Terminal Restriction Fragment Length Polymorphism Profiles Research Articles

The Endophytic Plant Growth Promoting Methylobacterium oryzae CBMB20 Integrates and Persists into the Seed-Borne Endophytic Bacterial Community of Rice

Endophytic persistence of inoculated plant growth promoting bacteria (PGPB) involves interaction with the host plant and the host’s indigenous endophytic bacterial communities. This study investigated the persistence of Methylobacterium oryzae CBMB20 into the rice endosphere together with the impact of inoculation on the diversity and community structure of the root and shoot bacterial endophytes in Oryza sativa L. spp. indica cv. IR29. Terminal Restriction Fragment Length Polymorphism (T-RFLP) analysis of the root and shoot showed that M. oryzae CBMB20 was able to integrate and persist in the rice endosphere without causing drastic shifts in bacterial endophytic diversity and community composition. The bacterial communities in the root and shoot are very similar to the seeds of IR29, suggesting that most of them are seed-borne. The root endosphere bacterial communities of inoculated and uninoculated IR29 plants are more diverse compared to the shoots in terms of richness and diversity indices. The dominant bacterial T-RFs of the root endosphere of IR29 belong to Microbacterium, Delftia, Pseudomonas, Xanthomonas and Stenotrophomonas, Herbaspirillum, Enterobacter, and Sphingomonas, as observed in the three restriction enzyme T-RFLP profiles. Bacterial clades identified as Curtobacterium, Enterobacter, Stenotrophomonas, and Xanthomonas were distinctly observed in both the root and shoot communities, and these bacterial groups are also the dominant endophytes of the shoot endosphere. This study showed that Methylobacterium oryzae CBMB20 could persist and incorporate into the endophytic bacterial community of the endosphere without causing long-term antagonistic interactions with its host plant and with the native microbiota.

Control of Apple Replant Disease Using Mixed Cropping with Brassica juncea or Allium fistulosum

Evidence indicates that Allium and Brassica species which release bioactive compounds are widely used in bio-fumigation to suppress soil-borne diseases. However, the active molecules of such plant residues are easily volatilized. In this study, we conducted mixed cropping of the apple tree with Allium fistulosum or Brassica juncea; the results demonstrated that such mixed cropping significantly improved the growth of the grafted apple seedlings and alleviated apple replant disease (ARD) for two years. The terminal-restriction fragment length polymorphism profile results showed that the soil fungal community demonstrated distinct variation and diversity in terms of composition. A. fistulosum and B. juncea significantly improved the Margalef, Pielou, and Shannon indices. In addition, the analyses of clone libraries showed that A. fistulosum and B. juncea promoted the proliferation of antagonistic fungi such as Mortierella, Trichoderma, and Penicillium, and inhibited the proliferation of pathogens such as Fusarium. Fusarium. Proliferatum(F. proliferatum) was abundant in replanted soil and proved to be an aggressive pathogen of apple seedlings. Our findings thus indicate that apple tree mixed cropping with A. fistulosum and B. juncea was an effective long-term method for modifying the resident fungal community and alleviating ARD.

Relationships between root morphology, root exudate compounds and rhizosphere microbial community in durum wheat

A multidisciplinary approach was adopted to investigate multiple relationships between root morphology, rhizosphere soil exudates and soil microbial community in eight durum wheat [Triticum turgidum ssp. durum (Desf.)] cultivars previously characterized in terms of their ability to interact with mycorrhizal fungi. Six plants from each cultivar were grown to the five-leaf stage in a growth chamber. Root system architecture was characterized by root scans, root exudates were identified and quantified by gas chromatography–mass spectrometry (GC–MS), and the rhizosphere bacterial, archaeal and fungal community structure was assessed using terminal-restriction fragment length polymorphism (T-RFLP). Wheat cultivars were very different in terms of the morphological root traits. Cappelli, Grecale, Ofanto and PR22D89 cultivars showed the most developed root system. Root exudates included sugars, amino acids, polyalcohols and organic acids, with carbohydrates accounting for 87.3% of the whole data set. All the compounds showed significant differences among cultivars, with Grecale and Claudio showing the highest exudation ability of metabolites. T-RFLP profiles showed a greater relative abundance of bacterial taxa (55.8% on average) than those of archaea and fungi (35.6% and 8.6%, on average, respectively) in all cultivars, and responded positively to root exudation. Principal component analysis identified root dry weight, surface area and volume, carbohydrate exudation, bacterial and archaeal Shannon–Weaver diversity indices and the fungal evenness index as the traits that mainly contributed to the cultivar variability. A larger root system did not always promote a higher diversity of the rhizosphere microbial biomass. This then suggests complex interactions between traits, probably due to the differences in the quality and/or composition of the root exudates. Based on our results, we propose that durum wheat cultivars could be classified according to the different plant resource-use strategies employed, such as conservative (i.e. Cappelli, with a large root system but low exudation ability) and explorative/competitive (i.e. Grecale and Claudio, with a high exudation ability) cultivars. The results are discussed with reference to the complex relationships between the plant and the rhizospheric microorganisms in order to determine the best combinations that would enhance root efficiency.

