Genetic structure of Metarhizium species in western USA: Finite populations composed of divergent clonal lineages with limited evidence for recent recombination.

Globally distributed, soil associated Metarhizium species used in insect biological control are evidently facultatively sexual and obligately outcrossing, yet sexual morphs have not been observed for most species and corroboration that they recombine in nature remains limited. Community-wide genetic diversity of Metarhizium species among 480 soil isolates from 14 states of western USA was investigated to assess the contributions of clonality and recombination in determining each species' population structure. Seven species, varying greater than 100-fold in relative abundance, were identified by phylogenetic analysis of 5' EF1-α (5TEF), including M. robertsii (n = 372), M. guizhouense (n = 37), M. brunneum (n = 37), M. lepidiotae (n = 14), M. pemphigi (n = 11), M. anisopliae (n = 7) and M. pingshaense (n = 2). Analyses of composite multilocus genotypes integrating 5TEF sequences, multilocus microsatellites and mating type idiomorphs conducted on a subset of 239 isolates revealed that all species populations display pronounced clonal structure. Following clone-correction procedures to remove redundant clonal genotypes and collapse clonal lineages, each species' population sample was determined to be composed of a dozen or fewer genetically unique individuals. Thus, the Metarhizium community inhabiting western USA is conservatively estimated to comprise as few as 34 distinct genetic individuals, with a single, geographically ubiquitous clonal lineage of M. robertsii constituting 45% of total isolates. M. robertsii was the only population determined to be in linkage equilibrium. However, the high proportion of private alleles differentiating most M. robertsii clonal lineages argues against contemporary panmixia, thus the recombination signal detected may be historical. Nevertheless, within M. robertsii, M. brunneum and M. guizhouense there are closely related genotypes of opposite mating type, which suggests that if recombination is contemporary, it likely occurs between closely related individuals. The restricted number of genetic individuals observed throughout western North American Metarhizium species may signify that these represent peripheral populations descended from limited numbers of founders among which there has been little recombination relative to the extent of clone expansion and within-clone genetic divergence.

Molecular evolution of Pr1 proteases depicts ongoing diversification in Metarhizium spp.

The Pr1 family of serine endopeptidases plays an important role in pathogenicity and virulence of entomopathogens such as Metarhizium anisopliae (Ascomycota: Hypocreales). These virulence factors allow for the penetration of the host cuticle, a vital step in the infective process of this fungus, which possesses 11 Pr1 isoforms (Pr1A through Pr1K). The family is divided into two classes with Class II (proteinase K-like) comprising 10 isoforms further split into three subfamilies. It is believed that these isoforms act synergistically and with other virulence factors, allowing pathogenicity to multiple hosts. As virulence coevolves through reciprocal selection with hosts, positive selection may lead to the evolution of new protease families or isoforms of extant ones that can withstand host defenses. This work tests this hypothesis in Class II Pr1 proteins, focusing on M. anisopliae, employing different methods for phylogenetic inference in amino acid and nucleotide datasets in multiple arrangements for Metarhizium spp. and related species. Phylogenies depict groups that match the taxonomy of their respective organisms with high statistical support, with minor discrepancies. Positively selected sites were identified in six out of ten Pr1 isoforms, most of them located in the proteolytic domain and spatially close to the catalytic residues. Moreover, there was evidence of functional divergence in the majority of pairwise comparisons. These results imply the existence of differential selective pressure acting on Pr1 proteins and a potential new isoform, likely affecting host specificities, virulence, or even adapting the organism to different host-independent lifestyles.

Identification of hypocrealean reptile pathogenic isolates with MALDI-TOF MS.

