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1.  Combined Analyses of Bacterial, Fungal and Nematode Communities in Andosolic Agricultural Soils in Japan 
Microbes and Environments  2012;27(1):72-79.
We simultaneously examined the bacteria, fungi and nematode communities in Andosols from four agro-geographical sites in Japan using polymerase chain reaction-denaturing gradient gel electrophoresis (PCR-DGGE) and statistical analyses to test the effects of environmental factors including soil properties on these communities depending on geographical sites. Statistical analyses such as Principal component analysis (PCA) and Redundancy analysis (RDA) revealed that the compositions of the three soil biota communities were strongly affected by geographical sites, which were in turn strongly associated with soil characteristics such as total C (TC), total N (TN), C/N ratio and annual mean soil temperature (ST). In particular, the TC, TN and C/N ratio had stronger effects on bacterial and fungal communities than on the nematode community. Additionally, two-way cluster analysis using the combined DGGE profile also indicated that all soil samples were classified into four clusters corresponding to the four sites, showing high site specificity of soil samples, and all DNA bands were classified into four clusters, showing the coexistence of specific DGGE bands of bacteria, fungi and nematodes in Andosol fields. The results of this study suggest that geography relative to soil properties has a simultaneous impact on soil microbial and nematode community compositions. This is the first combined profile analysis of bacteria, fungi and nematodes at different sites with agricultural Andosols.
doi:10.1264/jsme2.ME11281
PMCID: PMC4036027  PMID: 22223474
bacteria-fungi-nematode community; andosols; PCR-DGGE; two-way cluster analysis
2.  Effects of the nematicide imicyafos on soil nematode community structure and damage to radish caused by Pratylenchus penetrans 
Journal of Nematology  2011;43(1):1-6.
The effects of the non-fumigant nematicide imicyafos on soil nematode community structure and damage to radish caused by Pratylenchus penetrans were evaluated in two field experiments in consecutive years (2007 and 2008). Nematode densities in soil at 0 - 10 cm (the depth of nematicide incorporation) and 10 - 30 cm were measured. The application of imicyafos had a significant impact on the density of P. penetrans at 0 - 10 cm but had no effect on free-living nematode density. PCR-DGGE analysis conducted using extracted nematodes showed that the nematode community structure 12 d after application in 2007 was altered by the application of imicyafos at the 0 - 10 cm depth, but not at 10 - 30 cm. No significant differences were observed in the diversity of the nematode community at harvest (89 and 91 d after application) between the control and imicyafos treatments in both depths and both years. In both years, the damage to radish caused by P. penetrans was markedly suppressed by the nematicide. Overall, the nematicide imicyafos decreased populations of P. penetrans in soil and thereby decreased damage to radish, while having little impact on the soil nematode community.
PMCID: PMC3380483  PMID: 22791909
free-living nematode; granular nematicide; imicyafos; lesion nematode; management; non-target effect; PCR-DGGE
3.  Granite Rock Outcrops: An Extreme Environment for Soil Nematodes? 
Journal of Nematology  2009;41(1):84-91.
We studied soil nematode communities from the surface of granite flatrock outcrops in the eastern Piedmont region of the United States. The thin soils that develop here experience high light intensity and extreme fluctuations in temperature and moisture and host unique plant communities. We collected soils from outcrop microsites in Virginia (VA) and North Carolina (NC) in various stages of succession (Primitive, Minimal, and Mature) and compared soil properties and nematode communities to those of adjacent forest soils. Nematodes were present in most outcrop soils, with densities comparable to forest soils (P > 0.05). Nematode communities in Mature and Minimal soils had lower species richness than forest soils (P < 0.05) and contained more bacterial-feeders and fewer fungal-feeders (P < 0.05). Primitive soils contained either no nematodes (NC) or only a single species (Mesodorylaimus sp., VA). Nematode communities were similar between Mature and Minimal soils, according to trophic group representation, MI, PPI, EI, SI, and CI (P > 0.05). Forest soils had a higher PPI value (P < 0.05), but otherwise community indices were similar to outcrop soils (P > 0.05). Outcrop nematode communities failed to group together in a Bray-Curtis cluster analysis, indicating higher variability in community structure than the Forest soils, which did cluster together. A high proportion of the nematodes were extracted from outcrop soils in coiled form (33-89%), indicating that they used anhydrobiosis to persist in this unique environment.
PMCID: PMC3365297  PMID: 22661780
Anhydrobiosis; community structure; diversity; ecology; granite flatrock outcrops; Maturity index; nematode survival; primary succession
4.  Impact of Fumigants on Soil Microbial Communities 
Agricultural soils are typically fumigated to provide effective control of nematodes, soilborne pathogens, and weeds in preparation for planting of high-value cash crops. The ability of soil microbial communities to recover after treatment with fumigants was examined using culture-dependent (Biolog) and culture-independent (phospholipid fatty acid [PLFA] analysis and denaturing gradient gel electrophoresis [DGGE] of 16S ribosomal DNA [rDNA] fragments amplified directly from soil DNA) approaches. Changes in soil microbial community structure were examined in a microcosm experiment following the application of methyl bromide (MeBr), methyl isothiocyanate, 1,3-dichloropropene (1,3-D), and chloropicrin. Variations among Biolog fingerprints showed that the effect of MeBr on heterotrophic microbial activities was most severe in the first week and that thereafter the effects of MeBr and the other fumigants were expressed at much lower levels. The results of PLFA analysis demonstrated a community shift in all treatments to a community dominated by gram-positive bacterial biomass. Different 16S rDNA profiles from fumigated soils were quantified by analyzing the DGGE band patterns. The Shannon-Weaver index of diversity, H, was calculated for each fumigated soil sample. High diversity indices were maintained between the control soil and the fumigant-treated soils, except for MeBr (H decreased from 1.14 to 0.13). After 12 weeks of incubation, H increased to 0.73 in the MeBr-treated samples. Sequence analysis of clones generated from unique bands showed the presence of taxonomically unique clones that had emerged from the MeBr-treated samples and were dominated by clones closely related to Bacillus spp. and Heliothrix oregonensis. Variations in the data were much higher in the Biolog assay than in the PLFA and DGGE assays, suggesting a high sensitivity of PLFA analysis and DGGE in monitoring the effects of fumigants on soil community composition and structure. Our results indicate that MeBr has the greatest impact on soil microbial communities and that 1,3-D has the least impact.
