Dynamics of Bacterial and Fungal Communities on Decaying Salt Marsh Grass

ABSTRACT Both bacteria and fungi play critical roles in decomposition processes in many natural environments, yet only rarely have they been studied as an integrated microbial community. Here we describe the bacterial and fungal assemblages associated with two decomposition stages of Spartina alterniflora detritus in a productive southeastern U.S. salt marsh. 16S rRNA genes and 18S-to-28S internal transcribed spacer (ITS) regions were used to target the bacterial and ascomycete fungal communities, respectively, based on DNA sequence analysis of isolates and environmental clones and by using community fingerprinting based on terminal restriction fragment length polymorphism (T-RFLP) analysis. Seven major bacterial taxa (six affiliated with the α-Proteobacteria and one with the Cytophagales) and four major fungal taxa were identified over five sample dates spanning 13 months. Fungal terminal restriction fragments (T-RFs) were informative at the species level; however, bacterial T-RFs frequently comprised a number of related genera. Amplicon abundances indicated that the salt marsh saprophyte communities have little-to-moderate variability spatially or with decomposition stage, but considerable variability temporally. However, the temporal variability could not be readily explained by either successional shifts or simple relationships with environmental factors. Significant correlations in abundance (both positive and negative) were found among dominant fungal and bacterial taxa that possibly indicate ecological interactions between decomposer organisms. Most associations involved one of four microbial taxa: two groups of bacteria affiliated with the α-Proteobacteria and two ascomycete fungi (Phaeosphaeria spartinicola and environmental isolate “4clt”).

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672 IDENTIFICATION OF POLYBACTERIAL 16S RRNA GENES FROM ASCITIC FLUIDS WITH T-RFLP ANALYSIS AND DIRECT SEQUENCING

Journal of Hepatology ◽

10.1016/s0168-8278(12)60685-0 ◽

2012 ◽

Vol 56 ◽

pp. S266

Author(s):

S. Krohn ◽

J. Hartmann ◽

A. Brodzinski ◽

A. Chatzinotas ◽

S. Bohm ◽

...

Keyword(s):

16S Rrna ◽

Direct Sequencing ◽

Rflp Analysis ◽

16S Rrna Genes ◽

Rrna Genes

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Microbial Community Composition Analysis in Spring Aerosols at Urban and Remote Sites over the Tibetan Plateau

Atmosphere ◽

10.3390/atmos11050527 ◽

2020 ◽

Vol 11 (5) ◽

pp. 527

Author(s):

Prakriti Sharma Ghimire ◽

Shichang Kang ◽

Wasim Sajjad ◽

Barkat Ali ◽

Lekhendra Tripathee ◽

...

Keyword(s):

Tibetan Plateau ◽

Bacterial Load ◽

Microbial Community Composition ◽

16S Rrna Genes ◽

Rrna Genes ◽

Composition Analysis ◽

The Tibetan Plateau ◽

Culturable Bacteria ◽

Remote Sites ◽

Bacteria And Fungi

This study presents features of airborne culturable bacteria and fungi from three different sites (Lanzhou; LZ; 1520 m ASL, Lhasa; LS; 3640 m ASL and Qomolangma; ZF; 4276 m ASL) representing urban (LZ and LS) and remote sites (ZF) over the Tibetan Plateau (TP). Total suspended particle (TSP) samples were collected with an air sampler (Laoying 2030, China) on a quartz filter. Community structures of bacteria and fungi were studied and compared among three different locations. The average levels of bacterial load in the outdoor air ranged from approximately 8.03 × 101 to 3.25 × 102 CFU m–3 (Colony forming unit per m3). However, the average levels of fungal loads ranged from approximately 3.88 × 100 to 1.55 × 101 CFU m−3. Bacterial load was one magnitude higher at urban sites LZ (2.06 × 102–3.25 × 102 CFU m−3) and LS (1.96 × 102–3.23 × 102 CFU m−3) compared to remote sites ZF (8.03 × 101–9.54 × 101 CFU m−3). Similarly, the maximum fungal load was observed in LZ (1.02 × 101–1.55 × 101 CFU m−3) followed by LS (1.03 × 101–1.49 × 101 CFU m−3) and ZF (3.88 × 100–6.26 × 100 CFU m−3). However, the maximum microbial concentration was observed on the same day of the month, corresponding to a high dust storm in Lanzhou during the sampling period. The reported isolates were identified by phylogenetic analysis of 16S rRNA genes for bacteria and ITS sequences for fungi amplified from directly extracted DNA. Bacterial isolates were mostly associated with Proteobacteria, Eurotiomycetes and Bacillus, whereas fungal isolates were mostly Aspergillus and Alternaria. Overall, this is a pioneer study that provides information about the airborne microbial concentration and composition of three sites over the TP region depending on environmental parameters. This study provided preliminary insight to carry out more advanced and targeted analyses of bioaerosol in the sites presented in the study.

