Microbial Diversity and Community Structure in Two Different Agricultural Soil Communities

Abstract In this study, two different agricultural soils were investigated: one organic soil and one sandy soil, from Stend (south of Bergen), Norway. The sandy soil was a field frequently tilled and subjected to crop rotations. The organic soil was permanent grazing land, infrequently tilled. Our objective was to compare the diversity of the cultivable bacteria with the diversity of the total bacterial population in soil. About 200 bacteria, randomly isolated by standard procedures, were investigated. The diversity of the cultivable bacteria was described at phenotypic, phylogenetic, and genetic levels by applying phenotypical testing (Biolog) and molecular methods, such as amplified rDNA restriction analysis (ARDRA); hybridization to oligonucleotide probes; and REP-PCR. The total bacterial diversity was determined by reassociation analysis of DNA isolated from the bacterial fraction of environmental samples, combined with ARDRA and DGGE analysis. The relationship between the diversity of cultivated bacteria and the total bacteria was elucidated. Organic soil exhibited a higher diversity for all analyses performed than the sandy soil. Analysis of cultivable bacteria resulted in different resolution levels and revealed a high biodiversity within the population of cultured isolates. The difference between the two agricultural soils was significantly higher when the total bacterial population was analyzed than when the cultivable population was. Thus, analysis of microbial diversity must ultimately embrace the entire microbial community DNA, rather than DNA from cultivable bacteria.     

Culture-Dependent and -Independent Methods Capture Different Microbial Community Fractions in Hydrocarbon-Contaminated Soils

Bioremediation is a cost-effective and sustainable approach for treating polluted soils, but our ability to improve on current bioremediation strategies depends on our ability to isolate microorganisms from these soils. Although culturing is widely used in bioremediation research and applications, it is unknown whether the composition of cultured isolates closely mirrors the indigenous microbial community from contaminated soils. To assess this, we paired culture-independent (454-pyrosequencing of total soil DNA) with culture-dependent (isolation using seven different growth media) techniques to analyse the bacterial and fungal communities from hydrocarbon-contaminated soils. Although bacterial and fungal rarefaction curves were saturated for both methods, only 2.4% and 8.2% of the bacterial and fungal OTUs, respectively, were shared between datasets. Isolated taxa increased the total recovered species richness by only 2% for bacteria and 5% for fungi. Interestingly, none of the bacteria that we isolated were representative of the major bacterial OTUs recovered by 454-pyrosequencing. Isolation of fungi was moderately more effective at capturing the dominant OTUs observed by culture-independent analysis, as 3 of 31 cultured fungal strains ranked among the 20 most abundant fungal OTUs in the 454-pyrosequencing dataset. This study is one of the most comprehensive comparisons of microbial communities from hydrocarbon-contaminated soils using both isolation and high-throughput sequencing methods.     

Richness and bioactivity of culturable soil fungi from the Fildes Peninsula,Antarctica

Since the discovery of penicillin, fungi have been an important source of bioactive natural products. However, as a specific resource, the bioactive potentiality and specificity of fungal metabolites from the Antarctic region have had little attention. In this paper, we investigated the diversity patterns and biological activities of cultivable fungi isolated from soil samples in Fildes Peninsula, King George Island, Antarctica. Fungal communities showed low abundance and diversity; a total of 150 cultivable fungi were isolated from eight soil samples. After being dereplicated by morphological characteristics and chemical fingerprints, 47 fungal isolates were identified by ITS-rDNA sequencing. We confirmed that these isolates belonged to at least 11 different genera and clustered into nine groups corresponding to taxonomic orders in the phylogenetic analysis. Using two different fermentation conditions, 94 crude extracts acquired from the abovementioned different metabolite characteristic isolates were screened by bioactivity assay and 18 isolates produced biologically active compounds. Compared with HPLC–DAD–UV fingerprint analysis of culture extracts and standard compounds, two bioactive components secalonic acid and chetracins were identified. Our research suggests that the abundance and diversity of Antarctic cultivable fungal communities exhibit unique ecological characteristics and potential producers of novel natural bioactive products.     

