Application of polymerase chain reaction-denaturing gradient gel electrophoresis for comparison of direct and indirect extraction methods of soil DNA used for microbial community fingerprinting

 We used polymerase chain reaction-denaturing gradient gel electrophoresis (PCR-DGGE) to compare bacterial community patterns obtained with target DNA extracted from a soil by direct and indirect methods. For this purpose, two direct extraction methods, i.e. cell lysis by bead beating and cell disruption by grinding in liquid N, and two indirect methods, i.e. cell extraction followed by DNA extraction, and combined RNA/DNA extraction from the bacterial cell fraction, were performed. Crude extracts were purified and amplified using universal bacterial primers. PCR products were then analysed by DGGE, and similarity between the profiles obtained was determined by unweighted pair group with mathematical averages clustering. The results showed clear profiles that presumably represented the dominant bacterial fractions in the samples. The profiles generated by all four methods were similar, indicating that the methods were of approximately equal efficiency in the extraction of target DNA representative of the soil bacterial community. However, the patterns of clustering also indicated that different populations of bacteria could be detected in the same soil using different soil DNA extraction methods. The application of two dilution levels of DNA in PCR-DGGE showed that the most stable profile of the soil bacterial community could be generated by the direct methods. The indirect methods gave clustered profiles at both dilution levels. It is likely that these methods extracted DNA from a major, easily desorbed, bacterial fraction, consisting of low-density populations. PCR-DGGE was found to be a suitable technique with which to assess differences in methods for DNA extraction from soil, which can be further used for the determination of microbial community diversity at the molecular level. Received: 22 June 1999     

Soil DNA extraction and assessment of the fate of Mycobacterium chlorophenolicum strain PCP-1 in different soils by 16S ribosomal RNA gene sequence based most-probable-number PCR and immunofluorescence

Since Mycobacterium chlorophenolicum strain PCP-1 is not detectable in soil by selective plating, a specific tracking method was based on the polymerase chain reaction (PCR) using soil DNA as a target. A direct extraction protocol based on bead beating was adapted and used to obtain PCR-amplifiable DNA from five different soils. In one soil, the disruption of cells of PCP-1, of Pseudomonas fluorescens R2f and of Paenibacillus azotofixans P3L5, as well as of the indigenous bacteria increased with increasing bead beating times. After 4.5 min, lysis efficiency was about 90% or more in all cases. Total DNA yields varied between soils, from 2 to 35 μg g^–1. The purification steps needed to obtain amplifiable DNA were different per soil. To detect target DNA specifically in bacterial cells, a new indirect extraction protocol was developed, which efficiently dislodged bacterial cells from the soil matrix, and produced amplifiable DNA with high yield. To detect strain PCP-1 in soil, 16S ribosomal gene-based PCR combined with oligonucleotide hybridization was applied using a most-probable-number (MPN) set-up, whereas immunofluorescence was used for calibration. Strain PCP-1 was detected shortly after introduction into three soils at about the inoculum levels, as evidenced by both approaches. Both the direct and indirect DNA extraction methods yielded similar MPN estimates. The dynamics of M. chlorophenolicum PCP-1 was estimated in two soils over 14 days via MPN-PCR/oligonucleotide probing. PCP-1 showed good survival in both soils, and results obtained by MPN-PCR with directly and indirectly extracted DNA were internally consistent. Immunofluorescence cell enumerations supported the gross stability of PCP-1 in these two as well as in two additional soils. Received: 8 February 1996     

