Accumulation of pyrethroids induces changes in metabolism of the entomopathogenic fungus Beauveria bassiana-Proteomic and lipidomic background.

Advances in the agrochemical industry, such as using plant protection products e.g. pyrethroid insecticides, lead to environmental pollution via the accumulation of toxic compounds in soil. An interesting approach to overcoming this threat is using biopreparations based on entomopathogenic fungi that come into contact with the residues of the insecticides in the environment. The aim of this study was to determine whether the soil-dwelling entomopathogenic fungus Beauveria bassiana ARSEF 2860 is capable of accumulating pyrethroids (λ-cyhalothrin, α-cypermethrin and deltamethrin) and to identify the metabolomics and proteomic implications of this process. In this work, we demonstrated for the first time that the tested fungus accumulated pyrethroids as early as on day 2 of incubation with an average efficiency of 90%. Pyrethroids accumulated in large quantities in the mycelium of B. bassiana induced oxidative stress and interacted differently with the enzymes of the basic metabolic pathways, enzymes associated with the organization of the actin cytoskeleton and cell walls, as well as extracellular enzymes responsible for the infectious abilities (α-cypermethrin caused a 61% decrease in PR1, λ-cyhalothrin - a 31% decrease in PR2, which are proteolytic enzymes with a confirmed role in the infectious process). This study also revealed that the accumulated pyrethroids decreased the activity of phospholipase C, which increased the triacylglycerols/diacylglycerols (TAG/DAG) ratio, especially in mycelium in which α-cypermethrin was accumulated. It should be emphasized that the accumulation of pyrethroids in the environment is not fully understood, and current research suggests that entomopathogenic fungi may be part of the process.

Potential of Trichoderma harzianum and Its Metabolites to Protect Wheat Seedlings against Fusarium culmorum and 2,4-D.

Wheat is a critically important crop. The application of fungi, such as Trichoderma harzianum, to protect and improve crop yields could become an alternative solution to synthetic chemicals. However, the interaction between the fungus and wheat in the presence of stress factors at the molecular level has not been fully elucidated. In the present work, we exposed germinating seeds of wheat (Triticum aestivum) to the plant pathogen Fusarium culmorum and the popular herbicide 2,4-dichlorophenoxyacetic acid (2,4-D) in the presence of T. harzianum or its extracellular metabolites. Then, the harvested roots and shoots were analyzed using spectrometry, 2D-PAGE, and MALDI-TOF/MS techniques. Although F. culmorum and 2,4-D were found to disturb seed germination and the chlorophyll content, T. harzianum partly alleviated these negative effects and reduced the synthesis of zearalenone by F. culmorum. Moreover, T. harzianum decreased the activity of oxidoreduction enzymes (CAT and SOD) and the contents of the oxylipins 9-Hode, 13-Hode, and 13-Hotre induced by stress factors. Under the influence of various growth conditions, changes were observed in over 40 proteins from the wheat roots. Higher volumes of proteins and enzymes performing oxidoreductive functions, such as catalase, ascorbate peroxidase, cytochrome C peroxidase, and Cu/Zn superoxide dismutase, were found in the Fusarium-inoculated and 2,4-D-treated wheat roots. Additionally, observation of the level of 12-oxo-phytodienoic acid reductase involved in the oxylipin signaling pathway in wheat showed an increase. Trichoderma and its metabolites present in the system leveled out the mentioned proteins to the control volumes. Among the 30 proteins examined in the shoots, the expression of the proteins involved in photosynthesis and oxidative stress response was found to be induced in the presence of the herbicide and the pathogen. In summary, these proteomic and metabolomic studies confirmed that the presence of T. harzianum results in the alleviation of oxidative stress in wheat induced by 2,4-D or F. culmorum.

Trichoderma harzianum metabolites disturb Fusarium culmorum metabolism: Metabolomic and proteomic studies.

