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Zearalenone and radicicol are resorcylic acid lactones produced by numerous plant pathogenic fungi. Zearalenone is a non-steroidal estrogen mimic that can cause serious reproductive issues in livestock that consume contaminated feed. Radicicol is a potent inhibitor of the molecular chaperone Hsp90, which, in plants, has an important role in coordinating the host’s immune response during infection. Here, we describe the identification and characterization of a soil-borne strain of the Gram-positive bacterium Aeromicrobium sp. capable of hydrolyzing the macrolide ring of resorcylic acid lactones, including zearalenone and radicicol. Proteomic analysis of biochemically enriched fractions from the isolated and cultured bacterium identified an α/β-hydrolase responsible for this activity. A recombinantly expressed and purified form of the hydrolase (termed RALH) was active against both zearalenone and radicicol. Interpretation of high-resolution mass spectrometry and NMR data confirmed the structures of the enzymatic products as the previously reported non-toxic metabolite hydrolyzed zearalenone and hydrolyzed radicicol. Hydrolyzed radicicol was demonstrated to no longer inhibit the ATPase activity of the Saccharomyces cerevisiae Hsp90 homolog in vitro. Enzymatic degradation of resorcylic acid lactones will enable insight into their biological functions.

Mycotoxin emergence and co-occurrence trends in Canadian grains are dynamic and evolving in response to changing weather patterns within each growing season. The mycotoxins deoxynivalenol and zearalenone are the dominant mycotoxins detected in grains grown in Eastern Canada. Two potential emerging mycotoxins of concern are sterigmatocystin, produced by Aspergillus versicolor, and diacetoxyscirpenol, a type A trichothecene produced by a number of Fusarium species. In response to a call from the 83rd Joint Expert Committee on Food Additives and Contaminants, we conducted a comprehensive survey of samples from cereal production areas in Ontario, Canada. Some 159 wheat and 160 corn samples were collected from farms over a three-year period. Samples were extracted and analyzed by LC-MS/MS for 33 mycotoxins and secondary metabolites. Ergosterol was analyzed as an estimate of the overall fungal biomass in the samples. In wheat, the ratio of DON to its glucoside, deoxynivalenol-3-glucoside (DON-3G), exhibited high variability, likely attributable to differences among cultivars. In corn, the ratio was more consistent across the samples. Sterigmatocystin was detected in some wheat that had higher concentrations of ergosterol. Diacetoxyscirpenol was not detected in either corn or wheat over the three years, demonstrating a low risk to Ontario grain. Overall, there was some change to the mycotoxin profiles over the three years for wheat and corn. Ongoing surveys are required to reassess trends and ensure the safety of the food value chain, especially for emerging mycotoxins.

Accurate taxonomy is fundamental to the study and conservation of biodiversity. Because of their morphological similarities, most brook and river lampreys in western North America have been placed in the genus Lampetra along with lampreys from Eurasia and eastern North America. However, molecular-based phylogenetic studies dating back several decades indicate that lampreys from Pacific drainages are genetically distinct from Atlantic Lampetra. Reviewing previous phylogenetic analysis of two mitochondrial and two nuclear genes for Northern Hemisphere lampreys, we assign these western North American brook and river lampreys to a new genus, Occidentis. To assess species diversity within Occidentis, we performed a species delimitation analysis using all publicly available cytochrome b sequences of the genus. Similar to previous studies, O. ayresii and O. richardsoni were not reciprocally monophyletic and are best categorized as life history variants of a single species. In addition to O. pacifica, O. hubbsi, and the diverse O. ayresii species complex, as many as seven undescribed candidate species from Oregon and California were identified, supporting results from previous studies with more geographically limited datasets. One specimen from Paynes Creek, California, was identified as a candidate species, although this single individual showed minimal interspecific divergence (1.34%) with O. hubbsi. Further genetic assessment along with information on morphology and phylogeography is needed to determine whether the variation observed between groups of candidate species represents distinct species or divergent lineages within a species complex. Additional sampling will inform whether there are additional species not currently represented in this dataset. Thus, the number of species formally recognized under Occidentis is subject to change with new information. Systematic assessment of the distribution and phylogenetic complexity within Occidentis will enhance our understanding of its evolutionary history and taxonomic diversity, which will guide efforts to conserve the biodiversity of lampreys.

