化感水稻介导特异根际微生物抑草作用的过程与机制研究

The interactions between plant allelochemicals and rhizospheric microbes are highlighted recently and become a hot issue. To further understand the rhizosphere biological process and its underlying mechanism between the two factors is favorable for providing theory evidence to control weeds in practical uses. The protocol is designed based on our previous studies. Allelopathic rice cultivars PI 312777 and its counterpart Lemont as well as a specific myxobacteria were used as experimental materials to study the ecological roles of myxobacteria in the rhizosphere of allelopathic rice. The RNAi and overexpression of phenolics synthesis related-gene PAL-21 and momilactone synthesis related-gene sDPT-2 were performed in both allelopathic and non-allelopathic rice cultivars. The dynamics of allelopathic compounds of these transgenic rice plants would be determined along with their effects on the growth of myxobacteria, in order to confirm the positive effect of allelochemicals released by allelopathic rice on the growth of myxobacteria. Furthermore, the myxobacteria labeled with green fluorescence protein (GFP) would be incubated with key amino acid, phenolic acid, terpenoid and flavonoid in the sterile soil and then the amounts of myxobacteria would be measured using qRT-PCR in situ，and the content of soraphen A in the test strain will also detected by HPLC. Based on the researches mentioned above, the weed controlling effect of myxobacteria and its fermented liquid were assessed by bioassay method and the components of the fermented liquid were determined using GC-MS and HPLC. It is expected to further uncover the rhizospheric biological process and its underlying mechanism of allelopathic rice accession in the suppression of the target weeds.

作物化感物质与根际土壤微生物的互作关系是当前国际研究的热点，揭示这一生态过程与作用机制可以为建立生态安全防控杂草技术提供重要依据。本研究以化感水稻PI312777和非化感水稻Lemont为材料，并利用从化感水稻根际土壤分离与鉴定得到的一种特异粘细菌，研究该菌在水稻化感抑草作用的根际生物学过程与机制。试验拟分别RNA干扰和过量表达水稻酚酸类合成关键基因PAL-21以及萜类momilactone合成关键基因sDPT-2；在试验室条件下，动态检测转化后水稻较其野生型的化感物质变化对该菌生长的影响，确认是酚酸类还是萜类物质对该菌呈促进作用；进一步转绿色荧光蛋白基因入该菌，并接种到分别添加不同浓度的关键氨基酸、酚酸、萜类和黄酮类的无菌土壤中，原位和定量检测该菌数量和soraphen A含量，验证化感物质的作用；并分析该菌发酵液组分，评价发酵液及菌株的抑草效果，阐明作用机理。

作物化感物质与根际土壤微生物的互作关系是当前国际研究的热点，揭示这一生态过程与作用机制可以为建立生态安全防控杂草技术提供重要依据。本项目通过研究发现，苗期水稻随着叶龄的增加，其根际土壤中的粘细菌属微生物的数量增加，但其化感潜力强的水稻根际土壤中的数量明显高于化感潜力弱的水稻根际土壤中的数量。对水稻苗期酚酸类物质的检测发现，化感水稻根际土壤中的酚酸类物质变化多呈现出“高-低-高”趋势，并以2叶期，3叶期和5叶期酚酸类物质总量较高，该变化趋势与其根际土壤粘细菌种群数量的动态变化情况相一致，即粘细菌数量与酚酸含量呈正相关。外源添加酚酸类物质对土壤粘细菌属微生物有明显的促进作用。抑制化感水稻的PAL基因表达后，其分泌进入根际土壤酚酸类物质总含量明显降低，根际土壤中的粘细菌数量也减少。进一步从强化感水稻根际土壤中分离得到4株粘细菌，这4株粘细菌对稗草的发芽率和茎长生长均显示较强的抑制能力，且阿魏酸诱导能够显著提高粘细菌的抑草能力。GC-MS鉴定结果显示，阿魏酸诱导后的粘细菌中，其次级代谢产物的数量和浓度均较对照组存在明显差异，且在添加阿魏酸后的粘细菌次级代谢产物中发现含有多种具有生物作用的物质，表明酚酸类化感物质可刺激粘细菌的生长以及其有效代谢产物的合成。此外，阿魏酸诱导还使得粘细菌中与细胞表面的信号识别因子及受体、营养生长、运动、其他信号响应与转导、代谢过程、次级代谢物质产生、群体捕食、转运和胞内运输相关的蛋白均增强表达。对部分差异蛋白进行转录水平验证，结果显示不同浓度阿魏酸诱导下frzS、MXAN_2023、MXAN_3668和MXAN_3339基因在mRNA水平和相应蛋白水平的变化情况基本一致。进一步对Frz基因家族另外5个成员基因frzA、frzB、frzCD、frzE、frzG的表达变化模式进行检测，结果也显示0.10mM阿魏酸诱导下黄色粘球菌中5个Frz成员基因均明显上调表达。上述研究结果表明化感水稻通过根系分泌的酚酸类化感物质与其根际土壤中的粘细菌相互作用，促进了粘细菌的增殖及其对酚酸类化合物的转化与利用，两者最终协同作用抑制邻近稗草的生长。