doi:10.1128/AEM.67.7.3245-3257.2001
PMCID: PMC93007  PMID: 11425748
5.  Effect of Soybean Monoculture on the Bacterial Communities Associated with Cysts of Heterodera glycines 
Journal of Nematology  2013;45(3):228-235.
The soybean cyst nematode (SCN), Heterodera glycines, can cause significant reductions in soybean yield and quality in many parts of the world. Natural biological control may play an important role in regulating SCN population. In this study the bacterial communities associated with SCN cysts obtained from fields under different lengths of soybean monoculture were explored. Soil samples were collected in 2010 and 2011 from six fields that had been used for soybean monoculture for 2 to 41 yr. SCN population densities were determined and bacterial communities from SCN cysts were investigated by Biolog and PCR-DGGE methods. SCN population densities initially increased in the first 5 yr of soybean monoculture but then declined steeply as years of soybean monoculture increased. Catabolic diversity of bacterial communities associated with cysts tended to decline as number of years of monoculture increased. Some specific PCR-DGGE bands, mainly representing Streptomyces and Rhizobium, were obtained from the cysts collected from the long-term monoculture fields. Principal component analysis of Biolog and PCR-DGGE data revealed that bacterial communities associated with cysts could be divided into two groups: those from cysts obtained from shorter (< 8 yr) vs. longer (> 8 yr) monoculture. This research demonstrates that the composition of the bacterial communities obtained from SCN cysts changes with length of soybean monoculture; the suppressive impact of these bacterial communities to SCN is yet to be determined.
PMCID: PMC3792841  PMID: 24115788
bacterial community; biodiversity; biological control; cyst; Heterodera glycines; monoculture; soybean; soybean cyst nematode
6.  Changes in Plant Species Richness Induce Functional Shifts in Soil Nematode Communities in Experimental Grassland 
PLoS ONE  2011;6(9):e24087.
Background
Changes in plant diversity may induce distinct changes in soil food web structure and accompanying soil feedbacks to plants. However, knowledge of the long-term consequences of plant community simplification for soil animal food webs and functioning is scarce. Nematodes, the most abundant and diverse soil Metazoa, represent the complexity of soil food webs as they comprise all major trophic groups and allow calculation of a number of functional indices.
Methodology/Principal Findings
We studied the functional composition of nematode communities three and five years after establishment of a grassland plant diversity experiment (Jena Experiment). In response to plant community simplification common nematode species disappeared and pronounced functional shifts in community structure occurred. The relevance of the fungal energy channel was higher in spring 2007 than in autumn 2005, particularly in species-rich plant assemblages. This resulted in a significant positive relationship between plant species richness and the ratio of fungal-to-bacterial feeders. Moreover, the density of predators increased significantly with plant diversity after five years, pointing to increased soil food web complexity in species-rich plant assemblages. Remarkably, in complex plant communities the nematode community shifted in favour of microbivores and predators, thereby reducing the relative abundance of plant feeders after five years.
Conclusions/Significance
The results suggest that species-poor plant assemblages may suffer from nematode communities detrimental to plants, whereas species-rich plant assemblages support a higher proportion of microbivorous nematodes stimulating nutrient cycling and hence plant performance; i.e. effects of nematodes on plants may switch from negative to positive. Overall, food web complexity is likely to decrease in response to plant community simplification and results of this study suggest that this results mainly from the loss of common species which likely alter plant – nematode interactions.
doi:10.1371/journal.pone.0024087
PMCID: PMC3164708  PMID: 21909412
7.  Investigating the Impact of Storage Conditions on Microbial Community Composition in Soil Samples 
PLoS ONE  2013;8(7):e70460.
Recent advances in DNA sequencing technologies have allowed scientists to probe increasingly complex biological systems, including the diversity of bacteria in the environment. However, despite a multitude of recent studies incorporating these methods, many questions regarding how environmental samples should be collected and stored still persist. Here, we assess the impact of different soil storage conditions on microbial community composition using Illumina-based 16S rRNA V4 amplicon sequencing. Both storage time and temperature affected bacterial community composition and structure. Frozen samples maintained the highest alpha diversity and differed least in beta diversity, suggesting the utility of cold storage for maintaining consistent communities. Samples stored for intermediate times (three and seven days) had both the highest alpha diversity and the largest differences in overall beta diversity, showing the degree of community change after sample collection. These divergences notwithstanding, differences in neither storage time nor storage temperature substantially altered overall communities relative to more than 500 previously examined soil samples. These results systematically support previous studies and stress the importance of methodological consistency for accurate characterization and comparison of soil microbiological assemblages.
doi:10.1371/journal.pone.0070460
PMCID: PMC3729949  PMID: 23936206
8.  Soil Microbial Community Structure across a Thermal Gradient following a Geothermal Heating Event 
Applied and Environmental Microbiology  2002;68(12):6300-6309.
