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Fungal oxidative exo-enzymes lacking substrate specificity play a central role in the cycling of soil organic matter. Due to their broad ecological impact and available knowledge of their gene structure, laccases appeared to be appropriate markers to monitor fungi with this kind of oxidative potential in soils. A degenerate PCR-primer pair Cu1F/Cu2R, specific for basidiomycetes, was designed to assess directly the diversity of laccase genes in soils. PCR amplification of mycelial cultures and fruit-bodies of a wide spectrum of basidiomycetes, covering all functional groups (saprophytes, symbionts, and pathogens), produced multiple DNA fragments around 200 bp. A neighbor-joining tree analysis of the PCR-amplified laccase sequences showed a clear species-specificity, but also revealed that most fungal taxa possess several laccase genes showing a large sequence divergence. This sequence diversity precluded the systematic attribution of amplified laccase of unknown origin to specific taxa. Amplification of laccase sequences from DNA, extracted from a brown (moder) forest soil, showed a specific distribution of laccase genes and of the corresponding fungal species in the various soil horizons (Oh, Ah, Bv). The most organic Oh-horizon displayed the highest gene diversity. Saprophytic fungi appeared to be less widespread through the soil horizons and displayed a higher diversity of laccase genes than the mycorrhizal ones.  相似文献   

3.
Laccases- or laccase-like multicopper oxidases (LMCO) catalyze the oxidation of various substrates, such as phenols, diamines and metals, coupled with the reduction of molecular oxygen to water. Compared to studies on function and diversity of LMCO in plants and fungi, little is known about this enzyme type in bacteria and especially on their possible implication in degradation of organic matter in soils. This study presents a molecular investigation of the diversity and distribution of bacterial LMCO genes among three upper horizons of a forest Cambisol and in a grassland Cambisol. Some culture strains of soil bacteria were also analyzed at the molecular level and for their capability to oxidize naturally occurring 2,6-dimethoxyphenol, a LMCO substrate. A high LMCO gene diversity was found in the Cambisol soil samples with 16 distinct sequence type clades, of which approximately one half was not matching with any reference sequence of known bacteria. The highest richness of bacterial LMCO genes was observed in the organic horizon of the forest soil, which is concomitant with a previous analysis of the diversity of fungal laccase genes and corresponding soil laccase activity. Some clusters of sequence types showed a specific distribution in one of the soils or in horizons, while others appeared more ubiquist. Multiple bacterial LMCO genes were described in Agromyces salentinus and Sinorhizobium morelense, what so far was only known from fungi.  相似文献   

4.
Black soils (Mollisols) are one of the most important soil resources for maintaining food security in China, and they are mainly distributed in northeast China. A previous comprehensive study revealed the biogeographical distribution patterns of bacterial communities in the black soil zone. In this study, we used the same soil samples and analyzed the 454 pyrosequencing data for the nuclear ribosomal internal transcribed spacer (ITS) region to examine the fungal communities in these black soils. A total of 220,812 fungal ITS sequences were obtained from 26 soil samples that were collected across the black soil zone. These sequences were classified into at least 5 phyla, 20 classes, greater than 70 orders and over 350 genera, suggesting a high fungal diversity across the black soils. The diversity of fungal communities and distribution of several abundant fungal taxa were significantly related to the soil carbon content. Non-metric multidimensional scaling and canonical correspondence analysis plots indicated that the fungal community composition was most strongly affected by the soil carbon content followed by soil pH. This finding differs from the bacterial community results, which indicated that soil pH was the most important edaphic factor in determining the bacterial community composition of these black soils. A variance partitioning analysis indicated that the geographic distance contributed 20% of the fungal community variation and soil environmental factors that were characterized explained approximately 35%. A pairwise analysis revealed that the diversity of the fungal community was relatively higher at lower latitudes, which is similar to the findings for the bacterial communities in the same region and suggests that a latitudinal gradient of microbial community diversity might occur in the black soil zone. By incorporating our previous findings on the bacterial communities, we can conclude that contemporary factors of soil characteristics are more important than historical factor of geographic distance in shaping the microbial community in the black soil zone of northeast China.  相似文献   

