Scientia Agricultura Sinica ›› 2026, Vol. 59 ›› Issue (5): 996-1007.doi: 10.3864/j.issn.0578-1752.2026.05.006

• PLANT PROTECTION • Previous Articles     Next Articles

A Novel Plasmid pEA60 of Erwinia amylovora Enhances the Pathogenicity of Strains by Regulating the Synthesis of Virulence Factors

DONG Yu(), WU Qian, FENG Xuan, ZHENG YinYing, CUI BaiMing()   

  1. College of Life Sciences, Shihezi University, Shihezi 832003, Xinjiang
  • Received:2025-11-10 Accepted:2025-12-21 Online:2026-03-01 Published:2026-03-06
  • Contact: CUI BaiMing

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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

Table 1

Primers used in this study"

引物名称
Primer name		 引物序列
Sequence of primers (5′-3′)		 用途
Usage
EaU_F ACCAGTTGCACCGAAAAGT 检测解淀粉欧文氏菌染色体
Detection of E. amylovora chromosome
EaU_R ATGTAAACTGGTGCGCAGAC
p60_F AAGTGATGCTTCCGGTCGAA 检测pEA60质粒的复制起点
Detection of the origin of replication of the plasmid pEA60
p60_R GACGACCCAGCGCTTTATTT
p29_F CGTTTGCTGAACCTTGCTCT 检测pEA29质粒
Detection of the plasmid pEA29
p29_R CATCCAGTCAAGTGCCAGTG
p60ori_F ACTTCTCTGGCCGGGCT 扩增pEA60质粒的复制调控区
Amplification of the replication regulatory region of the plasmid pEA60
p60ori_R CCCCTGAATGTCTAACGGGG
COLONY_F CGGCTCGTATGTTGTGTGGA 检测p60MD2质粒
Detection of the plasmid p60MD2
COLONY_R GTAACGCCAGGGTTTTCCCA
p60C6_F ATCTGGCAAAGGGGGTGTTC 检测pEA60质粒
Detection of the plasmid pEA60
p60C6_R GAGGGTGGTTTCCTCACTGG
kanori_F ATCAGGAAGTCGGCATCAGAGCAGATTG 扩增pET28a(+)载体的卡那霉素抗性基因和复制起点
Amplification of the kanamycin resistance gene and ori of the vector pET28a(+)
kanori_R TCCTGTGTGATTCAGGTGGCACTTTTCG
19MCS_F GCCACCTGAATCACACAGGAAACAGCTATG 扩增pMD19载体的多克隆位点
Amplification of the multiple cloning site of the vector pMD19
19MCS_R GAATGATCAGCCAGTCACGACGTTGTAAAAC
29ori_F TCGTGACTGGCTGATCATTCCTCTTTTGTG 扩增pEA29质粒的复制起始区
Amplification of the replication origin region of the plasmid pEA29
29ori_R TCTGATGCCGACTTCCTGATGATCGTGC

Fig. 1

The genome map of plasmid pEA60 The inner side of the circle shows positions in kb, arrows on the outside of the circle display CDSs. Purple arrows represent replication related proteins, light green arrows represent type IV coupling proteins, dark green arrows represent the main proteins of type IV secretion system, yellow arrow represents relaxase, and brown arrows represent pilus proteins"

Fig. 2

Sequence comparison of the plasmid pEA60 Arrows indicate the predicted CDSs. Gray shading indicates nucleotide similarity, with darker shading indicating higher similarity"

Fig. 3

Stability detection of plasmid pEA60"

Fig. 4

Identification of the replication regulatory region in plasmid pEA60"

Fig. 5

Conjugation detection of plasmid pEA60"

Fig. 6

Determination of exopolysaccharide production"

Fig. 7

Inoculation of Korla fragrant pear immature fruits and detached shoots"

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