Fungal Endophytes: Discovering What Lies within Some of Canada's Oldest and Most Resilient Grapevines.

Plant diseases and pests reduce crop yields, accounting for global crop losses of 30% to 50%. In conventional agricultural production systems, these losses are typically controlled by applying chemical pesticides. However, public pressure is mounting to curtail agrochemical use. In this context, employing beneficial endophytic microorganisms is an increasingly attractive alternative to the use of conventional chemical pesticides in agriculture. A multitude of fungal endophytes are naturally present in plants, producing enzymes, small peptides, and secondary metabolites due to their bioactivity, which can protect hosts from pathogens, pests, and abiotic stresses. The use of beneficial endophytic microorganisms in agriculture is an increasingly attractive alternative to conventional pesticides. The aim of this study was to characterize fungal endophytes isolated from apparently healthy, feral wine grapes in eastern Canada that have grown without agrochemical inputs for decades. Host plants ranged from unknown seedlings to long-lost cultivars not widely propagated since the 1800s. HPLC-MS was used to identify unique endophyte-derived chemical compounds in the host plants, while dual-culture competition assays showed a range in endophytes' ability to suppress the mycelial growth of Botrytis, which is typically controlled in viticulture with pesticides. Twelve of the most promising fungal endophytes isolated were identified using multilocus sequencing and morphology, while DNA barcoding was employed to identify some of their host vines. These fungal endophyte isolates, which consisted of both known and putative novel strains, belonged to seven genera in six families and five orders of Ascomycota. Exploring the fungal endophytes in these specimens may yield clues to the vines' survival and lead to the discovery of novel biocontrol agents.

Poisoning by Baccharis coridifolia in Early-Weaned Beef Calves: Pathological Study and New Macrocyclic Trichothecene Identification.

This study investigated two outbreaks of spontaneous poisoning by Baccharis coridifolia (Asteraceae) in early-weaned beef calves in Tacuarembó, Uruguay. A total of 34 affected calves showed signs of salivation, anorexia, apathy, marked dehydration, and diarrhea. Deaths occurred 36-72 h after consumption and mortality varied from 37.5% to 43.3% for outbreak 1 and outbreak 2, respectively. The main pathological findings include diffuse severe necrosis of the prestomachs and lymphoid tissues. Ultrastructurally, epithelial cells of the rumen showed swelling, lysis of the organelles, degradation of intercellular attachments, and degradation of the nuclear chromatin. Using LC-MS with diagnostic fragmentation filtering, 56 macrocyclic trichothecenes including glycosyl and malonyl conjugates were identified. The total concentration of macrocyclic trichothecenes, including conjugates, was estimated to be 1.2 ± 0.1 mg/g plant material. This is the first report of these malonyl-glucose conjugates from Baccharis coridifolia.

Farm management practices and season dependent factors affect the microbial community and chemical profile of corn and grass-legume silages of farms in Ontario, Québec, and Northern New York.

The effects of farm management practices and seasonal variation on the microbial community and chemical composition of corn and grass-legume silage are largely understudied due to the advantages of controlled mini-silo experiments. This study aims to investigate the effects that some key farm factors (use of an inoculant, farm region, and bunker or tower silo) and seasonal variations have on corn and grass-legume silage from farms across Ontario, Quebec, and New York. The silage was either treated with a commercial inoculant (Lallemand Biotal Buchneri 500® or Chr Hansen SiloSolve FC®) or left untreated. The bacterial communities of silage were compared to those of raw bulk tank milk from the same farm to determine if they were similarly affected by management practices or seasonal variations. Family level analysis of the 16S rRNA V3-V4 gene amplicon bacterial community, the ITS1 amplicon fungal community, NMR water soluble metabolome, and mycotoxin LC-MS were performed on silage over a two-year period. Chemical compounds associated with the use of inoculants in corn and grass-legume silage were higher in inoculated corn (acetate, propane-1,2-diol, γ-aminobutyrate; p < 0.001) and grass-legume (propionate; p = 0.011). However, there was no significant difference in the relative abundance (RA) of Lactobacillaceae in either silage type. Leuconostocaceae was higher in non-inoculated corn (p < 0.001) and grass-legume (p < 0.001) silage than in inoculated silage. Tower silos had higher RA of Leuconostocaceae (p < 0.001) and higher pH (p < 0.001) in corn and grass-legume silage. The one farm that used liquid manure with no other fertilizer type had higher RA of Clostridiaceae (p = 0.045) and other rumen/fecal (p < 0.006) bacteria in grass-legume silage than all other farms. Seasonal variation affected most of the key silage microbial families, however the trends were rarely visible across both years. Few trends in microbial variation could be observed in both silage and bulk tank milk: two farms had higher Moraxellaceae (p < 0.001) in milk and either corn or grass-legume silage. In farms using an inoculant, lower Staphylococcaceae was observed in the raw bulk tank milk.