Biotyper analysis of Nannizziopsis guarroi, a fatal fungal pathogen in lizards, was described recently. Hypocrealean fungal infections in captive reptiles appear with an increasing frequency during the last decade. Therefore, the aim of this study was to proof Matrix-assisted laser desorption ionization-time-of-flight mass spectrometry (MALDI-TOF MS) as diagnostic tool for the identification of reptile pathogenic hypocrealean fungi. Ten fungal isolates obtained from nine reptiles with fungal glossitis, disseminated visceral mycosis, pneumomycosis, and fungal keratitis were analyzed. Phylogeny consisted of fragments of the large subunit of nuclear encoded ribosomal DNA (D1/D2, LSU) and the internal transcribed spacer region 1 of nuclear encoded ribosomal DNA (ITS1) as well as the protein coding gene translation elongation factor 1 alpha (TEF). Results revealed unanimously two Metarhizium granulomatis genotypes in a total of three isolates, various M. viride genotypes (n = 3), two different Purpureocillium lilacinum isolates as well as one isolate of each P. lavendulum and Beauveria bassiana. Purpureocillium lilacinum and B. bassiana are likewise frequently employed as a mycoinsecticide and mycoacaricide in agriculture on a worldwide scale and have occasionally been reported in man, causing fungal keratitis, sclerokeratitis, nosocomial infections in immunosuppressed patients, as well as cavitary pulmonary disease and cutaneous hyalohyphomycosis in immunocompetent patients. According to the results establishment of Biotyper analysis for faster differentiation of reptile-associated fungal pathogens is entirely justified.

Biocontrol potential and genetic diversity of Metarhizium anisopliae lineage in agricultural habitats.

AIMS: To assess phylogenetic and genotypic diversity of Metarhizium anisopliae lineage within diverse agroecosystems in the Karnataka State of India and to compare their chitinase activity and pathogenicity against insect pest of field crops subterranean termite, Odontotermes obesus. METHODS AND RESULTS: Three phylogenetic and 27 microsatellite markers were used to assess the genetic diversity of M. anisopliae lineage within multiple agroecosystems. Multilocus phylogeny of the Metarhizium isolates identified two species: Metarhizium pingshaense and Metarhizium guizhouense. Multilocus phylogeny and microsatellite markers resolved two phylogenetic species of M. pingshaense, Mp_1 and Mp_2, and one phylogenetic species of M. guizhouense, Mg_1. Phylogenetic species, Mp_2 and Mg_1, were detected with one genotype each and Mp_1 with eleven genotypes. Metarhizium pingshaense GKVK 02_16 isolate caused significantly high mortality of O. obesus in bioassays and detected with high chitinase activity. CONCLUSIONS: The study revealed phylogenetic and genotypic diversity of M. anisopliae lineage in agroecosystems of Karnataka State. Findings of pathogenicity and chitinase activity suggest that M. pingshaense GKVK 02_16 isolate provides effective control of O. obesus. SIGNIFICANCE AND IMPACT OF THE STUDY: The investigation provided an understanding of the genetic diversity and biocontrol efficiency of M. anisopliae lineage in agroecosystem. These data will serve as a resource in the future pest management strategies at a regional scale.

A species-specific multiplexed PCR amplicon assay for distinguishing between Metarhizium anisopliae, M. brunneum, M. pingshaense and M. robertsii.

The fungal species Metarhizium pingshaense, M. anisopliae, M. robertsii, and M. brunneum, a monophyletic group informally referred to as the PARB species complex, are well known facultative entomopathogens, including many commercialized strains used for biological pest control. Accurate and expedient species identification of Metarhizium isolates represents an important first step when addressing ecological as well as application-related questions involving these fungi. To this end, a species-specific multiplexed polymerase chain reaction (PCR) assay was developed for identification and discrimination among Metarhizium PARB complex species, based on unique sequence signature differences within the nuclear ribosomal intergenic spacer (rIGS) and nuclear intergenic spacer regions MzFG546 and MzIGS2. Species-specificities of the four primer pairs were assessed following a three-step approach including: (1) in silico verification of sequence signatures by BLASTN searches against publically available genome and amplicon sequence data, (2) corroboration of assay specificity and robustness by performing test PCR amplifications against a taxonomically curated reference strain collection of 68 Metarhizium strains representing 12 species, and (3) testing against a field collection of 19 unknown Metarhizium isolates from soil of a Swiss meadow. The specificity of these four primer pairs provide an efficient means to detect and discriminate PARB species in studies targeting ecological aspects of indigenous isolates, as well as efficacy, persistence and potential non-target effects of applied biocontrol strains.

Occurrence and diversity of entomopathogenic fungi (Beauveria spp. and Metarhizium spp.) in Australian vineyard soils.