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Phenotypic and molecular characterization of chickpea rhizobia isolated from different areas of Tunisia

Canadian Journal of Microbiology ◽

10.1139/w06-127 ◽

2007 ◽

Vol 53 (3) ◽

pp. 427-434 ◽

Cited By ~ 17

Author(s):

Boulbaba L’taief ◽

Bouaziz Sifi ◽

Maher Gtari ◽

Mainassara Zaman-Allah ◽

Mokhtar Lachaâl

Keyword(s):

Rflp Analysis ◽

16S Rrna Genes ◽

Rrna Genes ◽

Phenotypic Characteristics ◽

Cicer Arietinum L ◽

Mesorhizobium Ciceri ◽

Length Polymorphisms ◽

Reference Strains ◽

Tolerance To Salinity

Several phenotypic markers were used in this study to determine the biodiversity of rhizobial strains nodulating Cicer arietinum L. in various areas of Tunisia. They include symbiotic traits, the use of 21 biochemical substrates, and tolerance to salinity and pH. In addition, restriction fragment length polymorphisms (RFLPs) of PCR-amplified 16S rDNA were compared with those of reference strains. Numeric analysis of the phenotypic characteristics showed that the 48 strains studied fell into three distinct groups. This heterogeneity was highly supported by the RFLP analysis of 16S rRNA genes, and two ribotypes were identified. Chickpea rhizobia isolated from Tunisian soils are both phenotypically and genetically diverse. Results showed that 40 and 8 isolates were assigned, respectively, to Mesorhizobium ciceri and Mesorhizobium mediterraneum .

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Analysis of the Endophytic Actinobacterial Population in the Roots of Wheat (Triticum aestivum L.) by Terminal Restriction Fragment Length Polymorphism and Sequencing of 16S rRNA Clones

Applied and Environmental Microbiology ◽

10.1128/aem.70.3.1787-1794.2004 ◽

2004 ◽

Vol 70 (3) ◽

pp. 1787-1794 ◽

Cited By ~ 113

Author(s):

Vanessa M. Conn ◽

Christopher M. M. Franco

Keyword(s):

Restriction Fragment Length Polymorphism ◽

16S Rrna ◽

Restriction Fragment ◽

Length Polymorphism ◽

Fragment Length ◽

Fragment Length Polymorphism ◽

Rflp Analysis ◽

16S Rrna Genes ◽

Rrna Genes ◽

Restriction Fragment Length

ABSTRACT The endophytic actinobacterial population in the roots of wheat grown in three different soils obtained from the southeast part of South Australia was investigated by terminal restriction fragment length polymorphism (T-RFLP) analysis of the amplified 16S rRNA genes. A new, validated approach was applied to the T-RFLP analysis in order to estimate, to the genus level, the actinobacterial population that was identified. Actinobacterium-biased primers were used together with three restriction enzymes to obtain terminal restriction fragments (TRFs). The TRFs were matched to bacterial genera by the T-RFLP Analysis Program, and the data were analyzed to validate and semiquantify the genera present within the plant roots. The highest diversity and level of endophytic colonization were found in the roots of wheat grown in a dark loam from Swedes Flat, and the lowest were found in water-repellent sand from Western Flat. This molecular approach detected a greater diversity of actinobacteria than did previous culture-dependent methods, with the predominant genera being Mycobacterium (21.02%) in Swedes Flat, Streptomyces (14.35%) in Red Loam, and Kitasatospora (15.02%) in Western Flat. This study indicates that the soil that supported a higher number of indigenous organisms resulted in wheat roots with higher actinobacterial diversity and levels of colonization within the plant tissue. Sequencing of 16S rRNA clones, obtained using the same actinobacterium-biased PCR primers that were used in the T-RFLP analysis, confirmed the presence of the actinobacterial diversity and identified a number of Mycobacterium and Streptomyces species.