Non-targeted Screening of Natural Products from 288 Fungal Endophytes from Canadian Fruit Crops.

Many diverse species of fungi naturally occur as endophytes in plants. The majority of these fungi produce secondary metabolites of diverse structures and biological activities. Culture extracts from 288 fungi isolated from surface-sterilized blueberries, cranberries, raspberries, and grapes were analyzed by LC-HRMS/MS. Global Natural Products Social (GNPS) Molecular Networking modeling was used to investigate the secondary metabolites in the extracts. This technique increased the speed and simplicity of dereplicating the extracts, targeting new compounds that are structurally related. In total, 60 known compounds were dereplicated from this collection and seven new compounds were identified. These previously unknown compounds are targets for purification, characterization, and bioactivity testing in future studies. The fungal endophytes characterized in this study are potential candidates for providing bio-protection to the host plant with a reduced reliance on chemical pesticides.

Environmental Concentrations of the Type 2 Diabetes Medication Metformin and Its Transformation Product Guanylurea in Surface Water and Sediment in Ontario and Quebec, Canada.

Metformin, used to treat Type 2 diabetes, is the active ingredient of one of the most prescribed drugs in the world, with over 120 million yearly prescriptions globally. In wastewater-treatment plants (WWTPs), metformin can undergo microbial transformation to form the product guanylurea, which could have toxicological relevance in the environment. Surface water samples from 2018 to 2020 and sediment samples from 2020 were collected from six mixed-use watersheds in Quebec and Ontario, Canada, and analyzed to determine the metformin and guanylurea concentrations at each site. Metformin and guanylurea were present above their limits of quantification in 51.0% and 50.7% of all water samples and in 64% and 21% of all sediment samples, respectively. In surface water, guanylurea was often present at higher concentrations than metformin, while the inverse was true in sediment, with metformin frequently detected at higher concentrations than guanylurea. In addition, at all sites influenced solely by agriculture, concentrations of metformin and guanylurea were <1 µg/L in surface water, suggesting that agriculture is not a significant source of these compounds in the investigated watersheds. These data suggest that WWTPs and potentially septic system leaks are the most likely sources of the compounds in the environment. Guanylurea was detected at many of these sites above environmental concentrations of concern, where critical processes in fish may be affected. Due to the scarcity of available ecotoxicological data and the prominence of guanylurea across all sample sites, there is a need to perform more toxicological investigations of this transformation product and revisit regulations. The present study will help provide toxicologists with environmentally relevant concentration ranges in Canada. Environ Toxicol Chem 2023;42:1709-1720. © 2023 His Majesty the King in Right of Canada and The Authors. Environmental Toxicology and Chemistry published by Wiley Periodicals LLC on behalf of SETAC. Reproduced with the permission of the Minister of Agriculture and Agri-Food Canada.

Investigation of N,N,N-Trimethyl-L-alanyl-L-proline Betaine (TMAP) as a Biomarker of Kidney Function.