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

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.

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.

Anguraj Vadivel et al. show that a bZIP transcription factor GmbZIP5 interacts with an R1 MYB transcription factor GmMYB176 to regulate the biosynthesis of isoflavonoid in soybean. This study suggests biotechnological strategies to manipulate the levels of metabolites for plant defense or human health benefits in economically important crops.

Background: It is estimated that 20–30% of ginseng crops in Canada are lost to root rot each harvest. This disease is commonly caused by fungal infection with Ilyonectria, previously known as Cylindrocarpon. Previous reports have linked the virulence of fungal disease to the production of siderophores, a class of small-molecule iron chelators. However, these siderophores have not been identified in Ilyonectria. Methods: High-resolution LC–MS/MS was used to screen Ilyonectria and Cylindrocarpon strain extracts for secondary metabolite production. These strains were also tested for their ability to cause root rot in American ginseng and categorized as virulent or avirulent. The differences in detected metabolites between the virulent and avirulent strains were compared with a focus on siderophores. Results: For the first time, a siderophore N,N′,N″-triacetylfusarinine C (TAFC) has been identified in Ilyonectria, and it appears to be linked to disease virulence. Siderophore production was suppressed as the concentration of iron increased, which is in agreement with previous reports. Conclusion: The identification of the siderophore produced by Ilyonectria gives us further insight into the root rot disease that heavily affects ginseng crop yields. This research identifies a molecular pathway previously unknown for ginseng root rot and could lead to new disease treatment options.

The lamprey genus Lethenteron Creaser & Hubbs, 1922 is widespread across Eurasia and North America, but the number and distribution of its constituent species is not firmly established. After a morphological examination of extant type material of the currently recognized species and their synonyms, Lethenteron mitsukurii (Hatta, 1901) is resurrected with Le. matsubarai Vladykov & Kott, 1978 as its junior synonym. Amongst nonparasitic species Le. reissneri (Dybowski, 1869) and Le. mitsukurii are confirmed as present in Japan and the former is also present on Sakhalin. An in-depth study of large samples of nonparasitic lamprey adults from Japan and Sakhalin Island is needed to determine whether the lower trunk myomere (< 66) individuals from these areas represent one or more undescribed species, or Le. mitsukurii, or Le. reissneri, or a mixture of these three alternatives. The material from the Anadyr Estuary identified by Berg (1931, 1948) as Lampetra japonica kessleri has been re-identified as Le. camtschaticum and there is no evidence that Le. kessleri occurs there. Lethenteron reissneri is reported from the Angara River system, Yenisei River drainage, Russia. Lethenteron alaskense Vladykov & Kott, 1978 is provisionally considered to be a junior synonym of Le. kessleri (Anikin, 1905). Petromyzon ernstii Dybowski, 1872, Ammocoetes aureus Bean, 1881, Petromyzon dentex Anikin, 1905, Lampetra mitsukurii major Hatta, 1911, and Lampetra japonica septentrionalis Berg, 1931 are junior synonyms of Petromyzon marinus camtschaticus Tilesius, 1811. A key is provided to adults of the six species recognized as belonging in the genus Lethenteron.

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.

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.

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.

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.

The pouched lamprey, Geotria australis Gray, 1851, has long been considered monotypic in the Geotriidae family with a wide southern temperate distribution across Australasia and South America. Recent studies have provided molecular and morphological evidence for a second Geotria species in South America; Geotria macrostoma (Burmeister, 1868). The aim of this study was to determine morphometric and physical characteristics of adult G. macrostoma that further differentiate this re-instated species of Geotriidae from G. australis. The diagnostic features discriminating immature adult G. macrostoma from G. australis when entering fresh water, are distinct differences in dentition, oral papillae and fimbriae counts and differences in coloration. In addition, G. macrostoma display greater growth of the prebranchial region and oral disc and has a deeper body depth and higher condition factor. All current ecological knowledge of the genus Geotria is based on Australasian populations, which may not be applicable to G. macrostoma. To ensure the conservation and protection of the Patagonian lamprey as a re-identified species, further investigations are needed to understand its life history, biology and ecology throughout its range.