毛竹根际可培养微生物种群多样性分析

[目的]为了了解天然毛竹林根际可培养微生物种群的多样性信息,[方法]采用稀释平板法,对浙江天目山和重庆缙云山天然毛竹林根际细菌和放线菌进行了分离,并对其16S rDNA序列进行了分析.[结果]分别从天目山和缙云山天然毛竹林根际分离得到51株和31株菌落形态差异的细菌和放线菌.16S rDNA序列分析表明,天目山和缙云山毛竹根际细菌主要包括厚壁菌门(Firmicutes,分别为40%和58%)、放线菌门(Actinobacteria,分别为36.7%和10.52%)、变形菌门-亚群(Alphaproteobacteria,分别为10%和5.26%)和变形菌门 --亚群(Gammaproteobacteria,分别为10%和26.32%),其中芽孢杆菌属(Bacillus sp.)为共同的优势菌属(分别为34.38%和42.11%).分离的菌株中,B188、B171和B152等6株与GenBank中已报道16S rRNA基因序列的相似性从90%到96%不等,可能代表着新属或种.[结论]这表明,天然毛竹林根际具有较为丰富的可培养微生物种群多样性,并存在一些潜在的新的微生物菌种资源.     

Bacterial Diversity of East Calcutta Wet Land Area: Possible Identification of Potential Bacterial Population for Different Biotechnological Uses

The extent of microbial diversity in nature is still largely unknown, suggesting that there might be many more useful products yet to be identified from soil microorganisms. This insight provides the scientific foundation for a renewed interest in examining soil microorganisms for novel commercially important products. This has led us to access the metabolic potential of soil microorganisms via cultivation strategy. Keeping this in mind, we have performed a culture-dependent survey of important soil bacterial community diversity in East Calcutta Wetland area (Dhapa Landfill Area). We describe isolation of 38 strains, their phenotypic and biochemical characterization, and finally molecular identification by direct sequencing of polymerase chain reaction (PCR)-amplified 16S rRNA gene products. We have isolated and identified strains able to fix nitrogen, produce extracellular enzymes like protease, cellulase, xylanase, and amylase, and solubilize inorganic phosphates. Some isolates can synthesize extracellular insecticidal toxins. We find a good correlation between biochemical and phenotypic behavior and the molecular study using 16S rRNA gene of the isolates. Furthermore, our findings clearly indicate the composition of cultivable soil bacteria in East Calcutta Wetland Area.     

Impact of Ectomycorrhizosphere on the Functional Diversity of Soil Bacterial and Fungal Communities from a Forest Stand in Relation to Nutrient Mobilization Processes

The ectomycorrhizal symbiosis alters the physicochemical and biological conditions in the surrounding soil, thus creating a particular environment called ectomycorrhizosphere, which selects microbial communities suspected to play a role in gross production and nutrient cycling. To assess the ectomycorrhizosphere effect on the structure of microbial communities potentially involved in the mobilization of nutrients from the soil minerals in a poor-nutrient environment, we compared the functional diversity of soil and ectomycorrhizosphere bacterial communities in a forest stand. Two hundred and sixty-four bacterial strains and 107 fungal strains were isolated from the bulk soil of an oak (Quercus petraea) stand and from oak–Scleroderma citrinum ectomycorrhizosphere and ectomycorrhizae, in two soil organo-mineral horizons (0 to 3 cm and 5 to 10 cm). They were characterized using two in vitro tests related to their capacities to mobilize iron and phosphorus. We demonstrated that the oak–S. citrinum ectomycorrhizosphere significantly structures the culturable bacterial communities in the two soil horizons by selecting very efficient strains for phosphorus and iron mobilization. This effect was also observed on the diversity of the phosphate-solubilizing fungal communities in the lower soil horizon. A previous study already demonstrated that Laccaria bicolor–Douglas fir ectomycorrhizosphere structures the functional diversity of Pseudomonas fluorescens population in a forest nursery soil. Comparing to it, our work highlights the consistency of the mycorrhizosphere effect on the functional diversity of bacterial and fungal communities in relation to the mineral weathering process, no matter the fungal symbiont, the age and species of the host tree, or the environment (nursery vs forest). We also demonstrated that the intensity of phosphorus and iron mobilization by the ectomycorrhizosphere bacteria isolated from the lower soil horizon was significantly higher compared to that which was isolated from the upper horizon. This reveals for the first time a stratification of the functional diversity of the culturable soil bacterial communities as related to phosphorus and iron mobilization.     