An analysis of oral microbial flora by T-RFLP in patients undergoing hematopoietic stem cell transplantation

Even though the hematopoietic stem cell transplantation (HSCT) procedure has been improved, oral mucositis (OM) is still a severe complication of the conditioning regimen. We investigated the association between OM severity and the alteration of oral bacterial flora using 16S rRNA gene-based terminal restriction fragment length polymorphism (T-RFLP) analysis in 19 consecutive patients undergoing HSCT. Oral samples were collected at pre-transplantation, at the peak of mucositis and post-engraftment. T-RFLP profiles for each timepoint were constructed into an X-Y matrix, and the distances between timepoints were calculated. Patients with severe and moderate OM had larger changes in their oral bacterial flora from before HSCT to peak of mucositis than controls (p = 0.031 and 0.016, respectively). Moreover, severe mucositis was significantly associated with an extended period of fever until engraftment, high maximum C-reactive protein levels, and prolonged periods of opioid treatment and intravenous hyper-alimentation. These findings suggest that mucositis severity is associated with the magnitude of change in the oral bacterial flora. This novel finding may help advance strategies for the prevention or treatment of OM after HSCT.

Effects of environmental variables on abundance of ammonia-oxidizing communities in sediments of Luotian River, China.

Ammonia-oxidizing communities play important functional roles in the nitrification. However, environmental stresses can significantly affect this process by controlling the abundant communities of ammonia-oxidizing archaea (AOA) and ammonia-oxidizing bacteria (AOB) communities. In this study, we examined the abundance variations of ammonia-oxidizing communities using quantitative polymerase chain reaction (qPCR) and terminal-restriction fragment length polymorphism (T-RFLP) in a typical subtropical river, Luotian County, South Dabie Mountains, China. Clone libraries were conducted to evaluate the community structure and abundance of AOA and AOB in sediments. Results showed that Nitrososphaera sp and Nitrosopumilus sp were the most dominant AOA. The abundance of the AOA and AOB amoA gene ranged from 5.28 × 108 gene copies (g-soil−1) to 2.23 × 108 gene copies (g-soil−1) and 5.45 × 108 gene copies (g-soil−1) to 3.30 × 107 gene copies (g-soil−1), respectively. Five environmental variables, namely, ORP, DO, NOn}{}{}_{3}^{-}, Temp, and NHn}{}{}_{4}^{+} were played a major function in microbial communities of AOA and AOB in sediments. The T-RFLP profiles of AOA showed that 488 and 116 bp T-RFs were dominated. Overall, the results of this study showed that anthropogenic activities andenvironmental stress in rivers can alter the structure and function of microbes in their variable environment.

Initial effects of supplemental forages and feedstuffs on bovine rumen ecology in vitro as determined by DNA-based molecular procedures

ABSTRACT This research aided in determining the impacts on rumen microbial ecology when supplemental forages and feedstuffs were added for 48 h after an initial 24-h in vitro rumen fermentation of orchardgrass (Dactylis glomerata L.) hay. Short-term shifts in bovine rumen community structure (bacteria, archea, protozoa, and fungi) resulting from each separate forage and feedstuff addition were measured using unique operational taxonomic units (OTU)s contained in terminal-restriction fragment length polymorphism (T-RFLP) profiles. The Tukey Vacuum Cleaner Analysis (TVCA) model of bacteria accounted for 66.3% of the treatment variance and OTUs clustered into 3 distinct groupings. The model developed for TVCA analysis of archaea accounted for 76.6% of the treatment variance and OTUs clustered into 2 distinct groupings. The TVCA treatment variance of protozoa accounted for 75.8% and OTUs clustered into 3 distinct groupings. Additions of various forages and feedstuffs did not result in shifts in fungal community structure in the short-term experiment. Our results demonstrate that the rumen ecology using initial T-RFLP profiles in vitro of bacteria, archaea, and protozoa and a TVCA analysis can identify pattern groupings of forages and feedstuffs. Groupings of forages and feedstuffs can help refine supplementation for improved nutritional management of ruminants.

