Pathogen Identification And Rapid Detection Method Development For Soft Rot Of Amorphophallus Konjac

Konjac soft rot is the most serious disease in konjac production of Hubei Province.Once soft rot occurs,it is difficult to control,resulting in a serious reduction in the yield of konjac and causing serious economic losses.In this paper,we identified the species of konjac soft rot pathogens in Hubei Province using isolation,pathognicity test,physiological and biochemical identification,and sequence analysis methods.And a novel loop-mediated isothermal amplification(LAMP)method was developed for rapidly detecting soft rot bacteria using a comparative genomic approach.In addition,ethidium bromide(EMA)and the quantitave real-time PCR were combined for developing a viable cell quantitative detection method.These rapid detection methods provide an important basis for early diagnosis and control of konjac soft rot.The main results are described as follows:(1)Diseased samples of konjac soft rot were collected from main konjac planting areas,Dianjun District,Changyang City,Zhijiang City and Wufeng County in Yichang,Hubei Province.Total 110 bacterial isolates were obtained and 45 isolates were confirmed as konjac soft rot pathogens by Koch’s rule.Using morphological observation,physiological and biochemical determination and molecular biological identification(specific amplification,16S rDNA sequence analysis and multi-locus sequence analysis),the konjac soft rot pathogens were identified as P.aroidearum and D.fangzhongdai.A total of 30 strains of P.aroidearum accounted for 66.7%of all pathogenic bacteria,while D.fangzhongdai containing 15 strains,accounting for33.3%of all pathogens.The P.aroidearum is main konjac soft rot pathogen in Hubei Province.(2)A rapid loop-mediated isothermal amplification(LAMP)detection method of konjac soft rot pathogen P.aroidearum was developed using a comparative genomic approach.In the first-round screening,4393 gene sequences of P.aroidearum were used as queries against a 50-genome nucleotide database using Standalone TBLASTX.The results yielded 132 genes that had no matches in the database.These unmatched genes were then used as translated nucleotide queries against the GenBank NR protein database using Standalone BLASTX for the second-round screening.Among these,30 genes had no matches in the NR protein database.Finally,nine P.aroidearum genes were chosen as candidate targets for designing LAMP specific primers.After screening,primer set 1675-1 was chosen for the best LAMP detection primers of soft-rot bacteria P.aroidearum in konjac.The specificity and sensitivity of LAMP assay are both high for primer set 1675-1.The LAMP amplification products of 24 P.aroidearum strains with primer set 1675-1could be clearly observed with white precipitates,and showed green fluorescence with the addition of SYBR Green I,which was a positive reaction,while the LAMP amplification of other strains was negative reaction.The detection limit of LAMP assay was 50 fg/μL of P.aroidearum genomic DNA at a isothermal temperature of65°C in 40 min,which was 100-fold higher than conventional PCR.Also,The LAMP assay could detect P.aroidearum at a concentration as low as 1.2×10~4 CFU/g from artificially inoculated soil samples,which was 10 times higher than that of conventional PCR in soil samples.The LAMP assay also can be successfully applied to the detection of P.aroidearum in konjac and soil samples in the field,and it can detect the pathogens in latent period.Moreover,the field diseased rate had a certain positive correlation with the konjac tissue and soil bacterial detection rate.Therefore,LAMP detection of latent infections provides important information for early prevention and control of disease outbreaks.(3)The ethidium bromide(EMA)and quantitave real-time PCR were combined to develop a viable cell quantitative detection method for konjac soft rot pathogen P.aroidearum.The 1675-1F3/1675-1B3 primers were used to establish the real-time PCR detection system of P.aroidearum,and it can detect P.aroidearum genomic DNA as low as 300 fg/μL.The Ct value of real-time PCR amplification and the logarithm of genomic DNA concentration had a good linear relationship,thus establishing a standard curve,y=-3.4725x+41.184(R~2=0.9995).Also,the final concentration of EMA was optimized to completely inhibit the DNA amplification of dead cells of P.aroidearum,but had no effect on the DNA amplification of viable cells.The optimal final concentration of EMA was determined to be 8μg/mL,and the exposure time of EMA was optimized to be 3 min.In addition,When EMA-qPCR was used to detect P.aroidearum mixture containing dead and viable cells,it was able to amplify the DNA of viable cells selectively without false positives,whereas traditional qPCR could not effectively distinguish dead and viable cells.In this work,EMA-qPCR also can be applied to detect and quantify the viable P.aroidearum in konjac samples in the fields.