Response of bacterial community during bioremediation of an oil-polluted soil

AIM: To study the response of the bacterial community to bioremediation of a soil with an aged contamination of crude oil. METHODS AND RESULTS: The bacterial community in laboratory soil columns during a 72-day biostimulation treatment was followed by analysing the number of total cultivable hydrocarbon-degrading bacteria, soil respiratory activity and the 16S-23S rDNA internal transcribed spacer homoduplex heteroduplex polymorphisms (ITS-HHP) of total soil bacterial DNA. ITS-HHP permits an estimate of both length and sequence polymorphism in a 16S-23S rDNA spacer population, using to advantage the homoduplex and heteroduplex fragments that are generated during PCR. The treatment, made by air sparging and biostimulation with a mineral nutrient and surfactant solution, resulted in a 39.5% decrease of the total hydrocarbon content. Within 4 days of treatment onset the bacterial community underwent a first phase of activation that led to a substantial increase in the observable diversity. Subsequently, after a 12-day period of stability, another activation phase was observed with further shifts of the community structure and an increase in the abundance and diversity of catechol-2,3-dioxygenase (C23O) genes. CONCLUSIONS: The overall data suggest an important contribution of uncultivable bacteria to the soil bioremediation, since, during the second activation phase, the increases of the respiratory activity, bacterial diversity and C23O gene abundance and diversity were not accompanied by a corresponding increase of the cultivable bacteria number. SIGNIFICANCE AND IMPACT OF THE STUDY: This study shows that successive phases of activation of bacterial populations occur during a bioremediation treatment of oil-polluted soil.     

白马雪山云南黄芪与灰毛康定黄芪根际微生物物种多样性及抗生物膜活性菌株筛选

【背景】细菌生物膜是造成病原菌耐药性增强和持续感染的主要因素,但目前尚无针对抗菌膜的特效药物。特境植物根际微生物可产生大量具有提高宿主免疫功能的活性成分,极具抗生物膜药源开发潜力。【目的】了解滇西北高寒特境白马雪山分布的云南黄芪与灰毛康定黄芪植物根际微生物的物种多样性,并对可培养菌株进行抑菌与抗生物膜活性筛选。【方法】采用宏基因组技术结合传统微生物培养方法,对采自我国云南迪庆藏族自治州德钦县白马雪山的云南黄芪与灰毛康定黄芪的根际微生物进行物种多样性研究,并通过“孔板法”测定其可培养菌株发酵液乙酸乙酯粗浸膏的抗菌、抗生物膜活性。【结果】宏基因组测序结果显示,云南黄芪根际土壤样本中的微生物来自6门7纲8目8科9属10种,其中栖热菌属为优势菌群;灰毛康定黄芪根际土壤样本中的微生物来自6门8纲10目11科14属15种,其中慢生根瘤菌属为优势菌群。通过纯培养共获得145株可培养菌株,包括112株细菌和33株真菌。其中,云南黄芪根际细菌59株,共计16属35种,优势属为假单胞菌属和链霉菌属;根际真菌19株,共计4属5种,优势属为曲霉属;灰毛康定黄芪根际细菌53株,归属于16属29种,优势属为芽孢杆菌属与寡养单胞菌属;根际真菌14株,归属于3属4种,优势属为曲霉属。从不同种水平上选择51株细菌和7株真菌为代表菌株进行抗生素药源评估,发现5株细菌及1株真菌发酵液的乙酸乙酯粗浸膏具有中等至较强的抗革兰阳性菌活性,而且其中4株具有抗MRSA生物膜活性,最终确定了链霉属放线菌Streptomyces fulvissimus KTA1和曲霉属真菌Aspergillus fumigatus YNF5为潜力活性菌株。【结论】首次报道了滇西北地区高寒特境黄芪属植物根际微生物具有较好的物种多样性,而且具有一定的抗生素药用资源开发潜力。本研究对滇西北高寒特境特色植物来源的微生物资源开发利用与保护具有重要的借鉴意义。     

Spatial analysis of a hydrocarbon waste-remediating landfarm demonstrates influence of management practices on bacterial and fungal community structure

