模拟干湿交替对水稻土古菌群落结构的影响

干湿交替是自然界普遍存在的现象,但长期以来由于技术的限制,复杂土壤中微生物对水分变化的响应规律仍不清楚。针对我国江苏常熟湖泊底泥发育的典型水稻土,在室内开展湿润-风干以及风干-湿润各三次循环,每次循环中湿润、风干状态各维持7d,利用微生物核糖体rRNA的通用引物进行PCR扩增,通过高通量测序分析土壤古菌多样性变化,同时结合实时荧光定量PCR技术,在DNA和RNA水平研究古菌数量对干湿交替过程的响应规律。结果表明:水稻土湿润-风干过程中,在DNA水平土壤古菌数量降幅约为149倍~468倍,而在RNA水平降幅最高仅为2.06倍;水稻土风干-湿润过程中,在DNA水平古菌数量增幅在147倍~360倍之间,而在RNA水平增幅最高仅为2.95倍。表明在干湿交替过程中,DNA水平的古菌16S rRNA基因数量变化远高于RNA水平。基于高通量测序多样性的结果表明,在DNA和RNA水平,湿润土壤3次风干、以及风干土壤3次加水湿润7d恢复后,土壤古菌群落结构均发生统计显著性改变。在微生物门、纲、目、科和属的不同分类水平下,水稻土古菌主要包括3、10、13、14、10种不同的类群,在RNA和DNA水平的结果基本一致。干湿交替导致部分古菌类群发生显著变化,其中在微生物分类学目水平发生显著变化的古菌最高达到6种,主要包括产甲烷古菌和氨氧化古菌,如Methanobacteriales、Methanosarcinales、Methanomicrobiales和Nitrososphaerales等。这些研究结果表明,反复的干湿交替并未显著改变水稻土中古菌的主要类群组成,古菌类群的绝对数量和相对丰度发生了一定程度的变化,但这些变化与微生物生理作用的联系仍需进一步研究;风干土壤中古菌RNA序列极可能来自于完整的古菌细胞,暗示了这些古菌细胞能够较好地适应水稻土中水分的剧烈变化,风干状态的土壤在一定程度也可用于土壤古菌群落组成研究。     

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.     

Microbial activity and community structure of a soil after heavy metal contamination in a model forest ecosystem

We assessed the effects of chronic heavy metal (HM) contamination on soil microbial communities in a newly established forest ecosystem. We hypothesized that HM would affect community function and alter the microbial community structure over time and that the effects are more pronounced in combination with acid rain (AR). These hypotheses were tested in a model forest ecosystem consisting of several tree species (Norway spruce, birch, willow, and poplar) maintained in open top chambers. HMs were added to the topsoil as filter dust from a secondary metal smelter and two types of irrigation water acidity (ambient rain vs. acidified rain) were applied during four vegetation periods. HM contamination strongly impacted the microbial biomass (measured with both fumigation-extraction and quantitative lipid biomarker analyses) and community function (measured as basal respiration and soil hydrolase activities) of the soil microbial communities. The most drastic effect was found in the combined treatment of HM and AR, although soil pH and bioavailable HM contents were comparable to those of treatments with HM alone. Analyses of phospholipid fatty acids (PLFAs) and terminal restriction fragment length polymorphisms (T-RFLPs) of PCR-amplified 16S ribosomal DNA showed that HM treatment affected the structure of bacterial communities during the 4-year experimental period. Very likely, this is due to the still large bioavailable HM contents in the HM contaminated topsoils at the end of the experiment.     

纳米TiO2对黄瓜叶围细菌群落的影响

利用可培养和变性梯度凝胶电泳(DGGE)方法,对经不同浓度TiO2处理黄瓜(Cucumis sativus)叶围细菌群落结构的变化进行了分析。研究发现,TiO2处理后黄瓜叶围可培养细菌数量较对照显著降低。随着TiO2浓度从0.02 mg/mL提高至20 mg/mL,黄瓜叶围可培养细菌数量从1.8×107 cfu/g降低至3.1×106 cfu/g。DGGE指纹图谱分析表明,当TiO2的喷雾浓度超过0.02 mg/mL时,黄瓜叶围细菌的多样性显著降低。对DGGE条带克隆测序结果显示,在黄瓜叶围至少存在7种不同属的细菌,其中只有一种叶围细菌不受TiO2喷雾浓度影响。     

