Establishment Of A New Method To Assessment Of Quarantine Pests Of Disease Risk By EMA-PCR/qPCR

Citrus canker (causal pathogen, Xanthomnas citri subsp.citri) is one of the mostdestructive diseases of citrus, threatening the citrus industry worldwide. It was listed as"the entry plant quarantine pests" into the directory published by the People’s Republicof China in2007. Solanaceae bacterial wilt pathogen (Ralstonia solanacearum) has awide host range. It can survive in the soil for a long time and cause huge economiclosses. Conventional PCR and real-time PCR with high specificity, sensitivity and otheradvantages have been widely used in the detection of the above plant pathogenicbacteria. However, conventional PCR method detects targets based on DNA derivedfrom dead cells, live bacteria, viable but non-culturable bacteria (VBNC) and residualDNA; therefore, it cannot accurately detect viable cells. Indeed, distinguishing deadcells from live cells is often necessary in practical use, such as detecting the therapeuticeffect of infection in medicine, detecting phytosanitary pathogens for pesticidestreatment effect, and the plant disease risk assessment. Since only the live cells are ofhigh risk, traditional DNA-based PCR detection often has high false positive results.Recently, the VBNC of plant pathogenic bacteria has been being widely studied. Manyimportant plant pathogenic bacteria can enter the VBNC state under certain conditions,escaping from routine culture detection by traditional separation method as "hidden"source of infection, leading to the detection of false-negative results. Pathogens in theVBNC state may restore and regain pathogenicity as the primary infection source,causing occurrence and epidemic breakout of the disease in field. Therefore, there is anurgent need for a specific, accurate and rapid method to detect viable cells of plantpathogenic bacteria.The specific nucleic acid dye, ethidium monoazide bromide, is a fluorescent dyewhich can covalently bind to DNA molecules. EMA molecules enter into the dead andliving cells selectively. Only incomplete cell walls and membranes of dead cells allowthe entry of EMA, while the integrity of the cell membrane system in living cells canprevent the entry. Combining the EMA dye with PCR results in a rapid and sensitivemethod for detecting the genomic DNA from viable cells, and thus, overcoming thefalse negative and false positive results of conventional PCR, as well as the drawbacksof traditional plate count method when detecting pathogens at VBNC state. In this study,we firstly optimized EMA concentration and PCR conditions to detect viable cells in pure culture conditions by pre-treating the bacteria with EMA before DNA preparation.Secondly, the optimized EMA-PCR system was applied for the detection of pathogensat the VBNC state in pure culture conditions. Finally, EMA-PCR was used to detect thecontaminated samples from the field. The major findings are as below:①Establishment of EMA-PCR detection method of viable cells ofXanthomonas citri subsp. Citri: Firstly, according to the the citrus canker uniqueconserved protein gene, primers (Xcc R/Xcc F) specific to X. citri subsp. citri weredesigned to amplify a278-bp fragment. The minimum limit of detection was25cells/25μL PCR volume or2.75pg/25μL PCR volume. The results of EMA-PCR showed thatthe optimized light exposure time was at least1min, allowing cross-linking of DNA bythe EMA in dead cells and to photolyse the free EMA in solution. The minimum amountEMA to completely inhibit the PCR amplification of DNA derived from heat-killedcells (1.0×10~8CFU/mL) was1mg/L. EMA less than30mg/L did not inhibit the PCRamplification of DNA derived from viable cells of X. citri subsp. citri. A linearrelationship was found between the average fluorescent intensity of the DNA bands andthe log value of genomic targets derived from the viable cells in mixtures of viable anddead cells in the range of6.875×10~1-6.875×10~5CFU/PCR（R~2=0.934）．The data ofEMA-PCR detection on citrus filed samples indicated that2mg/L of EMA successfullyinhibited PCR amplification of DNA from dead bacteria in filed samples. Twelve citruscanker leaf and fruit samples with suspected symptoms were collected from Chongqingand Guangxi, and tested using EMA-PCR compared with conventional PCR and platecount method. The results indicate that EMA-PCR is a promising and accurate methodto prevent false positive results in detection of X. citri subsp. Citri.②Establishment of a new method to detect viable cells of Ralstoniasolanacearum by EMA-qPCR: Primers (RSF/RSR) were designed according to thethe Ralstonia solanacearum UDP-3-O-acyl-GlcNAc deacetylase, amplifying a fragmentof159bp. A final concentration of2.0mg/L EMA was demonstrated to completelyinhibit the PCR amplification from DNA derived from1.0×10~7CFU/mL dead cells, butno inhibition to viable and Viable but-Non-Culturable (VBNC) cells. For live bacteriaconcentration≦10~7CFU/mL and EMA concentration≧40mg/L, the target geneamplification living cells was significantly inhibited (P <0.05). As a result, the EMAconcentration (40mg/L) affecting PCR amplification from living cells is much greaterthan the minimum EMA concentration (1.5mg/L) inhibiting DNA amplification fromdead cells. A standard curve was generated relating the Ctvalues of the EMA-qPCR to the log number of genomic targets per PCR. A linear range of DNA amplification wasobserved from5.0×10~0to5×10~4genomic targets per PCR. EMA-qPCR method wasused to evaluate the survival rate of R.solanacearum treated with different temperaturesfor a short time, compared with the method of plate count. The results indicate thatsamples can be stored for a short time under room temperature and4℃. Artificialinoculation mimicking soil contamination test showed that the minimum detection limitof EMA-qPCR on the Ralstonia solanacearum soil samples was10~2CFU/g, and whenthe amount of bacteria in the soil was in the range of10~7CFU/g-10~2CFU/g of soil,bacterial amount of the value and the Ct value has a linear relationship (R~2=0.995). Theminimum detection limit of EMA-qPCR was10~3CFU/g on the Ralstonia solanacearumsoil samples with removal of particulate soil supernatant. The EMA final concentrationof50mg/L completely inhibited the DNA amplification from the dead cells of10~6CFU/g soil, but partially for the dead cells more than10~6CFU/g.In conclusion, this study established an EMA-PCR (qPCR) method to detect plantpathogens, by distinguishing dead cells from living cultured cells and VBNC state cells.The process of EMA-treatment was specifically optimized, and the method was appliedto detect field samples of citrus canker and soil samples of Ralstonia solanacearum. Theoptimized EMA-PCR (qPCR) method can provide more scientific and accuratetechnical support for the inspection and quarantine of plant pathogens, so as tocontribute to the management of plant diseases.