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.

DNA methyltransferase implicated in the recovery of conidiation, through successive plant passages, in phenotypically degenerated Metarhizium.

Metarhizium robertsii is a fungus with two lifestyles; it is a plant root symbiont and an insect pathogen. A spontaneously phenotypically degenerated strain of M. robertsii strain ARSEF 2575 (M. robertsii lc-2575; lc = low conidiation) showed a reduction in conidiation and fungal virulence after successive subculturing on agar medium. In order to recover conidiation, we experimentally passaged M. robertsii lc-2575 through plant (soldier bean and switchgrass) root or insect (Galleria mellonella) larvae. After five passages, the resultant strains had significantly increased conidial yields on agar and increased virulence in insect bioassays. Concomitantly, DNA methyltransferase, MrDIM-2 expression was downregulated in BR5 (a strain after 5 bean root passages) and isolates after switchgrass and insect passages. Bisulfite sequencing showed little difference in overall genomic DNA methylation levels (~ 0.37%) between M. robertsii lc-2575 and BR5. However, a finer comparison of the different methylated regions (DMRs) showed that DMRs of BR5 were more abundant in the intergenic regions (69.32%) compared with that of M. robertsii lc-2575 (33.33%). The addition of DNA methyltransferase inhibitor, 5-azacytidine, to agar supported the role of DNA methyltransferases and resulted in an increase in conidiation of M. robertsii lc-2575. Differential gene expression was observed in selected DMRs in BR5 when compared with M. robertsii lc-2575. Here we implicated epigenetic regulation in the recovery of conidiation through the effects of DNA methyltransferase and that plant passage could be used as a method to recover fungal conidiation and virulence in a phenotypically degenerated M. robertsii. KEY POINTS: • Passage of Metarhizium through plant root or insect results in increased conidiation. • DNA methyltransferase is downregulated after host passage. • Bisulfite sequencing identified potentially methylated genes involved in conidiation.

The multifunctional lifestyles of Metarhizium: evolution and applications.

The genus Metarhizium is comprised of a diverse group of common soil fungi that exhibit multifunctional lifestyles with varying degrees of saprotrophic, endophytic, and insect pathogenic modes of nutrient acquisition. The transcriptome of these species is modulated to reflect immediate needs of the fungus and availability of resources-a form of transcriptional plasticity that allows for physiological adaptation to environments with diverse and dynamic exploitable nutrient sources. In this review, we discuss the endophytic, insect pathogenic lifestyles of Metarhizium spp., including their symbiotic interface, origins, and evolution, and agricultural applications. Isotope labeling experiments have demonstrated that a mutually beneficial exchange of limiting nutrients occurs between the fungus and its host plant, with nitrogen derived via insect pathogenesis being translocated from Metarhizium to host plants in exchange for fixed carbon in the form of photosynthate. Thus, the endophytic and entomopathogenic abilities of Metarhizium spp. are not exclusive of one another, but rather are interdependent and reciprocal in nature. Although endophytic, insect pathogenic fungi (EIPF) could certainly have evolved from insect pathogenic fungi, phylogenomic evidence indicates that this genus is more closely related to plant-associated fungi than animal pathogens, suggesting that Metarhizium evolved from a lineage of plant symbionts, which subsequently acquired genes for insect pathogenesis. Entomopathogenicity may have been an adaptive trait, allowing for procurement of insect-derived nitrogen that could be translocated to host plants and bartered for fixed carbon, thereby improving the stability of fungal-plant symbioses. Given their ability to simultaneously parasitize soil insects, including a number of pests of agriculturally important crops, as well as promote plant health, growth, and productivity, Metarhizium spp. are considered promising alternatives to the chemical pesticides and fertilizers that have wreaked havoc on the health and integrity of ecosystems. KEY POINTS: • Metarhizium is a fungus that is an insect pathogen as well as a plant symbiont. • The genus Metarhizium has specialist and generalist insect pathogens. • Metarhizium is phylogenetically most closely related to plant endophytes.

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.

Hydrophobins contribute to root colonization and stress responses in the rhizosphere-competent insect pathogenic fungus Beauveria bassiana.

