Enrichment and molecular characterization of chloropicrin- and metam-sodium-degrading microbial communities

Chloropicrin (CP) and metam sodium are commonly used as fumigants in agricultural soils in order to provide effective control of nematodes, soil-borne pathogens, and weeds in preparation for planting of high-value cash crops. Repeated application of these compounds to agricultural soils for many years may result in the enrichment of microorganisms capable of degrading them. In this study, a microcosm-enrichment approach was used to investigate bacterial populations that may be components of metam-sodium- and CP-degrading microorganisms in compost-amended soils. After 6 months incubation, with repeated application of metam sodium and CP, degradation was 70% faster in compost-manure-amended (CM) soil compared to 50% in the unamended soils. The accelerated fumigant degradation may have been due to the addition of compost or to the development of new microbial populations with enhanced degradation capacity. Denaturing gradient gel electrophoresis (DGGE) profiles of PCR-amplified regions of 16S rRNA genes were used to identify dominant bacterial populations responsible for the accelerated fumigant degradation. The DGGE results indicated that specific bacterial types had been enriched and these were similar to strains isolated from basal minimal media. Fragments from DGGE bands and colonies were cloned, sequenced, and compared with published 16S rRNA sequences. Cloned sequences were dominated by Pseudomonas, Bacillus, Arthrobacter, Mycobacterium and uncultured bacterial species. The addition of organic amendment to soil during fumigation practices has the potential to increase the diversity of different microbial species, thereby accelerating fumigant degradation and reducing atmospheric emissions.     

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.     

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.     

Effect of phenylurea herbicides on soil microbial communities estimated by analysis of 16S rRNA gene fingerprints and community-level physiological profiles

The effect of three phenyl urea herbicides (diuron, linuron, and chlorotoluron) on soil microbial communities was studied by using soil samples with a 10-year history of treatment. Denaturing gradient gel electrophoresis (DGGE) was used for the analysis of 16S rRNA genes (16S rDNA). The degree of similarity between the 16S rDNA profiles of the communities was quantified by numerically analysing the DGGE band patterns. Similarity dendrograms showed that the microbial community structures of the herbicide-treated and nontreated soils were significantly different. Moreover, the bacterial diversity seemed to decrease in soils treated with urea herbicides, and sequence determination of several DGGE fragments showed that the most affected species in the soils treated with diuron and linuron belonged to an uncultivated bacterial group. As well as the 16S rDNA fingerprints, the substrate utilization patterns of the microbial communities were compared. Principal-component analysis performed on BIOLOG data showed that the functional abilities of the soil microbial communities were altered by the application of the herbicides. In addition, enrichment cultures of the different soils in medium with the urea herbicides as the sole carbon and nitrogen source showed that there was no difference between treated and nontreated soil in the rate of transformation of diuron and chlorotoluron but that there was a strong difference in the case of linuron. In the enrichment cultures with linuron-treated soil, linuron disappeared completely after 1 week whereas no significant transformation was observed in cultures inoculated with nontreated soil even after 4 weeks. In conclusion, this study showed that both the structure and metabolic potential of soil microbial communities were clearly affected by a long-term application of urea herbicides.     

Use of rpoB and 16S rRNA genes to analyse bacterial diversity of a tropical soil using PCR and DGGE

AIM: To evaluate the rpoB gene as a biomarker for PCR-DGGE microbial analyses using soil DNA from the Cerrado, Brazil. METHODS: DNA extraction from soil was followed by Polymerase Chain Reaction (PCR) amplification of rpoB and 16S rRNA genes. PCR products were compared by Denaturing Gradient Gel Electrophoresis (DGGE) to compare gene/community profiles. RESULTS: The rpoB DGGE profiles comprised fewer bands than the 16S rDNA profiles and were easier to delineate and therefore to analyse. Comparison of the community profiles revealed that the methods were complementary. CONCLUSIONS, SIGNIFICANCE AND IMPACT OF THE STUDY: The gene for the beta subunit of the RNA polymerase, rpoB, is a single copy gene unlike 16S rDNA. Multiple copies of 16S rRNA genes in bacterial genomes complicate diversity assessments made from DGGE profiles. Using the rpoB gene offers a better alternative to the commonly used 16S rRNA gene for microbial community analyses based on DGGE.     

