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.

Glyoxal oxidase-mediated detoxification of reactive carbonyl species contributes to virulence, stress tolerance, and development in a pathogenic fungus.

Reactive carbonyl and oxygen species (RCS/ROS), often generated as metabolic byproducts, particularly under conditions of pathology, can cause direct damage to proteins, lipids, and nucleic acids. Glyoxal oxidases (Gloxs) oxidize aldehydes to carboxylic acids, generating hydrogen peroxide (H2O2). Although best characterized for their roles in lignin degradation, Glox in plant fungal pathogens are known to contribute to virulence, however, the mechanism underlying such effects are unclear. Here, we show that Glox in the insect pathogenic fungus, Metarhizium acridum, is highly expressed in mycelia and during formation of infection structures (appressoria), with the enzyme localizing to the cell membrane. MaGlox targeted gene disruption mutants showed RCS and ROS accumulation, resulting in cell toxicity, induction of apoptosis and increased autophagy, inhibiting normal fungal growth and development. The ability of the MaGlox mutant to scavenge RCS was significantly reduced, and the mutant exhibited increased susceptibility to aldehydes, oxidative and cell wall perturbing agents but not toward osmotic stress, with altered cell wall contents. The ΔMaGlox mutant was impaired in its ability to penetrate the host cuticle and evade host immune defense resulting in attenuated pathogenicity. Overexpression of MaGlox promoted fungal growth and conidial germination, increased tolerance to H2O2, but had little to other phenotypic effects. Transcriptomic analyses revealed downregulation of genes related to cell wall synthesis, conidiation, stress tolerance, and host cuticle penetration in the ΔMaGlox mutant. These findings demonstrate that MaGlox-mediated scavenging of RCS is required for virulence, and contributes to normal fungal growth and development, stress resistance.

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.

Bioremediation of mercury-polluted soil and water by the plant symbiotic fungus Metarhizium robertsii.

Fungi are central to every terrestrial and many aquatic ecosystems, but the mechanisms underlying fungal tolerance to mercury, a global pollutant, remain unknown. Here, we show that the plant symbiotic fungus Metarhizium robertsii degrades methylmercury and reduces divalent mercury, decreasing mercury accumulation in plants and greatly increasing their growth in contaminated soils. M. robertsii does this by demethylating methylmercury via a methylmercury demethylase (MMD) and using a mercury ion reductase (MIR) to reduce divalent mercury to volatile elemental mercury. M. robertsii can also remove methylmercury and divalent mercury from fresh and sea water even in the absence of added nutrients. Overexpression of MMD and MIR significantly improved the ability of M. robertsii to bioremediate soil and water contaminated with methylmercury and divalent mercury. MIR homologs, and thereby divalent mercury tolerance, are widespread in fungi. In contrast, MMD homologs were patchily distributed among the few plant associates and soil fungi that were also able to demethylate methylmercury. Phylogenetic analysis suggests that fungi could have acquired methylmercury demethylase genes from bacteria via two independent horizontal gene transfer events. Heterologous expression of MMD in fungi that lack MMD homologs enabled them to demethylate methylmercury. Our work reveals the mechanisms underlying mercury tolerance in fungi, and may provide a cheap and environmentally friendly means of cleaning up mercury pollution.

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.

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.

Virulence and proteomic responses of Metarhizium anisopliae against Aedes albopictus larvae.

