Methods For Simultaneous Detection Of The Presences Of Three Kinds Of Quarantine Diseases In Rice

Rice bacterial blight caused by Xanthomonas oryzae pv. oryzae,Bacterial leaf streak caused by X. oryzae pv. oryzicola and Rice bacterialpanicle blight (bacterial grain rot) caused by B. glumae, spread mainlythrough reeds. They are listed as a quarantine pest in2007by ChineseMinistry of Agriculture. Polymerase chain reaction (PCR) and SYBR greenreal-time PCR are particularly useful for plant pathogen detection due to thecharacters of rapid, accuracy and sensitivity. However, little information isavailable for simultaneous detection of the presences of X. oryzae pv. oryzae,X. oryzae pv. oryzicola, and B. glumae. The unique PCR primer sets weredesigned from portions of a putative glycosyltransferase gene（NCBI No.AF169030.1） of X. oryzae pv. oryzae, an AvrRxo gene (NCBI No.AY395713.1) of X. oryzae pv. oryzicola, and internal transcribed spacer (ITS)sequence of16S rDNA(NCBI No. D87080.1) of B. glumae, the results showthat specificity of the three primer sets are good and the size of specificfragments are230bp,112bp and164bp, respectively. Using conventionalPCR assay，X. oryzae pv. oryzae, X. oryzae pv. oryzicola, and B. glumae weredetected with1pg/μl,0.5fg/μl, and1pg/μl, respectively. Using SY R greenreal-time PCR assays, X. oryzae pv. oryzae, X. oryzae pv. oryzicola, and B.glumae, were detected with1fg/μl,1fg/μl, and10fg/μl, respectively.Variety of pathogens can be specific amplified successfully by usingmultiple PCR assay in one PCR reaction. The study developed multiplex PCRmethods for simultaneous detection of the presences of X. oryzae pv. oryzae,X. oryzae pv. oryzicola, and B. glumae. The detection limits by the multiplexPCR assay were0.3pg/μl for X. oryzae pv. oryzae,0.167pg/μl for X. oryzaepv. oryzicola and16.7pg/μl for B. glumae in the20μl reaction. Thedetection limit of each pathogen in multiplex PCR was pretty similar withthat of single pathogen in the routine PCR.Because of the rice seeds with three pathogens do not be gained fromnature, so the rice seeds artificially infected by three pathogens were detected in this study. When the seeds were infected by the mixture of the threepathogens, the230bp,164bp and112bp fragments for X. oryzae pv. oryzae,X. oryzae pv. oryzicola and B. glumae were detected, respectively.Above all, the study developed multiple PCR methods for simultaneousdetection of the presences of X. oryzae pv. oryzae, X. oryzae pv. oryzicola,and B. glumae. This method is simple, rapid, sensitive and can be used forrapid detection of the pathogen.