Influence of soil fumigation by methyl bromide and methyl iodide on rhizosphere and phyllosphere microbial community structure.

Rhizosphere and phyllosphere microbial communities were evaluated on roots and leaves of growth chamber-grown lettuce (Lactuca sativa (L.) cv. Green Forest) plants by culture-dependent and -independent methods after soil fumigation. Denaturing gradient gel electrophoresis (DGGE) with 16S rRNA primers followed by cloning and sequencing was used to identify major rRNA bands from the rhizosphere and phyllosphere. Three weeks after fumigation, there were no differences (P = 0.16) in rhizosphere microbial communities between the fumigated treatments and the control. The same effect was observed during week seven after fumigation (P=0.49). Also, no significant differences (P=0.49) were found in the phyllosphere microbial communities between the fumigated treatments and the control during the growth period of the plant. A majority of the bands in the rhizosphere were related to known bacterial sequences with a 96 to 100 % sequence similarity. Some of the derived sequences were related to Pseudomonas syringae pv. tomato DC300 and Bradyrhizobium japonicum USDA 110. A total of 23 isolates were identified from leaf surface by both culture-dependent and independent methods, and only Photorhabdus luminescens was found on leaf surface using both techniques. All the Biolog isolates from phyllosphere were from the Proteobacteria phylum compared to the culture-independent bands from the leaves that were from different bacterial phyla. Based on our data, methyl bromide (MeBr) and methyl iodide (MeI) did not have any significant negative effects on rhizosphere and phyllosphere microbial communities throughout the growing period of lettuce.

Influence of fumigants on soil microbial diversity and survival of E. coli O157:H7.

The aim of this study was to assess the effects of soil fumigation with methyl bromide (MeBr; CH(3)Br) and methyl iodide (MeI, iodomethane; CH(3)I) on the microbial community structure and diversity in two soils and determine the effects of microbial diversity on the survival of Escherichia coli O157:H7 from contaminated irrigation water. Polymerase chain reaction (PCR) was used to amplify 16S rRNA from total bacterial community composition and the products were subjected to denaturing gradient gel electrophoresis (DGGE). The Shannon-Weaver index of diversity (H') was used to determine the effects of both fumigants on soil microbial diversity. The effect was more severe in sandy soil than in clay soil at the normal application rate of MeBr and MeI. Our results showed that MeBr and MeI have about the same effects on soil microbial diversity. The two fumigants had greater impact on microbial diversity in sandy soil than in clay soil and this resulted in higher survival of E. coli O157:H7 in sandy soil than in clay soil during the 50 days that the study was conducted. MeBr has been used as soil fumigant for >40 years with no serious detrimental effects on agricultural production and our research also suggests that the use of MeI may also produce no long-term detrimental effects on agricultural production since both fumigants had about the same effects on soil microbial communities. Therefore, soil systems with reduced microbial diversity may offer greater opportunities for the survival of pathogenic bacteria such as E. coli O157:H7.

Impact of treated wastewater for irrigation on soil microbial communities.

The use of treated wastewater (TWW) for irrigation has been suggested as an alternative to use of fresh water because of the increasing scarcity of fresh water in arid and semiarid regions of the world. However, significant barriers exist to widespread adoption due to some potential contaminants that may have adverse effects on soil quality and or public health. In this study, we investigated the abundance and diversity of bacterial communities and the presence of potential pathogenic bacterial sequences in TWW in comparison to synthetic fresh water (SFW) using pyrosequencing. The results were analyzed using UniFrac coupled with principal coordinate analysis (PCoA) to compare diversity and abundance of different bacterial groups in TWW irrigated soils to soils treated with SFW. Shannon diversity index values (H') suggest that microbial diversity was not significantly different (P<0.086) between soils with TWW and SFW. Pyrosequencing detected sequences of 17 bacterial phyla with Proteobacteria (32.1%) followed by Firmicutes (26.5%) and Actinobacteria (14.3%). Most of the sequences associated with nitrifying bacteria, nitrogen-fixing bacteria, carbon degraders, denitrifying bacteria, potential pathogens, and fecal indicator bacteria were more abundant in TWW than in SFW. Therefore, TWW effluent may contain bacterial that may be very active in many soil functions as well as some potential pathogens.

Bacterial community dynamics in surface flow constructed wetlands for the treatment of swine waste.

