Pathogenicity of Metarhizium rileyi (Hypocreales: Clavicipitaceae) against Tenebrio molitor (Coleoptera: Tenebrionidae).

Tenebrio molitor L., also known as the mealworm, is a polyphagous insect pest that infests various stored grains worldwide. Both the adult and larval stages can cause significant damage to stored grains. The present study focused on isolating entomopathogenic fungi from an infected larval cadaver under environmental conditions. Fungal pathogenicity was tested on T. molitor larvae and pupae for 12 days. Entomopathogenic fungi were identified using biotechnological methods based on their morphology and the sequence of their nuclear ribosomal internal transcribed spacer (ITS). The results of the insecticidal activity indicate that the virulence of fungi varies between the larval and pupal stages. In comparison to the larval stage, the pupal stage is highly susceptible to Metarhizium rileyi, exhibiting 100% mortality rates after 12 days (lethal concentration 50 [LC50] = 7.8 × 106 and lethal concentration 90 (LC90) = 2.1 × 1013 conidia/mL), whereas larvae showed 92% mortality rates at 12 days posttreatment (LC50 = 1.0 × 106 and LC90 = 3.0 × 109 conidia/mL). The enzymatic analyses revealed a significant increase in the levels of the insect enzymes superoxide dismutase (4.76-10.5 mg-1) and glutathione S-transferase (0.46-6.53 mg-1) 3 days after exposure to M. rileyi conidia (1.5 × 105 conidia/mL) compared to the control group. The findings clearly show that M. rileyi is an environmentally friendly and effective microbial agent for controlling the larvae and pupae of T. molitor.

Study on the virulence of Metarhizium anisopliae against Spodoptera frugiperda (J. E. Smith, 1797).

This study examined the impact of Metarhizium anisopliae (Hypocreales: Clavicipitaceae) conidia on the eggs, larvae, pupae, and adults of Spodoptera frugiperda. The results showed that eggs, larvae, pupae, and adults exhibited mortality rates that were dependent on the dose. An increased amount of conidia (1.5 × 109 conidia/mL) was found to be toxic to larvae, pupae, and adults after 9 days of treatment, resulting in a 100% mortality rate in eggs, 98% in larvae, 76% in pupae, and 85% in adults. A study using earthworms as bioindicators found that after 3 days of exposure, M. anisopliae conidia did not cause any harmful effects on the earthworms. In contrast, the chemical treatment (positive control) resulted in 100% mortality at a concentration of 40 ppm. Histopathological studies showed that earthworm gut tissues treated with fungal conidia did not show significant differences compared with those of the negative control. The gut tissues of earthworms treated with monocrotophos exhibited significant damage, and notable differences were observed in the chemical treatment. The treatments with 70 and 100 µg/mL solutions of Eudrilus eugeniae epidermal mucus showed no fungal growth. An analysis of the enzymes at a biochemical level revealed a decrease in the levels of acetylcholinesterase, α-carboxylesterase, and ß-carboxylesterase in S. frugiperda larvae after exposure to fungal conidia. This study found that M. anisopliae is effective against S. frugiperda, highlighting the potential of this entomopathogenic fungus in controlling this agricultural insect pest.

N-terminal zinc fingers of MaNCP1 contribute to growth, stress tolerance, and virulence in Metarhizium acridum.

C2H2 zinc finger proteins (ZFPs) are a class of important transcriptional regulators in eukaryotes involved in multiple biological regulation processes. Here, MaNCP1, a C2H2 ZFP, was functionally characterized in the model entomopathogenic fungus Metarhizium acridum. Deletion of MaNCP1 delayed conidial germination and hyphal growth, decreased the conidial yield and reduced the tolerances to UV-B irradiation and heat-shock. The N-terminal zinc fingers (ZFs) of MaNCP1 made the main contributions to these traits. In addition, disruption of MaNCP1 altered the conidial surface structure and decreased the conidial hydrophobicity. Bioassays showed that the virulence of the MaNCP1-disruption strain (ΔMaNCP1) was reduced in topical inoculation compared to the WT or the mutant complemented strain (CP), and the N-terminal C2H2 ZFs made a major contribution to virulence. Furthermore, the ΔMaNCP1 and C2H2 ZFs deletion mutants (MaNCP1∆N and MaNCP1∆N+C) impaired cuticular penetration. RNA-seq showed that several cuticle-degrading genes were down-regulated in the ΔMaNCP1 background, suggesting that MaNCP1 plays vital roles in regulating insect cuticle penetration. In summary, MaNCP1 affected the growth, stress tolerances and virulence of M. acridum, and the N-terminal C2H2 ZFs played indispensable roles in these important biocontrol traits. These results provide further insights into the functions of C2H2 ZFPs in entomopathogenic fungi.

