Diazotrophs in the digestive tract of termite Neotermes castaneus

Normal vital activity of termites Neotermes castaneus requires the presence and continuous replenishment of transient nitrogen-fixing bacteria in their digestive tract, which is realized by coprophagy and repeated utilization of substrate enriched in termite feces. This is the first demonstration of significant changes in the complex of aerobic and facultatively anaerobic nitrogen fixers mediated by an extension of microbial group composition and a shift in dominant taxa in termites that cannot reutilize their feces.     

Phylogenetic Diversity of Nitrogen Fixation Genes in the Intestinal Tract of Reticulitermes chinensis Snyder

Wood-feeding termites live on cellulolytic materials that typically lack of nitrogen sources. It was reported that symbiotic microbes play important roles in the maintenance of a normal nitrogen contents in termite by different metabolisms including nitrogen fixation. In this study, the diversity of nitrogen-fixing organisms in the symbiotic intestinal microflora of Reticulitermes chinensis Snyder was investigated with culture independent method. Fragments of the nifH genes, which encode dinitrogenase reductase, were directly amplified from the DNA of the mixed microbial population in the termite gut with four sets of primers corresponding to the conserved regions of the genes. Clones were randomly selected and analyzed by RFLP. Sequence analysis revealed that a large number of nifH sequences retrieved from the termite gut were most closely related to strict anaerobic bacteria such as clostridia and spirochetes, some of the others were affiliated with proteobacteria, bacteroides, or methanogenic archaea. The results showed that there was a remarkable diversity of nitrogenase genes in the gut of Reticulitermes chinensis Snyder.     

Extracting DNA from the gut microbes of the termite (Zootermopsis nevadensis)

Termites are among the few animals known to have the capacity to subsist solely by consuming wood. The termite gut tract contains a dense and species-rich microbial population that assists in the degradation of lignocellulose predominantly into acetate, the key nutrient fueling termite metabolism (Odelson & Breznak, 1983). Within these microbial populations are bacteria, methanogenic archaea and, in some ("lower") termites, eukaryotic protozoa. Thus, termites are excellent research subjects for studying the interactions among microbial species and the numerous biochemical functions they perform to the benefit of their host. The species composition of microbial populations in termite guts as well as key genes involved in various biochemical processes has been explored using molecular techniques (Kudo et al., 1998; Schmit-Wagner et al., 2003; Salmassi & Leadbetter, 2003). These techniques depend on the extraction and purification of high-quality nucleic acids from the termite gut environment. The extraction technique described in this video is a modified compilation of protocols developed for extraction and purification of nucleic acids from environmental samples (Mor et al., 1994; Berthelet et al., 1996; Purdy et al., 1996; Salmassi & Leadbetter, 2003; Ottesen et al. 2006) and it produces DNA from termite hindgut material suitable for use as template for polymerase chain reaction (PCR).     

Effect of Abandonment on Diversity and Abundance of Free-Living Nitrogen-Fixing Bacteria and Total Bacteria in the Cropland Soils of Hulun Buir,Inner Mongolia

In Inner Mongolia, steppe grasslands face desertification or degradation because of human over activity. One of the reasons for this condition is that croplands have been abandoned after inappropriate agricultural management. The soils in these croplands present heterogeneous environments in which conditions affecting microbial growth and diversity fluctuate widely in space and time. In this study, we assessed the molecular ecology of total and free-living nitrogen-fixing bacterial communities in soils from steppe grasslands and croplands that were abandoned for different periods (1, 5, and 25 years) and compared the degree of recovery. The abandoned croplands included in the study were natural restoration areas without human activity. Denaturing gradient gel electrophoresis and quantitative PCR (qPCR) were used to analyze the nifH and 16S rRNA genes to study free-living diazotrophs and the total bacterial community, respectively. The diversities of free-living nitrogen fixers and total bacteria were significantly different between each site (P<0.001). Neither the total bacteria nor nifH gene community structure of a cropland abandoned for 25 years was significantly different from those of steppe grasslands. In contrast, results of qPCR analysis of free-living nitrogen fixers and total bacteria showed significantly high abundance levels in steppe grassland (P<0.01 and P<0.03, respectively). In this study, the microbial communities and their gene abundances were assessed in croplands that had been abandoned for different periods. An understanding of how environmental factors and changes in microbial communities affect abandoned croplands could aid in appropriate soil management to optimize the structures of soil microorganisms.     

