Entomopathogenic Fungus Metarhizium Robertsii Research Articles

Potato productivity on the background of the entomopathogenic fungus <i>Metarhizium robertsii</i> application in production trials

For the first time in Western Siberia (Novosibirsk region), two-year production trials of an experimental prototype of a drug based on the entomopathogenic fungus Metarhizium robertsii have been conducted. Strain P-72 isolated from the dead larvae of Colorado potato beetle Leptinotarsa decemlineata Say from the collection of microorganisms of the Institute of Systematics and Ecology of Animals of the Siberian Branch of the Russian Academy of Sciences was used in this work. Species identification of the fungus was performed based on sequencing of the elongation factor gene region EF1α. The ability of the preparation to significantly increase the parameters of potato yield structure elements throughout the whole vegetation period was shown. A reliable increase in the number of surviving sprouts by 1.4 times, the number of stems and tubers by 1.5 times was found. The growth-stimulating effect of using the preparation based on entomopathogenic fungus was manifested in the increase of plant weight by the flowering phase. The average yield increase for two years amounted to 36.0% in the variant with application of M. robertsii significantly exceeding the productivity of potatoes in the control. The level of profitability of potato production with the use of entomopathogenic fungus M. robertsii increased to 92.0% (with 50.0% in the control) due to the increase in gross yield and improved quality of marketable products. Infestation of new harvest tubers with rhizoctoniose significantly decreased in the variant including treatment with entomopathogenic fungus: the number of tubers with sclerotia decreased 2.8 times compared to the control, which eventually led to an increase in the number of healthy tubers and a significant (2.5 times) decrease in the sclerotial index.

Can we manage alien invasive insects without altering native soil faunal communities? A field trial on Popillia japonica

Biological and integrated pest control are key assets to environmentally friendly management of cropland. Use of entomopathogens against target pests is common, yet the effects of such released organisms on native, non-target invertebrates are currently poorly known. This is particularly true for relatively inconspicuous components of agroecosystems such as soil biological biota. Popillia japonica, a polyphagous alien invasive scarab beetle native to Japan, is now present in Europe where huge efforts are being conducted to control the pest and slow its spread. In particular, entomopathogenic nematodes and fungi have been used for larval control. Here we test the effects of those agents on both P. japonica larvae and non-target soil biota by comparing soil arthropod assemblages before and after treatment of field plots in an irrigated perennial meadow.Application of an indigenous strain of the nematode Heterorhabditis bacteriophora resulted in significant reduction in numbers of P. japonica larvae. In contrast, an indigenous strain of the entomopathogenic fungus Metarhizium robertsii failed to provide significant control. We found no evident effect on non-target edaphic arthropods under either treatment in comparison to untreated control plots. Some taxa, namely Collembola and Acarina, showed a disproportionate increase in treated plots, suggesting that some changes may occur in the long term, at least in particular arthropod groups. Our results suggest the selected strain of H. bacteriophora is an excellent candidate for managing P. japonica larvae with minimal adverse impact on non-target species and ecosystem functioning.

Frequent horizontal chromosome transfer between asexual fungal insect pathogens

Entire chromosomes are typically only transmitted vertically from one generation to the next. The horizontal transfer of such chromosomes has long been considered improbable, yet gained recent support in several pathogenic fungi where it may affect the fitness or host specificity. To date, it is unknown how these transfers occur, how common they are, and whether they can occur between different species. In this study, we show multiple independent instances of horizontal transfers of the same accessory chromosome between two distinct strains of the asexual entomopathogenic fungus Metarhizium robertsii during experimental co-infection of its insect host, the Argentine ant. Notably, only the one chromosome-but no other-was transferred from the donor to the recipient strain. The recipient strain, now harboring the accessory chromosome, exhibited a competitive advantage under certain host conditions. By phylogenetic analysis, we further demonstrate that the same accessory chromosome was horizontally transferred in a natural environment between M. robertsii and another congeneric insect pathogen, Metarhizium guizhouense. Hence, horizontal chromosome transfer is not limited to the observed frequent events within species during experimental infections but also occurs naturally across species. The accessory chromosome that was transferred contains genes that may be involved in its preferential horizontal transfer or support its establishment. These genes encode putative histones and histone-modifying enzymes, as well as putative virulence factors. Our study reveals that both intra- and interspecies horizontal transfer of entire chromosomes is more frequent than previously assumed, likely representing a not uncommon mechanism for gene exchange.

