关键词: 解淀粉欧文氏菌, 库尔勒香梨, 火疫病, pEA60质粒, 胞外多糖, 致病力

Abstract:

【Background】 Fire blight poses a serious threat to the development of the Korla fragrant pear industry in Xinjiang. Its pathogen, Erwinia amylovora, often carries diverse plasmids, which are key factors leading to phenotypic differences among strains, such as pathogenicity and antibiotic resistance. Recently, a novel plasmid pEA60 was identified from the E. amylovora strain Ea102 which was isolated from Xinjiang Korla fragrant pear.【Objective】 The objective of this study is to investigate the origin, function of E. amylovora plasmid pEA60 and its impact on pathogenicity of the pathogen.【Method】 The genome of Ea102 was sequenced using PacBio and Illumina and assembled with SPAdes. Functional annotation was performed using tools such as PAGP, while BLASTN was used for comparative genomic analysis, and MLST for plasmid typing. The replication region was identified using plasmid incompatibility, and a pEA60-cured mutant was constructed. Conjugation experiments were conducted to assess its self-transfer capability. In terms of functional verification, the effect of the plasmid on bacterial pathogenicity was systematically evaluated by quantitative determination of exopolysaccharide production and immature fruit and in vitro shoots test of pear.【Result】 pEA60 is a conjugative plasmid with a size of 61 198 bp, containing 65 predicted CDSs primarily involved in three functional modules: replication and stability, pilus formation, and conjugation. No known antibiotic resistance genes or virulence-related genes were identified. Comparative analysis revealed that the pilus formation and conjugation functional regions of pEA60 are highly similar to those of the E. amylovora plasmid pEA68 (average nucleotide identity: 97.04%), while its replication and stability regions resemble those of the Erwinia aphidicola plasmid p1B06c, suggesting that pEA60 likely originated from a recombination event. pEA60 is an IncFII-type plasmid, and its 2 610 bp fragment harboring the replication protein gene and the origin of replication is sufficient to maintain plasmid viability. The plasmid influenced exopolysaccharide synthesis and pathogenicity in E. amylovora. Curing of pEA60 resulted in reduced amylovoran production and levansucrase activity, increased cellulose production, and attenuated symptoms on both pear shoots and immature fruits.【Conclusion】 pEA60 is a novel, conjugative IncFII plasmid in E. amylovora. It affects exopolysaccharide synthesis and enhances the pathogenicity of its host strain. This study provides new insights into the pathogenic mechanisms of E. amylovora and plasmid-mediated evolution of virulence.

Key words: Erwinia amylovora, Korla fragrant pear, fire blight, pEA60 plasmid, exopolysaccharide, pathogenicity