Assessing insecticide and fungicide effects on the culturable soil bacterial community by analyses of variance of their DGGE fingerprinting data

To assess the effects of three insecticides (aldicarb, chlorpyrifos, deltamethrin) and two fungicides (tebuconazole and metalaxyl + mancozeb) on the PCR-DGGE fingerprints of culturable soil bacterial communities (CSBC), a greenhouse experiment was carried out with soil samples from an Integrated System for Agroecological Production (ISAP), a Conventional Potato Production Area (CPPA) and a Secondary Forest Area (SFA) close to the CPPA. Samples were obtained at 15 day intervals starting at 32 until 77 days after sowing (DAS) to perform the PCR-DGGE analysis of the CSBC cultured on media amended with soil suspension. Analysis of variance from PCR-DGGE data indicated significant differences among treatments. Regardless the type of pesticide applied, CSBC was disturbed and similarity values varied from 5% to 90% in comparison to the control. Significant shifts on CSBC were only detected among treatments in the first two harvests, while CSBC tended to be more akin to each other at the last two harvest dates. The most significant responses observed were due to different soil sample origins, where values of 5% of similarity to the control were observed on CPPA soil. The use of analysis of variance on PCR-DGGE data was useful to a better understanding of the changes on CSBC induced by pesticides applications.     

Analysis of bacterial communities on alkaline phosphatase genes in soil supplied with organic matter

Abstract

We studied the effects of the application of organic matter (OM) and chemical fertilizer (CF) on soil alkaline phosphatase (ALP) activity and ALP-harboring bacterial communities in the rhizosphere and bulk soil in an experimental lettuce field in Hokkaido, Japan. The ALP activity was higher in soils with OM than in soils with CF, and activity was higher in the rhizosphere for OM than in the bulk soil. Biomass P and available P in the soil were positively related to the ALP activity of the soil. As a result, the P concentration of lettuce was higher in OM soil than in CF soil. We analyzed the ALP-harboring bacterial communities using polymerase chain reaction based denaturing gradient gel electrophoresis (DGGE) on the ALP genes. Numerous ALP genes were detected in the DGGE profile, regardless of sampling time, fertilizer treatment or sampled soil area, which indicated a large diversity in ALP-harboring bacteria in the soil. Several ALP gene fragments were closely related to the ALP genes of Mesorhizobium loti and Pseudomonas fluorescens. The community structures of the ALP-harboring bacteria were assessed using principal component analysis of the DGGE profiles. Fertilizer treatment and sampled soil area significantly affected the community structures of ALP-harboring bacteria. As the DGGE bands contributing to the principal component were different from sampling time, it is suggested that the major bacteria harboring the ALP gene shifted. Furthermore, there was, in part, a significant correlation between ALP activity and the community structure of the ALP-harboring bacteria. These results raise the possibility that different ALP-harboring bacteria release different amounts and/or activity of ALP, and that the structure of ALP-harboring bacterial communities may play a major role in determining overall soil ALP activity.     

A DGGE analysis shows that crop rotation systems influence the bacterial and fungal communities in soils

A better understanding of the relationships among different cropping systems, their effects on soil microbial ecology, and their effects on crop health and productivity is necessary for the development of more efficient, sustainable crop production systems. We used denaturing gradient gel electrophoresis (DGGE) to determine the impacts of crop rotations and crop types on bacterial and fungal communities in the soil. The communities of bacterial 16S rRNA genes and fungal 18S rRNA genes were analyzed in experimental field plots that were kept under 4 different crop rotation systems from 1999 to 2008 (continuous cabbage (Brassica oleracea var. capitata L.), cabbage–lettuce (Lactuca sativa L.) rotation, cabbage–radish (Raphanus sativus L. var. longipinnatus L.H. Bailey) rotation, and a 3-year crop rotation). A principal component analysis (PCA) and a canonical correspondence analysis (CCA) revealed that both the bacterial and fungal communities in bulk soils were influenced by the crop rotation systems. However, the primary factors influencing each community differed: bacterial communities were most affected by soil properties (especially carbon content), while fungal communities were influenced most strongly by rotation times. To elucidate factors that may cause differences in crop rhizosphere microbial communities, the microbial communities in the harvested cabbage rhizospheres were also analyzed. The results suggest that the fungal communities in bulk soil are related to the rhizosphere fungal communities. Our present study indicates that the microbial communities in bulk and rhizosphere soils could be managed by crop rotation systems.     