The homeobox transcription factor MrHOX7 contributes to stress tolerance and virulence in the entomopathogenic fungus Metarhizium robertsii

Entomopathogenic fungi, including Metarhizium species, represent promising environmentally friendly biopesticides. Understanding the molecular mechanisms governing their infection processes is vital for enhancing their effectiveness. Transcription factors (TFs) play critical roles in gene regulation, yet the functions of many TFs in M. robertsii remain unknown. Homeobox transcription factors, implicated in diverse cellular processes, have received limited attention in entomopathogenic fungi. Here, we identify and characterize, a homeobox TF, MrHOX7, in the model entomopathogenic fungus M. robertsii. Subcellular localization and transcriptional profiling revealed MrHOX7′s nuclear localization and high expression during conidia and appressoria formation. Deletion of Mrhox7 (ΔMrhox7) enhanced conidial tolerance to heat and UV-B stress, accompanying with upregulated stress-related gene expression. Intriguingly, ΔMrhox7 exhibits inhibited virulence exclusively through topical inoculation. Further investigations unveiled reduced conidial adhesion and appressorium formation, with downregulation of the adhesion gene Mad1 and appressorium-related genes, as the underlying causes of the reduced fungal virulence. Our findings illuminate the role of MrHOX7 in stress tolerance and virulence, providing insights into the molecular basis of fungal biopesticides.

Effect of Selected Entomopathogenic Fungal Species on Embryonic Development of Ascaris suum (Nematoda).

The aim of the study was to evaluate the potential of using five selected species of entomopathogenic fungi (Beauveria bassiana, B. brongniartii, Conidiobolus coronatus, Isaria fumosorosea, and Metarhizium robertsii) in the bioregulation of the dispersive stages of the parasitic nematode-Ascaris suum. Experimental cultures of each of the selected entomopathogenic fungi, as well as a control culture without fungi, were incubated with A. suum eggs at 26 °C for 28 days. Development of the A. suum eggs was observed using a light microscope on the 7th, 14th, 21st, and 28th days of incubation. The API-ZYM® test was used to determine, semiquantitatively, the activity of 19 hydrolytic enzymes from the entomopathogenic fungi. The cytotoxicity of the fungi was determined using tetrazole salt MTT. It was found that none of the five tested strains of entomopathogenic fungi showed an ovicidal effect, and none of them colonized the A. suum egg shells. However, ovistatic activity was observed mainly until the 14th day of incubation by I. fumosorosea, M. robertsii, and B. bassiana. In the MTT test, M. robertsii showed moderate cytotoxicity, while the other species showed low cytotoxicity. Among the strains tested, I. fumosorosea showed the highest spectrum of hydrolase production (13 out of 19 enzymes gave a positive reaction from 3 to 5; 20-40 nM or more). The absence of morphological changes in the A. suum egg shells suggests that the antagonistic effect of the studied entomopathogenic fungi may be due to their cytotoxicity, associated with the production of secondary metabolites-toxins (M. robertsii) and enzymatic activity (I. fumosorosea).

Biopriming of broad bean seeds with entomopathogenic fungus Metarhizium robertsii J.F. Bisch., S.A. Rehner et Humber, 2009 does not affect invertebrate communities of the agroecosystem

Biopriming of broad bean seeds with entomopathogenic fungus Metarhizium robertsii J.F. Bisch., S.A. Rehner et Humber, 2009 does not affect invertebrate communities of the agroecosystem

Assessment of bio-formulations of indigenous strains of Bacillus thuringiensis, Metarhizium robertsii and Metarhizium majus for management of the rhinoceros beetle, Oryctes rhinoceros L., in field