PCR-DGGE技术在农田土壤微生物多样性研究中的应用

变性梯度凝胶电泳技术(DGGE)在微生物生态学领域有着广泛的应用。研究采用化学裂解法直接提取出不同农田土壤微生物基因组DNA,并以此基因组DNA为模板,选择特异性引物F357GC和R515对16S rRNA基因的V3区进行扩增,长约230bp的PCR产物经变性梯度凝胶电泳(DGGE)进行分离后,得到不同数目且分离效果较好的电泳条带。结果说明,DGGE能够对土壤样品中的不同微生物的16S rRNA基因的V3区的DNA扩增片断进行分离,为这些DNA片断的定性和鉴定提供了条件。与传统的平板培养方法相比,变性梯度凝胶电泳(DGGE)技术能够更精确的反映出土壤微生物多样性,它是一种有效的微生物多样性研究技术。     

Effects of community-accessible biochar and compost on diesel-contaminated soil

Petroleum pollution is a global problem that requires effective and accessible remediation strategies that takes ecosystem functioning into serious consideration. Bioremediation can be an effective tool to address the challenge. In this study, we used a mesocosm experiment to evaluate the effects of locally sourced and community produced biochar and compost amendments on diesel-contaminated soil. At the end of the 90-day experiment, we quantified the effects of the amendments on total petroleum hydrocarbons (C9-C40) (TPH) and soil pH, organic matter, aggregate stability, soil respiration, extractable phosphorus, extractable potassium, and micronutrients (Mg, Fe, Mn, and Zn). We observed significantly higher TPH degradation in compost-amended soils than in controls and soils amended with biochar. We propose that the addition of compost improved TPH biodegradation by augmenting soil nutrient content and microbial activity. Our results suggest that community-accessible compost can improve TPH biodegradation, and that implementation is possible at the community level.     

Lime and compost promote plant re-colonization of metal-polluted, acidic soils

The revegetation of soils affected by historic depositions of an industrial complex in Central Chile was studied. The plant re-colonization from the existing soil seed bank and changes in the physico-chemical properties of the soil were evaluated in field plots amended with lime and/or compost. We found that the application of lime and/or compost decreased the Cu2+ ion activity in the soil solution and the exchangeable Cu in the soil, showing an effective Cu immobilization in the topsoil. Whereas lime application had no effect on plant productivity in comparison with the unamended control, the application of compost and lime+compost increased the plant cover and aboveground biomass due to the higher nutrient availability and water-holding capacity of the compost-amended soils. Although the Cu2+ activity and the exchangeable Cu were markedly lower in the amended soils than in the unamended control, the shoot Cu concentrations of Lolium spp. and Eschscholzia californica did not differ between the treatments.     

Effect of various amendments on heavy mineral oil bioremediation and soil microbial activity

To examine the effects of amendments on the degradation of heavy mineral oil, we conducted a pilot-scale experiment in the field for 105 days. During the experiment, soil samples were collected and analyzed periodically to determine the amount of residual hydrocarbons and evaluate the effects of the amendments on microbial activity. After 105 days, the initial level of contamination (7490+/-480 mg hydrocarbon kg(-1) soil) was reduced by 18-40% in amended soils, whereas it was only reduced by 9% in nonamended soil. Heavy mineral oil degradation was much faster and more complete in compost-amended soil than in hay-, sawdust-, and mineral nutrient-amended soils. The enhanced degradation of heavy mineral oil in compost-amended soil may be a result of the significantly higher microbial activity in this soil. Among the studied microbial parameters, soil dehydrogenase, lipase, and urease activities were strongly and negatively correlated with heavy mineral oil biodegradation (P<0.01) in compost-amended soil.     