Entomopathogenic Ascomycetes: Hypocreales fungi occur worldwide in the soil; however, the abundance and distribution of these fungi in a vineyard environment is unknown. A survey of Australian vineyards was carried out in order to isolate and identify entomopathogenic fungi. A total of 240 soil samples were taken from eight vineyards in two states (New South Wales and Victoria). Insect baiting (using Tenebrio molitor) and soil dilution methods were used to isolate Beauveria spp. and Metarhizium spp. from all soil samples. Of the 240 soil samples, 60% contained either Beauveria spp. (26%) or Metarhizium spp. (33%). Species of Beauveria and Metarhizium were identified by sequencing the B locus nuclear intergenic region (Bloc) and elongation factor-1 alpha (EFT1) regions, respectively. Three Beauveria species (B. bassiana, B. australis and B. pseudobassiana) and six Metarhizium species (M. guizhouense, M. robertsii, M. brunneum, M. flavoviride var. pemphigi, M. pingshaense and M. majus) were identified. A new sister clade made up of six isolates was identified within B. australis. Two potentially new phylogenetic species (six isolates each) were found within the B. bassiana clade. This study revealed a diverse community of entomopathogenic fungi in sampled Australian vineyard soils.

Isolation and characterization of Metarhizium anisopliae TK29 and its mycoinsecticide effects against subterranean termite Coptotermes formosanus.

The entomopathogenic fungus Metarhizium anisopliae is widely used as biocontrol agent against many insect pests. In the present study, the potential isolate of M. anisopliae TK29 was isolated from the agricultural soils in Thekkady, India. The taxonomic identity of the isolate was confirmed based on its morphology and 18S rDNA gene sequence homology. Phylogenetic analysis confirmed that the isolated strains were related to the same species. A potential isolate (TK29) was optimized for mass cultivation and conidial spore production was enhanced using three different raw substrates (Rice, Maize, black gram) by solid-state fermentation. The results showed higher conidial spore yield from rice (2.6 ±â€¯0.32%) compared to black gram (2.1 ±â€¯0.28%) and maize (1.9 ±â€¯0.23%) substrates. Dry green conidia were applied against Formosan subterranean termite, Coptotermes formosanus at three different concentrations (1 × 106, 1 × 107, and 1 × 108 conidia/ml-1). The highest mortality rate was obtained from 1 × 108 conidia/ml-1 at 120 h post-treatment. Our study indicated that M. anisopliae TK29 had desirable attributes for the development of a mycoinsecticide against C. formosanus.

Elevational distribution and morphological attributes of the entomopathogenic fungi from forests of the Qinling Mountains in China.

Entomopathogenic fungi are considered to be a safe microbiological pesticide alternative to chemical control. Efforts are underway to understand precisely their taxonomy and natural distribution through mycological and biodiversity studies based on molecular markers. Here, we present descriptions of the diversity of the entomopathogenic fungi in the genera Metarhizium and Beauveria found along the elevational gradients of the Qinling subtropical and temperate forests of Shaanxi province in China, using morphological aspects and molecular markers. Molecular characterization using the Mz_IGS3 intergenic region revealed that Metarhizium isolates phylogenetically clustered in the PARB clade with four different distinguishable species, but the 5'-TEF gene allowed only ambiguous delimitation of Metarhizium species. Beauveria isolates were characterized by sequence analyses of the translation elongation factor 1-α and the Bloc region. The richness of Metarhizium species decreased with increasing elevation, with Metarhizium robertsii s.l. being the most abundant species along the elevational gradient. Our bioassay suggests that certain species of Metarhizium are significantly pathogenic to the insect model Tenebrio molitor at both the adult and larvae stages and could potentially serve as a control of insect pests of forests.

Rhipicephalus microplus infected by Metarhizium: unveiling hemocyte quantification, GFP-fungi virulence, and ovary infection.

Hemocytes, cells present in the hemocoel, are involved in the immune response of arthropods challenged with entomopathogens. The present study established the best methodology for harvesting hemocytes from Rhipicephalus microplus and evaluated the number of hemocytes in addition to histological analysis from ovaries of fungus-infected females and tested the virulence of GFP-fungi transformants. Different centrifugation protocols were tested, and the one in which presented fewer disrupted cells and higher cell recovery was applied for evaluating the effect of Metarhizium spp. on hemocytes against R. microplus. After processing, protocol number 1 (i.e., hemolymph samples were centrifuged at 500×g for 3 min at 4 °C) was considered more efficient, with two isolates used (Metarhizium robertsii ARSEF 2575 and Metarhizium anisopliae ARSEF 549), both wild types and GFP, to assess their virulence. In the biological assays, the GFP-fungi were as virulent as wild types, showing no significant differences. Subsequently, hemocyte quantifications were performed after inoculation, which exhibited notable changes in the number of hemocytes, reducing by approximately 80% in females previously treated with Metarhizium isolates in comparison to non-treated females. Complementarily, 48 h after inoculation, in which hemolymph could not be obtained, histological analysis showed the high competence of these fungi to colonize ovary from ticks. Here, for the first time, the best protocol (i.e., very low cell disruption and high cell recovery) for R. microplus hemocyte obtaining was established aiming to guide directions to other studies that involves cellular responses from ticks to fungi infection.

