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题名: 三七根腐病根际微生态特征与调控技术研究
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.
内容类型: 学位论文
URI标识: http://ir.rcees.ac.cn/handle/311016/37025
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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.
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