In this study microbial species diversity was assessed across a landscape in Yellowstone National Park, where an abrupt increase in soil temperature had occurred due to recent geothermal activity. Soil temperatures were measured, and samples were taken across a temperature gradient (35 to 65°C at a 15-cm depth) that spanned geothermally disturbed and unimpacted soils; thermally perturbed soils were visually apparent by the occurrence of dead or dying lodgepole pine trees. Changes in soil microbial diversity across the temperature gradient were qualitatively assessed based on 16S rRNA sequence variation as detected by denaturing gradient gel electrophoresis (DGGE) using both ribosomal DNA (rDNA) and rRNA as PCR templates and primers specific for the Bacteria or Archaea domain. The impact of the major heating disturbance was apparent in that DGGE profiles from heated soils appeared less complex than those from the unaffected soils. Phylogenetic analysis of a bacterial 16S rDNA PCR clone library from a recently heated soil showed that a majority of the clones belonged to the Acidobacterium (51%) and Planctomyces (18%) divisions. Agar plate counts of soil suspensions cultured on dilute yeast extract and R2A agar media incubated at 25 or 50°C revealed that thermophile populations were two to three orders of magnitude greater in the recently heated soil. A soil microcosm laboratory experiment simulated the geothermal heating event. As determined by both RNA- and DNA-based PCR coupled with DGGE, changes in community structure (marked change in the DGGE profile) of soils incubated at 50°C occurred within 1 week and appeared to stabilize after 3 weeks. The results of our molecular and culture data suggest that thermophiles or thermotolerant species are randomly distributed in this area within Yellowstone National Park and that localized thermal activity selects for them.
doi:10.1128/AEM.68.12.6300-6309.2002
PMCID: PMC134386  PMID: 12450855
9.  Impact of swapping soils on the endophytic bacterial communities of pre-domesticated, ancient and modern maize 
BMC Plant Biology  2014;14(1):233.
Background
Endophytes are microbes that live within plants such as maize (corn, Zea mays L.) without causing disease. It is generally assumed that most endophytes originate from soil. If this is true, then as humans collected, domesticated, bred and migrated maize globally from its native Mexico, they moved the species away from its native population of endophyte donors. The migration of maize persists today, as breeders collect wild and exotic seed (as sources of diverse alleles) from sites of high genetic diversity in Mexico for breeding programs on distant soils. When transported to new lands, it is unclear whether maize permits only selective colonization of microbes from the Mexican soils on which it co-evolved, tolerates shifts in soil-derived endophytes, or prevents colonization of soil-based microbes in favour of seed-transmitted microbes. To test these hypotheses, non-sterilized seeds of three types of maize (pre-domesticated-Mexican, ancient-Mexican, modern-temperate) were planted side-by-side on indigenous Mexican soil, Canadian temperate soil or sterilized sand. The impact of these soil swaps on founder bacterial endophyte communities was tested using 16S-rDNA profiling, culturing and microbial trait phenotyping.
Results
Multivariate analysis showed that bacterial 16S-rDNA TRFLP profiles from young, surface-sterilized maize plants were more similar when the same host genotype was grown on the different soils than when different maize genotypes were grown on the same soil. There appeared to be two reasons for this result. First, the largest fraction of bacterial 16S-signals from soil-grown plants was shared with parental seeds and/or plants grown on sterilized sand, suggesting significant inheritance of candidate endophytes. The in vitro activities of soil-derived candidate endophytes could be provided by bacteria that were isolated from sterile sand grown plants. Second, many non-inherited 16S-signals from sibling plants grown on geographically-distant soils were shared with one another, suggesting maize can select microbes with similar TRFLP peak sizes from diverse soils. Wild, pre-domesticated maize did not possess more unique 16S-signals when grown on its native Mexican soil than on Canadian soil, pointing against long-term co-evolutionary selection. The modern hybrid did not reject more soil-derived 16S-signals than did ancestral maize, pointing against such rejection as a mechanism that contributes to yield stability across environments. A minor fraction of 16S-signals was uniquely associated with any one soil.
Conclusion
Within the limits of TRFLP profiling, the candidate bacterial endophyte populations of pre-domesticated, ancient and modern maize are partially buffered against the effects of geographic migration --- from a Mexican soil associated with ancestral maize, to a Canadian soil associated with modern hybrid agriculture. These results have implications for understanding the effects of domestication, migration, ex situ seed conservation and modern breeding, on the microbiome of one of the world’s most important food crops.
Electronic supplementary material
The online version of this article (doi:10.1186/s12870-014-0233-3) contains supplementary material, which is available to authorized users.
doi:10.1186/s12870-014-0233-3
PMCID: PMC4189167  PMID: 25227492
Endophyte; Zea; Maize; Bacteria; 16S; Domestication; Evolution; Microbial ecology; Root; Shoot; Seed; TRFLP; Soil; Teosinte; Parviglumis; Mixteco; Landrace; Vertical transmission; Yield stability; Corn hybrid; Maize hybrid; Breeding
10.  Molecular characterization and the effect of salinity on cyanobacterial diversity in the rice fields of Eastern Uttar Pradesh, India 
Saline Systems  2009;5:4.
