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

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

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

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

Metarhizium: jack of all trades, master of many.

The genus Metarhizium and Pochonia chlamydosporia comprise a monophyletic clade of highly abundant globally distributed fungi that can transition between long-term beneficial associations with plants to transitory pathogenic associations with frequently encountered protozoans, nematodes or insects. Some very common 'specialist generalist' species are adapted to particular soil and plant ecologies, but can overpower a wide spectrum of insects with numerous enzymes and toxins that result from extensive gene duplications made possible by loss of meiosis and associated genome defence mechanisms. These species use parasexuality instead of sex to combine beneficial mutations from separate clonal individuals into one genome (Vicar of Bray dynamics). More weakly endophytic species which kill a narrow range of insects retain sexuality to facilitate host-pathogen coevolution (Red Queen dynamics). Metarhizium species can fit into numerous environments because they are very flexible at the genetic, physiological and ecological levels, providing tractable models to address how new mechanisms for econutritional heterogeneity, host switching and virulence are acquired and relate to diverse sexual life histories and speciation. Many new molecules and functions have been discovered that underpin Metarhizium associations, and have furthered our understanding of the crucial ecology of these fungi in multiple habitats.

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.

The mitochondrial genome contribution to the phylogeny and identification of Metarhizium species and strains.

The genus Metarhizium is composed of entomopathogenic fungal biological control agents (BCAs) used for invertebrate pest control. The phylogenetic relationships of species within this genus are still under scrutiny as several cryptic species can be found. In this work, the mitochondrial (mt) genome of Metarhizium brunneum ARSEF 4556 was fully sequenced and a comparative genome analysis was conducted with 7 other available mt genomes, belonging to 5 Metarhizium species: M. anisopliae, M. brunneum, M. robertsii, M. guizhouense and M. majus. Results showed that Metarhizium demonstrates greater conserved stability than other fungal mt genomes. Furthermore, this analysis located 7 diverse regions in both intergenic domains and gene fragments which were ideal for species/strain discrimination. The sequencing of these regions revealed several SNPs among 38 strains tested, 11 of which were uncharacterized. Single gene phylogenies presented variable results which may be used further for intra-species discrimination. Phylogenetic trees based on the concatenation of mt domains and the nuclear ITS1-5.8S-ITS2 region showed discrimination of the species studied and allowed the identification of uncharacterized strains. These were mostly placed within species M. anisopliae and M. brunneum. Five strains clustered together in a clade related to M. brunneum, suggesting that they comprise a cryptic species.

Genetic diversity of the Metarhizium anisopliae complex in Colima, Mexico, using microsatellites.

Metarhizium anisopliae is a complex of cryptic species with wide geographical distribution and versatile lifestyles. In this study, 45 isolates of the Metarhizium genus harbored in the "Colección de Hongos Entomopatógenos" of the "Centro Nacional de Referencia de Control Biológico" from different substrates, insect-host, and localities from Colima, Mexico, were phylogenetically identified using the 5'end of translation elongation factor 1-α (5'TEF) and intergenic nuclear region MzFG543igs. Seven species were recognized, M. acridum (n = 26), M. pemphigi (n = 1), and within the PARB and MGT clades: M. anisopliae (N = 7; sensu stricto: n = 2; sensu lato: n = 5), M. brunneum (n = 2), M. guizhouense (n = 2), M. pingshaense (n = 2), and M. robertsii (n = 5). Twenty-nine SSR markers were developed for M. acridum; according to the analysis of 12 polymorphic SSR loci, M. acridum showed low genetic diversity, revealing five genotypes with a dominant one (n = 21). Based on the analysis of 13 specific SSR loci, 14 genotypes were identified within the PARB and MGT clades. This study contributes to generating valuable information about the community structure and genotypic diversity of Metharhizum species in the state of Colima, Mexico.

Natural occurrence in Argentina of a new fungal pathogen of cockroaches, Metarhizium argentinense sp. nov.

