Transfection of entomopathogenic Metarhizium species with a mycovirus confers hypervirulence against two lepidopteran pests.

Although most known viruses infecting fungi pathogenic to higher eukaryotes are asymptomatic or reduce the virulence of their host fungi, those that confer hypervirulence to entomopathogenic fungus still need to be explored. Here, we identified and studied a novel mycovirus in Metarhizium flavoviride, isolated from small brown planthopper (Laodelphax striatellus). Based on molecular analysis, we tentatively designated the mycovirus as Metarhizium flavoviride partitivirus 1 (MfPV1), a species in genus Gammapartitivirus, family Partitiviridae. MfPV1 has two double-stranded RNAs as its genome, 1,775 and 1,575 bp in size respectively, encapsidated in isometric particles. When we transfected commercial strains of Metarhizium anisopliae and Metarhizium pingshaense with MfPV1, conidiation was significantly enhanced (t test; P-value < 0. 01), and the significantly higher mortality rates of the larvae of diamondback moth (Plutella xylostella) and fall armyworm (Spodoptera frugiperda), two important lepidopteran pests were found in virus-transfected strains (ANOVA; P-value < 0.05). Transcriptomic analysis showed that transcript levels of pathogenesis-related genes in MfPV1-infected M. anisopliae were obviously altered, suggesting increased production of metarhizium adhesin-like protein, hydrolyzed protein, and destruxin synthetase. Further studies are required to elucidate the mechanism whereby MfPV1 enhances the expression of pathogenesis-related genes and virulence of Metarhizium to lepidopteran pests. This study presents experimental evidence that the transfection of other entomopathogenic fungal species with a mycovirus can confer significant hypervirulence and provides a good example that mycoviruses could be used as a synergistic agent to enhance the biocontrol activity of entomopathogenic fungi.

Molecular identification and related functional characterization of the FKBP52 gene in immunity of Locusta migratoria manilensis (Orthoptera: Oedipodidae).

Metarhizium anisopliae is an important class of entomopathogenic fungi used for the biocontrol of insects, but its virulence is affected by insect immunity. We identified a novel FK506 binding protein gene that was differentially expressed between control and Metarhizium-treated Locusta migratoria manilensis. We hypothesized that this protein played an important role in Metarhizium infection of L. migratoria and could provide new insights for developing highly efficient entomopathogenic fungi. We, therefore, cloned the specific gene and obtained its purified protein. The gene was then named FKBP52, and its dsRNA (dsFKBP52) was synthesized and used for gene interference. Bioassay results showed that the mortality of L. migratoria treated with dsFKBP52 + Metarhizium was significantly lower than that of other treatments. Furthermore, immune-related genes (MyD88, Dorsal, Cactus, and Defensin) in L. migratoria treated with dsFKBP52 + Metarhizium showed significant upregulation compared to that treated with Metarhizium only. However, the activities of peroxidase (POD), superoxide dismutase (SOD), and calcineurin (CaN) showed fluctuations. These results suggest that the FKBP52 gene may play a crucial role in the innate immunity of L. migratoria. The effect of its silencing indicated that this immunity-related protein might be a potential target for insect biocontrol.

The role of MrUbp4, a deubiquitinase, in conidial yield, thermotolerance, and virulence in Metarhizium robertsii.

Ubiquitin-specific proteases (UBPs), the largest subfamily of deubiquitinating enzymes, regulate ubiquitin homeostasis and play diverse roles in eukaryotes. Ubp4 is essential for the growth, development, and pathogenicity of various fungal pathogens. However, its functions in the growth, stress responses, and virulence of entomopathogenic fungi remain unclear. In this study, we elucidated the role of the homolog of Ubp4, MrUbp4, in the entomopathogenic fungus Metarhizium robertsii. Deletion of MrUbp4 led to a notable increase in ubiquitination levels, demonstrating the involvement of MrUbp4 in protein deubiquitination. Furthermore, the ΔMrUbp4 mutant displayed a significant reduction in conidial yield, underscoring the pivotal role of MrUbp4 in conidiation. Additionally, the mutant exhibited heightened resistance to conidial heat treatment, emphasizing the role of MrUbp4 in thermotolerance. Notably, insect bioassays unveiled a substantial impairment in the virulence of the ΔMrUbp4 mutant. This was accompanied by a notable decrease in cuticle penetration ability and appressorium formation upon further analysis. In summary, our findings highlight the essential role of MrUbp4 in regulating the conidial yield, thermotolerance, and contributions to the virulence of M. robertsii.