Glomerular filtration rate (GFR) is the most widely used tool for the measurement of kidney function, but endogenous biomarkers such as cystatin C and creatinine have limitations. A previous metabolomic study revealed N,N,N-trimethyl-L-alanyl-L-proline betaine (TMAP) to be reflective of kidney function. In this study, we developed a quantitative LCMS assay for the measurement of TMAP and evaluated TMAP as a biomarker of GFR. An assay to measure TMAP was developed using liquid chromatography-mass spectrometry. After validation of the method, we applied it to plasma samples from three distinct kidney disease patient cohorts: nondialysis chronic kidney disease (CKD) patients, patients receiving peritoneal and hemodialysis, and living kidney donors. We investigated whether TMAP was conserved in other mammalian and nonmammalian species, by analyzing plasma samples from Wistar rats with diet-induced CKD and searching for putative matches to the m/z for TMAP and its known fragments in the raw sample data repository "Metabolomics Workbench". The assay can measure plasma TMAP at a lower limit of quantitation (100 ng/mL) with an interday precision and accuracy of 12.8 and 12.1%, respectively. In all three patient cohorts, TMAP concentrations are significantly higher in patients with CKD than in controls with a normal GFR. Further, TMAP concentrations are also elevated in rats with CKD and TMAP is present in the sap produced from Acer saccharum trees. TMAP concentration is inversely related to GFR suggesting that it is a marker of kidney function. TMAP is present in nonmammalian species suggesting that it is part of a biologically conserved process.

Metabolomics Reveals Strain-Specific Cyanopeptide Profiles and Their Production Dynamics in Microcystis aeruginosa and M. flos-aquae.

Cyanobacterial blooms that release biologically active metabolites into the environment are increasing in frequency as a result of the degradation of freshwater ecosystems globally. The microcystins are one group of cyanopeptides that are extensively studied and included in water quality risk management frameworks. Common bloom-forming cyanobacteria produce incredibly diverse mixtures of other cyanopeptides; however, data on the abundance, distribution, and biological activities of non-microcystin cyanopeptides are limited. We used non-targeted LC-MS/MS metabolomics to study the cyanopeptide profiles of five Microcystis strains: four M. aeruginosa and one M. flos-aquae. Multivariate analysis and GNPS molecular networking demonstrated that each Microcystis strain produced a unique mixture of cyanopeptides. In total, 82 cyanopeptides from the cyanopeptolin (n = 23), microviridin (n = 18), microginin (n = 12), cyanobactin (n = 14), anabaenopeptin (n = 6), aeruginosin (n = 5), and microcystin (n = 4) classes were detected. Microcystin diversity was low compared with the other detected cyanopeptide classes. Based on surveys of the literature and spectral databases, most cyanopeptides represented new structures. To identify growth conditions yielding high amounts of multiple cyanopeptide groups, we next examined strain-specific cyanopeptide co-production dynamics for four of the studied Microcystis strains. When strains were cultivated in two common Microcystis growth media (BG-11 and MA), the qualitative cyanopeptides profiles remained unchanged throughout the growth cycle. For each of the cyanopeptide groups considered, the highest relative cyanopeptide amounts were observed in the mid-exponential growth phase. The outcomes of this study will guide the cultivation of strains producing common and abundant cyanopeptides contaminating freshwater ecosystems. The synchronous production of each cyanopeptide group by Microcystis highlights the need to make more cyanopeptide reference materials available to investigate their distributions and biological functions.

Optimization of Aflatoxin B1-Lysine Analysis for Public Health Exposure Studies.

Aflatoxin B1 is a potent human carcinogen produced by several species of Aspergillus mainly found on nuts and maize. Exposures in parts of Africa, Latin America and Asia can be at multiples, sometimes orders of magnitude above tolerable daily levels. Although human exposure to aflatoxin can be estimated by analysis of the diet, only determination of the serum albumin aflatoxin adduct provides a health-relevant exposure measure. The lack of a reference serum limits interlaboratory method validation and data comparisons. In this study, we synthetically produced AFB1-dialdehyde and covalently coupled it to serum albumin in human serum. This synthetic produced aflatoxin-serum reference material was used in conjunction with isotopically labelled internal standards to evaluate sample digestion methods. This showed using sufficient Pronase in the digestion step was critical to ensure complete proteolytic digestion, which occurs within 4 h. Increasing the digestion temperature from 37 °C to 50 °C also provided a benefit to the overall analysis. In addition, the use of dried blood spots and Volumetric Absorptive Microsampling (VAMS) were investigated showing samples stored with VAMS produced equivalent results to serum samples.