BackgroundAmong all the coconut pests, rhinoceros beetle causes acute and serious damage to coconut palm. Management of this pest is very difficult due to its nocturnal activity, and also, it damages the emerging leaf inside the bud. Management of rhinoceros beetle using entomopathogens will be of great importance as it is economical and ecofriendly. Studies were carried out to decipher the biocontrol potential of indigenous entomopathogenic bacterium (Bacillus thuringiensis) and entomopathogenic fungi (Metarhizium robertsii and M. majus) against Oryctes rhinoceros in the field, individually as well as in combination, by soil drench as well as by topical spray method.ResultsThe study showed that B. thuringiensis strain NBAIR-BTAN4 showed 24.8% mortality at 5th week in soil drench method and 24% mortality in topical spray method. M. robertsii showed 24% mortality at 5th week in soil drench method as well as topical spray method. Similarly, M. majus showed 24% mortality in soil drench method and 23.2% in topical spray method at 5th week. In combination, NBAIR-BTAN4 + M. robertsii showed 40.8 and 44% in soil drench and topical spray method at 5th week, respectively. Combination of NBAIR-BTAN4 + M. majus showed 44.8 and 40.8% in soil drench and topical spray method at 5th week, respectively. Combination of NBAIR-BTAN4 + M. robertsii + M. majus showed 52.8 and 57.6% mortality at 5th week in soil drench and topical spray method, respectively.ConclusionThe present study showed application B. thuringiensis in combination with M. robertsii and M. majus is effective in management rhinoceros beetle in coconut orchard. Study also indicated that soil drench method is more promising strategy than topical spray method in managing larval population of the beetles and also confirmed that B. thuringiensis, M. robertsii and M. majus are compatible and they seemed to have a synergistic effect in controlling the pest in coconut orchard.

Transcriptomic dissection of termite gut microbiota following entomopathogenic fungal infection.

Termites are social insects that live in the soil or in decaying wood, where exposure to pathogens should be common. However, these pathogens rarely cause mortality in established colonies. In addition to social immunity, the gut symbionts of termites are expected to assist in protecting their hosts, though the specific contributions are unclear. In this study, we examined this hypothesis in Odontotermes formosanus, a fungus-growing termite in the family Termitidae, by 1) disrupting its gut microbiota with the antibiotic kanamycin, 2) challenging O. formosanus with the entomopathogenic fungus Metarhizium robertsii, and finally 3) sequencing the resultant gut transcriptomes. As a result, 142531 transcripts and 73608 unigenes were obtained, and unigenes were annotated following NR, NT, KO, Swiss-Prot, PFAM, GO, and KOG databases. Among them, a total of 3,814 differentially expressed genes (DEGs) were identified between M. robertsii infected termites with or without antibiotics treatment. Given the lack of annotated genes in O. formosanus transcriptomes, we examined the expression profiles of the top 20 most significantly differentially expressed genes using qRT-PCR. Several of these genes, including APOA2, Calpain-5, and Hsp70, were downregulated in termites exposed to both antibiotics and pathogen but upregulated in those exposed only to the pathogen, suggesting that gut microbiota might buffer/facilitate their hosts against infection by finetuning physiological and biochemical processes, including innate immunity, protein folding, and ATP synthesis. Overall, our combined results imply that stabilization of gut microbiota can assist termites in maintaining physiological and biochemical homeostasis when foreign pathogenic fungi invade.

Rhizoctonia potato disease in production conditions in the forest-steppe of Western Siberia

The authors carried out a scientific study from 1990 to 2022 on 20 varieties of potatoes to study the pathogenesis of Rhizoctonia in crop production conditions and substantiate the methods of the system of protection against the disease. The fungus R. Solani persists annually in the soil and on seed tubers in the form of mycelium and sclerotia. It was found that more than 60% of the grounds of potato agroecosystems have the content of the causative agent of Rhizoctonia is many times higher than the established ETH (Economic threshold of harmfulness). Furthermore, monitoring of the phytosanitary state of potato seed material showed that, on average, about 60% of the tubers are inhabited by the causative agent of Rhizoctonia, including about 30% of the sclerotial form, which is six times higher than the phytosanitary regulations. Thus, in the region’s conditions, a double mechanism of phytopathogen transmission is provided annually. The causative agent of Rhizoctonia infects potato sprouts, subsequently causing their complete death (on average 12%) and shootouts, reducing the density of productive plants in crop plantings. At subsequent stages of growth and development of potatoes, significant expansion of the disease on the stems was noted (on average 31.4%), and stolons were damaged and died (9.3 and 5.5%, respectively), which disrupted the processes of stolon and tuberization. In the region’s conditions, Rhizoctonia occurs in the form of epiphytotic with a prevalence of 88% or more. Therefore, the potato protection strategy against Rhizoctonia should be based on methods that reduce the initial population density of the R. Solani fungus in the soils of agroecosystems and on seed tubers. Essential elements of potato cultivation technology are cropping rotation with a saturation of no more than 30% (biological efficiency - 43.2–79.5%); phytosanitary precursor, including green manure pairs (biological efficiency - 49.0–75.9%); mandatory etching with chemical preparations (biological efficiency - 54.9–98.8%) or natural origin. Using entomopathogenic fungi Metarhizium robertsii and Baeuveria bassiana can be a promising direction in reducing Rhizoctonia. Dressing seed tubers with conidia of entomopathogenic fungi suppressed the development of the disease in potato plantings by 1.8–3.7%, providing an increase in yield by 12.1–19.2%. The study of the influence of Metarhizium robertsii and Baeuveria bassiana on the pathogenesis of Rhizoctonia will be continued.

