Herbicidal efficacy of harzianums produced by the biofertilizer fungus, Trichoderma brevicompactum.

Herbicides are important tools for weed control in modern agriculture. In the search for potential herbicidal natural products from fungal species, harzianum A and B were identified from the biofertilizer fungus, Trichoderma brevicompactum. In the phytotoxicity assays on the dicot species Brassica chinensis, harzianum A and B reduced both shoot and root lengths at low concentrations and inhibited the seed germination at 2 µg mL-1. In addition, harzianum A and B also exhibited phytotoxicity against monocots, Oryza sativa L. cv. Nipponbare and Echinochloa crusgalli L. Beauv.. Compared with a common herbicide, 2,4-dichlorophenoxyacetic acid, harzianum A and B performed similar activity in a pot assay, and were more effective in post-emergence than pre-emergence conditions. Harzianum A and B have potential as efficient herbicide for controlling important dicotyledon and monocotyledon weeds at low concentrations. They can be sprayed in liquid form in both pre- and post-emergence conditions. Our results confirmed the importance of these molecules for the development of new herbicides.

The bZIP Transcription Factor AflRsmA Regulates Aflatoxin B1 Biosynthesis, Oxidative Stress Response and Sclerotium Formation in Aspergillus flavus.

Fungal secondary metabolites play important roles not only in fungal ecology but also in humans living as beneficial medicine or harmful toxins. In filamentous fungi, bZIP-type transcription factors (TFs) are associated with the proteins involved in oxidative stress response and secondary metabolism. In this study, a connection between a bZIP TF and oxidative stress induction of secondary metabolism is uncovered in an opportunistic pathogen Aspergillus flavus, which produces carcinogenic and mutagenic aflatoxins. The bZIP transcription factor AflRsmA was identified by a homology research of A. flavus genome with the bZIP protein RsmA, involved in secondary metabolites production in Aspergillusnidulans. The AflrsmA deletion strain (ΔAflrsmA) displayed less sensitivity to the oxidative reagents tert-Butyl hydroperoxide (tBOOH) in comparison with wild type (WT) and AflrsmA overexpression strain (AflrsmAOE), while AflrsmAOE strain increased sensitivity to the oxidative reagents menadione sodium bisulfite (MSB) compared to WT and ΔAflrsmA strains. Without oxidative treatment, aflatoxin B1 (AFB1) production of ΔAflrsmA strains was consistent with that of WT, but AflrsmAOE strain produced more AFB1 than WT; tBOOH and MSB treatment decreased AFB1 production of ΔAflrsmA compared to WT. Besides, relative to WT, ΔAflrsmA strain decreased sclerotia, while AflrsmAOE strain increased sclerotia. The decrease of AFB1 by ΔAflrsmA but increase of AFB1 by AflrsmAOE was on corn. Our results suggest that AFB1 biosynthesis is regulated by AflRsmA by oxidative stress pathways and provide insights into a possible function of AflRsmA in mediating AFB1 biosynthesis response host defense in pathogen A. flavus.

The MAP kinase AflSlt2 modulates aflatoxin biosynthesis and peanut infection in the fungus Aspergillus flavus.

Aflatoxin contamination in food and feed products has been brought into sharp focus over the last few decades in the world. However, there is no effective strategy for solving the problem thus far. Therefore, basic research on the aflatoxin-producer Aspergillus flavus is an urgent need. The vital role of mitogen-activated protein kinases (MAPKs) in signal transduction has been documented in various pathogenic fungi, but their functions in A. flavus have rarely been investigated. Herein, we characterized the detailed function of one of these MAPKs, AflSlt2. Targeted deletion of AflSlt2 gene indicates that this kinase is required for vegetative growth, conidia generation, and sclerotium formation. The analysis of AflSlt2 deletion mutant revealed hypersensitivity to cell wall-damaging chemicals and resistance against hydrogen peroxide. Interestingly, the ability of the ΔAflSlt2 mutant to generate aflatoxins in medium was significantly increased compared to wild type. However, a pathogenicity assay indicated that the ΔAflSlt2 mutant was deficient in peanut infection. Site-directed mutation study uncovered that the function of AflSlt2 was dependent on the phosphorylated residues (Thr-186 and Tyr-188) within the activation loop and the phosphotransfer residue (Lys-52) within the subdomain II. Interestingly, an autophosphorylation mutant of AflSlt2 (AflSlt2R66S) displayed wild type-like phenotypes. Bringing these observations together, we propose that Slt2-MAPK pathway is involved in development, stress response, aflatoxin biosynthesis, and pathogenicity in A. flavus. This study may be useful to unveil the regulation mechanism of aflatoxin biosynthesis and provide strategy to control A. flavus contamination.

