Diversity and phage sensitivity to phages of porcine enterotoxigenic Escherichia coli.

Enterotoxigenic Escherichia coli (ETEC) is a diverse and poorly characterized E. coli pathotype that causes diarrhea in humans and animals. Phages have been proposed for the veterinary biocontrol of ETEC, but effective solutions require understanding of porcine ETEC diversity that affects phage infection. Here, we sequenced and analyzed the genomes of the PHAGEBio ETEC collection, gathering 79 diverse ETEC strains isolated from European pigs with post-weaning diarrhea (PWD). We identified the virulence factors characterizing the pathotype and several antibiotic resistance genes on plasmids, while phage resistance genes and other virulence factors were mostly chromosome encoded. We experienced that ETEC strains were highly resistant to Enterobacteriaceae phage infection. It was only by enrichment of numerous diverse samples with different media and conditions, using the 41 ETEC strains of our collection as hosts, that we could isolate two lytic phages that could infect a large part of our diverse ETEC collection: vB_EcoP_ETEP21B and vB_EcoS_ETEP102. Based on genome and host range analyses, we discussed the infection strategies of the two phages and identified components of lipopolysaccharides ( LPS) as receptors for the two phages. Our detailed computational structural analysis highlights several loops and pockets in the tail fibers that may allow recognition and binding of ETEC strains, also in the presence of O-antigens. Despite the importance of receptor recognition, the diversity of the ETEC strains remains a significant challenge for isolating ETEC phages and developing sustainable phage-based products to address ETEC-induced PWD.IMPORTANCEEnterotoxigenic Escherichia coli (ETEC)-induced post-weaning diarrhea is a severe disease in piglets that leads to weight loss and potentially death, with high economic and animal welfare costs worldwide. Phage-based approaches have been proposed, but available data are insufficient to ensure efficacy. Genome analysis of an extensive collection of ETEC strains revealed that phage defense mechanisms were mostly chromosome encoded, suggesting a lower chance of spread and selection by phage exposure. The difficulty in isolating lytic phages and the molecular and structural analyses of two ETEC phages point toward a multifactorial resistance of ETEC to phage infection and the importance of extensive phage screenings specifically against clinically relevant strains. The PHAGEBio ETEC collection and these two phages are valuable tools for the scientific community to expand our knowledge on the most studied, but still enigmatic, bacterial species-E. coli.

Preparation and Electrochemical Dissolution of a Soluble Uranium Oxycarbide Anode.

A soluble uranium oxycarbide (UC0.4O0.6) anode was synthesized by a carbothermal reduction process, using U3O8, UO2, and graphite as raw materials, in a vacuum environment of 0.1 MPa at 1750 °C. The sintered UC0.4O0.6 exhibited excellent conductivity and stability in LiCl-KCl molten salt. The dissolution process of UC0.4O0.6 in molten salt was analyzed by linear sweep voltammetry (LSV), and the initial dissolution potential, rapid dissolution potential, and passivation potential were determined. The oxidation/reduction process of dissolved uranium ions in LiCl-KCl molten salt was studied by cyclic voltammetry (CV) and square wave voltammetry (SWV). The experimental results showed that the addition of Li2O significantly reduced the volatilization of U(VI) and increased the concentration of UO22+ in molten salt. Finally, the product was conducted by electrolysis at 1.0 V and was confirmed to be UO2 by XRD and SEM-EDS analysis.

Temporal encoding of bacterial identity and traits in growth dynamics.

In biology, it is often critical to determine the identity of an organism and phenotypic traits of interest. Whole-genome sequencing can be useful for this but has limited power for trait prediction. However, we can take advantage of the inherent information content of phenotypes to bypass these limitations. We demonstrate, in clinical and environmental bacterial isolates, that growth dynamics in standardized conditions can differentiate between genotypes, even among strains from the same species. We find that for pairs of isolates, there is little correlation between genetic distance, according to phylogenetic analysis, and phenotypic distance, as determined by growth dynamics. This absence of correlation underscores the challenge in using genomics to infer phenotypes and vice versa. Bypassing this complexity, we show that growth dynamics alone can robustly predict antibiotic responses. These findings are a foundation for a method to identify traits not easily traced to a genetic mechanism.

Climate factors affect forest biomass allocation by altering soil nutrient availability and leaf traits.

