多粘类芽胞杆菌KM2501-1杀南方根结线虫活性产物研究

Inhibition of Meloidogyne incognita by active compound from Panebacillus polymyxa KM2501-1

摘要:

[目的] 南方根结线虫（Meloidogyne incognita）是一种危害严重的土传性植物病原线虫，给农业生产造成了巨大的经济损失，前期研究发现多粘类芽胞杆菌（Panebacillus polymyxa）KM2501-1具有很好的温室防治南方根结线虫效果，且可产生多种挥发性杀线虫活性物质，但对其非挥发性产物是否有杀线虫活性没有研究。本研究拟进一步分离鉴定其产生的杀线虫活性代谢产物，发掘新的杀线虫药物。[方法] 对菌株KM2501-1进行液体发酵并离心收集发酵上清液，通过硅胶柱层析、高效液相色谱分离等方法得到高纯度的杀线虫活性物质，并通过液相色谱质谱联用分析、核磁共振等技术鉴定杀线虫活性物质的结构。[结果] 生物活性检测显示，多粘类芽胞杆菌KM2501-1发酵上清液具有较强的南方根结线虫触杀活性，并能有效抑制南方根结线虫卵孵化，体外杀线虫效率高达87.66%，抑制卵孵化效率达92.26%。结构鉴定结果显示多粘类芽胞杆菌产生的杀线虫活性物质为环二肽类物质cyclo（Pro-Phe），800 mg/L的cyclo（Pro-Phe）杀线虫效率达84.75%。进一步的显微观测结果表明，与对照组相比，活性物质cyclo（Pro-Phe）处理后的根结线虫肠道组织紊乱、结构发生破坏。[结论] 多粘类芽胞杆菌KM2501-1产生的cyclo（Pro-Phe）是一个具有杀线虫新功能的活性物质，其可能通过破坏线虫肠道杀死线虫。

Abstract:

[Objective] Meloidogyne incongnita is a soil transmission plant parasitic nematode that causes serious losses to agricultural production worldwide. Panebacillus polymyxa KM2501-1 inhibits Meloidogyne incongnita in greenhouse experiment, and could produce a variety of nematocidal volatile organic compounds. But whether its non-volatile products have nematocidal activity is still unknown. This study focused on identification of non-volatile nematocidal metabolites from Panebacillus polymyxa KM2501-1.[Methods] Strain KM2501-1 was fermented to collect supernatant. The high purity nematocidal substance was isolated and purified by silica gel column chromatography and high-performance liquid chromatography. The structure of the nematocidal substance was identified by liquid chromatography-mass spectrometry and nuclear magnetic resonance.[Results] The fermentation filtrate of Panebacillus polymyxa KM2501-1 inhibited Meloidogyne incongnita and the egg hatching of Meloidogyne incongnita. The nematocidal efficiency was 87.66% in vitro and the egg hatching inhibitory efficiency was 92.26%. The results of structural identification showed that the nematocidal active substance produced by Panebacillus polymyxa KM2501-1 was cyclo (Pro-Phe). The nematocidal efficiency of 800 mg/L cyclo (Pro-Phe) was up to 84.75% after 96 hours exposure. Microscopic observations show that the intestinal tissue of the Meloidogyne incongnita treated with the cyclo (Pro-Phe) was disordered compared with the control group.[Conclusion] The cyclo (Pro-Phe) produced by Panebacillus polymyxa KM2501-1 is a new nematocidal substance that may inhibit nematodes by destroying the nematode intestinal tract.