Phyllosphere Bacterial Community of Floating Macrophytes in Paddy Soil Environments as Revealed by Illumina High-Throughput Sequencing

The phyllosphere of floating macrophytes in paddy soil ecosystems, a unique habitat, may support large microbial communities but remains largely unknown. We took Wolffia australiana as a representative floating plant and investigated its phyllosphere bacterial community and the underlying driving forces of community modulation in paddy soil ecosystems using Illumina HiSeq 2000 platform-based 16S rRNA gene sequence analysis. The results showed that the phyllosphere of W. australiana harbored considerably rich communities of bacteria, with Proteobacteria and Bacteroidetes as the predominant phyla. The core microbiome in the phyllosphere contained genera such as Acidovorax, Asticcacaulis, Methylibium, and Methylophilus. Complexity of the phyllosphere bacterial communities in terms of class number and α-diversity was reduced compared to those in corresponding water and soil. Furthermore, the bacterial communities exhibited structures significantly different from those in water and soil. These findings and the following redundancy analysis (RDA) suggest that species sorting played an important role in the recruitment of bacterial species in the phyllosphere. The compositional structures of the phyllosphere bacterial communities were modulated predominantly by water physicochemical properties, while the initial soil bacterial communities had limited impact. Taken together, the findings from this study reveal the diversity and uniqueness of the phyllosphere bacterial communities associated with the floating macrophytes in paddy soil environments.     

不同品种酿酒葡萄根围、叶围微生物群落结构特点解析

【目的】了解不同品种酿酒葡萄根围、叶围细菌群落结构。【方法】以不同品种酿酒葡萄根围、叶围样品为分析材料,采用末端限制性片段长度多态性(T-RFLP)技术研究根围、叶围细菌群落结构,并对细菌群落多样性进行分析。【结果】不同品种葡萄根围微生物优势菌群为变形菌门,不同品种葡萄叶围微生物优势菌群为放线菌目。根围Simpson指数大小顺序为白福尔琼瑶浆白诗南赛美蓉白玉霓米勒长相思,Shannon指数与Simpson指数基本保持一致。叶围Simpson指数最大的为白诗南,最小的为米勒,与Shannon指数基本吻合。【结论】葡萄细菌群落结构综合多样性指数和品种以及定殖部位密切相关。     

Variability of Bacterial Community Composition on Leaves Between and Within Plant Species

The phyllosphere is one of the largest habitats for terrestrial microorganisms. To gain a better insight into the factors underlying the composition of bacterial communities inhabiting leaf surfaces we performed culture-dependent and independent (Denaturing Gradient Gel Electrophoresis) analyses on the bacteria associated with the leaves of three plant species: Amygdalus communis, Citrus paradisi, and Nicotiana glauca. We found that the culturable classes Bacilli and Actinobacteria were the predominant classes on the phyllosphere of all three plant species. In contrast to this consistency on the bacterial class level, we found a significant variation on the bacterial species-level based on the culturable methods. Although some variation was detected among individual plants within one plant species, the inter-specific variability exceeded the intra-specific variability. C. paradisi leaf surface had the highest predicted total species richness (Chao 2 and ICE) and the highest species diversity (βw) among the three plant species. Our findings demonstrate that environmental conditions, mainly the plant species within a site, govern the bacterial community composition on leaf surfaces.     

Resilience of the Natural Phyllosphere Microbiota of the Grapevine to Chemical and Biological Pesticides

The phyllosphere is colonized by complex microbial communities, which are adapted to the harsh habitat. Although the role and ecology of nonpathogenic microorganisms of the phyllosphere are only partially understood, leaf microbiota could have a beneficial role in plant growth and health. Pesticides and biocontrol agents are frequently applied to grapevines, but the impact on nontarget microorganisms of the phyllosphere has been marginally considered. In this study, we investigated the effect of a chemical fungicide (penconazole) and a biological control agent (Lysobacter capsici AZ78) on the leaf microbiota of the grapevine at three locations. Amplicons of the 16S rRNA gene and of the internal transcribed spacer were sequenced for bacterial and fungal identification, respectively. Pyrosequencing analysis revealed that the richness and diversity of bacterial and fungal populations were only minimally affected by the chemical and biological treatments tested, and they mainly differed according to grapevine locations. Indigenous microbial communities of the phyllosphere are adapted to environmental and biotic factors in the areas where the grapevines are grown, and they are resilient to the treatments tested. The biocontrol properties of phyllosphere communities against downy mildew differed among grapevine locations and were not affected by treatments, suggesting that biocontrol communities could be improved with agronomic practices to enrich beneficial populations in vineyards.     

