三七根腐病根际微生态特征与调控技术研究
文献类型：学位论文
| 作者 | 吴照祥 |
| 学位类别 | 博士 |
| 答辩日期 | 2016-11 |
| 授予单位 | 中国科学院研究生院 |
| 授予地点 | 北京 |
| 导师 | 陈保冬 ; 郭兰萍 |
| 关键词 | AM真菌，三七，根腐病，根际土壤，土壤微生物群落 AM fungi, Panax notoginseng, Root-rot disease, Rhizosphere soil, Soil microbial community |
| 其他题名 | Characterization and Regulation of the Rhizosphere Microecosystem of Panax notoginseng for Controlling Root- rot Disease |
| 学位专业 | 生态学 |
| 中文摘要 | 三七是我国特有的名贵中药材，由于对多种病症具有独特的疗效，国内外市场需求量日益增加。根腐病引起的三七产量大幅下降、品质严重恶化对三七生产带来巨大的经济损失，已经成为三七种植业发展的重要限制因素之一。目前，针对三七根腐病已经分离了几种致病菌，并开展针对性的化学防治工作，但是关于根腐病发生后土壤微生物群落变化以及环境因子的影响还未有报道。本研究通过三七发病株与健康株根际土壤以及根系内微生物群落结构组成和多样性的系统比较，确定三七根腐病发生与植株根际土壤和根系内微生物群落结构的关系以及环境因子的影响；从根际土壤微生态角度出发，通过分室装置栽培试验，研究三七种植土壤自毒物质以及微生物对三七生长和根腐病的影响，探讨自毒物质与土壤微生物群落的作用；构建毁坏柱孢霉菌（Cylindrocarpon destructans）定量检测方法，测定并比较植株根际土壤中该病原菌的数量，探讨毁坏柱孢霉与植株生长和 AM真菌侵染之间的数量效应关系，确认植株根际土壤C. destructans与三七根腐病发生之间的相关关系；通过室内模拟接菌试验，证实 AM真菌接种对三七幼苗的作用，初步探明 AM真菌作用的调控机理。该结果为三七根腐病根际微生态过程揭示以及生物防治技术研究提供新的理论依据和试验证据。论文主要研究结果如下： （1）三七健康与发病植株根际土壤微生物生物量组成差异 通过磷脂脂肪酸技术（phospholipid fatty acid，PLFA）定量测定土壤中不同类群微生物生物量组成，考查发病植株根际土壤微生物生物量组成的变化以及环境因子的影响。研究结果表明，三七发病植株根际土壤微生物生物量增加，其群落结构组成明显有别于健康植株。土壤有机碳、有效氮、土壤质地及丛枝丰度对土壤微生物群落结构组成具有显著性影响。这说明三七根腐病的发生与土壤微生物群落改变直接相关，并且受到土壤理化性质以及菌根侵染等因素的影响。 （2）三七健康与发病植株根际土壤以及根系内的微生物群落结构差异 通过微生物高通量测序技术 Illumina MiSeq对三七植株根际土壤以及根系内细菌和真菌群落组成进行测定，考查三七发病植株根际土壤以及根系内微生物群落组成的变化以及环境因子的影响。研究结果表明，三七根腐病发病植株根际土壤以及根系内细菌和真菌群落结构发生显著改变，微生物多样性明显降低。三七发病植株根际土壤细菌群落相似性降低，真菌群落相似性上升，但是根系内微生物群落相似性没有明显变化。菌根侵染强度、土壤粘粒含量和土壤无机氮对根际土壤细菌群落组成具有显著性影响，而对根际土壤真菌群落组成构成显著性影响的是菌根丛枝丰度和土壤粘粒含量。三七根腐病发病植株根系内微生物区系也表现出明显的差异。植株地上部硫浓度和碳浓度对根系内细菌群落组成具有显著性影响，而对根系内真菌群落组成具有显著性影响的是根际土壤 C/N和地上部氮浓度。这些结果说明，三七植株根际土壤以及根系内微生物群落结构与土壤理化性状以及植株营养状况密切相关。 （3）自毒物质与土壤微生物作用分析 通过分室装置栽培试验，研究三七种植土壤自毒物质和微生物对三七生长和根腐病发生的影响。研究结果表明轮作时间较短的种植土壤中自毒物质浓度显著的高于轮作时间长的种植土壤，两者微生物群落组成具有显著性差异。自毒物质对三七生长以及根腐病的发生没有影响，但是轮作时间短的种植土壤中微生物区系的引入显著的抑制三七根系的生长，加剧根腐病的发生。另外，我们发现自毒物质显著的改变根际土壤真菌群落组成。上述结果表明三七种植土壤微生物群落受到自毒物质的影响，其群落结构的改变与三七根腐病的发生紧密联系。 （4）三七植株根际土壤病原真菌 C.destructans与植物生长和AM真菌侵染的数量效应关系 基于实时定量 PCR技术建立一种快速、准确的三七根腐病病原真菌毁坏柱孢霉（C.destructans）的分子定量检测方法，探讨毁坏柱孢霉与植株生长和AM真菌侵染之间的数量效应关系。大田样品检测结果显示，发病植株根际土壤毁坏柱孢霉显著高于健康植株。三七根际土壤毁坏柱孢霉数量与植株地上部生物量以及菌根侵染强度（M%）呈显著负相关（P＜0.05），与根系生物量以及根内丛枝丰度（A%）相关性不显著。这表明三七植株根际土壤毁坏柱孢霉 C. destructans与三七根腐病发生之间存在相关关系，并且 AM真菌对该病原微生物具有潜在生防功能。 （5）AM真菌接种对三七生长以及代谢的调节作用 AM真菌侵染三七植株根系，形成良好的菌根共生体结构，包括致密的内生和外生菌丝、丛枝结构以及孢子体。通过室内模拟盆栽并接种 AM真菌试验，发现 AM真菌与三七共生关系建成后促进三七幼苗对P元素的吸收，改善其P营养水平。此外，AM真菌对三七生理代谢具有调节作用，提高三七幼苗叶绿素光系统Ⅱ最大光化学效率。研究结果显示 AM真菌能够侵染三七根系并形成紧密的共生关系，具有潜在的促生和生理调节作用，显著的提高三七种苗质量。 综上所述，本论文综合运用荧光定量 PCR、高通量测序等现代分子生态学研究方法与 PLFA表征技术比较三七健康和根腐病发病植株根际土壤、根系内微生物群落组成差异以及环境因子的影响；通过分室装置栽培试验区分连作土壤自毒物质与根际微生物的作用；室内模拟接种试验揭示 AM真菌接种对三七幼苗营养元素吸收、生理代谢、叶绿素光系统Ⅱ最大光化学效率以及生长的影响。本研究对全面认识三七根腐病发生过程中根际微生态变化具有重要意义，为三七栽培和田间管理提供科学依据，有利于三七产业的健康、可持续发展。 |
| 英文摘要 | Panax notoginseng, a well-known traditional Chinese medicinal herb, is unique to China and distributes in the region of Wenshan, Yunnan province. The international and domestic demand for P. notoginseng is increasing gradually for its unique curative effect on cardiovascular diseases, inflammation, different body pains, trauma, and internal and external bleeding due to injury. In practice, continuous cropping of P. notoginseng is almost infeasible due to serious root-rot disease, which could cause significant yield reduction and low content of active ingredients and subsequent economic losses. To reveal the microecological characteristics of the P. notoginseng root-rot disease, microbial community in rhizosphere soils and roots between diseased and healthy P. notoginseng were compared, and the effects