Insect Pathogenic Fungus Metarhizium Research Articles

How Metarhizium robertsii's mycelial consciousness gets its conidia Zen-ready for stress.

This memoir takes a whimsical ride through my professional adventures, spotlighting my fungal stress research on the insect-pathogenic fungus Metarhizium robertsii, which transformed many of my wildest dreams into reality. Imagine the magic of fungi meeting science and me, a happy researcher, arriving at Utah State University ready to dive deep into studies with the legendary insect pathologist, my advisor Donald W. Roberts, and my co-advisor Anne J. Anderson. From my very first "Aha!" moment in the lab, I plunged into a vortex of discovery, turning out research like a mycelium on a mission. Who knew 18h/day, seven days a week, could be so exhilarating? I was fueled by an insatiable curiosity, boundless creativity, and a perhaps slightly alarming level of motivation. Years later, I managed to bring my grandest vision to life: the International Symposium on Fungal Stress-ISFUS. This groundbreaking event has attracted 162 esteemed speakers from 29 countries to Brazil, proving that fungi can be both fun and globally fascinating. ISFUS is celebrating its fifth edition in 2024, a decade after its 2014 debut.

Potential of 3-octanone as a lure and kill agent for control of the Brown garden snail

The brown garden snail (Cornu aspersum) is a major agricultural pest, causing damage to a wide range of economically important crops. Withdrawal or restricted use of pollutant molluscicides like metaldehyde has prompted a search for more benign control products. This study investigated the response of snails to 3-octanone; a volatile organic compound (VOCs) produced by the insect pathogenic fungus Metarhizium brunneum. Concentrations of 1 – 1000 ppm of 3-octanone were first assessed in laboratory choice assays to determine behavioural response. Repellent activity was found at 1000 ppm whereas attractance was found for the lower concentrations of 1, 10 and 100 ppm. These three concentrations of 3-octanone were carried forward in field evaluations to assess potential for use in “lure and kill” strategies. The highest concentration (100 ppm) was the most attractive to the snails but also the most lethal. Even at the lowest concentration this compound proved toxic effects making 3-octanone an excellent candidate for the development as a snail attractant and molluscicide.

Asphyxiation of Metarhizium robertsii during mycelial growth produces conidia with increased stress tolerance via increased expression of stress-related genes

Little is known about the impact of hypoxia and anoxia during mycelial growth on tolerance to different stress conditions of developing fungal conidia. Conidia of the insect-pathogenic fungus Metarhizium robertsii were produced on potato dextrose agar (PDA) medium under normoxia (control = normal oxygen concentrations), continuous hypoxia, and transient anoxia, as well as minimal medium under normoxia. The tolerance of the conidia produced under these different conditions was evaluated in relation to wet heat (heat stress), menadione (oxidative stress), potassium chloride (osmotic stress), UV radiation, and 4-nitroquinoline-1-oxide (=4-NQO genotoxic stress). Growth under hypoxic condition induced higher conidial tolerance of M. robertsii to menadione, KCl, and UV radiation. Transient anoxic condition induced higher conidial tolerance to KCl and UV radiation. Nutritional stress (i.e., minimal medium) induced higher conidial tolerance to heat, menadione, KCl, and UV radiation. However, neither of these treatments induced higher tolerance to 4-NQO. The gene hsp30 and hsp101 encoding a heat shock protein was upregulated under anoxic condition. In conclusion, growth under hypoxia and anoxia produced conidia with higher stress tolerances than conidia produced in normoxic condition. The nutritive stress generated by minimal medium, however, induced much higher stress tolerances. This condition also caused the highest level of gene expression in the hsp30 and hsp101 genes. Thus, the conidia produced under nutritive stress, hypoxia, and anoxia had greater adaptation to stress.

Cytotoxic cytochalasans from cultures of the fungus Metarhizium brunneum TBRC-BCC 79240.

Fourteen new cytochalasans, brunnesins A-N (1-14), along with eleven known compounds, were isolated from the culture extracts of the insect pathogenic fungus Metarhizium brunneum strain TBRC-BCC 79240. The compound structures were established by spectroscopy, X-ray diffraction analysis, and electronic circular dichroism. Compound 4 exhibited antiproliferative activity against all cell lines tested (mammalian), with 50% inhibition concentration (IC50) values ranging from 2.09 to 16.8 μg mL-1. Compounds 6 and 16 were shown to be bioactive only against non-cancerous Vero cells (IC50 4.03 and 0.637 μg mL-1, respectively) whereas compounds 9 and 12 were bioactive only against NCI-H187 small-cell lung cancer cells (IC50 18.59 and 18.54 μg mL-1, respectively). Compounds 7, 13, and 14 showed cytotoxicity against NCI-H187 and Vero cell lines with IC50 values ranging from 3.98-44.81 μg mL-1.

