Profiling Destruxin Synthesis by Specialist and Generalist Metarhizium Insect Pathogens during Coculture with Plants.

Metarhizium is a genus of endophytic, insect-pathogenic fungi that is used as a biological control agent. The dual lifestyles of these fungi combine the parasitism of insect pests with the symbiotic association with plant roots. A major class of secreted metabolites by Metarhizium are cyclic depsipeptides called destruxins (DTXs). As prominent insecticidal compounds, their role during plant interactions is still largely unknown. Here, we examined the metabolomic profile of Metarhizium, with special emphasis on DTX production, using untargeted, liquid chromatography-tandem mass spectrometry (LC-MS/MS). Four Metarhizium species, two insect generalists (M. robertsii and M. brunneum), and two insect specialists (M. flavoviride and M. acridum) were inoculated onto agar plate cultures containing either bean (Phaseolus vulgaris) or corn (Zea mays) and grown for four and seven days. After methanol extraction, feature-based molecular networking (FBMN) was used to obtain DTX identification as defined by the Global Natural Products Social Molecular Networking (GNPS). A total of 25 DTX analogs were identified, with several DTX-like compounds in coculture that could not be identified. Metarhizium species differed in the amount and type of DTXs they produced, with the insect specialists producing far fewer amounts and types of DTXs than the insect generalists. The production of these metabolites varied between cultures of different ages and plant hosts. Conditions that influence the production of DTXs are discussed. As the genetic arsenal of natural products relates to the lifestyle of the organism, uncovering conditions with an ecological context may reveal strategies for producing novel compounds or precursors suitable for synthetic biology. IMPORTANCE The development of an intimate and beneficial association between fungi and plants requires an exchange of a complex mixture of chemical cues. These compounds are a means of communication, promoting or limiting the interaction, but can have numerous other biological and ecological functions. Determining how the metabolome, or a subset thereof, is linked to plant host preference and colonization has implications for future functional studies and may uncover novel therapeutic compounds whose production is elicited only under cocultivation. In this study, we performed an untargeted metabolomic analysis of plate cocultures with individual plant-fungal pairs. The identification of a major group of fungal metabolites, the destruxins, was examined for their role in plant specificity. The diversity of these metabolites and the production of numerous unidentified, structural analogs are evidence of the sensitivity of the methodology and the potential for future mining of this living data set.

Plant microbiome analysis after Metarhizium amendment reveals increases in abundance of plant growth-promoting organisms and maintenance of disease-suppressive soil.

The microbial community in the plant rhizosphere is vital to plant productivity and disease resistance. Alterations in the composition and diversity of species within this community could be detrimental if microbes suppressing the activity of pathogens are removed. Species of the insect-pathogenic fungus, Metarhizium, commonly employed as biological control agents against crop pests, have recently been identified as plant root colonizers and provide a variety of benefits (e.g. growth promotion, drought resistance, nitrogen acquisition). However, the impact of Metarhizium amendment on the rhizosphere microbiome has yet to be elucidated. Using Illumina sequencing, we examined the community profiles (bacteria and fungi) of common bean (Phaseolus vulgaris) rhizosphere (loose soil and plant root) after amendment with M. robertsii conidia, in the presence and absence of an insect host. Although alpha diversity was not significantly affected overall, there were numerous examples of plant growth-promoting organisms that significantly increased with Metarhizium amendment (Bradyrhizobium, Flavobacterium, Chaetomium, Trichoderma). Specifically, the abundance of Bradyrhizobium, a group of nitrogen-fixing bacteria, was confirmed to be increased using a qPCR assay with genus-specific primers. In addition, the ability of the microbiome to suppress the activity of a known bean root pathogen was assessed. The development of disease symptoms after application with Fusarium solani f. sp. phaseoli was visible in the hypocotyl and upper root of plants grown in sterilized soil but was suppressed during growth in microbiome soil and soil treated with M. robertsii. Successful amendment of agricultural soils with biocontrol agents such as Metarhizium necessitates a comprehensive understanding of the effects on the diversity of the rhizosphere microbiome. Such research is fundamentally important towards sustainable agricultural practices to improve overall plant health and productivity.