Community structure of bacteria on different types of mineral particles in a sandy soil

Various types of mineral particles in a soil probably provide different microenvironments for microorganisms. The purpose of this study is to investigate whether different types of mineral in a soil harbor different bacterial populations. DNA was extracted from five types (quartz, feldspar, pyroxene, magnetite, iron-coated reddish brown particles) of sand-size mineral particles separated from a sandy soil, and was amplified for partial 16 S rRNA gene by polymerase chain reaction (PCR). Twenty-nine to 69 amplicons per each type of mineral were cloned and sequenced, followed by phylogenetic affiliation of the sequences. As a result, some types of bacteria were detected on all of the types of mineral including the orders Rhizobiales, Bacillales, and Acidobacteriales. In the case of Acidobacteriales, higher percentages were found on magnetite and quartz. Some taxa were restricted to specific types of mineral; the class Actinobacteria was found on pyroxene but not on quartz, and rarely on magnetite and feldspar. Bacterial diversity at the order level estimated by Chao1 value was higher in feldspar and pyroxene than the other three types of mineral. The UniFrac Significance test indicated that the differences in bacterial communitiy structures among the particles were suggestive except that between feldspar and pyroxene. These results support the idea that different communities of bacteria were associated with each of the mineral types.     

Abundance and community composition of methanotrophs in a Chinese paddy soil under long-term fertilization practices

Background, aim, and scope  As the second most important greenhouse gas, methane (CH₄) is produced from many sources such as paddy fields. Methane-oxidizing bacteria (methanotrophs) consume CH₄ in paddy soil and, therefore, reduce CH₄ emission to the atmosphere. In order to estimate the contribution of paddy fields as a source of CH₄, it is important to monitor the effects of fertilizer applications on the shifts of soil methanotrophs, which are targets in strategies to combat global climate change. In this study, real-time polymerase chain reaction (PCR) and denaturing gradient gel electrophoresis (DGGE) based on 16S rRNA and pmoA genes, respectively, were used to analyze the soil methanotrophic abundance and community diversity under four fertilization treatments: urea (N), urea and potassium chloride (NK), urea, superphosphate, and potassium chloride (NPK), and urea, superphosphate, potassium chloride, and crop residues (NPK+C), compared to an untreated control (CON). The objective of this study was to examine whether soil methanotrophs responded to the long-term, different fertilizer regimes by using a combination of quantitative and qualitative molecular approaches. Materials and methods  Soil samples were collected from the Taoyuan Experimental Station of Agro-ecosystem Observation at Changde (28°55′ N, 111°26′ E), central Hunan Province of China, in July 2006. Soil DNAs were extracted from the samples, then the 16S rRNA genes were quantified by real-time PCR and the pmoA genes were amplified via general PCR followed by DGGE, cloning, sequencing, and phylogenetic analysis. The community diversity indices were assessed through the DGGE profile. Results  Except for NPK, other treatments of N, NK, and NPK+C showed significantly higher copy numbers of type I methanotrophs (7.0–9.6 × 10⁷) than CON (5.1 × 10⁷). The copy numbers of type II methanotrophs were significantly higher in NPK+C (2.8 × 10⁸) and NK (2.5 × 10⁸) treatments than in CON (1.4 × 10⁸). Moreover, the ratio of type II to type I methanotrophic copy numbers ranged from 1.88 to 3.32, indicating that the type II methanotrophs dominated in all treatments. Cluster analyses based on the DGGE profile showed that the methanotrophic community in NPK+C might respond more sensitively to the environmental variation. Phylogenetic analysis showed that 81% of the obtained pmoA sequences were classified as type I methanotrophs. Furthermore, the type I-affiliated sequences were related to Methylobacter, Methylomicrobium, Methylomonas, and some uncultured methanotrophic clones, and those type II-like sequences were affiliated with Methylocystis and Methylosinus genera. Discussion  There was an inhibitory effect on the methanotrophic abundance in the N and a stimulating effect in the NK and NPK+C treatments, respectively. During the rice-growing season, the type II methanotrophs might be more profited from such a coexistence of low O₂ and high CH₄ concentration environment than the type I methanotrophs. However, type I methanotrophs seemed to be more frequently detected. The relatively complex diversity pattern in the NPK+C treatment might result from the strong CH₄ production. Conclusions  Long-term fertilization regimes can both affect the abundance and the composition of the type I and type II methanotrophs. The inhibited effects on methanotrophic abundance were found in the N treatment, compared to the stimulated effects from the NK and NPK+C treatments. The fertilizers of nitrogen, potassium, and the crop residues could be important factors controlling the abundance and community composition of the methanotrophs in the paddy soil. Recommendations and perspectives  Methanotrophs are a fascinating group of microorganisms playing an important role in the biogeochemical carbon cycle and in the control of global climate change. However, it is still a challenge for the cultivation of the methanotrophs, although three isolates were obtained in the extreme environments very recently. Therefore, future studies will be undoubtedly conducted via molecular techniques just like the applications in this study.     