Annual and Seasonal Variation in the Phyllosphere Bacterial Community Associated with Leaves of the Southern Magnolia (Magnolia grandiflora)

The phyllosphere contains a diverse bacterial community that can be intimately associated with the host plant; however, few studies have examined how the phyllosphere community changes over time. We sampled replicate leaves from a single magnolia (Magnolia grandiflora) tree in the winter of three consecutive years (2007?C2009) as well as during four seasons of 1?year (2008) and used molecular techniques to examine seasonal and year-to-year variation in bacterial community structure. Multivariate analysis of denaturing gradient gel electrophoresis profiles of 16S rRNA gene fragments revealed minimal leaf to leaf variation and much greater temporal changes, with the summer (August 2008) leaf community being most distinct from the other seasons. This was confirmed by sequencing and analysis of 16S rRNA gene clone libraries generated for each sample date. All phyllosphere communities were dominated by Alphaproteobacteria, with a reduction in the representation of certain Beijerinckiaceae during the summer and a concurrent increase in the Methylobacteriaceae being the most significant seasonal change. Other important components of the magnolia phyllosphere included members of the Bacteroidetes, Acidobacteria, and Actinobacteria, with the latter two lineages also showing differences in their representation in samples collected at different times. While the leaf-associated bacterial community sampled at the same time of year in three separate years showed some similarities, generally these communities were distinct, suggesting that while there are seasonal patterns, these may not be predictable from year to year. These results suggest that seasonal differences do occur in phyllosphere communities and that broad-leafed evergreen trees such as M. grandiflora may present interesting systems to study these changes in the context of changing environmental conditions.     

Temporal and Spatial Diversity of Bacterial Communities in Coastal Waters of the South China Sea

Bacteria are recognized as important drivers of biogeochemical processes in all aquatic ecosystems. Temporal and geographical patterns in ocean bacterial communities have been observed in many studies, but the temporal and spatial patterns in the bacterial communities from the South China Sea remained unexplored. To determine the spatiotemporal patterns, we generated 16S rRNA datasets for 15 samples collected from the five regularly distributed sites of the South China Sea in three seasons (spring, summer, winter). A total of 491 representative sequences were analyzed by MOTHUR, yielding 282 operational taxonomic units (OTUs) grouped at 97% stringency. Significant temporal variations of bacterial diversity were observed. Richness and diversity indices indicated that summer samples were the most diverse. The main bacterial group in spring and summer samples was Alphaproteobacteria, followed by Cyanobacteria and Gammaproteobacteria, whereas Cyanobacteria dominated the winter samples. Spatial patterns in the samples were observed that samples collected from the coastal (D151, D221) waters and offshore (D157, D1512, D224) waters clustered separately, the coastal samples harbored more diverse bacterial communities. However, the temporal pattern of the coastal site D151 was contrary to that of the coastal site D221. The LIBSHUFF statistics revealed noticeable differences among the spring, summer and winter libraries collected at five sites. The UPGMA tree showed there were temporal and spatial heterogeneity of bacterial community composition in coastal waters of the South China Sea. The water salinity (P=0.001) contributed significantly to the bacteria-environment relationship. Our results revealed that bacterial community structures were influenced by environmental factors and community-level changes in 16S-based diversity were better explained by spatial patterns than by temporal patterns.     

Vertical profiles of microbial communities in perfluoroalkyl substance-contaminated soils

Poly- and perfluoroalkyl compounds (PFASs) are ubiquitous in the environment, but their influences on microbial community remain poorly known. The present study investigated the depth-related changes of archaeal and bacterial communities in PFAS-contaminated soils. The abundance and structure of microbial community were characterized using quantitative PCR and high-throughput sequencing, respectively. Microbial abundance changed considerably with soil depth. The richness and diversity of both bacterial and archaeal communities increased with soil depth. At each depth, bacterial community was more abundant and had higher richness and diversity than archaeal community. The structure of either bacterial or archaeal community displayed distinct vertical variations. Moreover, a higher content of perfluorooctane sulfonate (PFOS) could have a negative impact on bacterial richness and diversity. The rise of soil organic carbon content could increase bacterial abundance but lower the richness and diversity of both bacterial and archaeal communities. In addition, Proteobacteria, Actinobacteria, Chloroflexi, Cyanobacteria, and Acidobacteria were the major bacterial groups, while Thaumarchaeota, Euryarchaeota, and unclassified Archaea dominated in soil archaeal communities. PFASs could influence soil microbial community.     