Biomass in forests sequesters substantial amounts of carbon; although the contribution of aboveground biomass has been extensively studied, the contribution of belowground biomass remains understudied. Investigating the forest biomass allocation is crucial for understanding the impacts of global change on carbon allocation and cycling. Moreover, the question of how climate factors affect biomass allocation in natural and planted forests remains unresolved. Here, we addressed this question by collecting data from 384 planted forests and 541 natural forests in China. We evaluated the direct and indirect effects of climate factors on the belowground biomass proportion (BGBP). The average BGBP was 31.09% in natural forests and was significantly higher (38.75%) in planted forests. Furthermore, we observed a significant decrease in BGBP with increasing temperature and precipitation. Climate factors, particularly those affecting soil factors, such as pH, strongly affected the BGBP in natural and planted forests. Based on our results, we propose that future studies should consider the effects of forest type (natural or planted) and soil factors on BGBP.

Intravenous Injection of Coronavirus Disease 2019 (COVID-19) mRNA Vaccine Can Induce Acute Myopericarditis in Mouse Model.

BACKGROUND: Post-vaccination myopericarditis is reported after immunization with coronavirus disease 2019 (COVID-19) messenger RNA (mRNA) vaccines. The effect of inadvertent intravenous injection of this vaccine on the heart is unknown. METHODS: We compared the clinical manifestations, histopathological changes, tissue mRNA expression, and serum levels of cytokine/chemokine and troponin in Balb/c mice at different time points after intravenous (IV) or intramuscular (IM) vaccine injection with normal saline (NS) control. RESULTS: Although significant weight loss and higher serum cytokine/chemokine levels were found in IM group at 1-2 days post-injection (dpi), only IV group developed histopathological changes of myopericarditis as evidenced by cardiomyocyte degeneration, apoptosis, and necrosis with adjacent inflammatory cell infiltration and calcific deposits on visceral pericardium, although evidence of coronary artery or other cardiac pathologies was absent. Serum troponin level was significantly higher in IV group. Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike antigen expression by immunostaining was occasionally found in infiltrating immune cells of the heart or injection site, in cardiomyocytes and intracardiac vascular endothelial cells, but not skeletal myocytes. The histological changes of myopericarditis after the first IV-priming dose persisted for 2 weeks and were markedly aggravated by a second IM- or IV-booster dose. Cardiac tissue mRNA expression of interleukin (IL)-1ß, interferon (IFN)-ß, IL-6, and tumor necrosis factor (TNF)-α increased significantly from 1 dpi to 2 dpi in the IV group but not the IM group, compatible with presence of myopericarditis in the IV group. Ballooning degeneration of hepatocytes was consistently found in the IV group. All other organs appeared normal. CONCLUSIONS: This study provided in vivo evidence that inadvertent intravenous injection of COVID-19 mRNA vaccines may induce myopericarditis. Brief withdrawal of syringe plunger to exclude blood aspiration may be one possible way to reduce such risk.

Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) Infection by Intranasal or Intratesticular Route Induces Testicular Damage.

BACKGROUND: The role of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in the pathogenesis of testicular damage is uncertain. METHODS: We investigated the virological, pathological, and immunological changes in testes of hamsters challenged by wild-type SARS-CoV-2 and its variants with intranasal or direct testicular inoculation using influenza virus A(H1N1)pdm09 as control. RESULTS: Besides self-limiting respiratory tract infection, intranasal SARS-CoV-2 challenge caused acute decrease in sperm count, serum testosterone and inhibin B at 4-7 days after infection; and chronic reduction in testicular size and weight, and serum sex hormone at 42-120 days after infection. Acute histopathological damage with worsening degree of testicular inflammation, hemorrhage, necrosis, degeneration of seminiferous tubules, and disruption of orderly spermatogenesis were seen with increasing virus inoculum. Degeneration and death of Sertoli and Leydig cells were found. Although viral loads and SARS-CoV-2 nucleocapsid protein expression were markedly lower in testicular than in lung tissues, direct intratesticular injection of SARS-CoV-2 demonstrated nucleocapsid expressing interstitial cells and epididymal epithelial cells, While intranasal or intratesticular challenge by A(H1N1)pdm09 control showed no testicular infection or damage. From 7 to 120 days after infection, degeneration and apoptosis of seminiferous tubules, immune complex deposition, and depletion of spermatogenic cell and spermatozoa persisted. Intranasal challenge with Omicron and Delta variants could also induce similar testicular changes. This testicular damage can be prevented by vaccination. CONCLUSIONS: SARS-CoV-2 can cause acute testicular damage with subsequent chronic asymmetric testicular atrophy and associated hormonal changes despite a self-limiting pneumonia in hamsters. Awareness of possible hypogonadism and subfertility is important in managing convalescent coronavirus disease 2019 in men.