Leaf litter is the main driver for changes in bacterial community structures in the rhizosphere of ash and beech

The rhizosphere and the surrounding soil harbor an enormous microbial diversity and a specific community structure, generated by the interaction between plant roots and soil bacteria. The aim of this study was to address the influences of tree species, tree species diversity and leaf litter on soil bacterial diversity and community composition. Therefore, mesocosm experiments using beech, ash, lime, maple and hornbeam were established in 2006, and sampled in October 2008 and June 2009. Mesocosms were planted with one, three or five different tree species and treated with or without litter overlay.Cluster analysis of DGGE-derived patterns revealed a clustering of 2008 sampled litter treatments in two separated clusters. The corresponding treatments sampled in 2009 showed separation in one cluster. PCA analysis based on the relative abundance of active proteobacterial classes and other phyla in beech and ash single-tree species mesocosm indicated an effect of sampling time and leaf litter on active bacterial community composition. The abundance of next-generation sequencing-derived sequences assigned to the Betaproteobacteria was higher in the litter treatments, indicating a higher activity, under these conditions. The Deltaproteobacteria, Nitrospira and Gemmatimonadetes showed an opposite trend and were more active in the mesocosms without litter. The abundance of alphaproteobacterial sequences was higher in mesocosms sampled in 2009 (P = 0.014), whereas the Acidobacteria were more active in 2008 (P = 0.014). At the family level, we found significant differences of the litter vs. non-litter treated group. Additionally, an impact of beech and ash as tree species on soil bacterial diversity was confirmed by the Shannon and Simpson indices. Our results suggest that leaf litter decomposition in pH-stable soils affect the soil bacterial composition, while tree species influence the soil bacterial diversity.     

天山云杉林下土壤放线菌的分离与DNA指纹分析方法的初步研究

新疆天山土壤放线菌的分离和鉴定工作,对全面了解该地区放线菌分布状况具有重要意义。使用7种典型的放线菌分离培养基对土样中的放线菌进行分离,共得到8个属的放线菌分离株,并对分离培养基的分离效果进行比较。使用MseI、AluI、PstI和HhaI 4种限制性内切酶对20株链霉菌分离株的16S rRNA基因进行了PCR-RFLP分析。使用BOX-PCR方法构建了9株稀有放线菌菌株的指纹图谱。PCR-RFLP和BOX-PCR聚类分析结果分别与16S rRNA基因全序列聚类分析结果具有一致性。因此,PCR-RFLP和BOX-PCR指纹分析技术可在一定程度上代替16S rRNA基因全序列分析,用于新疆天山土壤放线菌的快速鉴定和多样性研究。     

Establishment of macro-aggregates and organic matter turnover by microbial communities in long-term incubated artificial soils