Proteomic Analysis of a Hypervirulent Mutant of the Insect-Pathogenic Fungus Metarhizium anisopliae Reveals Changes in Pathogenicity and Terpenoid Pathways.

Metarhizium anisopliae is a commercialized entomopathogenic fungus widely used for the control of insect pests. Significant efforts have been expended to screen and/or select for isolates that display increased virulence toward target insect hosts. UV-induced mutagenesis has resulted in the isolation of a number of hypervirulent M. anisopliae mutants; however, the underlying mechanisms that have led to the desired phenotype have yet to be characterized. Here, we performed a comparative proteomic analysis of an M. anisopliae UV-induced hypervirulent mutant (MaUV-HV) and its wild-type parent using tandem mass tag (TMT)-based quantitative proteomics. A total of 842 differentially abundant proteins were identified, with 360 being more abundant in the hypervirulent mutant and 482 in the wild-type parent. In terms of differential abundance, the critical pathways affected included those involved in secondary metabolite production, virulence, and stress response. In addition, a number of genes involved in terpenoid biosynthesis pathways were identified as significantly mutated in the MaUV-HV strain. In particular, mutations in the farnesyl pyrophosphate synthase (FPPS1) and geranylgeranyl diphosphate synthase (GGPPS5) genes were seen. The effects of the FPPS1 mutation were confirmed via the construction and characterization of a targeted gene knockout strain (ΔMaFPPS1). The overall effects of the mutations were increased resistance to UV stress, faster growth, and increased virulence. These results provide mechanistic insights and new avenues for modulating fungal virulence in efforts to increase the biological control potential of insect-pathogenic fungi. IMPORTANCE The mechanisms that underlie and contribute to microbial (fungal) virulence are known to be varied; however, the identification of contributing pathways beyond known virulence factors remains difficult. Using TMT-based proteomic analyses, changes in the proteomes of an M. anisopliae hypervirulent mutant and its wild-type parent were determined. These data revealed alterations in pathogenicity, stress, and growth/developmental pathways, as well as pathways not previously known to affect virulence. These include terpenoid pathways that can be manipulated to increase the efficacy of fungal insect biological control agents for increased sustainable pest control.

New Downstream Signaling Branches of the Mitogen-Activated Protein Kinase Cascades Identified in the Insect Pathogenic and Plant Symbiotic Fungus Metarhizium robertsii.

Fungi rely on major signaling pathways such as the MAPK (Mitogen-Activated Protein Kinase) signaling pathways to regulate their responses to fluctuating environmental conditions, which is vital for fungi to persist in the environment. The cosmopolitan Metarhizium fungi have multiple lifestyles and remarkable stress tolerance. Some species, especially M. robertsii, are emerging models for investigating the mechanisms underlying ecological adaptation in fungi. Here we review recently identified new downstream branches of the MAPK cascades in M. robertsii, which controls asexual production (conidiation), insect infection and selection of carbon and nitrogen nutrients. The Myb transcription factor RNS1 appears to be a central regulator that channels information from the Fus3- and Slt2-MAPK cascade to activate insect infection and conidiation, respectively. Another hub regulator is the transcription factor AFTF1 that transduces signals from the Fus3-MAPK and the membrane protein Mr-OPY2 for optimal formation of the infection structures on the host cuticle. Homologs of these newly identified regulators are found in other Metarhizium species and many non-Metarhizium fungi, indicating that these new downstream signaling branches of the MAPK cascades could be widespread.

Low- or high-white light irradiance induces similar conidial stress tolerance in Metarhizium robertsii.