Responses of soil nitrogen-fixing, ammonia-oxidizing, and denitrifying bacterial communities to long-term chlorimuron-ethyl stress in a continuously cropped soybean field in Northeast China

Chlorimuron-ethyl is a type of long-residual herbicide applied widely to soybean fields in China, but little information is available about the long-term impact of this herbicide on soil nitrogen-transforming microbial communities. Soil samples (0–20 cm) were collected from three treatments (no, 5-year and 10-year application of chlorimuron-ethyl) in a continuously cropped soybean field. Plate count (CFU), most probable number (MPN) count, and clone library analyses were conducted to investigate the abundance and composition of nitrogen-fixing, ammonia-oxidizing, and denitrifying bacterial communities, and a chlorate inhibition method was adopted to measure the soil nitrification potential. Long-term chlorimuron-ethyl application reduced the abundance of soil culturable nitrogen-fixing, ammonia-oxidizing, and denitrifying bacteria. Moreover, chlorimuron-ethyl decreased the diversity of nitrogen-fixing and ammonia-oxidizing bacteria but promoted that of denitrifying bacteria. Chlorimuron-ethyl restrained some uncultured nitrogen-fixing bacteria, ammonia-oxidizing bacteria Nitrosospira sp. cluster 3a and 3d, and some novel or putative denitrifying bacteria. The nitrogen-fixing bacteria were closely related to Bradyrhizobium sp., ammonia-oxidizing bacteria Nitrosospira sp. cluster 3b and 3c, and most denitrifying bacteria were resistant to chlorimuron-ethyl. There was a negative correlation between the nitrification potential and the residual amount of soil chlorimuron-ethyl (R²?=?0.88, n?=?3, P?<?0.05). Therefore, long-term application of chlorimuron-ethyl in the continuously cropped soybean field could seriously disturb soil N-transforming communities, and might impact soybean soil biological quality and soybean growth. Further studies should address rational amendment models of this herbicide to reduce the possible ecological risks of long-term application of this herbicide to soybean fields.     

喀斯特土壤固氮微生物群落与植被、土壤的关系

固氮微生物作为土壤中重要的功能微生物群之一,其与地上植物群落、土壤环境之间的关系尚不清楚。在桂西北的环江县、都安县和大化县选取喀斯特典型植被类型(草丛、灌丛、次生林)建立样方,通过植被调查、测定土壤理化性质和构建克隆文库的方法,研究了土壤固氮微生物群落的结构与组成,分析了固氮微生物群落与植物群落、土壤理化性质之间的关系。结果表明:研究区典型植被类型土壤中的优势固氮微生物为慢生根瘤菌。Mantel相关性分析表明植物群落与固氮微生物群落显著相关(r=0.6116,P=0.011);结合PCo A分析和Venn图可看出,植物群落组成与结构越相似,土壤固氮微生物群落结构与组成也越相似。CCA分析前两轴的解释率之和仅为22.72%,其中总氮、有效态钾、有效态钙对固氮微生物群落的影响显著,这表明本研究涵盖的土壤理化性质指标并不能完全解释固氮微生物群落的变异,需要补充更多的土壤数据进行更深入的研究。由此可见,在喀斯特生态恢复过程中,不仅要关注地上植被群落的恢复与重建,同时也应重视地下功能微生物群落的恢复与重建。     

Termite mounds contain soil-derived methanotroph communities kinetically adapted to elevated methane concentrations

