DNA Barcoding Method For Detection Of Plant Pathogenic Burkholderia

Plant pathogenic bacteria is a kind of very important plant pathogens,about 650 species of plant pathogenic bacteria in 30 genus have been reported around the world.Most bacteria can cause diseases on crops,flowers and other plants,resulting in severe economic losses.Eight different plant pathogenic bacteria of Burkholderia could induce various symptoms,including decay,withered and necrotic.Therefore a simple and highly-effective detection measurement appears particularly significant.DNA barcoding,which employs relatively short DNA fragment with sufficient variation,is an effective complement for traditional detection methods.With the advantage of accuracy and celerity,DNA barcoding breaks through the excessive dependence of traditional classification methods on the experience of classification and type specimen,thereby satisfying the requirements of the port quarantine on modern detection technology.In view of this,72 strains of Burkholderia were amplificated and sequenced in this paper using 6 optional DNA bar codes in order to screen the best DNA barcoding gene for this genus.And the paper makes a comparison analysis based on the amplification and sequencing success rate,the intraspecific genetic distance between species,the barcoding gap and the NJ tree.The result demonstrates that the rpoD is the most suitable gene for the distinction among species and pathovars of Burkholderia,and gyrB gene can be used as an effective supplement to the classification and identification.Three species and pathovars belonging to quarantine pathogens in Burkholderia involved in this paper have been listed on《The People’s Republic of China entry plant quarantine pest list》,it is undoubtly that once they failed to be detected efficiently,the incalculable damage and very serious consequences on domestic food industry,ecological environment security and free trade of goods will be an inevitable outcome.On the intercepted imported Vanda,local black brown spots and chlorosis on leaves were found and the affected leaves would fall after some time.In order to identify the pathogen,we isolated and purified the pathogen from Vanda and identified it first using traditional methods including morphological features,cultural characteristics,biochemical identification,pathogenicity test,Biolog analysis and other methods.The results showed that the unknown strain belonged to Burkholderia gladioli.Furthermore,DNA barcoding technique was applied to identify the strain on a pathovar level.Two barcoding genes for Burkholderia,rpoD and gyrB were used and the relevant NJ tree was built.The strain we isolated from Vanda,however,didn’t belong to any branches of the three B.gladioli pathovars analysed before.In conclusion,we considered this isolation to be a new pathovar of B.gladioli.