Prophage Hunter: an integrative hunting tool for active prophages.

Identifying active prophages is critical for studying coevolution of phage and bacteria, investigating phage physiology and biochemistry, and engineering designer phages for diverse applications. We present Prophage Hunter, a tool aimed at hunting for active prophages from whole genome assembly of bacteria. Combining sequence similarity-based matching and genetic features-based machine learning classification, we developed a novel scoring system that exhibits higher accuracy than current tools in predicting active prophages on the validation datasets. The option of skipping similarity matching is also available so that there's higher chance for novel phages to be discovered. Prophage Hunter provides a one-stop web service to extract prophage genomes from bacterial genomes, evaluate the activity of the prophages, identify phylogenetically related phages, and annotate the function of phage proteins. Prophage Hunter is freely available at https://pro-hunter.bgi.com/.

Effects of frequency and amount of stover mulching on the microbial community composition and structure in the endosphere and rhizosphere.

Stover mulching, as a sustainable agricultural conservation practice, has been shown to effectively increase soil organic matter and enhance crop yields. The impact of stover mulching on soil microorganisms has been extensively studied. However, less attention has been given to endophytic and rhizospheric microorganisms that have closer relationships with crops. How do the quality and frequency of stover mulching affect the composition and structure of these endosphere and rhizosphere microbial communities? And what is their influence on critical indicators of soil health such as bacterial plant pathogen and Rhizobiales? These questions remain unresolved. Therefore, we investigated the responses of the microbial functional guilds in the endosphere and rhizosphere to maize stover mulching qualities (0%, 33%, 67%, and total stover mulching every year) and frequencies (once every 3 years and twice every 3 years) under 10-year no-till management. Results showed significant correlations between Bacillales and Rhizobiales orders and soil SOC, NO3-N, and NH4+N; Hypocreales and Eurotiales orders were significantly correlated with soil NO3-N, with the Aspergillus genus also showing a significant correlation with soil SOC. The frequency and quality of stover mulching had a significant effect on root and rhizospheric microbial communities, with the lowest relative abundance of bacterial plant pathogens and highest relative abundance of nitrogen-fixing bacteria such as Rhizobiales and Hypocreales observed under F1/3 and F2/3 conditions. The most complex structures in endosphere and rhizospheric microbial communities were found under Q33 and Q67 conditions, respectively. This research indicates that from a soil health perspective, low-frequency high-coverage stover mulching is beneficial for the composition of endosphere and rhizosphere microbial communities, while moderate coverage levels are conducive to more complex structures within these communities. This study holds significant ecological implications for agricultural production and crop protection.

Chronic lung inflammation and CK14+ basal cell proliferation induce persistent alveolar-bronchiolization in SARS-CoV-2-infected hamsters.

BACKGROUND: Post-acute sequalae of COVID-19 defines a wide range of ongoing symptoms and conditions long after SARS-CoV-2 infection including respiratory diseases. The histopathological changes in the lung and underlying mechanism remain elusive. METHODS: We investigated lung histopathological and transcriptional changes in SARS-CoV-2-infected male hamsters at 7, 14, 42, 84 and 120dpi, and compared with A (H1N1)pdm09 infection. FINDINGS: We demonstrated viral residue, inflammatory and fibrotic changes in lung after SARS-CoV-2 but not H1N1 infection. The most prominent histopathological lesion was multifocal alveolar-bronchiolization observed in every SARS-CoV-2 infected hamster (31/31), from 42dpi to 120dpi. Proliferating (Ki67+) CK14+ basal cells accumulated in alveoli adjacent to bronchioles at 7dpi, where they proliferated and differentiated into SCGB1A+ club cell or Tubulin+ ciliated cells forming alveolar-bronchiolization foci. Molecularly, Notch pathway significantly upregulated with intensive Notch3 and Hes1 protein expression in alveolar-bronchiolization foci at 42 and 120dpi, suggesting Notch signaling involving the persistence of alveolar-bronchiolization. This is further demonstrated by spatial transcriptomic analysis. Intriguingly, significant upregulation of some cell-growth promoting pathways and genes such as Tubb4b, Stxbp4, Grb14 and Mlf1 were spatially overlapping with bronchiolization lesion. INTERPRETATION: Incomplete resolution of SARS-CoV-2 infection in lung with viral residue, chronic inflammatory and fibrotic damage and alveolar-bronchiolization impaired respiratory function. Aberrant activation of CK14+ basal cells during tissue regeneration led to persistent alveolar-bronchiolization due to sustained Notch signaling. This study advances our understanding of respiratory PASC, sheds light on disease management and highlights the necessity for monitoring disease progression in people with respiratory PASC. FUNDING: Funding is listed in the Acknowledgements section.

