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In order to identify qualitative markers of a potential mycoherbicide against Canada thistle based on the mycelium of the fungus Stagonospora cirsii VIZR 1.41, changes in its pathogenicity and lipid profiles during submerged cultivation on liquid sucrose-soybean medium were analyzed. The fatty-acid composition of major lipids and the identification of molecular species of structural lipids were determined. The lipid composition during fungal growth changed together with its pathogenicity. The highly pathogenic mycelium of the fungus (at the log phase of growth) was characterized with the relatively highest content of structural lipids: phosphatidylcholine (PC), phosphatidylethanolamine (PE), and ergosterol (ES). Their relative concentration decreased with culture age and a decrease in pathogenicity. The highest lipid yield was found at the beginning of stationary growth phase (at the maximal biomass yield) of S. cirsii; triacylglycerides (TAGs) were the most abundant. The double-bond index of fatty acids in glycerolipids decreased with culture age, and the substitution of molecular PC species (34:2 versus 36:4) and PE (36:4 versus 34:2) was observed as well. High levels of ES, TAG, PC (in particular, 34:2 forms) and PE (36:4 forms) were presumed to be qualitative markers of S. cirsii mycelium.

Secondary metabolites were isolated from cultures of four geographically distant isolates of the cosmopolitan fungus, Bipolaris sorokiniana for intense biological characterization. Analysis of extracts from their liquid and solid cultures with TLC and HPLC-MS showed high similarity of their composition despite on fungal origin. From the extracts of B. sorokiniana 14 metabolites (terpenes, quinones, xanthones etc.) were isolated using column chromatography and preparative HPLC; among them, 12 known substances were identified. Chloromonilinic acid B (CAB) showed the highest level phytotoxic activity on wheat leaves. The most of isolated compounds inhibited growth of Bacillus subtilis, of which methyl-8,3-hydroxy-3-methyl-4-chloroxanthon-1-carboxilate (MMC) demonstrated the strongest activity. Some metabolites of B. sorokiniana (CAB and MMC) were shown to have insecticidal activity against common cereal aphid. All the compounds were cytotoxic (especially, cochlioqionones and fusaroproliferin) when tested on Sf9 cell line. However, no one was acute toxic for infusoria. Evidently, the production of multiple bioactive compounds makes B. sorokiniana a strong competitor in different ecological niches and a dangerous contaminant of grain.

In order to determine the possible toxic properties of a potential mycoherbicide based on the fungus Stagonospora cirsii S-47, the effects of solid and liquid media composition on the yield of extractive matter (YEM), the spectrum of biological activity, and the chromatography profiles of extracts obtained from cultures of this fungus were studied. The maximal YEM (~250 mg/L) was obtained from filtrates taken from a 2-week culture of S. cirsii S-47 grown on a sucrose–soy meal medium. The same time was optimal for the production of maximal YEM (~1.5 g/kg) when the fungus was grown on pearl-barley substrate. The fungal extracts showed phytotoxic, antimicrobial, cytotoxic, and antiesterase activity. Extracts from culture filtrates generally demonstrated a higher level and wider spectrum of biological activity than extracts from solid S. cirsii S-47 culture. The maximal phytotoxic and antimicrobial activity was found in dichloromethane extracts from the filtrate of 3-week fungal culture on Czapek medium, while extracts from two-week-old culture strongly inhibited carboxyl esterase. The maximal cytotoxic activity against the U251 cell line was found in ethyl acetate extract from a 3-week culture of S. cirsii S-47 produced on YMG liquid medium. The extract compositions varied considerably depending on the culture substrate.

Ten-membered lactones (nonenolides) demonstrate phytotoxic, antimicrobial, and fungicidal activity promising for the development of natural product-derived pesticides. The fungus Stagonospora cirsii is able to produce phytotoxic stagonolides A (1), J (2), K (3) and herbarumin I (4) with high yield. The aim of this study was to create a set of structurally related nonenolides and to reveal the structural features that affect their biological activity. Stagonolide A (1) and C-7 oxidized stagonolide K (11) showed the highest phytotoxicity in leaf puncture assay and agar seedlings assay. The oxidation of C-7 hydroxyl group (as in 1, acetylstagonolide A (10) and (11) led to the manifestation of toxicity to microalgae, Bacillus subtilis and Sf9 cells regardless of the configuration of C-9 propyl chains (R in 1 and 10, S in 11). C-7 non-oxidized nonenolides displayed none or little non-target activity. Notably, 7S compounds were more phytotoxic than their 7R analogues. Due to the high inhibitory activity against seedling growth and the lack of side toxicity, mono- and bis(acetyl)- derivatives of herbarumin I were shown to be potent for the development of pre-emergent herbicides. The identified structural features can be used for the rational design of new herbicides.