The effect of long-term mercury pollution on the soil microbial community

The effect of long-term exposure to mercury on the soil microbial community was investigated in soil from three different sites along a pollution gradient. The amount of total and bioavailable mercury was negatively correlated to the distance from the center of contamination. The size of the bacterial and protozoan populations was reduced in the most contaminated soil, whereas there was no significant difference in fungal biomass measured as chitinase activity. Based on the number of colony morphotypes, moreover, the culturable bacterial population was structurally less diverse and contained a higher proportion of resistant and fast-growing forms. The profiles of amplified 16S rDNA sequences obtained from community DNA by denaturating gradient gel electrophoresis (DGGE) also reflected the altered community structure and decreased diversity along the mercury gradient as expressed in terms of the number and abundance of bands. The functional potential of the microbial population measured as sole carbon source utilization by Ecoplates((R)) differed between the soils, but there was no change in the number of substrates utilized. The observed changes in the different soil microbial populations are probably a combination of both direct and indirect effects of the mercury contamination.     

Effect of monoculture soybean on soil microbial community in the Northeast China

Monoculture (MC) soybean, a common practice in the Northeast China, causes significant declines in soybean yield and quality. The objective of this study was to evaluate the responses of the soil microbial community and soybean yield to different soybean cropping systems. Three cropping systems were compared, (1) corn-soybean rotation (corn-corn-soybean, CS), (2) MC soybean for 3 years (S3), (3) MC soybean for 9 years (S9). Both bulk and rhizosphere soil samples were collected at three growth stages: two trifoliate (V2), full bloom (R2), and full seed (R6), respectively. Soil microbial DNA was analyzed using polymerase chain reaction (PCR)—denaturing gradient gel electrophoresis (DGGE) to assess changes in composition of bacterial and fungal communities. Prominent DGGE bands were excised and sequenced to gain insight into the identities of the predominant microbial populations. Some prominent differences were observed in bacterial DGGE patterns of amplified 16S rDNA (V3 region) among rhizosphere soils. These major differences included one DGGE band (showing 100% similarity to Arthrobacter sp.) that was enriched at R2 stages in CS and S9, and another band with 97% sequence similarity to an uncultured actinobacterium was detected at R6 stage in CS, and at R2 and R6 stages in S9. The bacterial community from bulk soil showed no significant band change in DGGE patterns among different cropping systems. In fungal DGGE patterns of the amplified 18S rDNA partial fragment, one specific band (showing 98% similarity to Trichoderma viride) occurred in rhizosphere soil of treatment CS at V2 and R6 stages and treatment S9 at R6 stage. None of the above bands were detected in treatment S3. The soybean yields and plant heights from CS and S9 were greater than those from S3. Moreover, catalase activities from CS and S9 at V2 and R2 stages were higher than those tested from S3 at the corresponding times in rhizosphere soil. The present results showed that DGGE patterns were not able to detect significant differences in diversity or evenness among microbial communities, but significant differences were found in the composition of bacterial and fungal community structures. Some distinguished bands from bacterial and fungal DGGE patterns were only enriched in CS and S9 soil, which could potentially play an important role in soybean growth development.     

Effect of dissemination of 2,4-dichlorophenoxyacetic acid (2,4-D) degradation plasmids on 2,4-D degradation and on bacterial community structure in two different soil horizons

