抗青枯病型根际促生菌(PGPR)菌群构建及其生物防控机制

Construction of bacterial wilt-resistant and plant growth-promoting rhizobacteria (PGPR) and the mechanism of biocontrol

摘要:

【目的】根际促生菌（plant growth promoting rhizobacteria，PGPR）防控青枯病效果不稳定是目前有益微生物生防应用的瓶颈问题，构建稳定高效拮抗青枯菌的PGPR菌群是生物防控的关键。【方法】以前期筛选到的8株PGPR菌株（112、114、Ba-S、TLZZ、LX4、LX7、Ps-S和VC110）和青枯菌（Ralstonia solanacearum，Rs）为研究对象，在前期获得烟草根系分泌物组成的基础上，采用限菌微系统和生态微孔板结合绿色荧光蛋白标记、定量聚合酶链式反应（quantitative polymerase chain reaction，qPCR）、断棒模型设计等方法，探究PGPR菌群对青枯菌入侵烟草根际的抵御机制，并在田间进行抗病、促生效果验证。【结果】LX4、Ba-S、LX7可以充分利用烟草根系分泌物中的氨基酸、糖类碳源抑制青枯菌生长，LX7和112在所有酸类碳源下均对青枯菌有抑制作用，最高抑菌率分别为40.12%（LX7+乳酸）和35.15%（112+柠檬酸）。Ps-S、112和VC110的基础生态位宽度（basal niche breadth，Bsw）分别比Rs （Bsw=2.56）高41.9%、41.0%和38.1%，Ba-S的基础生态位重叠指数（niche overlap index，NOI）显著比其他PGPR菌株高4.88%–61.76%，与青枯菌存在明显的营养利用性竞争。4株PGPR组成的菌群组合中病情指数平均为27.01%，根际青枯菌数量平均为1.1×10⁴ CFU/g，显著低于单株和2株PGPR菌群组合，8株PGPR组成菌群处理烟株未发病，根际青枯菌数量平均为3.5×10² CFU/g。由LX4、Ba-S、LX7和VC110构成的菌群组合（菌群组合32）对不同碳源的利用效率显著高于青枯菌，尤其体现在醇类和糖类碳源利用方面，分别是青枯菌的1.62倍和1.41倍。菌群组合32田间防效显著高于其他处理，分别比菌群39（Ba-S、VC110、114和TLZZ）、40（LX4、LX7、VC110和112）和43（Ba-S、VC110、114和112）高27.18%、60.05%和54.80%，菌群32处理的产量和产值相对较好，分别较不施PGPR的对照增加67.50%和73.53%。【结论】可以利用不同PGPR菌株对青枯菌营养竞争或者拮抗竞争的特点构建PGPR菌群，多样性的PGPR菌群可以显著提高抵抗病原菌入侵的能力。

Abstract:

[Objective] The effect of plant growth-promoting rhizobacteria (PGPR) on bacterial wilt is unstable, which poses a challenge to the application of beneficial microorganisms. Thus, developing a stable and efficient PGPR flora against bacterial wilt is the key to the biocontrol. [Methods] The 8 anti-Ralstonia solanacearum (Rs) strains of PGPR (112, 114, Ba-S, TLZZ, LX4, LX7, Ps-S, and VC110) that had been screened out by our research group and Rs were studied. Based on the composition of tobacco root exudates, the microbial restriction microsystem, microplate, green fluorescent protein labeling, quantitative PCR, rod model design, and other methods were employed to explore the mechanism of PGPR against Rs in tobacco rhizosphere. In addition, experiment on the disease-resistant and growth-promoting effects was carried out in the field. [Results] LX4, Ba-S, and LX7 can make full use of amino acids and carbohydrates in tobacco root exudates as carbon sources to inhibit the growth of Rs. LX7 and 112 suppressed Rs with all acid carbon sources, and the highest antibacterial rates were 40.12% (LX7+lactic acid) and 35.15% (112+citric acid), respectively. The basal niche breadth (Bsw) of Ps-S, 112, and VC110 was 41.9%, 41.0%, and 38.1% higher than that of Rs (Bsw=2.56), respectively. The basal niche overlap index (NOI) of Ba-S was 4.88%-61.76% significantly higher than that of any other PGPR strains, and this strain obviously competed with Rs for nutrients. The average disease index was 27.01% and the average count of Rs in tobacco rhizosphere was 1.1×10⁴ CFU/g in the treatments with the combinations of four PGPR strains, which were significantly lower than those in the treatments with one PGPR strain and two strains. The tobacco plants treated with 8 strains of PGPR were free from the wilt, with the average Rs count of 3.5×10² CFU/g. The utilization efficiency of different carbon sources by combination 32 (consisting of LX4, Ba-S, LX7 and VC110) was significantly higher than that by Rs and particularly the utilization efficiency of alcohols and carbohydrate by the combination was 1.62 and 1.41 folds that by Rs. The field effect of combination 32 against Rs was significantly stronger than that of other treatments. Especially, the Rs-controlling rate was 27.18%, 60.05%, and 54.80% higher than that of combinations 39 (Ba-S, VC110, 114, and TLZZ), 40 (LX4, LX7, VC110, and 112), and 43 (Ba-S, VC110, 114, and 112), respectively. The yield and output value in the treatment with combination 32 were the highest among all treatments, which were 67.50% and 73.53% higher than those of the control treatment, separately. [Conclusion] Via the nutrient competition or antagonistic characteristics, different PGPR strains can be fully utilized to develop PGPR flora against R. solanacearum. The diverse PGPR floras have significantly stronger ability to resist the invasion of pathogenic bacteria.