The hyd1/hyd2 hydrophobins are important constituents of the conidial cell wall of the insect pathogenic fungus Beauveria bassiana. This fungus can also form intimate associations with several plant species. Here, we show that inactivation of two Class I hydrophobin genes, hyd1 or hyd2, significantly decreases the interaction of B. bassiana with bean roots. Curiously, the ∆hyd1/∆hyd2 double mutant was less impaired in root association than Δhyd1 or Δhyd2. Loss of hyd genes affected growth rate, conidiation ability and oosporein production. Expression patterns for genes involved in conidiation, cell wall integrity, insect virulence, signal transduction, adhesion, hydrophobicity and oosporein production were screened in the deletion mutants grown in different conditions. Repression of the major MAP-Kinase signal transduction pathways (Slt2 MAPK pathway) was observed that was more pronounced in the single versus double hyd mutants under certain conditions. The ∆hyd1/∆hyd2 double mutant showed up-regulation of the Hog1 MAPK and the Msn2 transcription factor under certain conditions when compared to the wild-type or single hyd mutants. The expression of the bad2 adhesin and the oosporein polyketide synthase 9 gene was severely reduced in all of the mutants. On the other hand, fewer changes were observed in the expression of key conidiation and cell wall integrity genes in hyd mutants compared to wild-type. Taken together, the data from this study indicated pleiotropic consequences of deletion of hyd1 and hyd2 on signalling and stress pathways as well as the ability of the fungus to form stable associations with plant roots.

Diversity and abundance of entomopathogenic fungi at ant colonies.

The purpose of this study was to identify whether entomopathogenic fungi in the genera Metarhizium and Beauveria were found at ant nests. These fungi have been used in studies of ant social immunity, however experimental conditions used may not normally be representative of that found within ant colonies. The presence of insect pathogenic fungi including Metarhizium and Beauveria was assessed in soils at 22 ant nests in Ontario, Canada. Soil samples were plated onto selective agar, fungi were isolated and DNA extracted and the fungi identified by amplifying the internal transcribed spacer region (ITS) and comparing sequences to those found in GenBank. We found that Metarhizium species were found in soils in and around most ant nests. Concentrations of Metarhizium in the soil were not influenced by the presence of ant nests suggesting co-existence rather than avoidance or seeking behaviour. Thus, Metarhizium appears to be a good pathogen to study ant-fungal interactions. Beauveria on the other hand, was not found in any of the samples indicating a decreased likelihood that ants encounter this pathogen. Other fungi found at relatively high concentrations at ant nests include Pochonia and Purpureocillium species, both recognized as nematode pathogens.

Genome-wide identification of pathogenicity, conidiation and colony sectorization genes in Metarhizium robertsii.

Metarhizium robertsii occupies a wide array of ecological niches and has diverse lifestyle options (saprophyte, insect pathogen and plant symbiont), that renders it an unusually effective model for studying genetic mechanisms for fungal adaptation. Here over 20,000 M. robertsii T-DNA mutants were screened in order to elucidate genetic mechanism by which M. robertsii replicates and persists in diverse niches. About 287 conidiation, colony sectorization or pathogenicity loci, many of which have not been reported in other fungi were identified. By analysing a series of conidial pigmentation mutants, a new fungal pigmentation gene cluster, which contains Mr-Pks1, Mr-EthD and Mlac1 was identified. A conserved conidiation regulatory pathway containing Mr-BrlA, Mr-AbaA and Mr-WetA regulates expression of these pigmentation genes. During conidiation Mr-BlrA up-regulates Mr-AbaA, which in turn controls Mr-WetA. It was found that Hog1-MAPK regulates fungal conidiation by controlling the conidiation regulatory pathway, and that all three pigmentation genes exercise feedback regulation of conidiation. This work provided the foundation for deeper understanding of the genetic processes behind M. robertsii adaptive phenotypes, and advances our insights into conidiation and pigmentation in this fungus.

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.

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.

Root isolations of Metarhizium spp. from crops reflect diversity in the soil and indicate no plant specificity.

Metarhizium spp. have recently been shown to be associated with the roots of different plants. Here we evaluated which Metarhizium species were associated with roots of oat (Avena sativa), rye (Secale cereale) and cabbage (Brassica oleracea), common crop plants in Denmark. Thirty-six root samples from each of the three crops were collected within an area of approximately 3ha. The roots were rinsed with sterile water, homogenized and the homogenate plated onto selective media. A subset of 126 Metarhizium isolates were identified to species by sequencing of the 5' end of the gene translation elongation factor 1-alpha and characterized by simple sequence repeat (SSR) analysis of 14 different loci. Metarhizium brunneum was the most common species isolated from plant roots (84.1% of all isolates), while M. robertsii (11.1%) and M. majus (4.8%) comprised the remainder. The SSR analysis revealed that six multilocus genotypes (MLGs) were present among the M. brunneum and M. robertsii isolates, respectively. A single MLG of M. brunneum represented 66.7%, 79.1% and 79.2% of the total isolates obtained from oat, rye and cabbage, respectively. The isolation of Metarhizium spp. and their MLGs from roots revealed a comparable community composition as previously reported from the same agroecosystem when insect baiting of soil samples was used as isolating technique. No specific MLG association with a certain crop was found. This study highlights the diversity of Metarhizium spp. found in the rhizosphere of different crops within a single agroecosystem and suggests that plants either recruit fungal associates from the surrounding soil environment or even govern the composition of Metarhizium populations.

