DNA Barcoding And Molecular Detection Of Plant Pathogenic Species Of Burkholderia And Botryosphaeria

DNA barcoding, a new technology for rapid the identification of species, attracts widespread attention worldwide. It can make up the shortage of traditional morphological identification, and promote the development of biodiversity research. The genus Burkholderia belong to Proteobacteria is a kind of important gram negative bacteria. It contains more than 60 species and exists widely in the soil environment, air and water, some of which are important pathogens of humans, animals and plants. Some species also can be used to prevent plant diseases and promote plant growth. Three species (B. caryophylli, B. gladioli pv. alliicola, B. glumae) are quarantine bacteria of China. The classfication of Botryosphaeria genus is very complex. As the morphological characteristics were very limted and not stable, it is also difficult to accurately identify the species of Botryosphaeria by morphological features. Based on the metioned problems, we collected Botryosphaeria sp. and Burkholderia sp. srains from different sources and conducted related research in this study. The main results were as follows:(1) DNA barcoding of Burkholderia: In total,87 strains, which belong to 29 species, were collected in this study. Four genes (16S rRNA, gyrB, rpoD and lepA) were used as the candidated DNA barcodes. We used genetic distance analysis, phylogenetic tree construction and TaxonDNA method to evaluate the feasibility of the 4 candidate genes used as DNA barcoding for Burkholderia. The results showed that the intrapecific and interspecific variation in 16S rRNA gene was the smallest, the intraspecific variation of gyrB gene was the biggest, and interspecific variation of rpoD gene was the biggest. The success rate of identification results based on the phylogenetic tree and TaxonDNA method showed that the lepA gene had the highest identification success rate. lepA gene could be used as the DNA barcode for the identification of Burkholderia. But Burkholderia cepacia complex species was very difficult to be identified using lepA gene, and it need further research on Burkholderia cepacia complex. Comparing the DNA barcoding methods in species identification:Comparing the usefulness of distance, ABGD, monophyly and character-based (BLOG) DNA barcoding methods in Burkholderia species identification, it showed that the identification success rate of character-based method for the 75 strains was 100%, higher than the other three methods. The results indicated that character-based method was the most useful method for the identification of Burkholderia cepacia complex species.(2) Loop-mediated isothermal amplification (LAMP) detection method was developed for detection of Burkholderia sp.:We designed four LAMP primers based on the ITS sequence of B. gladioli pv. alliicola. The real-time LAMP and visual LAMP methods were established by optimizing the reaction temperature. It has high specificity, and the detection sensitivity was 1 fg/μL for the genomic DNA of B. gladioli pv. alliicola, which was 100 times higher than the ordinary PCR. It suggest that the established LAMP detection method can be used for rapid detection of B. gladioli pv. alliicola.(3) DNA barcoding of Botryosphaeria:Four genes including ITS, LSU, TUB and EF-la were used as candidated the DNA barcodes. We used genetic distance analysis, phylogenetic tree construction, TaxonDNA method and the characteristic method to evaluate the 4 candidate genes. The results showed that the intrapecific and interspecific variation of LSU gene was the smallest, and the intraspecific and interspecific variation of EF-la gene was the biggest. The identification success rate results based on the TaxonDNA method and characteristic method showed that the EF-la gene has the highest success rate. The results indicated that EF-la gene can be used as the DNA barcode for the identification of Botryosphaeria species.(4) Real-time PCR for detection of three Botryosphaeria species associated with apple rot:We designed three sets of primers and probes for detection of B. dothidea, B. obtusa and B. stevensii using real-time PCR. The detection limit were 10-3 ng/μL for B. dothidea,10-3 ng/μL for B. obtusa,10-4 ng/μL for B. stevensii using simplex real-time PCR, respectively. And the detection limit was 10-3 ng/μL for the DNA samples of B. dothidea, B. obtusa and B.stevensii using mutiplex real-time PCR. It suggected that the mutiplex real-time PCR can be used for the disease detection in the field.