枯草芽孢杆菌对水稻根际微界面邻苯二甲酸酯降解及其累积的影响机制

Phthalate acid esters (PAEs) are widely used as plasticizers, but have endocrine disrupting effects. Agricultural soils polluted by PAEs and uptake and accumulation of PAEs by crops result in high human health risks. We have selected and obtained the low-PAE accumulating cultivar of rice (Oryza sativa) and efficient PAE-degrading endophytic bacterium Bacillus subtilis N-1. Nevertheless, whether the interaction of endophytic bacterium and rice plant can lower the residual concentrations of PAEs in soil and PAE accumulation by rice and its mechanism remain unknown. In this project, rhizobox experiments of low-PAE accumulating rice cultivar inoculated with strain N-1 will be conducted to investigate PAE migration and degradation in soil-microorganism-crop system. Bacillus subtilis strain N-1 will be labeled with green fluorescent protein and its colonization in rice was observed by two-photon confocal microscopy. The effects of strain N-1 on PAE accumulation and metabolism in vivo by rice plants will be investigated. Moreover, with the help of high-throughput sequencing and Geo-chip analysis, the effects of interaction between Bacillus subtilis N-1 and rice plant on microbial community structure in rhizosphere and endophytic community structure in rice plant will be analyzed to identify the predominant microorganisms and the key microbial consortium for PAE degradation/metabolism in vivo within rice-microbial system. The key genes of PAE degradation and promotion of plant growth as well as resistance will be identified to explore the correlation between PAE degradation rates and the relative abundance of PAE-degrading consortium and their genes. The results of the study will elucidate the mechanism of microorganism-crop interaction on PAE degradation and rice accumulation, which will provide scientific basis for the safety of agricultural products.

邻苯二甲酸酯（PAEs）广泛用作塑料增塑剂，具有环境内分泌干扰效应。农田土壤PAEs污染和农作物吸收累积PAEs导致人体高暴露风险。课题组前期筛选获得高效降解PAEs植物内生菌枯草芽孢杆菌N-1和低累积PAEs基因型水稻品种丰优丝苗，但植物内生菌与水稻互作能否降低土壤PAEs残留和水稻累积PAEs及其影响机制尚未清楚。本项目拟采用根箱培养水稻并接种内生菌N-1，采用绿色荧光蛋白标记和双光子共聚焦显微镜观察N-1菌在水稻体内定殖情况；研究PAEs的迁移和降解；探讨N-1菌对水稻累积PAEs及体内代谢的影响；采用高通量测序和基因芯片技术分析菌N-1与水稻互作对根际和水稻体内微生物群落结构和功能的影响，明确优势菌群和PAEs降解/代谢关键菌群，查明PAEs降解关键基因和促生抗性基因。从而阐明水稻-微生物互作对PAEs降解和水稻累积的作用机制，为保障农产品质量安全提供科学依据。

农田土壤邻苯二甲酸酯（PAEs）污染和农作物吸收累积PAEs导致人体高暴露风险，微生物降解可以有效去除土壤和作物中有机污染物的残留。然而，植物内生菌降解土壤-作物系统PAE以及内生降解菌与土著微生物互作机制尚未清楚。本项目采用根箱培养接种高效降解菌于高/低累积PAEs基因型水稻品种，研究PAEs在土壤微生物-作物系统中的迁移和降解；采用高通量测序技术分析菌N-1与水稻互作对根际和水稻体内微生物群落结构和功能的影响。成功获得了绿色荧光蛋白标记的枯草芽孢杆菌N-1-gfp，并构建了功能菌群JQ。采用激光共聚焦扫描显微镜和qPCR多手段证实菌株N-1-gfp可以的水稻根际和植株中很好地定殖（菌落数为103~106 cfu/g组织）。标记菌N-1-gfp在培养液中5天高效降解99.7%的DBP（200 mg/L），且接种N-1-gfp显著促进土壤和植物中DBP的去除（75.6%~93.3%和67.8%~73.1%）。接种菌群JQ能够显著降低不同根际区PAEs的残留（62%~77% DBP、51%~56% DEHP）、PAEs消减规律为近根际区>根生长区>非根际区、显著提高根际微界面PAEs生物有效性，还能维持污染胁迫下水稻根尖和叶肉细胞形态正常、显著提高根系分泌物含量（1.86~4.24倍）、光合作用速率（5.8%~43.8%）、蒸腾作用速率（17.0%~24.6%）和水稻生物量（23.9%~84.5%），接种菌减少了PAEs在水稻根系的吸收（52.2%~62.2%）、阻挡了PAEs向水稻地上部的迁移（57.6%~73.5%）、显著降低了水稻籽粒PAEs残留量（55.2%~76.4%）。此外，接种N-1-gfp能够调节水稻根际和植株中的微生物群落，其中芽孢杆菌属的相对丰度显著增加了6.5%~34.6%。菌株N-1-gfp定殖还有助于水稻招募特定功能性细菌（促生菌、降解菌），功能菌相对丰度提高了0.63~26.6倍，且群落的KEGG功能活性显著提高了17.2%~48.1%（包括营养物质代谢、污染物降解）。本项目揭示了水稻-微生物互作对PAEs降解和水稻累积的作用机制，为保障农产品质量安全提供科学依据。