The study of fungal antibiotics in their competitive interactions with arthropods may lead to the development of novel biorational insecticides. Extracts of Alternaria tenuissima MFP253011 obtained using various methods showed a wide range of biological activities, including entomotoxic properties. Analysis of their composition and bioactivity allowed us to reveal several known mycotoxins and unidentified compounds that may be involved in the entomotoxic activity of the extracts. Among them, tenuazonic acid (TeA), which was the major component of the A. tenuissima extracts, was found the most likely to have larvicidal activity against Galleria mellonella. In the intrahaemocoel injection bioassay, TeA was toxic to G. mellonella and of Zophobas morio with an LT50 of 6 and 2 days, respectively, at the level of 50 µg/larva. Administered orally, TeA inhibited the growth of G. mellonella larvae and caused mortality of Acheta domesticus adults (LT50 7 days) at a concentration of 250 µg/g of feed. TeA showed weak contact intestinal activity against the two phytophages, Tetranychus urticae and Schizaphis graminum, causing 15% and 27% mortality at a concentration of 1 mg/mL, respectively. TeA was cytotoxic to the Sf9 cell line (IC50 25 µg/mL). Thus, model insects such as G. mellonella could be used for further toxicological characterization of TeA.

Study of fungal antibiotics in their competitive interactions with arthropods may lead to development novel biorational insecticides. Extracts of Alternaria tenuissima MFP253011 obtained by various methods showed a wide range of biological activity, including entomotoxic properties. Analysis of their composition and bioactivity allowed to reveal several known mycotoxins and unidentified compounds that may be involved in entomotoxic activity of the extracts. Among them, tenuazonic acid (TeA), which was the major component of the A. tenuissima extracts, was found the most likely to have larvicidal activity against Galleria mellonella. In the intrahaemocoel injection bioassay, TeA was toxic to G. mellonella and of Zophobas morio with LT50 6 and 2 days, respectively, at the level of 50 µg/larva. Administered orally, TeA inhibited growth of G. mellonella larvae and caused mortality of Acheta domesticus imagines (LT50 7 days) at a concentration of 250 µg/g of feed. TeA showed weak contact-intestinal activity against the two phytophages, Tetranychus urticae and Schizaphis graminum, causing the 12 and 40% of mortality at a concentration of 1 mg/mL. TeA was cytotoxic to Sf9 cell line (IC50 25 µg/mL). Thus, model insect G. mellonella and cell line Sf9 could be used for a further toxicological characterization of TeA.

The study of fungal antibiotics in their competitive interactions with arthropods may lead to the development of novel biorational insecticides. Extracts of Alternaria tenuissima MFP253011 obtained using various methods showed a wide range of biological activities, including entomotoxic properties. Analysis of their composition and bioactivity allowed us to reveal several known mycotoxins and unidentified compounds that may be involved in the entomotoxic activity of the extracts. Among them, tenuazonic acid (TeA), which was the major component of the A. tenuissima extracts, was found the most likely to have larvicidal activity against Galleria mellonella. In the intrahaemocoel injection bioassay, TeA was toxic to G. mellonella and of Zophobas morio with an LT50 of 6 and 2 days, respectively, at the level of 50 µg/larva. Administered orally, TeA inhibited the growth of G. mellonella larvae and caused mortality of Acheta domesticus adults (LT50 7 days) at a concentration of 250 µg/g of feed. TeA showed weak contact intestinal activity against the two phytophages, Tetranychus urticae and Schizaphis graminum, causing 15% and 27% mortality at a concentration of 1 mg/mL, respectively. TeA was cytotoxic to the Sf9 cell line (IC50 25 µg/mL). Thus, model insects such as G. mellonella could be used for further toxicological characterization of TeA.