Differences in gut microbial patterns associated with salivary biomarkers in young Japanese adults.

Recent evidence suggests that psychological stress is associated with gut microbiota; however, there are no reports of its association with gut microbial structure. This cross-sectional study examined the relationship between psychological stress and gut microbial patterns in young Japanese adults. Analysis of fecal microbiota was performed using terminal restriction fragment length polymorphism (T-RFLP). Psychological stress was assessed using salivary biomarkers, including cortisol, alpha-amylase, and secretory IgA (S-IgA). Fecal microbial patterns were defined using principal component analysis of the T-RFLP profile and were classified into two enterotype-like clusters, which were defined by the B (microbiota dominated by Bacteroides) and BL patterns (microbiota dominated by Bifidobacterium and Lactobacillales), respectively. The Simpson index was significantly higher for the BL pattern than for the B pattern. The salivary cortisol level was significantly lower for the BL pattern than for the B pattern. Salivary alpha-amylase and S-IgA levels showed a negative correlation with the Simpson index. Our results raise the possibility that salivary biomarkers may be involved in the observed differences in microbial patterns.

Terminal restriction fragment length polymorphism is an \u201cold school\u201d reliable technique for swift microbial community screening in anaerobic digestion

The microbial community in anaerobic digestion has been analysed through microbial fingerprinting techniques, such as terminal restriction fragment length polymorphism (TRFLP), for decades. In the last decade, high-throughput 16S rRNA gene amplicon sequencing has replaced these techniques, but the time-consuming and complex nature of high-throughput techniques is a potential bottleneck for full-scale anaerobic digestion application, when monitoring community dynamics. Here, the bacterial and archaeal TRFLP profiles were compared with 16S rRNA gene amplicon profiles (Illumina platform) of 25 full-scale anaerobic digestion plants. The α-diversity analysis revealed a higher richness based on Illumina data, compared with the TRFLP data. This coincided with a clear difference in community organisation, Pareto distribution, and co-occurrence network statistics, i.e., betweenness centrality and normalised degree. The β-diversity analysis showed a similar clustering profile for the Illumina, bacterial TRFLP and archaeal TRFLP data, based on different distance measures and independent of phylogenetic identification, with pH and temperature as the two key operational parameters determining microbial community composition. The combined knowledge of temporal dynamics and projected clustering in the β-diversity profile, based on the TRFLP data, distinctly showed that TRFLP is a reliable technique for swift microbial community dynamics screening in full-scale anaerobic digestion plants.

Model development: establishing pigs with homogenous microbial profile in the hind gut

The considerable animal-to-animal variation in microbial profiles is a challenge in elucidating the role of gut microbiota in host metabolism. The main purpose of this study was, therefore, to develop a pig model with reduced animal-to-animal variation in gut microbial profile. Twelve piglets from four sows were reared conventionally and 12 piglets from four sows were reared artificially in high efficiency particulate air (HEPA) filtered isolators. All isolator-reared piglets were given an artificial colostrum formula containing the combined fecal material from all eight sows. All piglets were killed at 21 d of age and intestinal contents subjected to 16s rRNA gene-based terminal restriction fragment length polymorphism (T-RFLP) profiling. Resulting T-RFLP profiles clustered into two distinct groups representing the two treatment groups. Furthermore, Bray–Curtis dissimilarity distance values and Dice similarity indices showed reduced beta diversity in isolator-reared pigs indicating animal-to-animal variation was reduced in isolator-reared compared to conventional piglets. However, surprisingly, increased alpha diversity was observed in isolator-reared piglets compared with conventional piglets. In conclusion, the study demonstrated that rearing of piglets under conditions of controlled environment reduced animal-to-animal variation in the hindgut microbiota while paradoxically increasing within animal microbial diversity. Isolator rearing may be useful as a model to improve detection of treatment effects on gut microbiota.