Utilization of plant-derived sugars and lipids are coupled during colonization of rhizoplane and rhizosphere by the fungus Metarhizium robertsii.

Plant-derived sugars and lipids are key nutritional sources for plant associated fungi. However, the relationship between utilization of host-derived sugars and lipids during development of the symbiotic association remains unknown. Here we show that the fungus Metarhizium robertsii also needs plant-derived lipids to develop symbiotic relationship with plants. The fatty acid binding proteins FABP1 and FABP2 are important for utilization of plant-derived lipids as the deletion of Fabp1 and Fabp2 significantly reduced the ability of M. robertsii to colonize rhizoplane and rhizosphere of maize and Arabidopsis thaliana. Deleting Fabp1 and Fabp2 increased sugar utilization by upregulating six sugar transporters, and this explains why deleting the monosaccharide transporter gene Mst1, which plays an important role in utilization of plant-derived sugars, had no impact on the ability of the double-gene deletion mutant ΔFabp1::ΔFabp2 to colonize plant roots. FABP1 and FABP2 were also found in other plant-associated Metarhizium species, and they were highly expressed in the medium using the tomato root exudate as the sole carbon and nitrogen source, suggesting that they could be also important for these species to develop symbiotic relationship with plants. In conclusion, we discovered that utilization of plant-derived sugars and lipids are coupled during colonization of rhizoplane and rhizosphere by M. robertsii.

Genetic diversity of the entomopathogenic fungus Metarhizium rileyi based on de novo microsatellite markers.

Epizootics of the entomopathogenic fungus Metarhizium rileyi regulate lepidopteran populations in soybean, cotton, and peanut agroecosystems to the point that insecticide applications could be unnecessary. However, the contribution and how different strains operate during the epizootic are unknown. Several unanswered questions remain: 1. How many genotypes of M. rileyi are present during an epizootic? 2. Which genotype is the most common among them? 3. Are the genotypes involved in annual epizootics at the same location the same? Therefore, the development of molecular markers to accurately identify these genotypes is very important to answer these questions. SSR primers were designed by prospecting in silico to discriminate genotypes and infer the genetic diversity of M. rileyi isolates from the collection kept at Embrapa Soybean. We tested 13 SSR markers on 136 isolates to identify 43 clones and 12 different genetic clusters, with genetic diversity ranging from Hs = 0.15 (cluster I) to Hs = 0.41 (cluster IV) and an average diversity of 0.24. No clusters were categorically distinguished based on hosts or geographical origin using Bayesian clustering analysis. Nonetheless, some clusters comprised most of the isolates with a common geographic origin; for example, cluster VIII was mainly composed of isolates from Central-western Brazil, cluster II from Southern Brazil, and cluster XII from Quincy, Northern Florida, in the United States. Underrepresented regions (few isolates) from Pacific Island nations of Japan, the Philippines, and Indonesia (specifically from Java) were placed into clusters IX and X. Although the analyzed isolates displayed evidence of clonal structure, the genetic diversity indices suggest a potential for the species to adapt to different environmental conditions.

Involvement of a catalase gene in lignin catalysis and immune defense against pathogenic fungus in Coptotermes formosanus: a potential new target for termite control.