Insect Fungus Metarhizium robertsii Is a Rare Causative Agent of Keratitis.

PURPOSE: This study aimed to describe a case of keratitis secondary to Metarhizium robertsii, a soil-dwelling fungus with typical insect pathogenicity. METHODS: A Case report. RESULTS: A 58-year-old man with a ocular history of soft contact lens overwear, poor contact lens hygiene, and ocular exposure to ground well water supplying his house was referred to our university practice for a central corneal ulcer with 40% thinning. Same-day rapid microscopic detection with Giemsa stain identified the presence of many hyphae elements, and he was ultimately diagnosed with M. robertsii keratitis. The patient's course involved severe and progressive corneal thinning that stabilized over a 3-month course of antifungal therapy. CONCLUSIONS: Metarhizium robertsii seems more aggressive than other forms of fungal keratitis; therefore, this infection may be watched more closely with the risk for quick progressive corneal thinning, even while on antifungal management.

A fungus infected environment does not alter the behaviour of foraging ants.

Eusocial insects are exposed to a wide range of pathogens while foraging outside their nest. We know that opportunistic scavenging ants are able to assess the sanitary state of food and to discriminate a prey which died from infection by the entomopathogenic fungus Metarhizium brunneum. Here, we investigate whether a contamination of the environment can also influence the behaviour of foragers, both at the individual and collective level. In a Y-maze, Myrmica rubra ants had the choice to forage on two prey patches, one of which containing sporulating items. Unexpectedly, the nearby presence of sporulating bodies did not deter foragers nor prevent them from retrieving palatable prey. Ant colonies exploited both prey patches equally, without further mortality resulting from foraging on the contaminated area. Thus, a contamination of the environment did not prompt an active avoidance by foragers of which the activity depended primarily on the food characteristics. Generalist entomopathogenic fungi such as M. brunneum in the area around the nest appear more to be of a nuisance to ant foragers than a major selective force driving them to adopt avoidance strategies. We discuss the cost-benefit balance derived from the fine-tuning of strategies of pathogen avoidance in ants.

Metarhizium anisopliae blastospores are highly virulent to adult Aedes aegypti, an important arbovirus vector.

BACKGROUND: The use of entomopathogenic fungi (EPF) for the control of adult mosquitoes is a promising alternative to synthetic insecticides. Previous studies have only evaluated conidiospores against adult mosquitoes. However, blastospores, which are highly virulent against mosquito larvae and pupae, could also be effective against adults. METHODS: Metarhizium anisopliae (ESALQ 818 and LEF 2000) blastospores and conidia were first tested against adult Aedes aegypti by spraying insects with spore suspensions. Blastospores were then tested using an indirect contact bioassay, exposing mosquitoes to fungus-impregnated cloths. Virulence when using blastospores suspended in 20% sunflower oil was also investigated. RESULTS: Female mosquitoes sprayed with blastospores or conidia at a concentration of 108 propagules ml-1 were highly susceptible to both types of spores, resulting in 100% mortality within 7 days. However, significant differences in virulence of the isolates and propagules became apparent at 107 spores ml-1, with ESALQ 818 blastospores being more virulent than LEF 2000 blastospores. ESALQ 818 blastospores were highly virulent when mosquitoes were exposed to black cotton cloths impregnated with blastospores shortly after preparing the suspensions, but virulence declined rapidly 12 h post-application. The addition of vegetable oil to blastospores helped maintain virulence for up to 48 h. CONCLUSION: The results showed that blastospores were more virulent to adult female Ae. aegypti than conidia when sprayed onto the insects or applied to black cloths. Vegetable oil helped maintain blastospore virulence. The results show that blastospores have potential for use in integrated vector management, although new formulations and drying techniques need to be investigated.