Background
Salinity is known to affect almost half of the world's irrigated lands, especially rice fields. Furthermore, cyanobacteria, one of the critical inhabitants of rice fields have been characterized at molecular level from many different geographical locations. This study, for the first time, has examined the molecular diversity of cyanobacteria inhabiting Indian rice fields which experience various levels of salinity.
Results
Ten physicochemical parameters were analyzed for samples collected from twenty experimental sites. Electrical conductivity data were used to classify the soils and to investigate relationship between soil salinity and cyanobacterial diversity. The cyanobacterial communities were analyzed using semi-nested 16S rRNA gene PCR and denaturing gradient gel electrophoresis. Out of 51 DGGE bands selected for sequencing only 31 which showed difference in sequences were subjected to further analysis. BLAST analysis revealed highest similarity for twenty nine of the sequences with cyanobacteria, and the other two to plant plastids. Clusters obtained based on morphological and molecular attributes of cyanobacteria were correlated to soil salinity. Among six different clades, clades 1, 2, 4 and 6 contained cyanobacteria inhabiting normal or low saline (having EC < 4.0 ds m-1) to (high) saline soils (having EC > 4.0 ds m-1), however, clade 5 represented the cyanobacteria inhabiting only saline soils. Whilst, clade 3 contained cyanobacteria from normal soils. The presence of DGGE band corresponding to Aulosira strains were present in large number of soil indicating its wide distribution over a range of salinities, as were Nostoc, Anabaena, and Hapalosiphon although to a lesser extent in the sites studied.
Conclusion
Low salinity favored the presence of heterocystous cyanobacteria, while very high salinity mainly supported the growth of non-heterocystous genera. High nitrogen content in the low salt soils is proposed to be a result of reduced ammonia volatilization compared to the high salt soils. Although many environmental factors could potentially determine the microbial community present in these multidimensional ecosystems, changes in the diversity of cyanobacteria in rice fields was correlated to salinity.
doi:10.1186/1746-1448-5-4
PMCID: PMC2680867  PMID: 19344531
11.  SSU Ribosomal DNA-Based Monitoring of Nematode Assemblages Reveals Distinct Seasonal Fluctuations within Evolutionary Heterogeneous Feeding Guilds 
PLoS ONE  2012;7(10):e47555.
Soils are among the most complex, diverse and competitive habitats on Earth and soil biota are responsible for ecosystem services such as nutrient cycling, carbon sequestration and remediation of freshwater. The extreme biodiversity prohibits the making of a full inventory of soil life. Hence, an appropriate indicator group should be selected to determine the biological condition of soil systems. Due to their ubiquity and the diverse responses to abiotic and biotic changes, nematodes are suitable indicators for environmental monitoring. However, the time-consuming microscopic analysis of nematode communities has limited the scale at which this indicator group is used. In an attempt to circumvent this problem, a quantitative PCR-based tool for the detection of a consistent part of the soil nematofauna was developed based on a phylum-wide molecular framework consisting of 2,400 full-length SSU rDNA sequences. Taxon-specific primers were designed and tested for specificity. Furthermore, relationships were determined between the quantitative PCR output and numbers of target nematodes. As a first field test for this DNA sequence signature-based approach, seasonal fluctuations of nematode assemblages under open canopy (one field) and closed canopy (one forest) were monitored. Fifteen taxa from four feeding guilds (covering ∼ 65% of the free-living nematode biodiversity at higher taxonomical level) were detected at two trophic levels. These four feeding guilds are composed of taxa that developed independently by parallel evolution and we detected ecologically interpretable patterns for free-living nematodes belonging to the lower trophic level of soil food webs. Our results show temporal fluctuations, which can be even opposite within taxa belonging to the same guild. This research on nematode assemblages revealed ecological information about the soil food web that had been partly overlooked.
doi:10.1371/journal.pone.0047555
PMCID: PMC3480422  PMID: 23112818
12.  Soil Respiration and Organic Carbon Dynamics with Grassland Conversions to Woodlands in Temperate China 
PLoS ONE  2013;8(8):e71986.
Soils are the largest terrestrial carbon store and soil respiration is the second-largest flux in ecosystem carbon cycling. Across China's temperate region, climatic changes and human activities have frequently caused the transformation of grasslands to woodlands. However, the effect of this transition on soil respiration and soil organic carbon (SOC) dynamics remains uncertain in this area. In this study, we measured in situ soil respiration and SOC storage over a two-year period (Jan. 2007–Dec. 2008) from five characteristic vegetation types in a forest-steppe ecotone of temperate China, including grassland (GR), shrubland (SH), as well as in evergreen coniferous (EC), deciduous coniferous (DC) and deciduous broadleaved forest (DB), to evaluate the changes of soil respiration and SOC storage with grassland conversions to diverse types of woodlands. Annual soil respiration increased by 3%, 6%, 14%, and 22% after the conversion from GR to EC, SH, DC, and DB, respectively. The variation in soil respiration among different vegetation types could be well explained by SOC and soil total nitrogen content. Despite higher soil respiration in woodlands, SOC storage and residence time increased in the upper 20 cm of soil. Our results suggest that the differences in soil environmental conditions, especially soil substrate availability, influenced the level of annual soil respiration produced by different vegetation types. Moreover, shifts from grassland to woody plant dominance resulted in increased SOC storage. Given the widespread increase in woody plant abundance caused by climate change and large-scale afforestation programs, the soils are expected to accumulate and store increased amounts of organic carbon in temperate areas of China.