Diversity of Diatom Communities in Delaware Tidal Wetland and Their Relationship to Water Quality

Diatoms are strongly influenced by water quality and serve as indicators of water quality degradation in freshwater systems. Here, sediment and water samples were collected from four sites in Blackbird Creek, DE, a salt marsh characterized as mostly freshwater to low saline brackish (<8ppt). Recent changes in land use resulted in increased agricultural activity, suggesting the need to develop water quality indicators in this region. To test the hypothesis that diatom community composition changes seasonally with variations in water quality parameters, sediment and water samples were collected in 2009 and 2010 for analysis. Water temperature, salinity, pH, and dissolved oxygen were measured as well as water and sediment dissolved nutrient concentrations (nitrate, ammonia, and total and reactive phosphorous). DNA was extracted from sediments and changes in diatom community composition were evaluated by amplification of 18S rRNA gene using diatom-specific primers, followed by Terminal Restriction Fragment Length Polymorphism (TRFLP) analysis. Shannon (H') index for TRFLP profiles ranged from 2.5 to 3.0 and Simpson (Ds) index was 0.9 which infers moderate levels of diatom species richness and high diversity in these study sites. Although there were no water quality parameters that were significantly correlated with diatom community composition as determined by TRFLP patterns, temperature was the most highly correlated (r=0.203). Dissolved oxygen, salinity, and pH of water also had moderate but insignificant impacts on the diatom community. Further analysis of cloned 18S rRNA sequences revealed the presence of diatom taxa that tolerate wide salinity ranges, and included Navicula, Cyclotella, Thalassiosira and Skeletonema. Entomoneis sp. were also present in the spring and fall seasons. Overall, results in this study demonstrate significant differences in water qualities among the study years but little change in diatom community composition between study sites and seasons, but may serve as a baseline for future studies.

Identification of metolachlor mineralizing bacteria in aerobic and anaerobic soils using DNA-stable isotope probing.

The influence of soil environmental factors such as aeration on the ecology of microorganisms involved in the mineralization and degradation of the popular soil-applied pre-emergent herbicide, metolachlor is unknown. To address this knowledge gap, we utilized DNA-based stable isotope probing (SIP) where soil microcosms were incubated aerobically or anaerobically and received herbicide treatments with unlabeled metolachlor or 13C-metolachlor. Mineralization of metolachlor was confirmed as noted from the evolution of 14CO2 from 14C-metolachlor-treated microcosms and clearly demonstrated the efficient utilization of the herbicide as a carbon source. Terminal restriction fragment length polymorphisms (T-RFLP) bacterial community profiling performed on soil DNA extracts indicated that fragment 307bp from aerobic soil and 212bp from anaerobic soil were detected only in the herbicide-treated (both unlabeled metolachlor and 13C-metolachlor) soils when compared to the untreated control microcosms. T-RFLP profiles from the ultracentrifugation fractions illustrated that these individual fragments experienced an increase in relative abundance at a higher buoyant density (BD) in the labeled fractions when compared to the unlabeled herbicide amendment fractions. The shift in BD of individual T-RFLP fragments in the density-resolved fractions suggested the incorporation of 13C from labeled herbicide into the bacterial DNA and enabled the identification of organisms responsible for metolachlor uptake from the soil. Subsequent cloning and 16S rRNA gene sequencing of the 13C-enriched fractions implicated the role of organisms closely related to Bacillus spp. in aerobic mineralization and members of Acidobacteria phylum in anaerobic mineralization of metolachlor in soil.

Enrichment of lignocellulose-degrading microbial communities from natural and engineered methanogenic environments.

The aim of this study was to develop an effective bioaugmentation concept for anaerobic digesters treating lignocellulosic biomass such as straw. For that purpose, lignocellulose-degrading methanogenic communities were enriched on wheat straw from cow and goat rumen fluid as well as from a biogas reactor acclimated to lignocellulosic biomass (sorghum as mono-substrate). The bacterial communities of the enriched cultures and the different inocula were examined by 454 amplicon sequencing of 16S rRNA genes while the methanogenic archaeal communities were analyzed by terminal restriction fragment length polymorphism (T-RFLP) fingerprinting of the mcrA gene. Bacteroidetes was the most abundant phylum in all samples. Within the Bacteroidetes phylum, Bacteroidaceae was the most abundant family in the rumen-derived enrichment cultures, whereas Porphyromonadaceae was the predominant one in the reactor-derived culture. Additionally, the enrichment procedure increased the relative abundance of Ruminococcaceae (phylum: Firmicutes) in all cultures. T-RFLP profiles of the mcrA gene amplicons highlighted that the ruminal methanogenic communities were composed of hydrogenotrophic methanogens dominated by the order Methanobacteriales regardless of the host species. The methanogenic communities changed significantly during the enrichment procedure, but still the strict hydrogenotrophic Methanobacteriales and Methanomicrobiales were the predominant orders in the enrichment cultures. The bioaugmentation potential of the enriched methanogenic cultures will be evaluated in further studies.