Transfer of the 2,4-dichlorophenoxyacetic acid (2,4-D) degradation plasmids pEMT1 and pJP4 from an introduced donor strain, Pseudomonas putida UWC3, to the indigenous bacteria of two different horizons (A horizon, depth of 0 to 30 cm; B horizon, depth of 30 to 60 cm) of a 2,4-D-contaminated soil was investigated as a means of bioaugmentation. When the soil was amended with nutrients, plasmid transfer and enhanced degradation of 2,4-D were observed. These findings were most striking in the B horizon, where the indigenous bacteria were unable to degrade any of the 2,4-D (100 mg/kg of soil) during at least 22 days but where inoculation with either of the two plasmid donors resulted in complete 2,4-D degradation within 14 days. In contrast, in soils not amended with nutrients, inoculation of donors in the A horizon and subsequent formation of transconjugants (10(5) CFU/g of soil) could not increase the 2,4-D degradation rate compared to that of the noninoculated soil. However, donor inoculation in the nonamended B-horizon soil resulted in complete degradation of 2,4-D within 19 days, while no degradation at all was observed in noninoculated soil during 89 days. With plasmid pEMT1, this enhanced degradation seemed to be due only to transconjugants (10(5) CFU/g of soil), since the donor was already undetectable when degradation started. Denaturing gradient gel electrophoresis (DGGE) of 16S rRNA genes showed that inoculation of the donors was followed by a shift in the microbial community structure of the nonamended B-horizon soils. The new 16S rRNA gene fragments in the DGGE profile corresponded with the 16S rRNA genes of 2,4-D-degrading transconjugant colonies isolated on agar plates. This result indicates that the observed change in the community was due to proliferation of transconjugants formed in soil. Overall, this work clearly demonstrates that bioaugmentation can constitute an effective strategy for cleanup of soils which are poor in nutrients and microbial activity, such as those of the B horizon.     

Physiological and molecular characterization of a microbial community established in unsaturated, petroleum-contaminated soil

The microbial communities established in soil samples from an unsaturated, petroleum-contaminated zone and from an adjacent uncontaminated site were characterized by physiological and molecular approaches. Possible electron acceptors such as sulfate and nitrate had been completely depleted in these soil samples. Slurries of these soil samples were incubated in bottles in the presence of hydrocarbon indicators (benzene, toluene, xylene and decane), and the degradation of these compounds was examined. Supplementation with electron acceptors stimulated hydrocarbon degradation, although the stimulatory effect was small in the contaminated soil. The initial degradation rates in the contaminated soil under fermentative/methanogenic conditions were comparable to those under aerobic conditions. The microbial populations in the original soil samples were analysed by cloning and sequencing of polymerase chain reaction (PCR)-amplified bacterial and archaeal 16S rRNA gene fragments, showing that the sequences retrieved from these soils were substantially different. For instance, Epsilonproteobacteria, Gammaproteobacteria, Crenarchaeota and Methanosarcinales could only be detected at significant levels in the contaminated soil. Denaturing gradient gel electrophoresis (DGGE) analyses of 16S rRNA gene fragments amplified by PCR from the incubated soil-slurry samples showed that supplementation of the electron acceptors resulted in a shift in the major populations, while the DGGE profiles after incubating the contaminated soil under the fermentative/methanogenic conditions were not substantially changed. These results suggest that petroleum contamination of the unsaturated zone resulted in the establishment of a fermentative/methanogenic community with substantial hydrocarbon-degrading potential.     

Bulk and rhizosphere soil bacterial communities studied by denaturing gradient gel electrophoresis: plant-dependent enrichment and seasonal shifts revealed

The bacterial rhizosphere communities of three host plants of the pathogenic fungus Verticillium dahliae, field-grown strawberry (Fragaria ananassa Duch.), oilseed rape (Brassica napus L.), and potato (Solanum tuberosum L.), were analyzed. We aimed to determine the degree to which the rhizosphere effect is plant dependent and whether this effect would be increased by growing the same crops in two consecutive years. Rhizosphere or soil samples were taken five times over the vegetation periods. To allow a cultivation-independent analysis, total community DNA was extracted from the microbial pellet recovered from root or soil samples. 16S rDNA fragments amplified by PCR from soil or rhizosphere bacterium DNA were analyzed by denaturing gradient gel electrophoresis (DGGE). The DGGE fingerprints showed plant-dependent shifts in the relative abundance of bacterial populations in the rhizosphere which became more pronounced in the second year. DGGE patterns of oilseed rape and potato rhizosphere communities were more similar to each other than to the strawberry patterns. In both years seasonal shifts in the abundance and composition of the bacterial rhizosphere populations were observed. Independent of the plant species, the patterns of the first sampling times for both years were characterized by the absence of some of the bands which became dominant at the following sampling times. Bacillus megaterium and Arthrobacter sp. were found as predominant populations in bulk soils. Sequencing of dominant bands excised from the rhizosphere patterns revealed that 6 out of 10 bands resembled gram-positive bacteria. Nocardia populations were identified as strawberry-specific bands.     