Application of Aspergillus niger Fumonisin Amine Oxidase (AnFAO) to Detoxify Fumonisin-Contaminated Maize.

Fumonisin mycotoxins are a family of secondary metabolites produced by Fusarium verticillioides and related species, as well as some strains of Aspergillus niger. Fumonisin contamination of maize is a concern when grown under hot, dry conditions. When present above regulatory levels, there can be effects on animal health. New tools to reduce the toxicity of maize and maize products with high concentrations of fumonisin are needed. Recently, we reported an amine oxidase (AnFAO) from a fumonisin-producing Aspergillus niger strain capable of oxidatively deaminating intact fumonisins. In this study, AnFAO was used to reduce intact fumonisin concentrations in milled maize flour, whole kernel maize inoculated with fumonisin-producing Fusarium verticillioides, and dried distillers' grains with solubles (DDGS). The data showed that milled maize flour incubated with 1 µM AnFAO for 1 h resulted in complete deamination of FB1 and FB2. A greater than 90% reduction in FB1-3 concentrations was observed following a simple washing procedure of whole kernel maize in the presence of 1 µM AnFAO for 1 h. Similarly, a ≥86% reduction in FB1-3 concentrations was observed in DDGS after 4 h incubation with 1 µM AnFAO. Finally, we engineered the methylotrophic yeast Pichia pastoris to produce functional AnFAO in both a secreted and intracellular form. These results support the further development and application of AnFAO as a promising tool to remediate fumonisin-contaminated maize and maize products.

Investigation of Metabolic Resistance to Soybean Aphid (Aphis glycines Matsumura) Feeding in Soybean Cultivars.

Soybean aphid (Aphis glycines) is a major soybean (Glycine max) herbivore pest in many soybean growing regions. High numbers of aphids on soybean can cause severe reductions in yield. The management of soybean aphids includes monitoring, insecticide applications when required, and the use of resistant cultivars. Soybean aphid-resistant soybean varieties are associated with genes that confer one or more categories of resistance to soybean aphids, including antibiosis (affects survival, growth, and fecundity), antixenosis (affects behaviour such as feeding), and tolerance (plant can withstand greater damage without economic loss). The genetic resistance of soybean to several herbivores has been associated with isoflavonoid phytoalexins; however, this correlation has not been observed in soybean varieties commonly grown in southern Ontario, Canada. Isoflavonoids in the leaves of 18 cultivars in the early growth stage were analyzed by HPLC and the concentration by fresh weight was used to rate the potential resistance to aphids. Greenhouse and growth cabinet trials determined that the cultivars with greater resistance to aphids were Harosoy 63 and OAC Avatar. The most susceptible cultivar was Maple Arrow, whereas Pagoda and Conrad were more tolerant to aphid feeding damage. Overall, there was a low correlation between the number of aphids per leaf, feeding damage, and leaf isoflavonoid levels. Metabolite profiling by high-resolution LC-MS determined that the most resistant cultivar had on average lower levels of certain free amino acids (Met, Tyr, and His) relative to the most susceptible cultivar. This suggests that within the tested cultivars, nutritional quality stimulates aphid feeding more than isoflavonoids negatively affect aphid feeding or growth. These findings provide a better understanding of soybean host plant resistance and suggest ways to improve soybean resistance to aphid feeding through the breeding or metabolic engineering of leaf metabolites.

A live yeast supplementation to gestating ewes improves bioactive molecule composition in colostrum with no impact on its bacterial composition and beneficially affects immune status of the offspring.