A Toll pathway effector protects Drosophila specifically from distinct toxins secreted by a fungus or a bacterium

The Drosophila systemic immune response against many Gram-positive bacteria and fungi is mediated by the Toll pathway. How Toll-regulated effectors actually fulfill this role remains poorly understood as the known Toll-regulated antimicrobial peptide (AMP) genes are active only against filamentous fungi and not against Gram-positive bacteria or yeasts. Besides AMPs, two families of peptides secreted in response to infectious stimuli that activate the Toll pathway have been identified, namely Bomanins and peptides derived from a polyprotein precursor known as Baramicin A (BaraA). Unexpectedly, the deletion of a cluster of 10 Bomanins phenocopies the Toll mutant phenotype of susceptibility to infections. Here, we demonstrate that BaraA is required specifically in the host defense against Enterococcus faecalis and against the entomopathogenic fungus Metarhizium robertsii, albeit the fungal burden is not altered in BaraA mutants. BaraA protects the fly from the action of distinct toxins secreted by these Gram-positive and fungal pathogens, respectively, Enterocin V and Destruxin A. The injection of Destruxin A leads to the rapid paralysis of flies, whether wild type (WT) or mutant. However, a larger fraction of wild-type than BaraA flies recovers from paralysis within 5 to 10 h. BaraAs' function in protecting the host from the deleterious action of Destruxin is required in glial cells, highlighting a resilience role for the Toll pathway in the nervous system against microbial virulence factors. Thus, in complement to the current paradigm, innate immunity can cope effectively with the effects of toxins secreted by pathogens through the secretion of dedicated peptides, independently of xenobiotics detoxification pathways.

Efficiency of the Use of Entomopathogenic Fungus <i>Metarhizium robertsii</i> Against Potato Rhizoctoniosis

For the first time in Western Siberia, production tests of an experimental prototype of the drug based on the entomopathogenic fungus Metarhizium robertsii were carried out. Its ability to significantly restrain the development of potato rhizoctoniosis throughout the entire growing season is shown. A significant decrease in the death of sprouts and the development of rhizoctoniosis on the stems was established in comparison with the control and chemical protectant. In the area with the use of M. robertsii, a significant increase in the number of healthy tubers (by 2.5 times) was noted, the number of tubers with sclerotia decreased by 2.8 times compared to the control. The effectiveness of M. robertsii in the suppression of rhizoctoniosis was high at all stages of potato ontogeny and amounted to 52–84%. In laboratory experiments in the presence of M. robertsii, a significant inhibition of the formation of Rhizoctonia solani sclerotia on tubers was established, as well as a decrease in their viability and size.

Metarhizium robertsii MrAbaA affects conidial pigmentation via regulating MrPks1 and MrMlac1 expression

Pigments of conidia play a crucial role in fungal defense against environmental stressors such as UV radiation. The molecular basis of conidial pigmentation has been studied in the entomopathogenic fungus Metarhizium robertsii, while limited information been reported on function mechanisms transcription factors governing conidial pigmentation. Here, we identified transcription factor MrAbaA binding to the promoter regions of both MrPks1 and MrMlac1 in M. robertsii using yeast one-hybrid technology. Chromatin immunoprecipitation quantitative PCR assays further confirmed the interaction. Furthermore, overexpression of MrAbaA in M. robertsii resulted in increased conidial pigment accumulation and enhanced tolerances to UV stress by upregulated the MrPks1 and MrMlac1 expression. Taken together, MrAbaA affects conidial pigmentation by interacting with the promoter regions of both MrPks1 and MrMlac1 in M. robertsii. This work advances the understanding of the regulation mechanism for conidial pigmentation in entomopathogenic fungi.

