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Figure S1. m/z distribution diagrams and Base Peak Chromatograms of C. truncatum isolate C428 (from lentil) extracts. (a) PDA extract; (b) PDB extract; (c) Rice extract; (d) Richard extract. Figure S2. m/z distribution diagrams and Base Peak Chromatograms of C. trucatum isolate C431 (from soybean) extracts. (a) PDA extract; (b) PDB extract; (c) Rice extract; (d) Richard extract. Figure S3. m/z distribution diagrams and Base Peak Chromatograms of C. trifolii isolate C436 (from clover extracts. (a) PDA extract; (b) PDB extract; (c) Rice extract; (d) Richard extract. Figure S4. Structure of: validated metabolites with pure standards (level A; red); putatively identified and produced by Colletotrichum spp. (level B(i), blue); putatively identified and produced by other fungal species (level B(ii), black); Structures of Curvupallide A and B (Level C(i), brown). Table S1. Parameters Metaboanalyst 5.0 for LC-MS spectra processing. Table S2. Features lists transformation and scaling for PLS – DA analysis. Q2 and R2 values of PLS – DA models in cross validation. Table S3. Metabolites dereplicated by targeted and untargeted metabolomics analysis, organised according to identification level. Table S4. Metabolites dereplicated by targeted and untargeted metabolomics analysis, organised according to identification level.

Supplementary Figure S1. 1H-NMR spectrum of ascochlorin (1) (CDCl3, 400 MHz); Supplementary Figure S2. ESI/MS (+) spectrum of ascochlorin (1); Supplementary Figure S3. 1H-NMR spectrum of ascofuranol (2) (CDCl3, 400 MHz); Supplementary Figure S4. ESI/MS (+) spectrum of ascofuranol (2); Supplementary Figure S5. 1H-NMR spectrum of (R)-mevalonolactone (3) (CDCl3, 400 MHz); Supplementary Figure S6. ESI/MS (+) spectrum of (R)-mevalonolactone (3); Supplementary Figure S7. 1H-NMR spectrum of ascosalipyrone (4) (CDCl3, 400 MHz); Supplementary Figure S8. ESI/MS (+) spectrum of ascosalipyrone (4); Supplementary Figure S9. 1H-NMR spectrum of benzoic acid (5) (CDCl3, 400 MHz); Supplementary Figure S10. ESI/MS (+) spectrum of benzoic acid (5); Supplementary Figure S11. 1H-NMR spectrum of tyrosol (6) (CDCl3, 400 MHz); Supplementary Figure S12. ESI/MS (+) spectrum of tyrosol (6); Supplementary Figure S13. 1H-NMR spectrum of ascosalitoxin (7) (CDCl3, 400 MHz); Supplementary Figure S14. ESI/MS (+) spectrum of ascosalitoxin (7); Supplementary Figure S15. Images of the symptoms of each of the compounds. Supplementary Table S1. Diseased area (mm2) measured on detached leaves of several legume crops with exudates from the fungus A. fabae growth in vitro on 3 different culture media (Czapek-Dox = CD, potato dextrose broth = PDB, and rice) at concentrations of 0.5, 1, and 2 mg/mL. Negative (blank untreated, water and MeOH 5%) controls were also included. The experiment was repeated four times. Supplementary Table S2. Diseased area (mm2) measured in leaves detached from various legume crops with metabolites produced by the exudate of the A. fabae fungus from the three-growth media at concentrations of 1, 10, and 100 µM. Negative controls (untreated blank, water, and MeOH 5%) were also included. p-value compared with value from MeOH 5% control.

Different fungal species belonging to the Colletotrichum genus cause anthracnose disease in a range of major crops, resulting in huge economic losses worldwide. Typical symptoms include dark, sunken lesions on leaves, stems, or fruits. Colletotrichum spp. have synthesized, in vitro, a number of biologically active and structurally unusual metabolites that are involved in their host’s infection process. In this study, we applied a one strain many compounds (OSMAC) approach, integrated with targeted and non-targeted metabolomics profiling, to shed light on the secondary phytotoxic metabolite panels produced by pathogenic isolates of Colletotrichum truncatum and Colletotrichum trifolii. The phytotoxicity of the fungal crude extracts was also assessed on their primary hosts and related legumes, and the results correlated with the metabolite profile that arose from the different cultural conditions. To the best of our knowledge, this is the first time that the OSMAC strategy integrated with metabolomics approaches has been applied to Colletotrichum species involved in legume diseases.