Cultivation of dedicated soil plots called ‘landfarms' is an effective technology for bioremediation of hydrocarbon waste generated by various industrial practices. To understand the influence of soil conditions on landfarm microbial communities, analysis of bacterial and fungal community structure using next-generation sequencing at different sections and depths was performed across a hydrocarbon-waste landfarm in Regina, Saskatchewan, Canada. While a core set of hydrocarbon-associated bacterial and fungal taxa are present throughout the landfarm, unique bacterial and fungal operational taxonomic units are differentially abundant at sections within the landfarm, which correlate with differences in soil physiochemical properties and management practices. Increased frequency of waste application resulted in strong positive correlations between bacterial community assemblages and elevated amounts of oil, grease and F3 – F4 hydrocarbon fractions. In areas of standing water and lower application of hydrocarbon, microbial community structure correlated with soil pH, trace nutrients and metals. Overall, diversity and structure of bacterial communities remain relatively stable across the landfarm, while in contrast, fungal community structure appears more responsive to soil oxygen conditions. Results are consistent with the hypothesis that years of bioremediation activity have shaped microbial communities; however, several management practices can be undertaken to increase efficiency of remediation, including the removal of standing water and soil tilling across the landfarm.     

Biodiversity of a Burkholderia cepacia population isolated from the maize rhizosphere at different plant growth stages.

A Burkholderia cepacia population naturally occurring in the rhizosphere of Zea mays was investigated in order to assess the degree of root association and microbial biodiversity at five stages of plant growth. The bacterial strains isolated on semiselective PCAT medium were mostly assigned to the species B. cepacia by an analysis of the restriction patterns produced by amplified DNA coding for 16S rRNA (16S rDNA) (ARDRA) with the enzyme AluI. Partial 16S rDNA nucleotide sequences of some randomly chosen isolates confirmed the ARDRA results. Throughout the study, B. cepacia was strictly associated with maize roots, ranging from 0.6 to 3.6% of the total cultivable microflora. Biodiversity among 83 B. cepacia isolates was analyzed by the random amplified polymorphic DNA (RAPD) technique with two 10-mer primers. An analysis of RAPD patterns by the analysis of molecular variance method revealed a high level of intraspecific genetic diversity in this B. cepacia population. Moreover, the genetic diversity was related to divergences among maize root samplings, with microbial genetic variability markedly higher in the first stages of plant growth; in other words, the biodiversity of this rhizosphere bacterial population decreased over time.     

Diversity and PAH growth abilities of bacterial strains isolated from a contaminated soil in Slovakia

Different abandoned industrial areas contaminated by polycyclic aromatic hydrocarbons (PAHs) are present in Slovakia. These environmental burdens are very dangerous to the health of human and environment. The bioremediation, based on the use of hydrocarbons degrading microorganisms, is a promising strategy to sanitize these polluted sites. The aim of this investigation was to assess the bacterial diversity of a PAHs-contaminated soil and to select the potential hydrocarbonoclastic bacteria which can be used for different bioremediation approaches. The bacterial strains were isolated on minimal medium agar supplemented with a mixture of PAHs. Seventy-three isolated strains were grouped by ribosomal interspacer analysis in 15 different clusters and representatives of each cluster were identified by 16S rRNA sequencing. The PAHs degradation abilities of all bacterial isolates were estimated by the 2,6-dichlorophenol indophenol assay and by their growth on minimal broth amended with a mixture of PAHs. Different kinds of strains, members of the genus Pseudomonas, Enterobacter, Bacillus, Arthrobacter, Acinetobacter and Sphingomonas, were isolated from the contaminated soil. Four isolates (Pseudomonas putida, Arthrobacter oxydans, Sphingomonas sp. and S. paucimobilis) showed promising PAHs-degrading abilities and therefore their possible employing in bioremediation strategies.     