The tropical climate in Malaysia provides an ideal environment for the rapid proliferation of Aedes mosquitoes, notably Aedes aegypti and Aedes albopictus, prominent vectors of dengue fever. Alarmingly, these species are increasingly developing resistance to conventional pesticides. This study aimed to evaluate the efficacy of Metarhizium anisopliae isolate HSAH5 spores, specifically on conidia (CO) and blastospores (BL), against Ae. albopictus larvae. The study centered on evaluating their pathogenic effects and the resultant changes in protein expression. Spore suspensions with varying concentrations were prepared for larvicidal bioassays, and protein expressions were analysed using liquid chromatography-mass spectrometry. Subsequently, protein annotation and network analysis were conducted to elucidate infection mechanisms and the proteomic response. Based on the lethal concentrations and time frames, CO exhibited faster larval mortality than BL at lower concentrations. Despite this, both spore types demonstrated comparable overall pathogenic effects. Results from the proteomic profiling revealed 150 proteins with varied expressions following exposure to Ae. albopictus extract, shedding light on distinct infection strategies between the spores. Gene Ontology enrichment and network analysis illustrated the diverse metabolic adaptations of M. anisopliae and interactions with mosquito larvae. This highlighted the complexity of host-pathogen dynamics and the significance of biosynthetic processes, energy storage, and cellular interaction pathways in disease progression. The BL network, consisting 80 proteins and 74 connections, demonstrates the intricate fungal mechanisms triggered by host stimuli. Conversely, the CO network, though smaller, displayed notable interconnectivity and concentrated involvement at the cell periphery, suggesting a deliberate strategy for initial host contact. This study offers valuable insights into proteome dynamics of M. anisopliae's BL and CO for managing mosquito populations and combating disease transmission, thereby significantly advancing public health and environmental conservation efforts.

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.

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.

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.

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.

Molecular characterization of a novel victorivirus (order Ghabrivirales, family Totiviridae) infecting Metarhizium anisopliae.

Here, we report the occurrence and complete genome sequence of a novel victorivirus infecting Metarhizium anisopliae, named "Metarhizium anisopliae victorivirus 1" (MaVV1). The genome is 5353 bp in length and contains two open reading frames (ORFs), encoding a coat protein and an RNA-dependent RNA polymerase (RdRp), that overlap at the octanucleotide sequence AUGAGUAA. These ORFs showed sequence similarity to the corresponding ORFs of Ustilaginoidea virens RNA virus L (68.23%) and Ustilaginoidea virens RNA virus 13 (58.11%), respectively, both of which belong to the family Totiviridae. Phylogenetic analysis based on RdRp sequences revealed that MaVV1 clustered with members of the genus Victorivirus. This is the first genome sequence reported for a virus belonging to the genus Victorivirus infecting the entomopathogenic fungus M. anisopliae.

swnk plays an important role in the biosynthesis of swainsonine in Metarhizium anisopliae.

OBJECTIVE: Swainsonine (SW) is the principal toxic ingredient of locoweeds, and is produced by multiple fungi. A key enzyme in the SW synthesis pathway is a hybrid swnk/nrps. To analyze the role of swnk in the SW biosynthesis pathway of Metarhizium anisopliae. RESULTS: The concentration of SW and the swnk expression in M. anisopliae fermentation from 1st to 7th day were determined using LC-MS and RT-qPCR, respectively. M. anisopliae had the highest SW content and swnk expression on the 5th day of fermentation; Mutant strain (MT) were obtained by PEG-mediated homologous recombination (HR) which knocked out swnk in the wild-type (WT) strain. Complemented-type (CT) strain were obtained by transforming a modified PUC19 complementation vector containing the geneticin (G418) resistance gene and swnK. SW was not detected in the MT strain and reverted to its original level in the CT strain; A Psilent-1 plasmid with Benomyl (ben)-resistant that was used interfered with swnk of WT strain. The level of SW was markedly diminished in the RNAi strain. RNAi of swnk affects the formation of the cell wall in M. anisopliae. CONCLUSION: These results indicate that swnk plays a crucial role in the SW biosynthesis of M. anisopliae.

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

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

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.

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.

Tryptamine accumulation caused by deletion of MrMao-1 in Metarhizium genome significantly enhances insecticidal virulence.