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.

O-mannosyltransferase MaPmt2 contributes to stress tolerance, cell wall integrity and virulence in Metarhizium acridum.

As a conserved post-translational modification, O-mannosyltransferase families play important roles in many cellular processes. Three subfamilies (MaPmt1, MaPmt2 and MaPmt4) are grouped in Metarhizium acridum according to sequence homology. The functions of MaPmt1 and MaPmt4 have been characterized in M. acridum previously. In this study, the functions of another member belonging to the Pmt2 subfamily, MaPmt2, were identified through RNAi strategy. The three MaPmt2 knockdown mutants showed dramatically decreased expression of MaPmt2. Phenotypic analyses showed that the mutants exhibited decreased tolerances to wet-heat, UV-B irradiation and cell wall perturbing chemicals. Further studies revealed that the mutants presented thinner cell walls observed by transmission electron microscope combined with changed cell wall components. Besides, knockdown of MaPmt2 decelerated conidial germination and decreased conidial yield. Compared with the wild-type strain, the MaPmt2 knockdown mutants caused impaired virulence only by topical inoculation. Results illustrated that the decreased virulence by inoculation could result from the delayed conidial germination on locust wings, reduced appressorium formation, as well as reduced turgor pressure in MaPmt2 knockdown mutants.

A Baeyer-Villiger Monooxygenase Gene Involved in the Synthesis of Lysergic Acid Amides Affects the Interaction of the Fungus Metarhizium brunneum with Insects.

Several fungi, including the plant root symbiont and insect pathogen Metarhizium brunneum, produce lysergic acid amides via a branch of the ergot alkaloid pathway. Lysergic acid amides include important pharmaceuticals and pharmaceutical lead compounds and have potential ecological significance, making knowledge of their biosynthesis relevant. Many steps in the biosynthesis of lysergic acid amides have been determined, but terminal steps in the synthesis of lysergic acid α-hydroxyethylamide (LAH)-by far the most abundant lysergic acid amide in M. brunneum-are unknown. Ergot alkaloid synthesis (eas) genes are clustered in the genomes of fungi that produce these compounds, and the eas clusters of LAH producers contain two uncharacterized genes (easO and easP) not found in fungi that do not produce LAH. Knockout of easO via a CRISPR-Cas9 approach eliminated LAH and resulted in accumulation of the alternate lysergic acid amides lysergyl-alanine and ergonovine. Despite the elimination of LAH, the total concentration of lysergic acid derivatives was not affected significantly by the mutation. Complementation with a wild-type allele of easO restored the ability to synthesize LAH. Substrate feeding studies indicated that neither lysergyl-alanine nor ergonovine were substrates for the product of easO (EasO). EasO had structural similarity to Baeyer-Villiger monooxygenases (BVMOs), and labeling studies with deuterated alanine supported a role for a BVMO in LAH biosynthesis. The easO knockout had reduced virulence to larvae of the insect Galleria mellonella, indicating that LAH contributes to virulence of M. brunneum on insects and that LAH has biological activities different from ergonovine and lysergyl-alanine. IMPORTANCE Fungi in the genus Metarhizium are important plant root symbionts and insect pathogens. They are formulated commercially to protect plants from insect pests. Several Metarhizium species, including M. brunneum, were recently shown to produce ergot alkaloids, a class of specialized metabolites studied extensively in other fungi because of their importance in agriculture and medicine. A biological role for ergot alkaloids in Metarhizium species had not been demonstrated previously. Moreover, the types of ergot alkaloids produced by Metarhizium species are lysergic acid amides, which have served directly or indirectly as important pharmaceutical compounds. The terminal steps in the synthesis of the most abundant lysergic acid amide in Metarhizium species and several other fungi (LAH) have not been determined. The results of this study demonstrate the role of a previously unstudied gene in LAH synthesis and indicate that LAH contributes to virulence of M. brunneum on insects.