The study of interactions between minerals, organic matter (OM) and microorganisms is essential for the understanding of soil functions such as OM turnover. Here, we present an interdisciplinary approach using artificial soils to study the establishment of the microbial community and the formation of macro-aggregates as a function of the mineral composition by using artificial soils. The defined composition of a model system enables to directly relate the development of microbial communities and soil structure to the presence of specific constituents. Five different artificial soil compositions were produced with two types of clay minerals (illite, montmorillonite), metal oxides (ferrihydrite, boehmite) and charcoal incubated with sterile manure and a microbial community derived from a natural soil. We used the artificial soils to analyse the response of these model soil systems to additional sterile manure supply (after 562 days). The artificial soils were subjected to a prolonged incubation period of more than two years (842 days) in order to take temporally dynamic processes into account. In our model systems with varying mineralogy, we expected a changing microbial community composition and an effect on macro-aggregation after OM addition, as the input of fresh substrate will re-activate the artificial soils. The abundance and structure of 16S rRNA gene and internal transcribed spacer (ITS) fragments amplified from total community DNA were studied by quantitative real-time PCR (qPCR) and denaturing gradient gel electrophoresis (DGGE), respectively. The formation of macro-aggregates (>2 mm), the total organic carbon (OC) and nitrogen (N) contents, the OC and N contents in particle size fractions and the CO₂ respiration were determined. The second manure input resulted in higher CO₂ respiration rates, 16S rRNA gene and ITS copy numbers, indicating a stronger response of the microbial community in the matured soil-like system. The type of clay minerals was identified as the most important factor determining the composition of the bacterial communities established. The additional OM and longer incubation time led to a re-formation of macro-aggregates which was significantly higher when montmorillonite was present. Thus, the type of clay mineral was decisive for both microbial community composition as well as macro-aggregation, whereas the addition of other components had a minor effect. Even though different bacterial communities were established depending on the artificial soil composition, the amount and quality of the OM did not show significant differences supporting the concept of functional redundancy.     

Thirteen years of continued application of composted organic wastes in a vineyard modify soil quality characteristics

A solution for environmentally wiser agriculture is the use of composted organic wastes as soil amendments. Just as this alleviates the problem of recycling organic residues, it provides necessary nutrient input for food production. The objective of this work was to study the effect that 13 years of applying three different composted organic wastes or organic amendments have had on soil quality, GHG emissions and the dynamics of its microbial communities 15 days after the annual application. For this purpose, in 1996 a field trial was set up in a Tempranillo vineyard. Since 1998, the applied organic amendments have been as follows: 1. a pelletized organic compost (PEL) made from plant, animal and sewage sludge residues; 2. a compost made from the organic fraction of municipal solid waste (OF-MSW); 3. a compost made of stabilized sheep manure (SMC); 4. a mineral fertilizer (NPK); and 5. an unaltered control. The mean annual doses applied since 1998 have been 3700 kg ha⁻¹ fresh weight (FW) of PEL, 4075 kg ha⁻¹ FW of OF-MSW, 4630 kg ha⁻¹ FW of SMC, and 340 kg ha⁻¹ of NPK treatment. Soil quality was consistently enhanced by amendment application over the 13 years. Total nitrogen was significantly increased in PEL (0.1%), OF-MSW (0.09%) and SMC (0.1%) compared to control (0.06%). Nutrient content was also improved in a similar way, e.g. the most significant increase in P Olsen (80.7 mg kg⁻¹) and K₂O (473.8 mg kg⁻¹) was found on SMC. The overall enzyme activity was also increased 15 days after the annual application and OF-MSW had the highest rate (95.9) compared to control (51.3). This increase in metabolic activity was also recorded in GHG emissions. CO₂ equivalents per hectare were 1745 kg for OF-MSW and it was the only significant difference found. PEL with 1598 kg and SMC with 1591 kg were not different from the Control (1104 kg). Even though GHG emissions in the soil increased because of the application, soil organic matter content increased significantly (at least 35% more in all organic treatments compared to control) and this rise in organic matter was consistent over the years. According to the results, 85% of the sequences corresponded to 5 main phyla: Proteobacteria, Actinobacteria, Bacteroidetes, Acidobacteria and Gemmatimonadetes, with unclassified material making up for 10.9% (average) of the sequences. Bacterial diversity by Shannon and Chao1 indices was not affected 15 days after the application. However, slight changes in the bacterial community were recorded 15 days after application only in OF-MSW treatment. Assessing soil quality using these three factors allows the relevant agronomical capabilities of the soil to be integrated with the potential effect of this practise on global warming.     

