丛枝菌根真菌的资源和竞争侵染及土壤微生物的量热研究
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摘要
丛枝菌根存在于80％以上的维管植物中，为真菌与植物的严格共生体，具有促进磷等营养元素吸收、抗逆抗病和维护植物群落稳定性等重要作用。本论文主要以丛枝菌根真菌(AMF)为研究对象，对湖北等地区的AM真菌菌株资源进行收集、筛选及田间应用试验；运用nested-PCR技术和AM真菌特异性引物，进行了多种AM真菌在植物根部竞争性侵染的研究；运用量热技术探讨了生产活动对土壤微生物活动的影响。为深入研究或开发AM真菌生物资源，追踪土壤微生物的动态活动，发展可持续农业管理模式提供了有用的科学依据。论文在以下几个方面开展工作并取得相应研究结果。
以玉米和紫云英为宿主植物，对来自于湖北农业耕作土壤地区的80多份土样中的AM真菌进行分离，获得完全纯化的AM真菌菌株25株，其中绝大部分为为Glomus属成员。基于孢子形态学和PCR分子鉴定手段的检测，15株纯化的AM真菌鉴定为我国新记录种，17株被国际丛枝菌根真菌保藏中心(IBG)收藏。
通过盆栽试验筛选具有显著促长作用的菌株HAU-01(G. constrictum Trappe)和HAU-E4(G. etunicatum Becker & Gerdemann)进行田间试验，并将AM真菌的预接种技术和农业生产上的营养钵育苗技术相结合，对玉米籽粒产量和籽粒淀粉的含量有显著增加效果。其中HAU-01促进玉米籽粒产量增加10.84％，籽粒淀粉含量增加4.23％；HAU-E4促进玉米籽粒产量增加5.06％，籽粒淀粉含量增加3.54％。
运用nested-PCR技术和AM真菌特异性引物，建立了用新鲜植物根段直接检测AM真菌的分子生物学方法。追踪了多种AM真菌的竞争侵染动态及相互关系，解决了单纯的形态学研究难以解决的难题。对混合接种AM真菌的根样进行DNA的粗提，以真核生物通用引物进行第1次扩增，再分别以不同真菌的种特异性引物进行第2次扩增。结果显示：(1)G. intraradices和G. mosseae双接种时，前者的侵染率明显高于后者，在新的侵染区域差异更明显。(2)混合接种时，3种真菌可在1cm长的根段中同时侵染。(3)混合接种时，3种真菌的侵染率随取样时间的改变而发生变化，G. intraradices和G. mosseae存在着明显的竞争关系，而A.laevis与其它真菌的相互作用不明显。三者发挥菌根效应的时期各有特点，G. intraradices持续时间长，G. mosseae次之，A. laevis最晚。
此外，运用特异性分子探针和nested-PCR技术，从田间接种AM真菌G.intraradices和G.mosseae的玉米根样中成功检测到特定的接种AM真菌。该工作从分子水平为评价高效AM真菌的应用潜力，研究AM真菌之间及其与其它微生物之间的相互关系奠定了基础。
采用量热技术对武汉地区几种农业用地的土壤微生物的代谢活动进行了研究。结果表明，(1)所有土样的热输出曲线均表现为典型的生长曲线，明显区分为森林和农田来源的两大类群；(2)绝大多数土样中微生物的生长速率与细菌数量呈高度正相关(r=0.85，p＜0.05)；(3)土样F1中的微生物表现出最高热发散值，是微生物生长的胁迫环境，也表明植被是影响土壤微生物活性的重要因素；(4)热输出总量和植被的丰度相一致，可能与微生物的多样性有关。上述研究证明，土壤中微生物的代谢活动受人类生产活动的影响很大，量热技术可以灵敏、快速、定量地进行检测，为人类合理可持续开发利用农业用地提供了科学依据。
The absolute symbiosis between majority of land plants and arbuscular mycorrhizalfungi (AMF) is probably the most abundant and wide spread mutualism on earth.AMF not only enhance plant productivity and plant nutrition, AMF can also promoteplant disease suppression, drought resistance and influence plant community structure,plant diversity and ecosystem functioning. The aim of this studies are (1) to isolateAM fungal species from Hubei cultivated soil, screen efficient isolates for inoculationin agricultural practice, and detect AM fungi in fields at molecular level; (2) toinvestigate the competitive behavior of several AM fungi using nested PCR withspecies-specific primers, which cannot be distinguished based on fungal structures; (3)to investigate the influence of agricultural practices on soil microbial activitymeasured by calorimetry.