BACKGROUND: Detoxifying enzymes are likely involved in lignin feeding and immune defense mechanisms within termites, rendering them potential targets for biological control. However, investigations into the dual functionality of termite detoxification enzymes in vivo have not been documented. RESULTS: In this study, the complete cDNA of the catalase gene (Cfcat) derived from Coptotermes formosanus Shiraki was amplified. CFCAT comprises an open reading frame spanning 1527 bp, encoding a 508-amino acid sequence. The highest expression was observed in the epidermal tissues (including the fat body and hemolymph) followed by the foregut/salivary gland. Furthermore, we confirmed the catalase activity of the recombinant Cfcat protein. Using RNA interference (RNAi) technology, the importance of Cfcat in the lignin-feeding of C. formosanus was demonstrated, and the role of Cfcat in innate immunity was investigated. Survival assays showed that Cfcat RNAi significantly increased the susceptibility of C. formosanus to Metarhizium anisopliae. Irrespective of the infection status, Cfcat inhibition had a significant impact on multiple factors of humoral and intestinal immunity in C. formosanus. Notably, Cfcat RNAi exhibited a more pronounced immunosuppressive effect on humoral immunity than on intestinal immunity. CONCLUSION: Cfcat plays an important role in the regulation of innate immunity and lignin feeding in C. formosanus. Cfcat RNAi can weaken the immune response of termites against M. anisopliae, which may aid the biocontrol efficiency of M. anisopliae against C. formosanus. This study provides a theoretical basis and technical reference for the development of a novel biocontrol strategy targeting detoxifying enzymes of termites. © 2024 Society of Chemical Industry.

Metarhizium rileyi with broad-spectrum insecticidal ability confers resistance against phytopathogens and insect pests as a phytoendophyte.

BACKGROUND: Entomophagous fungi (EPF) not only directly kill insect pests, but also colonize plants and improve their resistance against pests. However, most previous research has focused on Beauveria bassiana and Metarhizium anisopliae, and there are few reports on whether other EPF can enhance resistance against pests via endogenous colonization. Herein, an EPF strain was isolated from diseased larvae of Spodoptera litura in a soybean field, and subjected to genome-wide sequencing at the chromosomal level. The pathogenicity of the isolate toward various pest insects was evaluated, and the ability to colonize plants and induce resistance against phytopathogens and insect pests was tested. RESULTS: The purified isolate was identified as M. rileyi and designated MrS1Gz1-1. Biological assays revealed its strong pathogenicity toward five insect pests belonging to Lepidoptera and Hemiptera. Furthermore, the strain inhibited the growth of soil-borne plant disease caused by Sclerotinia sclerotiorum in vitro. It colonized plants as an endophyte via soil application, thereby inducing plant resistance-related genes against phytopathogen infection, and it disrupted the feeding selectivity of S. litura larvae. CONCLUSION: M. rileyi MrS1Gz1-1 has potential as a broad-spectrum microbial control agent that can induce resistance against phytopathogens and insect pests feeding as an endotype. The complete genome provides a valuable resource for exploring host interactions. © 2024 Society of Chemical Industry.

MicroRNA Targets PAP1 to Mediate Melanization in Plutella xylostella (Linnaeus) Infected by Metarhizium anisopliae.