Negative Linear or Unimodal: Why Forest Soil Fungal Latitudinal Diversity Differs across China.

To identify the reasons for the inconsistency in patterns of latitudinal gradients of forest soil fungal biodiversity in China, a reanalysis of data was performed. Causes are linked to the different environments of continents and islands and the inconsistency between different classification standards. The following three suggestions are made for future studies: sites on the mainland and islands should be distinguished in these types of studies, the Shannon index should be used to represent fungal diversity instead of operational taxonomic unit (OTU) richness, and using the diversity of higher taxa (such as family level) instead of OTU level represents a potential proxy for species-level diversity. IMPORTANCE Latitudinal gradients of forest soil fungal biodiversity in China have been previously investigated; however, the results of these studies were inconsistent. In the present study, I reanalyzed the data from these studies on all forest types in China and showed that the differences in forest soil fungal latitudinal diversity were caused by the different environments of continents and islands, as well as by the inconsistency between different classification standards. Accordingly, three suggestions were outlined for future studies on this and similar topics. This study makes a significant contribution to the literature because these findings can be used to improve our understanding of the forest soil fungal latitudinal diversity and as a basis for future studies.

Structure and function of microbiomes in the rhizosphere and endosphere response to temperature and precipitation variation in Inner Mongolia steppes.

Introduction: Owing to challenges in the study of complex rhizosphere and endophytic microbial communities, the composition and function of such microbial communities in steppe ecosystems remain elusive. Here, we studied the microbial communities of the rhizosphere and endophytic microbes of the dominant plant species across the Inner Mongolian steppes using metagenomic sequencing and investigated their relationships with changes in mean annual temperature (MAT) and mean annual precipitation (MAP). Methods: Metagenomic sequencing based on Illumina high-throughput sequencing, using the paired end method to construct a small fragment library for sequencing. Results: Adaptation of root systems to the environment affected the composition and function of rhizosphere and endophytic microbial communities. However, these communities exhibited distinct community assembly and environmental adaptation patterns. Both rhizosphere and endophytic microbial communities can be divided into two unrelated systems based on their ecological niches. The composition and function of the rhizosphere microbial communities were mainly influenced by MAT, while those of the endophytic microbial communities were mainly influenced by MAP. MAT affected the growth, reproduction, and lipid decomposition of rhizosphere microorganisms, whereas MAP affected reverse transcription and cell wall/membrane/envelope biogenic functions of endophytic microorganisms. Conclusion: Our findings reveal the composition and function of the rhizosphere and endophytic microbial communities in response to changes in MAP and MAT, which has important implications for future biogeography and climate change research.

Characterization of a novel genus of jumbo phages and their application in wastewater treatment.

Phages widely exist in numerous environments from wastewater to deep ocean, representing a huge virus diversity, yet remain poorly characterized. Among them, jumbo phages are of particular interests due to their large genome (>200 kb) and unusual biology. To date, only six strains of jumbo phages infecting Klebsiella pneumoniae have been described. Here, we report the isolation and characterization of two jumbo phages from hospital wastewater representing the sixth genus: φKp5130 and φKp9438. Both phages showed lytic activity against broad range of clinical antibiotic-resistant K. pneumoniae strains and distinct physiology including long latent period, small burst size, and high resistance to thermal and pH stress. The treatment of sewage water with the phages cocktail resulted in dramatic decline in K. pneumoniae population. Overall, this study provides detailed molecular and genomics characterization of two novel jumbo phages, expands viral diversity, and provides novel candidate phages to facilitate environmental wastewater treatment.