Effect of Eichhornia crassipes on Ammoxidation and Denitrification Microorganisms in Eutrophic Freshwaters

The water hyacinth, Eichhornia crassipes, is widely used for the ecological restoration of eutrophic freshwater bodies, but little is known about its microbial interactions and nitrogen removal potential. In this study, we compared the relative importance of E. crassipes and bacteria in nitrogen removal. We also examined the plant's ability to modulate the abundance and diversity of nitrifying and denitrifying bacteria. Nitrogen removal and the genetic potential for nitrification and denitrification, determined using quantitative polymerase chain reactions (qPCRs) of the nitrification gene amoA and the denitrification genes nirS/K, were evaluated in microcosms containing water from a eutrophic lake with or without E. crassipes. The results showed that total nitrogen (TN) losses on day 70 of the experiment were similar in all treatments but the uptake by E. crassipes resulted in the rapid (within 24 h) removal of TN and NH4+-N. In microcosms containing E. crassipes, the abundance of amoA increased whereas the abundances of nirS/K decreased. The T-RFLP (terminal restriction fragment length polymorphism) profiles showed that Nitrosomonas dominated the ammonia-oxidizing prokaryotes, based on the DNA and RNA levels of the targeted genes. The E. crassipes cultivation can be used to achieve fast and efficient reductions in NH4+-N concentrations in eutrophic water bodies. While this aquatic macrophyte may not be essential to N removal, considering the potential toxicity of NH4+-N, both aquaphytes and microbes should be fully exploited in the restoration of freshwater ecosystems.

Effect of abattoir and cut on variations in microbial communities of vacuum-packaged beef

This report builds on the earlier studies of the shelf-life of chilled Australian vacuum packaged (VP) beef primals (striploin and cube roll), products distinguished in the global marketplace for unusually long shelf-life. Notable findings in those studies were a shelf-life of at least 26weeks at −0.5°C, low microbial counts, and relatively high sensory scores. However, growth rates for total viable counts (TVC) and lactic acid bacteria (LAB) varied among the different abattoirs. The present study adds to these findings, by providing greater definition about temporal changes in bacterial communities using terminal restriction fragment length polymorphism (TRFLP) and clone library analyses of 16S ribosomal RNA (16S rRNA) gene, and measuring statistical associations among abattoir, beef cut, storage time and sensory attributes. Bacterial communities changed over time, with Carnobacterium spp. typically predominating (29–97%) at the end of storage. Variation in TRFLP profiles showed that different Carnobacterium strains predominated in different abattoirs, and that additional variation was due to the presence of other taxa typical of VP meat microbiomes. TRFLP-based community structure correlated significantly (P≤0.01) with sensorial characteristics, such as vacuum integrity, confinement odour, and intact pack appearance of beef. This study shows that Carnobacterium spp. predominate on extended shelf-life VP beef primals, while other taxa may produce subtle effects on shelf-life duration.

Effect of bioaugmentation by cellulolytic bacteria enriched from sheep rumen on methane production from wheat straw

The aim of this study was to determine the potential of bioaugmentation with cellulolytic rumen microbiota to enhance the anaerobic digestion of lignocellulosic feedstock. An anaerobic cellulolytic culture was enriched from sheep rumen fluid using wheat straw as substrate under mesophilic conditions. To investigate the effects of bioaugmentation on methane production from straw, the enrichment culture was added to batch reactors in proportions of 2% (Set-1) and 4% (Set-2) of the microbial cell number of the standard inoculum slurry. The methane production in the bioaugmented reactors was higher than in the control reactors. After 30 days of batch incubation, the average methane yield was 154 mLN CH4 gVS−1 in the control reactors. Addition of 2% enrichment culture did not enhance methane production, whereas in Set-2 the methane yield was increased by 27%. The bacterial communities were examined by 454 amplicon sequencing of 16S rRNA genes, while terminal restriction fragment length polymorphism (T-RFLP) fingerprinting of mcrA genes was applied to analyze the methanogenic communities. The results highlighted that relative abundances of Ruminococcaceae and Lachnospiraceae increased during the enrichment. However, Cloacamonaceae, which were abundant in the standard inoculum, dominated the bacterial communities of all batch reactors. T-RFLP profiles revealed that Methanobacteriales were predominant in the rumen fluid, whereas the enrichment culture was dominated by Methanosarcinales. In the batch rectors, the most abundant methanogens were affiliated to Methanobacteriales and Methanomicrobiales. Our results suggest that bioaugmentation with sheep rumen enrichment cultures can enhance the performance of digesters treating lignocellulosic feedstock.

