Detection Of Important Pathogenic Bacteria In Rice Using Real-time Fluorescent Quantitative PCR And Digital PCR

Bacterial panicle blight,leaf blight,leaf streak,and foot rot are important rice diseases caused by bacterial pathogens.Using precise and sensitive methods to detect and identify these pathogens are critical for quarantine and control of these diseases.This study verified previous primers and newly designed primers based on analyses of bacterial whole genome sequences(WGS)and established precise and sensitive real-time fluorescent quantitative PCR(qPCR)and digital PCR(dPCR)methods to detect these bacterial pathogens.Burkholderia glumae is the primary pathogen of bacterial panicle blight of rice and B.gladioli is the secondary pathogen.Phylogenomic analysis revealed that B.gladioli contains five phylogroups.The primer pair BG1-F/BG1-R was designed to target the near 3’-end sequence of a B.glumae gene encoding a Rhs family protein.Analyses on WGS revealed its specificity to B.glumae and two of the five B.gladioli phylogroups.A 138-bp DNA fragment was amplified only from B.glumae and B.gladioli strains.dPCR methods with the EvaGreen fluorescent dye for specific and sensitive detection of B.glumae and B.gladioli were established.EvaGreen dPCR methods showed the same sensitivity on detection of B.glumae and B.gladioli.The detection limit on the two pathogens was 2.0×103 colony forming units(CFU)/mL from bacterial suspensions and 200 CFU/seed from rice seeds.Xanthomonas oryzae(Xo)contains five distinct phylogroups:X.oryzae pv.oryzae(Xoo)Asian lineage(XooAsia),Xoo African lineage(XooAfrica),Xo US lineage(XoUS),X.oryzae pv.oryzicola(Xoc),and X.oryzae pv.leersiae(Xol).Genomic analysis revealed that XoUS forms a subspecies distinct to the other four phylogroups;XooAsia and XooAfrica showed DDH similarities(78.6-79.8%)of genome relatedness equivalent to two subspecies(DDH similarity threshold 7980%).The DDH similarities between Xoc and XooAsia or XooAfrica are slightly higher than those between two subspecies.Xoo-specific primers were not obtained by targeting genes present in Xoo but absent in Xoc and vice versa.The genomes of Xo phylogroups contain high levels(10-15.6%)of pseudogenes.The coding sequence of the ferric-rhodotorulic acid/ferric-coprogen receptor FhuE in Xoc and Xoo genomes became pseudogenes due to differential deletion.Xoc-specific primers XocFhuE-F/XocFhuE-R targeting the region of Xoc fhuE pseudogene missing in Xoo genomes were obtained.PCR amplification produced only a 159-bp DNA fragment from only Xoc strains using the primers XocFhuE-F/XocFhuE-R.SYBR Green qPCR and EvaGreen dPCR methods using the Xoc-specific primers to detect Xoc were established.The detection limit of qPCR on Xoc was 1.6×104 CFU/mL from bacterial suspensions and 1.2×103 CFU/seed from rice seeds.The detection limit of dPCR on Xoc was 1.6×103 CFU/mL from bacterial suspensions and 120 CFU/seed from rice seeds.Eight pseudogenes present in Xoo genomes but absent in Xoc genomes were found.Three of the correspond genes encode a serine/threonine protein phosphatase,a CDP-alcohol phosphatidyltransferase,and a sensor histidine kinase.XooAsia-specific primers XooTPP1F/XooTPP1R(amplicon 121 bp)and XooTPP4F/XooTPP4R(amplicon 179 bp)targeting the serine/threonine protein phosphatase pseudogene and Xoo-specific primers XooCDP1F/XooCDP1R(amplicon 101 bp)targeting the CDP-alcohol phosphatidyltransferase pseudogene、XooSHK1F/XooSHK1R(amplicon 153 bp)and XooSHK2F/XooSHK2R(amplicon 138 bp)targeting the sensor histidine kinase pseudogene were obtained.A diagnostic multiplex PCR method using the primer sets Xoc-specific XocFhuE-F/XocFhuE-R,XooAsia-specific XooTPP4F/XooTPP4R,and Xoo-specific XooCDP1F/XooCDP1R was developed to distinguish Xoc,XooAsia and XooAfrica strains.A SYBR Green qPCR method using primers XooTPP4F/XooTPP4R and XooSHK2F/XooSHK2R was developed to detect XooAsia and Xoo.The detection limit of qPCR on XooAsia and Xoo was 1.5×104 CFU/mL from bacterial suspensions and 2.0×103 CFU/seed from rice seeds.Phylogenomic and genome relatedness analyses revealed that Dickeya oryzae contains four subspecies and the rice foot rot pathogens belong to two of the four subspecies.Seven genes present only in Dickeya oryzae among genus Dickeya were found by a pangenomic analysis.Among them,a gene encoding a carboxylesterase(CES)was present only in genomes of the rice foot rot pathogens.Primers were designed to target the seven genes and a rice-foot-rot-pathogen-specific primer pair DoCES-1F/DoCES-1R(amplicon 128 bp)was obtained.A qPCR method using the primers DoCES-1F/DoCES-1R and SYBR Green I was developed to detect rice foot rot pathogens.The detection limit of qPCR on the pathogen was 1.8×104 CFU/mL from bacterial suspension and 1.4×103 CFU/seed from rice seeds.In summary,this study extended the B.glumae-specific primers BG1-F/BG1-R to B.gladioli and developed the first dPCR method to detect the rice panicle blight pathogens.Moreover,this study for the first time developed Xoc,Xoo,and XooAsia specific primers targeting pseudogenes or differential regions in pseudogenes,a diagnostic multiplex PCR method based on these primers to distinguish Xoc,XooAsia,and XooAfrica strains,a qPCR method to detect Xoc、Xoo and XooAsia strains and a dPCR method to detect strains.This study also developed the first rice-foot-rot-pathogen-specific primers and a SYBR Green qPCR method to detect the rice foot rot pathogens.