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Natural diversity and distribution of species within the entomopathogenic fungal genus Metarhizium in forest ecosystems in Sichuan Province, China
Abstract
To understand the ecological roles and life history of the entomopathogenic fungi Metarhizium, its natural distribution must be known. The ability to recognize a limited species using robust regions of the genome to illustrate species boundaries diagnostically and phylogenetically is a crucial step. To investigate the phylogenetic relationship within Chinese Metarhizium isolates, an extensive sampling survey was conducted at 19 geographically separated locations in diverse forest regions of Sichuan Province. Isolation was accomplished using the insect susceptible-bait method with Tenebrio molitor larvae (Coleoptera, Tenebrionidae). Of the 173 Metarhizium isolates obtained, the maximum likelihood of both nuclear intergenic sequence markers Mz_IGS3 and Mz_FG543 revealed that 75 belonged to M. pingshaense, 44 to M. robertsii, 10 to M. brunneum, 4 to M. guizhouense, 3 to the M. flavoviride species complex (M. bibionidarum) and 37 to a segregated, unknown Metarhizium cluster. M. pingshaense was the predominant Metarhizium spp. isolate, followed by M. robertsii. Further molecular characterization was performed using GCPSR-based multilocus (6796 bp segments composed of whole translation elongation factor-1 alpha 5' and 3'-TEF, β-tubulin, ITS, and two intergenic spacers) phylogenetic analyses to illustrate the unexplained Metarhizium clade with reference sequences. The results revealed this cluster is a sister to the M. lepidiotae clade in the M. anisopliae species complex and may limit the geographical distributions of distinct Metarhizium spp., which contrasts with the large-scale distribution of the PARB (M. pingshaense, M. anisopliae, M. robertsii and M. brunneum) clade. These results indicate this cluster possesses a different ecological distribution and/or dispersal ability, which could lead to novel communities and interactions.
GP64 is the key membrane fusion protein of Group I baculovirus, and while the Bombyx mori nucleopolyhedrovirus (BmNPV) GP64 contains a longer n-region (18 amino acid) of the signal peptide than does the Autographa californica multiple nucleopolyhedrovirus (AcMNPV), the function of the n-region has not been determined. In this study, we first showed that n-region is required for membrane protein localization in BmN cells, then the transcriptome sequencing was conducted on proteins guided by different signal peptide regions, and the results were analyzed and validated by quantitative PCR and luciferase assays. The results indicated that 1049 differentially expressed genes (DEGs) were identified among the different region of signal peptides and the control. With the n-region, the protein export pathway was upregulated significantly, the Wnt-1 signaling pathway was downregulated, and BiP was significantly activated by the GP64 full-length signal peptide. Furthermore, RNA interference on BiP efficiently increased luciferase secretion. These results indicate that the GP64 n-region plays a key role in protein expression and regulation.
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.
Since the Guideline of Strengthen Preservation and Development of Traditional Village enacted, the traditional villages gradually gain academic concern and many achievements are acquired. But the study about outdoor wind environment is still insufficient. In this paper, the outdoor wind environment of Liyuanba Village in Bazhong City of Sichuan Province – a typical traditional village in the northeastern Sichuan Basin, is simulated by CFD methods. According to the simulation result, the outdoor wind environment in winter is comfortable for people in general, but there are still eddies and corner flows in some areas. To improve the outdoor wind environment, the suggestions such as changing the direction of courtyard and building are put forward. Furthermore, the optimization is also simulated and these two simulation results are compared in the end.
Metarhizium Sorokīn (Hypocreales: Clavicipitaceae) is a genus of facultative parasites of insects found in soils from various environments and is used for pest management. Understanding the habitat selection of Metarhizium spp. is critical to improve the efficacy and persistence of these fungi as microbial insecticides. This study sought to determine the habitat preferences of Metarhizium spp. in Japan. We identified 302 isolates of Metarhizium spp. as eight species by a combination of PCR–RFLP and phylogenetic analysis of DNA sequences. M. pingshaense was the predominant species in Japan and was most frequently isolated from both forest and agricultural environments. On the other hand, M. brunneum and M. pemphigi were comparatively restricted to forest environments. A similar species association was detected in a small area that was intensively investigated, where 7 species including 14 genotypes were isolated from soil. The results of this study have revealed different habitat preferences among Metarhizium spp. in Japan.
