高产吲哚乙酸东乡野生稻内生微杆菌KlspL 18分离及鉴定

Isolation and identification of a novel IAA high-producing endophytic strain KlspL18 from Oryza rufipogon

摘要:

【目的】对分离自东乡野生稻叶组织的高产吲哚乙酸菌株KlspL18进行分类学鉴定。【方法】采用菌株形态、生理生化指标结合16S rRNA基因序列同源性比对以及全基因组测序等方法，对该菌株进行多相分类鉴定，并采用高效液相(HPLC)法测定其产吲哚乙酸的产量。【结果】菌株KlspL18为革兰氏阳性、不形成孢子的棒杆状，接触酶实验阳性，其温度、NaCl浓度和pH值生长范围分别为15–40 °C (最适为28 °C)、1%–10% (最适为1%)及6.0–11.0 (最适为7.0)，能利用多种糖和有机酸作为碳源。菌株细胞壁氨基酸主要为鸟氨酸，细胞壁多糖为半乳糖和甘露糖，极性脂类为二磷脂酰甘油、磷脂酰甘油和2种未鉴定的糖脂，细胞脂肪酸主要为anteiso-C_15:0 (30.33%)、anteiso-C_17:0 (31.53%)和iso-C_16:0 (14.32%)，萘醌类主要为MK-10和MK-11。16S rRNA基因序列分析表明，该菌株与Microbacterium proteolyticum RZ36^T相似度为97.64%；全基因组测序分析显示，其G+C含量70.2%，与近缘标准菌株核苷酸同源性(ANI)和数字DNA-DNA杂交值(dDDH)分别为83.35%和26.4%，均低于种间同源性的临界值。菌株KlspL18产IAA可达291.7 mg/L。【结论】菌株KlspL18是高产吲哚乙酸微杆菌属的一个新种，命名为Microbacterium dongxiang sp. nov.，模式菌株为KlspL18 (=CCTCC M2022446)，具有应用于农业生产促进植物生长的潜能。

Abstract:

[Objective] To confirm the taxonomic status of the novel strain KlspL18 with high yield of indole acetic acid (IAA) isolated from the leaves of Oryza rufipogon. [Methods] KlspL18 was identified based on morphological, physiological, and biochemical characteristics, phylogenetic analysis, and whole genome sequencing. The production of IAA was determined by high performance liquid chromatography (HPLC). [Results] KlspL18 was Gram-positive, non-spore-forming, rod-shaped, and catalase-positive. It grew at 15–40 °C (optimum: 28 °C), pH 6.0–12.0 (optimum: 7.0), and in the presence of 0%–15% NaCl (optimum: 1.0%), and could use multiple carbohydrates and organic acids as carbon source. The main amino acid in the cell wall of KlspL18 was ornithine, and the dominant polysaccharides in the cell wall were galactose and mannose. The stain mainly had the polar lipids of diphosphatidyl glycerol, phosphatidyl glycerol, and two unidentified glycolipids, fatty acids of anteiso-C_15:0 (30.33%), iso-C_16:0 (31.53%), and anteiso-C_17:0 (14.32%), and naphthoquinones of MK-10 and MK-11. Phylogenetic analysis of 16S rRNA gene sequence showed the highest similarity (97.64%) to the type strain of Microbacterium proteolyticum RZ36^T. The genome of KlspL18 had the G+C content of 70.2%. The average nucleotide identity (ANI) and digital DNA-DNA hybridization (dDDH) between KlspL18 and the closely related strain of Microbacterium were 83.35% and 26.4%, respectively, both below the threshold for species discrimination. HPLC analysis suggested that KlspL18 had high yield of IAA (291.7 mg/L). [Conclusion] KlspL18, a novel high-IAA-producing species of Microbacterium, was named Microbacterium dongxiang sp. nov., which is a model strain KlspL18 (=CCTCC M2022446). It has the potential of improving soil fertility and promoting plant growth in agricultural production.