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.

The deletion of chiMaD1, a horizontally acquired chitinase of Metarhizium anisopliae, led to higher virulence towards the cattle tick (Rhipicephalus microplus).

The first line of the Arthropods defense against infections is the hard-structured exoskeleton, a physical barrier, usually rich in insoluble chitin. For entomopathogenic fungi that actively penetrate the host body, an arsenal of hydrolytic enzymes (as chitinases and N-acetylglucosaminidases), that break down chitin, is essential. Notably, twenty-one putative chitinase genes have been identified in the genome of Metarhizium anisopliae, a generalist entomopathogenic fungus. As a multigenic family, with enzymes that, presumably, perform redundant functions, the main goal is to understand the singularity of each one of such genes and to discover their precise role in the fungal life cycle. Specially chitinases that can act as virulence determinants are of interest since these enzymes can lead to more efficient biocontrol agents. Here we explored a horizontally acquired chitinase from M. anisopliae, named chiMaD1. The deletion of this gene did not lead to phenotypic alterations or diminished supernatant's chitinolytic activity. Surprisingly, chiMaD1 deletion enhanced M. anisopliae virulence to the cattle tick (Rhipicephalus microplus) larvae and engorged females, while did not alter the virulence to the mealworm larvae (Tenebrio molitor). These results add up to recent reports of deleted genes that enhanced entomopathogenic virulence, showing the complexity of host-pathogen interactions.

The G-protein coupled receptor GPRK contributes to fungal development and full virulence in Metarhizium robertsii.

G-protein-coupled receptor K (GPRK), which is a class VI fungal G-protein-coupled receptor (GPCR), plays a critical role in plant immunity against pathogens by mediating the endocytic pathway, influencing metabolism in response to environmental signals, and regulating asexual reproduction and pathogenic development. However, the function of these proteins in entomopathogenic fungi has rarely been investigated. Accordingly, we characterized MrGPRK, a GPCR in the entomopathogenic fungus Metarhizium robertsii containing a C-terminal seven-transmembrane and a conserved regulator of G protein signaling domain, and found that it localized to endosomes. Mutant phenotype assays showed that a ΔMrGprk strain displayed increased defects in radial growth (~28%) and decreased conidial production (~80%) compared with a wild-type strain. Decreased conidiation rates coincided well with the repression of conidiation-related regulatory genes, including three key conidial transcription factors: brlA, abaA, and wetA. MrGprk deficiency impaired full virulence (both topical and injectable inoculations). Further analysis demonstrated that deleting fungal MrGprk decreased the rates of appressorium formation and suppressed the transcription of several genes contributing to appressorial turgor pressure, cuticle penetration, and pH regulation. Additionally, the ΔMrGprk strain showed higher cyclic (cAMP) levels, suggesting that this GPCR is critical for cAMP signal transduction. In summary, MrGPRK was found to contribute to vegetative growth, conidial production, and full virulence of M. robertsii. These findings are conducive to a better understanding of the roles of GPCRs in the development and pathogenicity of entomopathogenic fungi.

Earthworms and their cutaneous excreta can modify the virulence and reproductive capability of entomopathogenic nematodes and fungi.