White light during mycelial growth influences high conidial stress tolerance of the insect-pathogenic fungus Metarhizium robertsii, but little is known if low- or high-white light irradiances induce different stress tolerances. The fungus was grown either in the dark using two culture media: on minimal medium (Czapek medium without sucrose = MM) or on potato dextrose agar (PDA) or PDA medium under five different continuous white light irradiances. The stress tolerances of conidia produced on all treatments were evaluated by conidial germination on PDA supplemented with KCl for osmotic stress or on PDA supplemented with menadione for oxidative stress. Conidia produced on MM in the dark were more tolerant to osmotic and oxidative stress than conidia produced on PDA in the dark or under the light. For osmotic stress, growth under the lower to higher irradiances produced conidia with similar tolerances but more tolerant than conidia produced in the dark. For oxidative stress, conidia produced under the white light irradiances were generally more tolerant to menadione than conidia produced in the dark. Moreover, conidia produced in the dark germinated at the same speed when incubated in the dark or under lower irradiance treatment. However, at higher irradiance, conidial germination was delayed compared to germination in the dark, which germinated faster. Therefore, growth under light from low to high irradiances induces similar conidial higher stress tolerances; however, higher white light irradiances cause a delay in germination speed.

The Sugar Transporter MST1 Is Involved in Colonization of Rhizosphere and Rhizoplane by Metarhizium robertsii.

ABSTRACTIt is widely recognized that plant-symbiotic fungi are supported by photosynthates; however, little is known about the molecular mechanisms underlying the utilization of plant-derived sugars by rhizospheric fungi. In the insect-pathogenic and plant-symbiotic fungus Metarhizium robertsii, we previously showed that the utilization of oligosaccharides by the transporter MRT (Metarhizium raffinose transporter) is important for rhizosphere competency. In this study, we identified a novel monosaccharide transporter (MST1) that is involved in the colonization of the rhizoplane and acts additively with MRT to colonize the rhizosphere. MST1 is not involved in infection of insects by M. robertsii. MST1 is an H+ symporter and is able to transport a broad spectrum of monosaccharides, including glucose, sorbose, mannose, rhamnose, and fructose. Deletion of the Mst1 gene impaired germination and mycelial growth in medium containing the sugars that it can transport. Homologs of MST1 were widely found in many fungi, including plant symbionts such as Trichoderma spp. and mycorrhizal fungi and plant pathogens such as Fusarium spp. This work significantly advances insights into the development of symbiotic relationships between plants and fungi.IMPORTANCE Over 90% of all vascular plant species develop an intimate symbiosis with fungi, which has an enormous impact on terrestrial ecosystems. It is widely recognized that plant-symbiotic fungi are supported by photosynthates, but little is known about the mechanisms for fungi to utilize plant-derived carbon sources. In the fungus Metarhizium robertsii, we identified a novel monosaccharide transporter (MST1) that is an H+ symporter and can transport a broad spectrum of monosaccharides, including glucose, sorbose, mannose, rhamnose, and fructose. MST1 is involved in the colonization of the rhizoplane and acts additively with the previously characterized oligosaccharide transporter MRT to colonize the rhizosphere. Homologs of MST1 were found in many fungi, including plant symbionts and plant pathogens, suggesting that the utilization of plant-derived sugars by MST1 homologs could also be important for other fungi to develop a symbiotic or parasitic relationship with their respective plant hosts.

A novel cascade allows Metarhizium robertsii to distinguish cuticle and hemocoel microenvironments during infection of insects

Pathogenic fungi precisely respond to dynamic microenvironments during infection, but the underlying mechanisms are not well understood. The insect pathogenic fungus Metarhizium robertsii is a representative fungus in which to study broad themes of fungal pathogenicity as it resembles some major plant and mammalian pathogenic fungi in its pathogenesis. Here we report on a novel cascade that regulates response of M. robertsii to 2 distinct microenvironments during its pathogenesis. On the insect cuticle, the transcription factor COH2 activates expression of cuticle penetration genes. In the hemocoel, the protein COH1 is expressed due to the reduction in epigenetic repression conferred by the histone deacetylase HDAC1 and the histone 3 acetyltransferase HAT1. COH1 interacts with COH2 to reduce COH2 stability, and this down-regulates cuticle penetration genes and up-regulates genes for hemocoel colonization. Our work significantly advances the insights into fungal pathogenicity in insects.