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.

Sensing of a spore surface protein by a Drosophila chemosensory protein induces behavioral defense against fungal parasitic infections

In addition to innate immunity in a physiological context, insects have evolved behavioral defenses against parasite attacks. Here, we report that Drosophila can sense the CFEM (common in fungal extracellular membrane) protein Mcdc9, which acts as a negative virulence factor of the entomopathogenic fungus Metarhizium robertsii. The individual deletions of 18 CFEM genes in Metarhizium followed by fly infection identified three null mutants that could kill the flies more quickly than the wild-type strain, among which Mcdc9 can coat fungal spores and interact with the fly chemosensory protein CheA75a. The deletion of Mcdc9 in the fungus or the knockdown of CheA75a in flies had a similar effect, in which a greater number of fungal spores were left on flies than on the respective controls after topical infection. Thus, similar to the accelerated death of the wild-type flies treated with ΔMcdc9, the CheA75aRNAi flies succumbed more quickly than the control insects topically challenged with the wild-type strain. The CheA75a gene is highly transcribed in fly legs and wings, and positive electrophysiological responses were evidenced in tarsal sensilla after stimulation with the Mcdc9 protein. The results imply that this CFEM protein could be sensed as a contact elicitor inducing the hygienic behavior of flies against fungal parasitic infection, which reveals a previously unsuspected mechanism of fungus-insect interactions.

The Effect of Bean Seed Treatment with Entomopathogenic Fungus Metarhizium robertsii on Soil Microarthropods (Acari, Collembola).

Simple SummaryPlant pest control is essential for agriculture. The treatment of agricultural crops with entomopathogenic fungi protects plants against pests and can also increase plant growth, but at the same time may disturb the community structure of non-target beneficial invertebrates, in particular soil microarthropods. This can have a negative impact on soil fertility and ultimately on crop yields. The effect of the treatment of broad bean (Vicia faba L.) seeds, with the entomopathogenic fungus Metarhizium robertsii, on the abundance and structure of soil microarthropod communities in the rhizosphere was assessed. Compared with the control, no adverse effect was revealed on both the abundance of soil microarthropods, including mites (Mesostigmata, Oribatida, Astigmata, Prostigmata) and springtails (Collembola), and the structure of microarthropod communities. Therefore, dressing seeds with a conidial suspension for plant inoculation, with entomopathogenic fungi (at least M. robertsii), can be assumed as a potentially safe method of plant protection for soil microarthropods.The treatment of agricultural crops with entomopathogenic fungi may disturb the structure of soil microarthropod communities, which can have an adverse impact on soil fertility and, ultimately, on the yield. The effect of the treatment of broad bean (Vicia faba L.) seeds, with the entomopathogenic fungus Metarhizium robertsii, on the abundance and community structure of soil microarthropods in the rhizosphere was assessed in different phases of plant vegetation in a two-year experiment. Under the conditions of the gradually decreased abundance of M. robertsii both in the soil cenoses and in the plants during summer, no adverse effect was revealed of the bean seed treatment, with the entomopathogenic fungus, on the abundance of soil microarthropods (Acari: Mesostigmata, Prostigmata, Oribatida and Astigmata; Collembola) and the structure of their communities. Similar results were obtained in the analysis, taking into account the positive colonization of plants. Some changes in the microarthropod community structure were explained primarily by the spatial heterogeneity of the field, the hydrothermal regime, and the features of the microarthropod life cycles. The results indicate the possibility of using dressing seeds with conidial suspension for plant inoculation with entomopathogenic fungus (at least M. robertsii) as a potentially safe plant protection method for non-target soil microarthropods.

Orchestrated Biosynthesis of the Secondary Metabolite Cocktails Enables the Producing Fungus to Combat Diverse Bacteria.

