Real-time PCR detection and dynamic analysis of Erwinia amylovora in the soils of pear orchards

Abstract: 【Objective】The pear fire blight is induced by Erwinia amylovora (E. amylovora). It first appeared in May, 2016 in Huocheng County, Xinjiang Yili, China. It has spread to 14 Xinjiang regions, posing danger to pears, apples, hawthorns, quince, and other fruit trees, particularly in Korla. The disease has been considered an enormous risk to the Xinjiang fruit industry. Since 2019, we have discovered that the main stem of the pear tree exhibits noticeable lesions and bacterial fluids that spread from the root to the stem and ultimately induce the tree to die. Thus, we assessed the E. amylovora concentrations in the soils of the pear orchards and monitor the dynamic variation trend of E. amylovora by realtime fluorescence quantitative PCR in order to get insight into the occurence and control of the pear fire blight.【Methods】Six samples were collected from the each point following the random diagonal fivepoint sampling method. From March to November 2021, we collected 810 soil samples from the diseased pear orchards in Korla, with a sampling depth of 0-20 cm and a sampling volume of around 100 gat every point. The soil was sieved in order to obtain a 2.0 mm fraction, air-dried at room temperature for 2 days and stored in paper bags. The total DNA was extracted from the each soil sample at a dry weight of 0.25 g using the MOBIO Soil Genomic DNA Extraction Kit. 0.25 g dry soil was taken out, and a standard bacterial suspension of 2.0×109 CFU·mL^-1 was used for serial dilution of quantitative soil DNA using sterile water. The standard curve was generated using different amounts of standard DNA dilutions, and the concentration of E. amylovora in soil samples was detected by the real-time fluorescence quantitative PCR established by our lab.【Results】The real time fluorescence quantitative PCR reaction was conducted under conditions of initial 5 min denaturation at 95 ℃, 45 cycles of 95 ℃ for 10 s, 60 ℃ for 30 s. The equation of standard curve was y=-3.458x + 46.033, there was a good linear relationship between the CT value and the logarithm of pathogen concentration. The evaluation standard was based on the CT value and the fluorescence enhancement signal, with a positive result when the CT value was less than 40 and there was a clear fluorescence enhancement signal and a negative result when the CT value was larger than 40 or there was no amplification signal. E. amylovora accounted for a small proportion of total microorganisms in the soils, and the CT value of positive samples ranged from 30 to 40 in general. The carrier rate was 24.2%, 8.9%, 22.2%, 42.2%, 44.4%, 17.8%, 23.3%, 2.2% and 2.2%, and the average concentration of E. amylovora was 515, 82, 428, 914, 965, 277, 408, 15 and 15 CFU · g- 1 from March to November respectively. E. amylovora levels were significantly higher in June and July than those in April, October, and November. The highest concentration of E. amylovora in the 810 soil samples was 5.29×104 CFU · g^{- 1} , with a CT value of 31.8. 13 soil samples had concentrations greater than 103 CFU·g^-1 in June, and 26 soil samples had concentrations greater than 103 CFU·g-1 in July. The results of the study suggested that June and July would have a higher risk of diseases. Since April, the concentration of E. amylovora in the soils had gradually increased with the growing period of pear plants. Because diseased residues such as diseased leaves and fruits were not cleaned in time after the picking season in 2020, the average concentration of E. amylovora in March was higher than those in April and May. The carrier rate and average concentration of E. amylovora in the soils were at their maximum in July and gradually decreased later. Because diseased leftovers such as diseased leaves and fruits were cleaned in time according to prevention and control requirements following the picking period in 2021, the average concentration of E. amylovora was reduced significantly in October and November 2021. In accordance with the survey data, the average concentration and carrier rate of E. amylovora fluctuated with the disease index. The pear orchard disease index was highest in May, whereas the average concentration and carrier rate of E. amylovora were at their maximum in July. The average E. amylovora concentration and carrier rate in the soils had a time lag, due to the sedimentation and accumulation.【Conclusion】The average concentration and carrier rate of E. amylovora were at their maximum in June and July in the soils of the pear orchards, which had a time lag with the disease index due to the sedimentation and accumulation of the pathogen. The diseased residues should be cleaned out from the orchard in time for effective control of the disease.