Termite mounds have recently been confirmed to mitigate approximately half of termite methane (CH₄) emissions, but the aerobic CH₄ oxidising bacteria (methanotrophs) responsible for this consumption have not been resolved. Here, we describe the abundance, composition and CH₄ oxidation kinetics of the methanotroph communities in the mounds of three distinct termite species sampled from Northern Australia. Results from three independent methods employed show that methanotrophs are rare members of microbial communities in termite mounds, with a comparable abundance but distinct composition to those of adjoining soil samples. Across all mounds, the most abundant and prevalent methane monooxygenase sequences were affiliated with upland soil cluster α (USCα), with sequences homologous to Methylocystis and tropical upland soil cluster (TUSC) also detected. The reconstruction of a metagenome-assembled genome of a mound USCα representative highlighted the metabolic capabilities of this group of methanotrophs. The apparent Michaelis–Menten kinetics of CH₄ oxidation in mounds were estimated from in situ reaction rates. Methane affinities of the communities were in the low micromolar range, which is one to two orders of magnitude higher than those of upland soils, but significantly lower than those measured in soils with a large CH₄ source such as landfill cover soils. The rate constant of CH₄ oxidation, as well as the porosity of the mound material, were significantly positively correlated with the abundance of methanotroph communities of termite mounds. We conclude that termite-derived CH₄ emissions have selected for distinct methanotroph communities that are kinetically adapted to elevated CH₄ concentrations. However, factors other than substrate concentration appear to limit methanotroph abundance and hence these bacteria only partially mitigate termite-derived CH₄ emissions. Our results also highlight the predominant role of USCα in an environment with elevated CH₄ concentrations and suggest a higher functional diversity within this group than previously recognised.Subject terms: Soil microbiology, Biogeochemistry     

Diversity of diazotrophic gut inhabitants of pikas (Ochotonidae) revealed by PCR-DGGE analysis

Diazotrophic gut symbionts are considered to act as nitrogen providers for their hosts, as was shown for various termite species. Although the diet of lagomorphs, like pikas or rabbits, is very poor in nitrogen and energy, their fecal matter contains 30–40% of protein. Since our hypothesis was that pikas maintained a diazotrophic consortium in their gastrointestinal tract, we conducted the first investigation of microbial diversity in pika guts. We obtained gut samples from animals of several Ochotona species, O. hyperborea (Northern pika), O. mantchurica (Manchurian pika), and O. dauurica (Daurian pika), in order to retrieve and compare the nitrogen-fixing communities of different pika species. The age and gender of the animals were taken into consideration. We amplified 320-bp long fragments of the nifH gene using the DNA extracted directly from the colon and cecum samples of pika’s gut, resolved them by DGGE, and performed phylogenetic reconstruction of 51 sequences obtained from excised bands. No significant difference was detected between the nitrogen-fixing gut inhabitants of different pika species. NifH sequences fell into two clusters. The first cluster contained the sequences affiliated with NifH Cluster I (Zehr et al., 2003) with similarity to Sphingomonas sp., Bradyrhizobium sp., and various uncultured bacteria from soil and rhizosphere. Sequences from the second group were related to Treponema sp., Fibrobacter succinogenes, and uncultured clones from the guts of various termites and belonged to NifH Cluster III. We suggest that diazotrophic organisms from the second cluster are genuine endosymbionts of pikas and provide nitrogen for further synthesis processes thus allowing these animals not to be short of protein.     

Diversity of Nitrogen Fixation Genes in the Symbiotic Intestinal Microflora of the Termite Reticulitermes speratus

The diversity of nitrogen-fixing organisms in the symbiotic intestinal microflora of a lower termite, Reticulitermes speratus, was investigated without culturing the resident microorganisms. Fragments of the nifH gene, which encodes the dinitrogenase reductase, were directly amplified from the DNA of the mixed microbial population in the termite gut and were clonally isolated. The phylogenetic analysis of the nifH product amino acid sequences showed that there was a remarkable diversity of nitrogenase genes in the termite gut. A large number of the termite nifH sequences were most closely related to those of a firmicute, Clostridium pasteurianum, with a few being most closely related to either the (gamma) subclass of the proteobacteria or a sequence of Desulfovibrio gigas. Some of the others were distantly related to those of the bacteria and were seemingly derived from the domain Archaea. The phylogenetic positions of these nifH sequences corresponded to those of genera found during a previous determination of rRNA-based phylogeny of the termite intestinal microbial community, of which a majority consisted of new, yet-uncultivated species. The results revealed that we have little knowledge of the organisms responsible for nitrogen fixation in termites.     

