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.

Surface water and groundwater are continuously used as sources of drinking water in many metropolitan areas of the United States. The quality of water from these sources may be reduced due to increases in contaminants such asEscherichia colifrom urban and agricultural runoffs. In this study, a multiplex fluorogenic PCR assay was used to quantifyE. coliO157:H7 in soil, manure, cow and calf feces, and dairy wastewater in an artificial wetland. Primers and probes were designed to amplify and quantify the Shiga-like toxin 1 (stx1) and 2 (stx2) genes and the intimin (eae)gene ofE. coliO157:H7 in a single reaction. Primer specificity was confirmed with DNA from 33E. coliO157:H7 and related strains with and without the three genes. A direct correlation was determined between the fluorescence threshold cycle (C_T) and the starting quantity ofE. coliO157:H7 DNA. A similar correlation was observed between theC_Tand number of CFU per milliliter used in the PCR assay. A detection limit of 7.9 × 10⁻⁵pg ofE. coliO157:H7 DNA ml⁻¹equivalent to approximately 6.4 × 10³CFU ofE. coliO157:H7 ml⁻¹based on plate counts was determined. Quantification ofE. coliO157:H7 in soil, manure, feces, and wastewater was possible when cell numbers were ≥3.5 × 10⁴CFU g⁻¹.E. coliO157:H7 levels detected in wetland samples decreased by about 2 logs between wetland influents and effluents. The detection limit of the assay in soil was improved to less than 10 CFU g⁻¹with a 16-h enrichment. These results indicate that the developed PCR assay is suitable for quantitative determination ofE. coliO157:H7 in environmental samples and represents a considerable advancement in pathogen quantification in different ecosystems.

Constructed wetlands have been recognized as a removal treatment option for high concentrations of contaminants in agricultural waste before land application. The goal of this study was to characterize microbial composition in two constructed wetlands designed to remove contaminants from dairy washwater. Water samples were collected weekly for 11 months from two wetlands to determine the efficiency of the treatment system in removal of chemical contaminants and total and fecal coliforms. The reduction by the treatment was greatest for biological oxygen demand, suspended solids, chemical oxygen demand, nitrate, and coliforms. There was only moderate removal of total nitrogen and phosphorus. Changes in the total bacterial community and ammonia-oxidizing bacterial composition were examined by using denaturing gradient gel electrophoresis (DGGE) and sequencing of PCR-amplified fragments of the gene carrying the α subunit of the ammonia monooxygenase gene ( amoA ) recovered from soil samples and DGGE bands. DGGE analysis of wetlands and manure samples revealed that the total bacterial community composition was dominated by bacteria from phylogenetic clusters related to Bacillus , Clostridium , Mycoplasma , Eubacterium , and Proteobacteria originally retrieved from the gastrointestinal tracts of mammals. The population of ammonia-oxidizing bacteria showed a higher percentage of Nitrosospira -like sequences from the wetland samples, while a higher percentage of Nitrosomonas -like sequences from manure, feces, raw washwater, and facultative pond was found. These results show that the wetland system is a natural process dependent upon the development of healthy microbial communities for optimal wastewater treatment.

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 sample...