Molecular assessment on impact of uranium ore contamination in soil bacterial diversity

Impact of uranium (U) ore and soluble uranium (at pH 4.0) contamination on agricultural soil bacterial diversity was assessed by using laboratory microcosms for one year. Diversity and abundance of metabolically active bacterial populations in periodically collected microcosm’s samples were analyzed by extracting total RNA and preparation of cDNA followed by analysis of 16S rRNA gene by DGGE and real time PCR. DGGE analysis revealed prominent shift of soil bacterial population due to uranium ore contamination within 12 months while uranium ore along with soluble U completely destroyed the soil bacterial diversity within first six months. Real time PCR based analysis indicated 100–200 folds increase in 16S rRNA gene copies of total as well as individual bacterial taxa in both U ore amended and unamended soils in first six months while increase in incubation period upto 12 months showed reduction of the same only in U ore amended soil. Antagonistic effect of U ore contamination on soil bacterial diversity indicated the severe impact of U mining likely to have on nearby ecosystems. Role of U at acidic pH in destroying the diversity completely is noteworthy as it corroborated the disastrous consequence of acid mine drainage generated from U mine sites.     

The microbial degradation of azimsulfuron and its effect on the soil bacterial community

AIMS: Azimsulfuron is a recently introduced sulfonylurea herbicide useful in controlling weeds in paddy fields. To date very little information is available on the biodegradation of this pesticide and on its effect on the soil microbial community. The aim of this work was to study its biodegradation both in slurry soil microcosms and in batch tests with mixed and pure cultures. METHODS AND RESULTS: Azimsulfuron was applied to forest bulk soil in order to study its effect on the structure of the bacterial soil community, as detectable by denaturant gradient gel electrophoresis (DGGE) analyses. Biodegradation and abiotic processes were investigated by HPLC analyses. In addition, a microbial consortium was selected, that was able to use azimsulfuron as the sole energy and carbon source. One of the metabolites produced by the consortium was isolated and identified through LC-MS analyses. Cultivable bacteria of the consortium were isolated and identified by 16S rDNA sequencing (1400 bp). CONCLUSIONS: Azimsulfuron treatment seems to have the ability to cause changes in the bacterial community structure that are detectable by DGGE analyses. It is easily biodegraded both in microcosms and in batch tests, with the formation of an intermediate that was identified as 2-methyl-4-(2-methyl-2H-tetrazol-5-yl)-2H-pyrazole-3-sulfonamide. SIGNIFICANCE AND IMPACT OF THE STUDY: The study increases the knowledge on the biodegradation of azimsulfuron and its effects on the soil microbiota.     

Effect of Medicago sativa L. and compost on organic and inorganic pollutant removal from a mixed contaminated soil and risk assessment using ecotoxicological tests

Several Gentle Remediation Options (GRO), e.g., plant-based options (phytoremediation), singly and combined with soil amendments, can be simultaneously efficient for degrading organic pollutants and either stabilizing or extracting trace elements (TEs). Here, a 5-month greenhouse trial was performed to test the efficiency of Medicago sativa L., singly and combined with a compost addition (30% w/w), to treat soils contaminated by petroleum hydrocarbons (PHC), Co and Pb collected at an auto scrap yard. After 5 months, total soil Pb significantly decreased in the compost-amended soil planted with M. sativa, but not total soil Co. Compost incorporation into the soil promoted PHC degradation, M. sativa growth and survival, and shoot Pb concentrations [3.8 mg kg^?1 dry weight (DW)]. Residual risk assessment after the phytoremediation trial showed a positive effect of compost amendment on plant growth and earthworm development. The O₂ uptake by soil microorganisms was lower in the compost-amended soil, suggesting a decrease in microbial activity. This study underlined the benefits of the phytoremediation option based on M. sativa cultivation and compost amendment for remediating PHC- and Pb-contaminated soils.     

