Establishment of rapid quantitative detection of viable Erwinia amylovora

Abstract:【Objective】Fire blight caused by Erwinia amylovora is a very severe bacterial disease of ap- ple, pear and many other plant species, mainly of the family Rosaceae. It has spread to more than 50 countries in the world and causes damage of economic importance. The disease affects all aboveground plant organs, causing their dieback. The common symptoms of fire blight are burn-like damage to flow- ers, leaves, fruits and terminal shoots, and necrotic lesions and cankers develop on woody tissues. A sin- gle severe outbreak of the disease can not only greatly reduce crop yield and marketing ability in the current season by infecting blossoms and killing fruit spurs, but also cause the loss of entire trees and even whole orchards, resulting in great economic losses due to stringent quarantine and international trade regulation. Although the disease has not been reported in China yet, recent reports of the disease in China’s neighboring countries, including South Korea, Kyrgyzstan, and Kazakhstan, poses great threat to the apple and pear industries. Strict quarantine measures for importing fruits and seedlings should be executed, and fast and quick diagnosis tools should be developed and disseminated as soon as possible. Once the disease is found, strict eradication measures should be imposed to prevent further spread and appropriate control measures should be performed. Propidium monoazide (PMA) is a kind of photosensitive dye that can pass through the damaged cell membrane and has high affinity with DNA molecules. The cross-linking of PMA and real-time fluorescent quantitative PCR (qPCR) can detect via- ble cells and inhibit PCR amplification reactions from the dead cells. The present experiment aimed at developing a rapid detection method for live bacteria of Erwinia amylovora by means of combining propidium monoazide (PMA) with real-time fluorescence quantitative PCR (qPCR), so as to provide technical support for the disease quarantine, early diagnosis, etiological and epidemiological researches, as well as to formulate the control measures to prevent its invasion.【Methods】The specific primer pair EaP29F1/EaP29R1 was designed with Erwinia amylovora plasmid pEA29. The optimal reaction conditions for PMA pretreatment was determined to distinguish between live and dead bacteria by optimizing the mass concentration and exposure time of PMA. Then the specific qPCR was performed by using via- ble bacteria DNA as a template. A mixed system was set up with different ratio of dead and viable bacteria to verify the reliability of the PMA-qPCR reaction.【Results】When the concentration of Erwinia amylovora was 1×108 CFU per mL, the concentration of PMA was 25 μmol·L-1, and the exposure time was 10 min, the amplification of dead bacteria could be completely inhibited, and only the DNA of living bacteria was targeted for selective amplification. When the proportion of viable bacteria was 10%-100% (concentration of 6.0×107-6.0×108 CFU per mL), the number of live bacteria measured by PMA-qPCR and the theoretical value of live bacteria were in the same of magnitude, and both had a linear relation- ship (R2=0.992 8). The sensitivity test showed that there was a linear correlation between the number of live bacteria and Ct value (R2=0.994 7) within the scope of 6.0×103-6.0×108 CFU·mL-1, and with 6×103CFU·mL-1 (12 CFU/qPCR reaction) as the lower limit of detection sensitivity. The established PMA-qP- CR method was also used to detect the artificially inoculated twigs of Fragrant Pear, apple, and Pyrus bet- ulifolia, which showed that PMA-qPCR could accurately detect the viable bacteria, and the results were consistent with those of the plate counting.【Conclusion】The established method could quantitatively de- tect the viable bacteria of Erwinia amylovora, with advantages of high accuracy, sensitivity and efficien- cy.“False positive”caused by conventional qPCR detection of dead bacteria can thus be avoided.