Species Diversity and Population Dynamics of Entomopathogenic Fungal Species in the Genus Metarhizium-a Spatiotemporal Study.

We studied the species diversity and population genetic structure of isolates of fungi from the entomopathogenic genus Metarhizium that had been isolated from sugarcane crops and surrounding grass. Soil and leaf samples were taken on four sampling occasions over 13 months (October 2014-October 2015). Isolations were made using the Galleria mellonella baiting method and selective media. Phylogenetic placement of isolates was done by sequencing a fragment of the 5' of the elongation factor 1-α gene (EF1-α). Population genetic structure was determined by analysing this sequence information using AMOVA and Haplotype network analyses. Genotypic diversity was studied using microsatellite genotyping. The most abundant species was M. anisopliae s.s. (80 isolates), then M. pingshaense (three isolates), and M. guizhouense (one isolate). More than 50% of the genetic variation was explained by the time the samples were collected regardless of plant host association. Some haplotypes were found on the first sampling date and then not found on subsequent sampling dates, while other haplotypes were found initially, disappeared, but then found again on the last sampling date. To the best of our knowledge, this is the first report of the population genetic structure of M. anisopliae species in time and space. The effect of abiotic factors is discussed.

Trajectory and genomic determinants of fungal-pathogen speciation and host adaptation.

Much remains unknown regarding speciation. Host-pathogen interactions are a major driving force for diversification, but the genomic basis for speciation and host shifting remains unclear. The fungal genus Metarhizium contains species ranging from specialists with very narrow host ranges to generalists that attack a wide range of insects. By genomic analyses of seven species, we demonstrated that generalists evolved from specialists via transitional species with intermediate host ranges and that this shift paralleled insect evolution. We found that specialization was associated with retention of sexuality and rapid evolution of existing protein sequences whereas generalization was associated with protein-family expansion, loss of genome-defense mechanisms, genome restructuring, horizontal gene transfer, and positive selection that accelerated after reinforcement of reproductive isolation. These results advance understanding of speciation and genomic signatures that underlie pathogen adaptation to hosts.

Secondary metabolite gene clusters in the entomopathogen fungus Metarhizium anisopliae: genome identification and patterns of expression in a cuticle infection model.

BACKGROUND: The described species from the Metarhizium genus are cosmopolitan fungi that infect arthropod hosts. Interestingly, while some species infect a wide range of hosts (host-generalists), other species infect only a few arthropods (host-specialists). This singular evolutionary trait permits unique comparisons to determine how pathogens and virulence determinants emerge. Among the several virulence determinants that have been described, secondary metabolites (SMs) are suggested to play essential roles during fungal infection. Despite progress in the study of pathogen-host relationships, the majority of genes related to SM production in Metarhizium spp. are uncharacterized, and little is known about their genomic organization, expression and regulation. To better understand how infection conditions may affect SM production in Metarhizium anisopliae, we have performed a deep survey and description of SM biosynthetic gene clusters (BGCs) in M. anisopliae, analyzed RNA-seq data from fungi grown on cattle-tick cuticles, evaluated the differential expression of BGCs, and assessed conservation among the Metarhizium genus. Furthermore, our analysis extended to the construction of a phylogeny for the following three BGCs: a tropolone/citrinin-related compound (MaPKS1), a pseurotin-related compound (MaNRPS-PKS2), and a putative helvolic acid (MaTERP1). RESULTS: Among 73 BGCs identified in M. anisopliae, 20 % were up-regulated during initial tick cuticle infection and presumably possess virulence-related roles. These up-regulated BGCs include known clusters, such as destruxin, NG39x and ferricrocin, together with putative helvolic acid and, pseurotin and tropolone/citrinin-related compound clusters as well as uncharacterized clusters. Furthermore, several previously characterized and putative BGCs were silent or down-regulated in initial infection conditions, indicating minor participation over the course of infection. Interestingly, several up-regulated BGCs were not conserved in host-specialist species from the Metarhizium genus, indicating differences in the metabolic strategies employed by generalist and specialist species to overcome and kill their host. These differences in metabolic potential may have been partially shaped by horizontal gene transfer (HGT) events, as our phylogenetic analysis provided evidence that the putative helvolic acid cluster in Metarhizium spp. originated from an HGT event. CONCLUSIONS: Several unknown BGCs are described, and aspects of their organization, regulation and origin are discussed, providing further support for the impact of SM on the Metarhizium genus lifestyle and infection process.