Inducible Antibacterial Activity in the Bacillales by Triphenyl Tetrazolium Chloride.

The world is in the midst of an antimicrobial resistance crisis, driving a need to discover novel antibiotic substances. Using chemical cues as inducers to unveil a microorganism's full metabolic potential is considered a successful strategy. To this end, we investigated an inducible antagonistic behavior in multiple isolates of the order Bacillales, where large inhibition zones were produced against Ralstonia solanacearum only when grown in the presence of the indicator triphenyl tetrazolium chloride (TTC). This bioactivity was produced in a TTC-dose dependent manner. Escherichia coli and Staphylococcus sp. isolates were also inhibited by Bacillus sp. strains in TTC presence, to a lesser extent. Knockout mutants and transcriptomic analysis of B. subtilis NCIB 3610 cells revealed that genes from the L-histidine biosynthetic pathway, the purine, pyrimidine de novo synthesis and salvage and interconversion routes, were significantly upregulated. Chemical space studied through metabolomic analysis, showed increased presence of nitrogenous compounds in extracts from induced bacteria. The metabolites orotic acid and L-phenylalaninamide were tested against R. solanacearum, E. coli, Staphylococcus sp. and B. subtilis, and exhibited activity against pathogens only in the presence of TTC, suggesting a biotransformation of nitrogenous compounds in Bacillus sp. cells as the plausible cause of the inducible antagonistic behavior.

Fungal Seed Pathogens of Wild Chili Peppers Possess Multiple Mechanisms To Tolerate Capsaicinoids.

The wild chili pepper Capsicum chacoense produces the spicy defense compounds known as capsaicinoids, including capsaicin and dihydrocapsaicin, which are antagonistic to the growth of fungal pathogens. Compared to other microbes, fungi isolated from infected seeds of C. chacoense possess much higher levels of tolerance of these spicy compounds, having their growth slowed but not entirely inhibited. Previous research has shown capsaicinoids inhibit microbes by disrupting ATP production by binding NADH dehydrogenase in the electron transport chain (ETC) and, thus, throttling oxidative phosphorylation (OXPHOS). Capsaicinoids may also disrupt cell membranes. Here, we investigate capsaicinoid tolerance in fungal seed pathogens isolated from C. chacoense We selected 16 fungal isolates from four ascomycete genera (Alternaria, Colletotrichum, Fusarium, and Phomopsis). Using relative growth rate as a readout for tolerance, fungi were challenged with ETC inhibitors to infer whether fungi possess alternative respiratory enzymes and whether effects on the ETC fully explained inhibition by capsaicinoids. In all isolates, we found evidence for at least one alternative NADH dehydrogenase. In many isolates, we also found evidence for an alternative oxidase. These data suggest that wild-plant pathogens may be a rich source of alternative respiratory enzymes. We further demonstrate that these fungal isolates are capable of the breakdown of capsaicinoids. Finally, we determine that the OXPHOS theory may describe a weak primary mechanism by which dihydrocapsaicin, but not capsaicin, slows fungal growth. Our findings suggest that capsaicinoids likely disrupt membranes, in addition to energy poisoning, with implications for microbiology and human health.IMPORTANCE Plants make chemical compounds to protect themselves. For example, chili peppers produce the spicy compound capsaicin to inhibit pathogen damage and animal feeding. In humans, capsaicin binds to a membrane channel protein, creating the sensation of heat, while in microbes, capsaicin limits energy production by binding respiratory enzymes. However, some data suggest that capsaicin also disrupts membranes. Here, we studied fungal pathogens (Alternaria, Colletotrichum, Fusarium, and Phomopsis) isolated from a wild chili pepper, Capsicum chacoense By measuring growth rates in the presence of antibiotics with known respiratory targets, we inferred that wild-plant pathogens might be rich in alternative respiratory enzymes. A zone of clearance around the colonies, as well as liquid chromatography-mass spectrometry data, further indicated that these fungi can break down capsaicin. Finally, the total inhibitory effect of capsaicin was not fully explained by its effect on respiratory enzymes. Our findings lend credence to studies proposing that capsaicin may disrupt cell membranes, with implications for microbiology, as well as human health.