Dipeptidase PEPDA Is Required for the Conidiation Pattern Shift in Metarhizium acridum

Filamentous fungi conduct two types of conidiation, typical conidiation from mycelia and microcycle conidiation (MC). Fungal conidiation can shift between the two patterns, which involves a large number of genes in the regulation of this process. In this study, we investigated the role of a dipeptidase gene pepdA in conidiation pattern shift in Metarhizium acridum, which is upregulated in MC pattern compared to typical conidiation. Results showed that disruption of the pepdA resulted in a shift of conidiation pattern from MC to typical conidiation. Metabolomic analyses of amino acids showed that the levels of 19 amino acids significantly changed in ΔpepdA mutant. The defect of MC in ΔpepdA can be rescued when nonpolar amino acids, α-alanine, β-alanine, or proline, were added into sucrose yeast extract agar (SYA) medium. Digital gene expression profiling analysis revealed that PEPDA mediated transcription of sets of genes which were involved in hyphal growth and development, sporulation, cell division, and amino acid metabolism. Our results demonstrated that PEPDA played important roles in the regulation of MC by manipulating the levels of amino acids in M. acridum. IMPORTANCE Conidia, as the asexual propagules in many fungi, are the start and end of the fungal life cycle. In entomopathogenic fungi, conidia are the infective form essential for their pathogenicity. Filamentous fungi conduct two types of conidiation, typical conidiation from mycelia and microcycle conidiation. The mechanisms of the shift between the two conidiation patterns remain to be elucidated. In this study, we demonstrated that the dipeptidase PEPDA, a key enzyme from the insect-pathogenic fungus Metarhizium acridum for the hydrolysis of dipeptides, is associated with a shift of conidiation pattern. The conidiation pattern of the ΔpepdA mutant was restored when supplemented with the nonpolar amino acids rather than polar amino acids. Therefore, this report highlights that the dipeptidase PEPDA regulates MC by manipulating the levels of amino acids in M. acridum.

Activation of microlipophagy during early infection of insect hosts by Metarhizium robertsii

ABSTRACT The requirement of macroautophagic/autophagic machinery for filamentous fungal development and pathogenicity has been recognized, but the underlying effects and mechanisms remain elusive. The insect pathogenic fungus Metarhizium robertsii infects hosts by cuticular penetration through the formation of the infection structure appressoria. Here, we show that autophagic fluxes were highly activated during the appressorial formation of M. robertsii. Genome-wide deletion of the autophagy-related genes and insect bioassays identified 10 of 23 encoded MrATG genes with requirements for topical fungal infection of insect hosts. Besides the defect in forming appressoria on insects (two null mutants), these virulence-reduced mutants were largely impaired in penetrating cellophane membrane and insect cuticles, suggesting their failures in generating proper appressorium turgor. We found that the conidial storage of lipid droplets (LDs) had no obvious difference between strains, but autophagic LD degradation was impaired in different mutants. After induction of cell autophagy by nitrogen starvation, we found that LD entry into vacuoles was unaffected in the selected mutant cells with potential failures in forming autophagosomes. The finding therefore reveals a microlipophagy machinery employed in this fungus and that the direct engulfment of LDs occurs without inhibition by the downstream defective lipolysis. Our data first unveil the activation and contribution of microlipophagy to fungal infection biology. The obtained technique may benefit future detection of microlipophagy in different organisms by examining vacuolar or lysosomal engulfment of LDs in core autophagic gene deletion mutants. Abbreviations: AIM: Atg8-family interacting motif; ATG: autophagy-related; CM: complete medium; CMAC: 7-amino-4-chloromethylcoumarin; DTT: dithiothreitol; ER: endoplasmic reticulum; GFP: green fluorescent protein; LD, lipid droplet; MM: minimum medium; MM-N: minimum medium without nitrogen source; PDA: potato dextrose agar; PMSF: phenylmethylsulfonyl fluoride; RFP: red fluorescent protein; SDB: Sabouraud dextrose broth; SDS-PAGE: sodium dodecyl sulfate-polyacrylamide gel electrophoresis; TAG: triacylglycerol; TEM: transmission electron microscopy; WT, wild type.

Conservative production of galactosaminogalactan in Metarhizium is responsible for appressorium mucilage production and topical infection of insect hosts.