Secondary metabolites from hypocrealean entomopathogenic fungi: novel bioactive compounds.

Covering: 2014 up to the third quarter of 2019 Entomopathogens constitute a unique, specialized trophic subgroup of fungi, most of whose members belong to the order Hypocreales (class Sordariomycetes, phylum Ascomycota). These Hypocrealean Entomopathogenic Fungi (HEF) produce a large variety of secondary metabolites (SMs) and their genomes rank highly for the number of predicted, unique SM biosynthetic gene clusters. SMs from HEF have diverse roles in insect pathogenicity as virulence factors by modulating various interactions between the producer fungus and its insect host. In addition, these SMs also defend the carcass of the prey against opportunistic microbial invaders, mediate intra- and interspecies communication, and mitigate abiotic and biotic stresses. Thus, these SMs contribute to the role of HEF as commercial biopesticides in the context of integrated pest management systems, and provide lead compounds for the development of chemical pesticides for crop protection. These bioactive SMs also underpin the widespread use of certain HEF as nutraceuticals and traditional remedies, and allowed the modern pharmaceutical industry to repurpose some of these molecules as life-saving human medications. Herein, we survey the structures and biological activities of SMs described from HEF, and summarize new information on the roles of these metabolites in fungal virulence.

Regulation of Morphology, Aflatoxin Production, and Virulence of Aspergillus flavus by the Major Nitrogen Regulatory Gene areA.

Aspergillus flavus is a renowned plant, animal and human pathogen. areA is a global nitrogen regulatory gene of the GATA transcription factor family, shown to be the major nitrogen regulator. In this study, we identified areA in A. flavus and studied its function. The AreA protein contained a signatory zinc finger domain, which is extremely conserved across fungal species. Gene deletion (ΔareA) and over-expression (OE::areA) strains were constructed by homologous recombination to elucidate the role of areA in A. flavus. The ΔareA strain was unable to efficiently utilize secondary nitrogen sources for growth of A. flavus, and it had poorly developed conidiophores, when observed on complete medium, resulting in the production of significantly less conidia than the wild-type strain (WT). Aflatoxin B1 (AFB1) production was reduced in ΔareA compared with the WT strain in most conditions tested, and ΔareA had impaired virulence in peanut seeds. areA also played important roles in the sensitivity of A. flavus to osmotic, cell wall and oxidative stresses. Hence, areA was found to be important for the growth, aflatoxin production and pathogenicity of A. flavus. This work sheds light on the function of areA in the regulation of the nitrogen metabolism of A. flavus, and consequently aims at providing new ways for controlling the crossover pathogen, A. flavus.

Cyclase-Associated Protein Cap with Multiple Domains Contributes to Mycotoxin Biosynthesis and Fungal Virulence in Aspergillus flavus.

In Aspergillus, the cyclic adenosine monophosphate (cAMP) signaling modulates asexual development and mycotoxin biosynthesis. Here, we characterize the cyclase-associated protein Cap in the pathogenic fungus Aspergillus flauvs. The cap disruption mutant exhibited dramatic reduction in hyphal growth, conidiation, and spore germination, while an enhanced production of the sclerotia was observed in this mutant. Importantly, the cap gene was found to be important for mycotoxin biosynthesis and virulence. The domain deletion study demonstrated that each domain played an important role for the Cap protein in regulating cAMP/protein kinase A (PKA) signaling, while only P1 and CARP domains were essential for the full function of Cap. The phosphorylation of Cap at S35 was identified in A. flavus, which was found to play a negligible role for the function of Cap. Overall, our results indicated that Cap with multiple domains engages in mycotoxin production and fungal pathogenicity, which could be designed as potential control targets for preventing this fungal pathogen.

Study on the bio-function of lipA gene in Aspergillus flavus.