Two new species of Metarhizium, M. bibionidarum and M. purpureogenum are described from Japan. Metarhizium bibionidarum is the phylogenetic sister species of M. pemphigi and a member of the M. flavoviride species complex. It is distinguished morphologically from M. pemphigi by its larger conidia. The species is based on a collection of an infected March fly larva (Diptera: Bibionidae) but is also known to occur on fruit beetle (Coleoptera: Scarabaeidae) encountered in France. Metarhizium purpureogenum was isolated from soil by plating and insect baiting methods and represents a unique phylogenetic lineage placed outside the M. anisopliae and M. flavoviride species complexes. Three isolates of M. purpureogenum excreted a distinctive red-purple pigment into agar medium when co-cultured with M. robertsii or Aspergillus oryzae.
An essential first step to elucidating the ecology and life histories of Metarhizium anisopliae-group species as entomopathogens, endophytes and soil-adapted fungi is the ability to define species limits and confidently infer a species phylogeny. In a multilocus phylogeny of the core Metarhizium anisopliae species complex, the majority of isolates sampled herein group within the currently defined limits of M. pingshaense, M. anisopliae, M. robertsii and M. brunneum, designated informally as the “PARB” clade. Multilocus phylogenetic analyses reveal pervasive congruent hierarchical structure among the genomic regions analyzed, which suggest that current PARB species delimitations likely encompass additional cryptic complexes. Further, the interpolation of isolates from different continents throughout each species lineage indicates periodic inter-continental dispersals. Although no PARB species has yet been confirmed to produce a sexual state, we demonstrate the mutually exclusive incidence of the MAT1 and MAT2 mating type idiomorphs among individuals in all PARB species. This configuration of mating type is diagnostic of a heterothallic, obligately outcrossing mating system, indicating the conservation of and ongoing potential for sexual reproduction in all PARB species. As one example of the utility of IGS markers, the commercially registered M. anisopliae strain F52, which is widely used for pest control in North America, Canada and Europe, is shown to be a member of the M. brunneum complex. While current PARB species delimitations evidently encompass cryptic partitions, formal recognition of segregate species should be approached cautiously until further evidence of their phylogenetic exclusivity, ecological distinctiveness or other unique attributes is demonstrated. Nevertheless, acknowledgment of these intraspecific partitions will provide a useful conceptual framework to guide future investigations of the community structure, phylogeography, population genetics, ecology and reproductive biology of this recent species radiation.
Feeding an increasing global population requires continued improvements in agricultural efficiency and productivity. Meeting estimated future production levels requires the adoption of practices that increase output without environmental degradation associated with external inputs to supplement nutrition or control pests. Enriching the community of microbes associated with plants in agricultural systems for those providing ecosystem services such as pest control is one possible component towards achieving sustainable productivity increases. In this review we explore the current state of knowledge for Hypocreales fungi used in biological control. Advances in understanding the field ecology, diversity and genetic determinants of host range and virulence of hypocrealean fungi provide the means to improve their efficacy.
Abstract 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 specie
Metarhizium robertsii is a common soil fungus that occupies a specialized ecological niche as an endophyte and an insect pathogen. Previously, we showed that the endophytic capability and insect pathogenicity of Metarhizium are coupled to provide an active method of insect-derived nitrogen transfer to a host plant via fungal mycelia. We speculated that in exchange for this insect-derived nitrogen, the plant would provide photosynthate to the fungus. By using 13 CO 2 , we show the incorporation of 13 C into photosynthate and the subsequent translocation of 13 C into fungal-specific carbohydrates (trehalose and chitin) in the root/endophyte complex. We determined the amount of 13 C present in root-associated fungal biomass over a 21-day period by extracting fungal carbohydrates and analysing their composition using nuclear magnetic resonance (NMR) spectroscopy. These findings are evidence that the host plant is providing photosynthate to the fungus, likely in exchange for insect-derived nitrogen in a tripartite, and symbiotic, interaction.