Contrasting diurnal variations in soil organic carbon decomposition and root respiration due to a hysteresis effect with soil temperature in a Gossypium s. (cotton) plantation

Cool-season grasses commonly harbor fungal endophytes in their aerial tissues. However the effects of these symbionts on soil microbial communities have rarely been investigated. Our objective was to explore microbial community responses in soils conditioned by plants of the annual grass Lolium multiflorum with contrasting levels of infection with the endophyte Neotyphodium occultans. At the end of the host growing season, we estimated the functional capacity of soil microbial communities (via catabolic response profiles), the contribution of fungi and bacteria to soil activity (via selective inhibition with antibiotics), and the structure of both microbial communities by molecular analyses. Soil conditioning by highly infected plants affected soil catabolic profiles and tended to increase soil fungal activity. We detected a shift in bacterial community structure while no changes were observed for fungi. Soil responses became evident even without changes in host plant biomass or soil organic carbon or total nitrogen content, suggesting that the endophyte modified host rhizodepositions during the conditioning phase. Our results have implications for the understanding of the reciprocal interactions between above and belowground communities, suggesting that plant-soil feedbacks can be mediated by this symbiosis.     

Abundance and Genetic Diversity of nifH Gene Sequences in Anthropogenically Affected Brazilian Mangrove Sediments

Although mangroves represent ecosystems of global importance, the genetic diversity and abundance of functional genes that are key to their functioning scarcely have been explored. Here, we present a survey based on the nifH gene across transects of sediments of two mangrove systems located along the coast line of São Paulo state (Brazil) which differed by degree of disturbance, i.e., an oil-spill-affected and an unaffected mangrove. The diazotrophic communities were assessed by denaturing gradient gel electrophoresis (DGGE), quantitative PCR (qPCR), and clone libraries. The nifH gene abundance was similar across the two mangrove sediment systems, as evidenced by qPCR. However, the nifH-based PCR-DGGE profiles revealed clear differences between the mangroves. Moreover, shifts in the nifH gene diversities were noted along the land-sea transect within the previously oiled mangrove. The nifH gene diversity depicted the presence of nitrogen-fixing bacteria affiliated with a wide range of taxa, encompassing members of the Alphaproteobacteria, Betaproteobacteria, Gammaproteobacteria, Firmicutes, and also a group of anaerobic sulfate-reducing bacteria. We also detected a unique mangrove-specific cluster of sequences denoted Mgv-nifH. Our results indicate that nitrogen-fixing bacterial guilds can be partially endemic to mangroves, and these communities are modulated by oil contamination, which has important implications for conservation strategies.     

Microecology of the termite gut: structure and function on a microscale

Long considered simply as anoxic fermentors, termite guts are in fact axially and radially structured environments with physicochemically distinct microhabitats. Recent developments in termite gut microecology, which combined traditional and modern techniques, have focused on the spatial organization of important microbial populations and their in situ activities, and have significantly furthered our understanding of functional interactions within highly structured microenvironments.     

Comparison of Euryarchaea Strains in the Guts and Food-Soil of the Soil-Feeding Termite Cubitermes fungifaber across Different Soil Types

Termites are an important component of tropical soil communities and have a significant effect on the structure and nutrient content of soil. Digestion in termites is related to gut structure, gut physicochemical conditions, and gut symbiotic microbiota. Here we describe the use of 16S rRNA gene sequencing and terminal-restriction fragment length polymorphism (T-RFLP) analysis to examine methanogenic archaea (MA) in the guts and food-soil of the soil-feeder Cubitermes fungifaber Sjostedt across a range of soil types. If these MA are strictly vertically inherited, then the MA in guts should be the same in all individuals even if the soils differ across sites. In contrast, gut MA should reflect what is present in soil if populations are merely a reflection of what is ingested as the insects forage. We show clear differences between the euryarchaeal communities in termite guts and in food-soils from five different sites. Analysis of 16S rRNA gene clones indicated little overlap between the gut and soil communities. Gut clones were related to a termite-derived Methanomicrobiales cluster, to Methanobrevibacter and, surprisingly, to the haloalkaliphile Natronococcus. Soil clones clustered with Methanosarcina, Methanomicrococcus, or rice cluster I. T-RFLP analysis indicated that the archaeal communities in the soil samples differed from site to site, whereas those in termite guts were similar between sites. There was some overlap between the gut and soil communities, but these may represent transient populations in either guts or soil. Our data do not support the hypothesis that termite gut MA are derived from their food-soil but also do not support a purely vertical transmission of gut microflora.     