5.
This study is the first approach to evaluate the diversity of bacterial β-glucosidase-encoding gene sequences, aiming to identify the main environmental factors structuring bacterial β-glucosidase genetic diversity in semiarid soils. Two agricultural management systems, soils under spontaneous cover vegetation vs. noncovered herbicide-treated soils, were tested. The weed biomass generated in the former was estimated around 2,600 kg?ha?1?year?1, whereas leaves and root exudates from olive trees were the only input of C biomass in the latter. Dendrograms generated from polymerase chain reaction–denaturing gradient gel electrophoresis profiles of bacterial β-glucosidase-encoding genes revealed two clusters determined by soil treatment and sharing <20 % similarity. The sequences of a total of 59 DNA fragments, representing 39 operational taxonomic units, were successfully determined. The Proteobacteria phylum clearly dominated all the soil samples, but representatives of Chloroflexi, Deinococci, Actinobacteria, Thermotogae, and Firmicutes class were also detected. Management strategies favoring the presence of spontaneous vegetation determined a higher genetic diversity of β-glucosidase-encoding genes of soil bacteria. However, since there is little information of β-glucosidase gene sequences available in databases, it is difficult to establish particular relationships between bacterial networks for C degradation and land use. Results from canonical correspondence analysis indicated that bacterial metabolic networks for oligomeric C substrates utilization were affected by the physicochemical properties of the soil; the uppermost 10 cm of covered soil clustered together and were positively correlated with some chemical properties related to soil fertility, whereas less influence of soil texture was observed for the deeper layers of bare soils.  相似文献   

6.
土壤微生物对无机氮的固持作用是构成土壤保氮机制的重要组成。作为土壤微生物的两大主要类群,真菌和细菌是微生物固持无机氮作用的主要参与者。然而,由于土壤微生物的高度复杂多变性,如何有效区分和量化土壤中真菌和细菌各自对无机氮的固持作用是个难题。针对该问题,本文采用“氨基糖稳定同位素探针(AS-SIP)”技术来区分和表征土壤中真菌、细菌各自对无机氮的固持速率。基于此进一步揭示了农业利用和外源碳输入分别对土壤真菌、细菌各自固持硝态氮作用的影响及原因,构建了土壤中真菌、细菌各自固持无机氮实际速率的估算模型,为区分和量化土壤中真菌、细菌各自对无机氮的实际固持速率提供了更为可信的新方法。本文介绍了AS-SIP 技术原理、主要技术优势、应用案例、不足之处以及改进对策,以期推进该方法的应用和发展。  相似文献   

7.
Revegetation of the tailings dumps produced by various industrial activities is necessary to prevent dust storms and erosion and represents a great challenge for ecological restoration. Little is known about the microbial colonisation and community structure of revegetated tailings following site exploitation. Here, we report the sequencing of 16S rRNA and internal transcribed spacer (ITS) fungal RNA gene amplicons from chlor-alkali residue and from an adjacent undisturbed soil to define the composition and assembly of the rhizosphere microbial communities. After quality filtering, a total of 72,373 and 89,929 bacterial sequences and 122,618 and 111,209 fungal sequences remained for community analysis from undisturbed soil and tailings dump samples, respectively. These reads were affiliated with 45 bacterial and 9 fungal phyla and 113 bacterial and 35 fungal classes. We observed a clear dominance of Gammaproteobacteria at our study site (24% of total sequences), especially of the Pseudomonas genera (72% of Gammaproteobacteria sequences), together with the dominance of a few fungal taxa, such as Hebeloma and Geopora. However, we also noticed that the core microbiome comprised 64.4% and 62.4% of the bacterial and fungal genera, respectively, despite marked differences in soil physico-chemical properties. A heatmap of correlations between soil parameters and taxa confirmed that approximately 50% of the 33 dominant taxa colonised both types of soil. We further demonstrated that the global bacterial-fungal network topology of the dump approximated that of the undisturbed soil. Our approach illuminates the importance of studying more than just a single component of the microbial community and represents a step forward in uncovering the microbial ecology of disturbed environments beyond what is generally found in conventional studies. Our study also provides novel global community proxies that have led us to conclude that environmental filtering is more likely to occur through the activity of tree roots rather than as a result of specific soil characteristics and could be an important force in the assembly of at least some microbial communities.  相似文献   