Effects of Dry Olive Residue Transformed by Coriolopsis floccosa (Polyporaceae) on the Distribution and Dynamic of a Culturable Fungal Soil Community

Dry olive residue (DOR) is an abundant waste product resulting from a two-phase olive oil extraction system. Due to its high organic and mineral content, this material has been proposed as an organic soil amendment; however, it presents phytotoxic and microtoxic properties. Thus, a pretreatment is necessary before its application to soil. Among the strategies for the bioremediation of DOR is treatment with ligninolytic fungi, e.g. Coriolopsis floccosa. This work aimed to assess the diversity of culturable fungi in a soil of the southeast Iberian Peninsula and to evaluate the short-term impact of untransformed and C. floccosa-transformed DOR on soil mycobiota. A total of 1,733 strains were isolated by the particle filtration method and were grouped among 109 different species using morphological and molecular methods. The majority of isolates were ascomycetes and were concentrated among three orders: Hypocreales, Eurotiales and Capnodiales. The soil amendment with untransformed DOR was associated with a depression in fungal diversity at 30 days and changes in the proportions of the major species. However, when C. floccosa-transformed DOR was applied to the soil, changes in fungal diversity were less evident, and species composition was similar to unamended soil.     

Bacterial Diversity in Linglong Gold Mine,China

Bacteria have been actively regulating cycles of various elements in the environment. To explore the potential bacterial role in gold biogeochemical cycling, this study analyzed the bacterial diversity of mine rock (MR) and surface soil (SS) samples from Linglong gold mine using 16S rRNA gene clone library analysis and cultivation method. From MR, 24 operational taxonomic units (OTUs) were identified from MR, covering 3 phyla and 18 genera. Meanwhile, 24 OTUs were identified from SS, including 4 phyla and 18 genera. Compared with 16S rRNA gene clone library analysis, 28 aerobic and 34 anaerobic isolates were obtained, whereas 26 aerobic and 71 anaerobic strains were isolated from SS. The cultivable bacteria were affiliated with Firmicutes, Proteobacteria and Actinobacteria phyla, and dominated by Firmicutes. These results underscore the high level of bacterial diversity in the gold mine. Our study provides information on the microbial diversity in Linglong gold mine and sheds light on the existence and potential function of bacteria in the gold biogeochemical cycling.     

Endophyte microbiome diversity in micropropagated Atriplex canescens and Atriplex torreyi var griffithsii

Microbial diversity associated with micropropagated Atriplex species was assessed using microscopy, isolate culturing, and sequencing. Light, electron, and confocal microscopy revealed microbial cells in aseptically regenerated leaves and roots. Clone libraries and tag-encoded FLX amplicon pyrosequencing (TEFAP) analysis amplified sequences from callus homologous to diverse fungal and bacterial taxa. Culturing isolated some seed borne endophyte taxa which could be readily propagated apart from the host. Microbial cells were observed within biofilm-like residues associated with plant cell surfaces and intercellular spaces. Various universal primers amplified both plant and microbial sequences, with different primers revealing different patterns of fungal diversity. Bacterial and fungal TEFAP followed by alignment with sequences from curated databases revealed 7 bacterial and 17 ascomycete taxa in A. canescens, and 5 bacterial taxa in A. torreyi. Additional diversity was observed among isolates and clone libraries. Micropropagated Atriplex retains a complex, intimately associated microbiome which includes diverse strains well poised to interact in manners that influence host physiology. Microbiome analysis was facilitated by high throughput sequencing methods, but primer biases continue to limit recovery of diverse sequences from even moderately complex communities.     

Nickel Tolerance and Accumulation by Filamentous Fungi From Sludge of Metal Finishing Industry

Industrialization, urbanization and increased vehicular traffic have resulted in increased contamination of our environment by heavy metals. The long persistence of heavy metals in nature has in turn resulted in development of metal resistant microbial strains. These strains are minimizing heavy metals toxicity, either by metal complexation or precipitation and other mechanisms. Characterization of fungal diversity was done in contaminated soil of the Wazirpur industrial area throughout the year. In this area highly acidic hazardous solid waste produced high concentration of heavy metals (Ni, Cu, Cr, Fe, Mn). Nickel toxicity is a major environmental concern. Due to long persistence of this waste in the environment without any treatment, many fungal isolates from the surrounding environment settle on the upper surface of waste. Few of them are capable of growing in the toxic conditions. More than 20 strains were isolated, most of them belonging to species of Aspergillus, Penicillium, Fusarium and Mucor genera. Seasonal variation in fungal diversity was significant. Four filamentous fungal isolates were found to be resistant for nickel (II) and a strain of Papulaspora sepedonoides reported first time for bioremediation of Ni (II) in this investigation, which is absorbing 62.33 μmol Ni gr^?1. These fungal isolates showed a high level (100–10000 mg kg^?1) of resistance for Ni (II) salt and removing Ni (II) from solution. Metal uptake varied with fungi. The toxicity also was influenced by different factors like pH and composition of growth medium.     