The ecology of the phyllosphere: geographic and phylogenetic variability in the distribution of bacteria on tree leaves

Large populations of bacteria live on leaf surfaces and these phyllosphere bacteria can have important effects on plant health. However, we currently have a limited understanding of bacterial diversity on tree leaves and the inter‐ and intra‐specific variability in phyllosphere community structure. We used a barcoded pyrosequencing technique to characterize the bacterial communities from leaves of 56 tree species in Boulder, Colorado, USA, quantifying the intra‐ and inter‐individual variability in the bacterial communities from 10 of these species. We also examined the geographic variability in phyllosphere communities on Pinus ponderosa from several locations across the globe. Individual tree species harboured high levels of bacterial diversity and there was considerable variability in community composition between trees. The bacterial communities were organized in patterns predictable from the relatedness of the trees as there was significant correspondence between tree phylogeny and bacterial community phylogeny. Inter‐specific variability in bacterial community composition exceeded intra‐specific variability, a pattern that held even across continents where we observed minimal geographic differentiation in the bacterial communities on P. ponderosa needles.     

The Ecological Perspective of Microbial Communities in Two Pairs of Competitive Hawaiian Native and Invasive Macroalgae

Marine macroalgae are known to harbor large populations of microbial symbionts, and yet, microbe symbiosis in invasive macroalgae remains largely unknown. In this study, we applied molecular methods to study microbial communities associated with two invasive algae Acanthophora spicifera and Gracilaria salicornia and the two native algae Gracilaria coronopifolia and Laurencia nidifica at spatial and temporal scales in Hawaiian coral reef ecosystems. Bacterial communities of both the invasive and native macroalgae displayed little spatial and temporal variations, suggesting consistent and stable bacterial associations with these macroalgae. Results of this study identified three types of bacterial populations: nonspecific (present in both algal and water samples); algae-specific (found in all algal species); and species-specific (only found in individual species). The bacterial diversity of invasive algae was lower than that of their native counterparts at phylum and species levels. Notably, the vast majority (71 %) of bacterial communities associated with the invasive algae G. salicornia were representatives of Cyanobacteria, suggesting a potential ecological significance of symbiotic Cyanobacteria.     

Bacterial Communities Associated with Surfaces of Leafy Greens: Shift in Composition and Decrease in Richness over Time

The phyllosphere is colonized by a wide variety of bacteria and fungi; it harbors epiphytes, as well as plant-pathogenic bacteria and even human pathogens. However, little is known about how the bacterial community composition on leafy greens develops over time. The bacterial community of the leafy-green phyllosphere obtained from two plantings of rocket salad (Diplotaxis tenuifolia) and three plantings of lettuce (Lactuca sativa) at two farms in Norway were profiled by an Illumina MiSeq-based approach. We found that the bacterial richness of the L. sativa samples was significantly greater shortly (3 weeks) after planting than at harvest (5 to 7 weeks after planting) for plantings 1 and 3 at both farms. For the second planting, the bacterial diversity remained consistent at the two sites. This suggests that the effect on bacterial colonization of leaves, at least in part must, be seasonally driven rather than driven solely by leaf maturity. The distribution of phyllosphere communities varied between D. tenuifolia and L. sativa at harvest. The variability between these species at the same location suggests that the leaf-dwelling bacteria are not only passive inhabitants but interact with the host, which shapes niches favoring the growth of particular taxa. This work contributes to our understanding of host plant-specific microbial community structures and shows how these communities change throughout plant development.     