8.
Background, aim, and scope  Fertilization is an important agricultural practice for increasing crop yields. In order to maintain the soil sustainability, it is important to monitor the effects of fertilizer applications on the shifts of soil microorganisms, which control the cycling of many nutrients in the soil. Here, culture-dependent and culture-independent approaches were used to analyze the soil bacterial and fungal quantities and community structure under seven fertilization treatments, including Control, Manure, Return (harvested peanut straw was returned to the plot), and chemical fertilizers of NPK, NP, NK, and PK. The objective of this study was to examine the effects on soil microbial composition and diversity of long-term organic and chemical fertilizer regimes in a Chinese upland red soil. Materials and methods  Soil samples were collected from a long-term experiment station at Yingtan (28°15′N, 116°55′E), Jiangxi Province of China. The soil samples (0–20 cm) from four individual plots per treatment were collected. The total numbers of culturable bacteria and fungi were determined as colony forming units (CFUs) and selected colonies were identified on agar plates by dilution plate methods. Moreover, soil DNAs were extracted and bacterial 16S rRNA genes and fungal 18S rRNA genes were polymerase chain reaction amplified, and then analyzed by denaturing gradient gel electrophoresis (DGGE), cloning, and sequencing. Results  The organic fertilizers, especially manure, induced the least culturable bacterial CFUs, but the highest bacterial diversity ascertained by DGGE banding patterns. Chemical fertilizers, on the other hand, had less effect on the bacterial composition and diversity, with the NK treatment having the lowest CFUs. For the fungal community, the manure treatment had the largest CFUs but much fewer DGGE bands, also with the NK treatment having the lowest CFUs. The conventional identification of representative bacterial and fungal genera showed that long-term fertilization treatments resulted in differences in soil microbial composition and diversity. In particular, 42.4% of the identified bacterial isolates were classified into members of Arthrobacter. For fungi, Aspergillus, Penicillium, and Mucor were the most prevalent three genera, which accounted for 46.6% of the total identified fungi. The long-term fertilization treatments resulted in different bacterial and fungal compositions ascertained by the culture-dependent and also the culture-independent approaches. Discussion  It was evident that more representative fungal genera appeared in organic treatments than other treatments, indicating that culturable fungi were more sensitive to organic than to chemical fertilizers. A very notable finding was that fungal CFUs appeared maximal in organic manure treatments. This was quite different from the bacterial CFUs in the manure, indicating that bacteria and fungi responded differently to the fertilization. Similar to bacteria, the minimum fungal CFUs were also observed in the NK treatment. This result provided evidence that phosphorus could be a key factor for microorganisms in the soil. Thus, despite the fact that culture-dependent techniques are not ideal for studies of the composition of natural microbial communities when used alone, they provide one of the more useful means of understanding the growth habit, development, and potential function of microorganisms from soil habitats. A combination of culture-dependent and culture-independent approaches is likely to reveal more complete information regarding the composition of soil microbial communities. Conclusions  Long-term fertilization had great effects on the soil bacterial and fungal communities. Organic fertilizer applications induced the least culturable bacterial CFUs but the highest bacterial diversity, while chemical fertilizer applications had less impact on soil bacterial community. The largest fungal CFUs were obtained, but much lower diversity was detected in the manure treatment. The lowest bacterial and also fungal CFUs were observed in the NK treatment. The long-term fertilization treatments resulted in different bacterial and fungal compositions ascertained by the culture-dependent and also the culture-independent approaches. Phosphorus fertilizer could be considered as a key factor to control the microbial CFUs and diversity in this Chinese upland red soil. Recommendations and perspectives  Soil fungi seem to be a more sensitive indicator of soil fertility than soil bacteria. Since the major limitation of molecular methods in soil microbial studies is the lack of discrimination between the living and dead, or active and dormant microorganisms, both culture-dependent and culture-independent methods should be used to appropriately characterize soil microbial diversity.  相似文献   

9.
Fungal denitrification in soils is receiving considerable attention as one of the dominant N2O production processes. However, because of the lack of a methodology to detect fungal denitrification-related genes, the diversity and ecological behavior of denitrifying fungi in soil remains unknown. Thus, we designed a primer set to detect the fungal nitrite reductase gene (nirK) and validated its sensitivity and specificity. Through clone library analyses, we identified congruence between phylogenies of the 18S rRNA gene and nirK of denitrifying fungal isolates obtained from the surface-fertilized cropland soil and showed that fungi belonging to Eurotiales, Hypocreales, and Sordariales were primarily responsible for N2O emissions in the soil.  相似文献   

