Catherine M. Grieve

Multiplex Fluorogenic Real-Time PCR for Detection and Quantification of Escherichia coli O157:H7 in Dairy Wastewater Wetlands

ABSTRACT 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 as Escherichia coli from urban and agricultural runoffs. In this study, a multiplex fluorogenic PCR assay was used to quantify E. coli O157: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 of E. coli O157:H7 in a single reaction. Primer specificity was confirmed with DNA from 33 E. coli O157:H7 and related strains with and without the three genes. A direct correlation was determined between the fluorescence threshold cycle (CT) and the starting quantity of E. coli O157:H7 DNA. A similar correlation was observed between the CT and number of CFU per milliliter used in the PCR assay. A detection limit of 7.9 × 10−5 pg of E. coli O157:H7 DNA ml−1 equivalent to approximately 6.4 × 103 CFU of E. coli O157:H7 ml−1 based on plate counts was determined. Quantification of E. coli O157:H7 in soil, manure, feces, and wastewater was possible when cell numbers were ≥3.5 × 104 CFU g−1. E. coli O157: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−1 with a 16-h enrichment. These results indicate that the developed PCR assay is suitable for quantitative determination of E. coli O157:H7 in environmental samples and represents a considerable advancement in pathogen quantification in different ecosystems.

Characterization of Microbial Communities and Composition in Constructed Dairy Wetland Wastewater Effluent

ABSTRACT 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.

Development of Salinity‐Tolerant Wheat Recombinant Lines from a Wheat Disomic Addition Line Carrying a Thinopyrum junceum Chromosome

Three Triticum aestivum L. × Thinopyrum junceum (L.) A. Löve partial amphidiploids (2n=8x=56; 21″ ABD + 7″ Eb/Ee) and 11 derived disomic addition lines (2n=44) were screened for salt tolerance in hydroponic solutions. One addition line (AJDAj5, 21″ ABD + 1″ Eb) had salt tolerance comparable to that in partial amphidiploids. It was crossed to a wheat line having the PhI allele from Aegilops speltoides Tausch to induce homoeologous pairing. F2 plants were subjected to salt screening and advanced to 30 F3 families, which were screened again. Four F3 lines were more tolerant than AJDAj5 when screened in a final electrical conductivity of 42 dS/m. Because one of the four lines was sterile, only three lines were further verified for their salinity tolerance and were cytologically and molecularly analyzed. These lines were translocation lines with 42 chromosomes having tiny fluorescent hybridization signals detected at interstitial positions of less condensed chromosomes using the genomic in situ hybridization technique. Amplified fragment length polymorphism analyses revealed the presence of very few (ca. 4%) putative markers specific to the Eb‐chromosome addition line. These lines also had from 2% to 14% of markers specific to the Ph inhibitor line and a few new AFLP markers that were not found in the two parental lines and the common wheat background, cv. Chinese Spring. Two recombinant lines were more salt tolerant than either parent, while the third one was as tolerant as either parent, which was more tolerant than Chinese Spring. The former two lines are valuable germplasm for breeding salt‐tolerant wheat cultivars.

Salinity Effects on Emergence, Survival, and Ion Accumulation of Limonium perezii

Abstract Saline wastewaters may provide a valuable water source for the irrigation of selected salt-tolerant floriculture crops as water quality and quantity becomes limited and as demand for quality water increases. A 2 × 7 factorial design with three replications was used to test the effects of water ionic composition and salinity, respectively, on emergence, survival, and mineral accumulation of the salt-tolerant cut flower Limonium perezii (Stapf) F. T. Hubb cv. ‘Blue Seas.’ Seeds (n = 100) were sown in each of 42 presalinized greenhouse sand tanks. Irrigation water composition represented saline drainage waters typically present in either the San Joaquin Valley (SJV) or the Imperial/Coachella Valleys (ICV) of California. Electrical conductivity levels of the treatment waters were 2.5 (control), 6, 8, 10, 12, 16, and 20 dS m− 1, respectively. Seedling emergence was monitored daily and leaf mineral concentrations (total sulfur (S), total phosphorus (P), Ca2 +, Mg2 +, Na+, K+, and Cl−) were determined three months after planting. A two-way ANOVA revealed that salinity had a significant effect on seedling emergence (F = 27.56; P < 0.01). Cumulative emergence showed a marked decrease above 12 dS m− 1 for both water treatments and tended to be greater from 6–10 dS m− 1 than at 2.5 dS m− 1. A significant interaction of salinity and water composition was found for survival (F = 2.90; P < 0.05). Survival approximated 90% in ICV and SJV treatments up to 8 and 10 dS m− 1, respectively, yet continued to decrease below 70% in both water treatments at 12 dS m− 1 and higher. Differences in leaf-mineral concentrations between ICV and SJV water composition can be attributed to differences in composition of irrigation waters. As salinity increased, leaf Ca2 +, K+, and total P decreased, whereas Mg2 +, Na2 +, Cl−, and total S increased. Stem length showed a marked decrease above 2.5 and 6 dS m− 1 under ICV and SJV treatments, respectively, with SJV waters producing ∼ 30% more marketable flowers at salinities up to 6 dS m− 1. Thus, Limonium perezii may be produced commercially with moderately saline wastewaters.