of environmental factors on microbial community were also analysed. Based on the quantitative real-time PCR,C.destructans in rhizosphere soil of P. notoginseng was quantified, and its relationships with plant biomass and AM fungal infection were established. In order to elucidate interactions between soil allelochemicals and microbial community, effects of allelochemicals and microbial community on plant growth and root-rot disease were identified through a compartmentation cultivation experiment. A following greenhouse experiment indicated that AM fungi regulated the physiological metabolism of P.notoginseng, enhanced absorbtion of mineral nutrients from soil and promoted the photosynthetic capacity. These results provided theoretical basis and experimental evidence for bio-control research of root-rot disease on P. notoginseng. Main results are summarized as following: (1) Comparison on the rhizosphere microbial biomass composition between healthy and root-rot diseased P. notoginseng. Microbial community in rhizosphere soils was compared between diseased and healthy P.notoginseng, and the effects of environmental factors on microbial community were analysed with PLFA indicating microbial structural composition. Microbial community from rhizosphere soils of diseased P. notoginseng differed significantly from those of healthy plants. Compared to those of healthy P. notoginseng,bacterial, fungal and total microbial PLFAs and relative abundance of negative-gram bacteria were higher in rhizosphere soils of diseased plants, and relative abundance of positive-gram bacteria, actinomycetes and AM fungi were much lower. For microbial community structural composition, soil organic carbon, arbuscule abundance, inorganic nitrogen and soil texture were identified to be the significant predictors across samples by the RDAmodel selection procedure. All of these results indicated that P. notoginseng root-rot disease closely related with soil microbial community composition, which was affected by soil physicochemical properties and mycorrhizal colonization. (2) Molecular characterization of microbial communitiesy in the rhizosphere soils and roots of diseased and healthy P. notoginseng. To compare microbial community in rhizosphere soils and roots between diseased and healthy p.notoginseng, and analyse the effects of environmental factors on microbial community, bacterial and fungal community were studied with Illumina MiSeqs technology. Microbial community in rhizosphere soils and roots of diseased P.notoginseng differed significantly from those of healthy plants. Microbial diversity in rhizosphere soils and roots of diseased P. notoginseng was decreased significantly. Similarity of bacterial community in rhizosphere soil of diseased P. notoginseng was lower, but of fungi was higher. Mycorrhizal colonization, soil clay content and inorganic nitrogen significantly affected rhizosphere bacterial community, but the significant environmental factors affecting rhizosphere fungal community were arbuscule abundance and soil clay content. Similarity of microbial community in plant roots showed no significant difference. Plant shoot sulfur and carbon content significantly affected bacterial community in roots, and the significant environmental factors affecting fungal community in roots were soil C/N ratio and shoot nitrogen content. These results showed that microbial community in rhizosphere soils and plant roots correlated with soil physicochemical characteristics and plant nutrition