Colostrum quality is of paramount importance in the management of optimal ruminant growth and infectious disease prevention in early life. Live yeast supplementation effect during the last month of gestation was evaluated on ewes' colostrum composition. Two groups of ewes (n = 14) carrying twin lambs were constituted and twins were separated into groups (mothered or artificially fed) 12 h after birth. Nutrient, oligosaccharides (OS), IgG and lactoferrin concentrations were measured over 72 h after lambing, and bacterial community was described in colostrum collected at parturition (T0). Immune passive transfer was evaluated through IgG measurement in lamb serum. In both groups, colostral nutrient, OS concentrations and IgG concentrations in colostrum and lamb serum decreased over time (P < 0⋅01), except for lactose, which slightly increased (P < 0⋅001), and lactoferrin, which remained stable. Bacterial population was stable over time with high relative abundances of Aerococcaceae, Corynebacteriaceae, Moraxellaceae and Staphylococcaceae in T0 colostrum. No effect of supplementation was observed in nutrient and lactoferrin concentrations. In supplemented ewes, the level of colostral IgG was higher at T0 and a higher level of serum IgG was observed in lambs born from supplemented mothers and artificially fed, while no effect of supplementation was observed in the mothered lamb groups. Using a metabolomic approach, we showed that supplementation affected OS composition with significantly higher levels of colostral Neu-5Gc compounds up to 5 h after birth. No effect of supplementation was observed on bacterial composition. Our data suggest that live yeast supplementation offsets the negative impact of early separation and incomplete colostrum feeding in neonate lambs.

Simplified Synthesis and Stability Assessment of Aflatoxin B1-Lysine and Aflatoxin G1-Lysine.

Aflatoxins B1 (AFB1) and G1 (AFG1) are carcinogenic mycotoxins that contaminate crops such as maize and groundnuts worldwide. The broadly accepted method to assess chronic human aflatoxin exposure is by quantifying the amount of aflatoxin adducted to human serum albumin. This has been reported using ELISA, HPLC, or LC-MS/MS to measure the amount of AFB1-lysine released after proteolysis of serum albumin. LC-MS/MS is the most accurate method but requires both isotopically labelled and unlabelled AFB1-lysine standards, which are not commercially available. In this work, we report a simplified synthetic route to produce unlabelled, deuterated and 13C6 15N2 labelled aflatoxin B1-lysine and for the first-time aflatoxin G1-lysine. Additionally, we report on the stability of these compounds during storage. This simplified synthetic approach will make the production of these important standards more feasible for laboratories performing aflatoxin exposure studies.

Monitoring of Environmental Contaminants in Mixed-Use Watersheds Combining Targeted and Nontargeted Analysis with Passive Sampling.

Understanding the environmental fate, transport, and occurrence of pesticides and pharmaceuticals in aquatic environments is of utmost concern to regulators. Traditionally, monitoring of environmental contaminants in surface water has consisted of liquid chromatography-tandem mass spectrometry analyses for a set of targeted compounds in discrete samples. These targeted approaches are limited by the fact that they only provide information on compounds within a target list present at the time and location of sampling. To address these limitations, there has been considerable interest in suspect screening and nontargeted analysis (NTA), which allow for the detection of all ionizable compounds in the sample with the added benefit of data archiving for retrospective mining. Even though NTA can detect a large number of contaminants, discrete samples only provide a snapshot perspective of the chemical disposition of an aquatic environment at the time of sampling, potentially missing episodic events. We evaluated two types of passive chemical samplers for nontargeted analysis in mixed-use watersheds. Nontargeted data were processed using MS-DIAL to screen against our in-house library and public databases of more than 1300 compounds. The data showed that polar organic chemicals integrative samplers (POCIS) were able to capture the largest number of analytes with better reproducibility than organic compound-diffusive gradients in thin film (o-DGT), resulting from the greater amount of binding sorbent. We also showed that NTA combined with passive sampling gives a more representative picture of the contaminants present at a given site and enhances the ability to identify the nature of point and nonpoint pollution sources and ecotoxicological impacts. Environ Toxicol Chem 2022;41:1131-1143. © 2021 Her Majesty the Queen in Right of Canada Environmental Toxicology and Chemistry © 2021 SETAC. Reproduced with the permission of the Minister of Agriculture and Agri-Food Canada.

Multilaboratory Collaborative Study of a Nontarget Data Acquisition for Target Analysis (nDATA) Workflow Using Liquid Chromatography-High-Resolution Accurate Mass Spectrometry for Pesticide Screening in Fruits and Vegetables.