Survival of Escherichia coli O157:H7 in soil and on lettuce after soil fumigation

Long-term survival of Escherichia coli O157:H7 in soil and in the rhizosphere of many crops after fumigation is relatively unknown. One of the critical concerns with food safety is the transfer of pathogens from contaminated soil to the edible portion of the plants. Multiplex fluorogenic polymerase chain reaction was used in conjunction with plate counts to quantify the survival of E. coli O157:H7 in soil after fumigation with methyl bromide and methyl iodide in growth chamber and microcosm laboratory experiments. Plants were grown at 20 degrees C in growth chambers during the first experiment and soils were irrigated with water contaminated with E. coli O157:H7. For the second experiment, soil microcosms were used in the laboratory without plants and were inoculated with E. coli O157:H7 and spiked with the two fumigants. Primers and probes were designed to amplify and quantify the Shiga-like toxin 1 (stx1) and 2 (stx2) genes and the intimin (eae) gene of E. coli O157:H7. Both fumigants were effective in reducing pathogen concentrations in soil, and when fumigated soils were compared with nonfumigated soils, pathogen concentrations were significantly higher in the nonfumigated soils throughout the study. This resulted in a longer survival of the pathogen on the leaf surface especially in sandy soil than observed in fumigated soils. Therefore, application of fumigant may play some roles in reducing the transfer of E. coli O157:H7 from soil to leaf. Regression models showed that survival of the pathogen in the growth chamber study followed a linear model while that of the microcosm followed a curvilinear model, suggesting long-term survival of the pathogen in soil. Both experiments showed that E. coli O157:H7 can survive in the environment for a long period of time, even under harsh conditions, and the pathogen can survive in soil for more than 90 days. This provides a very significant pathway for pathogen recontamination in the environment.

Mineral nutrition of leafy vegetable crops irrigated with saline drainage water.

ABSTRACT Nine leafy vegetable crops were grown in outdoor sand plots to determine the effects of salinity and the timing of salt stress on leaf-ion concentration. Vegetable species were: radicchio (Cichorium intybus L.), curly endive (C. endivia L.), pac choi (Brassica rapa L., chinensis group), tatsoi (B. rapa L., narinosa group), kale (B. oleracea, acephala group), cooking greens (B. rapa L.), mustard greens (B. juncea (L.) Czerniak), spinach (Spinacia oleracea L.), and Swiss chard (Beta vulgaris L.). All species were planted at the same time and irrigated with a complete nutrient solution. Three weeks after planting, six saline treatments were imposed on half of the plants; the remaining plants were salinized four weeks later. Saline solution compositions were prepared to simulate the high-sodium, high-sulfate drainage waters typically found in the San Joaquin Valley of California. Electrical conductivities of the irrigation waters (ECi) were 3 (control), 7, 11, 15, 19, and 23 dS.m−1. Mineral ion concentrations in leaves were significantly affected by increasing salinity, but not by the stage of growth when salinity was applied. With increasing salinity, Ca2+ and K+ decreased in the leaves of all species, whereas Na+ and total-S significantly increased. Magnesium in leaves of the composites and the crucifers also increased with salinity, but treatment had no effect on Mg2+ concentration in the chenopods. Increases in salinity caused significant increases in Cl− in leaves of the crucifers and spinach but had no influence on the Cl− relations in Swiss chard and the composites. The use of moderately saline irrigation waters for the production of these leafy vegetable crops did not adversely affect crop quality as rated by color, texture and the mineral nutrient content available to consumers.

Quantification of Persistence of Escherichia coli O157:H7 in Contrasting Soils

Persistence of Escherichia coli (E. coli) O157:H7 in the environment is a major concern to vegetable and fruit growers where farms and livestock production are in close proximity. The objectives were to determine the effects of preplant fumigation treatment on the survival of E. coli O157:H7 in two soils and the effects of indigenous bacterial populations on the survival of this pathogen. Real-time PCR and plate counts were used to quantify the survival of E. coli O157:H7 in two contrasting soils after fumigation with methyl bromide (MeBr) and methyl iodide (MeI). Ten days after fumigation, E. coli O157:H7 counts were significantly lower (P = .0001) in fumigated soils than in the non-fumigated. Direct comparison between MeBr and MeI within each soil indicated that these two fumigants showed similar impacts on E. coli O157:H7 survival. Microbial species diversity as determined by DGGE was significantly higher in clay soil than sandy soil and this resulted in higher initial decline in population in clay soil than in sandy soil. This study shows that if soil is contaminated with E. coli O157:H7, fumigation alone may not eliminate the pathogen, but may cause decrease in microbial diversity which may enhance the survival of the pathogen.