MicroRNAs (miRNAs) play a pivotal role in important biological processes by regulating post-transcriptional gene expression and exhibit differential expression patterns during development, immune responses, and stress challenges. The diamondback moth causes significant economic damage to crops worldwide. Despite substantial advancements in understanding the molecular biology of this pest, our knowledge regarding the role of miRNAs in regulating key immunity-related genes remains limited. In this study, we leveraged whole transcriptome resequencing data from Plutella xylostella infected with Metarhizium anisopliae to identify specific miRNAs targeting the prophenoloxidase-activating protease1 (PAP1) gene and regulate phenoloxidase (PO) cascade during melanization. Seven miRNAs (pxy-miR-375-5p, pxy-miR-4448-3p, pxy-miR-279a-3p, pxy-miR-3286-3p, pxy-miR-965-5p, pxy-miR-8799-3p, and pxy-miR-14b-5p) were screened. Luciferase reporter assays confirmed that pxy-miR-279a-3p binds to the open reading frame (ORF) and pxy-miR-965-5p to the 3' untranslated region (3' UTR) of PAP1. Our experiments demonstrated that a pxy-miR-965-5p mimic significantly reduced PAP1 expression in P. xylostella larvae, suppressed PO activity, and increased larval mortality rate. Conversely, the injection of pxy-miR-965-5p inhibitor could increase PAP1 expression and PO activity while decreasing larval mortality rate. Furthermore, we identified four LncRNAs (MSTRG.32910.1, MSTRG.7100.1, MSTRG.6802.1, and MSTRG.22113.1) that potentially interact with pxy-miR-965-5p. Interference assays using antisense oligonucleotides (ASOs) revealed that silencing MSTRG.7100.1 and MSTRG.22113.1 increased the expression of pxy-miR-965-5p. These findings shed light on the potential role of pxy-miR-965-5p in the immune response of P. xylostella to M. anisopliae infection and provide a theoretical basis for biological control strategies targeting the immune system of this pest.

Fungal infection of insects: molecular insights and prospects.

Entomopathogenic fungi (EPF) distribute in different fungal phyla with variable host ranges and play essential role in regulating insect populations by infecting hosts via cuticle penetration. The representative ascomycete EPF of Metarhizium and Beauveria species have been widely used in mechanistic investigations of fungus-insect interactions and as ecofriendly mycoinsecticides. Here, we review the function of diverse genes, pathways, and secondary metabolites associated with EPF stepwise infections. In particular, emerging evidence has shown that EPF have to outcompete insect ectomicrobiotas prior to penetrating cuticles, and subvert or evade host antifungal immunity by using effector-like proteins and chemicals like plant pathogens. Future prospects are discussed for a better understanding of fungal pathobiology, which will provide novel insights into microbe-animal interactions.

Rad2, Rad14 and Rad26 recover Metarhizium robertsii from solar UV damage through photoreactivation in vivo.

Rad2, Rad14 and Rad26 recover ultraviolet (UV) damage by nucleotide excision repair (NER) in budding yeast but their functions in filamentous fungi have not been elucidated. Here, we report mechanistically different anti-UV effects of nucleus-specific Rad2, Rad14 and Rad26 orthologs in Metarhizium robertsii, an insect-pathogenic fungus. The null mutants of rad2, rad14 and rad26 showed a decrease of ∼90% in conidial resistance to UVB irradiation. When conidia were impaired at a UVB dose of 0.15 J/cm2, they were photoreactivated (germinated) by only 6-13% through a 5-h light plus 19-h dark incubation, whereas 100%, 80% and 70% of the wild-type conidia were photoreactivated at 0.15, 0.3 and 0.4 J/cm2, respectively. The dose-dependent photoreactivation rates were far greater than the corresponding 24-h dark reactivation rates and were largely enhanced by the overexpression (OE) of rad2, rad14 or rad26 in the wild-type strain. The OE strains exhibited markedly greater activities in photoreactivation of conidia inactivated at 0.5-0.7 J/cm2 than did the wild-type strain. Confirmed interactions of Rad2, Rad14 and Rad26 with photolyase regulators and/or Rad1 or Rad10 suggest that each of these proteins could have evolved into a component of the photolyase regulator-cored protein complex to mediate photoreactivation. The interactions inhibited in the null mutants resulted in transcriptional abolishment or repression of those factors involved in the complex. In conclusion, the anti-UV effects of Rad2, Rad14 and Rad26 depend primarily on DNA photorepair-dependent photoreactivation in M. robertsii and mechanistically differ from those of yeast orthologs depending on NER.

A Drosophila melanogaster model shows that fast growing Metarhizium species are the deadliest despite eliciting a strong immune response.