Optimizing the indirect extraction of prokaryotic DNA from soils

The objective of this work was to develop protocols to selectively extract prokaryotic DNA from soils, representative of the whole community, amenable to high-throughput whole genome shotgun sequencing. Prokaryotic cells were extracted from soils by blending, followed by gradient centrifugation. Detergent (sodium deoxycholate) was required for complete dispersion of soil aggregates and detachment of prokaryotic cells from a broad range of soil types. Repeated extractions of a given soil sample were critical to maximize cell yield. Furthermore, cells obtained through repeated extractions captured unique prokaryotic assemblages that would otherwise have been missed in single-pass extractions. DNA was isolated from extracted cells using one of the following treatments: i) lysozyme-SDS-proteinase K (enzymatic) digestion; ii) potassium ethyl xanthogenate digestion; or iii) enzymatic digestion of cells embedded in agarose plugs. In addition, these methods were compared to a commercial bead-beating extraction kit (MoBio UltraClean). Of the indirect DNA extraction methods, plug digestion generated the largest yields (up to 70% of yields obtained by direct DNA extraction) of high-molecular weight DNA (>400 kb). Thus, plug digestion is amenable to large-insert metagenomic library construction and analysis. Comparisons of banding patterns generated by RAPD-PCR and DGGE indicated that sequence composition and inferred community composition of a given extract varied greatly with DNA isolation method. While overall diversity did not change significantly with the cell lysis method, analysis of 16S rRNA gene clone libraries revealed that each extraction procedure produced unique distributions of prokaryotic phyla within the sample population.     

Investigating microbial community structure in soils by physiological, biochemical and molecular fingerprinting methods

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.     

Distribution of microbial communities in a forest soil profile investigated by microbial biomass, soil respiration and DGGE of total and extracellular DNA

We studied the distribution of the indigenous bacterial and fungal communities in a forest soil profile. The composition of bacterial and fungal communities was assessed by denaturing gradient gel electrophoresis (DGGE) of total and extracellular DNA extracted from all the soil horizons. Microbial biomass C and basal respiration were also measured to assess changes in both microbial biomass and activity throughout the soil profile. The 16S rDNA-DGGE revealed composite banding patterns reflecting the high bacterial diversity as expected for a forest soil, whereas 18S rDNA-DGGE analysis showed a certain stability and a lower diversity in the fungal communities. The banding patterns of the different horizons reflected changes in the microbial community structure with increasing depth. In particular, the DGGE analysis evidenced complex banding patterns for the upper A1 and A2 horizons, and a less diverse microflora in the deeper horizons. The low diversity and the presence of specific microbial communities in the B horizons, and in particular in the deeper ones, can be attributed to the selective environment represented by this portion of the soil profile. The eubacterial profiles obtained from the extracellular DNA revealed the presence of some bands not present in the total DNA patterns. This could be interpreted as the remainders of bacteria not any more present in the soil because of changes of edaphic conditions and consequent shifting in the microbial composition. These characteristic bands, present in all the horizons with the exception of the A1, should support the concept that the extracellular DNA is able to persist within the soil. Furthermore, the comparison between the total and extracellular 16S rDNA-DGGE profiles suggested a downwards movement of the extracellular DNA.     

The impact of increasing heavy metal stress on the diversity and structure of the bacterial and actinobacterial communities of metallophytic grassland soil

A study was undertaken to investigate the bacterial community found in metallophytic grassland soil contaminated with Zn and Pb. We hypothesised that such communities would be tolerant of additional heavy metal stress due to phylogenetic and functional adaptation. In microcosm experiments, lasting 51 days, denaturing gradient gel electrophoresis (DGGE) analyses was used to compare the total bacterial and actinobacterial communities in non-amended soils and those to which additional Pb and Zn concentrations were added. There was a decrease in total bacterial diversity with each addition of Pb and Zn; in contrast, the actinobacterial community diversity remained unaffected. The community structures were analysed using multivariate analyses of the DGGE profiles. Total bacterial community profiles showed two distinct groups sharing less than 80% similarity, irrespective of Pb and Zn addition. The first contained profiles sampled during the first 7 days of the experiment; the second contained those sampled from day 10 onwards. Actinobacterial profiles from those that were non-amended showed a similar distribution to those of the total bacterial community. However, in soil amended with fivefold additional Pb and Zn, all the profiles shared more than 80% similarity. Raup and Crick analyses suggested that total bacterial soil communities were subject deterministic selection becoming significantly similar as the experiment progressed, but this was inhibited by the highest concentration of additional Pb and Zn. Actinobacterial communities showed a similar response but were less affected by elevated Pb and Zn concentrations. These data indicate that the diversity of the actinobacterial community was not negatively affected by additional heavy metal stress in contrast to total bacterial community diversity.     