doi:10.1371/journal.pone.0071986
PMCID: PMC3751950  PMID: 24058408
13.  Occurrence and Phylogenetic Diversity of Sphingomonas Strains in Soils Contaminated with Polycyclic Aromatic Hydrocarbons 
Bacterial strains of the genus Sphingomonas are often isolated from contaminated soils for their ability to use polycyclic aromatic hydrocarbons (PAH) as the sole source of carbon and energy. The direct detection of Sphingomonas strains in contaminated soils, either indigenous or inoculated, is, as such, of interest for bioremediation purposes. In this study, a culture-independent PCR-based detection method using specific primers targeting the Sphingomonas 16S rRNA gene combined with denaturing gradient gel electrophoresis (DGGE) was developed to assess Sphingomonas diversity in PAH-contaminated soils. PCR using the new primer pair on a set of template DNAs of different bacterial genera showed that the method was selective for bacteria belonging to the family Sphingomonadaceae. Single-band DGGE profiles were obtained for most Sphingomonas strains tested. Strains belonging to the same species had identical DGGE fingerprints, and in most cases, these fingerprints were typical for one species. Inoculated strains could be detected at a cell concentration of 104 CFU g of soil−1. The analysis of Sphingomonas population structures of several PAH-contaminated soils by the new PCR-DGGE method revealed that soils containing the highest phenanthrene concentrations showed the lowest Sphingomonas diversity. Sequence analysis of cloned PCR products amplified from soil DNA revealed new 16S rRNA gene Sphingomonas sequences significantly different from sequences from known cultivated isolates (i.e., sequences from environmental clones grouped phylogenetically with other environmental clone sequences available on the web and that possibly originated from several potential new species). In conclusion, the newly designed Sphingomonas-specific PCR-DGGE detection technique successfully analyzed the Sphingomonas communities from polluted soils at the species level and revealed different Sphingomonas members not previously detected by culture-dependent detection techniques.
doi:10.1128/AEM.70.4.1944-1955.2004
PMCID: PMC383131  PMID: 15066784
14.  Low-technology cooling box for storage of malaria RDTs and other medical supplies in remote areas 
Malaria Journal  2010;9:31.
Background
With the increase in use of point-of-care diagnostic tests for malaria and other diseases comes the necessity of storing the diagnostic kits and the drugs required for subsequent management, in remote areas, where temperatures are high and electricity supply is unreliable or unavailable.
Methods
To address the lack of temperature-controlled storage during the introduction of community-based malaria management in Cambodia, the Cambodian National Centre for Parasitology, Entomology and Malaria Control (CNM) developed prototype evaporative cooling boxes (Cambodian Cooler Boxes - CCBs) for storage of perishable medical commodities in remote clinics. The performance of these CCBs for maintaining suitable storage temperatures was evaluated over two phases in 2005 and 2006-7, comparing conditions in CCBs using water as designed, CCBs with no water for evaporation, and ambient storage room temperatures. Temperature and humidity was monitored, together with the capacity of the RDTs recommended for storage between 2 to 30 degree Celsius to detect low-density malaria parasite samples after storage under these conditions.
Results
Significant differences were recorded between the proportion of temperatures within the recommended RDT storage conditions in the CCBs with water and the temperatures in the storage room (p < 0.001) and maximum temperatures were lower. RDTs stored at ambient temperatures were negative when tested with parasitized blood (2,000 parasites per micro litre) at 210 days, while the field RDTs kept in CCBs with water gave positive results until 360 days.
Discussion and Conclusions
The CCB was an effective tool for storage of RDTs at optimal conditions, and extended the effective life-span of the tests. The concept of evaporative cooling has potential to greatly enhance access to perishable diagnostics and medicines in remote communities, as it allows prolonged storage at low cost using locally-available materials, in the absence of electricity.
doi:10.1186/1475-2875-9-31
PMCID: PMC2834699  PMID: 20096130
15.  Direct Assessment of Viral Diversity in Soils by Random PCR Amplification of Polymorphic DNA 
Applied and Environmental Microbiology  2013;79(18):5450-5457.
Viruses are the most abundant and diverse biological entities within soils, yet their ecological impact is largely unknown. Defining how soil viral communities change with perturbation or across environments will contribute to understanding the larger ecological significance of soil viruses. A new approach to examining the composition of soil viral communities based on random PCR amplification of polymorphic DNA (RAPD-PCR) was developed. A key methodological improvement was the use of viral metagenomic sequence data for the design of RAPD-PCR primers. This metagenomically informed approach to primer design enabled the optimization of RAPD-PCR sensitivity for examining changes in soil viral communities. Initial application of RAPD-PCR viral fingerprinting to soil viral communities demonstrated that the composition of autochthonous soil viral assemblages noticeably changed over a distance of meters along a transect of Antarctic soils and across soils subjected to different land uses. For Antarctic soils, viral assemblages segregated upslope from the edge of dry valley lakes. In the case of temperate soils at the Kellogg Biological Station, viral communities clustered according to land use treatment. In both environments, soil viral communities changed along with environmental factors known to shape the composition of bacterial host communities. Overall, this work demonstrates that RAPD-PCR fingerprinting is an inexpensive, high-throughput means for addressing first-order questions of viral community dynamics within environmental samples and thus fills a methodological gap between narrow single-gene approaches and comprehensive shotgun metagenomic sequencing for the analysis of viral community diversity.
doi:10.1128/AEM.00268-13
PMCID: PMC3754152  PMID: 23793630
16.  Nematode Assemblages in Native Plant Communities of Molokai, Hawaii 
Journal of Nematology  2005;37(2):242-248.