Nontarget data acquisition for target analysis (nDATA) workflows using liquid chromatography-high-resolution accurate mass (LC-HRAM) spectrometry, spectral screening software, and a compound database have generated interest because of their potential for screening of pesticides in foods. However, these procedures and particularly the instrument processing software need to be thoroughly evaluated before implementation in routine analysis. In this work, 25 laboratories participated in a collaborative study to evaluate an nDATA workflow on high moisture produce (apple, banana, broccoli, carrot, grape, lettuce, orange, potato, strawberry, and tomato). Samples were extracted in each laboratory by quick, easy, cheap, effective, rugged, and safe (QuEChERS), and data were acquired by ultrahigh-performance liquid chromatography (UHPLC) coupled to a high-resolution quadrupole Orbitrap (QOrbitrap) or quadrupole time-of-flight (QTOF) mass spectrometer operating in full-scan mass spectrometry (MS) data-independent tandem mass spectrometry (LC-FS MS/DIA MS/MS) acquisition mode. The nDATA workflow was evaluated using a restricted compound database with 51 pesticides and vendor processing software. Pesticide identifications were determined by retention time (tR, ±0.5 min relative to the reference retention times used in the compound database) and mass errors (δM) of the precursor (RTP, δM ≤ ±5 ppm) and product ions (RTPI, δM ≤ ±10 ppm). The elution profiles of all 51 pesticides were within ±0.5 min among 24 of the participating laboratories. Successful screening was determined by false positive and false negative rates of <5% in unfortified (pesticide-free) and fortified (10 and 100 µg/kg) produce matrices. Pesticide responses were dependent on the pesticide, matrix, and instrument. The false negative rates were 0.7 and 0.1% at 10 and 100 µg/kg, respectively, and the false positive rate was 1.1% from results of the participating LC-HRAM platforms. Further evaluation was achieved by providing produce samples spiked with pesticides at concentrations blinded to the laboratories. Twenty-two of the 25 laboratories were successful in identifying all fortified pesticides (0-7 pesticides ranging from 5 to 50 µg/kg) for each produce sample (99.7% detection rate). These studies provide convincing evidence that the nDATA comprehensive approach broadens the screening capabilities of pesticide analyses and provide a platform with the potential to be easily extended to a larger number of other chemical residues and contaminants in foods.

Soybean (Glycine max L Merr) host-plant defenses and resistance to the two-spotted spider mite (Tetranychus urticae Koch).

In southern Ontario, Canada, the two-spotted spider mite (Tetranychus urticae) is an emerging pest of soybean (Glycine max) due to the increasing incidence of warmer, drier weather conditions. One key strategy to manage soybean pests is breeding resistant cultivars. Resistance to pathogens and herbivores in soybean has been associated with isoflavonoid phytoalexins, a group of specialized metabolites commonly associated with root, leaf and seed tissues. A survey of 18 Ontario soybean cultivars for spider mite resistance included evaluations of antibiosis and tolerance in relation to isoflavonoid and other metabolites detected in the leaves. Ten-day and 4-week trials beginning with early growth stage plants were used to compare survival, growth, fecundity as well as damage to leaves. Two-spotted spider mite (TSSM) counts were correlated with HPLC measurements of isoflavonoid concentration in the leaves and global metabolite profiling by high resolution LC-MS to identify other metabolites unique to the most resistant (R) and susceptible (S) cultivars. Within 10 days, no significant difference (P>0.05) in resistance to TSSM was determined between cultivars, but after 4 weeks, one cultivar, OAC Avatar, was revealed to have the lowest number of adult TSSMs and their eggs. Other cultivars showing partial resistance included OAC Wallace and OAC Lakeview, while Pagoda was the most tolerant to TSSM feeding. A low, positive correlation between isoflavonoid concentrations and TSSM counts and feeding damage indicated these compounds alone do not explain the range of resistance or tolerance observed. In contrast, other metabolite features were significantly different (P<0.05) in R versus S cultivars. In the presence of TSSM, the R cultivars had significantly greater (P<0.05) concentrations of the free amino acids Trp, Val, Thr, Glu, Asp and His relative to S cultivars. Furthermore, the R cultivar metabolites detected are viable targets for more in-depth analysis of their potential roles in TSSM defense.