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Several biochemical and molecular methods are used to investigate the microbial diversity and changes in microbial community structure in rhizospheres and bulk soils resulting from changes in management. We have compared the effects of plants on the microbial community, using several methods, in three different types of soils. Pots containing soil from three contrasting sites were planted with Lolium perenne (rye grass). Physiological (Biolog), biochemical (PLFA) and molecular (DGGE and TRFLP) fingerprinting methods were employed to study the change in soil microbial communities caused by the growth of rye grass. Different methods of DNA extraction and nested PCR on TRFLP profiles were examined to investigate whether they gave different views of community structure. Molecular methods were used for both fungal and bacterial diversity. Principal component analysis of Biolog data suggested a significant effect of the plants on the microbial community structure. We found significant effects of both soil type and plants on microbial communities in PLFA data. Data from TRFLP of soil bacterial communities showed large effects of soil type and smaller but significant effects of plants. Effects of plant growth on soil fungal communities were measured by TRFLP and DGGE. Multiple Procrustes analysis suggested that both methods gave similar results, with only soil types having a significant effect on fungal communities. However, TRFLP was more discriminatory as it generated more ribotype fragments for each sample than the number of bands detected by DGGE. Neither methods of DNA extraction nor the nested PCR had any effect on the evaluation of soil microbial community structure. In conclusion, the different methods of microbial fingerprinting gave qualitatively similar results when samples were processed consistently and compatible statistical methods used. However, the molecular methods were more discriminatory than the physiological and biochemical approaches. We believe results obtained from this experiment will have a major impact on soil microbial ecology in general and rhizosphere–microbial interaction studies in particular, as we showed that the different fingerprinting methods for microbial communities gave qualitatively similar results.  相似文献   

12.
Verrucomicrobia are ubiquitous in soil, but members of this bacterial phylum are thought to be present at low frequency in soil, with few studies focusing specifically on verrucomicrobial abundance, diversity, and distribution. Here we used barcoded pyrosequencing to analyze verrucomicrobial communities in surface soils collected across a range of biomes in Antarctica, Europe, and the Americas (112 samples), as well as soils collected from pits dug in a montane coniferous forest (69 samples). Data collected from surface horizons indicate that Verrucomicrobia average 23% of bacterial sequences, making them far more abundant than had been estimated. We show that this underestimation is likely due to primer bias, as many of the commonly used PCR primers appear to exclude verrucomicrobial 16S rRNA genes during amplification. Verrucomicrobia were detected in 180 out of 181 soils examined, with members of the class Spartobacteria dominating verrucomicrobial communities in nearly all biomes and soil depths. The relative abundance of Verrucomicrobia was highest in grasslands and in subsurface soil horizons, where they were often the dominant bacterial phylum. Although their ecology remains poorly understood, Verrucomicrobia appear to be dominant in many soil bacterial communities across the globe, making additional research on their ecology clearly necessary.  相似文献   

13.
Different primer systems have been developed to characterize arbuscular mycorrhizal fungal (AMF) communities; however, a direct comparison of their specificity, potential to describe diversity and representation of different phylogenetic lineages is lacking. Using seven root samples, we compared four routinely used AMF-specific primer systems for nuclear ribosomal DNA covering i) the partial small subunit (SSU), ii) the partial large subunit (LSU), iii) the partial SSU and internal transcribed spacer (ITS; “Redecker”) and iv) the partial SSU–ITS–partial LSU region (“Krüger”). In addition, a new primer combination v) covering the ITS2 region (ITS2) was included in the comparison. The “Krüger” primers tended to yield the highest AMF diversity and showed a significantly higher Shannon diversity index than the SSU primers. We found a strong bias towards the Glomeraceae in the LSU and SSU primer systems and differences in the composition of AMF communities based on the “Redecker” primer system. Our results confirm the crucial role of the choice of target rRNA marker region for analysing AMF communities. We also provide evidence that nested-PCR based data can be interpreted semi-quantitatively and that the extent of observed AMF community overdominance largely depends on the choice of primer.  相似文献   

