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.

Initial stages of endophytic colonization by Metarhizium involves rhizoplane colonization.

Here we assessed the time course of rhizoplane colonization by the endophytic insect pathogenic fungus Metarhizium robertsii. We describe a method of quantifying root colonization of bean plants by M. robertsii using quantitative polymerase chain reaction (qPCR). Results of this method were compared to the standard plate count method using colony-forming units (c.f.u.). Both the c.f.u. and qPCR methods were used to monitor the time-course of haricot bean (Phaseolus vulgaris) colonization by a strain of M. robertsii that expresses the green fluorescent protein (ARSEF 2575-GFP) for colony verification. There was a strong correlation between the results of the c.f.u. and qPCR methods, indicating that both methods are well suited for the determination of colonization of P. vulgaris roots by M. robertsii. Primers for a catalase gene (cat) amplified DNA from M. robertsii, M. brunneum and M. guizhouense. Primers for a nitrogen response-regulator (nrr) additionally detected M. acridum and M. flavoviride, whereas Metarhizium perilipin-like protein (mpl) primers were specific to M. robertsii alone. However, cat was the only target that specifically amplified Metarhizium in experiments utilizing non-sterile soil. Endophytic colonization of P. vulgaris at 60 days post-inoculation with M. robertsii was detected from surface-sterilized roots with more sensitivity using our qPCR technique over the c.f.u. method. Our results suggest that there is a prolonged period of rhizoplane colonization by Metarhizium with transient, low-level endophytic colonization of the root system of P. vulgaris that persists for the entirety of the plant life cycle.

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.

Tripartite interactions of an endophytic entomopathogenic fungus, Asian corn borer, and host maize under elevated carbon dioxide.

BACKGROUND: Biological control of insect pests is encountering an unprecedented challenge in agricultural systems due to the ongoing rise in carbon dioxide (CO2) level. The use of entomopathogenic fungi (EPF) in these systems is gaining increased attention, and EPF as crop endophytes hold the potential for combining insect pest control and yield enhancement of crops, but the effects of increased CO2 concentration on this interaction are poorly understood. Here, the introduction of endophytic EPF was explored as an alternative sustainable management strategy benefiting crops under elevated CO2, using maize (Zea mays), Asian corn borer (Ostrinia furnacalis), and EPF (Beauveria bassiana) to test changes in damage to maize plants from O. furnacalis, and the nutritional status (content of carbon, nitrogen, phosphorus, potassium), biomass, and yield of maize. RESULTS: The results showed that endophytic B. bassiana could alleviate the damage caused by O. furnacalis larvae for maize plants under ambient CO2 concentration, and this effect was enhanced under higher CO2 concentration. Inoculation with B. bassiana effectively counteracted the adverse impact of elevated CO2 on maize plants by preserving the nitrogen content at its baseline level (comparable with ambient CO2 conditions without B. bassiana). Both simultaneous effects could explain the improvement of biomass and yield of maize under B. bassiana inoculation and elevated CO2. CONCLUSION: This finding provides key information about the multifaceted benefits of B. bassiana as a maize endophyte. Our results highlight the promising potential of incorporating EPF as endophytes into integrated pest management strategies, particularly under elevated CO2 concentrations. © 2024 The Authors. Pest Management Science published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.

Flexible metabolism in Metarhizium anisopliae and Beauveria bassiana: role of the glyoxylate cycle during insect pathogenesis.

Insect pathogenic fungi such as Metarhizium anisopliae and Beauveria bassiana have an increasing role in the control of agricultural insect pests and vectors of human diseases. Many of the virulence factors are well studied but less is known of the metabolism of these fungi during the course of insect infection or saprobic growth. Here, we assessed enzyme activity and gene expression in the central carbon metabolic pathway, including isocitrate dehydrogenase, aconitase, citrate synthase, malate synthase (MLS) and isocitrate lyase (ICL), with particular attention to the glyoxylate cycle when M. anisopliae and B. bassiana were grown under various conditions. We observed that ICL and MLS, glyoxylate cycle intermediates, were upregulated during growth on 2-carbon compounds (acetate and ethanol) as well as in insect haemolymph. We fused the promoter of the M. anisopliae ICL gene (Ma-icl) to a marker gene (mCherry) and showed that Ma-icl was upregulated when M. anisopliae was grown in the presence of acetate. Furthermore, Ma-icl was upregulated when fungi were engulfed by insect haemocytes as well as during appressorium formation. Addition of the ICL inhibitor 3-nitroproprionate delayed conidial germination and inhibited appressorium formation. These results show that these insect pathogenic fungi have a flexible metabolism that includes the glyoxylate cycle as an integral part of germination, pathogenesis and saprobic growth.

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.

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.

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.

Zika Virus: Quantification, Propagation, Detection, and Storage.

Zika virus (ZIKV), belonging to the family Flaviviridae, genus Flavivirus, is an arthropod-borne virus that was first discovered from the Zika forest in Uganda in 1947. Recent outbreaks in South America have linked ZIKV to cases of microcephaly and Guillain-Barré syndrome in humans. With the increased interest in ZIKV, protocols must be established to facilitate proper research. Here we describe the laboratory techniques required to quantify, propagate, and store ZIVK. We also review the proper safety protocol for the handling of ZIKV, which is classified as a Biosafety Level 2 pathogen by the United States Centers for Disease Control and Prevention. © 2016 by John Wiley & Sons, Inc.

Differential expression of insect and plant specific adhesin genes, Mad1 and Mad2, in Metarhizium robertsii.

Metarhizium robertsii is an entomopathogenic fungus that is also plant rhizosphere competent. Two adhesin-encoding genes, Metarhizium adhesin-like protein 1 (Mad1) and Mad2, are involved in insect pathogenesis or plant root colonization, respectively. Here we examined the differential expression of the Mad genes when grown on a variety of soluble (carbohydrates and plant root exudate) and insoluble substrates (locust, tobacco hornworm, and cockroach cuticle, chitin, tomato stems, cellulose, and starch) and during insect, Plutella xylostella, infection. On insect cuticles Mad1 was up regulated, whereas bean root exudate and tomato stems resulted in the up regulation of Mad2. During the early stages of insect infection Mad1 was expressed while Mad2 was not expressed until fungal hyphae emerged and conidiated on the insect cadaver. The regulation of Mad2 was compared to that of other stress-related genes (heat shock protein (Hsp)30, Hsp70, and starvation stress gene A (ssgA)). Mad2 was generally up regulated by nutrient starvation (similar to ssgA) but not by pH, temperature, oxidative or osmotic stresses. Whereas Hsp30 and Hsp70 were generally up regulated at 37 °C or by oxidative stress even under nutrient enriched conditions. We fused the promoter of the Mad2 gene to a marker gene (green fluorescent protein (GFP)) and confirmed that Mad2 was up regulated when M. robertsii was grown in the presence of nutrient starvation. Examination of the promoter region of Mad2 revealed that it possessed two copies of a stress-response element (STRE) known to be regulated under the general stress-response pathway.