Single particle analysis reveals that bacterial community structures are semi-specific to the type of soil particle

Abstract

With the aim of understanding how diverse bacteria are distributed within a heterogeneous soil, we compared local bacterial communities on individual soil particles. We picked up 11 coarse sand-sized particles (quartz, whitish feldspar, yellow feldspar or unidentified brown particles) from a sandy soil, extracted DNA from each particle, and carried out partial 16S rDNA polymerase chain reaction denaturing gradient gel electrophoresis analysis. Bacterial communities located on soil particles of the same type were more similar in composition than communities located on particles of the other types. Thus, the local structure of a bacterial community is related to the type of soil particle, which suggests that a high diversity of soil bacteria emerges through a combination of local bacterial communities on different types of soil particles.     

Culture-dependent and culture-independent microbial investigation of pine litters and soil in subtropical Australia

Background, aim, and scope  Forest plantations, widely grown for wood production, involve the selective promotion of single-tree species or replacement of natural species by exotic tree species. Slash pine (Pinus elliottii) has been chosen for reforestation of infertile sandy soils in southeast Queensland, Australia. These exotic pine plantations minimize soil and water losses and are important scientific study sites. The soil environment of these plantations, though devoid of sufficient nutrients, organic carbon and other factors, harbors innumerable bacteria that may play a crucial role in maintaining soil quality and ecosystem functions. These soil microorganisms also have the potential for use as sensitive biological indicators to reflect environmental changes. It is therefore essential to understand the interrelationships among bacterial communities and their environment by assessing their structural and functional diversity and their responses to disturbances. The main aim of our investigation was to determine the diversity of bacterial communities in forest litters and soil during the forest leaf litter decomposition using culture-dependent and culture-independent techniques. Materials and methods  A 25-cm (diameter) × 40-cm core sample was collected and fractionated into three subsamples designated E1 (L leaf litter layer), E2 (F leaf litter layer), and E5 (0–10 cm soil layer). Both culture-dependent and culture-independent methods were applied in this study. In the culture-independent study, a strategy of whole-community DNA extraction, polymerase chain reaction (PCR) amplification followed by cloning and 16S rDNA sequence analysis was used; for culture-dependent study, the strategy included sample plating and bacteria isolating, DNA extraction, PCR amplification, and 16S rDNA sequence analysis. The diversity similarities between two bacterial communities and two methods are quantified using Jensen–Shannon divergence. Results  From culture-dependent study, 336 colonies in total were isolated and grouped from the three subsamples, and the 16S rRNA sequence analysis from a representative isolate from each morphogroup (21 isolates) indicated that they belonged to the phyla Actinobacteria, Firmicutes, and Proteobacteria. Culture-independent assessment based on 16S rRNA gene library comprising 194 clones revealed that members of the phylum Actinobacteria were absent in the culture-independent studies. Clones in libraries from E1 consisted exclusively of members of the Firmicutes. The majority of clones from E2 were related to Firmicutes (79%) and Proteobacteria (21%). Clones derived from E5 were mostly affiliated with Acidobacterium (42%), followed by unclassified bacteria (27%), Verrucomicrobiales (12%), Proteobacteria (11%), and Planctomycetes (8%). Discussion  This study showed that bacterial culturabilities in different fractions of leaf litters were similar, and both of them were higher than the bacterial culturability in the soil. Unculturable bacterial diversity in the soil, however, was much higher than the leaf litter bacterial diversity. The bacterial diversity on the top layer of leaf litters was slightly less than that on the bottom layer of leaf litters. This might indicate that forest soils are a more complex environment than leaf litters are and also that they might inhabit more unculturable microorganisms in the forest soils, which would need to be further investigated. The leaf litter layer samples also demonstrate the significant difference between the bacterial community diversity discovered by these two methods in this study. The information provided by assessing the different fractions of leaf litters and forest soil has improved our understanding of the bacterial community distributions within the forest soil and the above-leaf litters in an exotic pine plantation of subtropical Australia. Conclusions  This study represents the first attempt to examine the bacterial community in the different fractions of forest leaf litters and soil in subtropical Australia. The data from this study show that the 16S rDNA clone libraries provided more comprehensive phylogenetic diversity in the soil and leaf litter samples than the culture collections provided, and both the culture-dependent and culture-independent studies revealed that the bacterial diversity present in the leaf litters was very different to that present in the soil. The comparative analysis of bacterial communities in different fractions of leaf litters and soil samples has also provided important baseline information about the bacterial diversity and composition in the exotic pine forest plantations. Recommendations and perspectives  The experimental data provided important information on the bacterial diversity in forest leaf litter and soil samples, though additional surveys and comparisons at different locations would be needed to further characterize. In addition, combined methods that can provide different parts of information on bacterial diversity are encouraged to be used in bacterial community study. The established libraries of diverse 16S rRNA gene fragments from slash pine leaf litters and forest soil can be used to construct specific DNA primers and probes to target bacterial groups of interest. It may then be possible to study the ecology of these bacterial communities and the role of specific bacterial groups that contribute to the many interesting properties of these environments.     

