Broad-Spectrum Antimicrobial Activity of Synthetic Peptides GV185 and GV187.

Optimizing synthetic antimicrobial peptides for safe and enhanced activity against fungal and bacterial pathogens is useful for genetic engineering of plants for resistance to plant pathogens and their associated mycotoxins. Nine synthetic peptides modeled after lytic peptides tachyplesin 1, D4E1 from cecropin A, and protegrin 1 were added to germinated spores of fungal species Aspergillus flavus, Rhizopus stolonifer, Fusarium oxysporum f. sp. vasinfectum, F. verticillioides, F. graminearum, Claviceps purpurea, Verticillium dahliae, and Thielaviopsis basicola and bacterial cultures of Pseudomonas syringae pv. tabaci and Xanthomonas campestris pv. campestris at different doses and inhibitory dose response curves, and were modeled to assess antimicrobial activity. Peptides GV185 and GV187, modified from tachyplesin 1, had superior abilities to inhibit fungal and bacterial growth (50% inhibitory concentrations [IC50] ranging from 0.1 to 8.7 µM). R. stolonifer (IC50 = 8.1 µM), A. flavus (IC50 = 3.1 µM), and F. graminearum (IC50 = 2.2 µM) were less inhibited by GV185 and GV187 than all the remaining fungi (IC50 = 1.4 µM) and bacteria (IC50 = 0.1 µM). Of the remaining peptides, GV193, GV195, and GV196 (IC50 range of 0.9 to 6.6 µM) inhibited fungal growth of A. flavus, F. verticillioides, and F. graminearum less than GV185 and GV187 (IC50 range of 0.8 to 3.9 µM), followed by GV197 (IC50 range of 0.8 to 9.1 µM), whereas GV190 and GV192 inhibited poorly (IC50 range of 28.2 to 36.6 µM and 15.5 to 19.4 µM, respectively) and GV198 stimulated growth. GV185 and GV187 had slightly weaker hydrophobic and cationic residues than other tachyplesin 1 modified peptides but still had unexpectedly high lytic activity. Germinated fungal spores of R. stolonifer and F. graminearum exposed to these two peptides and D4E1 and AGM182 appeared wrinkled, with perforations near potential cytoplasmic leakage, which provided evidence of plasma membrane and cell wall lysis. We conclude that peptides GV185 and GV187 are promising candidates for genetic engineering of crops for resistance to plant-pathogenic bacteria and fungi, including A. flavus and aflatoxin contamination.

Intraspecific Growth and Aflatoxin Inhibition Responses to Atoxigenic Aspergillus flavus: Evidence of Secreted, Inhibitory Substances in Biocontrol.

The fungus Aspergillus flavus infects corn, peanut, and cottonseed, and contaminates seeds with acutely poisonous and carcinogenic aflatoxin. Aflatoxin contamination is a perennial threat in tropical and subtropical climates. Nonaflatoxin-producing isolates (atoxigenic) are deployed in fields to mitigate aflatoxin contamination. The biocontrol competitively excludes toxigenic A. flavus via direct replacement and thigmoregulated (touch) toxin inhibition mechanisms. To understand the broad-spectrum toxin inhibition, toxigenic isolates representing different mating types and sclerotia sizes were individually cocultured with different atoxigenic biocontrol isolates. To determine whether more inhibitory isolates had a competitive advantage to displace or touch inhibit toxigenic isolates, biomass accumulation rates were determined for each isolate. Finally, to determine whether atoxigenic isolates could inhibit aflatoxin production without touch, atoxigenic isolates were grown separated from a single toxigenic isolate by a membrane. Atoxigenic isolates 17, Af36, and K49 had superior abilities to inhibit toxin production. Small (<400 µm) sclerotial, Mat1-1 isolates were not as completely inhibited as others by most atoxigenic isolates. As expected for both direct replacement and touch inhibition, the fastest-growing atoxigenic isolates inhibited aflatoxin production the most, except for atoxigenic Af36 and K49. Aflatoxin production was inhibited when toxigenic and atoxigenic isolates were grown separately, especially by slow-growing atoxigenic Af36 and K49. Additionally, fungus-free filtrates from atoxigenic cultures inhibited aflatoxin production. Toxin production inhibition without direct contact revealed secretion of diffusible chemicals as an additional biocontrol mechanism. Biocontrol formulations should be improved by identifying isolates with broad-spectrum, high-inhibition capabilities and production of secreted inhibitory chemicals.

