烟草青枯病劳尔氏菌与拮抗菌对根系分泌物的竞争作用

Competitive use of plant root exudates by Ralstonia solanacearum causing tobacco bacterial wilt and its antagonistic bacterium LX4

摘要:

[目的]研究青枯病病原菌与拮抗菌的营养特性及其对烟草根系分泌物的响应差异，对提高拮抗菌定殖能力、有效生物防控烟草青枯病具有非常重要的意义。[方法]本研究通过筛选与鉴定贵州烟区青枯病病原菌株及拮抗菌株，通过Biolog表型芯片技术分别检测病原菌与拮抗菌的特征性碳、氮源，利用气质联用（GC-MS）检测烟草主栽品种K326根系分泌物的主要物质，在此基础上进行病原菌与拮抗菌对其利用能力、利用强度以及共培养的研究。[结果]经鉴定，分离、筛选到的病原菌株和拮抗菌株分别为青枯劳尔氏菌（Ralstonia solanacearum）和枯草芽孢杆菌（Bacillus subtilis）；在含量为0.01μg/mL以上的根系分泌物中，12种物质的含量从高到低排序为：果胶 > 葡萄糖 > 木糖 > 阿拉伯糖 > 半乳糖 > 核糖 > 蔗糖 > 苯甲酸 > 果糖=D-甘露醇 > 棕榈酸 > 富马酸，果胶含量最高且明显高于其他物质；拮抗菌（LX4）对碳源利用能力高于病原菌（Rs）的碳源有阿拉伯糖、木糖和核糖，分别是病原菌利用能力的1.22、1.95和2.17倍；前12 h拮抗菌利用果糖强度高于病原菌，不同碳源共培养24h后LX4对gfp-Rs（绿色荧光蛋白标记后的青枯病病原菌）抑制率为18.34%（阿拉伯糖）、53.23%（木糖）、63.53%（核糖）和52.09%（果糖）。[结论]拮抗菌对烟草根系分泌物的利用不及病原菌，但在特定碳源条件下拮抗菌能够利用根系分泌物中的某些碳源产生某种拮抗物质抑制病原菌，拮抗菌与病原菌之间同时存在利用性竞争和干扰性竞争关系，研究结果为进一步研究烟草青枯病的生物防控提供了新的理论依据。

Abstract:

[Objective] It is important to study the nutrition relationship between tobacco bacterial wilt pathogen (Ralstonia solanacearum, Rs) and its antagonistic bacterial isolate LX4. The difference in their use of secreted chemicals from tobacco root plays a crucial role for increasing the colonization of antagonist and efficiently bio-controlling tobacco bacterial wilt. [Methods] Pathogen Rs and the antagonistic strain LX4 from tobacco field soil were isolated and identified. We examined the use of characteristic carbon and nitrogen sources in tobacco root exudates by Rs and LX4. Main chemicals in root exudates were identified with a gas chromatography-mass spectrometer (GC-MS). We compared and analyzed the capacity and intensity of utilizing the major nutrients by Rs and strain LX4 by co-culturing the two microorganisms. [Results] The isolated pathogen was identified as R. solanacearum and isolate LX4 was identified as Bacillus subtilis via phylogenetic tree analysis based on their 16S rDNA sequences. In the exudates of tobacco roots, the substances with contents of > 0.01 μg/mL were ordered descendingly as pectin > glucose > xylose > arabinose > galactose > ribose > sucrose > benzoic acid > fructose=D-mannitol > cetylic acid > fumaric acid. The content of pectin was the highest and significantly higher than all the other substances. Strain LX4 could use arabinose, xylose and ribose significantly better than R. solanacearum. The usage capacity of the former was 1.22, 1.95 and 2.17 times of those of the latter, respectively. Besides, the fructose usage by LX4 was higher than that by R. solanacearum in the first 12 h. After 24 h of co-culturing them on different substrates of carbon sources, the suppression rates of strain LX4 to R. solanacearum marked by green fluorescent protein were 18.34% (arabinose), 53.23% (xylose), 63.53% (ribose) and 52.09% (fructose). [Conclusion] In conclusion, the capacity of root exudates from LX4 was lower than that from Rs, which suggested the antagonist had disadvantage in its nutrition competition with the pathogen. However, the antagonist took the advantage of certain carbon substances secreted from tobacco roots to produce antagonistic substances, which were suppressive or toxic to the pathogen. There were both exploitation and interference competitions between the antagonist and pathogen. These results provide some theoretical evidences for the bio-control of tobacco bacterial wilt caused by R. solanacearum in the near future.