25 AM fungal isolates were obtained from more than 80 soil samples. The majorityof them were the members of Glomus. 15 isolates were identified as new species inChina and 17 species were registered in International Bank for the Glomeromycota(IBG).
High efficient isolates HAU-01 (Glomus constrictum) and HAU-E4 (G. etunicatum)were screened as species for filed trial by pot culture in glasshouse conditions. Thefield trials were carried out by means of producing pre-inoculated seedlings prior totransplanting. The yield of the maize inoculated with HAU-01 and HAU-E4 wassignificantly enhanced 10.84% and 5.06%, respectively. The content of starch ingrains was also increased 4.23% and 3.54%, respectively.
Three species of AM fungi, G. intraradices, G. mosseae and Acaulospora laeviswere detected in fresh root fragments by means of nested PCR with species-specificprimers. The competitive behavior of three AM fungi cannot be distinguished basedon fungal structures within same root system was monitored in this work. Roughlyextracted DNA from fresh root was amplified by using universal eukaryotic primers.The PCR products were diluted and used as template for the second PCRamplification using species specific primers respectively. The results showed below.(1) The infection rate of G. intraradices was higher than the rate of G. mosseae whenthey were co-inoculated plant, the difference was larger especially in new colonizedroots. (2) Three AM fungi were detected within same root fragment 1 cm in length. (3)The infection rate of three fungi was changed at different harvested stage. Thecompetitive interaction between G. intraradices and G. mosseae was observed, but the interaction between fungus of A. laevis and other fungi was not tight when three fungico-existed in root system. In addition, the ability of mycorrhizal effect of these fungiis different. The effect of G. intraradices is persistent compared with slow increaseshowed by other fungi.
Two introduced species of AM fungi, G. intraradices and G. mosseae weresuccessfully detected in the plant roots from fields using nested PCR technique. Thisstudy hasmade it possible to assess potential ability of introduced efficient species ofAM fungi at molecular level and understand the interactions between AM fungi andother microorganisms in fields.
To investigate the influence of different agricultural practices and vegetations onsoil microbial activity, soil samples from different land were studied by calorimetry.All power-time curves presented typical changes of microbial activity. The curves ofsoil samples could be divided into two groups differing in agricultural practices andvegetations. The most soil samples showed a significant positive correlation betweenthe values of microbial growth rate and the number of bacteria. A very highdissipative metabolism for sample F1 was observed, which suggest F1 is degradingorganic matter under stress. The values of total heat agreed with vegetation abundanceand probably with microbial diversity. In conclusion, microbial activity of the soilsamples determined by calorimetry reflected differences in soil due to agriculturalmanagement.
以玉米和紫云英为宿主植物，对来自于湖北农业耕作土壤地区的80多份土样中的AM真菌进行分离，获得完全纯化的AM真菌菌株25株，其中绝大部分为为Glomus属成员。基于孢子形态学和PCR分子鉴定手段的检测，15株纯化的AM真菌鉴定为我国新记录种，17株被国际丛枝菌根真菌保藏中心(IBG)收藏。
通过盆栽试验筛选具有显著促长作用的菌株HAU-01(G. constrictum Trappe)和HAU-E4(G. etunicatum Becker & Gerdemann)进行田间试验，并将AM真菌的预接种技术和农业生产上的营养钵育苗技术相结合，对玉米籽粒产量和籽粒淀粉的含量有显著增加效果。其中HAU-01促进玉米籽粒产量增加10.84％，籽粒淀粉含量增加4.23％；HAU-E4促进玉米籽粒产量增加5.06％，籽粒淀粉含量增加3.54％。
运用nested-PCR技术和AM真菌特异性引物，建立了用新鲜植物根段直接检测AM真菌的分子生物学方法。追踪了多种AM真菌的竞争侵染动态及相互关系，解决了单纯的形态学研究难以解决的难题。对混合接种AM真菌的根样进行DNA的粗提，以真核生物通用引物进行第1次扩增，再分别以不同真菌的种特异性引物进行第2次扩增。结果显示：(1)G. intraradices和G. mosseae双接种时，前者的侵染率明显高于后者，在新的侵染区域差异更明显。(2)混合接种时，3种真菌可在1cm长的根段中同时侵染。(3)混合接种时，3种真菌的侵染率随取样时间的改变而发生变化，G. intraradices和G. mosseae存在着明显的竞争关系，而A.laevis与其它真菌的相互作用不明显。三者发挥菌根效应的时期各有特点，G. intraradices持续时间长，G. mosseae次之，A. laevis最晚。
此外，运用特异性分子探针和nested-PCR技术，从田间接种AM真菌G.intraradices和G.mosseae的玉米根样中成功检测到特定的接种AM真菌。该工作从分子水平为评价高效AM真菌的应用潜力，研究AM真菌之间及其与其它微生物之间的相互关系奠定了基础。
采用量热技术对武汉地区几种农业用地的土壤微生物的代谢活动进行了研究。结果表明，(1)所有土样的热输出曲线均表现为典型的生长曲线，明显区分为森林和农田来源的两大类群；(2)绝大多数土样中微生物的生长速率与细菌数量呈高度正相关(r=0.85，p＜0.05)；(3)土样F1中的微生物表现出最高热发散值，是微生物生长的胁迫环境，也表明植被是影响土壤微生物活性的重要因素；(4)热输出总量和植被的丰度相一致，可能与微生物的多样性有关。上述研究证明，土壤中微生物的代谢活动受人类生产活动的影响很大，量热技术可以灵敏、快速、定量地进行检测，为人类合理可持续开发利用农业用地提供了科学依据。
