达乌里胡枝子产铁载体根瘤内生细菌的鉴定及其促生抗逆效应

Siderophore-producing endophytic bacteria in the root nodules of Lespedeza daurica: identification and examination of growth-promoting effect and stress resistance

摘要:

【背景】根瘤是植物-微生物共生的特殊结构，蕴含丰富的微生物资源。产铁载体根瘤内生细菌对促进结瘤固氮具有重要作用，但目前关于其是否对植物具有促生作用尚不明确。【目的】筛选鉴定达乌里胡枝子(Lespedeza daurica)根瘤产铁载体内生细菌，明确其促生作用，为丰富植物促生菌资源提供新的途径。【方法】利用铬天青(chrome azurol S, CAS)培养基初筛产铁载体根瘤内生细菌，对目的菌株进行鉴定；通过液体发酵试验确定其产铁载体能力；利用促生功能培养基测定其他促生特性；利用不同盐浓度(0.01%−9.00%)、不同pH值(3.0−12.0)的酵母甘露醇(yeast mannitol, YMA)液体培养基和不同培养温度(4−60 ℃)确定其抗逆特性；通过盆栽试验初步验证其对植物的促生作用。【结果】48株根瘤内生细菌可产铁载体，其中9株为高产铁载体细菌，分别为TG4、TG9、TG13、TG15、TG28、TG32、TG38、TG76和TG78，铁载体相对表达量为26%−93%；经鉴定，除了TG28为巨大普里斯特氏菌(Priestia megaterium)，其余8株菌均为耐寒短杆菌(Brevibacterium frigoritolerans)；TG15、TG28、TG76和TG78具有产生长素(indole-3-acetic acid, IAA) (7.62−64.90 μg/mL)、1-氨基环丙烷-1-羧酸(1-aminocyclopropane-1-carboxylic acid, ACC)脱氨酶和固氮能力；而且在盐浓度0.01%−5.00%、pH 4.0−12.0和温度4−37 ℃均可生长；TG15和TG28显著提高了达乌里胡枝子的茎粗，且TG28显著提高了株高。【结论】达乌里胡枝子根瘤中定殖有促生和抗逆效应较强的产铁载体细菌，但对植株的促生作用仅表现在形态发育方面，预测其分泌的铁载体等促生物质可能对共生的结瘤固氮细菌产生积极作用。

Abstract:

[Background] As a special structure of plant-microorganism symbiosis, the root nodules of legumes contain abundant microbial resources. The siderophore-producing endophytic bacteria in the root nodules can promote nodulation and nitrogen fixation. However, it remains unclear whether these bacteria have a direct growth-promoting effect on plants. [Objective] To screen out, identify, and examine the growth-promoting effect of the siderophore-producing endophytic bacteria in the root nodules of Lespedeza daurica, so as to provide a new way for enriching plant growth-promoting bacterial resources. [Methods] The CAS medium was used for the preliminary screening of efficient siderophore-producing endophytic bacteria in the root nodules. The liquid fermentation was carried out to examine the siderophore production. The growth-promoting functional media were used to measure the growth-promoting effects of the strains. The stress resistance of the strains was determined with the YMA liquid medium with different salt concentrations (0.01%–9.00%), pH 3.0–12.0, and culture temperatures (4–60 ℃). The pot experiment was carried out to preliminarily verify the direct growth-promoting effect. [Results] Forty-eight strains possessed siderophore-producing ability, including 9 efficient strains, which were TG4, TG9, TG13, TG15, TG28, TG32, TG38, TG76, and TG78, respectively. The siderophore production of the strains varied within 26%–93%. Except that TG28 was identified as Priestia megaterium, the other 8 strains were identified as Brevibacterium frigoritolerans. TG15, TG28, TG76, and TG78 could secrete indole-3-acetic acid (7.62–64.90 μg/mL) and ACC deaminase, fix nitrogen, and grow at 0.01%–5.00% NaCl, pH 4.0–12.0, and 4–37 ℃. TG15 and TG28 significantly increased the stem diameter, and TG28 significantly increased the plant height of L. daurica.[Conclusion] There were siderophore-producing bacteria with growth-promoting effects and stress resistance in the root nodules of L. davurica. However, the growth-promoting effect on plants was only reflected on the morphological development. The results indicate that the growth-promoting substances such as siderophores may have a positive effect on the nitrogen-fixing bacteria.