Independent origins of diploidy in Metarhizium.

In fungi, stable diploid genome arrangements are rare. Here we present evidence from nuclear intergenic DNA sequencing, microsatellite genotyping, and configuration of the mating-type locus to demonstrate two independent origins of persistent diploid genome organization in the Metarhizium majus species complex. Most taxa in the complex are genotypically haploid, with individual isolates consistently displaying a single allele across all nuclear loci, as well as having a single mating-type locus. In contrast, individuals of M. majus and the clade designated here MGT1 are shown to be diploid, based on a consistent finding of heterozygosity and the presence of both MAT1 and MAT2 mating-type loci. In single locus phylogenies, nuclear intergenic alleles of M. majus and MGT1 each form monophyletic groups, indicating that diploidy in both taxa likely originated by the union of conspecific individuals. Sequence divergence in the APN2/MAT1-1-3 and APN2/MAT2-1 intergenic spacers indicate the two MAT loci are physically separated in the genomes of both diploid taxa, although the linkage relationship of the MAT loci to one another is unknown. The presence of both mating genes in a single nucleus suggests these diploid genomes may represent a mating event that failed to complete meiosis. Whether or not these isolates are able to complete the sexual cycle under any conditions and form ascospores remains an open question.

Community composition and population genetics of insect pathogenic fungi in the genus Metarhizium from soils of a long-term agricultural research system.

Fungi in the genus Metarhizium are insect pathogens able to function in other niches, including soil and plant rhizosphere habitats. In agroecosystems, cropping and tillage practices influence soil fungal communities with unknown effects on the distribution of Metarhizium, whose presence can reduce populations of crop pests. We report results from a selective media survey of Metarhizium in soils sampled from a long-term experimental farming project in the mid-Atlantic region. Field plots under soybean cultivation produced higher numbers of Metarhizium colony-forming units (cfu) than corn or alfalfa. Plots managed organically and via chisel-till harboured higher numbers of Metarhizium cfu than no-till plots. Sequence typing of Metarhizium isolates revealed four species, with M. robertsii and M. brunneum predominating. The M. brunneum population was essentially fixed for a single clone as determined by multilocus microsatellite genotyping. In contrast, M. robertsii was found to contain significant diversity, with the majority of isolates distributed between two principal clades. Evidence for recombination was observed only in the most abundant clade. These findings illuminate multiple levels of Metarhizium diversity that can be used to inform strategies by which soil Metarhizium populations may be manipulated to exert downward pressure on pest insects and promote plant health.

Diversity within the entomopathogenic fungal species Metarhizium flavoviride associated with agricultural crops in Denmark.

BACKGROUND: Knowledge of the natural occurrence and community structure of entomopathogenic fungi is important to understand their ecological role. Species of the genus Metarhizium are widespread in soils and have recently been reported to associate with plant roots, but the species M. flavoviride has so far received little attention and intra-specific diversity among isolate collections has never been assessed. In the present study M. flavoviride was found to be abundant among Metarhizium spp. isolates obtained from roots and root-associated soil of winter wheat, winter oilseed rape and neighboring uncultivated pastures at three geographically separated locations in Denmark. The objective was therefore to evaluate molecular diversity and resolve the potential population structure of M. flavoviride. RESULTS: Of the 132 Metarhizium isolates obtained, morphological data and DNA sequencing revealed that 118 belonged to M. flavoviride, 13 to M. brunneum and one to M. majus. Further characterization of intraspecific variability within M. flavoviride was done by using amplified fragment length polymorphisms (AFLP) to evaluate diversity and potential crop and/or locality associations. A high level of diversity among the M. flavoviride isolates was observed, indicating that the isolates were not of the same clonal origin, and that certain haplotypes were shared with M. flavoviride isolates from other countries. However, no population structure in the form of significant haplotype groupings or habitat associations could be determined among the 118 analyzed M. flavoviride isolates. CONCLUSIONS: This study represents the first in-depth analysis of the molecular diversity within a large isolate collection of the species M. flavoviride. The AFLP analysis confirmed a high level of intra-specific diversity within the species and lack of apparent association patterns with crop or location indicates limited ecological specialization. The relatively infrequent isolation of M. flavoviride directly from crop roots suggests low dependence of root association for the species.

