Data_Sheet_1_Isolation and characterization of bacteriophages for controlling Rhizobium radiobacter – causing stem and crown gall of highbush blueberry.pdf

IntroductionStem and crown gall disease caused by the plant pathogen Rhizobium radiobacter has a significant impact on highbush blueberry (Vaccinium corymbosum) production. Current methods for controlling the bacterium are limited. Lytic phages, which can specifically target host bacteria, have been widely gained interest in agriculture.
MethodsIn this study, 76 bacteriophages were recovered from sewage influent and screened for their inhibitory effect against Rhizobium spp. The phages were genetically characterized through whole-genome sequencing, and their lytic cycle was confirmed.
ResultsFive potential candidate phages (isolates IC12, IG49, AN01, LG08, and LG11) with the ability to lyse a broad range of hosts were chosen and assessed for their morphology, environmental stability, latent period, and burst size. The morphology of these selected phages revealed a long contractile tail under transmission electron microscopy. Single-step growth curves displayed that these phages had a latent period of 80–110 min and a burst size ranging from 8 to 33 phages per infected cell. None of these phages contained any antimicrobial resistance or virulence genes in their genomes. Subsequently, a combination of two-, three- and four-phage cocktails were formulated and tested for their efficacy in a broth system. A three-phage cocktail composed of the isolates IC12, IG49 and LG08 showed promising results in controlling a large number of R. radiobacter strains in vitro. In a soil/peat-based model, the three-phage cocktail was tested against R. radiobacter PL17, resulting in a significant reduction (p < 0.05) of 2.9 and 1.3 log10 CFU/g after 24 and 48 h of incubation, respectively.
DiscussionThese findings suggest that the three-phage cocktail (IC12, IG49 and LG08) has the potential to serve as a proactive antimicrobial solution for controlling R. radiobacter on blueberry.

引言
由植物病原菌放射根瘤菌（Rhizobium radiobacter）引发的茎冠瘿病，对高丛蓝莓（Vaccinium corymbosum）的产业生产造成了显著负面影响。当前针对该病原菌的防治手段极为有限。能够特异性靶向宿主细菌的裂解性噬菌体（lytic phage），已在农业领域受到广泛关注。

材料与方法
本研究从污水进水中分离得到76株噬菌体（bacteriophages），并针对其对根瘤菌属（Rhizobium spp.）细菌的抑菌活性开展筛选。通过全基因组测序（whole-genome sequencing）对这些噬菌体进行遗传特征解析，并验证了其裂解周期。

结果
选取5株具备广谱宿主裂解能力的候选噬菌体（分离株IC12、IG49、AN01、LG08及LG11），对其形态学特征、环境稳定性、潜伏期及裂解量进行了系统性评估。透射电子显微镜（transmission electron microscopy）观测显示，所选噬菌体均具备长收缩尾的形态结构。一步生长曲线（single-step growth curve）结果表明，该批噬菌体的潜伏期为80~110分钟，单感染细胞裂解量介于8~33个噬菌体颗粒之间。所有候选噬菌体的基因组中均未检测到抗生素抗性基因（antimicrobial resistance gene）与毒力基因（virulence gene）。随后，分别构建了由2株、3株及4株噬菌体组成的混合噬菌体制剂（phage cocktail），并在液体培养基体系中测试其抑菌效能。由IC12、IG49与LG08三株噬菌体组成的混合制剂，在体外对大量放射根瘤菌菌株均展现出良好的抑菌效果。在土壤/泥炭基质模型中，该三株噬菌体混合制剂针对放射根瘤菌PL17的抑菌试验结果显示：培养24小时与48小时后，菌落形成单位（Colony-Forming Unit, CFU）分别显著降低2.9 log₁₀与1.3 log₁₀ CFU/g（p<0.05）。

讨论
本研究结果表明，由IC12、IG49与LG08组成的三株噬菌体混合制剂，具备作为主动型抑菌方案的潜力，可用于防治蓝莓上的放射根瘤菌病害。