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

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.

Abstract Background Expression of economically relevant proteins in alternative expression platforms, especially plant expression platforms, has gained significant interest in recent years. A special interest in working with plants as bioreactors for the production of pharmaceutical proteins is related to low production costs, product safety and quality. Among the different properties that plants can also offer for the production of recombinant proteins, protein glycosylation is crucial since it may have an impact on pharmaceutical functionality and/or stability. Results The pharmaceutical glycoprotein human Granulocyte-Colony Stimulating Factor was transiently expressed in Nicotiana benthamiana plants and subjected to mammalian-specific mucin-type O-glycosylation by co-expressing the pharmaceutical protein together with the glycosylation machinery responsible for such post-translational modification. Conclusions The pharmaceutical glycoprotein human Granulocyte-Colony Stimulating Factor can be expressed in N. benthamiana plants via agroinfiltration with its native mammalian-specific mucin-type O-glycosylation.

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.

Purified phenolic extracts of cooked regular-darkening (RR) and non-darkening (CND) cranberry beans were evaluated for their phenolic content, antioxidant and anti-inflammatory activity and bioavailability using a human colonic carcinoma (Caco-2) cell model. Major compounds included flavanols such as catechin, epicatechin and proanthocyanidins which were predominant to RR extract. RR and CND both contained p-coumaric acid, ferulic acid and tryptophan. Our results showed strong cellular antioxidant activity in RR and dose-dependent attenuation of TNF-α-induced pro-inflammatory cytokine Interleukin-8 (IL-8) secretion. In addition, the extracts displayed protective benefit to endogenous antioxidant enzymes such as SOD, CAT, GPx and GR as well as GSH. Flavanols displayed poor bioavailability suggesting the beneficial effects occur via adsorption on the cell surface leading to cell signalling and localized radical scavenging activity. Our results support the beneficial effects of cranberry bean phenolics in their ability to ameliorate oxidative stress conditions notably in intestinal inflammatory responses.

Maple syrup, made by boiling the sap of Acer saccharum, is an important agriculture commodity in eastern Canada and New England. Although the collection season is relatively short, a rich progression in the sensory qualities of maple syrup can occur throughout the season. A risk associated with maple syrup production at the end of a season is the development of off-flavors that result in syrup with little to no commercial value. Maple syrup producers in Canada and the USA call this 'buddy syrup'. In this study, sugar maple (Acer saccharum) sap was collected in sequential samples through the harvest season from stands across Ontario. Metabolomics analysis of the sap samples was performed by high-resolution mass spectrometry. This revealed an evolution of the chemical composition, mainly occurring 30 days prior to leaf emergence. The major chemical constituent of maple syrup, sucrose, decreased sharply in late season sap, driven by microbial activity. The alditol mannitol increased in late season sap to concentrations ≥2 mg/mL and is likely an indicator of the start of photosynthesis. Amino acids, notably methionine and asparagine were present in higher amounts in late season sap. Non-targeted analysis revealed a series of related compounds that contained quaternary ammonium moieties including choline, hercynine, trigonelline, glycine betaine and carnitine increased in late season sap. These classes of compounds could act as methyl donors during the heating/evaporation of sap into syrup, affecting taste. Based on descriptions of the nature of buddy syrup and an extensive literature on flavors in foods, the amino acids methionine and asparagine were found as likely precursors to the compounds responsible for buddy syrup.