14.
Microbial ecology is the key to understanding the function of biodiversity for organic matter cycling in the soil. We have investigated the impacts of farmyard manure added over 120 years on organic matter content, enzyme activities, total microbial biomass and structure of microbial populations in several particle‐size fractions of a Luvic Phaeozem a few kilometres northeast of Halle, Germany. We compared two treatments: no fertilization (control) and 12 t farmyard manure (FYM) ha?1 year?1 since 1878. The fine fractions contained most C and N, microbial biomass, total amount of phospholipid fatty acids (PLFAs) and greatest invertase activity. Xylanase activity as well as fungal biomass increased only gradually with diminishing particle size, whereas the relative abundance of fungi decreased with diminishing particle size. The least diversity of the soil microbial community, indicated by the smallest Shannon index based on the abundance and amount of different PLFAs and small number of terminal restriction fragments (T‐RFs) of 16S rRNA genes, was in the sand fractions. The results supported the hypothesis that this microhabitat is colonized by a less complex bacterial community than the silt and clay fractions. Addition of FYM had enhanced the amount of organic matter, total microbial biomass, and xylanase and invertase activity, and induced a shift of the microbial community towards a more bacteria‐dominated community in the coarse sand fraction. Microbial communities in finer fractions were less affected by addition of FYM.  相似文献   

15.
陈利军  孙波  金辰  蒋瑀霁  陈玲 《土壤》2015,47(2):340-348
施用有机肥是快速培育瘠薄土壤的一个重要措施。针对中亚热带第四纪红黏土发育的红壤旱地,建立了玉米和花生单作系统等碳量投入有机肥和生物炭的田间试验,利用聚合酶链式反应—变性梯度凝胶电泳(polymerase chain reaction-denaturing gradient gel electrophoresis,PCR-DGGE)方法研究了土壤细菌和真菌群落组成和多样性的变化,分析了土壤呼吸速率(CO2通量)的变化及其与微生物多样性的关系。两年的试验表明,不同施肥方式导致微生物群落结构显著分异,施用有机肥和生物炭显著增加了细菌多样性,但施肥第二年真菌多样性有下降趋势。秸秆和猪粪配施显著增加了土壤呼吸速率,土壤呼吸速率与细菌和真菌多样性呈显著正相关,细菌多样性对土壤呼吸的影响(相对贡献率为71%)显著高于真菌(29%)。土壤磷素(全磷和速效磷)含量的变化是驱动红壤微生物多样性变化的主导因素,其对细菌和真菌多样性的相对贡献率分别为44.8%和47.4%。因此,合理配施秸秆和猪粪可以快速提高瘠薄红壤的生物功能。  相似文献   

16.
The abundance and diversity of soil bacterial and fungal communities in a wheat field under elevated atmospheric CO2 concentrations and increased air temperatures were investigated using qPCR and pyrosequencing. Elevated CO2 concentrations significantly increased the abundances of bacteria and fungi, and an increase of air temperatures significantly reduced fungal abundance. We found that Proteobacteria, Bacteroidetes, Chloroflexi, and Ascomycota were the most abundant bacterial and fungal phyla in the wheat field soil. Elevated CO2 concentrations and increased air temperatures had no significant effect on the bacterial alpha diversity, whereas fungal richness was reduced under warming treatments. Moreover, we note that certain bacterial and fungal groups responded differentially to elevated CO2 concentrations and increased air temperatures, and fungal species were highly sensitive to climatic changes.  相似文献   

17.
土壤微生物多样性研究的新方法   总被引:34,自引:6,他引:34       下载免费PDF全文
传统的分离培养和鉴定土壤微生物方法所具有的困难性和局限性 ,是造成难以深入了解土壤微生物生态学特性和多样性组成方面的主要障碍。本文运用分子生物学技术 ,以澳大利亚两种主要森林类型的土壤微生物多样性研究为实例 ,介绍了从土壤中直接提取土壤微生物DNA的方法以及末端限制性酶切片段长度多态性 (T RFLP)分析的基本原理和方法。作者认为 ,用该方法提取的土壤真菌DNA的纯度高 ,完全适合PCR扩增和T RFLP分析的要求。T RFLP已成为国外深入研究土壤微生物多样性的理想方法之一  相似文献   