We used Drosophila melanogaster to investigate how differences between Metarhizium species in growth rate and mechanisms of pathogenesis influence the outcome of infection. We found that the most rapid germinators and growers in vitro and on fly cuticle were the fastest killers, suggesting that pre-penetration competence is key to Metarhizium success. Virulent strains also induced the largest immune response, which did not depend on profuse growth within hosts as virulent toxin-producing strains only proliferated post-mortem while slow-killing strains that were specialized to other insects grew profusely pre-mortem. Metarhizium strains have apparently evolved resistance to widely distributed defenses such as the defensin Toll product drosomycin, but they were inhibited by Bomanins only found in Drosophila spp. Disrupting a gene (Dif), that mediates Toll immunity has little impact on the lethality of most Metarhizium strains (an exception being the early diverged M. frigidum and another insect pathogen Beauveria bassiana). However, disrupting the sensor of fungal proteases (Persephone) allowed rapid proliferation of strains within hosts (with the exception of M. album), and flies succumbed rapidly. Persephone also mediates gender differences in immune responses that determine whether male or female flies die sooner. We conclude that some strain differences in growth within hosts depend on immune-mediated interactions but intrinsic differences in pathogenic mechanisms are more important. Thus, Drosophila varies greatly in tolerance to different Metarhizium strains, in part because some of them produce toxins. Our results further develop D. melanogaster as a tractable model system for understanding insect-Metarhizium interactions.

Divergent roles of Rad4 and Rad23 homologs in Metarhizium robertsii's resistance to solar ultraviolet damage.

The anti-ultraviolet (UV) role of a Rad4-Rad23-Rad33 complex in budding yeast relies on nucleotide excision repair (NER), which is mechanistically distinct from photorepair of DNA lesions generated under solar UV irradiation but remains poorly known in filamentous fungi. Here, two nucleus-specific Rad4 paralogs (Rad4A and Rad4B) and nucleocytoplasmic shuttling Rad23 ortholog are functionally characterized by multiple analyses of their null mutants in Metarhizium robertsii, an entomopathogenic fungus lacking Rad33. Rad4A was proven to interact with Rad23 and contribute significantly more to conidial UVB resistance (90%) than Rad23 (65%). Despite no other biological function, Rad4A exhibited a very high activity in photoreactivation of UVB-impaired/inactivated conidia by 5-h light exposure due to its interaction with Rad10, an anti-UV protein clarified previously to have acquired a similar photoreactivation activity through its interaction with a photolyase in M. robertsii. The NER activity of Rad4A or Rad23 was revealed by lower reactivation rates of moderately impaired conidia after 24-h dark incubation but hardly observable at the end of 12-h dark incubation, suggesting an infeasibility of its NER activity in the field where nighttime is too short. Aside from a remarkable contribution to conidial UVB resistance, Rad23 had pleiotropic effect in radial growth, aerial conidiation, antioxidant response, and cell wall integrity but no photoreactivation activity. However, Rad4B proved redundant in function. The high photoreactivation activity of Rad4A unveils its essentiality for M. robertsii's fitness to solar UV irradiation and is distinct from the yeast homolog's anti-UV role depending on NER. IMPORTANCE Resilience of solar ultraviolet (UV)-impaired cells is crucial for the application of fungal insecticides based on formulated conidia. Anti-UV roles of Rad4, Rad23, and Rad33 rely upon nucleotide excision repair (NER) of DNA lesions in budding yeast. Among two Rad4 paralogs and Rad23 ortholog characterized in Metarhizium robertsii lacking Rad33, Rad4A contributes to conidial UVB resistance more than Rad23, which interacts with Rad4A rather than functionally redundant Rad4B. Rad4A acquires a high activity in photoreactivation of conidia severely impaired or inactivated by UVB irradiation through its interaction with Rad10, another anti-UV protein previously proven to interact with a photorepair-required photolyase. The NER activity of either Rad4A or Rad23 is seemingly extant but unfeasible under field conditions. Rad23 has pleiotropic effect in the asexual cycle in vitro but no photoreactivation activity. Therefore, the strong anti-UV role of Rad4A depends on photoreactivation, unveiling a scenario distinct from the yeast homolog's NER-reliant anti-UV role.