Xerophilic fungi grow at low water activity or low equilibrium relative humidity and are an important part of the indoor fungal community, of which Aspergillus is one of the dominant genera. A survey of xerophilic fungi isolated from Canadian and Hawaiian house dust resulted in the isolation of 1039 strains; 296 strains belong to Aspergillus and represented 37 species. Reference sequences were generated for all species and deposited in GenBank. Aspergillus sect. Aspergillus (formerly called Eurotium) was one of the most predominant groups from house dust with nine species identified. Additional cultures deposited as Eurotium were received from the Canadian Collection of Fungal Cultures and were also re-identified during this study. Among all strains, two species were found to be new and are introduced here as A. mallochii and A. megasporus. Phylogenetic comparisons with other species of section Aspergillus were made using sequences of ITS, β-tubulin, calmodulin and RNA polymerase II second largest subunit. Morphological observations were made from cultures grown under standardized conditions. Aspergillus mallochii does not grow at 37 °C and produces roughened ascospores with incomplete equatorial furrows. Aspergillus megasporus produces large conidia (up to 12 µm diam) and roughened ascospores with equatorial furrows. Echinulin, quinolactacin A1 & A2, preechinulin and neoechinulin A & B were detected as major extrolites of A. megasporus, while neoechinulin A & B and isoechinulin A, B & C were the major extrolites from A. mallochii.

Alternaria spp. occur as plant pathogens worldwide under field and storage conditions. They lead to food spoilage and also produce several classes of secondary metabolites that contaminate the food production chain. From a food safety perspective, the major challenge of assessing the risk of Alternaria contamination is the lack of a clear consensus on their species-level taxonomy. Furthermore, there are currently no reliable DNA sequencing methods to allow for differentiation of the toxigenic potential of these fungi. Our objective was to determine which species of Alternaria exist in Canada, and to describe the compounds they make. To address these issues, we performed metabolomic profiling using liquid chromatography high-resolution mass spectrometry (LC-HRMS) on 128 Canadian strains of Alternaria to determine their chemotaxonomy. The Alternaria strains were analyzed using principal component analysis (PCA) and unbiased k-means clustering to identify metabolites with significant differences (p < 0.001) between groups. Four populations or ‘chemotypes’ were identified within the strains studied, and several known secondary metabolites of Alternaria were identified as distinguishing metabolites, including tenuazonic acid, phomapyrones, and altenuene. Though species-level identifications could not be concluded for all groups through metabolomics alone, A. infectoria was able to be identified as a distinct population.

Fusarium head blight (FHB) caused by Fusarium graminearum is a disease that results in yield loss and mycotoxin contamination in wheat globally. This study assessed the effect of a plant biostimulant prepared from a brown macroalga Ascophyllum nodosum (Liquid Seaweed Extract; LSE) alone and in combination with chitosan in controlling Fusarium. Wheat seedlings drenched with LSE and chitosan in combination showed reduced severity of F. graminearum infection on leaves as evidenced by a significant reduction in necrotic area and fewer number of conidia produced in the necrotic area. Gene expression studies showed that the combination of LSE and chitosan amplified the response of pathogenesis-related genes (TaPR1.1, TaPR2, TaPR3, TaGlu2) in wheat seedlings infected with Fusarium spores above that observed for the individual treatments. The combination treatments were more effective in enhancing the activity of various defense related enzymes such as peroxidase and polyphenol oxidase. FHB studies on adult plants showed a reduction of bleached spikes in wheat heads treated with the combination of LSE and chitosan. Mycotoxin content appeared to be correlated with FHB severity. Combination treatments of LSE and chitosan reduced the levels of mycotoxins deoxynivalenol and sambucinol in wheat grains. Systemic disease resistance appears to be induced by LSE and chitosan in response to F. graminearum in wheat by inducing defense genes and enzymes.