18.
As part of the restoration of biodiversity on former agricultural land there has been focused on methods to enhance the rate of transition from agricultural land towards natural grasslands or forest ecosystems. Management practices such as sowing seed mixtures and inoculating soil of later successional stages have been used. The aim of this study was to determine the effects of a managed plant community on the diversity of soil fungi in a newly abandoned agricultural land. A field site was set up consisting of 20 plots where the plant diversity was managed by either sowing 15 plant species, or natural colonization was allowed to occur. The plant mixture contained five species each of grasses, legumes and forbs that all were expected to occur at the site. A subset of the plots (five from each treatment) was inoculated with soil cores from a late successional stage. The plant community composition was subject to a principal component analysis based on the coverage of each species. Five years after abandonment, soil samples were taken from the plots, DNA was extracted and the ITS region of the rDNA gene was amplified using fluorescently labelled fungal specific primers (ITS 1F/ITS 4). The PCR products were digested using HinfI and TaqI and sequenced. Results from both restriction enzymes were combined and a principal component analysis performed on the presence/absence of fragments. Also the fungal diversity expressed as number of restriction fragments were analysed. There was significantly higher fungal species richness in the experimental plots compared to the forest and field soils, but no differences between sown and naturally colonized plots. The different plant treatments did not influence the below ground fungal community composition. Soil water content on the other hand had an impact on the fungal community composition.  相似文献   

19.
Lichens form the dominant plant cover in extreme environments and participate in mineral weathering, fine-earth stabilization and primary accumulation of soil organic matter. However, biochemical role of lichens in soil processes has never been investigated. Recently, laccases and tyrosinases have been discovered in representatives of the order Peltigerales (Laufer et al., 2006a, b; Zavarzina and Zavarzin, 2006). Laccases from most species had unusually large molecular weights (Laufer et al., 2009). Together with oligomeric laccases, we have found monomeric enzymes in Solorina crocea and Peltigera aphthosa (Lisov et al., 2007). In the present work we have purified homodimeric (large) and monomeric (small) laccases of the soil-stabilizing lichen S. crocea, determined their physico-chemical and catalytic properties and studied their reactions with soil humic acids. Our results suggest that oligomeric nature of lichen laccases can be artifactual, because homodimeric laccase was transformed into the monomeric form following hydrophobic interaction chromatography. We hypothesize that large laccase consists of two monomeric enzymes, each of which is bound with additional hydrophobic component(s). Small laccase is similar in its properties to the laccases of basidiomycetes. It is more resistant to elevated temperature and storage than the large form, showed a higher oxidation potential, had different pH-optima in oxidizing substrates and was less inhibited by humic acids. Despite these differences, both laccases depolymerized and decolorized humic acids from soils at comparable rates, with small laccase being slightly more effective. This finding suggests that lichens have a potential to participate in transformation of soil organic matter.  相似文献   

20.
In the southern Great Plains (USA), encroachment of grassland ecosystems by Prosopis glandulosa (honey mesquite) is widespread. Mesquite encroachment alters net primary productivity, enhances stores of C and N in plants and soil, and leads to increased levels of soil microbial biomass and activity. While mesquite’s impact on the biogeochemistry of the region is well established, it effects on soil microbial diversity and function are unknown. In this study, soils associated with four plant types (C3 perennial grasses, C4 midgrasses, C4 shortgrasses, and mesquite) from a mesquite-encroached mixed grass prairie were surveyed to in an attempt to characterize the structure, diversity, and functional capacity of their soil microbial communities. rRNA gene cloning and sequencing were used in conjunction with the GeoChip functional gene array to evaluate these potential differences. Mesquite soil supported increased bacterial and fungal diversity and harbored a distinct fungal community relative to other plant types. Despite differences in composition and diversity, few significant differences were detected with respect to the potential functional capacity of the soil microbial communities. These results may suggest that a high level of functional redundancy exists within the bacterial portion of the soil communities; however, given the bias of the GeoChip toward bacterial functional genes, potential functional differences among soil fungi could not be addressed. The results of this study illustrate the linkages shared between above- and belowground communities and demonstrate that soil microbial communities, and in particular soil fungi, may be altered by the process of woody plant encroachment.  相似文献   

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