Isolation, Identification And Establishment Of Viable Cells Detection Of The Pathogen Of Mulberry Bacterial Wilt

Mulberry bacterial wilt, caused by Ralstonia solanacearum race 5, is a damaging bacterial crop disease in China. Early detection is necessary for preventing disease outbreak, and effective control strategies are limited. The pathogenic strain was isolated from diseased mulberry roots in Guangdong province. After identification and pathogenicity test, strain RS-5 was identified to be a pathogenic R. solanacearum. A specific primer was designed to detect R. solanacearum race 5 by fluorescence quantitative PCR(qPCR), and qPCR method combined with propidium monoazide(PMA) was established to detect viable cells of R. solanacearum race 5.Three strains RS-5, JX-3, GD-3 with typical morphological characteristics of R. solanacearum were isolated form diseased mulberry roots among which RS-5 showed pathogenicity to mulberry after pathogenicity test. A blast analysis of the sequence indicated that the amplified gene of the re-isolated strain shared over 99% similarity with the 16 S r DNA gene sequences from R. solanacearum. And the strain RS-5 was identified to be R. solanacearum.A specific primer pair RS-72F/RS-312 R was designed based on the subtracted gene of R. solanacearum race 5 and other races. The primer pair can amplify a 241 bp fragment of R. solanacearum race 5 and without any amplification of other R. solanacearum races or other species in the soil. Detect limit of conventional PCR was 50 pg/μL to DNA and 1.0×104 CFU/ml to cell suspension. Standard curve analysis showed that log(ng/μL from 10-4 to 102 ng/μL) versus Ct was y =-2.747 x + 21.834(R2 = 0.993), while log(CFU/mL from 103 to 107) versus Ct was y =-3.418 x + 45.447(R2 = 0.998).Bacterial samples were pretreated with propidium monoazide, amplification of DNA from dead cells in a 107 CFU/mL concentration can be totally inhibited when treated with 15 ng/μL PMA for 10 min in the dark followed by 5 min halogen light exposure, and with little influence to DNA in live cells. Standard curve analysis of PMA-qPCR showed that log CFU/mL(from 103 to 107) versus Ct was y =-3.477 x + 47.489(R2 = 0.997). The method can distinguish live cells from dead cells in mixed culture.This study is the first report of viable cells detection of R. solanacearum race 5 which supplies a technical support for early detection and control of bacterial wilt, and has significance in reducing losses of bacterial wilt and keeping sustainable development of sericulture industry.