Microbiological characterization of the structures built by earthworms and ants in an agricultural field

The genesis and architecture of the structures built by ants and earthworms differ markedly, suggesting that—in addition to having different physical and chemical properties—the resident microbial community should also have unique properties. We characterized the inorganic N, biomass C, C mineralization rate, and functional diversity of the microbial communities of earthworm casts, earthworm burrow soil, ant mounds, and bulk soil from an agricultural field. Mound soil was most enriched in inorganic N and had the lowest pH, moisture content, and C mineralization rate. Functional diversity was evaluated by determining the ability of microorganisms to grow on 31 substrates using Biolog^®EcoPlates in combination with a most probable number (MPN) approach. Casts had MPNs that were one to two orders of magnitude higher than burrow, mound and bulk soil for most substrates. Casts also had the highest MPNs for particular substrate guilds relative to bulk soil, followed by mound and burrow soil. Indices of substrate diversity and evenness were highest for casts, followed by burrow, mound, and bulk soil. Differences in the type of habitat provided by the structures built by ants and earthworms result in the differential distribution of nutrients, microbial activity, and metabolic diversity of soils within an agricultural field that affect soil fertility and quality.     

Functional and molecular responses of soil microbial communities under differing soil management practices

The effects of soil management on some microbiological properties and soil bacterial community structure were evaluated. Two field sites with the same soil type, located on the same geographic area adjacent to one other, have received different soil management practices and cultivation. One site has been subjected for 20 years to intensive horticulture under conventional tillage and irrigation with low quality salt-rich water; the second field site has been uncultivated for a long period and was turned to organic farming practices over the last 5 years and is currently cultivated with fruit orchard. Total bacterial counts, microbial ATP, microbial community metabolic (BIOLOG^®) profiles, and DNA fingerprinting by PCR-DGGE were determined. Two-way ANOVA revealed that total bacterial counts were not significantly (P>0.3) affected by the two different management practices; ATP content was consistently and significantly (P<0.001) lower in salt-water irrigated soil than in organic soil at the three sampling times. The cluster analysis of community level physiological profiles indicated that microbial communities were much more uniform in organic soil than in irrigated one, suggesting that salt-water irrigation could have affected the size of the microbial population, its metabolic activities, as well as its composition. Molecular patterns fitted the BIOLOG^® profile diversity. In particular, at any sampling time, PCR-DGGE patterns of bacterial DNA, extracted by an indirect method, significantly discriminated irrigated from organic soil samples. The PCR-DGGE patterns of total soil DNA, extracted by a direct method, showed a moderate to significant variation among irrigated and organic soil samples. Biochemical, microbiological and molecular data contributed to evidence a significantly different response of indigenous microflora to soil management by using saline water or organic farming.     

Estimates of viral abundance in soils are strongly influenced by extraction and enumeration methods

Viruses are highly abundant in temperate soils, ranging from 10⁷ to 10⁹?g^?1, and outnumbering soil bacteria from 5- to over 1,000-fold. In order to determine the potential impacts of viruses on soil microbial communities, it is important to establish reliable methods for comparing changes in viral abundances within and across soil samples. The goals of this study were to optimize extraction-enumeration methods to accurately determine viral abundances in a range of soil types, to evaluate the feasibility of simultaneously enumerating bacterial cells and virus particles using a single extraction procedure, and to assess the utility of flow cytometry (FCM) for enumerating virus particles in soil extracts. Comparisons of extraction approaches indicated that sonication or blender extraction of soils with potassium citrate buffer yielded the highest viral abundances for most soil types. Combined viral and bacterial extractions underestimate abundances compared to separately-optimized extractions for each. Flow cytometric counts were anywhere between 350- and 1,400-fold higher than epifluorescence microscopy (EFM)-based counts for the same soil. Trends in viral abundance across soil types were different from those via EFM, and different relationships between viral abundance and soil properties were observed depending on the enumeration method. Thus, FCM is not currently recommended for enumeration of viruses in soil extracts. Based on EFM results, soil moisture and organic matter content were the most important factors determining viral abundance in soils.     

