蔬菜叶际有机磷农药降解菌的多样性及其影响因素

Organophosphate pesticides are widely used for plant protection in the world, however the pesticide residues in vegetable are harmful for food safty. The results of our previous study showed that using pesticides could change the microbial community structure and increase some bacterial populations in phyllosphere significantly. We supposed that the increased bacteria could degrade the pesticides and use the residues as nutriment for growth. The aims of this application are exploring the bacterial diversity of organophosphate pesticides degraders, understanding what factors could effect the metabolic activity of the pesticides degraders, and developing new methods for rapid remove pesticide residues from vegetables. High throughput sequencing with bioinformatics analysis will be used for investition of the bacterial communities increased by organophosphate pesticides in phyllosphere. DNA-based isotope probing with clone library will be used for detection of the bacterial diversity of pesticides degraders. Pesticide residues will be tested by GC/HPLC-MS in this process. The effect of biofertilizers and surfactants on the bacterial divesity and the metabolic activity of the pesticides degraders will be investigated.

有机磷农药是目前用量最大的一类农药，不当的使用导致我国蔬菜农药残留问题突出，易引发食品安全问题。我们前期研究结果表明，一些农药喷施到植物叶面后，会引起叶际微生物群落结构发生显著变化，有些细菌种群数量急剧增加，我们推测这些细菌可能降解农药并以农药残留为营养物生长。本申请的研究目标是为了认识蔬菜叶际能够降解有机磷农药的细菌多样性，探明这些农药降解菌代谢活性的主要影响因素，为加快蔬菜上农药残留降解提供理论参考，为叶际微生物的生态功能研究提供新思路。研究内容：以高通量测序技术结合生物信息学分析研究施用有机磷农药所引起的叶际种群数量增加的细菌群落特征；采用稳定性同位素核酸探针技术结合克隆文库技术和培养方法，解析能够降解农药的叶际功能菌群；采用定量PCR技术分析降解菌的种群动态，采用色谱-质谱联用技术分析农药残留动态，考察喷施农用菌剂、表面活性剂等调控措施对叶际农药降解菌群落结构和代谢活性的影响。

有机磷农药不当使用会导致蔬菜农药残留超标问题，危害食品安全。蔬菜叶际生存有数量巨大且多样性丰富的微生物，其中有许多微生物能够降解农药残留。本项目采用培养方法，从蔬菜叶际分离培养有机磷农药降解菌，研究了叶际可培养农药降解菌的多样性。筛选了有机磷农药高效降解菌：红细菌（Rhodobacter sp.）EBL0706菌株、枯草芽孢杆菌（Bacillus subtilis）3374菌株、乡间布丘氏菌（Buttiauxella sp.）CPF5菌株等10多株，设计了降解菌的定量PCR引物。通过16S rDNA高通量测序方法解析了有机磷农药（敌敌畏、毒死蜱等）在叶类蔬菜（油菜、白菜等）上喷施后，蔬菜叶际细菌群落结构的变化。采用色谱-质谱联用技术分析了农药残留的降解动态，揭示了有机磷农药在蔬菜叶面的残留规律，证实了喷施农药降解菌剂能够显著加快蔬菜叶面的农药残留降解。研究了不同环境条件（温度、pH、营养物质等）对叶际农药降解菌群及代谢活性的影响。采用高通量测序和定量PCR技术，研究了不同处理方式（喷施农用微生物菌剂、灭活菌剂、水等）对叶际农药降解菌群落结构及代谢活性的影响。采用转录组分析，推测了有机磷农药降解菌EBL0706菌株降解敌敌畏和毒死蜱的代谢途径，初步明确了有机磷农药能够被微生物完全降解或矿化。探明了生物强化加速蔬菜叶面农药残留降解的可行性，为无公害蔬菜的生产提供了科学依据。