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.
Beauveria bassiana (Balsamo) Vuillemin and Metarhizium anisopliae var. anisopliae (Metschnikoff) Sorokin are the most common entomopathogenic fungi used in microbial control programs all over the world. Assessments of the genetic variability of these 2 important species are useful for the development of effective biocontrol strategies and for evaluating the impact of artificial epizootics. In this study, the genetic diversity of 13 B. bassiana and 33 M. anisopliae var. anisopliae strains isolated from the hazelnut-growing region of Turkey was determined by using the amplified fragment length polymorphism (AFLP) and α- and β-isoenzyme analyses. Cluster analysis of AFLP data clearly separated both B. bassiana and M. anisopliae var. anisopliae strains into 3 and 4 different groups, respectively. While α- and β-esterase banding patterns clearly separated M. anisopliae var. anisopliae strains, it did not give enough information about B. bassiana strains. We also assessed the growing ability of all isolates at different temperatures (8, 16, 25, and 37 °C) and UV exposures (30 and 60 min), and virulence against Tenebrio molitor. These results indicated that there is a significant variability within the B. bassiana and M. anisopliae var. anisopliae populations in this region. Although the diversity of B. bassiana isolates is associated with geographic location, it is not associated with habitat type. There is also no association amongst B. bassiana isolates in terms of the ability to grow at different temperatures and UV exposures, or virulence against T. molitor. The diversity of M. anisopliae var. anisopliae strains is neither associated with habitat type nor geographic location, and there is no association amongst M. anisopliae var. anisopliae strains in terms of the ability to grow at different temperatures (except for 16 °C) and UV exposure, or virulence against T. molitor. The data presented here might be useful for controlling some hazelnut pests in this region.
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.
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.
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.
The development and use of entomopathogens as classical, conservation and augmentative biological control agents have included a number of successes and some setbacks in the past 15 years. In this forum paper we present current information on development, use and future directions of insect-specific viruses, bacteria, fungi and nematodes as components of integrated pest management strategies for control of arthropod pests of crops, forests, urban habitats, and insects of medical and veterinary importance.
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 (CFUs) than corn or alfalfa. Plots managed organically and via chisel-till harbored higher numbers of Metarhizium CFUs 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.
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 51 strains by sequencing of the 5' 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 acid-hydrolyzed 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. magus (n = 3), M. lepidiotae (n = 1), M. acrid (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.
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.
Phylogenies are increasingly used in all fields of medical and biological research. Moreover, because of the next generation sequencing revolution, datasets used for conducting phylogenetic analyses grow at an unprecedented pace. RAxML (Randomized Axelerated Maximum Likelihood) is a popular program for phylogenetic analyses of large datasets under maximum likelihood. Since the last RAxML paper in 2006, it has been continuously maintained and extended to accommodate the increasingly growing input datasets and to serve the needs of the user community.
I present some of the most notable new features and extensions of RAxML, such as, a substantial extension of substitution models and supported data types, the introduction of SSE3, AVX, and AVX2 vector intrinsics, techniques for reducing the memory requirements of the code and a plethora of operations for conducting post-analyses on sets of trees. In addition, an up-to-date, 50 page user manual covering all new RAxML options is available.
The code is available under GNU GPL at https://github.com/stamatak/standard-RAxML.
Alexandros.Stamatakis@h-its.org.
