Screening Of DNA Barcode For Xanthomonas And Detection Techniques For Important Pathovars

The bacterial genus Xanthomonas,which comprises a 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.Indeed,fourteen Xanthomonas species/pathovars have been regarded as official quarantine bacteria in China.The complexity of the genus makes it difficult to resolve this genus at the species or pathovar level.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.Therefore,in this study,we evaluated the capacity of DNA barcoding as a digital identification method for discriminating Xanthomonas at the species or pathovar level in the context of biosecurity.Meanwhile,we also investigated the digital PCR detection method of some important pathovars of Xanthomonas in order to improve the accuracy of identification.(1)Screening of DNA barcode for Xanthomonas In total,327 strains,comprising different species or pathovars,were collected for use in this study.Here,we selected the 16 S rRNA gene,cpn60,avrBs2,hrp,hpaA,gyrB and rpoD as candidate barcode genes.Tree-based methods and distance-based methods were applied to test the efficacy of the DNA barcode candidates for the identification of Xanthomonas species/pathovars.The results show that,the cpn60 gene exhibited the highest success rate of amplification and sequencing of all the genes tested in our study.Moreover,NJ tree analysis of a region of this gene provided the most efficient identification of Xanthomonas species/pathovars.Furthermore,the cpn60 gene exhibiting the best barcode gap performance and a high identification rate in the best close match test.Thus,we recommend cpn60 for use in barcode identification of the Xanthomonas.(2)The establishment and application of DNA barcoding for identification of Xanthomonas Based on the DNA barcode and all sequence data we obtained in this study,the DNA barcoding identification method for Xanthomonas has been established.In addition,we also used the DNA barcode identification method as well as some other traditional methods to identify the isolates of the suspected Xanthomonas,and the results were all consistent with the expectations.In contrast,the identification results of DNA barcode identification method are more accurate,the operation process is more simple and faster,and easy to popularize.(3)Detection techniques for important pathovar Two digital PCR methods for quantifying X.oryzae pv.oryzicola based on putative membrane protein gene and X.oryzae pv.oryzae based on rhs family gene was developed respectively.The specificity,sensitivity and repeatability were tested.The rice seed of artificial contaminated and authentic samples were tested and validated.The detection results suggest that the dPCR methods established in this studys are specific,sensitive,and highly reproducible,and have been satisfied with the application of artificial contaminated seed and natural seed samples.In summary,our studies have provided a new and reliable molecular method for the detection and identification of Xanthomonas.It is of great significance to protect the domestic agricultural and ecological security,as well as prevent the spread of disease.