Lipoic acid synthase (LipA) plays a role in lipoic acid synthesis and potentially affects the levels of acetyl-CoA, the critical precursor of tricarboxylic acid (TCA) cycle. Considering the potential effect of LipA on TCA cycle, whether the enzyme is involved in the growth and aflatoxin B1 (AFB1) biosynthesis, the significant events in Aspergillus flavus is yet known. The study was designed to explore the role of lipA gene in A. flavus, including growth rate, conidiation, sclerotia formation, and biosynthesis of AFB1. LipA coding lipoic acid synthetase was knocked out using homologous recombination. The role of lipA gene in A. flavus morphogenesis (including colony size, conidiation, and sclerotia formation) was explored on various media, and the bio-function of lipA gene in the biosynthesis of AFB1 was analyzed by thin layer chromatography analysis. The growth was suppressed in △lipA. The formation of conidia and sclerotia was also reduced when lipA gene was deleted. Moreover, AFB1 was down-regulated in ΔlipA compared with WT controls. LipA plays a role in the development of A. flavus and AFB1 biosynthesis, contributing to the full understanding of the lipA bio-function in A. flavus.

Contribution of peroxisomal protein importer AflPex5 to development and pathogenesis in the fungus Aspergillus flavus.

Peroxisomes are important organelles that have diverse metabolic functions and participate in the pathogenicity of fungal pathogens. Previous studies indicate that most functions of peroxisomes are dependent on peroxisomal matrix proteins, which are delivered from the cytoplasm into peroxisomes by peroxisomal protein importers. In this study, the roles of peroxisomal protein importer AflPex5 were investigated in Aspergillus flavus with the application of gene disruption. AflPex5 deletion mutants failed to localize the fluorescently fused peroxisomal targeting signal 1 (PTS1) proteins to peroxisomes. Deletion of AflPex5 caused defects in sporulation, sclerotial formation, aflatoxin biosynthesis, stress response, and plant infection. Moreover, AflPex5 null mutants exhibited a significant defect in carbon metabolism and oxidants' clearance. These results indicate that the PTS1 pathway mediated by AflPex5 serves as an important role in the development, metabolism, and pathogenesis of A. flavus.

Carbon catabolite repression gene creA regulates morphology, aflatoxin biosynthesis and virulence in Aspergillus flavus.

Carbon catabolite repression (CCR) is a very important mechanism employed in the utilization of carbon as an energy source, required for the regulation of growth, development and secondary metabolite production in fungi. Despite the wide study of this mechanism in fungi, little is known about the major CCR gene creA in A. flavus. Hence, we report identification of A. flavus carbon catabolite repression gene creA, which is responsible for the repression of secondary carbon sources. Gene deletion and over-expression was employed to explicate the role of creA in the morphology, pathogenicity, and secondary metabolite production in A. flavus. We investigated these factors using three carbon sources including glucose, sucrose and maltose. Gene deletion mutant (ΔcreA) had a significant growth defect on complete medium and minimal medium containing maltose. Conidia production in ΔcreA was significantly impaired irrespective of the carbon source available, while sclerotia production was significantly increased, compared to wild type (WT) and over-expression strain (OE::creA). Importantly, ΔcreA produced insignificant amount of aflatoxin in complete medium, and its ability to colonize hosts was also impaired. Concisely, we showed that creA played an important role in the morphology, pathogenicity and secondary metabolite production of A. flavus.

Histone Methyltransferase aflrmtA gene is involved in the morphogenesis, mycotoxin biosynthesis, and pathogenicity of Aspergillus flavus.

Arginine methyltransferases catalyze the posttranslational methylation of arginine, which is involved in a range of important biological processes. aflrmtA gene, an arginine methyltransferase was deleted from Aspergillus flavus in this study by homologous recombination. In morphogenesis assay, aflrmtA was found to down-regulate conidiation by regulating the activity of brlA and abaA genes. It was also found to increase sclerotia formation by up-regulating the expression of nsdC and nsdD genes. In mycotoxin biosynthesis, aflrmtA gene was found to significantly up-regulate the biosynthesis of AFB1 in PDA and PDB media by improving the expression of aflR, aflC and aflK, but it was of no effect in YES medium. aflrmtA was further found to be an important regulator of response to plasma membrane lesion, osmotic, and H2O2 - induced oxidative stresses. In pathogenicity analysis, aflrmtA was found to repress conidiation and up-regulate the AFB1 biosynthesis of A. flavus on peanut and corn seeds and also the activities of protease and lipase, but the activity of amylase was down-regulated. It was concluded that aflrmtA gene played important roles in the morphogenesis, mycotoxin biosynthesis and pathogenicity of A. flavus, and it could be a potential target in the prevention and control of crop contamination by A. flavus.