Effects of Salinity, Imazethapyr, and Chlorimuron Application on Soybean Growth and Yield

Abstract Soybean is an important crop worldwide. Soybean cultivars differ in their sensitivity to soil salinity and herbicide damage. In these experiments, we examined the impact of salinization and herbicide (imazethapyr and chlorimuron) application on the growth and yield of two soybean cultivars, Essex and Manokin. Experiments were conducted in small pots in the greenhouse, in outdoor sand cultures, and in drip‐irrigated field plots. Plants were irrigated with non‐saline water or saline water (electrical conductivity, EC=7 dS/m) with a composition typical of those in areas affected by sulfate‐dominated salinity. Morphological changes resulted from herbicide application, including leaf elongation and formation of large shoots at the cotyledonary node. Herbicide treatment significantly reduced main stem height, number of nodes on the main stem, and stem diameter relative to the controls (not treated with herbicide); responses from the two herbicides were not significantly different. Salinity had a significant effect on seed weight: yield for “Essex” (a Cl accumulator) was significantly higher when irrigated with fresh water, while yield for “Manokin” (a Cl excluder) was significantly larger for plants irrigated with saline water. Yield for “Essex” was greater than that for “Manokin” when the plants were irrigated with fresh water; but at the higher irrigation water salinity (7 dS/m), “Manokin” produced significantly greater yield than “Essex.” Although herbicide application significantly impacted several growth variables, herbicide treatment had no significant impact on yield.

Salinity effects on growth, leaf-ion content and seed production of Lesquerella fendleri (Gray) S. Wats

Abstract Little information is available on the response of lesquerella grown under saline conditions. This field study was initiated to determine the responses of lesquerella to salinity relative to growth, seed yield and oil content. Six salinity treatments were imposed on a Holtville silty clay (clayey over loamy, montmorillonitic (calcareous), hyperthermic Typic Torrifluvent). The plots were irrigated with Colorado River water artificially salinized with NaCl and CaCl2 (1:1 by weight). Electrical conductivities of the irrigation treatments in both years were 1.4, 2.2, 4.0, 6.0, 8.0, and 10.0 dS m−1. Vegetative growth, leaf-ion content, seed yield, oil content and fatty acid composition of the seed were measured. Seed yield decreased from 2100 kg ha−1 under nonsaline control conditions to 650 kg ha−1 at the highest salinity level. Analysis of the combined two-year data showed a 19% reduction in seed yield for each unit increase in soil salinity above a threshold of 6.1 dS m−1. Based on these results, lesquerella can be classified as a salt tolerant crop. In response to increases in salinity, leaf Ca and Cl increased, whereas Mg decreased. Sodium was effectively excluded from leaf tissue at all levels of salinity. Concentrations of Na were generally an order of magnitude lower in lesquerella leaves than has been reported in leaves of other cruciferous crops grown under saline field conditions. Total oil content of the seeds increased slightly, but significantly, with increases in soil salinity. The fatty acid composition showed a minor, but again significant, increase in linolenic acid (C18:3) content as salinity increased. With this exception, oil composition did not change with salinity level, including the content of lesquerolic acid in the oil.

RESPONSE OF ORNAMENTAL SUNFLOWER CULTIVARS ‘SUNBEAM’ AND ‘MOONBRIGHT’ TO IRRIGATION WITH SALINE WASTEWATERS

To explore the possibility that saline wastewaters may be used to grow commercially acceptable floriculture crops, a study was initiated to determine the effects of salinity on two pollen-free cultivars of ornamental sunflower (Helianthus annuus L.). ‘Moonbright’ and ‘Sunbeam’ were grown in greenhouse sand cultures irrigated with waters prepared to simulate wastewaters commonly present in two inland valley regions of California: 1) San Joaquin Valley (SJV) where saline-sodic drainage waters are dominated by sodium (Na+) and sulfate (SO 2− 4 ) and 2) Coachella Valley (CV) where major ions in tailwaters are Na+, chloride (Cl−), SO 2− 4 , magnesium (Mg2+), calcium (Ca2+), predominating in that order. Ten-day-old seedlings were subjected to five salinity treatments of each water composition, each replicated three times. Electrical conductivities (EC) of the irrigation waters were 2.5, 5, 10, 15, and 20 dS·m−1. Flowering stems were harvested when about 75% of the ray flowers were nearly horizontal. Stem length and fresh weight, flower and stem diameter were measured. Mineral ion concentrations in upper and lower stems, upper and lower leaves were determined. Sodium was excluded from the young tissues in the upper portions of the shoot and retained in the basal stem tissue. Inasmuch as sunflower is also a strong potassium (K)-accumulator, K+/Na+ selectivity coefficients were unusually high in the younger shoot organs. Despite a five-fold increase in substrate Ca2+ in both solutions, shoot-Ca decreased as salinity increased and this cation was retained in the older leaves. A few of the lower leaves of plants irrigated with ICV waters at EC = 10 dS·m−1 and higher, exhibited necrotic margins which were undoubtedly caused by high concentrations of Cl− in the tissues. Flowering stems produced in all treatments met florist quality standards in terms of diameters for stems (0.5 to 1.5 cm) and blooms (8 to 15 cm). Across treatments, stem lengths ranged from 60 to 175 cm. Both ornamental sunflower cultivars proved to be good candidates for production of marketable flowering stems using moderately saline wastewaters.