The study of symbiotic nitrogen transfer in soil has largely focused on nitrogen-fixing bacteria. Vascular plants can lose
a substantial amount of their nitrogen through insect herbivory. Previously, we showed that plants were able to reacquire
nitrogen from insects through a partnership with the endophytic, insect-pathogenic fungus Metarhizium robertsii. That is, the endophytic capability and insect pathogenicity of M. robertsii are coupled so that the fungus acts as a conduit to provide insect-derived nitrogen to plant hosts. Here, we assess the ubiquity
of this nitrogen transfer in five Metarhizium species representing those with broad (M. robertsii, M. brunneum, and M. guizhouense) and narrower insect host ranges (M. acridum and M. flavoviride), as well as the insect-pathogenic fungi Beauveria bassiana and Lecanicillium lecanii. Insects were injected with 15N-labeled nitrogen, and we tracked the incorporation of 15N into two dicots, haricot bean (Phaseolus vulgaris) and soybean (Glycine max), and two monocots, switchgrass (Panicum virgatum) and wheat (Triticum aestivum), in the presence of these fungi in soil microcosms. All Metarhizium species and B. bassiana but not L. lecanii showed the capacity to transfer nitrogen to plants, although to various degrees. Endophytic association by these fungi increased
overall plant productivity. We also showed that in the field, where microbial competition is potentially high, M. robertsii was able to transfer insect-derived nitrogen to plants. Metarhizium spp. and B. bassiana have a worldwide distribution with high soil abundance and may play an important role in the ecological cycling of insect
nitrogen back to plant communities.
Phyllophaga capillata (Blanchard) was recently described as a soybean pest in Brazil. The occurrence of Metarhizium spp. associated with adults and their pathogenicity were investigated. Natural prevalence of Metarhizium anisopliae was 0.51% in adults collected by light trap. Most Metarhizium isolates were identified as Metarhizium robertsii, although M. anisopliae sensu stricto and a few unidentified isolates were also found. Bioassays with representative isolates selected from different genetic groups resulted in ≤5.6% confirmed mortality against larvae and adults, suggesting low potential of microbial control of this pest by use of indigenous Metarhizium isolates.
Insect-pathogenic fungi are important natural enemies of insect pests but their dynamics in the soils of different agroecosystems are incompletely understood. In the present study, the seasonal occurrence and species diversity of insect-associated fungi in soil were investigated by baiting soil samples with larvae of Galleria mellonella. The survey included insect-pathogenic fungi, opportunistic insect pathogens, and secondary colonizers of insect cadavers. Soil samples from two habitat types (field crops and orchards) were collected from 2004 to 2005 at 2-month intervals except during wintertime. A total of 29 species were isolated and identified, with 25 species in field crop soils and 20 species in orchard soils. Although the common insect-pathogenic species, such as Beauveria bassiana, Metarhizium anisopliae var. anisopliae, and Paecilomyces fumosoroseus (Ascomycota: Hypocreales), were detected in both field crop and orchard soils, their frequency in the two agroecosystems differed significantly: B. bassiana and P. fumosoroseus were more frequently detected in orchard soils, while M. anisopliae var. anisopliae was more frequently detected in field crop soils. The diversity of insect-pathogenic fungi was greater in field crop soil than in orchard soil. The number of Galleria larvae that died from fungal infection was larger in orchard soil than in field crop soil. B. bassiana was the most abundant species in orchard soil, and the number of larvae killed by B. bassiana was larger in June and December than in April and August. However, Mucor spp. (opportunistic pathogens) were the most abundant species in field crop soils, and the number of larvae killed by them was larger in August than at any other time of the year.
We report a major update of the MAFFT multiple sequence alignment program. This version has several new features, including
options for adding unaligned sequences into an existing alignment, adjustment of direction in nucleotide alignment, constrained
alignment and parallel processing, which were implemented after the previous major update. This report shows actual examples
to explain how these features work, alone and in combination. Some examples incorrectly aligned by MAFFT are also shown to
clarify its limitations. We discuss how to avoid misalignments, and our ongoing efforts to overcome such limitations.
Penicillium chrysogenum is a filamentous fungus of major medical and historical importance, being the original and present-day industrial source of the antibiotic penicillin. The species has been considered asexual for more than 100 y, and despite concerted efforts, it has not been possible to induce sexual reproduction, which has prevented sexual crosses being used for strain improvement. However, using knowledge of mating-type (MAT) gene organization, we now describe conditions under which a sexual cycle can be induced leading to production of meiotic ascospores. Evidence of recombination was obtained using both molecular and phenotypic markers. The identified heterothallic sexual cycle was used for strain development purposes, generating offspring with novel combinations of traits relevant to penicillin production. Furthermore, the MAT1-1-1 mating-type gene, known primarily for a role in governing sexual identity, was also found to control transcription of a wide range of genes with biotechnological relevance including those regulating penicillin production, hyphal morphology, and conidial formation. These discoveries of a sexual cycle and MAT gene function are likely to be of broad relevance for manipulation of other asexual fungi of economic importance.
