噬菌体-有益菌协同消减土壤青枯菌生物障碍的效应初探

The Synergistic Effect of a Phage-Probiotic Combination on Suppressing Bacterial Wilt Disease

摘要:

由土传青枯菌入侵引起的土壤青枯菌生物障碍造成番茄、马铃薯、烟草等粮经作物大面积减产甚至绝收，严重威胁了农产品的安全供给。根际微生物在消减土壤生物障碍的过程中发挥重要作用，其中有益细菌能够通过拮抗或营养竞争抑制病原菌，噬菌体能够通过专性侵染精准靶向病原菌。但在实际应用中，有益细菌或噬菌体单一施用通常消减效果不稳定，且菌种高密度发酵的成本较高。为建立高效稳定的土壤生物障碍消减技术，构建了拮抗竞争型和营养竞争型两类有益细菌与噬菌体的组合，探究有益菌-噬菌体协同消减土壤青枯菌生物障碍的效果，并进一步探究二者协同抑制青枯菌的潜在机制。结果发现，大部分噬菌体与有益菌组合具有协同效应，能够显著提升对病原青枯菌生长和土壤青枯菌生物障碍的抑制消减效果。温室试验结果表明，与对照相比，协同效应平均降低病情指数58.18%，其中营养竞争型有益菌WR21与噬菌体组合后病情指数显著降低67.28%。进一步研究发现在有益菌浓度较低时噬菌体-有益菌协同仍表现出良好的消减效果，其中T-5在104 CFU?g-1 基质浓度下，与噬菌体组合后较单独施用有益菌的病情指数降低21.56%，根际病原菌数量降低19.21%。研究发现噬菌体-营养竞争型有益菌组合时能够显著降低病原菌的碳源利用能力，同时发现病原菌在噬菌体-拮抗竞争型有益菌双重胁迫下，对T-5产生的拮抗物质的敏感性提高64.10%。本研究明确了噬菌体-有益菌组合消减土壤青枯菌生物障碍的潜力，并初步揭示了噬菌体-有益菌协同消减土壤青枯菌生物障碍的潜在机制，为建立高效消减土壤生物障碍的技术体系提供了理论和技术支撑。

Abstract:

【Objective】Bacterial wilt disease, caused by soil-borne Ralstonia solanacearum, has led to significant yield reductions or even crop failures in tomatoes, potatoes, and tobacco, severely limiting the safe supply of agricultural products. Rhizosphere microorganisms play a crucial role in mitigating soil biotic impediments. Probiotics can inhibit pathogens through antagonism or nutrient competition, while phages can target pathogens through specific infections. However, the single use of either probiotics or phages often results in unstable effects, and the high-density fermentation cost of these strains is considerable. 【Method】To establish an efficient and stable technology for reducing soil biotic impediments, this study constructed combinations of antagonistic-competitive and nutrient-competitive beneficial bacteria with phages. It explored their synergistic effects in reducing soil wilt disease through in vitro microplate and greenhouse pot experiments. The potential mechanisms of synergy between probiotics and phages were also investigated. 【Result】 The results showed that most combinations of phages and probiotics exhibited synergistic effects, significantly enhancing the inhibition of pathogenic R. solanacearum growth and the reduction of bacterial wilt disease. Notably, the combination of the antagonistic-competitive beneficial bacterium T-5 with phages resulted in an 89.19% reduction in pathogenic bacterial growth compared to the control. Greenhouse experiments indicated that the synergistic effect reduced the disease index by an average of 58.18%, with the combination of the nutrient-competitive beneficial bacterium WR21 and phages significantly reducing the disease index by 67.28%. Further studies revealed that even at lower concentrations of beneficial bacteria, the phage-bacteria synergy remained effective. At a concentration of 104 CFU?g-1 substrate, the combination of T-5 with phages reduced the disease index by 21.56% and the number of rhizosphere pathogenic bacteria by 19.21% compared to the application of beneficial bacteria alone, demonstrating a strong synergistic effect. Additionally, the study explored the impact of phage-beneficial bacteria combinations on the characteristics of pathogenic R. solanacearum. The results showed that the phage-nutrient-competitive beneficial bacteria combination significantly reduced the pathogen""s carbon source utilization ability, with the WR21 and phage combination reducing the number of carbon sources utilized by the pathogen by 87.9%. Furthermore, under the dual stress of phages and antagonistic-competitive beneficial bacteria, the sensitivity of the pathogen to the antagonistic substances produced by T-5 increased by 64.10%. 【Conclusion】This study highlights the potential of phage-beneficial bacteria combinations in mitigating bacterial wilt disease and preliminarily elucidates the potential mechanisms behind their synergistic effects. These findings provide theoretical and technical support for the development of efficient soil biological obstacle reduction techniques.