Comparative genome analysis of entomopathogenic fungi reveals a complex set of secreted proteins.

BACKGROUND: Metarhizium anisopliae is an entomopathogenic fungus used in the biological control of some agricultural insect pests, and efforts are underway to use this fungus in the control of insect-borne human diseases. A large repertoire of proteins must be secreted by M. anisopliae to cope with the various available nutrients as this fungus switches through different lifestyles, i.e., from a saprophytic, to an infectious, to a plant endophytic stage. To further evaluate the predicted secretome of M. anisopliae, we employed genomic and transcriptomic analyses, coupled with phylogenomic analysis, focusing on the identification and characterization of secreted proteins. RESULTS: We determined the M. anisopliae E6 genome sequence and compared this sequence to other entomopathogenic fungi genomes. A robust pipeline was generated to evaluate the predicted secretomes of M. anisopliae and 15 other filamentous fungi, leading to the identification of a core of secreted proteins. Transcriptomic analysis using the tick Rhipicephalus microplus cuticle as an infection model during two periods of infection (48 and 144 h) allowed the identification of several differentially expressed genes. This analysis concluded that a large proportion of the predicted secretome coding genes contained altered transcript levels in the conditions analyzed in this study. In addition, some specific secreted proteins from Metarhizium have an evolutionary history similar to orthologs found in Beauveria/Cordyceps. This similarity suggests that a set of secreted proteins has evolved to participate in entomopathogenicity. CONCLUSIONS: The data presented represents an important step to the characterization of the role of secreted proteins in the virulence and pathogenicity of M. anisopliae.

Occurrence of entomopathogenic fungi in tejocote (Crataegus mexicana) orchard soils and their pathogenicity against Rhagoletis pomonella.

AIMS: To determine the abundance and diversity of entomopathogenic fungi in tejocote orchard soils and evaluate their ability to infect Rhagoletis pomonella Walsh., the main pest of tejocote. METHODS AND RESULTS: Surveys were made in two locations in Mexico state and two in Puebla state. Soil from selected locations was baited for entomopathogenic fungi with Galleria mellonella (L.). All isolates were identified morphologically to genus level and to species level using Bloc and elongation factor 1-α gene sequence information, respectively; Beauveria bassiana ((Bals.-Criv.) Vuill.), B. pseudobassiana (S.A. Rehner & Humber) and Metarhizium robertsii (J.F. Bisch., Rehner & Humber) were found, with B. bassiana being the most abundant and widely distributed. Pathogenicity of five selected B. bassiana isolates and three M. robertsii isolates was evaluated against larvae and pupae of R. pomonella. All isolates infected larvae resulting in an average mortality of 35%. Pupae were not susceptible; however, adults emerging from inoculated pupae did die due to infection. CONCLUSIONS: At least three species of entomopathogenic fungi are present in the soil from tejocote orchards, with B. bassiana being the most abundant and widely distributed. Rhagoletis pomonella larvae were more susceptible to infection than pupae. SIGNIFICANCE AND IMPACT OF THE STUDY: Our study has produced new information about the distribution of entomopathogenic fungi in cultivated soils from this region of North America, contributing to a better understanding of their natural occurrence and underpinning the development of biological control approaches.

Clarification of generic and species boundaries for Metarhizium and related fungi through multigene phylogenetics.