Fusarium graminearum is responsible for production of the mycotoxin deoxynivalenol (DON) on maize and wheat in Ontario, Canada. It has been understood since the early 1980s that in most parts of Canada, the predominant chemotype of F. graminearum is 15ADON, and not the 3ADON chemotype mainly found in Europe and Asia. The discovery of F. graminearum strains that did not produce DON but the structurally related 7-α hydroxy, 15-deacetylcalonectrin (3ANX) and its hydrolysis product 7-α hydroxy, 3,15-dideacetylcalonectrin to (NX) demonstrated that we still have a lot to learn about this well studied but complicated fungus. We conducted a survey of maize and wheat samples from Ontario farms. In the 2015 crop year, we isolated 86 strains and tested a representative subset of 20 using the published genetic probes for assessing genotype. We also developed a targeted LC-MS/MS method for the identification and quantitation of known toxins from this species to determine chemotype. The results showed that 80% of our strains produced some 3ANX in addition to 15ADON and one strain produced 3ANX and no 15ADON. Comparison of chemical data with genotyping revealed that in more than 50% of the cases there was no clear agreement. These data demonstrate the importance of chemical analysis for understanding the toxigenic potential of strains, especially using a LC-MS method that is capable of differentiating 3ADON and 15ADON. For this collection, genotyping of isolates did not produce reliable information on the chemotype. This is the first report of 3ANX toxin production concurrently with 15ADON and suggests that the 3ANX producers in North America likely originated from the 15ADON background.

Surveys of foliar endophytes from the Acadian forest region over the past three decades have identified numerous phylogenetically diverse fungi producing natural products toxic to forest pests and diseases. The life histories of some conifer endophytes can be restricted to plant foliage or may include saprotrophic phases on other plants tissues or even alternate hosts. Considering the potentially broad host preferences of conifer endophytes we explored fungi isolated from understory species and their metabolites as part of an ongoing investigation of fungal biodiversity from the Acadian forest. We report a hitherto unidentified Xylariomycetidae species isolated from symptomatic Labrador tea (Rhododendron groenlandicum) leaves and mountain laurel (Kalmia latifolia) collected in coastal southern New Brunswick, Canada. Morphological and phylogenetic evidence demonstrated the unknown species was a novel Synnemapestaloides (Sporocadaceae) species, described here as Syn. ericacearum. A preliminary screening assay indicated that the culture filtrate extract of the new species was potently antifungal towards the biotrophic pathogen Microbotryum violaceum, warranting an investigation of its natural products. Two natural products possessing a rare 1,3-benzodioxin-4-one scaffold, synnemadoxins A-B (1-2), and their postulated precursor, synnemadiacid A (3), were characterized as new structures and assessed for antimicrobial activity. All isolated compounds elicited in vitro inhibitory antifungal activity towards M. violaceum at 2.3 μg mL-1 and moderate antibiotic activity. Further, the characterization of synnemadoxins A-B provided a perspective on the biosynthesis of some related 1,3-benzodioxin-4-ones produced by other fungi within the Xylariales.

Ginseng root is an economically valuable crop in Canada at high risk of yield loss caused by the pathogenic fungus Ilyonectria mors-panacis, formerly known as Cylindrocarpon destructans. While this pathogen has been well-characterized from morphological and genetic perspectives, little is known about the secondary metabolites it produces and their role in pathogenicity. We used an untargeted tandem liquid chromatography-mass spectrometry (LC-MS)-based approach paired with global natural products social molecular networking (GNPS) to compare the metabolite profiles of virulent and avirulent Ilyonectria strains. The ethyl acetate extracts of 22 I. mors-panacis strains and closely related species were analyzed by LC-MS/MS. Principal component analysis of LC-MS features resulted in two distinct groups, which corresponded to virulent and avirulent Ilyonectria strains. Virulent strains produced more types of compounds than the avirulent strains. The previously reported I. mors-panacis antifungal compound radicicol was present. Additionally, a number of related resorcyclic acid lactones (RALs) were putatively identified, namely pochonins and several additional derivatives of radicicol. Pochonins have not been previously reported in Ilyonectria spp. and have documented antimicrobial activity. This research contributes to our understanding of I. mors-panacis natural products and its pathogenic relationship with ginseng.