长叶红砂叶际细菌和真菌群落对季节变化的响应特征

【背景】植物叶际(phyllosphere)定殖着丰富多样的微生物,叶际微生物通过发挥特定功能在逆境下生存,影响寄主植物的生理生态特性并受环境异质性的影响。【目的】植物叶际微生物群落是动态的,认识季节更替对植物叶际微生物群落结构的影响,对于加深对植物-微生物-环境相互作用的理解具有积极意义。【方法】以鄂尔多斯荒漠草原泌盐盐生植物长叶红砂为研究对象,分别测定春季、秋季植物叶片表面理化特性,并结合叶际细菌、真菌高通量测序结果进行综合分析。【结果】长叶红砂冠下土壤含水率、pH、电导率等指标在季节更替下存在显著差异,叶片表面Na+、K+和电导率值存在显著差异;进一步分析发现,叶际细菌分类操作单元(operational taxonomic unit,OTU)、Shannon、Chao1和ACE (abundance-based coverage estimator)指数与土壤和叶片表面盐分含量呈正相关;春季叶际蓝细菌门和拟杆菌门保持了较高的相对丰度,而秋季叶际变形菌门、放线菌门、子囊菌门的相对丰度则高于春季;其中,叶际Bradyrhizobium、Novosphingobium和Edaphob...     

Transgenic tobacco revealing altered bacterial diversity in the rhizosphere during early plant development

The rhizosphere constitutes a complex niche that may be exploited by a wide variety of bacteria. Bacterium–plant interactions in this niche can be influenced by factors such as the expression of heterologous genes in the plant. The objective of this work was to describe the bacterial communities associated with the rhizosphere and rhizoplane regions of tobacco plants, and to compare communities from transgenic tobacco lines (CAB1, CAB2 and TRP) with those found in wild-type (WT) plants. Samples were collected at two stages of plant development, the vegetative and flowering stages (1 and 3 months after germination). The diversity of the culturable microbial community was assessed by isolation and further characterization of isolates by amplified ribosomal RNA gene restriction analysis (ARDRA) and 16S rRNA sequencing. These analyses revealed the presence of fairly common rhizosphere organisms with the main groups Alphaproteobacteria, Betaproteobacteria, Actinobacteria and Bacilli. Analysis of the total bacterial communities using PCR-DGGE (denaturing gradient gel electrophoresis) revealed that shifts in bacterial communities occurred during early plant development, but the reestablishment of original community structure was observed over time. The effects were smaller in rhizosphere than in rhizoplane samples, where selection of specific bacterial groups by the different plant lines was demonstrated. Clustering patterns and principal components analysis (PCA) were used to distinguish the plant lines according to the fingerprint of their associated bacterial communities. Bands differentially detected in plant lines were found to be affiliated with the genera Pantoea, Bacillus and Burkholderia in WT, CAB and TRP plants, respectively. The data revealed that, although rhizosphere/rhizoplane microbial communities can be affected by the cultivation of transgenic plants, soil resilience may be able to restore the original bacterial diversity after one cycle of plant cultivation.     

Bacterial Succession on the Leaf Surface: A Novel System for Studying Successional Dynamics

Succession is a widely studied process in plant and animal systems, but succession in microbial communities has received relatively little attention despite the ubiquity of microorganisms in natural habitats. One important microbial habitat is the phyllosphere, or leaf surface, which harbors large, diverse populations of bacteria and offers unique opportunities for the study of succession and temporal community assembly patterns. To explore bacterial community successional patterns, we sampled phyllosphere communities on cottonwood (Populus deltoides) trees multiple times across the growing season, from leaf emergence to leaf fall. Bacterial community composition was highly variable throughout the growing season; leaves sampled as little as a week apart were found to harbor significantly different communities, and the temporal variability on a given tree exceeded the variability in community composition between individual trees sampled on a given day. The bacterial communities clearly clustered into early-, mid-, and late-season clusters, with early- and late-season communities being more similar to each other than to the mid-season communities, and these patterns appeared consistent from year to year. Although we observed clear and predictable changes in bacterial community composition during the course of the growing season, changes in phyllosphere bacterial diversity were less predictable. We examined the species–time relationship, a measure of species turnover rate, and found that the relationship was fundamentally similar to that observed in plant and invertebrate communities, just on a shorter time scale. The temporal dynamics we observed suggest that although phyllosphere bacterial communities have high levels of phylogenetic diversity and rapid turnover rates, these communities follow predictable successional patterns from season to season.     