Shifts in bacterial community structure associated with inputs of low molecular weight carbon compounds to soil

Low molecular weight carbon (C) substrates are major drivers of bacterial activity and diversity in the soil environment. However, it is not well understood how specific low molecular weight C compounds, which are frequently found in root exudates and litter leachates, influence bacterial community structure or if there are specific groups of soil bacteria that preferentially respond to these C inputs. To address these knowledge gaps, we added three simple C substrates representative of common root exudate compounds (glucose, glycine, and citric acid) to microcosms containing three distinct soils from a grassland, hardwood forest, and coniferous forest. CO₂ production was assessed over a 24 h incubation period and, at the end of the incubation, DNA was extracted from the samples for assessment of bacterial community structure via bar-coded pyrosequencing of the 16S rRNA gene. All three C substrates significantly increased CO₂ production in all soils; however, there was no relationship between the magnitude of the increase in CO₂ production and the shift in bacterial community composition. All three substrates had significant effects on overall community structure with the changes primarily driven by relative increases in β-Proteobacteria, γ-Proteobacteria, and Actinobacteria. Citric acid additions had a particularly strong influence on bacterial communities, producing a 2-5-fold increase in the relative abundance of the β-Proteobacteria subphylum. These results suggest that although community-level responses to substrate additions vary depending on the substrate and soil in question, there are specific bacterial taxa that preferentially respond to the substrate additions across soil types.     

枣园生草处理对土壤养分和细菌群落的影响

为了明确枣园种植长柔毛野豌豆(Vicia villosa Roth.)对土壤养分和细菌群落的影响,本试验以5年生金丝4号(Zizyphus jujuba Mill.)为试材,研究了枣园不同生草处理(清耕、自然生草、长柔毛野豌豆)对土壤基本理化指标和土壤细菌群落的影响.结果表明,与清耕(对照)相比,枣园生草提高了 土壤含...     

Microbial dynamics in Mediterranean Moder humus

There is a growing interest in the links between humus forms and soil biota, and little is known about these links in Mediterranean ecosystems. Culture-independent techniques, such as DNA extraction followed by DGGE and enzyme activities, allowed us to compare microbial communities in two horizons of a forest soil in different seasonal conditions. Direct in situ lysis was applied for extraction of DNA from soil; intracellular DNA was separated from extracellular and used to represent the composition of microflora. The aims were to describe how biochemical and microbiological parameters correlate with topsoil properties in typical Mediterranean Moder humus. Changes in bacterial and fungal community composition were evident from DGGE profiles. Degrees of similarity and clustering correlation coefficients showed that the seasonal conditions may affect the composition and activity of bacterial and fungal communities in the OH horizon, while in the E horizon the two communities were hardly modified. In the same season, OH and E horizons showed a different composition of bacterial and fungal communities and different enzyme activities, suggesting similar behaviour of eubacteria and fungi relatively to all the variables analysed. Evidently, different organic carbon content in soil horizons influenced microflora composition and microbial activities involved in the P and N cycles.     

Benefactor and allelopathic shrub species have different effects on the soil microbial community along an environmental severity gradient