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.

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.

Utilization of Metarhizium as an insect biocontrol agent and a plant bioinoculant with special reference to Brazil.

Brazil has a long history of using biological control and has the largest program in sugarcane agriculture to which a biocontrol program has been applied. This achievement is at least partly due to the utilization of the entomopathogenic fungus Metarhizium. This well-known fungal genus exhibits pathogenicity against a broad range of arthropod hosts and has been used globally as a biocontrol agent. This fungus is also a root symbiont, and in this capacity, it is a plant growth promoter. However, this feature (i.e., as a plant symbiont) has yet to be fully explored and implemented in Brazil, although the number of reports demonstrating Metarhizium's utility as a plant bioinoculant is increasing. The Brazilian bioproduct industry targets agricultural pests, and is limited to two Metarhizium species represented by four fungal isolates as active ingredients. Entomopathogenic fungi have also been successful in controlling arthropods of public health concern, as shown in their control of mosquitoes, which are vectors of diseases. The isolation of new indigenous Metarhizium isolates from a variety of substrates such as soil, insects, and plants shows the wide genetic diversity within this fungal genus. In this review, we emphasize the significance of Metarhizium spp. for the biological control of insects in Brazil. We also suggest that the experience and success of biological control with fungi in Brazil is an important resource for developing integrated pest management and sustainable strategies for pest control worldwide. Moreover, the future implementation prospects of species of Metarhizium being used as bioinoculants and possible new advances in the utility of this fungus are discussed.

The AreA Nitrogen Catabolite Repression Activator Balances Fungal Nutrient Utilization and Virulence in the Insect Fungal Pathogen Beauveria bassiana.

In many fungi, the AreA GATA-type transcription factor mediates nitrogen catabolite repression affecting fungal development and, where applicable, virulence. Here, we investigated the functions of AreA in the fungal entomopathogen and plant endophyte Beauveria bassiana using knockdown of gene expression. The antiAreA mutants were impaired in nitrogen utilization and showed increased sensitivities to osmotic stressors but increased tolerances to oxidative/hypoxia stresses. Repression of BbAreA caused overall minimal effects on fungal virulence. The minor effects on virulence appeared to be due in part to competing secondary effects where host defense phenoloxidase activity was significantly decreased, but production of the fungal metabolite oosporein was increased and hyphal body development was impaired. Knockdown of BbAreA expression also resulted in impairment in ability of the fungus to associate with host plants. These data implicate that BbAreA likely acts as a regulator to balance fungal nutrient utilization, pathogenicity, and mutualism, facilitating the fungal occupation of host niches.

The insect-pathogenic fungus Metarhizium robertsii (Clavicipitaceae) is also an endophyte that stimulates plant root development.

PREMISE OF THE STUDY: The soil-inhabiting insect-pathogenic fungus Metarhizium robertsii also colonizes plant roots endophytically, thus showing potential as a plant symbiont. Metarhizium robertsii is not randomly distributed in soils but preferentially associates with the plant rhizosphere when applied in agricultural settings. Root surface and endophytic colonization of switchgrass (Panicum virgatum) and haricot beans (Phaseolus vulgaris) by M. robertsii were examined after inoculation with fungal conidia. METHODS: We used light and confocal microscopy to ascertain the plant endophytic association with GFP-expressing M. robertsii. Root lengths, root hair density, and lateral roots emerged were also observed. KEY RESULTS: Initially, M. robertsii conidia adhered to, germinated on, and colonized roots. Furthermore, plant roots treated with Metarhizium grew faster and the density of plant root hairs increased when compared with control plants. The onset of plant root hair proliferation was initiated before germination of M. robertsii on the root (within 1-2 d). Plants inoculated with M. robertsii ΔMAD2 (plant adhesin gene) took significantly longer to show root hair proliferation than the wild type. Cell free extracts of M. robertsii did not stimulate root hair proliferation. Longer-term (60 d) associations showed that M. robertsii endophytically colonized cortical cells within bean roots. Metarhizium appeared as a mycelial aggregate within root cortical cells as well as between the intercellular spaces with no apparent damage to the plant. CONCLUSIONS: These results suggest that M. robertsii is not only rhizosphere competent but also displays a beneficial endophytic association with plant roots that results in the proliferation of root hairs.

A PCR-based method to identify Entomophaga spp. infections in North American grasshoppers.