Earthworms are ecological engineers that can contribute to the displacement of biological control agents such as the entomopathogenic nematodes (EPNs) and fungi (EPF). However, a previous study showed that the presence of cutaneous excreta (CEx) and feeding behavior of the earthworm species Eisenia fetida (Haplotaxida: Lumbricidae) compromise the biocontrol efficacy of certain EPN species by reducing, for example, their reproductive capability. Whether this phenomenon is a general pattern for the interaction of earthworms-entomopathogens is still unknown. We hypothesized that diverse earthworm species might differentially affect EPN and EPF infectivity and reproductive capability. Here we investigated the interaction of different earthworm species (Eisenia fetida, Lumbricus terrestris, and Perionyx excavatus) (Haplotaxida) and EPN species (Steinernema feltiae, S. riojaense, and Heterorhabditis bacteriophora) (Rhabditida) or EPF species (Beauveria bassiana and Metarhizium anisopliae) (Hypocreales), in two independent experiments. First, we evaluated the application of each entomopathogen combined with earthworms or their CEx in autoclaved soil. Hereafter, we studied the impact of the earthworms' CEx on entomopathogens applied at two different concentrations in autoclaved sand. Overall, we found that the effect of earthworms on entomopathogens was species-specific. For example, E. fetida reduced the virulence of S. feltiae, resulted in neutral effects for S. riojaense, and increased H. bacteriophora virulence. However, the earthworm P. excavates increased the virulence of S. feltiae, reduced the activity of H. bacteriophora, at least at specific timings, while S. riojaense remained unaffected. Finally, none of the EPN species were affected by the presence of L. terrestris. Also, the exposure to earthworm CEx resulted in a positive, negative or neutral effect on the virulence and reproduction capability depending on the earthworm-EPN species interaction. Concerning EPF, the impact of earthworms was also differential among species. Thus, E. fetida was detrimental to M. anisopliae and B. bassiana after eight days post-exposure, whereas Lumbricus terrestris resulted only detrimental to B. bassiana. In addition, most of the CEx treatments of both earthworm species decreased B. bassiana virulence and growth. However, the EPF M. anisopliae was unaffected when exposed to L. terrestris CEx, while the exposure to E. fetida CEx produced contrasting results. We conclude that earthworms and their CEx can have positive, deleterious, or neutral impacts on entomopathogens that often coinhabit soils, and that we must consider the species specificity of these interactions for mutual uses in biological control programs. Additional studies are needed to verify these interactions under natural conditions.

Exposure to a sublethal menadione concentration modifies the mycelial secretome and conidial enzyme activities of Metarhizium anisopliae sensu lato and increases its virulence against Rhipicephalus microplus.

Menadione (MND) is known to induce oxidative stress in fungal cells. Here, we explore how exposure to this molecule alters conidial enzyme activities, fungal efficacy against Rhipicephalus microplus, and mycelial secretion (secretome) of an isolate of Metarhizium anisopliae sensu lato. First, the fungus was exposed to different MND concentrations in potato-dextrose-agar (PDA) to determine the LC50 by evaluating conidia germination (38µM). To ensure high cell integrity, a sublethal dose of MND (half of LC50) was added to solid (PDA MND) and liquid media (MS MND). Changes in colony growth, a slight reduction in conidia production, decreases in conidial surface Pr1 and Pr2 activities as well as improvements in proteolytic and antioxidant (catalase, superoxide dismutase, and peroxidase) conidial intracellular activities were observed for PDA MND conidia. Additionally, PDA MND conidia had the best results for killing tick larvae, with the highest mortality rates until 15 days after treatment, which reduces both LC50 and LT50, particularly at 108 conidia mL-1. The diversity of secreted proteins after growth in liquid medium + R. microplus cuticle (supplemented or not with half of MND LC50), was evaluated by mass spectrometry-based proteomics. A total of 654 proteins were identified, 31 of which were differentially regulated (up or down) and mainly related to antioxidant activity (catalase), pathogenicity (Pr1B, Pr1D, and Pr1K), cell repair, and morphogenesis. In the exclusively MS MND profile, 48 proteins, mostly associated with cellular signaling, nutrition, and antioxidant functions, were distinguished. Finally, enzymatic assays were performed to validate some of these proteins. Overall, supplementation with MND in the solid medium made conidia more efficient at controlling R. microplus larvae, especially by increasing, inside the conidia, the activity of some infection-related enzymes. In the liquid medium (a consolidated study model that mimics some infection conditions), proteins were up- and/or exclusively-regulated in the presence of MND, which opens a spectrum of new targets for further study to improve biological control of ticks using Metarhizium species.

Ovicidal Efficacy of Metarhizium anisopliae (Hypocreales: Clavicipitaceae) towards Rhipicephalus sanguineus (Acari: Ixodidae) Eggs.

