三七根腐病株根际土壤真菌群落组成与碳源利用特征研究

根腐病是药用植物三七Panax notoginseng的多发性病害,而土壤真菌群落组成与根腐病的发生存在一定的联系。利用末端限制性酶切片段长度多态性分析和Biolog技术研究了云南文山三七种植园健康和发病三七植株根际土壤真菌群落结构和碳源利用特征,结果表明健康和发病植株的根际土壤真菌多样性和代谢活性差异不显著,但是真菌群落组成存在差异。健康和发病植株根际土壤都以小茎点霉Phoma exigua及镰孢菌属Fusarium真菌为主要种群,而健康植株根际土壤中火丝菌属Pyronema和被孢霉属Mortierella等真菌类群的相对丰度显著高于发病植株根际土壤,尖孢镰孢菌F.ox‐ysporum和刺座霉属的Volutella colletotrichoides的相对丰度则显著低于发病植株。     

Basidiomycete fungal communities in Australian sclerophyll forest soil are altered by repeated prescribed burning

Soil basidiomycetes play key roles in forest nutrient and carbon cycling processes, yet the diversity and structure of below ground basidiomycete communities remain poorly understood. Prescribed burning is a commonly used forest management practice and there is evidence that single fire events can have an impact on soil fungal communities but little is known about the effects of repeated prescribed burning. We have used internal transcribed spacer (ITS) terminal restriction fragment length polymorphism (T-RFLP) analysis to investigate the impacts of repeated prescribed burning every two or four years over a period of 30 years on soil basidiomycete communities in an Australian wet sclerophyll forest. Detrended correspondence analysis of ITS T-RFLP profiles separated basidiomycete communities in unburned control plots from those in burned plots, with those burned every two years being the most different from controls. Burning had no effect on basidiomycete species richness, thus these differences appear to be due to changes in community structure. Basidiomycete communities in the unburned control plots were vertically stratified in the upper 20 cm of soil, but no evidence was found for stratification in the burned plots, suggesting that repeated prescribed burning results in more uniform basidiomycete communities. Overall, the results demonstrate that repeated prescribed burning alters soil basidiomycete communities, with the effect being greater with more frequent burning.     

牛场肥水灌溉对土壤nirK、nirS型反硝化微生物群落结构的影响

以徐水县梁家营长期定位施肥试验田为研究对象,利用末端限制性片段长度多态性(T-RFLP)分析和克隆文库构建,研究了5种施肥处理(清水灌溉CK、无机肥灌溉CF、牛场肥水不同浓度、不同次数灌溉T4、T5和T11)下土壤中nirK、nirS型反硝化细菌群落多样性及其群落结构的演变。结果表明,不同施肥处理下nirK、nirS型反硝化细菌群落多样性无显著差异,但群落结构却有明显变化:nirK型反硝化细菌群落结构既受施肥种类又受施肥量影响,优势种群尤其对施肥种类和施肥量响应显著;nirS型反硝化细菌则主要受施肥种类影响,施肥量影响微弱。牛场肥水处理和无机肥处理分别促进和抑制不同的nirS型反硝化细菌,群落主成分受无机肥促进、牛场肥水抑制。系统发育分析结果表明,土壤中nirK型反硝化细菌主要与假单胞菌属(Pseudomonas)、产碱杆菌属(Alcaligenes)和根瘤菌属(Rhizobium)的反硝化细菌具有较近的亲缘关系;nirS型反硝化细菌主要与劳尔氏菌(Ralstonia)和红长命菌属(Rubrivivax)有较近的亲缘关系。试验土壤中反硝化微生物多与目前已报道的好氧反硝化细菌亲缘关系较近,这可能与微生物分析取自表层土有关。     

Host effects on ectomycorrhizal fungal communities: insight from eight host species in mixed conifer-broadleaf forests

To advance our understanding of host effects on the community structure of ectomycorrhizal fungi (EMF), EMF communities were compared among different host species, genera and families in two mixed conifer-broadleaf forests in Japan. Using molecular identification methods we examined EMF root tips of eight coexisting species belonging to six genera (three families): Abies and Tsuga (Pinaceae), Betula and Carpinus (Betulaceae) and Fagus and Quercus (Fagaceae). In total, 205 EMF species were detected, and the total richness was estimated to exceed 300 species using major estimators. Of the 55 EMF species occurring three or more times, eight showed significantly biased host preference. A Mantel test showed a significantly negative correlation between EMF community similarity and host taxonomic distance. Detrended correspondence analysis separated EMF communities mainly by host taxonomic and successional status. Thus, EMF communities are similar on hosts with similar taxonomic and successional status. A significant proportion of EMF exhibited host specificity, which may contribute to the extremely diverse EMF community in conifer-broadleaf forests.     