辽宁盘锦三角洲翅碱蓬根系及内生细菌群落多样性

【目的】翅碱蓬(Suaeda heteroptera)是一种典型的盐碱指示物,对重金属和石油污染的盐碱土壤有一定的修复作用,但是关于翅碱蓬根系与根系内生微生物之间的关系、微生物的多样性以及根系微生物在生物修复中所起作用的研究较少。本文以盘锦"红海滩"的翅碱蓬为例,研究翅碱蓬根系及根系内生细菌菌群种类和结构。【方法】通过传统的培养方法和非培养的高通量测序方法对翅碱蓬根系土壤样品、根系附着物样品以及根系匀浆样品中微生物群落多样性进行分析。【结果】传统方法共分离得到67株细菌,选择代表菌28株,根据其形态特征和生理生化特征,结合16S r RNA基因序列比对进行鉴定,它们分别属于盐单胞菌属(Halomonas)、海细菌属(Marinobacterium)、芽孢杆菌属(Bacillus)、副球菌属(Paracoccus)、假单胞菌属(Pseudomonas)、游动球菌属(Planococlus)、沙雷氏菌属(Serratia)、刘志恒菌属(Zhihengliuella)等。利用高通量测序技术对样品进行多样性分析,其中根系附着物样品的菌群丰度和多样性最高,依次分别为根系土壤样品和根系匀浆样品。3个样品中有效序列群落结构可分为12个门,包括酸杆菌门(Acidobacteria)、放线菌门(Actinobacteria)、拟杆菌门(Bacteroidetes)、绿菌门(Chlorobi)、绿弯菌门(Chloroflexi)、蓝菌门(Cyanobacteria)、厚壁菌门(Firmicutes)、芽单胞菌门(Gemmatimonadetes)、浮霉菌门(Planctomycetes)、变形菌门(Proteobacteria)、螺旋体门(Spirochaetes)和疣微菌门(Verrucomicrobia)。根系匀浆样品中蓝菌门为优势门类,占整个菌群的42%,变形菌门为次优势类群,占33%。变形菌门在根系附着物样品中为优势门类,占46%,拟杆菌门为次优势门类,占16%。根系土壤样品中拟杆菌门为优势门类,占整个菌群的37%,次优势类群为变形菌门,占20%。【结论】翅碱蓬根系和内生菌具有丰富的多样性,其根系微生物可能会在重金属和石油污染土壤的生物修复中起一定的修复作用。     

Quorum quenching in cultivable bacteria from dense marine coastal microbial communities

Acylhomoserine lactone (AHLs)-mediated quorum-sensing (QS) processes seem to be common in the marine environment and among marine pathogenic bacteria, but no data are available on the prevalence of bacteria capable of interfering with QS in the sea, a process that has been generally termed 'quorum quenching' (QQ). One hundred and sixty-six strains isolated from different marine dense microbial communities were screened for their ability to interfere with AHL activity. Twenty-four strains (14.4%) were able to eliminate or significantly reduce N-hexanoyl-l-homoserine lactone activity as detected by the biosensor strain Chromobacterium violaceum CV026, a much higher percentage than that reported for soil isolates, which reinforces the ecological role of QS and QQ in the marine environment. Among these, 15 strains were also able to inhibit N-decanoyl-l-homoserine lactone activity and all of them were confirmed to enzymatically inactivate the AHL signals by HPLC-MS. Active isolates belonged to nine different genera of prevalently or exclusively marine origin, including members of the Alpha- and Gammaproteobacteria (8), Actinobacteria (2), Firmicutes (4) and Bacteroidetes (1). Whether the high frequency and diversity of cultivable bacteria with QQ activity found in near-shore marine isolates reflects their prevalence among pelagic marine bacterial communities deserves further investigation in order to understand the ecological importance of AHL-mediated QS and QQ processes in the marine environment.     