Metarhizium is a group of insect-pathogenic fungi that can produce insecticidal metabolites, such as destruxins. Interestingly, the acridid-specific fungus Metarhizium acridum (MAC) can kill locusts faster than the generalist fungus Metarhizium robertsii (MAA) even without destruxin. However, the underlying mechanisms of different pathogenesis between host-generalist and host-specialist fungi remain unknown. This study compared transcriptomes and metabolite profiles to analyze the difference in responsiveness of locusts to MAA and MAC infections. Results confirmed that the detoxification and tryptamine catabolic pathways were significantly enriched in locusts after MAC infection compared with MAA infection and that high levels of tryptamine could kill locusts. Furthermore, tryptamine was found to be capable of activating the aryl hydrocarbon receptor of locusts (LmAhR) to produce damaging effects by inducing reactive oxygen species production and immune suppression. Therefore, reducing LmAhR expression by RNAi or inhibitor (SR1) attenuates the lethal effects of tryptamine on locusts. In addition, MAA, not MAC, possessed the monoamine oxidase (Mao) genes in tryptamine catabolism. Hence, deleting MrMao-1 could increase the virulence of generalist MAA on locusts and other insects. Therefore, our study provides a rather feasible way to design novel mycoinsecticides by deleting a gene instead of introducing any exogenous gene or domain.

Intra-hemocoel injection of pseurotin A from Metarhizium anisopliae, induces dose-dependent reversible paralysis in the Greater Wax Moth (Galleria mellonella).

Species from the Metarhizium genus are the causal agents of the green muscardine disease of insects. These fungi have been successfully employed for the biological control of pests over decades. Besides the biocontrol applications, recent efforts for genome sequencing of species in this genus have revealed a great diversity of biosynthetic gene clusters potentially associated with secondary metabolite synthesis. Amongst such molecules are the pseurotins, compounds with several activities, as chitin synthase inhibitors, and immunoglobulin E suppressors. Here, we report, for the first time, the isolation of pseurotin A from the culture broth of M. anisopliae, as well as the characterization of the effects of this compound over the model-arthropod Galleria mellonella. Pseurotin A displayed dose-dependent reversible paralysis effects when injected into the larvae hemocoel. However, the posterior challenge of the treated insects with M. anisopliae conidia did not lead to increased mortality, suggesting that pseurotin A treatment did not increase larvae susceptibility to the green muscardine disease. Although apparent insecticidal effects were not observed for pseurotin A, the paralysis effect observed can be important in M. anisopliae infection development.

Elucidating the Effect of Endophytic Entomopathogenic Fungi on Bread Wheat Growth through Signaling of Immune Response-Related Hormones.

Entomopathogenic fungi (EF) provide a potent biocontrol tool; also, their endophytic behavior has broadened their contribution to integrated pest management (IPM) and crop production. In this work, Beauveria bassiana and Metarhizium brunneum were applied to bread wheat (Triticum aestivum) seedlings to elucidate how fungal colonization influences plant growth and the relative expression of 24 genes involved in hormonal syntheses and plant immune mechanisms. A preliminary assay was used to determine the time needed for fungal colonization and assess its effect on wheat growth. Then, plant material collected at various times after inoculation (viz., 2, 8, 20, and 36 h and 9 and 15 days) was used to investigate gene expression by quantitative reverse transcription PCR (RT-qPCR). During the colonization time, B. bassiana and M. brunneum caused strong downregulation of most genes associated with plant immunity and the synthesis of hormones like auxin, cytokinin, and gibberellin. This effect was concomitant with a slowdown of endophytic-colonization-related plant growth until 19 days postinoculation (dpi). However, the wheat started to recover at 15 dpi, simultaneously with upregulation of auxin- and gibberellin-related genes. The results suggest that the EF trigger induced systemic resistance rather than acquired systemic resistance during early plant-microbe cross talk in wheat. Also, they confirm that the hormone and immune responses of wheat triggered by EF inoculation influenced plant growth, which can be useful with a view to optimizing management of these microorganisms for sustainable agriculture. IMPORTANCE Microbial control of insect and mite pests is a key tool to develop integrated pest management (IPM) and sustainable agriculture. Entomopathogenic fungi (EF) may have associations with the plants, playing additional ecological roles in the rhizosphere, in the phylloplane, and as plant endophytes. Beauveria bassiana 04/01TIP and Metarhizium brunneum 01/58Su are two strains that showed very good results either in pest control or plant growth promotion and would be good candidates to develop mycoinsecticides as an alternative to pesticides. However, deep knowledge about their interaction with the plant would let farmers optimize their use and understand the plant response, enhancing and promoting their broader contribution to IPM and crop production.