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Distribution and Diversity of Archaeal and Bacterial Ammonia Oxidizers in Salt Marsh Sediments

Applied and Environmental Microbiology ◽

10.1128/aem.01001-09 ◽

2009 ◽

Vol 75 (23) ◽

pp. 7461-7468 ◽

Cited By ~ 90

Author(s):

Nicole S. Moin ◽

Katelyn A. Nelson ◽

Alexander Bush ◽

Anne E. Bernhard

Keyword(s):

Salt Marsh ◽

16S Rrna ◽

New England ◽

Water Column ◽

Salt Marshes ◽

16S Rrna Genes ◽

Rrna Genes ◽

Group I ◽

Marsh Sediments ◽

Salt Marsh Sediments

ABSTRACT Diversity and abundance of ammonia-oxidizing Betaproteobacteria (β-AOB) and archaea (AOA) were investigated in a New England salt marsh at sites dominated by short or tall Spartina alterniflora (SAS and SAT sites, respectively) or Spartina patens (SP site). AOA amoA gene richness was higher than β-AOB amoA richness at SAT and SP, but AOA and β-AOB richness were similar at SAS. β-AOB amoA clone libraries were composed exclusively of Nitrosospira-like amoA genes. AOA amoA genes at SAT and SP were equally distributed between the water column/sediment and soil/sediment clades, while AOA amoA sequences at SAS were primarily affiliated with the water column/sediment clade. At all three site types, AOA were always more abundant than β-AOB based on quantitative PCR of amoA genes. At some sites, we detected 109 AOA amoA gene copies g of sediment−1. Ratios of AOA to β-AOB varied over 2 orders of magnitude among sites and sampling dates. Nevertheless, abundances of AOA and β-AOB amoA genes were highly correlated. Abundance of 16S rRNA genes affiliated with Nitrosopumilus maritimus, Crenarchaeota group I.1b, and pSL12 were positively correlated with AOA amoA abundance, but ratios of amoA to 16S rRNA genes varied among sites. We also observed a significant effect of pH on AOA abundance and a significant salinity effect on both AOA and β-ΑΟΒ abundance. Our results expand the distribution of AOA to salt marshes, and the high numbers of AOA at some sites suggest that salt marsh sediments serve as an important habitat for AOA.

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Effect of Temperature on Carbon and Electron Flow and on the Archaeal Community in Methanogenic Rice Field Soil

Applied and Environmental Microbiology ◽

10.1128/aem.66.11.4790-4797.2000 ◽

2000 ◽

Vol 66 (11) ◽

pp. 4790-4797 ◽

Cited By ~ 172

Author(s):

Axel Fey ◽

Ralf Conrad

Keyword(s):