status significantly. (3) Role of allelochemicals and soil microbial community. To identify the relevance of allelochemicals and soil microbial community in root-rot disease, a compartmentation experiment was conducted to compare the effects of allelochemicals and soil microbial community on plant growth and root-rot disease. Allelochemicals were much more in cropping soil rotated by maize for 3 years than those for 10 years, and microbial community differed significantly between each other.No effects of allelochemicals on plant growth and occurance of root-rot disease were found, however microbial community from soil rotated just for 3 years inhibited the growth of root, and increased root-rot disease. Additionally, allelochemicals from soil rotated just for 3 years were found to change soil fungal community significantly. Related results indicated that soil microbial community was affected by allelochemicals, and the change of soil microbial community was highly relevant to P. notoginseng root-rot disease. (4) The relationships of C. destructans in rhizosphere soils of P. notoginseng with plant biomass and AM fungi colonization. Based on the quantitative real-time PCR, C. destructans in rhizosphere soils of P.notoginseng was quantified and its relationships with plant biomass and AM fungi colonization were studied. C. destructans was more abundant in rhizosphere soils of diseased P. notoginseng than that of healthy plants, but AM colonization was more obvious in healthy plants. The quantity of C. destructans negatively correlated with plants shoot biomass and mycorrhizal colonization rate (M%). These results suggested that C. destructans was identified to be correlated with root-rot disease in field. (5) Growth promoting effect of AM fungi The arbuscular mycorrhizal association between AM fungi and P. notoginseng was well established, and typical mycorrhizal structures including dense external and internal hyphae, arbuscule and spore were formed. Inoculation of AM fungi improved P. notoginseng seedling P absorption, enhanced plant P nutrition status. AM fungi regulated physiological metabolism, and increased the maximal photochemical efficiency of chlorophyll photosystem. AM fungi established arbuscular mycorrhizal association with P. notoginseng seedling, possessed plant growth promotion and physiology regulation effects, and significantly improved seedling healthy status. In summary, modern molecular methods include quantitative real-time PCR and high-throughput sequencing, and biochemical approaches such as PLFA were employed to study the difference in microbial community in rhizosphere soils and plant roots between healthy and diseased P. notoginseng, and also the effects of environmental factors on microbial community. Relative importance of allelochemicals and soil microbial community were revealed by compartmentation cultivation experiment. In addition, effects of AM fungi inoculation on physiological metabolism,mineral nutrition absorbtion, potential photosynthetic capacity and plant growth of P.notoginseng seedling were also studied. The study substantially contributed to rhizosphere microecology of P. notoginseng, and provided scientific and technological supports for sustainable cultivation of P. notoginseng. |
| 源URL | [http://ir.rcees.ac.cn/handle/311016/37025]  |
| 专题 | 生态环境研究中心_城市与区域生态国家重点实验室 |
| 推荐引用方式 GB/T 7714 | 吴照祥. 三七根腐病根际微生态特征与调控技术研究[D]. 北京. 中国科学院研究生院. 2016. |
入库方式： OAI收割
来源：生态环境研究中心
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