Application of DGGE analysis to the study of bacterial community structure in plant roots and in nonrhizosphere soil

To analyze the structure of bacterial communities in spinach roots and in the nonrhizosphere soil, we used PeR-amplified 16S rRNA gene fragments separated by denaturing gradient gel electrophoresis (DGGE). DGGE revealed a large number of band patterns, which were ascribed to various bacterial species composing each of the bacterial communities. The pattern from the roots was less complex than that from the soil. It is considered that DGGE analysis is suitable for studies of bacterial community structure in soil-plant ecosystems.     

Impact of sulfadiazine and chlorotetracycline on soil bacterial community structure and respiratory activity

Veterinary medicines enter agricultural soils by the use of animal excrements as fertilizers. To study their impact on soil bacterial communities, microcosms containing orthic luvisol soil were spiked with the antimicrobial agents sulfadiazine (SDZ) and chlorotetracycline (CTC) at three different concentrations (1, 10, 50 mg kg⁻¹ soil) and incubated for 48 days at 20 °C. The impact on the microbial respiratory activity was measured continuously in a respirometer (Sapromat). Changes in bacterial community structure were visualized by means of PCR-denaturing gradient gel electrophoresis (DGGE) of 16S rDNA derived from soil samples after 1, 7, 11 and 48 days. Additionally, growth inhibitory effects of SDZ and CTC on bacteria previously isolated from the same soil were tested in agar diffusion tests. In microcosms with soil and antibiotics only, no effects could be observed, either on respiratory activity or on bacterial population structure. Therefore, further incubations were conducted in the presence of an additional assimilable carbon source (5 g glucose kg⁻¹ soil). In the presence of glucose, SDZ affected soil respiration as well as the bacterial community structure: Additional bands appeared and some bands already visible at the beginning of incubations increased in intensity. A clear relationship between SDZ concentrations and changes in DGGE patterns became visible. During 48 days of incubation, changes in DGGE patterns were minimal in microcosms with 50 mg SDZ kg⁻¹soil indicating an inhibition of strains, which were capable of growing on glucose in the presence of lower SDZ concentrations. Only a few soil bacterial isolates (5 out of 47 strains tested) were weakly inhibited by SDZ in agar diffusion disk tests. Contrastingly, CTC inhibited growth of 12 soil bacterial isolates significantly in disk tests, but no effects on soil respiration and bacterial community structure could be observed. In the presence of the soil matrix the growth inhibitory potential of CTC decreased due to adsorption or complexation. This was confirmed in growth inhibition experiments with soil suspensions and time-dependent sampling.     

Bacterial community in plant residues in a Japanese paddy field estimated by RFLP and DGGE analyses

Plant residues (PRs) are “hot spots” of microbial activities in soil. PRs with the size more than 0.5 mm were collected from a Japanese paddy field during rice cultivation period (from May to September) and fractionated into four categories by size (>4, 2-4, 1-2, and 0.5-1 mm) using sieves. Restriction fragment length polymorphism (RFLP) and denaturing gradient gel electrophoresis (DGGE) patterns were compared among the fractions after DNA extraction from the PRs and PCR amplification. The total amount of PRs with the size over 0.5 mm decreased in the field with the first-order kinetics (r²=0.810, p<0.01) with time from rice transplanting to harvest. RFLP analysis showed that the bacterial community structure in PRs with the 0.5-2 mm fraction was different from that in PRs with the >2 mm fraction and the latter community structure changed after the midseason drainage. In contrast, the DGGE patterns of the bacterial community in the PRs indicated the succession from June to September during rice cultivation forming three major groups irrespective of the fraction size. Sequence analysis of DGGE bands showed that Firmicutes (clostridia), α-, γ-, δ-Proteobacteria (myxobacteria), Nitrospira, Acidobacteria, Bacteroidetes, Verrucomicrobia and Spirochaetes were predominant members in the PRs irrespective of fraction size.     