Availability of carbon and nitrogen in soil affects Metarhizium robertsii root colonization and transfer of insect-derived nitrogen.

The endophytic, insect pathogenic fungus, Metarhizium, exchanges insect-derived nitrogen for photosynthate as part of a symbiotic association similar to well-known mycorrhizal relationships. However, little is known about this nitrogen transfer in soils where there is an abundance of nitrogen and/or carbon. Here, we applied D-glucose and ammonium nitrate to soil to examine the effect on root colonization and transfer of labelled nitrogen (15N) from an insect (injected with 15N-ammonium sulfate) to Metarhizium robertsii, into leaves of the common bean, Phaseolus vulgaris, over the course of 28 days. Application of exogenous carbon and/or nitrogen to soils significantly reduced detectable 15N in plant leaves. Metarhizium root colonization, quantified with real-time PCR, revealed colonization persisted under all conditions but was significantly greater on roots in soil supplemented with glucose and significantly lower in soil supplemented with ammonium nitrate. Fungal gene expression analysis revealed differential expression of sugar and nitrogen transporters (mrt, st3, nrr1, nit1, mep2) when Metarhizium was grown in pure broth culture or in co-culture with plant roots under various carbon and nitrogen conditions. The observation that Metarhizium maintained root colonization in the absence of nitrogen transfer, and without evidence of plant harm, is intriguing and indicates additional benefits with ecological importance.

Microbes and associated soluble and volatile chemicals on periodically wet household surfaces.

BACKGROUND: Microorganisms influence the chemical milieu of their environment, and chemical metabolites can affect ecological processes. In built environments, where people spend the majority of their time, very little is known about how surface-borne microorganisms influence the chemistry of the indoor spaces. Here, we applied multidisciplinary approaches to investigate aspects of chemical microbiology in a house. METHODS: We characterized the microbial and chemical composition of two common and frequently wet surfaces in a residential setting: kitchen sink and bathroom shower. Microbial communities were studied using culture-dependent and independent techniques, including targeting RNA for amplicon sequencing. Volatile and soluble chemicals from paired samples were analyzed using state-of-the-art techniques to explore the links between the observed microbiota and chemical exudates. RESULTS: Microbial analysis revealed a rich biological presence on the surfaces exposed in kitchen sinks and bathroom shower stalls. Microbial composition, matched for DNA and RNA targets, varied by surface type and sampling period. Bacteria were found to have an average of 25× more gene copies than fungi. Biomass estimates based on qPCR were well correlated with measured total volatile organic compound (VOC) emissions. Abundant VOCs included products associated with fatty acid production. Molecular networking revealed a diversity of surface-borne compounds that likely originate from microbes and from household products. CONCLUSIONS: Microbes played a role in structuring the chemical profiles on and emitted from kitchen sinks and shower stalls. Microbial VOCs (mVOCs) were predominately associated with the processing of fatty acids. The mVOC composition may be more stable than that of microbial communities, which can show temporal and spatial variation in their responses to changing environmental conditions. The mVOC output from microbial metabolism on kitchen sinks and bathroom showers should be apparent through careful measurement, even against a broader background of VOCs in homes, some of which may originate from microbes in other locations within the home. A deeper understanding of the chemical interactions between microbes on household surfaces will require experimentation under relevant environmental conditions, with a finer temporal resolution, to build on the observational study results presented here.

Carbon translocation from a plant to an insect-pathogenic endophytic fungus.