Fire severity shapes plant colonization effects on bacterial community structure,microbial biomass,and soil enzyme activity in secondary succession of a burned forest

The increasing frequency and severity of wildfires has led to growing attention to the effects of fire disturbance on soil microbial communities and biogeochemical cycling. While many studies have examined fire impacts on plant communities, and a growing body of research is detailing the effects of fire on soil microbial communities, little attention has been paid to the interaction between plant recolonization and shifts in soil properties and microbial community structure and function. In this study, we examined the effect of a common post-fire colonizer plant species, Corydalis aurea, on soil chemistry, microbial biomass, soil enzyme activity and bacterial community structure one year after a major forest wildfire in Colorado, USA, in severely burned and lightly burned soils. Consistent with past research, we find significant differences in soil edaphic and biotic properties between severe and light burn soils. Further, our work suggests an important interaction between fire severity and plant effects by demonstrating that the recolonization of soils by C. aurea plants only has a significant effect on soil bacterial communities and biogeochemistry in severely burned soils, resulting in increases in percent nitrogen, extractable organic carbon, microbial biomass, β-glucosidase enzyme activity and shifts in bacterial community diversity. This work propounds the important role of plant colonization in succession by demonstrating a clear connection between plant colonization and bacterial community structure as well as the cycling of carbon in a post-fire landscape. This study conveys how the strength of plant–microbe interactions in secondary succession may shift based on an abiotic context, where plant effects are accentuated in harsher abiotic conditions of severe burn soils, with implications for bacterial community structure and enzyme activity.     

The influence of soil properties on the structure of bacterial and fungal communities across land-use types

Land-use change can have significant impacts on soil conditions and microbial communities are likely to respond to these changes. However, such responses are poorly characterized as few studies have examined how specific changes in edaphic characteristics do, or do not, influence the composition of soil bacterial and fungal communities across land-use types. Soil samples were collected from four replicated (n = 3) land-use types (hardwood and pine forests, cultivated and livestock pasture lands) in the southeastern US to assess the effects of land-use change on microbial community structure and distribution. We used quantitative PCR to estimate bacterial–fungal ratios and clone libraries targeting small-subunit rRNA genes to independently characterize the bacterial and fungal communities. Although some soil properties (soil texture and nutrient status) did significantly differ across land-use types, other edaphic factors (e.g., pH) did not vary consistently with land-use. Bacterial–fungal ratios were not significantly different across the land-uses and distinct land-use types did not necessarily harbor distinct soil fungal or bacterial communities. Rather, the composition of bacterial and fungal communities was most strongly correlated with specific soil properties. Soil pH was the best predictor of bacterial community composition across this landscape while fungal community composition was most closely associated with changes in soil nutrient status. Together these results suggest that specific changes in edaphic properties, not necessarily land-use type itself, may best predict shifts in microbial community composition across a given landscape. In addition, our results demonstrate the utility of using sequence-based approaches to concurrently analyze bacterial and fungal communities as such analyses provide detailed phylogenetic information on individual communities and permit the robust assessment of the biogeographical patterns exhibited by soil microbial communities.     

Community structure of methanogenic archaea in paddy field soil under double cropping (rice-wheat)

Community structure of methanogenic archaea in paddy field soil under double cropping (rice [Oryza sativa L.] and wheat [Triticum aestivum L.]) was studied by the denaturing gradient gel electrophoresis (DGGE) method. Soil samples under flooded and upland conditions were collected 7 and 6 times, respectively, from two paddy fields throughout a year, and two primer sets, 0357F-GC/0691R and newly designed 1106F-GC/1378R, were used for DGGE analysis. The 25 and 29 different bands were observed on the DGGE gels with the primers 0357F-GC/0691R and 1106F-GC/1378R, respectively. DGGE band patterns of the methanogenic archaeal community were stable throughout a year including the cultivation periods of rice under flooded conditions and of wheat under upland conditions. Cluster analysis and principal component analysis suggested that the difference in the soil type (sampling region) largely influenced the community structures of methanogenic archaea in paddy field soil, while the effects of sampling period and different fertilizer treatments on them were small. Most of the sequences obtained from the DGGE bands were closely related to Methanomicrobiales, Methanosarcinaceae, Methanosaetaceae and Rice cluster-I.     