The exopolysaccharide galactosaminogalactan (GAG) has been well characterized in Aspergilli, especially the human pathogen Aspergillus fumigatus. It has been found that a five-gene cluster is responsible for GAG biosynthesis in Aspergilli to mediate fungal adherence, biofilm formation, immunosuppression or induction of host immune defences. Herein, we report the presence of the conserved GAG biosynthetic gene cluster in the insect pathogenic fungus Metarhizium robertsii to mediate either similar or unique biological functions. Deletion of the gene cluster disabled fungal ability to produce GAG on germ tubes, mycelia and appressoria. Relative to the wild type strain, null mutant was impaired in topical infection but not injection of insect hosts. We found that GAG production by Metarhizium is partially acetylated and could mediate fungal adherence to hydrophobic insect cuticles, biofilm formation, and penetration of insect cuticles. In particular, it was first confirmed that this exopolymer is responsible for the formation of appressorium mucilage, the essential extracellular matrix formed along with the infection structure differentiation to mediate cell attachment and expression of cuticle degrading enzymes. In contrast to its production during A. fumigatus invasive growth, GAG is not produced on the Metarhizium cells harvested from insect hemocoels; however, the polymer can glue germ tubes into aggregates to form mycelium pellets in liquid culture. The results of this study unravel the biosynthesis and unique function of GAG in a fungal system apart from the aspergilli species.

MripacC regulates blastosphere budding and influences virulence of the pathogenic fungus Metarhizium rileyi

Fungal dimorphism is the ability of certain fungi to switch between two different cellular forms, yeast and mycelial forms, in response to external environmental factors. The pacC/Pal signal transduction pathway responds to neutral and alkaline environments and is also involved in the fungal dimorphic transition. In this study, we investigated the function of the pacC homolog, MripacC, which regulates the dimorphic transition and modulates virulence of the insect pathogenic fungus Metarhizium rileyi. MripacC expression was upregulated under alkaline condition, with increased number of yeast-like cells compared to the number of hyphae cells. A MripacC deletion mutant (ΔMripacC) was obtained by homologous replacement and exhibited decreased blastospore budding, with direct development of conidia into hyphae without entering the yeast-like stage when cultured on alkaline medium. Observation of host hemolymph morphology and analysis of samples to detect the main immune factors revealed a decreased ability of ΔMripacC to evade the host immune system. The results of insect bioassays showed that ΔMripacC had decreased virulence with extended median lethality time. Together, the results suggested that MripacC not only regulated adaptation to acidic and alkaline environments, but also influenced virulence by budding blastospores. This elucidation of the function of MripacC adds to our understanding of blastospore budding and virulence of this fungal pathogen.

The Intermediates in Branched-Chain Amino Acid Biosynthesis Are Indispensable for Conidial Germination of the Insect-Pathogenic Fungus Metarhizium robertsii.

Metarhizium spp. are well-known biocontrol agents used worldwide to control different insect pests. Keto-acid reductoisomerase (ILVC) is a key enzyme for branched-chain amino acid (BCAA) biosynthesis, and it regulates many physiological activities. However, its functions in insect-pathogenic fungi are poorly understood. In this work, we identified MrilvC in M. robertsii and dissected its roles in fungal growth, conidiation, germination, destruxin biosynthesis, environmental stress response, and insecticidal virulence. BCAA metabolism affects conidial yields and germination. However, BCAAs cannot recover the conidial germination of an MrilvC-deficient strain. Further feeding assays with intermediates showed that some conidia of the ΔMrilvC mutant start to germinate. Therefore, it is the germination defect that causes the complete failures of conidial penetration and pathogenicity in the ΔMrilvC mutant. In conclusion, we found intermediates in BCAA biosynthesis are indispensable for Metarhizium robertsii conidial germination. This study will advance our understanding of the fungal germination mechanism.IMPORTANCE Branched-chain amino acid (BCAA) metabolism plays a significant role in many biological activities beyond protein synthesis. Spore germination initiates the first stage of vegetative growth, which is critical for the virulence of pathogenic fungi. In this study, we demonstrated that the keto-acid reductoisomerase MrILVC, a key enzyme for BCAA biosynthesis, from the insect-pathogenic fungus Metarhizium robertsii is associated with conidial germination and fungal pathogenicity. Surprisingly, the germination of the ΔMrilvC mutant was restored when supplemented with the intermediates of BCAA metabolism rather than three BCAAs. The result was significantly different from that of plant-pathogenic fungi. Therefore, this report highlights that the intermediates in BCAA biosynthesis are indispensable for conidial germination of M. robertsii.