Long-term impact of fertilization on activity and composition of bacterial communities and metabolic guilds in agricultural soil

To explore long-term impact of organic and inorganic fertilizers on microbial communities, we targeted both the total bacterial community and the autotrophic ammonia oxidizing bacteria (AOB) in soil from six treatments at an experimental field site established in 1956: cattle manure, sewage sludge, Ca(NO₃)₂, (NH₄)₂SO₄, unfertilized and unfertilized without crops. All plots, except the bare fallows, were cropped with maize. Effects on activity were assessed by measuring the basal respiration and substrate induced respiration (SIR) rates, and the potential activity of the AOB. To determine the bacterial community composition, 16S rRNA genes were used to fingerprint total soil communities by terminal restriction fragment length polymorphism analysis and AOB communities by denaturing gradient gel electrophoresis. The fertilization regimes had clear effects on both activity and composition of the soil communities. Basal respiration and r, which was kinetically derived as the exponentially growing fraction of the SIR-response, correlated well with the soil organic C content (r=0.93 and 0.66, respectively). Soil pH ranged from 3.97 to 6.26 in the treatments and was found to be an important factor influencing all microbial activities. pH correlated negatively with the ratio between basal respiration and SIR (r=0.90), indicating a decreased efficiency of heterotrophic microorganisms to convert organic carbon into microbial biomass in the most acid soils with pH 3.97 and 4.68 ((NH₄)₂SO₄ and sewage sludge fertilized plots, respectively). The lowest SIR and ammonia oxidation rates were also found in these treatments. In addition, these treatments exhibited individually different community fingerprints, showing that pH affected the composition of AOB and total bacterial communities. The manure fertilized plots harbored the most diverse AOB community and the pattern was linked to a high potential ammonia oxidation activity. Thus, the AOB community composition appeared to be more strongly linked to the activity than the total bacterial communities were, likely explained by physiological differences in the populations present.     

Structural and functional diversity of bacterial community in soil treated with the herbicide napropamide estimated by the DGGE,CLPP and r/K-strategy approaches

Napropamide is one of the most commonly used herbicide in agricultural practice and can exhibit toxic effect to soil microorganisms. Therefore, the main objective of this study was to examine the genetic and functional diversity of microbial communities in soil treated with napropamide at field rate (FR, 2.25 mg kg⁻¹ of soil) and 10 times the FR (10 × FR, 22.5 mg kg⁻¹ of soil) by the denaturing gradient gel electrophoresis (DGGE) and the community level physiological profile (CLPP) methods. In addition, the r/K-strategy approach was used to evaluate the effect of this herbicide on the community structure of the culturable soil bacteria. DGGE patterns revealed that napropamide affected the structure of microbial community; however, the richness (S) and genetic diversity (H) values indicated that the FR dosage of napropamide experienced non-significant changes. In turn, the 10 × FR dosage of herbicide caused significant changes in the S and H values of dominant soil bacteria. DGGE profiles suggest an evolution of bacteria capable of degrading napropamide among indigenous microflora. Analysis of the CLPPs indicated that the catabolic activity of microbial community expressed as AWCD (average well-color development) was temporary positively affected after napropamide application and resulted in an increase of the substrate richness (S_R) as well as functional biodiversity (H) values. Analysis of the bacterial growth strategy revealed that napropamide affected the r- or K-type bacterial classes (ecotypes). In treated-soil samples K-strategists dominated the population, as indicated by the decreased ecophysiological (EP) index. Napropamide significantly affected the physiological state of culturable bacteria and caused a reduction in the rate of colony formation as well as a prolonged time of growth rate. Obtained results indicate that application of napropamide may poses a potential risk for soil functioning.     