Unraveling the Ergot Alkaloid and Indole Diterpenoid Metabolome in the Claviceps purpurea Species Complex Using LC-HRMS/MS Diagnostic Fragmentation Filtering.

The plant parasitic fungus Claviceps purpurea sensu lato produces sclerotia containing toxic ergot alkaloids and uncharacterized indole diterpenoids in grasses including cereals. The aim of this study was to detect as many peptide ergot alkaloids and indole diterpenoids in ergot sclerotia as possible by using a liquid chromatography-high-resolution mass spectrometry (LC-HRMS/MS) approach and applying filtering of diagnostic fragment ions for data extraction. The sample set consisted of 66 Claviceps sclerotia from four different geographic locations in southeastern Norway as well as Saskatchewan, Canada. The host plants included both wild grasses and important cereal grains such as rye. DNA sequencing showed that the sclerotia were from three Claviceps species, i.e., Claviceps purpurea sensu stricto (s.s.), Claviceps humidiphila, and Claviceps arundinis (former C. purpurea genotypes G1, G2, and G2a, respectively). All sclerotia from cereal grains were from C. purpurea s.s. Diagnostic fragment filtering was based on detecting specific product ions in MS/MS data sets that are well-conserved across the different ergot alkaloid subgroups and indole diterpenoids of the paspaline/paxilline type. The approach extracted mass spectra from 67 peptide ergot alkaloids (including C-8 epimers and lactam variants) and five indole diterpenoids. In addition, three clavines were detected by using targeted analysis. The sum of the peak areas for ergot alkaloids, which have been assigned as "major" analogues by the European Food Safety Authority (ergometrine, ergosine, ergotamine, α-ergocryptine, ergocornine, ergocristine, and their 8-S epimers), accounted for at least 50% of the extracted total ergot alkaloid metabolome. Univariate and multivariate statistical analyses showed that several of the alkaloids were specific for certain species within the C. purpurea species complex and could be used as chemotaxonomic markers for species assignment.

The Effects of Ditch Management in Agroecosystems on Embryonic and Tadpole Survival, Growth, and Development of Northern Leopard Frogs (Lithobates pipiens).

Agricultural drainage ditches help remove excess water from fields and provide habitat for wildlife. Drainage ditch management, which includes various forms of vegetation clearing and sediment dredging, can variably affect the ecological function of these systems. To determine whether ditch conditions following dredging/vegetation clearing management affected the survival, growth, and development of embryos and tadpoles of northern leopard frogs (Lithobates pipiens), we conducted three field studies using in situ cages over 2 years. We measured nutrients, pesticides, and other water quality properties in vegetated/unmanaged (i.e., no clearing or dredging) and newly cleared/dredged (i.e., treeless, then dredged), clay-bottomed drainage ditches in a river basin in Eastern Ontario, Canada. Nutrients, atrazine, and total neonicotinoid concentrations were generally lower at the cleared/dredged sites, whereas glyphosate was at higher concentrations. In contrast, water-quality variables measured in situ, particularly temperature, dissolved oxygen, and turbidity, tended to be higher in the cleared/dredged sites. Total phosphorous and total organic carbon concentrations at all sites were above the recommended limits for amphibian assays. No significant differences were detected in the survival, hatching success, or development of embryos among the ditch management treatments, but premature hatching was observed at one vegetated/unmanaged site where high specific conductivity may have been formative. We found the cleared/dredged sites supported earlier tadpole growth and development, likely as a result of the higher water temperatures. Increased temperature may have offset other growth/development stressors, such as those related to water chemistry. However, the long-term consequences of these differences on amphibian populations requires further study.

Structure Activity Relationship for Fumonisin Phytotoxicity.