水葫芦根际细菌群落结构多样性分析

【目的】了解水葫芦根际细菌群落结构。【方法】运用末端限制性片段长度多态性(Terminal restriction fragment length polymorphism,T-RFLP)技术分析富营养化水体中水葫芦根际和水葫芦近、远水样的细菌群落特征及多样性,结合克隆文库技术和培养法分析根际的细菌种群类型。【结果】同一时期水葫芦根际细菌多样性(Shannon-Weiner指数H′或Simpson指数D)更高,水葫芦近水样次之,远水样最小。10月份的细菌多样性高于5月份的。通过水葫芦根际细菌的克隆文库可知变形杆菌门(Proteobacteria)是水葫芦根际细菌的主要类群,占总群体的65.1%,包括噬菌弧菌(Bacteriovorax sp.)、Dechloromonas sp.、Leptothrix sp.、红螺菌科(Rhodospirillaceae)、Rhodoferax sp.和红环菌科(Rhodocyclaceae)等。T-RFLP图谱显示159 bp为最大优势菌,247 bp为第二大优势菌,对照克隆文库及培养结果分析247 bp属于γ-Proteobacteria,159 bp为不动杆菌(Acinetobacter sp.)。【结论】水葫芦根际细菌的群落结构丰富,不同时段水葫芦根际细菌的丰度略有变化,主要类群为变形杆菌门。     

Using terminal restriction fragment length polymorphism (T-RFLP) to identify mycorrhizal fungi: a methods review

Terminal restriction fragment length polymorphism (T-RFLP) is an increasingly widely used technique in mycorrhizal ecology. In this paper, we review the technique as it is used to identify species of mycorrhizal fungi and distinguish two different versions of the technique: peak-profile T-RFLP (the original version) and database T-RFLP. We define database T-RFLP as the use of T-RFLP to identify individual species within samples by comparison of unknown data with a database of known T-RFLP patterns. This application of T-RFLP avoids some of the pitfalls of peak-profile T-RFLP and allows T-RFLP to be applied to polyphyletic functional groups such as ectomycorrhizal fungi. The identification of species using database T-RFLP is subject to several sources of potential error, including (1) random erroneous matches of peaks to species, (2) shared T-RFLP profiles across species, and (3) multiple T-RFLP profiles within a species. A mathematical approximation of the risk of the first type of error as a function of experimental parameters is discussed. Although potentially less accurate than some other methods such as clone libraries, the high throughput of database T-RFLP permits much greater replication and may, therefore, be preferable for many ecological questions, particularly when combined with other techniques such as cloning.     

新疆低阶煤本源生物甲烷转化中微生物群落组成及变化

【目的】探究新疆低阶煤生物甲烷转化过程微生物群落组成及多样性。【方法】采用厌氧培养方法和末端限制性片段长度多态性技术(Terminal restriction fragment length polymorphism,T-RFLP)分析新疆低阶煤本源微生物对甲烷转化及有机酸含量的影响,分析新疆哈密大南湖长焰煤生物甲烷转化过程中微生物群落动态变化。【结果】研究表明长焰煤和褐煤对本源微生物产甲烷影响较小,随着低阶煤生物甲烷转化时间的延长,甲烷产量呈上升趋势,转化60 d后长焰煤甲烷产量高达10.28 m L/g,挥发性有机酸(VFA)浓度则最低;微生物多样性指数变化不明显,不同转化时间微生物主要类群为放线菌门(Actinobacteria),拟杆菌门(Bacteroidetes),厚壁菌门(Firmicutes),变形菌门(Proteobacteria);甲烷菌的群落结构相对于细菌较简单,在整个低阶煤生物转化产甲烷过程中共有古菌类群为甲烷八叠球菌属(Methanosarcina)、甲烷盐菌属(Methanohalobium)、甲烷叶菌属(Methanolobus)、甲烷食甲基菌属(Methanomethylovorans),它们是构成群落结构的基本菌群。【结论】低阶煤生物甲烷转化过程微生物群落具有丰富的多样性,且不同时期多样性有较大差异。甲烷菌群落结构相对于细菌较简单,共有类群明显。     