Four native plant community types (in decreasing elevation: montane bog, rain forest, wet mesic forest, drier forest) on Molokai were sampled for nematodes. Six samples of 10 cores each were gathered from each community. Nematodes were extracted from 200 cm³ soil by elutriation. All extracted nematodes were counted and identified to species-level taxa. Sixty-seven species were identified among the four plant communities; only eight species occurred in all four communities. Species diversity and evenness were greater in the rain forest and mesic forest than in the bog and the drier forest, but the drier forest and mesic forest had similar species communities. The bog nematode community was not similar to the other three sites. In a presence/absence cluster analysis, all six bog sample assemblages clustered together. The rain forest samples also clustered together but were associated with the mesic forest sample closest to the rain forest edge. Of 11 nematode orders collected, Tylenchida accounted for 40% to 73% of all individuals, followed by Dorylaimida (5% to 17%). Diplogasterida were absent. No plant-parasitic nematodes of known Hawaiian agricultural importance or occurrence were collected in these native plant communities. Calculated nematode densities (76,000 to 321,300/m²) were comparable to those reported for some other Pacific tropical forests.
PMCID: PMC2620952  PMID: 19262867
biodiversity; biogeography; bog; community structure; cluster analysis; hawaii; island; Molokai; nematode; rain forest; survey
17.  Effects of Manure Compost Application on Soil Microbial Community Diversity and Soil Microenvironments in a Temperate Cropland in China 
PLoS ONE  2014;9(10):e108555.
The long-term application of excessive chemical fertilizers has resulted in the degeneration of soil quality parameters such as soil microbial biomass, communities, and nutrient content, which in turn affects crop health, productivity, and soil sustainable productivity. The objective of this study was to develop a rapid and efficient solution for rehabilitating degraded cropland soils by precisely quantifying soil quality parameters through the application of manure compost and bacteria fertilizers or its combination during maize growth. We investigated dynamic impacts on soil microbial count, biomass, basal respiration, community structure diversity, and enzyme activity using six different treatments [no fertilizer (CK), N fertilizer (N), N fertilizer + bacterial fertilizer (NB), manure compost (M), manure compost + bacterial fertilizer (MB), and bacterial fertilizer (B)] in the plowed layer (0–20 cm) of potted soil during various maize growth stages in a temperate cropland of eastern China. Denaturing gradient electrophoresis (DGGE) fingerprinting analysis showed that the structure and composition of bacterial and fungi communities in the six fertilizer treatments varied at different levels. The Shannon index of bacterial and fungi communities displayed the highest value in the MB treatments and the lowest in the N treatment at the maize mature stage. Changes in soil microorganism community structure and diversity after different fertilizer treatments resulted in different microbial properties. Adding manure compost significantly increased the amount of cultivable microorganisms and microbial biomass, thus enhancing soil respiration and enzyme activities (p<0.01), whereas N treatment showed the opposite results (p<0.01). However, B and NB treatments minimally increased the amount of cultivable microorganisms and microbial biomass, with no obvious influence on community structure and soil enzymes. Our findings indicate that the application of manure compost plus bacterial fertilizers can immediately improve the microbial community structure and diversity of degraded cropland soils.
doi:10.1371/journal.pone.0108555
PMCID: PMC4193766  PMID: 25302996
18.  Long-term room temperature preservation of corpse soft tissue: an approach for tissue sample storage 
Background
Disaster victim identification (DVI) represents one of the most difficult challenges in forensic sciences, and subsequent DNA typing is essential. Collected samples for DNA-based human identification are usually stored at low temperature to halt the degradation processes of human remains. We have developed a simple and reliable procedure for soft tissue storage and preservation for DNA extraction. It ensures high quality DNA suitable for PCR-based DNA typing after at least 1 year of room temperature storage.
Methods
Fragments of human psoas muscle were exposed to three different environmental conditions for diverse time periods at room temperature. Storage conditions included: (a) a preserving medium consisting of solid sodium chloride (salt), (b) no additional substances and (c) garden soil. DNA was extracted with proteinase K/SDS followed by organic solvent treatment and concentration by centrifugal filter devices. Quantification was carried out by real-time PCR using commercial kits. Short tandem repeat (STR) typing profiles were analysed with 'expert software'.
Results
DNA quantities recovered from samples stored in salt were similar up to the complete storage time and underscored the effectiveness of the preservation method. It was possible to reliably and accurately type different genetic systems including autosomal STRs and mitochondrial and Y-chromosome haplogroups. Autosomal STR typing quality was evaluated by expert software, denoting high quality profiles from DNA samples obtained from corpse tissue stored in salt for up to 365 days.
Conclusions
The procedure proposed herein is a cost efficient alternative for storage of human remains in challenging environmental areas, such as mass disaster locations, mass graves and exhumations. This technique should be considered as an additional method for sample storage when preservation of DNA integrity is required for PCR-based DNA typing.
doi:10.1186/2041-2223-2-17
PMCID: PMC3170604  PMID: 21846338
19.  Detection and Investigation of Soil Biological Activity against Meloidogyne incognita 
Journal of nematology  2008;40(2):109-118.