Comparative transcriptome analysis to unveil genes affecting the host cuticle destruction in Metarhizium rileyi.

Insect pathogenic fungi, also known as entomopathogenic fungi, are one of the largest insect pathogenic microorganism communities, represented by Beauveria spp. and Metarhizium spp. Entomopathogenic fungi have been proved to be a great substitute for chemical pesticide in agriculture. In fact, a lot of functional genes were also already characterized in entomopathogenic fungi, but more depth of exploration is still needed to reveal their complicated pathogenic mechanism to insects. Metarhizium rileyi (Nomuraea rileyi) is a great potential biocontrol fungus that can parasitize more than 40 distinct species (mainly Lepidoptera: Noctuidae) to cause large-scale infectious diseases within insect population. In this study, a comparative analysis of transcriptome profile was performed with topical inoculation and hemolymph injection to character the infectious pattern of M. rileyi. Appressorium and multiple hydrolases are indispensable constituents to break the insect host primary cuticle defense in entomopathogenic fungi. Within our transcriptome data, numerous transcripts related to destruction of insect cuticle rather growth regulations were obtained. Most importantly, some unreported ribosomal protein genes and novel unannotated protein (hypothetical protein) genes were proved to participate in the course of pathogenic regulation. Our current data provide a higher efficiency gene library for virulence factors screen in M. rileyi, and this library may be also useful for furnishing valuable information on entomopathogenic fungal pathogenic mechanisms to host.

Metarhizium anisopliae E6 secretome reveals molecular players in host specificity and toxicity linked to cattle tick infection.

Although Metarhizium anisopliae is one of the most studied fungal biocontrol agents, its infection mechanism is far from being completely understood. Using multidimensional protein identification technology (MudPIT), we evaluated the differential secretome of M. anisopliae E6 induced by the host Rhipicephalus microplus cuticle. The proteomic result showed changes in the expression of 194 proteins after exposure to host cuticle, such as proteins involved in adhesion, penetration, stress and fungal defense. Further, we performed a comparative genomic distribution of differentially expressed proteins of the M. anisopliae secretome against another arthropod pathogen, using the Beauveria bassiana ARSEF2860 protein repertory. Among 47 analyzed protein families, thirty were overexpressed in the M. anisopliae E6 predicted genome compared to B. bassiana. An in vivo toxicity assay using a Galleria mellonella model confirmed that the M. anisopliae E6 secretome was more toxic in cattle tick infections compared to other secretomes, including B. bassiana with cattle ticks and M. anisopliae E6 with the insect Dysdereus peruvianus, which our proteomic results had also suggested. These results help explain molecular aspects associated with host infection specificity due to genetic differences and gene expression control at the protein level in arthropod-pathogenic fungi.

Genome-wide identification of Coptotermes formosanus immune genes and their potential roles in termite control.

In recent years, the use of microbes to control termites has attracted increasing attention. It was found that pathogenic bacteria, nematodes, and fungi effectively control termites under laboratory conditions. However, their effects have not been replicated in the field, and one reason for this is the complex immune defense mechanisms of termites, which are mainly regulated by immune genes. Therefore, altering the expression of immune genes may have a positive influence on the biocontrol efficacy of termites. Coptotermes formosanus Shiraki is one of the most economically important termite pests worldwide. Currently, the large-scale identification of immune genes in C. formosanus is primarily based on cDNA library or transcriptome data rather than at the genomic level. In this study, we identified the immune genes of C. formosanus according to genome-wide analysis. In addition, our transcriptome analysis showed that immune genes were significantly downregulated when C. formosanus was exposed to the fungus Metarhizium anisopliae or nematodes. Finally, we found that injecting dsRNA to inhibit three immune genes (CfPGRP-SC1, CfSCRB3, and CfHemocytin), which recognize infectious microbes, significantly increased the lethal effect of M. anisopliae on termites. These immune genes show great potential for C. formosanus management based on RNAi. These results also increase the number of known immune genes in C. formosanus which will provide a more comprehensive insight into the molecular basis of immunity in termites.