Patches where shrubs have either positive or negative effects on their understory plant community are common in arid ecosystems. The intensity and balance of these effects change along environmental severity gradients but, despite the major role of soil microbes in plant interactions, little is known about the differences among soil microbial communities under these species and their possible influence on such contrasting shrub effects. We hypothesized that microbial communities associated to benefactor and allelopathic shrubs would differ among them and that differences would increase with environmental severity. To test these hypotheses we characterized soil microbial biomass, activity and community composition under a benefactor shrub species, Retama sphaerocarpa, an allelopathic shrub species, Thymus hyemalis, and in bare soil among plants (gaps) at three sites along an environmental severity gradient. Shrubs promoted an increase in soil bacterial diversity, being bacterial communities associated to benefactor shrubs, allelopathic shrubs and gaps different in composition. Microbial enzymatic activity and biomass increased under shrubs and under more mesic conditions; nonetheless, they were highest under benefactor shrubs at the most arid site and under allelopathic shrubs at the less severe site. Compared to gaps, the presence of shrubs induced changes in microbial activity and community composition that were larger at the most severe site than at the less severe site. Along the gradient, benefactor shrubs enhanced the abundance of bacterial groups involved in organic matter decomposition and N fixation as well as plant pathogens, which could contribute to Retama's outstanding positive effects on understory plant biomass and diversity. Plant patches mitigate the effects of extreme conditions on associated plant and soil microbial communities and promote soil biodiversity and ecosystem functioning in arid ecosystems, with shrubs actively selecting for specific microbial groups in their understory.     

Analysis of the structure of microbial community in soils with different degrees of salinization using T-RFLP and real-time PCR techniques

Molecular methods were used to study variation in the taxonomic structure of bacterial, archaeal, and fungal communities in soil samples taken along a salinity gradient from a solonchak in the vicinity of Lake Akkol’ (Shingirlau, Kazakhstan). Soils from arable fields located 195 km from the solonchak served as the control. Total DNA was isolated from every sample and analyzed by T-RFLP and real-time PCR. Salinization was found to be the main ecological factor determining the structure of soil microbial community in the study region. The values of Simpson’s index characterizing the diversity of this community proved to be similar in all the samples, which, however, significantly differed in the taxonomic composition of microorganisms. A significantly increased content of archaea was revealed in the sample with the highest salinity. The results of this study show that the structure of soil microbial community reflects specific features of a given soil and can be used as an indicator of its ecological state.     

不同基肥对黄瓜根际土壤微生物群落多样性的影响

分别以RAPD分子生物学方法和BIOLOG生理学方法,研究了不同基肥对黄瓜根际土壤微生物群落DNA序列多样性和群落功能多样性的影响。结果表明,在本试验条件下,基肥为75000 kg/hm2有机肥处理和75000 kg/hm2有机肥加300 kg/hm2复合肥处理最好;基肥为600 kg/hm2复合肥处理而使土壤微生物群落DNA序列丰富度指数和多样性指数显著下降,与对照的DNA序列相似系数最低;有机肥处理有利于土壤微生物群落DNA序列多样性、均匀度和黄瓜产量的提高。此外,不同基肥处理改变了土壤微生物对单一碳源的利用能力。     

黄瓜与西芹间作土壤细菌多样性及其对黄瓜枯萎病发生的影响

本试验以黄瓜与西芹间作种植模式为处理,黄瓜单作和西芹单作种植模式为对照,利用Illumina公司Miseq平台对上述不同处理土壤进行16S rDNA细菌群落多样性高通量测序分析和田间接种黄瓜枯萎病菌,探讨黄瓜与西芹间作模式土壤细菌的多样性及其对田间黄瓜枯萎病发生的影响。16S rDNA测序结果表明,黄瓜与西芹间作土壤的细菌物种总数最多,群落多样性水平最高,与对照相比显著提高了土壤细菌observed species指数、Shannon指数和Chao1指数(P0.05);Beta多样性聚类分析表明,黄瓜与西芹间作土壤的环境群落物种与黄瓜单作和西芹单作有一定差异性。在门分类水平上,共检测到45个菌门,其中变形菌门占明显优势,其次为酸杆菌门和放线菌门等;黄瓜与西芹间作土壤细菌种类所占比例最高,达98.63%。在属水平上,共检测到428类菌属,GP6、GP16、GP4、芽单胞菌属、节细菌属5属的丰度值较大;黄瓜与西芹间作土壤的节细菌属分布比例最高,红游动菌属、鞘氨醇单胞菌属和芽球菌属丰度值较大,为黄瓜与西芹间作土壤细菌明显优势菌属。田间接种黄瓜枯萎病菌试验结果表明,采用上述3种不同种植模式土壤种植黄瓜,在黄瓜苗期接种黄瓜枯萎病菌,黄瓜与西芹间作处理的黄瓜枯萎病的田间发病率较西芹单作和黄瓜单作分别降低57.03%~63.54%和66.95%~72.15%。因此,黄瓜与西芹间作增加了土壤细菌群落多样性,降低了黄瓜枯萎病的发病率,对后茬黄瓜土传病害防控具有一定科学指导意义。     