ABSTRACTFungal secondary metabolites with antibiotic activities can promote fungal adaptation to diverse environments. Besides the global regulator, individual biosynthetic gene clusters (BGCs) usually contain a pathway-specific transcription factor for the tight regulation of fungal secondary metabolism. Here, we report the chemical biology mediated by a supercluster containing three BGCs in the entomopathogenic fungus Metarhizium robertsii. These clusters are jointly controlled by an embedded transcription factor that orchestrates the collective production of four classes of chemicals: ustilaginoidin, indigotide, pseurotin, and hydroxyl-ovalicin. The ustilaginoidin BGC is implicated as a late-acquired cluster in Metarhizium to produce both the bis-naphtho-γ-pyrones and the monomeric naphtho-γ-pyrone glycosides (i.e., indigotides). We found that the biosynthesis of indigotides additionally requires the functions of paired methylglucosylation genes located outside the supercluster. The pseurotin/ovalicin BGCs are blended and mesosyntenically conserved to the intertwined pseurotin/fumagillin BGCs of Aspergillus fumigatus. However, the former have lost a few genes, including a polyketide synthase gene responsible for the production of a pentaene chain used for assembly with ovalicin to form fumagillin, as observed in A. fumigatus. The collective production of chemical cocktails by this supercluster was dispensable for fungal virulence against insects and could enable the fungus to combat different bacteria better than the metabolite(s) produced by an individual BGC could. Thus, our results unveil a novel strategy employed by fungi to manage chemical ecology against diverse bacteria.

Parasitoid venom alters the lipid composition and development of microorganisms on the wax moth cuticle

AbstractEctoparasitoids may change host metabolism, making the environment more favorable for the development of their larvae. These alterations may also be suitable for growth and proliferation of commensal microorganisms and pathogens. This is especially important in relation to cuticular microbiota, with which ectoparasitoids interact directly. However, biochemical and microbiological changes occurring on insect cuticles in response to parasitoid venoms are insufficiently understood. We assayed the biochemical and microbiological changes in cuticles of larvae of the wax moth,Galleria mellonella(L.) (Lepidoptera: Pyralidae), after envenomation byHabrobracon brevicornis(Wesmael) (Hymenoptera: Braconidae).In particular, lipid composition in the epicuticle, total nitrogen (N) and carbon (C) amounts in the cuticle, bacterial colony‐forming unit (CFU) counts, and bacterial community structure (16S rRNA gene‐based metagenomics) on the insect surface were analyzed. The susceptibility of wax moth larvae to the entomopathogenic fungus (EPF)Metarhizium robertsiiJ.F. Bisch., S.A. Rehner & Humber (Hypocreales: Clavicipitaceae) was also assessed.Envenomation led to an increase in the hydrocarbon amount, as well as alterations in the fatty acid composition of the epicuticle; in particular, a large decrease in the amount of ω‐1 hydroxy fatty acids. The total N and C amounts in the cuticle also slightly increased after envenomation. These changes were correlated with a decrease in bacterial diversity and an increase inEnterococcusabundance on the surface of envenomated larvae. Envenomation also led to a substantial increase in larval susceptibility toM. robertsiiinfection; differences between the LC50values of envenomated and control larvae were 505 000‐fold. We suggest that the hyper‐lipidation of the cuticle is related to processes occurring in the host’s hemocoel after envenomation by the parasitoid. The effects of the changes in the lipid composition on the proliferation and development of microorganisms are discussed.

Role of Two G-Protein α Subunits in Vegetative Growth, Cell Wall Integrity, and Virulence of the Entomopathogenic Fungus Metarhizium robertsii.

Heterotrimeric G-proteins are crucial for fungal growth and differentiation. The α subunits of heterotrimeric G-proteins play an essential role in controlling signal transduction. However, the function of G-protein α subunits in entomopathogenic fungi remains poorly understood. Two group II Gα subunits (MrGPA2 and MrGPA4) were characterized in the entomopathogenic fungus, Metarhizium robertsii. Bioinformatics analysis showed that the relationship between MrGPA2 and MrGPA4 was closer than that of other MrGPAs. Both green fluorescent protein (GFP)-tagged MrGPA2 and MrGPA4 were localized at the cytoplasm. Furthermore, ∆MrGpa2∆MrGpa4 double mutants showed remarkably reduced vegetative growth compared to the wild-type and single-mutant strains, which was accompanied by the downregulation of several growth-related genes, such as ssk2, pbs2, stuA, hog1, and ac. Only the ∆MrGpa2∆MrGpa4 double mutant was sensitive to Congo red stress. The insect bioassay demonstrated significantly attenuated virulence for the ∆MrGpa2∆MrGpa4 double mutant compared to the wild-type and single-mutant strains. Further analysis indicated that double deletion of MrGpa2 and MrGpa4 had no effect on appressorium formation but suppressed the expression levels of several virulence-related genes in the insect hemocoel. These findings demonstrate that MrGpa2 and MrGpa4 exhibit functional redundancy and contribute to the vegetative growth, stress tolerance, and pest control potential in M. robertsii.