Genetic polymorphism and plant growth promotion traits of potent fungal entomopathogens of rice leaf folder.

Entomopathogenic fungal biocides are preferred for environment friendly sustainable management of insect pests due to their host specificity and harmlessness to non-target insects. Plant growth promotion (PGP) functions of the entomofungi are also important attributes but hitherto insignificantly explored. Therefore, virulence of 17 natural fungal entomocides (Cordyceps, Beauveria, Metarhizium, Nomuraea, Fusarium, Verticillium, Trichoderma and Paecilomyces spp.) were evaluated for pathogenicity against five rice pests (brown plant hopper (Nilaparvata lugens) and green leaf hopper (Nephotettix virescens) nymphs, leaf folder (Cnaphalocrosis medinalis) and yellow stem borer (Scirpophaga incertulas) larvae and swarming caterpillar (Spodoptera mauritia), respectively), and PGP traits of the potent leaf folder pathogens. Among the fungi, only the leaf folder pathogens (3 isolates each of Beauveria and Metarhizium spp.) infected > 50% (80-90%) larvae but other fungi were ineffective as infected < 50% (0-47%) insects. Besides, the leaf folder pathogens exhibited diverse PGP traits such as organic/inorganic phosphate solubilization (104.7-236.4 µg/ml), and siderophore, ammonia, hydrogen cyanide (HCN), indole production etc. Restriction fragment length polymorphism (RFLP), random amplified polymorphic DNA (RAPD), simple sequences repeat (SSR) and internal transcribed spacers (ITS) analysis ascertained strain identity and genetic (inter and intra-specific) diversity among the potent biocides Beauveria and Metarhizium spp. The virulent natural fungal pathogens of rice pests with polyvalent PGP traits may be prospected for rice growth promotion and biocontrol of leaf folder.

A novel partitivirus orchestrates conidiation, stress response, pathogenicity, and secondary metabolism of the entomopathogenic fungus Metarhizium majus.

Mycoviruses are widely present in all major groups of fungi but those in entomopathogenic Metarhizium spp. remain understudied. In this investigation, a novel double-stranded (ds) RNA virus is isolated from Metarhizium majus and named Metarhizium majus partitivirus 1 (MmPV1). The complete genome sequence of MmPV1 comprises two monocistronic dsRNA segments (dsRNA 1 and dsRNA 2), which encode an RNA-dependent RNA polymerase (RdRp) and a capsid protein (CP), respectively. MmPV1 is classified as a new member of the genus Gammapartitivirus in the family Partitiviridae based on phylogenetic analysis. As compared to an MmPV1-free strain, two isogenic MmPV1-infected single-spore isolates were compromised in terms of conidiation, and tolerance to heat shock and UV-B irradiation, while these phenotypes were accompanied by transcriptional suppression of multiple genes involved in conidiation, heat shock response and DNA damage repair. MmPV1 attenuated fungal virulence since infection resulted in reduced conidiation, hydrophobicity, adhesion, and cuticular penetration. Additionally, secondary metabolites were significantly altered by MmPV1 infection, including reduced production of triterpenoids, and metarhizins A and B, and increased production of nitrogen and phosphorus compounds. However, expression of individual MmPV1 proteins in M. majus had no impact on the host phenotype, suggesting insubstantive links between defective phenotypes and a single viral protein. These findings indicate that MmPV1 infection decreases M. majus fitness to its environment and its insect-pathogenic lifestyle and environment through the orchestration of the host conidiation, stress tolerance, pathogenicity, and secondary metabolism.