Constructed wetlands are generally used for the removal of waste from contaminated water. In the swine production system, wastes are traditionally flushed into an anaerobic lagoon which is then sprayed on agricultural fields. However, continuous spraying of lagoon wastewater on fields can lead to high N and P accumulations in soil or lead to runoff which may contaminate surface or ground water with pathogens and nutrients. In this study, continuous marsh constructed wetland was used for the removal of contaminants from swine waste. Using pyrosequencing, we assessed bacterial composition within the wetland using principal coordinate analysis (PCoA) which showed that bacterial composition from manure influent and lagoon water were significantly different (P=0.001) from the storage pond to the final effluent. Canonical correspondence analysis (CCA) showed that different bacterial populations were significantly impacted by ammonium--NH4 (P=0.035), phosphate--PO4(3-) (P=0.010), chemical oxygen demand--COD (P=0.0165), total solids--TS (P=0.030), and dissolved solids--DS (P=0.030) removal, with 54% of the removal rate explained by NH4+PO4(3-) according to a partial CCA. Our results showed that different bacterial groups were responsible for the composition of different wetland nutrients and decomposition process. This may be the major reason why most wetlands are very efficient in waste decomposition.

Potential pathogens, antimicrobial patterns and genotypic diversity of Escherichia coli isolates in constructed wetlands treating swine wastewater.

Escherichia coli populations originating from swine houses through constructed wetlands were analyzed for potential pathogens, antimicrobial susceptibility patterns, and genotypic diversity. Escherichia coli isolates (n = 493) were screened for the presence of the following virulence genes: stx1, stx2 and eae (Shiga toxin-producing E. coli [STEC]), heat-labile enterotoxin (LT) genes and heat stable toxin STa and STb (enterotoxigenic E. coli (ETEC), cytotoxin necrotizing factors 1 and 2 (cnf1 and cnf2 [necrotoxigenic E. coli- NTEC]), as well as O and H antigens, and the presence of the antibiotic resistance genes blaTEM, blaSHV, blaCMY-2, tet A, tet B, tet C, mph(A), aadA, StrA/B, sul1, sul2 and sul3. The commensal strains were further screened for 16 antimicrobials and characterized by BOX AIR-1 PCR for unique genotypes. The highest antibiotic resistance prevalence was for tetracycline, followed by erythromycin, ampicillin, streptomycin, sulfisoxazole and kanamycin. Our data showed that most of the isolates had high distribution of single or multidrug-resistant (MDR) genotypes. Therefore, the occurrence of MDR E. coli in the wetland is a matter of great concern due to possible transfer of resistance genes from nonpathogenic to pathogenic strains or vice versa in the environment.

Impact of plant density and microbial composition on water quality from a free water surface constructed wetland.

AIMS: To correlate microbial community composition and water quality changes within wetland cells containing varying plant densities and composition in a free water surface (FWS) constructed wetland. METHODS AND RESULTS: Water chemistry was monitored weekly for nitrate, orthophosphate, and suspended solids, at various sites throughout the wetland for 6 months. Treatment ponds with 50% plant cover had about a 96.3% nitrate removal. The average change between the influent and effluent was 50-60% nitrate removal and 40-50% orthophosphate removal. Community profile of total DNA, generated by using denaturing gradient gel electrophoresis (DGGE), was used to determine the major microbial composition associated with the wetland sediment, rhizosphere, and surface water. Bacterial cloned libraries were constructed, and 300 clones were analysed by amplified ribosomal DNA restriction analysis (ARDRA) and grouped into operational taxonomic units (OTUs). A total of 35, 31, and 36 different OTU were obtained from sediment, rhizosphere, and surface water, respectively. The bacterial members within the dominant group of our clone library belonged to unclassified taxa, while the second predominant group consisted of members of the phylum Proteobacteria. The dominant organisms within the class were in the gamma, beta, and delta classes. CONCLUSION: Microbial diversity as determined by Shannon-Weaver index (H) was higher in the wetland cells with 50% plant density than the 100%. This was in agreement with the most efficient wetland contaminant removal units. SIGNIFICANCE AND IMPACT OF THE STUDY: This study provides evidence that wetlands with 50% plant cover may promote the growth of diverse microbial communities that facilitate decomposition of chemical pollutants in surface water, and improve water quality.

Detection and quantification of Escherichia coli O157:H7 in environmental samples by real-time PCR.

AIMS: To apply the real-time Polymerase chain reaction (PCR) method to detect and quantify Escherichia coli O157:H7 in soil, manure, faeces and dairy waste washwater. METHODS AND RESULTS: Soil samples were spiked with E. coli O157:H7 and subjected to a single enrichment step prior to multiplex PCR. Other environmental samples suspected of harbouring E.coli O157:H7 were also analysed. The sensitivity of the primers was confirmed with DNA from E.coli O157:H7 strain 3081 spiked into soil by multiplex PCR assay. A linear relationship was measured between the fluorescence threshold cycle (C T ) value and colony counts (CFU ml(-1)) in spiked soil and other environmental samples. The detection limit for E.coli O157:H7 in the real-time PCR assay was 3.5 x 10(3) CFU ml(-1) in pure culture and 2.6 x 10(4) CFU g(-1) in the environmental samples. Use of a 16-h enrichment step for spiked samples enabled detection of <10 CFU g(-1) soil. E. coli colony counts as determined by the real-time PCR assay, were in the range of 2.0 x 10(2) to 6.0 x 10(5) CFU PCR (-1) in manure, faeces and waste washwater. CONCLUSIONS: The real-time PCR-based assay enabled sensitive and rapid quantification of E. coli O157:H7 in soil and other environmental samples. SIGNIFICANCE AND IMPACT OF THE STUDY: The ability to quantitatively determine cell counts of E.coli O157:H7 in large numbers of environmental samples, represents considerable advancement in the area of pathogen quantification for risk assessment and transport studies.