Quantitative Monitoring of Microbial Species during Bioleaching of a Copper Concentrate.

Monitoring of the microbial community in bioleaching processes is essential in order to control process parameters and enhance the leaching efficiency. Suitable methods are, however, limited as they are usually not adapted to bioleaching samples and often no taxon-specific assays are available in the literature for these types of consortia. Therefore, our study focused on the development of novel quantitative real-time PCR (qPCR) assays for the quantification of Acidithiobacillus caldus, Leptospirillum ferriphilum, Sulfobacillus thermosulfidooxidans, and Sulfobacillus benefaciens and comparison of the results with data from other common molecular monitoring methods in order to evaluate their accuracy and specificity. Stirred tank bioreactors for the leaching of copper concentrate, housing a consortium of acidophilic, moderately thermophilic bacteria, relevant in several bioleaching operations, served as a model system. The microbial community analysis via qPCR allowed a precise monitoring of the evolution of total biomass as well as abundance of specific species. Data achieved by the standard fingerprinting methods, terminal restriction fragment length polymorphism (T-RFLP) and capillary electrophoresis single strand conformation polymorphism (CE-SSCP) on the same samples followed the same trend as qPCR data. The main added value of qPCR was, however, to provide quantitative data for each species whereas only relative abundance could be deduced from T-RFLP and CE-SSCP profiles. Additional value was obtained by applying two further quantitative methods which do not require nucleic acid extraction, total cell counting after SYBR Green staining and metal sulfide oxidation activity measurements via microcalorimetry. Overall, these complementary methods allow for an efficient quantitative microbial community monitoring in various bioleaching operations.

Effects of Fertilization on Soil Microbial Abundance and Community Structure at DNA and cDNA Levels in Paddy Soils

Fertilizer applications have important effects on soil microbial abundance and community structure. In this study, total soil microbial DNA and RNA were directly extracted from paddy soils of N0 (control treatment, no nitrogen fertilizer), NPK (balanced fertilization), NPK+LS (balanced fertilization with additional 3.0 t·hm-2 rice straw incorporation) and NPK+HS (balanced fertilization with additional 6.0 t·hm-2 rice straw incorporation) treatments in a long-term fertilization experiment of double rice cropping system in Changsha County, Hunan Province. Soil bacteria community structures were evaluated by analyzing the 16S rRNA gene fragments at DNA and cDNA levels with Terminal Restriction Fragment Length Polymorphism (T-RFLP) and quantitative PCR techniques. Balancing fertilization with chemical fertilizers and rice straw incorporation significantly changed the composition of bulk (DNA-based) and potentially active (mRNA-based) soil bacterial community as shown in T-RFLP profiles, and also reduced the bulk soil microbial diversity, but not the potentially active ones, as compared with the control treatment. The DNA-based abundance of 16S rRNA gene was on average 377 times as many as the m-RNA based population size. Compared to N0,balanced fertilization with rice straw incorporation (NPK+LS and NPK+HS) increased the bulk and active copy numbers of 16S rRNA gene, but not for balanced fertilization (NPK). The abundance and microbial community structure were not significantly different between the NPK+LS and NPK+HS treatments. Redundancy analysis (RDA) showed that soil ammonium was the key environmental factor determining the bulk and active soil microbial community structure among the treatments. In conclusion, the effect of fertilization on soil microbial abundance and community structure could be indicated at both DNA and cDNA levels; the cDNA information could better reflect the adaptability of bacterial community to the environmental stress.

Long-Term Effects of Multiwalled Carbon Nanotubes and Graphene on Microbial Communities in Dry Soil.