Soil microbial community structure across a thermal gradient following a geothermal heating event

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 degrees 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 degrees 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 degrees 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.     

Effects of soil pH on the biodegradation of chlorpyrifos and isolation of a chlorpyrifos-degrading bacterium

We examined the role of microorganisms in the degradation of the organophosphate insecticide chlorpyrifos in soils from the United Kingdom and Australia. The kinetics of degradation in five United Kingdom soils varying in pH from 4.7 to 8.4 suggested that dissipation of chlorpyrifos was mediated by the cometabolic activities of the soil microorganisms. Repeated application of chlorpyrifos to these soils did not result in the development of a microbial population with an enhanced ability to degrade the pesticide. A robust bacterial population that utilized chlorpyrifos as a source of carbon was detected in an Australian soil. The enhanced ability to degrade chlorpyrifos in the Australian soil was successfully transferred to the five United Kingdom soils. Only soils with a pH of >/=6.7 were able to maintain this degrading ability 90 days after inoculation. Transfer and proliferation of degrading microorganisms from the Australian soil to the United Kingdom soils was monitored by molecular fingerprinting of bacterial 16S rRNA genes by PCR-denaturing gradient gel electrophoresis (DGGE). Two bands were found to be associated with enhanced degradation of chlorpyrifos. Band 1 had sequence similarity to enterics and their relatives, while band 2 had sequence similarity to strains of Pseudomonas. Liquid enrichment culture using the Australian soil as the source of the inoculum led to the isolation of a chlorpyrifos-degrading bacterium. This strain had a 16S rRNA gene with a sequence identical to that of band 1 in the DGGE profile of the Australian soil. DNA probing indicated that genes similar to known organophosphate-degrading (opd) genes were present in the United Kingdom soils. However, no DNA hybridization signal was detected for the Australian soil or the isolated degrader. This indicates that unrelated genes were present in both the Australian soil and the chlorpyrifos-degrading isolate. These results are consistent with our observations that degradation of chlorpyrifos in these systems was unusual, as it was growth linked and involved complete mineralization. As the 16S rRNA gene of the isolate matched a visible DGGE band from the Australian soil, the isolate is likely to be both prominent and involved in the degradation of chlorpyrifos in this soil.     

Despite Long-Term Compost Amendment Seasonal Changes are Main Drivers of Soil Fungal and Bacterial Population Dynamics in a Tuscan Vineyard

The effect of long-term (8 years) compost treatments (compost or compost plus mineral fertilizer) on genetic structure of bacterial and fungal populations in both bulk soil and rhizosphere of grapevine (Vitis vinifera) was analyzed in respect to a control constituted by the soil treated with mineral fertilization. Soils were sampled in early summer (July), mid-summer (August), and before harvest (October). Bacterial and fungal populations were characterized by genetic fingerprints generated by the application of 16S rDNA and ITS rDNA Multiplex Terminal Fragment Length Polymorphism (M-TRFLP) technique. Compost induced no significant differences at any time on microbial communities from bulk soil samples, whereas seasonal variations significantly affected both bacterial and fungal populations as indicated by the Multi Dimensional Scaling (MDS) ordination method of the M-TRFLPs results. MDS analysis of grapevine rhizosphere M-TRFLPs showed that temporal separation was significant for the bacterial population only. Results suggested that soil microbial populations in vineyard productive ecosystems may be sensitive to environmental changes induced by seasonal variations and show a certain degree of resilience to different agricultural practices.