The genus Metarhizium historically refers to green-spored asexual insect pathogenic fungi. Through culturing and molecular methods, Metarhizium has been linked to Metacordyceps sexual states. Historically fungal nomenclature has allowed separate names for the different life stages of pleomorphic fungi. However, with the move to one name for one fungus regardless of life stage, there is a need to determine which name is correct. For Metarhizium the situation is complicated by the fact that Metacordyceps sexual states are interspersed among additional asexual genera, including Pochonia, Nomuraea and Paecilomyces. Metarhizium has priority as the earliest available name, but delimiting the boundaries of this genus remains problematic. To clarify relationships among these taxa we have obtained representative material for each genus and established a molecular dataset of the protein-coding genes BTUB, RPB1, RPB2 and TEF. The resulting phylogeny supports Metarhizium combining the majority of species recognized in Metacordyceps as well as the green-spored Nomuraea species and those in the more recently described genus Chamaeleomyces. Pochonia is polyphyletic, and we restrict the definition of this genus to those species forming a monophyletic clade with P. chlamydosporia, and the excluded species are transferred to Metapochonia gen. nov. It is our hope that this unified concept of sexual and asexual states in Metarhizium will foster advances in communication and understanding the unique ecologies of the associated species.

MALDI-TOF mass spectrometry applied to identifying species of insect-pathogenic fungi from the Metarhizium anisopliae complex.

Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) has proven to be a powerful tool for taxonomic resolution of microorganisms. In this proof-of-concept study, we assessed the effectiveness of this technique to track the current gene sequence-based phylogenetic classification of species in the Metarhizium anisopliae complex. Initially the phylogenetic analysis of 5' strains by sequencing of the 59' end of the TEF-1α gene region revealed seven species within M. anisopliae sensu lato and two varieties outside this complex. Because initial studies on MS profiles from different cell types showed that mycelial fragments or conidia produced on nutrient-poor medium may yield too much background noise, all subsequent spectrometric analyses were performed with acidhydrolyzed conidia from 10-12 d old PDA cultures. The initial MALDI-TOF reference library included protein spectral profiles from nine taxonomically distinct, molecularly identified isolates sharing high genetic homology with the ex-type or ex-epitype isolates of these taxa in Metarhizium. A second reference library added one isolate each for M. anisopliae sensu stricto and M. robertsii. The second, larger reference library (including 11 taxa) allowed nearly perfect MALDI-TOF matching of DNA-based species identification for the 40 remaining isolates molecularly recognized as M. anisopliae sensu stricto (n = 19), M. robertsii (n = 6), M. majus (n = 3), M. lepidiotae (n = 1), M. acridum (n = 3), M. flavoviride var. pemphigi (n = 1), plus seven unidentified strains (six of them phylogenetically close to M. anisopliae sensu stricto and one outside the Metarhizium pingshaense-anisopliae-robertsii-brunneum clade). Due to the increasing frequency of phylogenetically (genomically) based taxonomic revisions of fungi, this approach is especially useful for culture collections, because once the protein profiles of Metarhizium isolates are obtained taxonomic updating of MALDI-TOF library data is easily accomplished by comparing stored profiles with those of newly proposed taxa.

Production of microsclerotia by Brazilian strains of Metarhizium spp. using submerged liquid culture fermentation.

We investigated the potential production and desiccation tolerance of microsclerotia (MS) by Brazilian strains of Metarhizium anisopliae (Ma), M. acridum (Mc) and M. robertsii (Mr). These fungi were grown in a liquid medium containing 16 g carbon l⁻¹ with a carbon:nitrogen ratio of 50:1. One hundred milliliters cultures were grown in 250 ml Erlenmeyer flasks in a rotary incubator shaker at 28 °C and 200 rpm for 5 days. Five-day-old MS were harvested, mixed with diatomaceous earth (DE) and air-dried for 2 days at 30 °C. The air-dried MS-DE granular preparations were milled by mortar + pestle and stored in centrifuged tubes at either 26 or -20 °C. Desiccation tolerance and conidia production were assessed for dried MS granules by measuring hyphal germination after incubation for 2 days on water agar plates at 26 °C and for conidia production following 7 days incubation. Yields of MS by all strains of Metarhizium were 6.1-7.3 × 106 l⁻¹ after 3 days growth with maximum MS yields (0.7-1.1 × 107 l⁻¹) after 5 days growth. No differences in biomass accumulation were observed after 3 days growth, whereas Ma-CG168 showed the highest biomass accumulation after 5 days growth. Dried MS-DE preparations of all fungal strains were equally tolerant to desiccation (≥93 % germination) and the highest conidia production was obtained by MS granules of Mc-CG423 (4 × 109 conidia g⁻¹). All MS granules showed similar stability after storage at either 26 or -20 °C for 3.5 months.