Identification Of Clavibacter Sp.from Wheat And Barley Seeds And The Study On Detection Technology Of Clavibacter

The bacterial genus Clavibacter,which comprises large number of plant pathogens that are responsible for diseases of many economically important crops,is one of the most important genera in plant bacteria.Until now Clavibacter michiganensis is the only species in Clavibacter,which contains five subspecies: C.michiganensis subsp.michiganensis,C.michiganensis subsp.insidiosus,C.michiganensis subsp.nebraskensis,C.michiganensis subsp.sepedonicus and C.michiganensis subsp.tessellarius,respectively.Among them,cmm,cmn,cmi and cms have been regarded as official quarantine bacteria in China,which will cause serious economic losses.In recent years,with the application of genome-wide analysis and multi-gene analysis in bacterial classification,new subspecies have been reported.At present,the classification of this genus is also controversial,so it is very important to improve the ability of inspection and identification of harmful organisms in this genus.In this paper,we reclassified strains isolated from wheat and barley seeds.On the base of phylogenetic analysis with 16 S r RNA gene sequences,the strains isolated from wheat and barley seed were grouped in a separate clade from other subspecies of C.michiganensis.Biochemical,physiological and genetic characteristics of strains DM1、DM2、DM4、YAN7、NDM-1and B3,which is the representative strain of the isolates from wheat and barley seed,were examined in this study.Based on multi-locus sequence typing and other biochemical and physiological features including colony color,utilization of carbon sources and enzyme activities.Final confirmation the strain NXM-1 belong to cmt,the rest of strains were categorically differentiated from eight subspecies of C.michiganensis.10 strains can be divided into six categories,namely the DM1(DM3),DM2,DM4(DM5,DM6),YAN7(YAN10),NDM-1 and B3,including DM4(DM5,DM6),YAN7(YAN10)were close relate to cmt,the strain B3 was close relatives to subspecies c.michiganensis subsp.capsici genetic relationship.However,whether to identify it as a new subspecies still needs to be further analyzed and discussed in combination with the whole genome sequencing results.DNA barcoding is a molecular technique that was developed as a practical method for identifying organisms using specific DNA sequences,which was fast,accurate and with high throughput.It effectively meets the requirements of the inspection and quarantine system and plant protection institutions.Which has been successfully applied to the molecular identification of animals and plants in recent years.In order to screen a suitable barcoding gene for Clavibacter,the 127 strains,including 8 subspecies of Clavibacter,were collected for use in this study.Here,we selected the 16 S r RNA gene,cpn60 gene,gyr B gene as candidate barcode genes.Tree-based methods was applied to test the efficacy of the DNA barcode candidates for the identification of Clavibacter.The results show that,gyr B gene is an ideal barcoding gene of barbar bacillus.While it distinguishes Clavibacter from its close genus,gyr B gene can also clearly distinguish eight subspecies of Clavibacter.gyr B gene is the best choice among three barcoding genes with the best performance.cpn60 gene was also able to distinguish the subspecies of Clavibacter,but its effect was less than that of gyr B gene,while 16 S r RNA gene was very advantageous in species differentiation,but could not distinguish subspecies well.The classification of plant pathogen Clavibacter has been changing with the development of research and the development of identification technology.In recent years,new subspecies of Clavibacter have been isolated and identified.Therefore,it is necessary to further explore whether the specific detection method for each subspecies of the genus Clavibacter is still applicable,especially for the important quarantine diseases of the genus Clavibacter(cmm、cms、cmn).Therefore,the main purpose of the study is to test,screen and evaluate the reported specific primers for three important quarantine diseases of the genus Clavibacter(cmm、cmn and cms).Finally,experimental results successfully screened against 1 pair,2 pairs and 1 pair of specific primers,which only amplified the target bands against the positive strains of black canker disease in tomato,pathogen of potato ring rot and goss’ s bacterial wilt and leaf blight of corn,but the other control strains did not.Bacterial canker diseases caused by Clavibacter michiganensis subsp.michiganensis(Cmm)is one of the most severe diseases of tomato in both the greenhouse and field,which has resulted in substantial economic losses in many countries.However,with conventional methods,it is difficult to quickly distinguish this pathogen from the other closely related subspecies of C.michiganensis,especially the non-pathogenic subspecies that were isolated and identified from tomato seeds recently.Here,we have developed a novel droplet digital polymerase chain reaction(dd PCR)method for the identification of this specific bacteria based on a Taq Man probe designed based on the pat-1 gene of Cmm.The newly designed primers and probe were highly specific that all Cmm strains were accurately identified from all the strains tested.The detection threshold of the method was 10.8 CFU/m L in both pure cell suspensions and infected tomato seed,which was 100 times higher than that of the realtime PCR performed using the same primers and probe.Therefore,the newly developed dd PCR method will facilitate the early detection and monitoring of black canker disease in tomato and facilitate the prevention of infection.