The aim of this study was to search for entomopathogenic fungi that infect wild cockroaches in forest ecosystems in two protected natural areas of Argentina. Two isolates of Metarhizium argentinense were obtained and identified from wild cockroaches (Blaberidae: Epilamprinae) through the use of morphological characteristics and molecular phylogenetic analyses. This novel species was found in Argentina and is a member of the Metarhizium flavoviride species complex. Phylogenetic analyses, based on sequence similarity analysis using internal transcribed spacer (ITS) and a set of four protein-coding marker sequences (EF1A, RPB1, RPB2 and BTUB), supported the status of this fungus as a new species. In addition, we tested the biological activity of the new species through assays against Blattella germanica nymphs and found that the two evaluated isolates were pathogenic. However, isolate CEP424 was more virulent and caused a confirmed mortality of 76 % with a median lethal time of 7.2 d. This study reports the southernmost worldwide location of a Metarhizium species that infects cockroaches and will help expand the knowledge of the biodiversity of pathogenic fungi of Argentine cockroaches.

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.

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.

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.

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.

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.

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.

Species of the Metarhizium anisopliae complex with diverse ecological niches display different susceptibilities to antifungal agents.

Species of the Metarhizium anisopliae complex are globally ubiquitous soil-inhabiting and predominantly insect-pathogenic fungi. The Metarhizium genus contains species ranging from specialists, such as Metarhizium acridum that only infects acridids, to generalists, such as M. anisopliae, Metarhizium brunneum, and Metarhizium robertsii that infect a broad range of insects and can also colonize plant roots. There is little information available about the susceptibility of Metarhizium species to clinical and non-clinical antifungal agents. We determined the susceptibility of 16 isolates comprising four Metarhizium species with different ecological niches to seven clinical (amphotericin B, ciclopirox olamine, fluconazole, griseofulvin, itraconazole, tebinafine, and voriconazole) and one non-clinical (benomyl) antifungal agents. All isolates of the specialist M. acridum were clearly more susceptible to most antifungals than the isolates of the generalists M. anisopliae sensu lato, M. brunneum, and M. robertsii. All isolates of M. anisopliae, M. brunneum, and M. robertsii were resistant to fluconazole and some were also resistant to amphotericin B. The marked differences in susceptibility between the specialist M. acridum and the generalist Metarhizium species suggest that this characteristic is associated with their different ecological niches, and may assist in devising rational antifungal treatments for the rare cases of mycoses caused by Metarhizium species.

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.

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.

Fungal dermatitis, glossitis and disseminated visceral mycosis caused by different Metarhizium granulomatis genotypes in veiled chameleons (Chamaeleo calyptratus) and first isolation in healthy lizards.

Metarhizium (M.) granulomatis (formerly Chamaeleomyces granulomatis) invariably causes fatal fungal glossitis and systemic mycosis in veiled chameleons (Chamaeleo calyptratus). Isolation of M. granulomatis in other lizards thus far has not been described. The aim of this study therefore was to obtain information on the presence of M. granulomatis in reptiles kept as pets, and to examine whether there was an association between specific genotypes and clinical/pathological outcomes. Besides 18S ribosomal (r) DNA (SSU) and internal transcribed spacer1-5.8S (ITS1-5.8S) rDNA, a fragment of the large subunit of the 28S rDNA (LSU), including the domains 1 (D1) and D2, were sequenced for identification of the fungus and phylogenetic analysis. Metarhizium granulomatis was isolated from 23 veiled chameleons, two panther chameleons (Furcifer pardalis) and one central bearded dragon (Pogona vitticeps). Only the veiled chameleons revealed corresponding pathological findings in the form of glossal hemorrhage, granulomatous glossitis, pharyngitis, dermatitis and/or visceral mycosis. The infection site correlated to survival times of infected veiled chameleons. Combined long-term treatment with terbinafine and nystatin based on susceptibility testing may be helpful for prevention of disease and visceral spreading of the fungus, but elimination of the fungal pathogen or successful treatment of diseased veiled chameleons have not been achieved yet. Sequencing of the ribosomal genes yielded five different genotypes, with genotype A being strongly correlated with dermatitis, and remaining genotypes with pharyngitis and glossitis. However, disseminated visceral mycosis developed irrespective of the genotypes.

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.

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.