Ottawa Research and Development Centre, Agriculture and Agri-Food Canada, Ottawa, ON, K1A 0C6, Canada.
Canadian Blood Service, Ottawa, ON, K1G 4J5, Canada.
Sci Rep. 2024 Sep 3;14(1):20517. doi: 10.1038/s41598-024-71418-y.
Long-term use of chemical fungicides to control plant diseases caused by fungi and oomycetes has led to pathogen resistance and negative impacts on public health and environment. There is a global search for eco-friendly methods and antagonistic bacteria are emerging as alternatives. We isolated a potent antagonistic bacterial strain (S1Bt23) from woodland soil in Québec, Canada. Taxonomic characterization by 16S rRNA, multi-locus sequence analysis, pairwise whole-genome comparisons, phylogenomics and phenotypic data identified strain S1Bt23 as a novel subspecies within Pseudomonas chlororaphis. In dual culture studies, strain S1Bt23 exhibited potent mycelial growth inhibition (60.2-66.7%) against Pythium ultimum. Furthermore, strain S1Bt23 was able to significantly bioprotect potato tuber slices from the development of necrosis inducible by P. ultimum. Annotations of the whole genome sequence of S1Bt23 revealed the presence of an arsenal of secondary metabolites including the complete phenazine biosynthetic cluster (phzABCDEFG). Thin-layer (TLC) and high-performance liquid (HPLC) chromatographic analyses of S1Bt23 extracts confirmed the production of phenazines, potent antifungal compounds. CRISPR/Cas9-mediated deletion of phzB (S1Bt23ΔphzB) or phzF (S1Bt23ΔphzF) gene abrogated phenazine production based on TLC and HPLC analyses. Also, S1Bt23ΔphzB and S1Bt23ΔphzF mutants lost antagonistic activity and bioprotection ability of potato tubers against P. ultimum. This demonstrated that phenazines are involved in the antagonistic activity of S1Bt23 against P. ultimum. Finally, based on genotypic and phenotypic data, we taxonomically conclude that S1Bt23 represents a novel subspecies for which the name Pseudomonas chlororaphis subsp. phenazini is proposed.
长期使用化学杀菌剂来控制真菌和卵菌引起的植物病害，导致病原体产生抗性，并对公共健康和环境产生负面影响。因此，人们正在全球范围内寻找环保的方法，而拮抗菌正成为替代品。我们从加拿大魁北克的林地土壤中分离到一株强效的拮抗菌株（S1Bt23）。通过 16S rRNA、多位点序列分析、成对全基因组比较、系统发育基因组学和表型数据的分类学特征鉴定，该菌株 S1Bt23 被鉴定为假单胞菌属中 Chlororaphis 的一个新亚种。在双层培养研究中，菌株 S1Bt23 对腐霉属的菌丝生长抑制率达到 60.2-66.7%。此外，该菌株 S1Bt23 还能显著保护马铃薯切片免受腐霉属诱导的坏死。S1Bt23 全基因组序列注释显示，该菌存在包括完整吩嗪生物合成簇（phzABCDEFG）在内的大量次生代谢产物。S1Bt23 提取物的薄层层析（TLC）和高效液相（HPLC）分析证实了吩嗪的产生，这是一种强效的抗真菌化合物。基于 TLC 和 HPLC 分析，CRISPR/Cas9 介导的 phzB（S1Bt23ΔphzB）或 phzF（S1Bt23ΔphzF）基因缺失导致吩嗪的产生被阻断。此外，S1Bt23ΔphzB 和 S1Bt23ΔphzF 突变体丧失了对腐霉属的拮抗活性和对马铃薯块茎的生物保护能力。这表明吩嗪参与了 S1Bt23 对腐霉属的拮抗作用。最后，根据基因型和表型数据，我们从分类学上得出结论，S1Bt23 代表了一个新的亚种，我们提议将其命名为假单胞菌属 Chlororaphis 亚种 phenazini。
