解淀粉欧文氏菌噬菌体phB78的分离、基因组序列分析及其防控效果

Isolation, genome sequencing, and biocontrol effect evaluation of Erwinia amylovora bacteriophage phB78

摘要:

【背景】 解淀粉欧文氏菌(Erwinia amylovora)是梨、苹果等果树梨火疫病的病原菌。目前，梨火疫病防控主要依靠抗生素和含铜农药，但噬菌体被认为是一类有前途的生防制剂。【目的】 分离鉴定新的解淀粉欧文氏菌噬菌体，为梨火疫病的生物防治提供噬菌体资源。【方法】 以解淀粉欧文氏菌Ea102为宿主菌，采用双层琼脂平板法从发生梨火疫病的香梨果园的土壤中分离纯化噬菌体。用透射电镜观察噬菌体形态。PacBio测序分析噬菌体基因组。通过噬菌斑计数法测定噬菌体的最佳感染复数、一步生长曲线、热稳定性和酸碱度稳定性等生物学特性，并评价其噬菌体在香梨幼果和杜梨苗上对解淀粉欧文氏菌Ea102的抑菌效果。【结果】 分离得到一株解淀粉欧文氏菌烈性噬菌体phB78。噬菌斑中部透亮，外围有晕圈。phB78为有尾噬菌体，头部成二十面体，直径(108±3) nm，尾部长(210±6) nm，基因组为长261 824 bp的环形DNA分子(GenBank登录号：PP942979)，G+C含量为49.3%，预测编码322个蛋白质，其中大部分功能未知。phB78与Alexandravirus属噬菌体pEa_SNUABM_30 (NC_061446.1)最相似，它们的平均核苷酸一致性为95.23%。在温度50 ℃以下及pH 6.0-10.0的环境条件下，phB78的裂解活性能够保持稳定状态。以Ea102为宿主菌时，噬菌体的最佳感染复数为0.1，潜伏期约50 min，暴发量为20 PFU/cell。phB78能够侵染从梨树和苹果树分离出的4株解淀粉欧文氏菌中的3株，14株成团泛菌(Pantoea agglomerans)中的7株。phB78可以显著降低解淀粉欧文氏菌Ea102在香梨幼果和杜梨幼苗上的致病力。【结论】 phB78是一株新的烈性解淀粉欧文氏菌噬菌体，对梨火疫病的生物防治极具价值。

Abstract:

[Background] Erwinia amylovora is the causative agent of fire blight, a bacterial disease that is destructive to pear and apple plants. At present, the control of fire blight mainly relies on antibiotics and copper-containing chemicals. Bacteriophages are considered to be promising biocontrol agents. [Objective] To isolate and characterize novel E. amylovora bacteriophages and contribute to the biocontrol of fire blight. [Methods] With E. amylovora strain Ea102 as the host, bacteriophages were isolated by the double-layer agar plate method from the soil of pear orchards suffering from fire blight. The bacteriophage particles were observed by transmission electron microscopy. PacBio sequencing was conducted to analyze the bacteriophage genome. Biological characteristics such as optimal multiplicity of infection, one-step growth curve, and thermal and pH stability were determined by the plaque counting method. The inhibitory effects of the bacteriophage against E. amylovora Ea102 on young fragrant pears and Pyrus betulaefolia seedlings were evaluated. [Results] A bacteriophage strain phB78 infecting E. amylovora was isolated. It formed clear plaques with translucent halo zones. phB78 was composed of an icosahedral head with a diameter of (108±3) nm and a long tail with a length of (210±6) nm. It had a circular genome of 261 824 bp (GenBank Accession No: PP942979) with the GC content of 49.3%. A total of 322 open reading frames were predicted, most of which had unknown functions. phB78 was closely related to the Alexandravirus bacteriophage pEa_SNUABM_30 (NC_061446.1), with 95.23% average nucleotide identity. phB78 kept stable lytic activity at pH 6-10 and temperatures below 50 ℃. With E. amylovora Ea102 as the host, phB78 showed the optimal multiplicity of infection of 0.1, the latent period of about 50 min, and the burst size of 20 PFU/cell. A host range analysis revealed that phB78 was able to infect 3 out of 4 E. amylovora strains and 7 out of 14 Pantoea agglomerans strains isolated from pear and apple plants. phB78 significantly reduced the pathogenicity of E. amylovora Ea102 on young fragrant pears and P. betulaefolia seedlings. [Conclusion] phB78 is a novel virulent bacteriophage of E. amylovora, demonstrating a great value for the biocontrol of pear fire blight.