COVID-19 mRNA vaccine protects against SARS-CoV-2 Omicron BA.1 infection in diet-induced obese mice through boosting host innate antiviral responses.

BACKGROUND: Obesity is a worldwide epidemic and is considered a risk factor of severe manifestation of Coronavirus Disease 2019 (COVID-19). The pathogenicity of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and host responses to infection, re-infection, and vaccination in individuals with obesity remain incompletely understood. METHODS: Using the diet-induced obese (DIO) mouse model, we studied SARS-CoV-2 Alpha- and Omicron BA.1-induced disease manifestations and host immune responses to infection, re-infection, and COVID-19 mRNA vaccination. FINDINGS: Unlike in lean mice, Omicron BA.1 and Alpha replicated to comparable levels in the lungs of DIO mice and resulted in similar degree of tissue damages. Importantly, both T cell and B cell mediated adaptive immune responses to SARS-CoV-2 infection or COVID-19 mRNA vaccination are impaired in DIO mice, leading to higher propensity of re-infection and lower vaccine efficacy. However, despite the absence of neutralizing antibody, vaccinated DIO mice are protected from lung damage upon Omicron challenge, accompanied with significantly more IFN-α and IFN-ß production in the lung tissue. Lung RNAseq and subsequent experiments indicated that COVID-19 mRNA vaccination in DIO mice boosted antiviral innate immune response, including the expression of IFN-α, when compared to the nonvaccinated controls. INTERPRETATION: Our findings suggested that COVID-19 mRNA vaccination enhances host innate antiviral responses in obesity which protect the DIO mice to a certain degree when adaptive immunity is suboptimal. FUNDING: A full list of funding bodies that contributed to this study can be found in the Acknowledgements section.

Intranasal infection by SARS-CoV-2 Omicron variants can induce inflammatory brain damage in newly weaned hamsters.

SUMMARY: Intranasal infection of newly-weaned Syrian hamsters by SARS-CoV-2 Omicron variants can lead to brain inflammation and neuron degeneration with detectable low level of viral load and sparse expression of viral nucleoprotein.

Effects of frequency and amount of stover mulching on soil nitrogen and the microbial functional guilds of the endosphere and rhizosphere.

Stover mulching as a conservation and sustainable agricultural practice is beneficial for maintaining soil nitrogen (N) requirements and plant health. The microbial functional guilds of the root and rhizosphere are important factors in the soil nitrogen cycle. However, it is unclear how the frequency and amount of stover mulching influence microbial functional guilds in the root and rhizosphere. Therefore, we investigated the responses of the microbial functional guilds in the endosphere and rhizosphere to maize stover mulching amounts (0, 1/3, 2/3, and total stover mulching every year) and frequencies (once every 3 years and twice every 3 years) under 10-year no-till management. The bacterial functional guilds of nitrogen fixation, nitrification, and anaerobic nitrate oxidation displayed the significantly correlation with C/N, total nitrogen, NO3 -, and NH4 +. The fungal functional guilds of plant pathogens and saprotrophs showed significantly correlations with C/N, total nitrogen, and NO3 -. Moreover, we found that bacterial guilds play a pivotal role in maintaining N requirements at the jointing stage, whereas root endophytic fungal guilds play a more important role than bacterial guilds in regulating plant health at the mature stage. The frequency and amount of stover mulching had significant effects on the microbial functional guilds in the root and rhizosphere. Our data suggest that stover mulch application twice every 3 years is the optimal mulching frequency because it yielded the lowest abundance of nitrifying and anaerobic nitrate-oxidising bacteria and the highest abundance of nitrogen-fixing bacteria at the jointing stage, as well as the lowest abundance of fungal plant pathogens in roots at the mature stage.

Linking Leaf Functional Traits with Soil and Climate Factors in Forest Ecosystems in China.