The role of complement in the tick cellular immune defense against the entomopathogenic fungus Metarhizium robertsii

Entomopathogenic fungi (EPF) have been widely explored for their potential in the biological control of insect pests and as an environmentally friendly alternative to acaricides for limiting tick infestation in the field. The arthropod cuticle is the main barrier against fungal infection, however, an understanding of internal defense mechanisms after EPF intrusion into the invertebrate hemocoel is still rather limited. Using an infection model of the European Lyme borreliosis vector Ixodes ricinus with the EPF Metarhizium robertsii, we demonstrated that ticks are capable of protecting themselves to a certain extent against mild fungal infections. However, tick mortality dramatically increases when the capability of tick hemocytes to phagocytose fungal conidia is impaired. Using RNAi-mediated silencing of tick thioester-containing proteins (TEPs), followed by in vitro and/or in vivo phagocytic assays, we found that C3-like complement components and α2-macroglobulin pan-protease inhibitors secreted to the hemolymph play pivotal roles in M. robertsii phagocytosis.

A Novel Nitrogen and Carbon Metabolism Regulatory Cascade Is Implicated in Entomopathogenicity of the Fungus Metarhizium robertsii.

ABSTRACTThe entomopathogenic fungus Metarhizium robertsii can switch among parasitic, saprophytic, and symbiotic lifestyles in response to changing nutritional conditions, which is attributed to its extremely versatile metabolism. Here, we found that the Fus3–mitogen-activated protein kinase (MAPK) and the transcription factor regulator of nutrient selection 1 (RNS1) constitute a novel fungal cascade that regulates the degradation of insect cuticular lipids, proteins, and chitin to obtain nutrients for hyphal growth and enter the insect hemocoel for subsequent colonization. On the insect cuticle, Fus3-MAPK physically contacts and phosphorylates RNS1, which facilitates the entry of RNS1 into nuclei. The phosphorylated RNS1 binds to the DNA motif BM2 (ACCAGAC) in its own promoter to self-induce expression, which then activates the expression of genes for degrading cuticular proteins, chitin, and lipids. We further found that the Fus3-MAPK/RNS1 cascade also activates genes for utilizing complex and less-favored nitrogen and carbon sources (casein, colloid chitin, and hydrocarbons) that were not derived from insects, which is repressed by favored organic carbon and nitrogen nutrients, including glucose and glutamine. In conclusion, we discovered a novel regulatory cascade that controls fungal nitrogen and carbon metabolism and is implicated in the entomopathogenicity of M. robertsii.IMPORTANCE Penetration of the cuticle, the first physical barrier to pathogenic fungi, is the prerequisite for fungal infection of insects. In the entomopathogenic fungus Metarhizium robertsii, we found that the Fus3–mitogen-activated protein kinase (MAPK) and the transcription factor regulator of nutrient selection 1 (RNS1) constitute a novel cascade that controls cuticle penetration by regulating degradation of cuticular lipids, proteins, and chitin to obtain nutrients for hyphal growth and entry into the insect hemocoel. In addition, during saprophytic growth, the Fus3-MAPK/RNS1 cascade also activates utilization of complex and less-favored carbon and nitrogen sources that are not derived from insects. The homologs of Fus3-MAPK and RNS1 are widely found in ascomycete filamentous fungi, including saprophytes and pathogens with diverse hosts, suggesting that the regulation of utilization of nitrogen and carbon sources by the Fus3-MAPK/RNS1 cascade could be widespread. Our work provides significant insights into regulation of carbon and nitrogen metabolism in fungi and fungal pathogenesis in insects.