烟草根际铁载体产生菌G-229-21T的筛选、鉴定及拮抗机理

Screening, identification and antagonistic activity of a siderophore-producing bacteria G-229-21T from rhizosphere of tobacco

摘要:

【目的】从烟草根际筛选烟草疫霉[Phytophthora parasitica var. nicotianae (Breda de Hann) Tucker]拮抗菌，探索其拮抗机理。【方法】限铁(2.0 mmol/L FeCl3)蔗糖-天冬酰胺平板对峙法筛选烟草疫霉拮抗菌；刃天青(CAS)法检测其铁载体的产生及其对铁离子的亲和能力。结合形态、生理生化、16S rRNA序列同源性和系统发育分析及种特异性分子法对其进行鉴定。XAD-2吸附层析法提取其铁载体，分光光度法检测其铁载体类型。不同铁离子浓度下，比较其铁载体对烟草疫霉的抑制作用。【结果】我们筛选到一株限铁条件下烟草疫霉拮抗菌G-229-21T，该菌产生高亲和力铁载体，被初步鉴定为Pseudomonas mediterranea。该菌产生的羧酸型铁载体，在低铁条件下(0.16 mmol/L~10 mmol/L FeCl3)对烟草疫霉的抑制率达92.3%以上，而在富铁条件下(100 mmol/L FeCl3)抑制率仅为2.0%。【结论】首次报道P. mediterranea G-229-21T产生高亲和力羧酸型铁载体，该铁载体在低铁条件下对烟草疫霉有显著的抑制作用。

关键词:Pseudomonas mediterranea;铁载体;筛选;鉴定;拮抗机理

Abstract:

[Objective] To screen antagonistic bacteria from rhizosphere of tobacco against Phytophthora parasitica var. nicotianae (Breda de Hann) Tucker and to analyze its antagonistic activity. [Methods] The antagonistic bacteria were screened by dual culture with P. parasitica var. nicotianae (Breda de Hann) Tucker on low iron (2μmol/L FeCl3) Sucrose-L-Asparagine (SA) plate. The chrome azurol S (CAS) assay was used to detect the siderophore production and its affinity to ferric ion. The strain was identified by using morphological, biochemical and physiological characteristics, 16S rRNA sequence homology, phylogenetics and specific species molecular analysis. The siderophore was isolated by column chromatography on Amberlite XAD-2 and analyzed by spectrophotometer. Antagonistic activity of the siderophore was confirmed by incubation of P. parasitica var. nicotianae (Breda de Hann) Tucker in liquid SA medium with different concentration of siderophore and FeCl3. [Results] We obtained an antagonistic bacterium G-229-21T against P. parasitica var. nicotianae (Breda de Hann) Tucker. The strain produced high-affinity siderophore under low iron conditions and was identified as Pseudomonas mediterranea. This strain produced carboxylate-type siderophore against P. parasitica var. nicotianae (Breda de Hann) Tucker. The suppression ratio was up to 92.3% under low iron conditions (0.16 mmol/L~10 mmol/L FeCl3). However, it was only 2.0% under Fe-replete conditions (100 mmol/L FeCl3). [Conclusion] P. mediterranea G-229-21T could produce high-affinity carboxylate-type siderophore against P. parasitica var. nicotianae (Breda de Hann) Tucker under low iron conditions.

Key words:Pseudomonas mediterranea; siderophore; screening; identification; antagonistic mechanism