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.

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.

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.

Identification of N,N',N″-triacetylfusarinine C as a key metabolite for root rot disease virulence in American ginseng.

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.

Metabolomic Profiling of Fungal Pathogens Responsible for Root Rot in American Ginseng.

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.