Effects of inoculation with ectomycorrhizal fungi on microbial biomass and bacterial functional diversity in the rhizosphere of Pinus tabulaeformis seedlings

This study investigated the effects of inoculation with three individual ectomycorrhizal (ECM) fungal species on soil microbial biomass carbon and indigenous bacterial community functional diversity in the rhizosphere of Chinese pine (Pinus tabulaeformis Carr.) seedlings under field experimental conditions. The results showed that ECM fungal inoculation significantly increased the ectomycorrhizal colonization compared with non-inoculated seedlings. ECM fungal inoculations have higher soil microbial biomass carbon than that of control, ranging from 49.6 μg C g^?1 dry soil in control to 134.02 μg C g^?1 dry soil in treatment inoculated with Boletus luridus Schaeff ex Fr. Multivariate analyses (PCA) of BIOLOG data revealed that the application of ECM fungi significantly influenced bacterial functional diversity in the rhizosphere of P. tabulaeformis seedlings. The highest average well-color development (AWCD) and functional diversity indices were also observed in treatment inoculated with B. luridus. A wider range of sole carbon sources were utilized by the bacterial community in the rhizosphere of inoculated seedlings. The data gathered from this study provides important information for utilization of ECM fungi in forest restoration project in the Northwestern China. The present study will also significantly broaden our understanding of practical importance in the application of ECM fungal inoculum to promote soil microbial community diversity of soil.     

Soil fumigation and compost amendment alter soil microbial community composition but do not improve tree growth or yield in an apple replant site

Apple replant disease (ARD) is a disease complex that reduces survival, growth and yield of replanted trees, and is often encountered in establishing new orchards on old sites. Methyl bromide (MB) has been the fumigant used most widely to control ARD, but alternatives to MB and cultural methods of control are needed. In this experiment, we evaluated the response of soil microbial communities and tree growth and yield to three pre-plant soil treatments (compost amendment, soil treatment with a broad-spectrum fumigant, and untreated controls), and use of five clonal rootstock genotypes (M.7, M.26, CG.6210, G.30 and G.16), in an apple replant site in Ithaca, New York. Polymerase chain reaction (PCR)—denaturing gradient gel electrophoresis (DGGE) analysis was used to assess changes in the community composition of bacteria and fungi in the bulk soil 8, 10, 18 and 22 months after trees were replanted. PCR-DGGE was also used to compare the community composition of bacteria, fungi and pseudomonads in untreated rhizosphere soil of the five rootstock genotypes 31 months after planting. Tree caliper and extension growth were measured annually in November from 2002 to 2004. Apple yield data were recorded in 2004, the first fruiting year after planting. Trees on CG.6210 rootstocks had the most growth and highest yield, while trees on M.26 rootstocks had the least growth and lowest yield. Tree growth and yield were not affected by pre-plant soil treatment except for lateral extension growth, which was longer in trees growing in compost-treated soil in 2003 as compared to those in the fumigation treatment. Bulk soil bacterial PCR-DGGE fingerprints differed strongly among the different soil treatments 1 year after their application, with the fingerprints derived from each pre-plant soil treatment clustering separately in a hierarchical cluster analysis. However, the differences in bacterial communities between the soil treatments diminished during the second year after planting. Soil fungal communities converged more rapidly than bacterial communities, with no discernable pattern related to pre-plant soil treatments 10 months after replanting. Changes in bulk soil bacterial and fungal communities in response to soil treatments had no obvious correlation with tree performance. On the other hand, rootstock genotypes modified their rhizosphere environments which differed significantly in their bacterial, pseudomonad, fungal and oomycete communities. Cluster analysis of PCR-DGGE fingerprints of fungal and pseudomonad rhizosphere community DNA revealed two distinct clusters. For both analyses, soil sampled from the rhizosphere of the two higher yielding rootstock genotypes clustered together, while the lower yielding rootstock genotypes also clustered together. These results suggest that the fungal and pseudomonad communities that have developed in the rhizosphere of the different rootstock genotypes may be one factor influencing tree growth and yield at this apple replant site.