Fumonisins are mycotoxins produced by a number of species of Fusarium and Aspergillus. They are polyketides that possess a linear polyol structure with two tricarballylic acid side chains and an amine moiety. Toxicity results from their inhibition of Ceramide Synthase (CerS), which perturbs sphingolipid concentrations. The tricarballylic side chains and amine group of fumonisins are key molecular features responsible for inhibiting CerS, however their individual contributions toward overall toxicity are not fully understood. We have recently reported novel, deaminated fumonisins produced by A. niger and have identified an enzyme (AnFAO) responsible for their synthesis. Here we performed a structure/function activity assay to investigate the individual contributions of the tricarballylic acid and amine toward overall fumonisin toxicity. Lemna minor was treated at 40 µM against FB1, hydrolyzed FB1 (hFB1), deaminated FB1 (FPy1), or hydrolyzed/deaminated (hFPy1). Four end points were monitored: plant dry weight, frond surface area, lipidomics, and metabolomics. Overall, hFB1 was less toxic than FB1 and FPy1 was less toxic than hFB1. hFPy1 which lacks both the amine group and tricarballylic side chains was also less toxic than FB1 and hFB1, however it was not significantly less toxic than FPy1. Lipidomic analysis showed that FB1 treatment significantly increased levels of phosphotidylcholines, ceramides, and pheophorbide A, while significantly decreasing the levels of diacylglycerides, sulfoquinovosyl diacylglycerides, and chlorophyll. Metabolomic profiling revealed a number of significantly increased compounds that were unique to FB1 treatment including phenylalanine, asymmetric dimethylarginine (ADMA), S-methylmethionine, saccharopine, and tyrosine. Conversely, citrulline, N-acetylornithine and ornithine were significantly elevated in the presence of hFB1 but not any of the other fumonisin analogues. These data provide evidence that although removal of the tricarballylic side chains significantly reduces toxicity of fumonisins, the amine functional group is a key contributor to fumonisin toxicity in L. minor and justify future toxicity studies in mammalian systems.

Imaging of Neurotransmitters and Small Molecules in Brain Tissues Using Laser Desorption/Ionization Mass Spectrometry Assisted with Zinc Oxide Nanoparticles.

Inorganic nanostructured materials such as silicon, carbon, metals, and metal oxides have been explored as matrices of low-background signals to assist the laser desorption/ionization (LDI) mass spectrometric (MS) analysis of small molecules, but their applications for imaging of small molecules in biological tissues remain limited in the literature. Titanium dioxide is one of the known nanoparticles (NP) that can effectively assist LDI MS imaging of low molecular weight molecules (LMWM). TiO2 NP is commercially available as dispersions, which can be applied using a chemical solution sprayer. However, aggregation of NP can occur in the dispersions, and the aggregated NP can slowly clog the sprayer nozzle. In this work, the use of zinc oxide (ZnO) NP for LDI MS imaging is investigated as a superior alternative due to its dissolution in acidic pH. ZnO NP was found to deliver similar or better results in the imaging of LMWM in comparison to TiO2 NP. The regular acid washes were effective in minimizing clogging and maintaining high reproducibility. High-quality images of mouse sagittal and rat coronal tissue sections were obtained. Ions were detected predominately as Na+ or K+ adducts in the positive ion mode. The number of LMWM detected with ZnO NP was similar to that obtained with TiO2 NP, and only a small degree of specificity was observed.

A combinatorial action of GmMYB176 and GmbZIP5 controls isoflavonoid biosynthesis in soybean (Glycine max).

GmMYB176 is an R1 MYB transcription factor that regulates multiple genes in the isoflavonoid biosynthetic pathway, thereby affecting their levels in soybean roots. While GmMYB176 is important for isoflavonoid synthesis, it is not sufficient for the function and requires additional cofactor(s). The aim of this study was to identify the GmMYB176 interactome for the regulation of isoflavonoid biosynthesis in soybean. Here, we demonstrate that a bZIP transcription factor GmbZIP5 co-immunoprecipitates with GmMYB176 and shows protein-protein interaction in planta. RNAi silencing of GmbZIP5 reduced the isoflavonoid level in soybean hairy roots. Furthermore, co-overexpression of GmMYB176 and GmbZIP5 enhanced the level of multiple isoflavonoid phytoallexins including glyceollin, isowighteone and a unique O-methylhydroxy isoflavone in soybean hairy roots. These findings could be utilized to develop biotechnological strategies to manipulate the metabolite levels either to enhance plant defense mechanisms or for human health benefits in soybean or other economically important crops.