Why Do Plant-Pathogenic Fungi Produce Mycotoxins? Potential Roles for Mycotoxins in the Plant Ecosystem.

For many plant-pathogenic or endophytic fungi, production of mycotoxins, which are toxic to humans, may present a fitness gain. However, associations between mycotoxin production and plant pathogenicity or virulence is inconsistent and difficult due to the complexity of these host-pathogen interactions and the influences of environmental and insect factors. Aflatoxin receives a lot of attention due to its potent toxicity and carcinogenicity but the connection between aflatoxin production and pathogenicity is complicated by the pathogenic ability and prevalence of nonaflatoxigenic isolates in crops. Other toxins directly aid fungi in planta, trichothecenes are important virulence factors, and ergot alkaloids limit herbivory and fungal consumption due to insect toxicity. We review a panel discussion at the American Phytopathological Society's Plant Health 2021 conference, which gathered diverse experts representing different research sectors, career stages, ethnicities, and genders to discuss the diverse roles of mycotoxins in the lifestyles of filamentous fungi of the families Clavicipitaceae, Trichocomaceae (Eurotiales), and Nectriaceae (Hypocreales).

Divergent Aspergillus flavus corn population is composed of prolific conidium producers: Implications for saprophytic disease cycle.

The ascomycete fungus Aspergillus flavus infects and contaminates corn, peanuts, cottonseed, and tree nuts with toxic and carcinogenic aflatoxins. Subdivision between soil and host plant populations suggests that certain A. flavus strains are specialized to infect peanut, cotton, and corn despite having a broad host range. In this study, the ability of strains isolated from corn and/or soil in 11 Louisiana fields to produce conidia (field inoculum and male gamete) and sclerotia (resting bodies and female gamete) was assessed and compared with genotypic single-nucleotide polymorphism (SNP) differences between whole genomes. Corn strains produced upward of 47× more conidia than strains restricted to soil. Conversely, corn strains produced as much as 3000× fewer sclerotia than soil strains. Aspergillus flavus strains, typified by sclerotium diameter (small S-strains, <400 µm; large L-strains, >400 µm), belonged to separate clades. Several strains produced a mixture (M) of S and L sclerotia, and an intermediate number of conidia and sclerotia, compared with typical S-strains (minimal conidia, copious sclerotia) and L-strains (copious conidia, minimal sclerotia). They also belonged to a unique phylogenetic mixed (M) clade. Migration from soil to corn positively correlated with conidium production and negatively correlated with sclerotium production. Genetic differences correlated with differences in conidium and sclerotium production. Opposite skews in female (sclerotia) or male (conidia) gametic production by soil or corn strains, respectively, resulted in reduced effective breeding population sizes when comparing male:female gamete ratio with mating type distribution. Combining both soil and corn populations increased the effective breeding population, presumably due to contribution of male gametes from corn, which fertilize sclerotia on the soil surface. Incongruencies between aflatoxin clusters, strain morphotype designation, and whole genome phylogenies suggest a history of sexual reproduction within this Louisiana population, demonstrating the importance of conidium production, as infectious propagules and as fertilizers of the A. flavus soil population.

Putative Core Transcription Factors Affecting Virulence in Aspergillus flavus during Infection of Maize.

Aspergillus flavus is an opportunistic pathogen responsible for millions of dollars in crop losses annually and negative health impacts on crop consumers globally. A. flavus strains have the potential to produce aflatoxin and other toxic secondary metabolites, which often increase during plant colonization. To mitigate the impacts of this international issue, we employ a range of strategies to directly impact fungal physiology, growth and development, thus requiring knowledge on the underlying molecular mechanisms driving these processes. Here we utilize RNA-sequencing data that are obtained from in situ assays, whereby Zea mays kernels are inoculated with A. flavus strains, to select transcription factors putatively driving virulence-related gene networks. We demonstrate, through growth, sporulation, oxidative stress-response and aflatoxin/CPA analysis, that three A. flavus strains with knockout mutations for the putative transcription factors AFLA_089270, AFLA_112760, and AFLA_031450 demonstrate characteristics such as reduced growth capacity and decreased aflatoxin/CPA accumulation in kernels consistent with decreased fungal pathogenicity. Furthermore, AFLA_089270, also known as HacA, eliminates CPA production and impacts the fungus's capacity to respond to highly oxidative conditions, indicating an impact on plant colonization. Taken together, these data provide a sound foundation for elucidating the downstream molecular pathways potentially contributing to fungal virulence.