Destruxin A (DA), a hexa-cyclodepsipeptidic mycotoxin produced by the entomopathogenic fungus Metarhizium anisopliae, exhibits insecticidal activities in a wide range of pests and is known as an innate immunity inhibitor. However, its mechanism of action requires further investigation. In this research, the interactions of DA with the six aminoacyl tRNA synthetases (ARSs) of Bombyx mori, BmAlaRS, BmCysRS, BmMetRS, BmValRS, BmIleRS, and BmGluProRS, were analyzed. The six ARSs were expressed and purified. The BLI (biolayer interferometry) results indicated that DA binds these ARSs with the affinity indices (KD) of 10−4 to 10−5 M. The molecular docking suggested a similar interaction mode of DA with ARSs, whereby DA settled into a pocket through hydrogen bonds with Asn, Arg, His, Lys, and Tyr of ARSs. Furthermore, DA treatments decreased the contents of soluble protein and free amino acids in Bm12 cells, which suggested that DA impedes protein synthesis. Lastly, the ARSs in Bm12 cells were all downregulated by DA stress. This study sheds light on exploring and answering the molecular target of DA against target insects.

A simple HPLC technique for the quantitative determination of phytotoxins (stagonolide A and herbarumin I) in liquid culture of the fungus Stagonospora cirsii S-47 has been developed. Some examples of its application are given. The technique was tested in a study of effects of the concentration of refined sunflower oil and the cultivation duration on the production level of these toxins. At an oil concentration of 0.5% or higher in modified Czapek medium with vitamins, the accumulation of S. cirsii biomass and the concentration of stagonolide A increased, while the concentration of herbarumin I decreased. Maximal amounts of both phytotoxins were obtained on the tenth day of submerged S. cirsii cultivation in flasks. When S. cirsii was grown in the same medium supplied with 1% of oil in a laboratory bioreactor, the maximal concentration of stagonolide A (116 mg/L) in the liquid culture was achieved after 5 days of fermentation followed by its considerable reduction after 6 days, while after 7 days of the submerged growth the maximal content of herbarumin I (116 mg/L) increased sharply. The technique of the phytotoxins analysis was used to monitor them during their isolation, as well as for preparative HPLC.

Two new natural 10-membered macrolides (1, 2) and one chromene-4,5-dione derivative (3), named stagonolides J and K and stagochromene A, respectively, were isolated from the phytopathogenic fungus Stagonospora cirsii S-47, together with two known compounds, stagonolide A (4) and herbarumin I (5). Stagonolides J and K and stagochromene A were characterized as (5E,7R*,8S*,9R*)-7,8-dihydroxy-9-propyl-5-nonen-9-olide, (5E,7R,9S)-7-hydroxy-9-propyl-5-nonen-9-olide, and (2R*,3R*)-3-hydroxy-2-propyltetrahydro-2H-chromene-4,5(3H,4aH)-dione, respectively, by spectroscopic (mostly by NMR and ESIMS) data. Compounds 1-5 showed different rates of phytotoxic activity on punctured leaf discs of Sonchus arvensis. The antimicrobial, cytotoxic, and antiprotozoal activity of isolated compounds was also evaluated. Based on our data, stagonolide K and herbarumin I can be proposed as a potential scaffold for the development of a new natural herbicide and estimated as possible selection/quality markers of a bioherbicide based on S. cirsii, while stagonolide A can be considered as a mycotoxin.

The effect of the nitrogen source (sodium nitrate and soybean meal) on the growth, pathogenicity, and lipid composition of mycelium of Stagonospora cirsii Davis VIZR 1.41, a potential mycoherbicide against Canada thistle (Cirsium arvens (L.) Scop.), was studied. The fungus grew significantly (two times) faster on sucrose-soybean meal medium (SSM) than on modified Czapek medium (CM). The fungal mycelium during the exponential growth phase demonstrated the maximal pathogenicity level on the third day on SSM and on the sixth day on CM. Canada thistle leaf tissues were more susceptible (25% higher development of necrotic lesion) to mycelium obtained on SSM than to mycelium obtained on CM. The nitrogen source strongly affected the lipid composition of S. cirsii mycelium. In S. cirsii mycelium obtained on SSM, the total lipid content was 1.7 times lower; the ratio of nonpolar lipids to polar lipids was three times lower than that for fungal mycelium grown on CM. Considerable differences were found in the composition of nonpolar (sterols and fatty acid) and polar (sphingolipids) lipids with respect to the nitrogen source in the culture medium. The higher contents of sterols, free fatty acids, and some glycoceramides in the mycelium of S. cirsii obtained on SSM may be related to its higher pathogenicity level and can serve as a marker of mycoherbicide quality.