Metarhizium indicum, a new species of entomopathogenic fungus infecting leafhopper, Busoniomimus manjunathi from India.

A new species of entomopathogenic fungus, Metarhizium indicum, which derives its species epithet after its Indian origin is reported here. The fungus was found to cause natural epizootics in leafhopper (Busoniomimus manjunathi) infesting Garcinia gummi-gutta (Malabar tamarind), an evergreen spice tree native to South and Southeast Asia, known for its use as a culinary flavourant, dietary supplement and traditional remedy for various human ailments. The fungus was found to cause more than 60% mortality in field collected insects. The identity of the new species was established based on its distinct morphological characteristics and multi-gene sequence data analyses. Phylogenetic analyses using internal transcribed spacer region (ITS), DNA lyase (APN2) and a concatenated set of four marker genes [translation elongation factor 1-alpha (TEF), ß-tubulin (BTUB), RNA polymerase II largest subunit (RPB1) and RNA polymerase II second largest subunit (RPB2)] along with marked differences in nucleotide composition and genetic distance unambiguously support our claim that the present fungus infecting Garcinia leafhopper is a new addition to the genus Metarhizium.

Transcriptomics Reveals the Killing Mechanism by Which Entomopathogenic Fungi Manipulate the RNA Expression Profiles of Termites and Provides Inspiration for Green Pest Management.

As chemical pesticides have caused serious environmental pollution, fungus-based biological control has become a developing alternative to chemical control. Here, we aimed to determine the molecular mechanism underlying how Metarhizium anisopliae facilitated invasive infection. We found that the fungus increased its virulence by downregulating glutathione S-transferase (GST) and superoxide dismutase (SOD) throughout termite bodies. Among 13 fungus-induced microRNAs throughout termite bodies, miR-7885-5p and miR-252b upregulation significantly downregulated several mRNAs in response to toxic substances to increase the fungal virulence [e.g., phosphoenolpyruvate carboxykinase (GTP) and heat shock protein homologue SSE1]. In addition, nanodelivered small interfering RNA of GST and SOD and miR-7885-5p and miR-252b mimics increased the virulence of the fungus. These findings provide new insights into the killing mechanism of entomopathogens and their utilization of the host miRNA machinery to reduce host defenses, laying the groundwork to enhance virulence of biocontrol agents for green pest management.

Detection of viral genes in Metarhizium anisopliae JEF-290-infected longhorned tick, Haemaphysalis longicornis using transcriptome analysis.

Ticks are carriers of viruses that can cause disease in humans and animals. The longhorned ticks (Haemaphysalis longicornis; LHT), for example, mediates the severe fever with thrombocytopenia syndrome virus (SFTSV) in humans, and the population of ticks is growing due to increases in temperature caused by climate change. As ticks carry primarily RNA viruses, there is a need to study the possibility of detecting new viruses through tick virome analysis. In this study, viruses in LHTs collected in Korea were investigated and virus titers in ticks exposed to the entomopathogenic fungus Metarhizium anisopliae JEF-290 were analyzed. Total RNA was extracted from the collected ticks, and short reads were obtained from Illumina sequencing. A total of 50,024 contigs with coding capacity were obtained after de novo assembly of the reads in the metaSPAdes genome assembler. A series of BLAST-based analyses using the GenBank database was performed to screen viral contigs, and three putative virus species were identified from the tick meta-transcriptome, such as Alongshan virus (ALSV), Denso virus and Taggert virus. Measurements of virus-expression levels of infected and non-infected LHTs failed to detect substantial differences in expression levels. However, we suggest that LHT can spread not only SFTSV, but also various other disease-causing viruses over large areas of the world. From the phylogenetic analysis of ALSV glycoproteins, genetic differences in the ALSV could be due to host differences as well as regional differences. Viral metagenome analysis can be used as a tool to manage future outbreaks of disease caused by ticks by detecting unknown viruses.