pathogens that infect stone fruit crops.In particular,CGMMV,PNRSV and PPV are listed as quarantine plant viruses in P.R.China.The increasing development of domestic and international trade leads to serious harm to crop production and ecological security of our country.Herein,to prevent the spread of P.syringae pv.Lachrymans,CGMMV,PNRSV and PPV,we developed the rapid detection method for these four pathogens by using cross-priming isothermal amplification(CPA)and recombinase polymerase isothermal amplification(RPA),and the results are as follows.1.Development of a CPA-based assay for P.syringae pv.lachrymansSpecific primers based on the encoding gene of glyceraldehyde-3-phosphate dehydrogenase 1(gap1)of P.syringae pv.Lachrymans were designed and screened,and were applied to develop a CPA-based detection method for this pathogen.The method can accurately distinguish P.syringae pv.lachrymans from the other13 pathogenic bacteria of Pseudomonas spp with detection limit of 0.54×10~3 CFU/mL.2.Development of a CPA-based assay for CGMMVSpecific primers were designed based on the encoding gene of coat protein(CP)which is located in 3’of CGMMV genome,and were used to develop a CPA-based method for CGMMV detection.The developed method can accurately distinguish CGMMV from the other 5 viral pathogens of Cucurbitaceae crops,including Cucumber mosaic virus,Pepper mild mottle virus,Tobacco mosaic virus,Tomato mosaic virus,Zucchini yellow mosaic virus,with the detection limit of 24.3 ng/μL total RNA.3.Development of a CPA-based assay for PNRSVSpecific primers used for the CPA-based assay for PNRSV were designed based on CP gene of PNRSV.The resulted method can distinguish PNRSV from the other pathogens of Prunus crops,including 3 viruses(Arabis mosaic virus,Plum pox virus,Apple mosaic virus)and 2 viroids(Apple scar skin viroid,Peach latent mosaic viroid),with the detection limit of 72 copies.4.Development of a RPA-based assay for PPVSpecific primers used for the PRA-based assay for PPV were designed based on CP gene of PPV.The developed RPA method can distinguish PPV from the other 3 potyviruses(Potato virus A,Potato virus Y,Zucchini yellow mosaic virus),as well as 5 pathogens of Prunus crops,including Prunus necrotic ringspot virus,Arabis mosaic virus,Apple mosaic virus,Apple scar skin viroid,Peach latent mosaic viroid.The detection limit is 0.53 ng/μL total RNA.In summary,we developed high-efficiency and specific isothermal amplification detection methods for P.syringae pv.lachrymans,CGMMV,PNRSV and PPV on the basis of CPA and RPA,which can amplify the target nucleotide sequence in a constant temperature.With comparison of the conventional detction methods,the detection methods have advantages of less time consuming,good specificity,high sensitivity and easy operation,thus providing valuable tools for quarantine and field test and showing great significance for protection of Cucurbitaceae and Prunus crops production.