Land Use History Shifts In Situ Fungal and Bacterial Successions following Wheat Straw Input into the Soil

Soil microbial communities undergo rapid shifts following modifications in environmental conditions. Although microbial diversity changes may alter soil functioning, the in situ temporal dynamics of microbial diversity is poorly documented. Here, we investigated the response of fungal and bacterial diversity to wheat straw input in a 12-months field experiment and explored whether this response depended on the soil management history (grassland vs. cropland). Seasonal climatic fluctuations had no effect on the diversity of soil communities. Contrastingly fungi and bacteria responded strongly to wheat regardless of the soil history. After straw incorporation, diversity decreased due to the temporary dominance of a subset of copiotrophic populations. While fungi responded as quickly as bacteria, the resilience of fungal diversity lasted much longer, indicating that the relative involvement of each community might change as decomposition progressed. Soil history did not affect the response patterns, but determined the identity of some of the populations stimulated. Most strikingly, the bacteria Burkholderia, Lysobacter and fungi Rhizopus, Fusarium were selectively stimulated. Given the ecological importance of these microbial groups as decomposers and/or plant pathogens, such regulation of the composition of microbial successions by soil history may have important consequences in terms of soil carbon turnover and crop health.     

Kimchi microflora: history,current status,and perspectives for industrial kimchi production

Kimchi, a traditional Korean food made by the fermentation of vegetables, has become popular globally because of its organoleptic, beneficial, and nutritional properties. Spontaneous kimchi fermentation in unsterilized raw materials leads to the growth of various lactic acid bacteria (LAB), which results in variations in the taste and sensory qualities of kimchi products and difficulties in the standardized industrial production of kimchi. Raw materials, kimchi varieties, ingredients, and fermentation conditions have significant effects on the microbial communities and fermentative characteristics of kimchi during fermentation. Heterofermentative LAB belonging to the genera Leuconostoc, Lactobacillus, and Weissella are likely to be key players in kimchi fermentation and have been subjected to genomic and functional studies to gain a better understanding of the fermentation process and beneficial effects of kimchi. The use of starter cultures has been considered for the industrial production of high quality, standardized kimchi. Here, we review the composition and biochemistry of kimchi microflora communities, functional and genomic studies of kimchi LAB, and perspectives for industrial kimchi production.     

Diverse bacteria related to the bacteroides subgroup of the CFB phylum within the gut symbiotic communities of various termites

Phylogenetically diverse clones of the partial 16S rDNA (ca. 850 bp) of bacteria belonging to the bacteroides subgroup of the cytophaga-flavobacter-bacteroides phylum were collected from the symbiotic microbial communities in the guts of six termite species without cultivation. Combined with the sequences reported previously, a total of thirty phylotypes of the subgroup were identified and classified into five phylogenetic clusters. One that was comprised of the phylotypes from a single termite species was related to the genus Rikenella. Two were clustered each with some cultured strains, genera of which have not been clearly defined yet. The remaining two clusters had no culturable representatives, suggesting the presence of yet-uncultivated genera within the termite guts. From these sequence data, we designed a specific primer for the bacteroides subgroup, which was successful in the terminal-restriction fragment length polymorphism analysis to detect the phylotypes of the subgroup in the termite gut.     