Analysis of bacterial communities in soil by PCR-DGGE targeting protease genes

We studied the effects of the application of organic (OM) and inorganic fertilizer (CF) on soil protease activity and proteolytic bacterial communities in rhizosphere and bulk soil on an experimental lettuce field in Hokkaido, Japan. The protease activity always was higher in soils of the OM than with the CF treatment, and also higher in the rhizosphere than in the bulk soil. We analyzed proteolytic bacterial communities by denaturing gradient gel electrophoresis (DGGE) of the alkaline metalloprotease (apr) and neutral metalloprotease (npr) genes. Most apr forms detected were closely related to apr of Pseudomonas fluorescens, and all npr variants closely resembled the gene of Bacillus megaterium. These results were consistent with findings from tests using cultured bacterial communities, indicating a high specificity of our PCR-DGGE for amplifying apr and npr genes. The community compositions of proteolytic bacteria were assessed by principal component analysis of the DGGE profiles. There were significant differences in the effects of CF and OM on the community compositions of apr- and npr-expressing bacteria, and the communities of the two types of bacteria played different roles in rhizosphere and bulk soil. We found significant correlations between the protease activity and the communities of the two types of bacteria. The results indicate that different proteolytic bacteria release different amounts or activities of protease, and that the composition of proteolytic bacterial communities may play a major role in determining overall soil protease activity.     

Bacterial communities from soil sediments of a mountain oasis in northern Oman

Paleoecological records of a 20 meter deep profile near an oasis settlement in northern Oman have yielded a chronosequence providing insights into relationships between vegetation, the environment and development of human settlements in this area over a period of time spanning 19,000 years. In conjunction with analysis of the chemical and physical properties of this profile, we hypothesized that bacterial community structures associated with this chronosequence may also constitute a part of the biogeochemical record of the climate history that has been preserved at this site. To examine this hypothesis, we studied the composition of the community as revealed by profiling of 16S rRNA genes at 1 meter intervals along the entire profile. The results of our study show distinct changes in bacterial communities with increasing depth that correspond with differences in the climatic record as indicated by the occurrence of micro-charcoal particles. Sequencing of 16S rRNA genes proved the presence of Acidobacteria, Actinobacteria, Proteobacteria, Gemmatimonadetes, Chloroflexi and representatives from the candidate divisions SPAM, NC10, and OP10. Differences in the communities support the hypothesis that the bacterial species compositions in the sediment reflect properties of the organic matter and vegetation at the time they were deposited.     

Parent material and vegetation influence bacterial community structure and nitrogen functional genes along deep tropical soil profiles at the Luquillo Critical Zone Observatory

Microbial communities mediate every step of the soil nitrogen cycle, yet the structure and associated nitrogen cycle functions of soil microbial communities remain poorly studied in tropical forests. Moreover, tropical forest soils are often many meters deep, but most studies of microbial nitrogen cycling have focused exclusively on surface soils. The objective of our study was to evaluate changes in bacterial community structure and nitrogen functional genes with depth in soils developed on two contrasting geological parent materials and two forest types that occur at different elevations at the Luquillo Critical Zone Observatory in northeast Puerto Rico. We excavated three soil pits to 140 cm at four different sites representing the four soil × forest combinations (n = 12), and collected samples at ten-centimeter increments from the surface to 140 cm. We used bacterial 16S rRNA gene-DGGE (denaturant gradient gel electrophoresis) to fingerprint microbial community structures, and quantitative PCR to measure the abundance of five functional genes involved in various soil nitrogen transformations: nifH (nitrogen fixation), chiA (organic nitrogen decomposition), amoA (ammonia oxidation), nirS (nitrite reduction) and nosZ (nitrous oxide reduction). Multivariate analyses of DGGE fingerprinting patterns revealed differences in bacterial community structure across the four soil × forest types that were strongly correlated with soil pH (r = 0.69, P < 0.01) and nutrient stoichiometry (r² ≥ 0.36, P < 0.05). Across all soil and forest types, nitrogen functional genes declined significantly with soil depth (P < 0.001). Denitrification genes (nirS and nosZ) accounted for the largest proportion of measured nitrogen functional genes. Measured nitrogen functional genes were positively correlated with soil carbon, nitrogen and phosphorus concentrations (P < 0.001) and all genes except amoA were significantly more abundant in the Inceptisol soil type compared with the Oxisol soil type (P < 0.03). Greater abundances and a stronger vertical zonation of nitrogen functional genes in Inceptisols suggest more dynamic nitrogen transformation processes in this soil type. As the first study to examine bacterial nitrogen functional gene abundances below the surface 20 cm in tropical forest soils, our work provides insight into how pedogenically-driven vertical gradients control the nitrogen-cycling capacity of soil microbial communities. While previous studies have shown evidence for redox-driven hotspots in tropical nitrogen cycling on a watershed scale, our study corroborates this finding on a molecular scale.     