Rhipicephalus sanguineus, commonly known as brown dog tick is a widespread species with considerable public health and economic importance. Tremendous efforts were performed to control the tick populations with the concern of resistance build-up and environmental issues. Alternative towards microbial control thus emerged as one option to reduce tick populations. In this study, the ovicidal efficacy of a native isolate entomopathogenic hyphomycetes fungi, Metarhizium anisopliae strain HSAH5 was evaluated against eggs of R. sanguineus. Spray applications with three different conidial concentrations of 105, 106 and 107 conidia mL-1; 40 ppm of Flumethrin and a negative control. The M. anisopliae strain was found highly virulent to R. sanguineus eggs by reducing the hatching percentages to ≈30% compared with 8.9% in Flumethrin eggs. The result showed a significantly higher mortality in M. anisopliae group than those of the control groups (F = 42.08, df = 32, P < 0.001) at 30 days post-infection. However, there are no significant differences within the M. anisopliae group, in which the mortality between different conidial concentrations is almost the same. The estimated LC50 of M. anisopliae against eggs of R. sanguineus is 1.36 × 103 conidia ml-1. Thus, these results suggest M. anisopliae strain HSAH5 could be a potential biocontrol agent of R. sanguineus in the integrated approach to managing ticks in the residential landscape by targeting on the eggs.

Functional and characteristic analysis of an appressorium-specific promoter PMagas1 in Metarhizium acridum.

Entomopathogenic fungi have been used as important biological control agents throughout the world. To improve the biocontrol efficacy of entomopathogenic fungi, many genes have been used to improve fungal virulence or tolerance to adverse conditions via modulating their expression with strong promoters. The Magas1 gene is specifically expressed during appressorium formation and contributes to the virulence in Metarhizium acridum. In this study, we analyzed the functional region of the promoter of Magas1 gene (PMagas1) in M. acridum using 5'-deletion technique with enhanced green fluoresces protein (EGFP) as a reporter. Results showed the full length of the PMagas1 was at least 897 bp. Two regions (-897 to -611 bp and -392 to -328 bp) were essential for the activity of PMagas1. An engineered M. acridum strain was constructed with PMagas1 driving the expression of a subtilisin-like proteinase gene Pr1A (PMagas1-PR1A). Bioassay showed that the virulence was significantly increased in PMagas1-PR1A strain compared to wild type strain. Pmagas1 promoter is suitable for the overexpression of some genes during the infection of entomopathogenic fungi, which avoids the waste of nutritional resources and the influence on other fungal characteristics during the saprophytic process of constitutive promoter.

Comparative Virulence of Metarhizium anisopliae and Four Strains of Beauveria bassiana Against House Fly (Diptera: Muscidae) Adults With Attempted Selection for Faster Mortality.

Entomopathogenic fungi such as Beauveria bassiana (Balsamo) Vuillemin and Metarhizium anisopliae/brunneum (Metchnikoff)/Petch have shown promising results for managing the house fly, Musca domestica L. A primary challenge of using these biological control agents (BCAs) in field situations is the time required to induce high adult house fly mortality, typically 6-7 d post-exposure. In this study, virulence of M. anisopliae (strain F52) and four B. bassiana strains were compared. The B. bassiana strains GHA and HF23 are used in commercial products and those were compared with two strains that were isolated from house flies on dairy farms (NFH10 and L90). Assays were conducted by exposing adult house flies to fungal-treated filter paper disks for 2 h. The lethal time to 50% mortality (LT50) at the high concentration of 1 × 109 conidia ranged from 3.8 to 5.2 d for all five strains. GHA, NFH10, and L90 killed flies faster than M. anisopliae strain F52; HF23 did not differ from either the M. anisopliae or the other B. bassiana strains. Attempts with the NFH10 strain to induce faster fly mortality through selection across 10 fungal to fly passages did not result in shorter time to fly death of the selected strain compared with the unselected strain.

Pathogenicity of Metarhizium rileyi against Spodoptera litura larvae: Appressorium differentiation, proliferation in hemolymph, immune interaction, and reemergence of mycelium.