Metarhizium robertsii is a common soil fungus that occupies a specialized ecological niche as an endophyte and an insect pathogen. Previously, we showed that the endophytic capability and insect pathogenicity of Metarhizium are coupled to provide an active method of insect-derived nitrogen transfer to a host plant via fungal mycelia. We speculated that in exchange for this insect-derived nitrogen, the plant would provide photosynthate to the fungus. By using 13CO2, we show the incorporation of 13C into photosynthate and the subsequent translocation of 13C into fungal-specific carbohydrates (trehalose and chitin) in the root/endophyte complex. We determined the amount of 13C present in root-associated fungal biomass over a 21-day period by extracting fungal carbohydrates and analysing their composition using nuclear magnetic resonance (NMR) spectroscopy. These findings are evidence that the host plant is providing photosynthate to the fungus, likely in exchange for insect-derived nitrogen in a tripartite, and symbiotic, interaction.

Fungi with multifunctional lifestyles: endophytic insect pathogenic fungi.

This review examines the symbiotic, evolutionary, proteomic and genetic basis for a group of fungi that occupy a specialized niche as insect pathogens as well as endophytes. We focus primarily on species in the genera Metarhizium and Beauveria, traditionally recognized as insect pathogenic fungi but are also found as plant symbionts. Phylogenetic evidence suggests that these fungi are more closely related to grass endophytes and diverged from that lineage ca. 100 MYA. We explore how the dual life cycles of these fungi as insect pathogens and endophytes are coupled. We discuss the evolution of insect pathogenesis while maintaining an endophytic lifestyle and provide examples of genes that may be involved in the transition toward insect pathogenicity. That is, some genes for insect pathogenesis may have been co-opted from genes involved in endophytic colonization. Other genes may be multifunctional and serve in both lifestyle capacities. We suggest that their evolution as insect pathogens allowed them to effectively barter a specialized nitrogen source (i.e. insects) with host plants for photosynthate. These ubiquitous fungi may play an important role as plant growth promoters and have a potential reservoir of secondary metabolites.

Molecules to Ecosystems: Actinomycete Natural Products In situ.

Actinomycetes, filamentous actinobacteria found in numerous ecosystems around the globe, produce a wide range of clinically useful natural products (NP). In natural environments, actinomycetes live in dynamic communities where environmental cues and ecological interactions likely influence NP biosynthesis. Our current understating of these cues, and the ecological roles of NP, is in its infancy. We postulate that understanding the ecological context in which actinomycete metabolites are made is fundamental to advancing the discovery of novel NP. In this review we explore the ecological relevance of actinomycetes and their secondary metabolites from varying ecosystems, and suggest that investigating the ecology of actinomycete interactions warrants particular attention with respect to metabolite discovery. Furthermore, we focus on the chemical ecology and in situ analysis of actinomycete NP and consider the implications for NP biosynthesis at ecosystem scales.

Nutrient transfer in plant-fungal symbioses.

Almost all plant species form symbioses with soil fungi, and nutrient transfer to plants is largely mediated through this partnership. Studies of fungal nutrient transfer to plants have largely focused on the transfer of limiting soil nutrients, such as nitrogen and phosphorous, by mycorrhizal fungi. However, certain fungal endophytes, such as Metarhizium and Beauveria, are also able to transfer nitrogen to their plant hosts. Here, we review recent studies that have identified genes and their encoded transporters involved in the movement of nitrogen, phosphorous, and nonlimiting soil nutrients between symbionts. These recent advances in our understanding could lead to applications in agricultural and horticultural settings, and to the development of model fungal systems that could further elucidate the role of fungi in these symbioses.

Ubiquity of insect-derived nitrogen transfer to plants by endophytic insect-pathogenic fungi: an additional branch of the soil nitrogen cycle.