Survival of bacterial DNA and culturable bacteria in archived soils from the Rothamsted Broadbalk experiment

Dried soil samples from many sources have been stored in archives world-wide over the years, but there has been little research on their value for studying microbial populations. Samples collected since 1843 from the Broadbalk field experiment on crop nutrition at Rothamsted have been used to document changes in the structure and composition of soils as agricultural practices evolve, also offering an invaluable record of environmental changes from the pre- to post-industrial era in the UK. To date, the microbial communities of these soils have not been studied, in part due to the well-documented drop in bacterial culturability in dried soils. However, modern molecular methods based on PCR amplification of DNA extracted directly from soil do not require bacterial cells to be viable or intact and may allow investigations into the legacy of bacteria that were present at the time of sample collection.

In a preliminary study, to establish if dried soils can provide a historical record of bacterial communities, samples from the Broadbalk soil archive dating back to 1868 were investigated and plots treated with either farmyard manure (FYM) or inorganic fertilizer (NPK) were compared. As anticipated, the processes of air-drying and milling greatly reduced bacterial viability whilst DNA yields declined less and may be preserved by desiccation. A higher proportion of culturable bacteria survived the archiving process in the FYM soil, possibly protected by the increased soil organic matter. The majority of surviving bacteria were firmicutes, whether collected in 2003 or in 1914, but a wide range of genera was detected in DNA extracted from the samples using PCR and DGGE of 16S rRNA genes. Analysis of DGGE band profiles indicated that the two plots maintained divergent populations. Sequence analysis of bands excised from DGGE gels, from a sample collected in 1914, revealed DNA from - and β-proteobacteria as well as firmicutes. PCR using primers specific for ammonia oxidizing bacteria showed similar band profiles across the two treatments in recently collected samples, however older samples from the NPK plot showed greater divergence. Primers specific for the genus Pseudomonas were designed and used in real-time quantitative PCR to indicate that archived soil collected in 1868 contained 10-fold less pseudomonad DNA than fresh soil, representing around 10⁵ genomes g⁻¹ soil. Prior to milling, dramatically less pseudomonad DNA was extracted from recently collected air-dried soil from the NPK compared to the FYM plot; otherwise, the two plots followed similar trends. Overall bacterial abundance, diversity and survival during the archiving process differed in the two soils, possibly due to differences in clay and soil organic matter content. Nevertheless, the results demonstrate that air-dried soils can protect microbial DNA for more than 150 years and offer an invaluable resource for future research.     

Bacterial communities in soybean rhizosphere in response to soil type, soybean genotype, and their growth stage

Three experiments were conducted in this study in order to investigate the impacts of soil type, soybean genotype, and the reproductive growth stage on bacterial communities in the soybean rhizosphere. Communities were evaluated by principal component analysis of denaturing gradient gel electrophoresis (DGGE) banding patterns and sequencing of partial 16S rDNA polymerase chain reaction (PCR) amplicons. A pot experiment analyzing three soybean genotypes grown in two different types of soil (Black soil and Dark Brown soil) indicated that soil type was the major factor in influencing the bacterial communities in the soybean rhizosphere, with a more significant effect observed in the Black soil samples than in the Dark Brown soil samples. A field experiment was conducted in Dark Brown soil using three soybean genotypes, and the results gleaned from both pot and field experiments indicated that bacterial communities in the soybean rhizosphere changed with growth stages, and higher number of DGGE bands observed in early reproductive growth stages, while surprisingly, a significant impact of genotype on the bacterial communities was not observed in these experiments. However, a plate culture experiment targeting the culturable bacterial communities detected a remarkable difference in the community structures of the rhizosphere between the two soybean genotypes, suggesting that a small portion of the total bacteria was influenced by genotype. Sequence analysis of DGGE bands indicated that bacterial phyla of Proteobacteria, Actinobacteria, Bacteroidetes, Nitrospirae, Firmicutes, Verrucomicrobia and Acidobacteria commonly inhabit the soybean rhizosphere.     