Steady State ◽

Rice Field ◽

Electron Flow ◽

Rflp Analysis ◽

Archaeal Community ◽

16S Rrna Genes ◽

Rrna Genes ◽

Field Soil ◽

Rice Field Soil ◽

Different Temperatures

ABSTRACT Temperature is an important factor controlling CH4production in anoxic rice soils. Soil slurries, prepared from Italian rice field soil, were incubated anaerobically in the dark at six temperatures of between 10 to 37°C or in a temperature gradient block covering the same temperature range at intervals of 1°C. Methane production reached quasi-steady state after 60 to 90 days. Steady-state CH4 production rates increased with temperature, with an apparent activation energy of 61 kJ mol−1. Steady-state partial pressures of the methanogenic precursor H2 also increased with increasing temperature from <0.5 to 3.5 Pa, so that the Gibbs free energy change of H2 plus CO2-dependent methanogenesis was kept at −20 to −25 kJ mol of CH4 −1 over the whole temperature range. Steady-state concentrations of the methanogenic precursor acetate, on the other hand, increased with decreasing temperature from <5 to 50 μM. Simultaneously, the relative contribution of H2 as methanogenic precursor decreased, as determined by the conversion of radioactive bicarbonate to 14CH4, so that the carbon and electron flow to CH4 was increasingly dominated by acetate, indicating that psychrotolerant homoacetogenesis was important. The relative composition of the archaeal community was determined by terminal restriction fragment length polymorphism (T-RFLP) analysis of the 16S rRNA genes (16S rDNA). T-RFLP analysis differentiated the archaeal Methanobacteriaceae,Methanomicrobiaceae, Methanosaetaceae,Methanosarcinaceae, and Rice clusters I, III, IV, V, and VI, which were all present in the rice field soil incubated at different temperatures. The 16S rRNA genes of Rice cluster I andMethanosaetaceae were the most frequent methanogenic groups. The relative abundance of Rice cluster I decreased with temperature. The substrates used by this microbial cluster, and thus its function in the microbial community, are unknown. The relative abundance of acetoclastic methanogens, on the other hand, was consistent with their physiology and the acetate concentrations observed at the different temperatures, i.e., the high-acetate-requiring Methanosarcinaceae decreased and the more modest Methanosaetaceae increased with increasing temperature. Our results demonstrate that temperature not only affected the activity but also changed the structure and the function (carbon and electron flow) of a complex methanogenic system.

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Formation of Pseudo-Terminal Restriction Fragments, a PCR-Related Bias Affecting Terminal Restriction Fragment Length Polymorphism Analysis of Microbial Community Structure

Applied and Environmental Microbiology ◽

10.1128/aem.69.5.2555-2562.2003 ◽

2003 ◽

Vol 69 (5) ◽

pp. 2555-2562 ◽

Cited By ~ 213

Author(s):

Markus Egert ◽

Michael W. Friedrich

Keyword(s):

Restriction Fragment Length Polymorphism ◽

16S Rrna ◽

Length Polymorphism ◽

Fragment Length Polymorphism ◽

Rflp Analysis ◽

Restriction Enzymes ◽

16S Rrna Genes ◽

Rrna Genes ◽

Restriction Fragments ◽

Restriction Fragment Length

ABSTRACT Terminal restriction fragment length polymorphism (T-RFLP) analysis of PCR-amplified genes is a widely used fingerprinting technique in molecular microbial ecology. In this study, we show that besides expected terminal restriction fragments (T-RFs), additional secondary T-RFs occur in T-RFLP analysis of amplicons from cloned 16S rRNA genes at high frequency. A total of 50% of 109 bacterial and 78% of 68 archaeal clones from the guts of cetoniid beetle larvae, using MspI and AluI as restriction enzymes, respectively, were affected by the presence of these additional T-RFs. These peaks were called “pseudo-T-RFs” since they can be detected as terminal fluorescently labeled fragments in T-RFLP analysis but do not represent the primary terminal restriction site as indicated by sequence data analysis. Pseudo-T-RFs were also identified in T-RFLP profiles of pure culture and environmental DNA extracts. Digestion of amplicons with the single-strand-specific mung bean nuclease prior to T-RFLP analysis completely eliminated pseudo-T-RFs. This clearly indicates that single-stranded amplicons are the reason for the formation of pseudo-T-RFs, most probably because single-stranded restriction sites cannot be cleaved by restriction enzymes. The strong dependence of pseudo-T-RF formation on the number of cycles used in PCR indicates that (partly) single-stranded amplicons can be formed during amplification of 16S rRNA genes. In a model, we explain how transiently formed secondary structures of single-stranded amplicons may render single-stranded amplicons accessible to restriction enzymes. The occurrence of pseudo-T-RFs has consequences for the interpretation of T-RFLP profiles from environmental samples, since pseudo-T-RFs may lead to an overestimation of microbial diversity. Therefore, it is advisable to establish 16S rRNA gene sequence clone libraries in parallel with T-RFLP analysis from the same sample and to check clones for their in vitro digestion T-RF pattern to facilitate the detection of pseudo-T-RFs.