施氮肥对华北平原土壤氨氧化细菌和古菌数量及群落结构的影响

利用荧光定量PCR、末端限制性片段长度多样性(T-RFLP)和基因克隆文库技术,比较了4种施氮水平(不施氮肥,0 kg N/hm~2,CK;施低水平氮肥,75 kg N/hm~2,N1;施中水平氮肥,150 kg N/hm~2,N2;施高水平氮肥,225 kg N/hm~2,N3)下华北平原地区小麦季表层(0—20 cm)土壤总细菌、氨氧化细菌(AOB)和氨氧化古菌(AOA)的丰度和群落结构。结果表明,土壤总细菌、AOB和AOA数量分别在每克干土5.74×10~9—7.50×10~9、8.89×10~6—2.66×10~7和3.83×10~8—7.78×10~8之间。不同施氮量土壤AOA数量均高于AOB数量,AOA/AOB值在81.72—14.38之间。增施氮肥显著显著提高AOB数量(P0.05),对总细菌和AOA数量的影响不显著(P0.05)。与CK相比,处理N1、N2和N3中AOB数量分别提高了0.64、1.50和1.99倍。增施氮肥显著改变了AOB和AOA的群落结构,且不同施氮量处理中AOB群落结构差异更大。系统进化分析显示,施氮肥小麦土壤AOB主要为Nitrosospira属类群,分布在Cluster 3的两个分支中;AOA分布在Cluster S的4个分支中。相关性分析显示,AOB数量与全氮和铵态氮含量呈显著正相关关系,与土壤pH和碳氮比呈显著负相关关系(P0.05);AOA数量与硝态氮含量和土壤pH呈显著正相关关系,与铵态氮含量呈显著负相关关系(P0.05)。研究结果表明:增施氮肥可显著改变华北平原地区碱性土壤AOB数量与群落结构,该地区小麦土壤中AOB比AOA对氮肥响应更敏感。     

毒死蜱乳油对辣椒根围土壤细菌群落结构的影响

【目的】了解有机磷杀虫剂毒死蜱对土壤细菌群落结构的作用。【方法】联合微生物平板计数法、末端限制性片段多态性分析,选择PRIMER 5进行群落结构分析,以研究不同浓度的毒死蜱对辣椒根围可培养和不可培养细菌群落结构的影响。【结果】毒死蜱施入后前30 d,3个处理组的可培养细菌较对照组具有显著差异(P0.05),但在第30天后,处理组可培养细菌数均能恢复到对照水平。采用PRIMER 5对T-RFLP数据进行多角度分析发现,HaeⅢ酶切片段中,对照组C3、处理组Y0和Z2的细菌群落结构较整体聚类较远。HhaⅠ酶切片段中,150μg/g的毒死蜱处理组(Z0)在第0天表现出最大的群落差异。ANOSIM表明,以不同浓度的毒死蜱分组,各组间细菌群落组成差异不显著(HaeⅢ:Global R=0.041,P=0.168;HhaⅠ:Global R=-0.04,P=0.842);以不同取样时间分组时,细菌群落组成差异显著(HaeⅢ:Global R=0.304,P=0.001;HhaⅠ:Global R=0.28,P=0.001)。经SIMPER分析所有TRFs可知,对群落丰度贡献最大的片段分别为TRF239、TRF240、TRF241。在线比对得到其代表菌群有芽孢杆菌属(Bacillus sp.)、梭菌属(Clostridium sp.)、葡萄球菌属(Staphylococcus sp.)、八叠球菌属(Sarcina sp.)、假单胞菌属(Pseudomonas sp.)等。【结论】高浓度的毒死蜱会对土壤细菌群落产生影响,抑制根围细菌生长,从而遏制植物的健康生长,因而有必要及时采取措施以减少大量重复使用毒死蜱所带来的危害。     

Chitinase genes revealed and compared in bacterial isolates, DNA extracts and a metagenomic library from a phytopathogen-suppressive soil

Soil that is suppressive to disease caused by fungal pathogens is an interesting source to target for novel chitinases that might be contributing towards disease suppression. In this study, we screened for chitinase genes, in a phytopathogen-suppressive soil in three ways: (1) from a metagenomic library constructed from microbial cells extracted from soil, (2) from directly extracted DNA and (3) from bacterial isolates with antifungal and chitinase activities. Terminal restriction fragment length polymorphism (T-RFLP) of chitinase genes revealed differences in amplified chitinase genes from the metagenomic library and the directly extracted DNA, but approximately 40% of the identified chitinase terminal restriction fragments (TRFs) were found in both sources. All of the chitinase TRFs from the isolates were matched to TRFs in the directly extracted DNA and the metagenomic library. The most abundant chitinase TRF in the soil DNA and the metagenomic library corresponded to the TRF¹⁰³ of the isolate Streptomyces mutomycini and/or Streptomyces clavifer . There were good matches between T-RFLP profiles of chitinase gene fragments obtained from different sources of DNA. However, there were also differences in both the chitinase and the 16S rRNA gene T-RFLP patterns depending on the source of DNA, emphasizing the lack of complete coverage of the gene diversity by any of the approaches used.     

