Understanding the microbiome shift and bacterial VOC-based control-mechanism in the Meloidogyne disease complex in Ugandan tomatoes

Background: Root-knot nematodes (RKNs, Meloidogyne spp.) are plant pests that cause severe crop losses worldwide, especially in tropical climates. When establishing a feeding site in the root endosphere, they change the physiological state of the root cells, which also increases the severity of secondary fungal and bacterial infections. Bacterial microbiome changes due to RKN infections in the root endosphere of Ugandan tomato (Solanum lycopersicum) were investigated using 16S rRNA gene amplicon analysis. Furthermore, we screened for autochthonic bacteria that may be suitable for biocontrol RKNs by the production of nematicidal volatile organic compounds (nVOCs). RKNs were identified using morphological and molecular techniques as M. javanica and M. incognita. Bacterial strains were isolated from the rhizosphere, as well as from the healthy and diseased endosphere of 20 different tomato plants.Results: Bacterial alpha diversity of galls differed both qualitatively and quantitatively from healthy roots, while composition of the bacteriome of the rhizosphere and healthy endosphere only differed in abundances. Pasteuriaceae and Rhizobiaceae were more abundant in diseased roots, whereas Enterobacteriaceae and Burkholderiaceae were less abundant. A set of 260 bacterial isolates were tested for nVOC production. By using SPME GC-MS analysis, sulfuric compounds (dimethyl disulfide, methanethiol), alkenes (mainly 1-undecene) and one pyrazine (3-methoxy-2,5-dimethylpyrazine) were found amongst others. Present alkenes and pyrazines were further tested as single compounds for their nematicidal activity. Accordingly, six nVOC-producing strains were identified as members of the genera Pseudomonas, Comamonas and Variovorax. Their relative abundance was highest in rhizosphere (Pseudomonas spp. and Comamonas spp.) or diseased root endosphere (Variovorax spp.). Additionally, all bacterial strains were tested in dual cultures for antagonistic activity towards Botrytis cinerea, Fusarium verticilloides, F. oxysporum, Sclerotium rolfsii and Verticillium dahliae. Five fungal antagonists were identified as Bacillus amyloliquefaciens, B. methylotrophicus and B. velezensis. Bacillus spp. mainly found in healthy root parts. Bacterial antagonists of V. dahliae were only found in rhizosphere isolates; they were less efficient against the other fungal pathogens and vice versa.

背景：根结线虫（Root-knot nematodes, RKNs，*Meloidogyne* spp.）是一类在全球范围内造成严重作物减产的植物害虫，在热带气候区域危害尤为突出。当它们在根内空间定植取食位点时，会改变根部细胞的生理状态，同时还会加重次生真菌与细菌侵染的病害严重程度。本研究采用16S rRNA基因扩增子测序技术，分析了乌干达番茄（*Solanum lycopersicum*）根内空间因根结线虫侵染发生的细菌菌群变化。此外，本研究还通过筛选可产生杀线虫挥发性有机化合物（nematicidal volatile organic compounds, nVOCs）的土著细菌，探索其用于根结线虫生物防治的潜力。研究人员通过形态学与分子生物学技术，将靶标根结线虫鉴定为爪哇根结线虫（*M. javanica*）和南方根结线虫（*M. incognita*）。细菌菌株分离自20株不同番茄植株的根际、健康根内空间以及发病根内空间。结果：发病根结的细菌α多样性在定性与定量层面均与健康根系存在显著差异，而根际与健康根内空间的细菌菌群组成仅在物种丰度上存在差异。发病根系中，巴斯德氏菌科（Pasteuriaceae）和根瘤菌科（Rhizobiaceae）的相对丰度更高，而肠杆菌科（Enterobacteriaceae）和伯克霍尔德氏菌科（Burkholderiaceae）的相对丰度更低。本研究共获得260株细菌分离株，对其产nVOC的能力进行了检测。通过固相微萃取-气相色谱-质谱联用（SPME GC-MS）分析，共检测到包括含硫化合物（二甲基二硫、甲硫醇）、烯烃类（主要为1-十一碳烯）以及一种吡嗪类物质（3-甲氧基-2,5-二甲基吡嗪）在内的多种挥发性成分。研究人员进一步选取其中的烯烃与吡嗪类化合物，单独测试其杀线虫活性。最终，6株产nVOC的细菌菌株被鉴定为假单胞菌属（Pseudomonas）、丛毛单胞菌属（Comamonas）和贪铜菌属（Variovorax）的成员。这些菌株的相对丰度在根际（假单胞菌属与丛毛单胞菌属）或发病根内空间（贪铜菌属）中最高。此外，本研究还通过双培养实验，测试了所有细菌菌株对灰葡萄孢（*Botrytis cinerea*）、串珠镰孢（*Fusarium verticilloides*）、尖孢镰孢（*F. oxysporum*）、齐整小核菌（*Sclerotium rolfsii*）以及大丽轮枝菌（*Verticillium dahliae*）的拮抗活性。5株具有真菌拮抗活性的细菌菌株被鉴定为解淀粉芽孢杆菌（*Bacillus amyloliquefaciens*）、甲基营养芽孢杆菌（*B. methylotrophicus*）和贝莱斯芽孢杆菌（*B. velezensis*）。芽孢杆菌属菌株主要分离自健康根系组织。针对大丽轮枝菌的细菌拮抗菌株仅分离自根际，它们对其他真菌病原菌的拮抗效果相对较弱，反之亦然。