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.     

长期施肥对黑土农田土壤微生物群落的影响

基于中国科学院海伦农业生态试验站长期定位试验区,应用实时荧光定量PCR(Real-time PCR)和变性梯度凝胶电泳(DGGE)技术研究了无施肥(NF)、单施N、P化肥(NP)以及化肥配施有机猪粪肥(NPM)等3种长期施肥措施对黑土区玉米田土壤微生物群落密度和结构的影响.Real-time PCR方法定量NF、NP及NPM措施土壤细菌群落基因组DNA质量分别为381、1 351和1 773 ng g-1干土,真菌群落基因组DNA质量分别113.3、127.3和20.6 ng g-1干土,真菌与细菌的比率分别为0.31、0.09和0.01,NPM措施显著低于另两种施肥方式(p＜0.05).DGGE方法研究表明,NP和NPM措施不能改善土壤细菌和真菌群落的多样性、均匀性及优势菌优势程度;但主成分分析结果显示NP和NPM措施均可改变土壤细菌和真菌群落的构成,且真菌群落的变化更为显著;聚类分析结果显示NP和NPM措施下细菌群落结构较相近,其相似系数为0.89,真菌群落中NP措施与NF措施相近,相似系数为0.63,高于NP与NPM措施的相似系数0.51.上述结果表明有机猪粪肥的长期施用可以显著降低黑土农田土壤真菌与细菌的比率,且明显地改变土壤细菌和真菌群落的结构.     

Microbial composition and diversity of an upland red soil under long-term fertilization treatments as revealed by culture-dependent and culture-independent approaches

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.     

施肥及秸秆还田对砂姜黑土细菌群落的影响

利用Illumina平台Miseq高通量测序技术对小麦分蘖期砂姜黑土耕层土壤细菌进行高通量测序,结合相关生物信息学分析,探讨了不施化肥秸秆不还田(CK)、施化肥秸秆不还田(F)以及不施化肥秸秆还田(W)3种处理土壤细菌群落组成、多样性和结构的变化。结果显示,测序共获得14 873个OTUs,计173 323条读数,平均读长439 bp。砂姜黑土细菌优势门(相对丰度10%)为变形菌门(Proteobacteria)、酸杆菌门(Acidobacteria)、放线菌门(Actinobacteria)和拟杆菌门(Bacteroidetes);优势纲(相对丰度10%)为α-变形菌纲(Alphaproteobacteria)、β-变形菌纲(Betaproteobacteria)、酸杆菌纲(Acidobacteria)、鞘脂杆菌纲(Sphingobacteriia)和γ-变形菌纲(Gammaproteobacteria);优势属(相对丰度1%)共47个,3个处理中均有分布的优势属21个,F处理的细菌优势属的种类最多,为39个。相对丰度最大的门、纲和属分别是变形菌门(38.7%~43.1%)、α-变形菌纲(14.5%~18.1%)和鞘氨醇单胞菌属(Sphingomonas)(4.6%~7.7%)。F处理细菌丰富度指数(Chao1指数和ACE指数)显著低于CK及W处理,W处理和CK处理土壤细菌丰富度指数无显著差异,与CK处理相比,F处理ACE指数降低22.8%。W处理土壤细菌Shannon多样性指数显著大于CK及F处理,W处理Shannon指数较CK处理提高4.1%,而F处理土壤细菌Shannon指数与CK处理无显著差异。F处理Simpson指数显著高于CK及W处理;F处理Simpson指数较CK处理提高38.1%,而W处理细菌Simpson指数最小,显著低于CK处理,较CK降低23.8%。分层聚类图显示在属的水平上,W处理和CK处理土壤细菌群落结构相似性较高,F处理与CK处理及W处理细菌群落结构差异较大。施化肥对土壤细菌优势类群组成、相对丰度及群落结构的影响大于秸秆还田,施化肥显著降低了土壤细菌丰富度,秸秆还田显著提高了土壤细菌的多样性。