Changes in developing plant microbial community structure as affected by contaminated water.

The effects of sand and clay soils and water contaminated by Escherichia coli O157:H7 on the development of rhizosphere and phyllosphere microbial communities were analyzed to determine the influence of plant age on microbial community structure and composition. Community bacterial nucleic acids were extracted from lettuce rhizosphere and phyllosphere samples at different stages of plant development after the soils were irrigated with water contaminated with E. coli O157:H7 at planting and 15 days after planting. PCR was used to amplify 16S ribosomal RNA (rRNA) for total bacterial community composition and the products were subjected to denaturing gradient gel electrophoresis (DGGE). Prominent DGGE bands were excised and sequenced to gain insight into the identities of predominant bacterial populations. The majority of DGGE band sequences were related to bacterial genera previously associated with the rhizosphere and phyllosphere, such as Pseudomonas, Acidobacterium, Bacillus and Agrobacterium. The PCR-DGGE patterns observed for rhizosphere samples were more complex than those obtained from the bulk soil and the phyllosphere. The Shannon index of diversity (H) was used to determine the complexity of the DGGE bands from the phyllosphere, rhizosphere and the bulk soils at different growth stages. A higher diversity was observed in the clay soil than sandy soil during the first week. Few changes in diversity were observed after the first week. The results show that microbial community development in lettuce may take about 7-12 days and this may be the most likely period for maximum pathogen contamination in plants.

Microbial diversity along a transect of agronomic zones.

The diversity of microbial communities constitutes a critical component of good soil-management practices. To characterize the effects of different management practices, molecular indicators such as phospholipid fatty acid (PLFA), denaturing gradient gel electrophoresis (DGGE) and composition of ammonia-oxidizing bacteria were used to analyze bacterial community structure and diversity from four eastern Washington State soils. Samples from four sites were collected representing a transect of high-precipitation to low-precipitation areas that covered different agronomic zones with different management and cropping practices. Biomass amounts estimated from extractable PLFA were significantly higher in the no-till (NT) soil than in the conventional-till (CT) soil. Similarities among the different 16S rDNA DGGE band profiles were analyzed quantitatively using correspondence analysis and this confirmed that the CT soil was the most dissimilar soil. DGGE analysis of 16S rDNA ammonia-oxidizing bacteria from the four soils revealed two identical bands, indicating little effect of agronomic practices and precipitation on these species. A second set of primers, specific for amoA (ammonia monooxygenase) genes, was used to examine ammonia oxidizers in the samples. Six banding patterns (clusters) from amplified rDNA restriction analysis of 16S rDNA fragments were observed after restriction analysis with HinfI. Sequencing of these clones revealed the presence of only Nitrosospira-like sequences. Analysis of the sequences showed that ammonia oxidizers from the NT soil were more diverse compared to those from the CT and conservation reserve program soils. Our data showed that management and agronomic practices had more impact on bacterial community structure than annual precipitation.

Microcosm enrichment of 1,3-dichloropropene-degrading soil microbial communities in a compost-amended soil.

AIMS: A microcosm-enrichment approach was used to investigate bacterial populations that may represent 1,3-dichloropropene (1,3-D)-degrading micro-organisms in compost-amended soil. METHODS AND RESULTS: After 8 weeks of incubation, with repeated application of 1,3-D, volatilization fluxes were much lower for compost-amended soil (CM) than with the unamended soils, indicating accelerated degradation due to addition of compost, or development of new microbial populations with enhanced degradation capacity. Denaturing gradient gel electrophoresis (DGGE) profiles of the PCR-amplified region of 16S rDNA genes were used to identify dominant bacterial populations in the fumigant-degrading soil. The DGGE results indicated that specific bacterial types had been enriched, and a more diverse fingerprint was observed in the community derived from the compost-amended soil compared with the unamended soil. Fragments from 16 different DGGE bands were cloned, sequenced and compared with published 16S rDNA sequences. Two clones, designated E1 and E4, were unique to all soils to which compost was added, and corresponded to strains of Pseudomonas and Actinomadura, respectively. CONCLUSIONS: The results show that the addition of compost to soil increases specific microbial populations and results in the accelerated degradation of fumigants. SIGNIFICANCE AND IMPACT OF THE STUDY: Application of compost manure to soil can help degrade soil fumigants at a faster rate.

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.