The study of symbiotic nitrogen transfer in soil has largely focused on nitrogen-fixing bacteria. Vascular plants can lose a substantial amount of their nitrogen through insect herbivory. Previously, we showed that plants were able to reacquire nitrogen from insects through a partnership with the endophytic, insect-pathogenic fungus Metarhizium robertsii. That is, the endophytic capability and insect pathogenicity of M. robertsii are coupled so that the fungus acts as a conduit to provide insect-derived nitrogen to plant hosts. Here, we assess the ubiquity of this nitrogen transfer in five Metarhizium species representing those with broad (M. robertsii, M. brunneum, and M. guizhouense) and narrower insect host ranges (M. acridum and M. flavoviride), as well as the insect-pathogenic fungi Beauveria bassiana and Lecanicillium lecanii. Insects were injected with (15)N-labeled nitrogen, and we tracked the incorporation of (15)N into two dicots, haricot bean (Phaseolus vulgaris) and soybean (Glycine max), and two monocots, switchgrass (Panicum virgatum) and wheat (Triticum aestivum), in the presence of these fungi in soil microcosms. All Metarhizium species and B. bassiana but not L. lecanii showed the capacity to transfer nitrogen to plants, although to various degrees. Endophytic association by these fungi increased overall plant productivity. We also showed that in the field, where microbial competition is potentially high, M. robertsii was able to transfer insect-derived nitrogen to plants. Metarhizium spp. and B. bassiana have a worldwide distribution with high soil abundance and may play an important role in the ecological cycling of insect nitrogen back to plant communities.

Nutrient transfer to plants by phylogenetically diverse fungi suggests convergent evolutionary strategies in rhizospheric symbionts.

Most land plants are able to form symbiotic associations with fungi, and in many cases these associations are necessary for plant and fungal survival. These plant/fungal associations are formed with mycorrhizal (arbuscular mycorrhizal or ectomycorrhizal) or endophytic fungi, fungi from distinct phylogenetic lineages. While it has been shown that mycorrhizal fungi are able to transfer nutrients to plant roots in exchange for carbon, endophytes have been thought as asymptomatic colonizers. Recently, however, it has been shown that some insect pathogenic endophytic fungi are able to transfer insect derived nitrogen to plant roots, likely in exchange for plant sugars. Here we explore potential convergent evolutionary strategies for nutrient transfer between insect pathogenic endophytes and mycorrhizal fungus.

Potential agricultural benefits through biotechnological manipulation of plant fungal associations.

Genes involved in beneficial plant-fungal associations are attractive targets for biotechnological applications in agriculture. We suggest that some endophytic ascomycetous insect pathogenic fungi (i.e., Metarhizium) may be good candidates for these biotechnological manipulations.

Insects as a Nitrogen Source for Plants.

Many plants have evolved adaptations in order to survive in low nitrogen environments. One of the best-known adaptations is that of plant symbiosis with nitrogen-fixing bacteria; this is the major route by which nitrogen is incorporated into plant biomass. A portion of this plant-associated nitrogen is then lost to insects through herbivory, and insects represent a nitrogen reservoir that is generally overlooked in nitrogen cycles. In this review we show three specialized plant adaptations that allow for the recovery of insect nitrogen; that is, plants gaining nitrogen from insects. First, we show specialized adaptations by carnivorous plants in low nitrogen habitats. Insect carnivorous plants such as pitcher plants and sundews (Nepenthaceae/Sarraceniaceae and Drosera respectively) are able to obtain substantial amounts of nitrogen from the insects that they capture. Secondly, numerous plants form associations with mycorrhizal fungi that can provide soluble nitrogen from the soil, some of which may be insect-derived nitrogen, obtained from decaying insects or insect frass. Finally, a specialized group of endophytic, insect-pathogenic fungi (EIPF) provide host plants with insect-derived nitrogen. These soil-inhabiting fungi form a remarkable symbiosis with certain plant species. They can infect a wide range of insect hosts and also form endophytic associations in which they transfer insect-derived nitrogen to the plant. Root colonizing fungi are found in disparate fungal phylogenetic lineages, indicating possible convergent evolutionary strategies between taxa, evolution potentially driven by access to carbon-containing root exudates.