A Three-Scale Analysis of Bacterial Communities Involved in Rocks Colonization and Soil Formation in High Mountain Environments

Alpha and beta diversities of the bacterial communities growing on rock surfaces, proto-soils, riparian sediments, lichen thalli, and water springs biofilms in a glacier foreland were studied. We used three molecular based techniques to allow a deeper investigation at different taxonomic resolutions: denaturing gradient gel electrophoresis, length heterogeneity-PCR, and automated ribosomal intergenic spacer analysis. Bacterial communities were mainly composed of Acidobacteria, Proteobacteria, and Cyanobacteria with distinct variations among sites. Proteobacteria were more represented in sediments, biofilms, and lichens; Acidobacteria were mostly found in proto-soils; and Cyanobacteria on rocks. Firmicutes and Bacteroidetes were mainly found in biofilms. UniFrac P values confirmed a significant difference among different matrices. Significant differences (P < 0.001) in beta diversity were observed among the different matrices at the genus–species level, except for lichens and rocks which shared a more similar community structure, while at deep taxonomic resolution two distinct bacterial communities between lichens and rocks were found.     

Co-habiting amphibian species harbor unique skin bacterial communities in wild populations

Although all plant and animal species harbor microbial symbionts, we know surprisingly little about the specificity of microbial communities to their hosts. Few studies have compared the microbiomes of different species of animals, and fewer still have examined animals in the wild. We sampled four pond habitats in Colorado, USA, where multiple amphibian species were present. In total, 32 amphibian individuals were sampled from three different species including northern leopard frogs (Lithobates pipiens), western chorus frogs (Pseudacris triseriata) and tiger salamanders (Ambystoma tigrinum). We compared the diversity and composition of the bacterial communities on the skin of the collected individuals via barcoded pyrosequencing of the 16S rRNA gene. Dominant bacterial phyla included Acidobacteria, Actinobacteria, Bacteriodetes, Cyanobacteria, Firmicutes and Proteobacteria. In total, we found members of 18 bacterial phyla, comparable to the taxonomic diversity typically found on human skin. Levels of bacterial diversity varied strongly across species: L. pipiens had the highest diversity; A. tigrinum the lowest. Host species was a highly significant predictor of bacterial community similarity, and co-habitation within the same pond was not significant, highlighting that the skin-associated bacterial communities do not simply reflect those bacterial communities found in their surrounding environments. Innate species differences thus appear to regulate the structure of skin bacterial communities on amphibians. In light of recent discoveries that some bacteria on amphibian skin have antifungal activity, our finding suggests that host-specific bacteria may have a role in the species-specific resistance to fungal pathogens.     

Characterization of rhizosphere and endophytic bacterial communities from leaves,stems and roots of medicinal Stellera chamaejasme L.

A diverse array of bacteria that inhabit the rhizosphere and different plant organs play a crucial role in plant health and growth. Therefore, a general understanding of these bacterial communities and their diversity is necessary. Using the 16S rRNA gene clone library technique, the bacterial community structure and diversity of the rhizosphere and endophytic bacteria in Stellera chamaejasme compartments were compared and clarified for the first time. Grouping of the sequences obtained showed that members of the Proteobacteria (43.2%), Firmicutes (36.5%) and Actinobacteria (14.1%) were dominant in both samples. Other groups that were consistently found, albeit at lower abundance, were Bacteroidetes (2.1%), Chloroflexi (1.9%), and Cyanobacteria (1.7%). The habitats (rhizosphere vs endophytes) and organs (leaf, stem and root) structured the community, since the Wilcoxon signed rank test indicated that more varied bacteria inhabited the rhizosphere compared to the organs of the plant. In addition, correspondence analysis also showed that differences were apparent in the bacterial communities associated with these distinct habitats. Moreover, principal component analysis revealed that the profiles obtained from the rhizosphere and roots were similar, whereas leaf and stem samples clustered together on the opposite side of the plot from the rhizosphere and roots. Taken together, these results suggested that, although the communities associated with the rhizosphere and organs shared some bacterial species, the associated communities differed in structure and diversity.     