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Community Structure of Denitrifiers, Bacteria, and Archaea along Redox Gradients in Pacific Northwest Marine Sediments by Terminal Restriction Fragment Length Polymorphism Analysis of Amplified Nitrite Reductase (nirS) and 16S rRNA Genes

Applied and Environmental Microbiology ◽

10.1128/aem.67.4.1893-1901.2001 ◽

2001 ◽

Vol 67 (4) ◽

pp. 1893-1901 ◽

Cited By ~ 195

Author(s):

Gesche Braker ◽

Héctor L. Ayala-del-Rı́o ◽

Allan H. Devol ◽

Andreas Fesefeldt ◽

James M. Tiedje

Keyword(s):

Community Structure ◽

Restriction Fragment Length Polymorphism ◽

16S Rrna ◽

Fragment Length Polymorphism ◽

Puget Sound ◽

Rflp Analysis ◽

16S Rrna Genes ◽

Rrna Genes ◽

Restriction Fragment Length ◽

Redox Gradients

ABSTRACT Steep vertical gradients of oxidants (O2 and NO3 −) in Puget Sound and Washington continental margin sediments indicate that aerobic respiration and denitrification occur within the top few millimeters to centimeters. To systematically explore the underlying communities of denitrifiers,Bacteria, and Archaea along redox gradients at distant geographic locations, nitrite reductase (nirS) genes and bacterial and archaeal 16S rRNA genes (rDNAs) were PCR amplified and analyzed by terminal restriction fragment length polymorphism (T-RFLP) analysis. The suitablility of T-RFLP analysis for investigating communities of nirS-containing denitrifiers was established by the correspondence of dominant terminal restriction fragments (T-RFs) of nirS to computer-simulated T-RFs ofnirS clones. These clones belonged to clusters II, III, and IV from the same cores and were analyzed in a previous study (G. Braker, J. Zhou, L. Wu, A. H. Devol, and J. M. Tiedje, Appl. Environ. Microbiol. 66:2096–2104, 2000). T-RFLP analysis ofnirS and bacterial rDNA revealed a high level of functional and phylogenetic diversity, whereas the level of diversity ofArchaea was lower. A comparison of T-RFLPs based on the presence or absence of T-RFs and correspondence analysis based on the frequencies and heights of T-RFs allowed us to group sediment samples according to the sampling location and thus clearly distinguish Puget Sound and the Washington margin populations. However, changes in community structure within sediment core sections during the transition from aerobic to anaerobic conditions were minor. Thus, within the top layers of marine sediments, redox gradients seem to result from the differential metabolic activities of populations of similar communities, probably through mixing by marine invertebrates rather than from the development of distinct communities.

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Coyotes (Canis latrans) as the Reservoir for a Human Pathogenic Bartonella sp.: Molecular Epidemiology ofBartonella vinsonii subsp. berkhoffii Infection in Coyotes from Central Coastal California

Journal of Clinical Microbiology ◽

10.1128/jcm.38.11.4193-4200.2000 ◽

2000 ◽

Vol 38 (11) ◽

pp. 4193-4200 ◽

Cited By ~ 62

Author(s):

Chao-Chin Chang ◽

Rickie W. Kasten ◽

Bruno B. Chomel ◽

Darren C. Simpson ◽

Carrie M. Hew ◽

...