重庆酸雨区不同林型对土壤酸化和真菌群落的影响

不同林型土壤的酸化缓冲能力不同,真菌在土壤系统中扮演着重要的角色,而对土壤真菌群落结构和组成与土壤酸化的关系缺乏深入研究。以重庆铁山坪林场的马尾松纯林（Pi）和经马尾松纯林改造后的香樟纯林（Ci）、木荷纯林（Sc）、马尾松-香樟混交林（Pi_Ci）以及马尾松-木荷混交林（Pi_Sc）为研究对象,每个林型分别设置4个20 m×20 m的样地,分别采集腐殖质层（O层）和淋溶层（A层）土壤进行土壤性质及真菌群落分析,以探讨酸雨区森林土壤真菌群落与缓解土壤酸化的关系。研究表明：（1）与Pi相比,Ci土壤酸化明显缓解（高pH低NH₄ ： NO₃）,且能有效提高土壤全磷（TP）含量;而Sc虽然土壤pH值与Pi没有显著差异,但显著（P<0.05）提高了NH₄ ： NO₃,且显著降低土壤TP和全钾（TK）含量（P<0.05）;（2）不同林型土壤真菌群落多样性以Ci最为丰富,且表征土壤酸化的指标pH值、阳离子交换量（CEC）与真菌多样性显著正相关（P<0.05）,NH₄ ： NO₃与多样性显著负相关（P<0.05）;（3）林型和土层都对真菌群落结构有显著影响（P<0.001）,且林型的影响大于土层的影响;而土壤酸化程度将五个林型的土壤真菌群落区分成两个大类：Ci和Pi_Ci;Pi,Sc以及Pi_Sc。（4） Ci中有益菌（如Mortierella）更多,Pi以外生菌根真菌占优势（Russulaceae、Russula、Tomentella以及Sebacina）;Sc以及Pi_Sc则含有更多的植物病原菌（Cladophialophora,Paecilomyces,Venturiales）、嗜酸菌及产酸菌（Paecilomyces,Penicillium）。在酸雨区受损马尾松林地种植香樟促进土壤真菌多样性提高,且产酸真菌、嗜酸菌丰度降低,而有益真菌丰度增加,可有效缓解土壤酸化;而种植木荷后土壤中的病原菌、嗜酸菌和产酸菌相对丰度增加,导致土壤进一步酸化。因此,通过将受酸雨损害严重的马尾松纯林改造成香樟纯林或马尾松-香樟混交林,有助于缓解土壤的酸化,实现酸雨区森林生态系统的可持续发展。     

再生水补水对河道底泥细菌群落结构的影响

以北京市永定河麻峪湿地再生水补水口附近河道底泥为研究对象,应用末端限制性片段长度多态性(Terminal Restriction Fragment Length Polymorphism,T-RFLP)技术探究再生水补水口附近底泥细菌群落结构的空间差异特征以及环境因子所产生的影响,并借助典范对应分析(Canonical Correspondence Analysis,CCA)方法分析麻峪湿地底泥细菌群落结构空间差异特征的形成原因。分析结果表明:河道底泥微生物群落对再生水的净化主要发生在距补水口1200m的范围内,随着再生水与上游来水径向混合净化渐变过程,在补水口2000m处河道底泥微生物群落结构与河道再生水补水口上游趋于相似。基于样点聚类结果的群落结构多样性分析表明随着再生水净化过程的实现微生物综合多样性指数呈现下降的趋势,从均匀度指数的变化趋势看,再生水补水口具有最高均匀度指数,补水口下游1200米处由于非优势菌群所占相对丰度最低以及偶见菌群缺失,导致群落结构比较单一、群落多样性和均匀度指数都最低。CCA方面分析结果表明再生水补水口上游细菌群落与因累积效应形成的重金属有密切关系,补水口出口底泥细菌群落则主要受到总磷和总有机碳的影响较大,而再生水补水与上游来水汇水径向渐变过程可能与氨氮净化具密切关系。研究区的主要优势菌种为假单胞菌属、贪食菌属和芽孢杆菌属,是与再生水中有机碳、氮降解具有密切关系的菌属。     

Spatial separation of litter decomposition and mycorrhizal nitrogen uptake in a boreal forest

Our understanding of how saprotrophic and mycorrhizal fungi interact to re-circulate carbon and nutrients from plant litter and soil organic matter is limited by poor understanding of their spatiotemporal dynamics. In order to investigate how different functional groups of fungi contribute to carbon and nitrogen cycling at different stages of decomposition, we studied changes in fungal community composition along vertical profiles through a Pinus sylvestris forest soil. We combined molecular identification methods with 14C dating of the organic matter, analyses of carbon:nitrogen (C:N) ratios and 15N natural abundance measurements. Saprotrophic fungi were primarily confined to relatively recently (< 4 yr) shed litter components on the surface of the forest floor, where organic carbon was mineralized while nitrogen was retained. Mycorrhizal fungi dominated in the underlying, more decomposed litter and humus, where they apparently mobilized N and made it available to their host plants. Our observations show that the degrading and nutrient-mobilizing components of the fungal community are spatially separated. This has important implications for biogeochemical studies of boreal forest ecosystems.