Isolation,Identification And Genetic Diversity Of Acidovorax Avenae Causing Red Stripe Of Sugarcane

Sugarcane red stripe is a bacterial disease caused by Acidovorax avenae subsp.Avenae(Aaa),which distributed in major sugarcane cultivation and regions worldwide including China.There are few reports on the molecular identification and genetic diversity of the causal pathogens of sugarcane red stripe in China.Thus,in this study,we collected 108 sugarcane leaf samples during 2013 to 2016 from six different sugarcane planting regions in the Guangxi,Yunnan,Guangdong,Hainan,Fujian,and Guizhou provinces of China.Of these collected samples,84 leaf samples were symptomatic(red stripe)and and other 24 leaf samples were asymptomatic.We designed a specific primers(RS-ITS-F1/RS-ITS-R1)targeted at the 16S-23S rDNA spacer regions of A.avenae.The DNA samples form 108 suagrcane leaves were detected by PCR assay with this primer pairs.Positive results of 96.4%(81/84)and 83.3%(20/24)were found in symptomatic(red stripe)and asymptomatic leaves,respectively.All the amplified fragments were cloned and sequenced,and a total of 142 sequences of the 16S-23S rDNA spacer regions were obtained.The size of these squences ranged from 436 bp to 454 bp,and shared 89.2-100%sequence identity with each other.Four insertion/deletion(InDel)mutation sites were obsvered among these sequences.Phylogenetic tree of neigbour-joining revealed that the 142 A.avenae sequences originated from this study were clustered into six phylogenetic groups H,HE,E1-1,E1-2,E2-1 and E2-2.In silico and polyacrylamide gel electrophoresis analysis of PCR products digested with Hiind? and EcoRI revealed that these sequences showed various restriction fragment length polymorphism(RFLP)profiles that were closely associated with phylogenetic grouping.The groups H,HE and E2-2 of Chinese A.avenae were prevalently occureed in China and accounted for 32.4%(46/142),29.6%(42/142)and 28.2%(40/142),respectively.We found the HE and E1-2 groups of A.avenae in the six provinces of China and the H and E2-2 groups of A.avenae in the five provinces.The E1-1 group of A.avenae only presented in Hainan and Guizhou provinces,while the E2-1 group of A.avenae was only occurred in Yunnan province.We isolated the causal bacteria SC-026(KY363762,HE group)strain from the field leaf sample of sugarcane cv.FN41.The causal bacterium was confirmed as A.avenae by colonial morphology,physiological and biochemical characteristics and molecular identification with the PCR assay.The isolated strain was inoculated into healthy plantlets of sugarcane cv.FN41.The typical symptoms of diseased leaf disease were stable at 6 days of post-inoculation(6 dpi)and the incidence rate was as high as 60%.This is the first report of the genetic diversity of Chinese A.avenae infecting sugarcane and the geographical distribution of different strains(phylogenetic groups)in China.Our findings provide technical supports for the ecological control of red strike disease of sugarcane.