ZLSY22菌株抑制紫茎泽兰的活性成分及其作用机理研究

Eupatorium adenophorum is a malignant invasive weed which has caused serious harm to agricultural and forestry production, but it still lacks effective control measures at present. The project applicant had done some preliminary research on Procecidochares utilis (one obligate parasitic natural enemy insect of E. adenophorum), and the symbiotic bacteria, Kluyvera ascorbata strain ZLSY22 had been isolated from the digestive tract of P. utilis. Moreover, strain ZLSY22 had been proved to have high herbicidal activity against E. adenophorum, and it had been initially identified that herbicidal substances were in the fermentation supernatant of strain ZLSY22. In view of this, the project intends to isolate the metabolites of strain ZLSY22 by chromatography, detect active substances through bioactivity tracking, identify active substances by mass spectrometry and nuclear magnetic resonance spectroscopy. The characteristics of the absorption, transport and distribution of the active component will be studied in E. adenophorum by liquid chromatography, which is helpful to preliminarily identify the target organs/tissues. After the target organs/tissues are treated with the active substance, the pathological changes of tissues and cells will be analyzed by microscopic examination and transmission electron microscopy, and the target organs/tissues will be further verified. The differences of genes and proteins in the target organ/tissue between the treatment with active substance and control will be analyzed by the transcriptional group and the proteome for understanding the biochemical and molecular mechanisms of the active substance. The results will find the herbicidal active compounds against E. adenophorum, will preliminarily clarify the action mechanism of herbicidal active compound, and will promote the development of the sustainable control technology against E. adenophorum.

紫茎泽兰是恶性入侵杂草，对农林业生产造成严重危害，但目前尚乏有效的防治措施。本项目申请人前期研究紫茎泽兰的专性寄生天敌泽兰实蝇，从其肠道中分离获得了一种对紫茎泽兰具有高效除草活性的抗坏血酸克吕沃尔菌ZLSY22菌株，初步明确该菌株的除草活性物质存在于其发酵液中。为此，本项目拟采用色谱技术分离ZLSY22菌株的代谢产物，通过生物活性跟踪测定发现杀草活性分子，借助质谱与核磁共振波谱技术鉴定活性物质，进而采用液质联用技术研究活性成分在紫茎泽兰中的吸收、输导和分布特点，初步明确其靶器官/组织；用活性物质处理植物器官组织，通过显微切片检查与透射电镜分析组织细胞病变，进一步明确靶器官/组织；通过转录组与蛋白质组分析靶器官/组织的基因与蛋白质差异，了解活性物质作用的生化和分子机理。研究结果将发现防除紫茎泽兰的活性化合物，并初步明确其作用机理，促进紫茎泽兰可持续防治技术的发展。

紫茎泽兰是恶性入侵杂草，目前尚乏有效的防治措施。本项目在从泽兰实蝇肠道中分离得到一株具有除草活性的抗坏血酸克吕沃尔菌ZLSY22的基础上，采用色谱、质谱、核磁共振等技术对ZLSY22菌株代谢物中的除草活性成分进行分离、纯化及鉴定，通过生物活性跟踪测定发现杀草活性物质，并通过组织切片观察与透射电镜、比色法、转录组等技术分析紫茎泽兰茎干经活性物质处理后的组织细胞、酶活性和基因的变化，从而初步明确了活性物质作用的作用机理，研究结果促进了紫茎泽兰可持续防治技术的发展。主要研究结果如下：.（1）从ZLSY22发酵液的沉淀及上清液中共分离得到41个化合物。利用种子萌发法、幼苗小杯法等方法筛选出具有除草活性的4个化合物，其中吲哚-3-乙酸甲酯（MeIAA）对紫茎泽兰及其幼苗抑制效果较好。.（2）经100 mM的MeIAA处理后，紫茎泽兰植株体内的维管束中木质化的组织在减少，髓细胞中细胞间的间隙增大，髓细胞壁有断裂的痕迹，叶绿体中白色体减少。.（3）经MeIAA处理后，紫茎泽兰体内过氧化物酶活性、丙二醛和可溶性糖的含量显著降低；超氧化物歧化酶、过氧化氢酶和乙酰辅酶A羧化酶的酶活均升高；高浓度MeIAA处理促进叶绿素含量显著升高，低浓度处理导致叶绿素含量显著降低。.（4）MeIAA处理导致紫茎泽兰茎干木质素的减少，且4-香豆酸：辅酶A连接酶、木质素过氧化物酶和苯丙氨酸解氨酶等与木质素生物合成相关酶的酶活性随着MeIAA浓度的增加而降低。MeIAA还能抑制5-烯醇式丙酮酰莽草酸-3-磷酸合酶、ATP磷酸核糖基转移酶、酮酸还原异构酶、异丙基苹果酸脱水酶和异丙基苹果酸异构酶等氨基酸合成相关酶的活性。.（5）MeIAA处理引起了紫茎泽兰茎干生长素响应、氧化还原酶活性、苯丙烷分解代谢过程、木质素分解代谢过程、木质素代谢过程和苯丙烷代谢过程等相关基因的表达变化，引起吲哚-3-乙酸和脱落酸信号转导基因的表达变化，还抑制了苯丙烷生物合成途径中与木质素合成相关酶的活性。