Genetic Responses and Aflatoxin Inhibition during Co-Culture of Aflatoxigenic and Non-Aflatoxigenic Aspergillus flavus.

Aflatoxin is a carcinogenic mycotoxin produced by Aspergillus flavus. Non-aflatoxigenic (Non-tox) A. flavus isolates are deployed in corn fields as biocontrol because they substantially reduce aflatoxin contamination via direct replacement and additionally via direct contact or touch with toxigenic (Tox) isolates and secretion of inhibitory/degradative chemicals. To understand touch inhibition, HPLC analysis and RNA sequencing examined aflatoxin production and gene expression of Non-tox isolate 17 and Tox isolate 53 mono-cultures and during their interaction in co-culture. Aflatoxin production was reduced by 99.7% in 72 h co-cultures. Fewer than expected unique reads were assigned to Tox 53 during co-culture, indicating its growth and/or gene expression was inhibited in response to Non-tox 17. Predicted secreted proteins and genes involved in oxidation/reduction were enriched in Non-tox 17 and co-cultures compared to Tox 53. Five secondary metabolite (SM) gene clusters and kojic acid synthesis genes were upregulated in Non-tox 17 compared to Tox 53 and a few were further upregulated in co-cultures in response to touch. These results suggest Non-tox strains can inhibit growth and aflatoxin gene cluster expression in Tox strains through touch. Additionally, upregulation of other SM genes and redox genes during the biocontrol interaction demonstrates a potential role of inhibitory SMs and antioxidants as additional biocontrol mechanisms and deserves further exploration to improve biocontrol formulations.

Draft Genome Sequences of 20 Aspergillus flavus Isolates from Corn Kernels and Cornfield Soils in Louisiana.

Aspergillus flavus is a common saprophyte and opportunistic fungal pathogen that infects plants, animals, and humans. It also produces numerous toxic and nontoxic secondary metabolites. Here, we report the draft genome sequences of 20 A. flavus isolates, belonging to 16 vegetative compatibility groups, from Louisiana corn kernels and cornfield soils.

Influence of Neighboring Clonal-Colonies on Aflatoxin Production by Aspergillus flavus.

Aspergillus flavus is an ascomycete fungus that infects and contaminates corn, peanuts, cottonseed, and treenuts with acutely toxic and carcinogenic aflatoxins. The ecological function of aflatoxin production is not well understood; though not phytotoxic, aflatoxin may be involved in resisting oxidative stress responses from infection or drought stress in plants. Observation of aflatoxin stimulation in 48-well plates in response to increasing inoculated wells sparked an investigation to determine if A. flavus volatiles influence aflatoxin production in neighboring colonies. Experiments controlling several culture conditions demonstrated a stimulation of aflatoxin production with increased well occupancy independent of pH buffer, moisture, or isolate. However, even with all wells inoculated, aflatoxin production was less in interior wells. Only one isolate stimulated aflatoxin production in a large Petri-dish format containing eight small Petri dishes with shared headspace. Other isolates consistently inhibited aflatoxin production when all eight Petri dishes were inoculated with A. flavus. No contact between cultures and only shared headspace implied the fungus produced inhibitory and stimulatory gases. Adding activated charcoal between wells and dishes prevented inhibition but not stimulation indicating stimulatory and inhibitory gases are different and/or gas is inhibitory at high concentration and stimulatory at lower concentrations. Characterizing stimulatory and inhibitory effects of gases in A. flavus headspace as well as the apparently opposing results in the two systems deserves further investigation. Determining how gases contribute to quorum sensing and communication could facilitate managing or using the gases in modified atmospheres during grain storage to minimize aflatoxin contamination.