The absolute symbiosis between majority of land plants and arbuscular mycorrhizalfungi (AMF) is probably the most abundant and wide spread mutualism on earth.AMF not only enhance plant productivity and plant nutrition, AMF can also promoteplant disease suppression, drought resistance and influence plant community structure,plant diversity and ecosystem functioning. The aim of this studies are (1) to isolateAM fungal species from Hubei cultivated soil, screen efficient isolates for inoculationin agricultural practice, and detect AM fungi in fields at molecular level; (2) toinvestigate the competitive behavior of several AM fungi using nested PCR withspecies-specific primers, which cannot be distinguished based on fungal structures; (3)to investigate the influence of agricultural practices on soil microbial activitymeasured by calorimetry.
25 AM fungal isolates were obtained from more than 80 soil samples. The majorityof them were the members of Glomus. 15 isolates were identified as new species inChina and 17 species were registered in International Bank for the Glomeromycota(IBG).
High efficient isolates HAU-01 (Glomus constrictum) and HAU-E4 (G. etunicatum)were screened as species for filed trial by pot culture in glasshouse conditions. Thefield trials were carried out by means of producing pre-inoculated seedlings prior totransplanting. The yield of the maize inoculated with HAU-01 and HAU-E4 wassignificantly enhanced 10.84% and 5.06%, respectively. The content of starch ingrains was also increased 4.23% and 3.54%, respectively.
Three species of AM fungi, G. intraradices, G. mosseae and Acaulospora laeviswere detected in fresh root fragments by means of nested PCR with species-specificprimers. The competitive behavior of three AM fungi cannot be distinguished basedon fungal structures within same root system was monitored in this work. Roughlyextracted DNA from fresh root was amplified by using universal eukaryotic primers.The PCR products were diluted and used as template for the second PCRamplification using species specific primers respectively. The results showed below.(1) The infection rate of G. intraradices was higher than the rate of G. mosseae whenthey were co-inoculated plant, the difference was larger especially in new colonizedroots. (2) Three AM fungi were detected within same root fragment 1 cm in length. (3)The infection rate of three fungi was changed at different harvested stage. Thecompetitive interaction between G. intraradices and G. mosseae was observed, but the interaction between fungus of A. laevis and other fungi was not tight when three fungico-existed in root system. In addition, the ability of mycorrhizal effect of these fungiis different. The effect of G. intraradices is persistent compared with slow increaseshowed by other fungi.
Two introduced species of AM fungi, G. intraradices and G. mosseae weresuccessfully detected in the plant roots from fields using nested PCR technique. Thisstudy hasmade it possible to assess potential ability of introduced efficient species ofAM fungi at molecular level and understand the interactions between AM fungi andother microorganisms in fields.
To investigate the influence of different agricultural practices and vegetations onsoil microbial activity, soil samples from different land were studied by calorimetry.All power-time curves presented typical changes of microbial activity. The curves ofsoil samples could be divided into two groups differing in agricultural practices andvegetations. The most soil samples showed a significant positive correlation betweenthe values of microbial growth rate and the number of bacteria. A very highdissipative metabolism for sample F1 was observed, which suggest F1 is degradingorganic matter under stress. The values of total heat agreed with vegetation abundanceand probably with microbial diversity. In conclusion, microbial activity of the soilsamples determined by calorimetry reflected differences in soil due to agriculturalmanagement.
引文
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