Crustaceous wastes as growth substrates for insect-pathogenic fungus Metarhizium majus UICC 295

The genus Metarhizium consists of a diverse group of species, which have a global distribution and a wide range of insect hosts. Metarhizium majus was reported to inflict 100 % mortality on Oryctes rhinoceros beetle. Feeding activity of the O. rhinoceros beetle causes major crop loss in many coconut and palm oil plantations. This study investigated the use of crustaceous wastes and colloidal chitin as substrates for Metarhizium majus UICC 295 and the fungal virulence on O. rhinoceros larvae. Morphology of M. majus was observed on Sabouraud Dextrose Agar with Yeast Extract (SDAY) added with 5, 10, and 15 % (w/v) powder prepared from crab shell, green mussel shell, shrimp shell, or colloidal chitin and without addition as control. Fungal cultures in 10 % (w/v) crustaceous wastes or colloidal chitin were selected and virulence of M. majus UICC 295 was assayed by dripping the larvae with cell suspension. Untreated larvae were dripped with sterile distilled water and served as control. Mortality was recorded daily for 15 days. Fungal culture prepared from powder of colloidal chitin, green mussel shell and crab shell inflicted 100 % larval mortality in 10, 12, and 13 days, respectively, compared to fungal culture prepared from SDAY in 11 days.

A M35 family metalloprotease is required for fungal virulence against insects by inactivating host prophenoloxidases and beyond

ABSTRACT A diverse family of metalloproteases (MPs) is distributed in eukaryotes. However, the functions of MPs are still understudied. We report that seven MPs belonging to the M35 family are encoded in the genome of the insect pathogenic fungus Metarhizium robertsii. By gene deletions and insect bioassays, we found that one of the M35-family MPs, i.e. MrM35-4, is required for fungal virulence against insect hosts. MrM35-4 is a secretable enzyme and shows a proteolytic activity implicated in facilitating fungal penetration of insect cuticles. After gene rescue and overexpression, insect bioassays indicated that MrM35-4 contributes to inhibiting insect cuticular and hemocyte melanization activities. Enzymatic cleavage assays revealed that the recombinant prophenoloxidases PPO1 and PPO2 of Drosophila melanogaster could be clipped by MrM35-4 in a manner differing from a serine protease that can activate PPO activities. In addition, it was found that MrM35-4 is involved in suppressing antifungal gene expression in insects. Consistent with the evident apoptogenic effect of MrM35-4 on host cells, we found that the PPO mutant flies differentially succumbed to the infections of the wild-type and mutant strains of M. robertsii. Thus, MrM35-4 plays a multifaceted role beyond targeting PPOs during fungus-insect interactions, which represents a previously unsuspected strategy employed by Metarhizium to outmaneuver insect immune defenses.

Metarhizium robertsii ammonium permeases (MepC and Mep2) contribute to rhizoplanecolonization and modulates the transfer of insect derived nitrogen to plants.

The endophytic insect pathogenic fungi (EIPF) Metarhizium promotes plant growth through symbiotic association and the transfer of insect-derived nitrogen. However, little is known about the genes involved in this association and the transfer of nitrogen. In this study, we assessed the involvement of six Metarhizium robertsii genes in endophytic, rhizoplane and rhizospheric colonization with barley roots. Two ammonium permeases (MepC and Mep2) and a urease, were selected since homologous genes in arbuscular mycorrhizal fungi were reported to play a pivotal role in nitrogen mobilization during plant root colonization. Three other genes were selected on the basis on RNA-Seq data that showed high expression levels on bean roots, and these encoded a hydrophobin (Hyd3), a subtilisin-like serine protease (Pr1A) and a hypothetical protein. The root colonization assays revealed that the deletion of urease, hydrophobin, subtilisin-like serine protease and hypothetical protein genes had no impact on endophytic, rhizoplane and rhizospheric colonization at 10 or 20 days. However, the deletion of MepC resulted in significantly increased rhizoplane colonization at 10 days whereas ΔMep2 showed increased rhizoplane colonization at 20 days. In addition, the nitrogen transporter mutants also showed significantly higher 15N incorporation of insect derived nitrogen in barley leaves in the presence of nutrients. Insect pathogenesis assay revealed that disruption of MepC, Mep2, urease did not reduce virulence toward insects. The enhanced rhizoplane colonization of ΔMep2 and ΔMepC and insect derived nitrogen transfer to plant hosts suggests the role of MepC and Mep2 in Metarhizium-plant symbiosis.

Fungal volatile organic compounds show promise as potent molluscicides.