Greenhouse experiments with two susceptible hosts of Meloidogyne incognita, a dwarf tomato and wheat, led to the identification of a soil in which the root-knot nematode population was reduced 5- to 16-fold compared to identical but pasteurized soil two months after infestation with 280 M. incognita J2/100 cm3 soil. This suppressive soil was subjected to various temperature, fumigation and dilution treatments, planted with tomato, and infested with 1,000 eggs of M. incognita/100 cm3 soil. Eight weeks after nematode infestation, distinct differences in nematode population densities were observed among the soil treatments, suggesting the suppressiveness had a biological nature. A fungal rRNA gene analysis (OFRG) performed on M. incognita egg masses collected at the end of the greenhouse experiments identified 11 fungal phylotypes, several of which exhibited associations with one or more of the nematode population density measurements (egg masses, eggs or J2). The phylotype containing rRNA genes with high sequence identity to Pochonia chlamydosporia exhibited the strongest negative associations. The negative correlation between the densities of the P. chlamydosporia genes and the nematodes was corroborated by an analysis using a P. chlamydosporia-selective qPCR assay.
PMCID: PMC2586535  PMID: 19259527
biological control; dwarf tomato; Meloidogyne incognita; Pochonia chlamydosporia; root-knot nematode; Solanum lycopersicon; suppressive soil; Triticum aestivum; wheat
20.  Differences between Bacterial Communities in the Gut of a Soil-Feeding Termite (Cubitermes niokoloensis) and Its Mounds▿ † 
Applied and Environmental Microbiology  2007;73(16):5199-5208.
In tropical ecosystems, termite mound soils constitute an important soil compartment covering around 10% of African soils. Previous studies have shown (S. Fall, S. Nazaret, J. L. Chotte, and A. Brauman, Microb. Ecol. 28:191-199, 2004) that the bacterial genetic structure of the mounds of soil-feeding termites (Cubitermes niokoloensis) is different from that of their surrounding soil. The aim of this study was to characterize the specificity of bacterial communities within mounds with respect to the digestive and soil origins of the mound. We have compared the bacterial community structures of a termite mound, termite gut sections, and surrounding soil using PCR-denaturing gradient gel electrophoresis (DGGE) analysis and cloning and sequencing of PCR-amplified 16S rRNA gene fragments. DGGE analysis revealed a drastic difference between the genetic structures of the bacterial communities of the termite gut and the mound. Analysis of 266 clones, including 54 from excised bands, revealed a high level of diversity in each biota investigated. The soil-feeding termite mound was dominated by the Actinobacteria phylum, whereas the Firmicutes and Proteobacteria phyla dominate the gut sections of termites and the surrounding soil, respectively. Phylogenetic analyses revealed a distinct clustering of Actinobacteria phylotypes between the mound and the surrounding soil. The Actinobacteria clones of the termite mound were diverse, distributed among 10 distinct families, and like those in the termite gut environment lightly dominated by the Nocardioidaceae family. Our findings confirmed that the soil-feeding termite mound (C. niokoloensis) represents a specific bacterial habitat in the tropics.
doi:10.1128/AEM.02616-06
PMCID: PMC1950997  PMID: 17574999
21.  Effects of Mixed Cropping on a Soil Nematode Community in Honduras 
Journal of Nematology  1993;25(4):666-673.
Nematode-resistant tropical legumes are effective in reducing populations of plant-parasitic nematodes when used in rotation systems. Mixed cropping is a common practice of many small farmers in Central America, but little is known about the effects of tropical legumes on nematode communities under these systems. To examine the effects of intercropping on the nematode fauna associated with squash (Cucurbita pepo) and cucumber (Cucumis sativa) in Honduras, two field experiments were conducted to compare nematode density and diversity in soil under cucurbits grown as a monocrop with that in soil under cucurbits intercropped with alfalfa (Medicago sativa) or hairy indigo (Indigofera hirsuta). A parallel series of field tests compared soil nematode communities associated with a cucurbit monocrop and a cucurbit intercropped with marigold (Tagetes patula), which may decrease nematode populations through the production of toxic root exudates. Among all four tests, over a period of 90 days, there were no consistent differences in densities of various nematode genera or trophic groups in intercropped versus monocropped plants, nor were there consistent differences in community diversities among treatments.
PMCID: PMC2619433  PMID: 19279824
agroecology; cropping system; ecology; intercropping; mixed cropping; nematode; nematode community
22.  Impacts of Inter- and Intralaboratory Variations on the Reproducibility of Microbial Community Analyses▿ †  
Applied and Environmental Microbiology  2010;76(22):7451-7458.
With the advent of molecular biological techniques, especially next-generation sequencing and metagenomics, the number of microbial biogeography studies is rapidly increasing. However, these studies involve the synthesis of data generated by different laboratories using different protocols, chemicals, etc., all with inherent biases. The aim of this study was to assess inter- as well as intralaboratory variations in microbial community composition when standardized protocols are applied to a single soil sample. Aliquots from a homogenized soil sample from a rice field in Italy were sent to five participating laboratories. DNA was extracted by two investigators per laboratory using an identical protocol. All DNA samples were sent to one laboratory to perform DNA quantification, quantitative PCR (QPCR), and microarray and denaturing gradient gel electrophoresis (DGGE) analyses of methanotrophic communities. Yields, as well as purity of DNA, were significantly different between laboratories but in some cases also between investigators within the same laboratory. The differences in yield and quality of the extracted DNA were reflected in QPCR, microarray, and DGGE analysis results. Diversity indices (Shannon-Wiener, evenness, and richness) differed significantly between laboratories. The observed differences have implications for every project in which microbial communities are compared in different habitats, even if assessed within the same laboratory. To be able to make sensible comparisons leading to valid conclusions, intralaboratory variation should be assessed. Standardization of DNA extraction protocols and possible use of internal standards in interlaboratory comparisons may help in rendering a “quantifiable” bias.
doi:10.1128/AEM.01595-10
PMCID: PMC2976186  PMID: 20870788
23.  Management of Paratrichodorus allius Damage to Onion in the Columbia Basin of Oregon 
Journal of Nematology  1999;31(4S):678-683.