Little is known about the long-term effects of engineered carbonaceous nanomaterials (ECNMs) on soil microbial communities, especially when compared to possible effects of natural or industrial carbonaceous materials. To address these issues, we exposed dry grassland soil for 1 year to 1 mg g(-1) of either natural nanostructured material (biochar), industrial carbon black, three types of multiwalled carbon nanotubes (MWCNTs), or graphene. Soil microbial biomass was assessed by substrate induced respiration and by extractable DNA. Bacterial and fungal communities were examined by terminal restriction fragment length polymorphism (T-RFLP). Microbial activity was assessed by soil basal respiration. At day 0, there was no treatment effect on soil DNA or T-RFLP profiles, indicating negligible interference between the amended materials and the methods for DNA extraction, quantification, and community analysis. After a 1-year exposure, compared to the no amendment control, some treatments reduced soil DNA (e.g., biochar, all three MWCNT types, and graphene; P < 0.05) and altered bacterial communities (e.g., biochar, carbon black, narrow MWCNTs, and graphene); however, there were no significant differences across the amended treatments. These findings suggest that ECNMs may moderately affect dry soil microbial communities but that the effects are similar to those from natural and industrial carbonaceous materials, even after 1-year exposure.

甲壳素对连作条件下平邑甜茶幼苗生长及土壤环境的影响

PDF HTML阅读 XML下载 导出引用 引用提醒 甲壳素对连作条件下平邑甜茶幼苗生长及土壤环境的影响 DOI: 10.5846/stxb201501270221 作者: 作者单位: 作者简介: 通讯作者: 中图分类号: 基金项目: 现代农业产业技术体系建设专项资金（CARS-28）；教育部长江学者和创新团队发展计划（IRT1155）；泰安市科技发展计划项目（32802） Effects of chitin on the growth of Malus hupehensis Rehd. seedlings and soil environment under replant condition Author: Affiliation: Fund Project: 摘要 | 图/表 | 访问统计 | 参考文献 | 相似文献 | 引证文献 | 资源附件 | 文章评论 摘要:研究在苹果连作土壤中添加甲壳素对苹果幼苗生长、土壤酶及土壤真菌群落结构的影响，探讨甲壳素缓解苹果连作障碍的可能性，为防控苹果连作障碍提供依据。盆栽条件下，以平邑甜茶幼苗为试材，在苹果连作土壤中分别添加0，0.5，1.0和2.5 g/kg的甲壳素，测定了连作土壤中添加不同量的甲壳素后，幼苗生物量、根系保护酶活性、土壤主要酶（蔗糖酶、脲酶、磷酸酶等）活性以及土壤中真菌群落结构的变化。9月份结果表明，与对照相比，1.0 g/kg的甲壳素处理连作土，可显著提高平邑甜茶幼苗株高和干鲜重，分别比对照增加了36.8%、82.1%和100.8%；甲壳素处理能增加幼苗根系保护酶活性，其中1.0 g/kg甲壳素处理SOD、POD和CAT活性最高，其次为0.5 g/kg，而2.5 g/kg甲壳素处理显著抑制了幼苗根系保护酶活性。1.0 g/kg甲壳素处理可提高土壤中细菌/真菌值，并且提高了土壤中蔗糖酶、脲酶、蛋白酶、磷酸酶、过氧化氢酶和多酚氧化酶活性，分别比对照提高了8.6%、40.5%、81.1%、15.3%、18.7%和49.8%，2.5 g/kg甲壳素处理则降低土壤酶活性或者使土壤酶活性与对照相当。根据T-RFLP的图谱中OUT的数量、种类及丰度，分别计算了不同处理土壤的真菌多样性，发现1.0 g/kg甲壳素处理的连作土具有最高的多样性、均匀度和丰富度指数，分别比对照增加了52.2%、8.0%和87.1%。主成分分析（PCA）结果显示，不同剂量甲壳素处理的连作土壤中真菌被PC2分成了两部分，其中0.5 g/kg和1.0 g/kg的甲壳素添加量分布在PC2的负方向上，而CK和2.5 g/kg的甲壳素处理分布在PC2的正方向上，这说明添加不同量的甲壳素对连作土壤真菌群落多样性有显著影响，添加量太多或者太少均会造成土壤真菌多样性下降，只有适量的甲壳素可提高真菌群落结构多样性。实验结果表明1.0 g/kg的甲壳素可提高连作平邑甜茶幼苗生物量，改善连作土壤环境，有效缓解平邑甜茶的连作障碍。 Abstract:The effects of different concentrations of chitin on the activity of enzymes and fungal communities existing in replant soils were explored, for enrichment of soils to alleviate apple replant disease (ARD). In this study, Malus hupehensis Rehd. seedlings potted in replant soil were treated with four concentrations of chitin (0, 0.5, 1.0, and 2.5 g/kg). The growth of seedlings was monitored though plant height, fresh and dry weight, antioxidant enzyme activities, such as superoxide dismutase (SOD), peroxidase (POD), and catalase (CAT), and lipid peroxidation in roots. Simultaneously, soil enzyme activities, soil microbial load, and fungal community structure, which was measured by terminal-restriction fragment length polymorphism (T-RFLP) profiles, were determined in soil samples subjected to the four chitin treatments. Compared with the control, seedlings treated with 1.0 g/kg chitin showed 36.8%, and 82.1% and 100.8% increase in plant height, and fresh and dry weight, respectively. The optimal dose of chitin could increase antioxidant enzyme activity in the root system of the seedlings, whereas high doses inhibited enzyme activity. The effectiveness of chitin treatment for inhibiting the activity of SOD, POD, and CAT in the root system of M. hupehensis seedlings decreased in the following order:1.0 g/kg > 0.5 g/kg > 0 g/kg > 2.5 g/kg. Chitin treatment at 1.0 g/kg enhanced the bacterial-fungal ratio, and resulted in a "bacteria-rich soil"; consequently, sucrase, urease, protease, phosphatase, catalase, and polyphenol oxidase activities increased by 8.6%, 40.5%, 81.1%, 15.3%, 18.7%, and 49.8%, respectively. Remarkable differences in T-RFLP profiles were observed among control, 0.5 g/kg, 1.0 g/kg, and 2.5 g/kg treatments. A significant difference in fungal community structure was observed with the application of different doses of chitin. The Shannon diversity, evenness, and richness indexes were the highest in replanted soil treated with 1.0 g/kg chitin and the least in soil treated with 2.5 g/kg chitin. Principal component analysis indicated that fungal community structure of soil treated with 1.0 g/kg chitin was considerably different from that of the control soil and soil treated with 2.5 g/kg chitin. The present study showed that low concentrations of chitin could alleviate ARD stress in M. hupehensis Rehd. seedlings, whereas high concentrations could aggravate ARD stress. Collectively, these findings suggest that chitin applied to replant soil at 1.0 g/kg obviously enhanced plant height, fresh and dry weight, improved the soil environment, and effectively alleviate ARD stress in M. hupehensis Rehd. seedlings, therefore, a chitin dose of 1.0 g/kg was considered optimal for replant soil. 参考文献 相似文献 引证文献