BACKGROUNDSlugs and snails constitute major crop pests. Withdrawal of metaldehyde has prompted a search for more environmentally friendly yet fast acting molluscicides. This study investigated the response of representative molluscs to conidia and volatile organic compounds (VOCs) of the insect pathogenic fungus Metarhizium brunneum Petch.RESULTSConidia of M. brunneum had antifeedant/repellent properties with repellency being dependent upon the fungal strain and conidia concentration. Three commonly produced fungal VOCs, 1‐octene, 3‐octanone and 1‐octen‐3‐ol, were repellent at low doses (1–5 μL) but could kill slugs and snails on contact or fumigation. At the highest dose tested (10 μL), 100% mortality was achieved for Cornu aspersum Muller (garden snail) and Derocerus reticulatum Muller (grey field slug) within 1 h post‐treatment with the first deaths being recorded in <11 min. Aqueous formulations (20% v/v) of the most potent VOCs, 3‐octanone and 1‐octen‐3‐ol, could be sprayed onto plants to kill or drive the pest of the crop with no phytotoxic effects.CONCLUSIONThe sensitivity of terrestrial molluscs to 3‐octanone and 1‐octen‐3‐ol and the ephemeral nature of these compounds makes these excellent candidates for development as mollusc repellents or molluscicides. © 2019 The Authors. Pest Management Science published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.

Disruption of an adenylate-forming reductase required for conidiation, increases virulence of the insect pathogenic fungus Metarhizium acridum by enhancing cuticle invasion.

Metarhizium acridum, is a specific acridid pathogen developed for use against the migratory locust (Locusta migratoria manilensis). Adenylate-forming reductases (AFRs) include enzymes that are involved in natural product biosynthesis. Here, we genetically characterize the functions of a class IV AFR in M. acridum (MaAfrIV ) on fungal development and virulence. Gene expression analyses indicated MaAfrIV was induced on locust wings early during the infection process. Surprisingly, loss of MaAfrIV increased virulence (25.20% decrease in the median lethal time) against the locust in topical bioassays but was no different than the wild type when the cuticle was bypassed by direct infection of conidia into the insect hemocoel. Virulence markers including protease (Pr1) expression and appressorial turgor pressure were higher in the mutant than the parent strain. No difference was seen in the expression of host immune genes (Toll pathway) or in polyphenol oxidase (PPO) activity in locusts infected by the ΔMaAfrIV or wild type strains. However, the ΔMaAfrIV strain was unable to successfully sporulate on dead cadavers. Disruption of MaAfrIV increased fungal virulence by promoting insect cuticle invasion without altering host immune response or fungal immune evasion. Although loss of MaAfrIV conferred an apparent benefit to the fungus in terms of enhanced virulence, a significant trade-off was seen in the inability of the fungus to sporulate on the cadaver. As conidiation on the cadaver is essential for subsequent propagation in the environment, loss of MaAfrIV can reduce the engineering strains survivability in the field and improve the safety. © 2019 Society of Chemical Industry.

Unveiling the function and regulation control of the DUF3129 family proteins in fungal infection of hosts.

Many prokaryotic and eukaryotic proteins contain domains of unknown function (DUFs). A DUF3129 family of proteins is widely encoded in the genomes of fungal pathogens. A few studies in plant and insect pathogens indicated that the DUF3129 genes are required for fungal penetration of host cuticles with an unclear mechanism. We found that a varied number of DUF3129 proteins is present in different fungal species and the proteins are evolutionarily diverged from each other at the inter- and intra-specific levels. By using the insect pathogenic fungus Metarhizium robertsii as a model, we performed experiments and found that the seven DUF3129 proteins encoded by this fungus are localized to cellular lipid droplets (LDs). Individual deletion of these genes did not affect fungal formation of the infection structure appressoria and the accumulation of LDs in fungal conidia. When compared with the wild-type (WT) strain, insect bioassays revealed that the virulence of most null mutants were significantly impaired during topical infection but not during injection of insects. Carbon starvation and the subsequent Western blot analysis indicated that the LD-specific perilipin protein was completely degraded in the WT cells whereas varied levels of perilipin could be detected in the mutant cells, which signified that depletion of LD content was delayed in mutant cells, and DUF3129 proteins are therefore involved in LD degradation. We also provided biochemical evidence that these DUF3129 genes are transcriptionally regulated by a yeast Ste12-like transcription factor. The findings of this study not only unveil the function of DUF3129 proteins but also better understand the diverse mechanism of fungus-host interactions. This article is part of the theme issue 'Biotic signalling sheds light on smart pest management'.