Biotic Stress Shifted Structure and Abundance of Enterobacteriaceae in the Lettuce Microbiome

Lettuce cultivars are not only amongst the most popular vegetables eaten raw, they are also involved in severe pathogen outbreaks world-wide. While outbreaks caused by Enterobacteriaceae species are well-studied, less is known about their occurrence in natural environments as well as the impact of biotic stress. Here, we studied the ecology of the human health-relevant bacterial family Enterobacteriaceae and assessed the impact of biotic disturbances by a soil-borne phytopathogenic fungus and Gastropoda on their structure and abundance in mesocosm and pot experiments. Using a polyphasic approach including network analyses of 16S rRNA gene amplicon libraries, quantitative PCR and complementary fluorescence in situ hybridization (FISH) microscopy we found substantial yet divergent Enterobacteriaceae communities. A similar spectrum of 14 genera was identified from rhizo- and phyllospheres but the abundance of Enterobacteriaceae was on average 3fold higher in phyllosphere samples. Both stress factors shifted the bacterial community of the leaf habitat, characterized by increases of species abundance and diversity. For the rhizosphere, we observed significant structural shifts of Enterobacteriaceae communities but also a high degree of resilience. These results could be confirmed by FISH microscopy but it was difficult to visualize phyllosphere communities. Additional inoculation experiments with Escherichia coli as model revealed their presence below the wax layer as well as in the endosphere of leaves. The observed presence influenced by stress factors and the endophytic life style of Enterobacteriaceae on lettuce can be an important aspect in relation to human health.     

Effects of the symbiosis between fungal endophytes and Atractylodes lancea on rhizosphere and phyllosphere microbial communities

Tissue-cultured plantlets of Atractylodes lancea were inoculated with the endophytes AL4 (Cunninghamella sp.) and AL12 (Gilmaniella sp.), and subsequently transplanted into soil after hardening of the tissue-cultured plantlets. We investigated rhizospheric and phyllospheric microbial communities using culture-based and culture-independent methods. Energy spectrum analysis, high performance liquid chromatography, and other assay methods were employed to quantify the elements in the leaves, and the soluble sugars, free amino acids and organic acids in the rhizosphere. The results showed that the endophytes enhanced the diversity and size of the rhizospheric microbial populations. In the phyllosphere, AL4 (Cunninghamella sp.) enhanced the diversity and size of bacterial populations, while AL12 (Gilmaniella sp.) enhanced the diversity and size of fungal populations. The dominant bacterial genera were Microbacterium, Kocuria and Sphingomon in the endophytes-inoculated groups, and Acinetobacter and Bacillus in the endophytes-free group. While Acremonium and Curvularia were the dominant fungal genera in the phyllosphere of endophytes-inoculated groups, Fusarium and Penicillum were most common in the endophytes-free group. AL4 (Cunninghamella sp.) enhanced the rhizospheric microbial population size and diversity by increasing rhizospheric free amino acids, while AL12 (Gilmaniella sp.) altered the rhizospheric microbes by changing concentration of soluble sugars in the rhizosphere. Elemental levels in the phyllosphere and the nutrients in the rhizosphere varied among the treatments and may also have influenced the microbial communities.     

Analysis of bacterial communities in sponges and coral inhabiting Red Sea,using barcoded 454 pyrosequencing

Microbial communities are linked with marine sponge are diverse in their structure and function. Our understanding of the sponge-associated microbial diversity is limited especially from Red Sea in Saudi Arabia where few species of sponges have been studied. Here we used pyrosequencing to study two marine sponges and coral species sampled from Obhur region from Red sea in Jeddah. A total of 168 operational taxonomic units (OTUs) were identified from Haliclona caerulea, Stylissa carteri and Rhytisma fulvum. Taxonomic identification of tag sequences of 16S ribosomal RNA revealed 6 different bacterial phyla and 9 different classes. A proportion of unclassified reads were was also observed in sponges and coral sample. We found diverse bacterial communities associated with two sponges and a coral sample. Diversity and richness estimates based on OUTs revealed that sponge H. caerulea had significantly high bacterial diversity. The identified OTUs showed unique clustering in three sponge samples as revealed by Principal coordinate analysis (PCoA). Proteobacteria (88–95%) was dominant phyla alonwith Bacteroidetes, Planctomycetes, Cyanobacteria, Firmicutes and Nitrospirae. Seventeen different genera were identified where genus Pseudoalteromonas was dominant in all three samples. This is first study to assess bacterial communities of sponge and coral sample that have never been studied before to unravel their microbial communities using 454-pyrosequencing method.