Prospects for the biological control of subterranean termites (Isoptera: rhinotermitidae), with special reference to Coptotermes formosanus

Costs associated with subterranean termite damage and control are estimated to approach $2 billion annually in the United States alone. The Formosan subterranean termite, Coptotermes formosanus Shiraki, is one of the more economically important subterranean species. In recent years, the shortcomings associated with conventional chemical control methods have prompted policymakers and scientists to evaluate the potential for biological control of subterranean termites (C. formosanus in particular), that is, to determine the potential for natural enemies - predators, parasitoids and pathogens - to suppress termite populations. Ants are the greatest predators of termites, and may have a considerable local impact on termite populations in some areas of the world. A few parasitoids of termites are known, but their potential for regulating termite populations seems negligible. Characteristics of the colony, such as a protected, underground location (and, for the C. formosanus nest, its modular and dispersed nature), are likely to limit the impact predators and parasitoids have on subterranean termites. Thus, there seems little potential for use of these agents for subterranean termite control. For various reasons, pathogenic organisms, such as viruses, bacteria, Protozoa, nematodes and most fungi, have shown little promise for use in biological termite control. However, research suggests that strains of two well-studied, endoparasitic fungi, Beauveria bassiana and Metarhizium anisopliae, when employed in baiting schemes, may offer the potential for at least some measure of subterranean termite control, although their successful use is compromised by a number of inherent biological limitations and logistical problems that have yet to be solved. Although not strictly in the realm of classical biological control, recent studies suggest that natural products, such as ant semiochemicals and fungal metabolites (siderophores), or their synthetic analogues, eventually might find a use in termite control programmes as repellents or insecticides in wood treatments or soil applications if stable formulations can be developed.     

Prokaryotic communities of the north-eastern Mongolian soda lakes

We have studied the activity and composition of several geochemically significant physiological groups of bacteria in more than twenty alkaline salt lakes of the north-east Mongolia steppe with water salinity from 3 to 390 g l^?1 and pH values ranging from 9.0 to 10.6. Active and diverse microbial communities have been found in most of the lakes. The methanotrophic bacteria were represented by the Type I members. Among the culturable forms of sulfur-oxidizing bacteria obligately chemolithoautotrophic and haloalkaliphilic representatives of the genera Thioalkalimicrobium and Thioalkalivibrio were detected in the sediments at high numbers (up to 10⁶ cells ml^?1). The largest population of anaerobic phototrophic bacteria was represented by purple sulfur bacteria of the Ectothiorhodospiraceae family. Salinity was the key factor in determining the activity and the composition of the microbial communities. The most diverse and active prokaryotic populations, including aerobic and anaerobic phototrophic, methanogenic, methanotrophic, sulfur-oxidizing, sulfate-reducing and nitrifying bacteria, were found in lakes with salinity less than 60 g l^?1. In hypersaline lakes with a salinity >100 g l^?1, the sulfur cycle remained active due to the activity of extremely halotolerant and alkaliphilic sulfur bacteria, while other important functional groups responsible for nitrification and methane oxidation processes were not detected. Overall, the prokaryotic communities of the Mongolian alkaline salt lakes represent an interesting new example of a diverse community of haloalkaliphilic bacteria well adopted to a broad salinity range.     

Extended disease resistance emerging from the faecal nest of a subterranean termite

Social insects nesting in soil environments are in constant contact with entomopathogens but have evolved a range of defence mechanisms, resulting in both individual and social immunity that reduce the chance for epizootics in the colony, as in the case of subterranean termites. Coptotermes formosanus uses its faeces as building material for its nest structure that result into a ‘carton material’, and here, we report that the faecal nest supports the growth of Actinobacteria which provide another level of protection to the social group against entomopathogens. A Streptomyces species with in vivo antimicrobial activity against fungal entomopathogens was isolated from the nest material of multiple termite colonies. Termite groups were exposed to Metarhizium anisopliae, a fungal entomopathogen, during their foraging activity and the presence of Streptomyces within the nest structure provided a significant survival benefit to the termites. Therefore, this report describes a non-nutritional exosymbiosis in a termite, in the form of a defensive mutualism which has emerged from the use of faecal material in the nesting structure of Coptotermes. The association with an Actinobacteria community in the termite faecal material provides an extended disease resistance to the termite group as another level of defence, in addition to their individual and social immunity.