Production of storage and dehydration (dehy) onions is increasing rapidly in the northcentral Oregon region of the Columbia Basin. Roughly circular patches of stunted onions have been observed in many fields and are often numerous and (or) extensive in area. Onions in these patches often exhibit symptoms typical of stubby-root nematodes, and Paratrichodorus allius has been collected from several damaged areas. This study examined the relationship of P. allius densities to onion yield reduction and benefit from oxamyl application. Transect samples and onions harvested across areas with varying P. allius densities revealed that the total number of onions was not affected by P. allius but that onions were in smaller, less valuable size classes. Yield of the largest size class of storage onions (colossals) declined substantially (57-61%) even at low (5-16/250 g soil) nematode densities. Treatment with oxamyl at 1.1 kg a.i./ha mitigated most of the damage, and the value of both storage and dehy onions was increased by more than $1,200/ha. Early applications were superior to later applications, whereas treatment at both times was no better than the early treatment alone. Damage at low (<10/250 g soil) nematode densities, low treatment cost ($86-172/ha), and high crop values suggest an economic benefit from treatment at population densities of four or more P. allius/250 g soil. However, stunted patches of onions may occur for reasons other than nematodes, and the presence of P. allius should be confirmed before treating fields. This is the first report of stubby-root nematode damage to dehy onions and to storage onions in the Columbia Basin.
PMCID: PMC2620406  PMID: 19270935
Allium cepa; management; nematode; onion; oxamyl; Paratrichodorus allius; stubby-root nematodes; threshold; yield loss
24.  Monitoring of Microbial Metabolites and Bacterial Diversity in Beef Stored under Different Packaging Conditions ▿ †  
Applied and Environmental Microbiology  2011;77(20):7372-7381.
Beef chops were stored at 4°C under different conditions: in air (A), modified-atmosphere packaging (MAP), vacuum packaging (V), or bacteriocin-activated antimicrobial packaging (AV). After 0 to 45 days of storage, analyses were performed to determine loads of spoilage microorganisms, microbial metabolites (by solid-phase microextraction [SPME]-gas chromatography [GC]-mass spectrometry [MS] and proton nuclear magnetic resonance [1H NMR]), and microbial diversity (by PCR–denaturing gradient gel electrophoresis [DGGE] and pyrosequencing). The microbiological shelf life of meat increased with increasing selectivity of storage conditions. Culture-independent analysis by pyrosequencing of DNA extracted directly from meat showed that Brochothrix thermosphacta dominated during the early stages of storage in A and MAP, while Pseudomonas spp. took over during further storage in A. Many different bacteria, several of which are usually associated with soil rather than meat, were identified in V and AV; however, lactic acid bacteria (LAB) dominated during the late phases of storage, and Carnobacterium divergens was the most frequent microorganism in AV. Among the volatile metabolites, butanoic acid was associated with the growth of LAB under V and AV storage conditions, while acetoin was related to the other spoilage microbial groups and storage conditions. 1H NMR analysis showed that storage in air was associated with decreases in lactate, glycogen, IMP, and ADP levels and with selective increases in levels of 3-methylindole, betaine, creatine, and other amino acids. The meat microbiota is significantly affected by storage conditions, and its changes during storage determine complex shifts in the metabolites produced, with a potential impact on meat quality.
doi:10.1128/AEM.05521-11
PMCID: PMC3194879  PMID: 21803905
25.  Soil Mineral Composition Matters: Response of Microbial Communities to Phenanthrene and Plant Litter Addition in Long-Term Matured Artificial Soils 
PLoS ONE  2014;9(9):e106865.
The fate of polycyclic aromatic hydrocarbons (PAHs) in soil is determined by a suite of biotic and abiotic factors, and disentangling their role in the complex soil interaction network remains challenging. Here, we investigate the influence of soil composition on the microbial community structure and its response to the spiked model PAH compound phenanthrene and plant litter. We used long-term matured artificial soils differing in type of clay mineral (illite, montmorillonite) and presence of charcoal or ferrihydrite. The soils received an identical soil microbial fraction and were incubated for more than two years with two sterile manure additions. The matured artificial soils and a natural soil were subjected to the following spiking treatments: (I) phenanthrene, (II) litter, (III) litter + phenanthrene, (IV) unspiked control. Total community DNA was extracted from soil sampled on the day of spiking, 7, 21, and 63 days after spiking. Bacterial 16S rRNA gene and fungal internal transcribed spacer amplicons were quantified by qPCR and subjected to denaturing gradient gel electrophoresis (DGGE). DGGE analysis revealed that the bacterial community composition, which was strongly shaped by clay minerals after more than two years of incubation, changed in response to spiked phenanthrene and added litter. DGGE and qPCR showed that soil composition significantly influenced the microbial response to spiking. While fungal communities responded only in presence of litter to phenanthrene spiking, the response of the bacterial communities to phenanthrene was less pronounced when litter was present. Interestingly, microbial communities in all artificial soils were more strongly affected by spiking than in the natural soil, which might indicate the importance of higher microbial diversity to compensate perturbations. This study showed the influence of soil composition on the microbiota and their response to phenanthrene and litter, which may increase our understanding of complex interactions in soils for bioremediation applications.
doi:10.1371/journal.pone.0106865
PMCID: PMC4164357  PMID: 25222697

Results 1-25 (591085)