Fungal phytotoxins can be defined as secondary metabolites toxic to host plants and are believed to be involved in the symptoms developed of a number of plant diseases by targeting host cellular machineries or interfering with host immune responses. As any crop, legumes can be affected by a number of fungal diseases, causing severe yield losses worldwide. In this review, we report and discuss the isolation, chemical, and biological characterization of fungal phytotoxins produced by the most important necrotrophic fungi involved in legume diseases. Their possible role in plant-pathogen interaction and structure-toxicity relationship studies have also been reported and discussed. Moreover, multidisciplinary studies on other prominent biological activity conducted on reviewed phytotoxins are described. Finally, we explore the challenges in the identification of new fungal metabolites and their possible applications in future experiments.

Ascochyta blight, caused by Ascochyta fabae, poses a significant threat to faba bean and other legumes worldwide. Necrotic lesions on stems, leaves, and pods characterize the disease. Given the economic impact of this pathogen and the potential involvement of secondary metabolites in symptom development, a study was conducted to investigate the fungus's ability to produce bioactive metabolites that might contribute to its pathogenicity. For this investigation, the fungus was cultured in three substrates (Czapek-Dox, PDB, and rice). The produced metabolites were analyzed by NMR and LC-HRMS methods, resulting in the dereplication of seven metabolites, which varied with the cultural substrates. Ascochlorin, ascofuranol, and (R)-mevalonolactone were isolated from the Czapek-Dox extract; ascosalipyrone, benzoic acid, and tyrosol from the PDB extract; and ascosalitoxin and ascosalipyrone from the rice extract. The phytotoxicity of the pure metabolites was assessed at different concentrations on their primary hosts and related legumes. The fungal exudates displayed varying degrees of phytotoxicity, with the Czapek-Dox medium's exudate exhibiting the highest activity across almost all legumes tested. The species belonging to the genus Vicia spp. were the most susceptible, with faba bean being susceptible to all metabolites, at least at the highest concentration tested, as expected. In particular, ascosalitoxin and benzoic acid were the most phytotoxic in the tested condition and, as a consequence, expected to play an important role on necrosis's appearance.

[Background] Rusts and powdery mildews are severe fungal diseases of major crops worldwide, including cereals and legumes. They can be managed by chemical fungicide treatments, with negative consequences as environmental pollution and risk for human and animal health. Bioactive natural products could be the safest alternative for pest control. The family of anthraquinones, as well as analogue compounds containing an anthraquinone moiety or some modified anthraquinone rings, has been reported to exhibit certain antibiotic activity. Thus, the potential antifungal activity of some anthraquinones isolated from Ascochyta lentis, was assayed in this study for their effectiveness to reduce rust and powdery mildew diseases on pea and oat. Their effect on fungal development was macro- and microscopically assessed on inoculated leaves, and compared to the control achieved by the chemical fungicide (Tetraconazol 12.5% and Azoxystrobin 25%). In addition, the most promising compound was also tested at different concentrations in inoculated whole plants in order to evaluate its preventive and curative potential against fungal infection., [Results] All metabolites studied strongly reduced the development of rust and powdery mildews in both pea and oat, being pachybasin and lentiquinone C the most effective ones in hampering fungal spore germination and appressoria formation. Some of them also affected post-penetration events reducing colony size and number of haustoria per colony. Results were confirmed for pachybasin in whole plants assays, showing an efficacy similar to the commercial fungicide to control fungal diseases, both in preventive and curative applications. [Conclusions] Some fungal anthraquinones and close metabolites, especially pachybasin, could be very promising molecules with effective potential as antifungal agents against both rust and powdery mildew of both pea and oat. Some structure activity-relationships feature have also been evaluated. © 2022 The Authors. Pest Management Science published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.

[Background] Pea powdery mildew incited by Erysiphe pisi represents a major constraint for pea crops worldwide. Crop protection is largely based on chemical control, although recently a renewed interest in the discovery of natural products as alternatives to synthetic fungicides application has emerged. Thus, 12 bioactive plant and fungal metabolites belonging to different class of natural compounds were evaluated, together with a commercial fungicide, at different concentrations on detached pea leaves for their potential to inhibit spore germination and subsequent stages of fungal growth. The most effective metabolites were tested at different concentrations in planta under controlled conditions to evaluate the level of control achieved by treatments before, concurrently and after pathogen inoculation. Pathogen development was macroscopically scored on whole plants as percentage of disease severity and area under the disease progress curve., [Results] Cavoxin, inuloxin C and sphaeropsidin A strongly inhibited E. pisi germination and haustoria formation and reduced colony size. This effect was dose dependent. These results were further confirmed in whole plants by spraying the metabolites on plant leaves for preventive or curative control, which reduced fungal developmental of E. pisi at levels comparable with those obtained by application of the fungicide., [Conclusions] Cavoxin, inuloxin C and sphaeropsidin A have potential as alternatives to synthetic fungicides for the control of crop pathogens of economic importance such as powdery mildew. © 2019 Society of Chemical Industry