小蓬竹根际土壤微生物及内生真菌多样性分析

为了解喀斯特典型物种-小蓬竹根际土壤微生物及不同部位内生真菌多样性,采用沿等高线等距离取样法采集小蓬竹根际土壤及健康植株,通过可培养对根际土微生物及内生菌进行分离,利用分子技术对其进行鉴定,根据鉴定结果构建系统发育树,并计算小蓬竹根际土壤微生物和根茎叶内生真菌多样性。结果如下：（1）共从根际土壤、根、茎、叶分离得到139个真菌菌株,隶属于27属,其中根际土壤分离得到34个真菌菌株隶属于12属,根部分离得到的63个内生真菌菌株隶属于17个属,茎部分离得到的14个内生真菌菌株隶属于8个属,叶部分离得到28个内生真菌菌株隶属于9个属;（2）根际土壤共分离得到41株细菌菌株,隶属于7个属26个种,20株放线菌菌株,隶属于1属15种;从Shannon-Wiener多样性指数、均匀度指数、Simpson指数排序来看,真菌主要表现为根 > 根际土壤 > 茎 > 叶,细菌和放线菌多样性均较低。（3）按层次聚类分析可分别将真菌、细菌、放线菌聚为3支。小蓬竹根际土壤、根、茎和叶具有丰富的微生物多样性,不同部位菌群组成存在差异性（P<0.05）,且存在以假单胞菌属、芽孢杆菌属等为优势属的抗盐耐旱菌群,这有助于揭示小蓬竹对喀斯特生境的适应性,以及为微生物-植物群落之间相互关系提供一定基础数据,为后期寻找小蓬竹相关耐性功能菌奠定基础。     

Diversity of bacteria associated with Bursaphelenchus xylophilus and other nematodes isolated from Pinus pinaster trees with pine wilt disease

The pinewood nematode (PWN), Bursaphelenchus xylophilus, has been thought to be the only causal agent of pine wilt disease (PWD), however, since bacteria have been suggested to play a role in PWD, it is important to know the diversity of the microbial community associated to it. This study aimed to assess the microbial community associated with B. xylophilus and with other nematodes isolated from pine trees, Pinus pinaster, with PWD from three different affected forest areas in Portugal. One hundred and twenty three bacteria strains were isolated from PWN and other nematodes collected from 14 P. pinaster. The bacteria strains were identified by comparative analysis of the 16S rRNA gene partial sequence. All except one gram-positive strain (Actinobacteria) belonged to the gram-negative Beta and Gammaproteobacteria. Most isolates belonged to the genus Pseudomonas, Burkholderia and to the family Enterobacteriaceae. Species isolated in higher percentage were Pseudomonas lutea, Yersinia intermedia and Burkholderia tuberum. The major bacterial population associated to the nematodes differed according to the forest area and none of the isolated bacterial species was found in all different forest areas. For each of the sampled areas, 60 to 100% of the isolates produced siderophores and at least 40% produced lipases. The ability to produce siderophores and lipases by most isolates enables these bacteria to have a role in plant physiological response. This research showed a high diversity of the microbial community associated with B. xylophilus and other nematodes isolated from P. pinaster with PWD.     

Culture-Dependent and Independent Studies of Microbial Diversity in Highly Copper-Contaminated Chilean Marine Sediments

Cultivation and molecular-based approaches were used to study microbial diversity in two Chilean marine sediments contaminated with high (835 ppm) and very high concentrations of copper (1,533 ppm). The diversity of cultivable bacteria resistant to copper was studied at oxic and anoxic conditions, focusing on sulfate-, thiosulfate-, and iron-reducing bacteria. For both sediments, the cultivable bacteria isolated at oxic conditions were mostly affiliated to the genus Bacillus, while at anoxic conditions the majority of the cultivable bacteria found were closely related to members of the genera Desulfovibrio, Sphingomonas, and Virgibacillus. Copper resistance was between 100 and 400 ppm, with the exception of a strain affiliated to members of the genus Desulfuromonas, which was resistant up to 1,000 ppm of copper. In parallel, cloning and sequencing of 16S rRNA was performed to study the total bacterial diversity in the sediments. A weak correlation was observed between the isolated strains and the 16S rRNA operational taxonomic units detected. The presence of copper resistance genes (copA, cusA, and pcoA) was tested for all the strains isolated; only copA was detected in a few isolates, suggesting that other copper resistance mechanisms could be used by the bacteria in those highly copper-contaminated sediments.