Genetic diversity of Rhizobium present in nodules of Phaseolus vulgaris L. cultivated in two soils of the central region in Chile

Although Phaseolus vulgaris L. is native from the Americas and is currently cultured in diverse areas, very little is known about the diversity of symbiotic nitrogen fixing Rhizobium (mycrosymbiont) in many of those cultures. Therefore, the aim of this study was to assess the genetic diversity of Rhizobium present in nodules of P. vulgaris in the central region of Chile. A method to extract DNA from surface-sterilized nodules was applied to two populations of the same seed variety grown in different fields. The 16S rRNA and nifH genes were amplified directly from the DNA extracted. DGGE analysis and clone libraries showed a restricted genetic diversity of the microsymbiotic populations that nodulate P. vulgaris. Both molecular markers revealed the presence of a microsymbiont closely related to Rhizobium etli in all the plants from the soils studied, indicating that the populations of Rhizobium sp. nodulating P. vulgaris in the central region of Chile displayed an extremely low genetic diversity. The level of genetic diversity in microsymbiont populations in plants grown in soils with different origin suggested that other factors rather than the indigenous soil rhizobial populations play a major role in the selection of the symbiotic partner in P. vulgaris.     

Rhizosphere microbiota assemblage associated with wild and cultivated soybeans grown in three types of soil suspensions

Soil microbial community composition is determined by the soil type and the plant species. By sequencing the V3-V4 region of the bacterial 16S rRNA gene amplicons, the current study assessed the bacterial community assemblage in rhizosphere and bulks soils of wild (Glycine soja) and cultivated (Glycine max) soybeans grown in the suspensions of three important soil types in China, including black, red and soda-saline-alkali soils. The alpha-diversity of the bacterial community in the rhizosphere was significantly higher than that of the bulk soils suggesting that bulk soil lacks plant nurturing effect under the current study conditions. Black and red soils were enriched with nitrifying and nitrogen-fixing bacteria but the soda-saline-alkali soil suspension had more denitrifying bacteria, which may reflect agronomic unsuitability of the latter. We also observed a high abundance of Bradyrhizobium and Pseudomonas, enriched cellulolytic bacteria, as well as a highly connected molecular ecological network in the G. soja rhizosphere soil. Taken all, the current study suggest that wild soybeans may have evolved to recruit beneficial microbes in its rhizosphere that can promote nutrients requisition, biostasis and disease-resistance, therefore ecologically more resilient than cultivated soybeans.     

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.     

Diversity of endophytic actinomycetes in mandarin grown in northern Thailand,their phytohormone production potential and plant growth promoting activity

Abstract

The rapid expansion of mandarin (Citrus reticulata L.) production areas with high agrochemical input in the highland areas of northern Thailand has resulted in negative effects in terms of production, environment, soil quality, and public health. The use of microorganisms as plant growth promoters is an alternative method to reduce agrochemical input. Thus, we studied the diversity of endophytic actinomycetes in mandarin and their potential as plant growth promoters. A total of 252 endophytic actinomycete isolates were recovered from mandarin. Based on spore chain morphology, cell wall type, and 16S rRNA gene sequence, the isolates were classified into six genera: Streptomyces, Nocardia, Nocardiopsis, Spirillospora, Microbispora and Micromonospora. The most frequent isolates recovered were members of Streptomyces (85.3%). Selected isolates (64 isolates) from these genera were evaluated for their indole-3-acetic acid (IAA) production potential in a medium with 2 mg mL^?1 tryptophan, and all the selected isolates showed the potential to produce IAA, with average values of IAA production of 13.34, 3.36, 140.38, 12.55, 1.40, and 6.19 µg IAA mL^?1, respectively. Isolates of genus Nocardiopsis showed a very high ability to produce IAA that was the highest among all the genera, with values ranging from 62.23 to 222.75 µg mL^?1. Twelve isolates selected from these genera were inoculated onto mandarin seedlings, and the results indicated that the shoot height, fresh shoot weight and fresh root weight of the seedlings were promoted by the inoculation of endophytic actinomycetes, with values ranging from 20.2 to 49.1%, 14.9 to 53.6%, and 1.6 to 102% over the control, respectively.     

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.