Plant leaf functional traits can reflect the adaptive strategies of plants to environmental changes. Exploring the patterns and causes of geographic variation in leaf functional traits is pivotal for improving ecological theory at the macroscopic scale. In order to explore the geographical variation and the dominant factors of leaf functional traits in the forest ecosystems of China, we measured 15 environmental factors on 16 leaf functional traits in 33 forest reserves in China. The results showed leaf area (LA), carbon-to-nitrogen ratio (C/N), carbon-to-phosphorus ratio (C/P), nitrogen-to-phosphorus ratio (N/P), phosphorus mass per area (Pa) and nitrogen isotope abundance (δ15N)) were correlated with latitude significantly. LA, Pa and δ15N were also correlated with longitude significantly. The leaf functional traits in southern China were predominantly affected by climatic factors, whereas those in northern China were mainly influenced by soil factors. Mean annual temperature (MAT), mean annual precipitation (MAP) and mean annual humidity (MAH) were shown to be the important climate factors, whereas available calcium (ACa), available potassium (AK), and available magnesium (AMg) were shown to be the important climate factors that affect the leaf functional traits of the forests in China. Our study fills the gap in the study of drivers and large-scale geographical variability of leaf functional traits, and our results elucidate the operational mechanisms of forest-soil-climate systems. We provide reliable support for modeling global forest dynamics.

Age-associated SARS-CoV-2 breakthrough infection and changes in immune response in a mouse model.

Older individuals are at higher risk of SARS-CoV-2 infection and severe outcomes, but the underlying mechanisms are incompletely understood. In addition, how age modulates SARS-CoV-2 re-infection and vaccine breakthrough infections remain largely unexplored. Here, we investigated age-associated SARS-CoV-2 pathogenesis, immune responses, and the occurrence of re-infection and vaccine breakthrough infection utilizing a wild-type C57BL/6N mouse model. We demonstrated that interferon and adaptive antibody response upon SARS-CoV-2 challenge are significantly impaired in aged mice compared to young mice, which results in more effective virus replications and severe disease manifestations in the respiratory tract. Aged mice also showed increased susceptibility to re-infection due to insufficient immune protection acquired during the primary infection. Importantly, two-dose COVID-19 mRNA vaccination conferred limited adaptive immune response among the aged mice, making them susceptible to SARS-CoV-2 infection. Collectively, our findings call for tailored and optimized treatments and prevention strategies against SARS-CoV-2 among older individuals.

Harnessing stepping-stone hosts to engineer, select, and reboot synthetic bacteriophages in one pot.

Advances in synthetic genomics have led to a great demand for genetic manipulation. Trimming any process to simplify and accelerate streamlining of genetic code into life holds great promise for synthesizing and studying organisms. Here, we develop a simple but powerful stepping-stone strategy to promote genome refactoring of viruses in one pot, validated by successful cross-genus and cross-order rebooting of 90 phages infecting 4 orders of popular pathogens. Genomic sequencing suggests that rebooting outcome is associated with gene number and DNA polymerase availability within phage genomes. We integrate recombineering, screening, and rebooting processes in one pot and demonstrate genome assembly and genome editing of phages by stepping-stone hosts in an efficient and economic manner. Under this framework, in vitro assembly, yeast-based assembly, or genetic manipulation of native hosts are not required. As additional stepping-stone hosts are being developed, this framework will open doors for synthetic phages targeting more pathogens and commensals.

Rapid residue analysis of four triazolopyrimidine herbicides in soil, water, and wheat by ultra-performance liquid chromatography coupled to tandem mass spectrometry.

A sensitive and effective method for simultaneous determination of triazolopyrimidine sulfonamide herbicide residues in soil, water, and wheat was developed using ultra-performance liquid chromatography coupled with tandem mass spectrometry. The four herbicides (pyroxsulam, flumetsulam, metosulam, and diclosulam) were cleaned up with an off-line C18 SPE cartridge and detected by tandem mass spectrometry using an electrospray ionization source in positive mode (ESI+). The determination of the target compounds was achieved in <2.0 min. The limits of detection were below 1 µg kg(-1), while the limits of quantification did not exceed 3 µg kg(-1) in different matrices. Quantitation was determined from calibration curves of standards containing 0.05-100 µg L(-1) with r(2) > 0.997. Recovery studies were conducted at three spiked levels (0.2, 1, and 5 µg kg(-1) for water; 5, 10, and 100 µg kg(-1) for soil and wheat). The overall average recoveries for this method in water, soil, wheat plants, and seeds at three levels ranged from 75.4% to 106.0%, with relative standard deviations in the range of 2.1-12.5% (n = 5) for all analytes.