Keyword(s):

16S Rrna ◽

Canis Latrans ◽

Bacterial Genome ◽

Clinical Signs ◽

Rflp Analysis ◽

Domestic Dog ◽

16S Rrna Genes ◽

Rrna Genes ◽

Intergenic Spacer Region ◽

Pcr Rflp

Bartonella vinsonii subsp. berkhoffii was originally isolated from a dog suffering infectious endocarditis and was recently identified as a zoonotic agent causing human endocarditis. Following the coyote bite of a child who developed clinical signs compatible with Bartonella infection in Santa Clara County, Calif., this epidemiological study was conducted. Among 109 coyotes (Canis latrans) from central coastal California, 31 animals (28%) were found to be bacteremic with B. vinsonii subsp.berkhoffii and 83 animals (76%) had B. vinsonii subsp. berkhoffii antibodies. These findings suggest these animals could be the wildlife reservoir of B. vinsonii subsp. berkhoffii. PCR-restriction fragment length polymorphism (PCR-RFLP) analysis of the gltA and 16S rRNA genes for these 31 isolates yielded similar profiles that were identical to those of B. vinsonii subsp.berkhoffii. Partial sequencing of the gltA and 16S rRNA genes, respectively, indicated 99.5 and 100% homology between the coyote isolate and B. vinsonii subsp.berkhoffii (ATCC 51672). PCR-RFLP analysis of the 16S-23S intergenic spacer region showed the existence of two different strain profiles, as has been reported in dogs. Six (19%) of 31Bartonella bacteremic coyotes exhibited the strain profile that was identified in the type strain of a canine endocarditis case (B. vinsonii subsp. berkhoffii ATCC 51672). The other 25 bacteremic coyotes were infected with a strain that was similar to the strains isolated from healthy dogs. Based on whole bacterial genome analysis by pulsed-field gel electrophoresis (PFGE) with SmaI restriction endonuclease, there was more diversity in fingerprints for the coyote isolates, which had at least 10 major variants compared to the two variants described for domestic dog isolates from the eastern United States. By PFGE analysis, threeBartonella bacteremic coyotes were infected by a strain identical to the one isolated from three healthy dog carriers. Further studies are necessary to elucidate the mode of transmission of B. vinsonii subsp. berkhoffii, especially to identify potential vectors, and to determine how humans become infected.

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Detection, in silico analysis and molecular diversity of phytoplasmas from solanaceous crops in Turkey

Plant Protection Science ◽

10.17221/115/2021-pps ◽

2021 ◽

Vol 58 (No. 1) ◽

pp. 31-39

Author(s):

Mustafa Usta ◽

Abdullah Güller ◽

Hikmet Murat Sipahioglu

Keyword(s):

Geographical Area ◽

Solanum Melongena ◽

In Silico Analysis ◽

Rflp Analysis ◽

16S Rrna Genes ◽

Rrna Genes ◽

Natural Occurrence ◽

Similarity Coefficients ◽

Universal Primer ◽

Virtual Rflp

Phytoplasma-like symptoms of leaf yellowing and calyx malformation were observed in eggplant (Solanum melongena L.), upward leaves and fruit malformation in pepper (Capsicum annuum L.), and aerial tuber formation in potato (S. tuberosum L.) during the survey performed in the late season (August to September) of 2015 and 2016 in Van province (Turkey). A total of 100 samples were tested by nested-PCR using universal primer pairs to assess the sanitary status of the solanaceous crops and to characterise the phytoplasma isolates. Among them, seven samples resulted in a 1.25 kb DNA fragment, and five (two eggplants, two peppers, and one potato) were molecularly characterised (Accession No.: KY579357, KT595210, MF564267, MF564266, and MH683601). BLAST and the virtual restriction fragment length polymorphism (RFLP) analysis of 16S rRNA genes revealed the presence of two distinct phytoplasma infections in solanaceous crops: ‘Candidatus Phytoplasma trifolii’ a member of the clover proliferation group (16SrVI) and subgroup A and ‘Candidatus P. solani’ a member of the stolbur group (16SrXII) and subgroup A. The virtual RFLP analysis and calculated coefficients of RFLP pattern similarities further revealed a remarkable genetic diversity among the ‘Candidatus P. solani’ isolates infecting pepper (similarity coefficient of 0.90) and eggplant (similarity coefficients of 0.98 and 1.00) at the same geographical area. This is the first report of the natural occurrence of ‘Candidadtus P. trifolii’ in potato from the Eastern Anatolia region, Turkey.

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