A PCR-based method was developed for the detection and identification of two species of grasshopper-specific pathogens belonging to the genus Entomophaga in North America, Entomophaga calopteni and Entomophaga macleodii. Two separate sets of primers specific for amplification of a DNA product from each species of Entomophaga as well as a positive control were utilized. Grasshoppers were collected from two sites in Mexico during an epizootic with grasshoppers found in "summit disease", typical of Entomophaga infections. There was a preponderance of Melanopline grasshoppers infected by E. calopteni. The described method is an accurate tool for identification of North American grasshopper infections by Entomophaga species.

Three Methods Assessing the Association of the Endophytic Entomopathogenic Fungus Metarhizium robertsii with Non-Grafted Grapevine Vitis vinifera.

Characterizing the association of endophytic insect pathogenic fungi (EIPF) with plants is an important step in order to understand their ecology before using them in biological control programs. Since several methods are available, it is challenging to identify the most appropriate for such investigations. Here, we used two strains of Metarhizium robertsii: EF3.5(2) native to the French vineyard environment and ARSEF-2575-GFP a laboratory strain expressing a green fluorescent protein, to compare their potential of association with non-grafted grapevine Vitis vinifera. Three methods were used to evaluate the kinetics of rhizosphere and grapevine endospheric colonization: (i) Droplet Digital (ddPCR), a sensitive molecular method of M. robertsii DNA quantification in different plant parts, (ii) culture-based method to detect the live fungal propagules from plant tissues that grew on the medium, (iii) confocal imaging to observe roots segments. Both strains showed evidence of establishment in the rhizosphere of grapevines according to the culture-based and ddPCR methods, with a significantly higher establishment of strain EF3.5(2) (40% positive plants and quantified median of exp(4.61) c/µL) compared to strain ARSEF-2575-GFP (13% positive plants and quantified median of exp(2.25) c/µL) at 96-98 days post-inoculation. A low incidence of association of both strains in the grapevine root endosphere was found with no significant differences between strains and evaluation methods (15% positive plants inoculated with strain EF3.5(2) and 5% with strain ARSEF-2575-GFP according to culture-based method). ddPCR should be used more extensively to investigate the association between plants and EIPF but always accompanied with at least one method such as culture-based method or confocal microscopy.

Three sympatrically occurring species of Metarhizium show plant rhizosphere specificity.

Here we tested the hypothesis that species of the soil-inhabiting insect-pathogenic fungus Metarhizium are not randomly distributed in soils but show plant-rhizosphere-specific associations. We isolated Metarhizium from plant roots at two sites in Ontario, Canada, sequenced the 5' EF-1α gene to discern Metarhizium species, and developed an RFLP test for rapid species identification. Results indicated a non-random association of three Metarhizium species (Metarhizium robertsii, Metarhizium brunneum and Metarhizium guizhouense) with the rhizosphere of certain types of plant species (identified to species and categorized as grasses, wildflowers, shrubs and trees). M. robertsii was the only species that was found associated with grass roots, suggesting a possible exclusion of M. brunneum and M. guizhouense. Supporting this, in vitro experiments showed that M. robertsii conidia germinated significantly better in Panicum virgatum (switchgrass) root exudate than did M. brunneum or M. guizhouense. M. guizhouense and M. brunneum only associated with wildflower rhizosphere when co-occurring with M. robertsii. With the exception of these co-occurrences, M. guizhouense was found to associate exclusively with the rhizosphere of tree species, predominantly Acer saccharum (sugar maple), while M. brunneum was found to associate exclusively with the rhizosphere of shrubs and trees. These associations demonstrate that different species of Metarhizium associate with specific plant types.

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.

Abscisic acid implicated in differential plant responses of Phaseolus vulgaris during endophytic colonization by Metarhizium and pathogenic colonization by Fusarium.

Metarhizium robertsii is an insect pathogen as well as an endophyte, and can antagonize the phytopathogen, Fusarium solani during bean colonization. However, plant immune responses to endophytic colonization by Metarhizium are largely unknown. We applied comprehensive plant hormone analysis, transcriptional expression and stomatal size analysis in order to examine plant immune responses to colonization by Metarhizium and/or Fusarium. The total amount of abscisic acid (ABA) and ABA metabolites decreased significantly in bean leaves by plant roots colonized by M. robertsii and increased significantly with F. solani compared to the un-inoculated control bean plant. Concomitantly, in comparison to the un-inoculated bean, root colonization by Metarhizium resulted in increased stomatal size in leaves and reduced stomatal size with Fusarium. Meanwhile, expression of plant immunity genes was repressed by Metarhizium and, alternately, triggered by Fusarium compared to the un-inoculated plant. Furthermore, exogenous application of ABA resulted in reduction of bean root colonization by Metarhizium but increased colonization by Fusarium compared to the control without ABA application. Our study suggested that ABA plays a central role in differential responses to endophytic colonization by Metarhizium and pathogenic colonization by Fusarium and, we also observed concomitant differences in stomatal size and expression of plant immunity genes.