Characterization of the infectious colonization and expansion with GFP tagged strain of Erwinia amylovora in Kuerlexiangli pear (Pyrus sinkiangensis Yu) shoots

Abstract: 【Objective】Fire blight is a major international quarantine bacterial disease caused by Erwinia amylovora, a destructive bacterium that infests pears, apples and other kinds of rosaceae nut fruit trees. In 2016 and 2017, it was found for the first time in Yili and Bazhou in Xinjiang, China, harming pear, apple, hawthorn, begonia, quince and other fruit trees, especially spreading extremely fast on Kuerlexiangli pear (Pyrus sinkiangensis Yu), resulting in severe economic loss to the pear industry. The infestation characteristic that E. amylovora can transfer rapidly in the host tissues is an important reason for the fast epidemic speed, destructive damage and great difficulty of control. Timely pruning of diseased fruit trees can not only greatly reduce the amount of pathogenic bacteria, but also prevent its migration and expansion in the tree, which is an important measure for disease prevention and control and easy toimplement and cost-effective. The artificial inoculation of tracing pathogenic bacteria in pear branch tissue infestation colonization and migration characteristics was employed to deeply understand the pathogenic bacteria in the host tissue infestation expansion process and mechanism in order to guide the field pruning of diseased branches, disease resistant varieties selection and breeding and the development of integrated control measures【Methods】In this study, the virulent pathogenic strain E. a001 was isolated the shoots infested by fire blight, and the Green Fluorescent Protein (GFP) plasmid phc60-gfp was transformed into E. a001 by thermal excitation method, and the strong green fluorescent strain E. a001-gfp was successfully obtained. The inoculations of different concentrations of E. a001-gfp suspension were conducted with two methods, needle prick and spray, to detect and observe the dynamics of infestation and expansion in the tissues of pear shoots.【Results】The GFP- labeled E. a001- gfp strain emitted strong green fluorescence under fluorescence microscopy, and a target fragment of about 700 bp was amplified from the genomic DNA of the labeled bacteria. The morphology, growth curve and pathogenicity of the tagged strain E. a001-gfp were not significantly different from those of the wild type, and its green fluorescence was stably inherited. The lowest concentrations of E. a001-gfp for inducing shoot disease were 106 cfu ·mL^-1 and 107 cfu ·mL^-1 for needle inoculation and spray inoculation, respectively. The expansion rate of the necrotic spots produced by the pathogen-infested shoots increased with the concentration of the pathogen, and the expansion rate of spots on shoots treated with 109 cfu·mL-1 inoculation was the fastest, and the migration rate was up to 3.96 cm· d^-1 . The migration time of the necrotic spots produced by the E. a001-gfp inoculation in the pear shoots was 12-15 d (average 13.5 d). The expansion distance of spots in 1-year-old branches was 37.72-47.27 cm (average 42.77 cm), and in 2-yearold branches was 2.05-3.86 cm (mean 3.21 cm) respectively, which were significantly different. The results of spray inoculation treatment were similar to those of the needle prick method. The laser confocal scanning microscope observation showed that the pathogenic bacteria mainly presented in the branch vascular system after invasion, and a large number of them were distributed in the cortical thin-walled cells and the interstices of the bast thin-walled cells, and a small number of them distributed in the xylem ducts. The E. a001-gfp was colonized at the necrotic lesions, disease-healthy junction and 0-10 cm of the non-significant part of the branch, and it could still be isolated at 40 d. The highest number of viable bacteria was found at the disease- healthy junction, which could reach 108 cfu · g^{- 1} tissue, and the highest number of pathogenic bacteria was 105 cfu · g^-1 tissue at 0-10 cm of the undeveloped part of the branch, but no pathogenic bacteria were isolated at 10- 15 cm of the undeveloped part of the pear branch outside the diseased spot. It is suggested that pruning the diseased branch at 20-30 cm of the visible spot of the fire blight susceptible pear branch could effectively remove the pathogen, which is a safe and effective pruning length; the pathogenic bacteria could survive in the diseased branches for a long period of time, and the viable bacteria count of fire blight bacteria detected in the diseased branches stored at room temperature for 6 months was 105 cfu·g-1 .【Conclusion】The study clarified the expansion distance, migration time and rate, distribution and colonization amount of the E. amylovora, in the branch tissue of pear, which provided a scientific basis for the accurate pruning of diseased branches and the formulation of comprehensive prevention and control measures of the disease.