The pathogenicity of Metarhizium rileyi is a multi-faceted process that depends on many factors. This study attempts to decipher those factors of M. rileyi by investigating its pathogenicity against Spodoptera litura (Lepidoptera: Noctuidae) larvae. Through morphogenesis analysis, we for the first time demonstrated the infection structure, appressorium, of M. rileyi that can generate a more than 4 MPa turgor pressure. The Mrpmk1 gene was found to be essential for appressorium differentiation and mycelium reemerging, ΔMrpmk1 mutant exhibited no pathogenicity towards S. litura by natural infection process. Delayed appressorium formation time, decreased appressorium formation rate and turgor pressure of ΔMrpbs2 mutant manifested itself in postponed death time and lower mortality against S. litura. Following invasion into the larval hemocoel, M. rileyi cells transformed into blastospores, which may be conducive to dispersal and propagation, moreover, the blastospore form M. rileyi may subverted phagocytic defenses. Then M. rileyi cells morphed into extended hyphal body to cope with elongated hemocytes that participated in encapsulation. In the end, M. rileyi mycelia reemerged from the larval cadaver evenly to form muscardine cadaver. Eventually, conidia were produced to complete the infection cycle. During the infection, M. rileyi triggered both cellular and humoral immunity of S. litura. Besides morphological changes, stage-specifically produced oxalic acid and F-actin arrangement may play roles in nutrient acquisition and mycelium reemerging, respectively.

Identification of a key G-protein coupled receptor in mediating appressorium formation and fungal virulence against insects.

Fungal G-protein coupled receptors (GPCRs) play essential roles in sensing environmental cues including host signals. The study of GPCR in mediating fungus-insect interactions is still limited. Here we report the evolution of GPCR genes encoded in the entomopathogenic Metarhizium species and found the expansion of Pth11-like GPCRs in the generalist species with a wide host range. By deletion of ten candidate genes MrGpr1-MrGpr10 selected from the six obtained subfamilies in the generalist M. robertsii, we found that each of them played a varied level of roles in mediating appressorium formation. In particular, deletion of MrGpr8 resulted in the failure of appressorium formation on different substrates and the loss of virulence during topical infection of insects but not during injection assays when compared with the wild-type (WT) strain. Further analysis revealed that disruption of MrGpr8 substantially impaired the nucleus translocation of the mitogen-activated protein kinase (MAPK) Mero-Fus3 but not the MAPK Mero-Slt2 during appressorium formation. We also found that the defect of AMrGpr8 could not be rescued with the addition of cyclic AMP for appressorium formation. Relative to the WT, differential expression of the selected genes have also been detected in AMrGpr8. The results of this study may benefit the understanding of fungus-interactions mediated by GPCRs.

Characterization and biocontrol potential of a naturally occurring isolate of Metarhizium pingshaense infecting Conogethes punctiferalis.

An entomopathogenic fungus was isolated from an infected larva of Conogethes punctiferalis (Guenée) (Crambidae: Lepidoptera), a highly polyphagous pest recorded from more than 120 plants and widely distributed in Asia and Oceanic countries. The fungus was identified as Metarhizium pingshaense Q.T. Chen & H.L. Guo (Ascomycota: Hypocreales) based on morphological characteristics and molecular studies. Scanning electron microscopic studies were conducted to study the infection of C. punctiferalis by M. pingshaense. Bioassay studies with purified conidial suspension proved that the isolate was highly virulent to C. punctiferalis, causing more than 86 % mortality to fifth instar larvae at 1 × 108 spores/mL, under laboratory conditions. The median lethal concentration (LC50) of the fungus against late instar larvae was 9.1 × 105 conidia/mL and the median survival time (MST) of late instar larvae tested at the doses of 1 × 108 and 1 × 107 conidia/mL were 4.7 and 6.4 days, respectively. The optimal temperature for fungal growth and sporulation was found to be 25 ± 1 °C. This is the first report of M. pingshaense naturally infecting C. punctiferalis. Isolation of a highly virulent strain of this fungus holds promise towards development of a potential mycoinsecticide against this pest.