Diversity of Methane-Oxidizing Bacteria in Soils from "Hot Lands of Medolla" (Italy) Featured by Anomalous High-Temperatures and Biogenic CO2 Emission.

“Terre Calde di Medolla” (TCM) (literally, “Hot Lands of Medolla”) refers to a farming area in Italy with anomalously high temperatures and diffuse emissions of biogenic CO2, which has been linked to CH4 oxidation processes from a depth of 0.7 m to the surface. We herein assessed the composition of the total bacterial community and diversity of methane-oxidizing bacteria (MOB) in soil samples collected at a depth at which the peak temperature was detected (0.6 m). Cultivation-independent methods were used, such as: i) a clone library analysis of the 16S rRNA gene and pmoA (coding for the α-subunit of the particulate methane monooxygenase) gene, and ii) Terminal Restriction Fragment Length Polymorphism (T-RFLP) fingerprinting. The 16S rRNA gene analysis assessed the predominance of Actinobacteria, Acidobacteria, Proteobacteria, and Bacillus in TCM samples collected at a depth of 0.6 m along with the presence of methanotrophs (Methylocaldum and Methylobacter) and methylotrophs (Methylobacillus). The phylogenetic analysis of pmoA sequences showed the presence of MOB affiliated with Methylomonas, Methylocystis, Methylococcus, and Methylocaldum in addition to as yet uncultivated and uncharacterized methanotrophs. Jaccard’s analysis of T-RFLP profiles at different ground depths revealed a similar MOB composition in soil samples at depths of 0.6 m and 0.7 m, while this similarity was weaker between these samples and those taken at a depth of 2.5 m, in which the genus Methylocaldum was absent. These results correlate the anomalously high temperatures of the farming area of “Terre Calde di Medolla” with the presence of microbial methane-oxidizing bacteria.