Trichoderma species are well known for producing various secondary metabolites in response to different fungal pathogens. This paper reports the effects of the metabolites produced during one-day cultivation of Trichoderma harzianum on the growth and development of the popular pathogen Fusarium culmorum. Inhibition of the growth of the pathogen and production of secondary metabolites including zearalenone was observed on Petri dishes. The presence of proteins such as cytochrome c oxidase subunit 4, glutathione-independent glyoxalase HSP31, and putative peroxiredoxin pmp20 in the extract-treated culture indicated oxidative stress, which was confirmed by the presence of a higher amount of catalase and dismutase in the later hours of the culture. A larger amount of enolase and glyceraldehyde 3-phosphate dehydrogenase resulted in faster growth, and the overexpression of stress protein and Woronin body major protein indicated the activation of defense mechanisms. In addition, a cardinal reduction in major mycotoxin production was noted.

Atrazine biodegradation by mycoinsecticide Metarhizium robertsii: Insights into its amino acids and lipids profile.

Atrazine, is one of major concern pesticides contaminating agricultural areas and ground water. Its microbial biodegradation seems to be the most efficient in terms of economic and environmental benefits. In the present work the cometabolic biodegradation of atrazine by the fungus Metarhizum robertsii IM 6519 during 10-day batch cultures was characterized. The herbicide was transformed to several hydroxy-, dechlorinated or dealkylated metabolites with the involvement of cytochrome P450 monooxygenases. The obtained metabolomics data revealed that atrazine induced oxidative stress (increased the levels of L-proline, L-ornithine, L-arginine, GABA and L-methionine), disruptions of the carbon and nitrogen metabolism (L-aspartic acid, L-asparagine, L-tyrosine, L-threonine, L-isoleucine, L-phenylalanine, 1-methyl-L-histidine, L-tryptophan, L-valine, L-alanine, O-phospho-L-serine, L-sarcosine or L-lysine) and caused an increase in the membrane fluidity (a rise in the phosphatidylcholines/phosphatidylethanolamines (PC/PE) ratio together with the growth of the taurine level). The increased level of hydroxyl derivatives of linoleic acid (9-HODE and 13-HODE) confirmed that atrazine induced lipid peroxidation. The presented results suggesting that M. robertsii IM 6519 might be applied in atrazine biodegradation and may bring up the understanding of the process of triazine biodegradation by Metarhizum strains.

Lipids, proteins and extracellular metabolites of Trichoderma harzianum modifications caused by 2,4-dichlorophenoxyacetic acid as a plant growth stimulator.

Strains of Trichoderma harzianum are well-known producers of bioactive secondary metabolites and have a beneficial effect on plants. However, to the best of our knowledge, the effect of the commonly used pesticides on the activity of this fungus is not yet investigated. Therefore, in the present study, the effect of the herbicide 2,4-dichlorophenoxyacetic acid (2,4-D) on the lipidome and selected extracellular compounds synthesized by T. harzianum IM 0961 was examined. It was observed that the herbicide 2,4-D caused changes in the lipid composition of the mycelium and that the herbicide exhibited lipophilic properties. In addition, the herbicide disturbed the phosphatidylcholine (PC)/phosphatidylethanolamine (PE) ratio and increased membrane permeability. The higher amount of cardiolipin CL 72:7 and the lower amount of CL 72:8 could have been associated with a decreased ratio of 18:2 and 18:1 fatty acids in the herbicide-treated samples. Moreover, in the presence of 2,4-D, an increased lipid peroxidation (twofold), as well as a higher content of oxylipin (9-HODE and 13-HODE) and phosphatidic acid (PA), was noted, confirming that 2,4-D induced lipid peroxidation in the mycelium. The herbicide also exerted its toxic effect on the production of 14-aminoacid peptaibols and two compounds, harzianic acid and t22-azaphilone, exhibiting antibiotic and plant growth-promoting activity. During proteomic analysis, the synthesis of some proteins, such as calcineurin-like phosphoesterase metallophosphatases (MPPs), which modulate the properties of cell walls, was found to be inhibited by the herbicide. These presented findings may be of significant value in understanding the effect of 2,4-D on the activity of T. harzianum.