Entomopathogenic fungi in the genus Metarhizium are useful for biological control programmes against economically important arthropod pests worldwide. However, understanding the true diversity and ecology of these organisms is hampered by convergent morphologies between species. The application of molecular techniques has enabled greater resolution of species than allowed by morphology alone. In particular, the commonly used biocontrol agent M. anisopliae was found to be a species complex composed of nine species. This prior work was conducted with commonly used markers in fungal phylogenetics (BTUB, RPB1, RPB2 and TEF), which likely under-represent diversity in the M. anisopliae complex. Using sequence data from nuclear genomes of M. acridum and M. robertsii we identified regions of conserved gene synteny and developed primers to amplify intergenic regions of seven loci. Using ex-type and authenticated tissue specimens for species in the M. anisopliae complex, we demonstrate that sequence data derived from intergenic loci is more variable and phylogenetically informative than previously available markers. These new markers will facilitate investigations at or below the species level for the M. anisopliae complex. The method of marker development employed here should be extendable to any group with sufficiently divergent genome data available.
Based on the internal transcribed spacer and inter-simple sequence repeats (ISSR), the phylogenetic relationship and genetic diversity of Metarhizium spp., pathogens found in Chinese burrower bugs, Schiodtella formosana, were analyzed. The results showed that the causative agents of the epizootic green muscardine disease in populations of S. formosana were actually composed of M. anisopliae and its sister species, M. robertsii. The genetic structure of Metarhizium spp. populations were assessed using ten ISSR. A 3D principal component analysis of 51 isolates sampled on different occasions revealed that the Metarhizium spp. populations were temporally heterogeneous. They differentiated into two main clades including over 71 % of all strains causing epizootics, with a similarity of 83 %. The population differentiation was relatively low (G ( ST ), 0.2080), reflecting a large proportion of gene differentiation (79.2 %) within the populations. Further knowledge of the complex species and heterogeneous populations of Metarhizium spp. may be necessary for sustainable control methods of S. formosana.
White grub larvae are important soil-dwelling pests in many regions of Mexico as they attack many important crops such as maize. The use of synthetic chemicals is currently the main control strategy, but they are not always effective; thus, other alternatives are needed. Microbial control using entomopathogenic fungi represents an important alternative strategy, and species within the genera Beauveria and Metarhizium are considered amongst the most promising candidates. Seventeen Beauveria spp. and two Metarhizium spp. isolates were obtained in surveys of white grub larvae from different regions of Guanajuato, Mexico. All isolates were capable of infecting healthy larvae of the white grub Phyllophaga polyphilla in laboratory assays, but mortality never exceeded 50 %. Isolates were identified using morphological and molecular methods. Based on elongation factor1-α and ITS partial gene sequence data, all Beauveria isolates were identified as Beauveria pseudobassiana. Elongation factor1-α and β-tubulin sequence data identified the Metarhizium isolates to be Metarhizium pingshaense. In contrast, three additional Metarhizium isolates obtained the previous year in the same region were identified as M. pingshaense, Metarhizium anisopliae and Metarhizium robertsii. Microsatellite genotyping showed that all B. pseudobassiana isolates were the same haplotype. Enterobacterial Repetitive Intergenic Consensus fingerprinting information confirmed no significant variation amongst the B. pseudobassiana isolates. The ecological role of these isolates and their impact on white grub larvae populations are discussed.
The biodiversity of entomopathogenic fungi in tropical ecosystems is still little investigated, and the objective of this study was to isolate and identify fungi of the entomopathogenic genus Metarhizium (Hypocreales: Clavicipitaceae) present in undisturbed soils of the Central Brazilian Cerrado. A total of 107 Metarhizium isolates was obtained from soils collected from Cerrado sites in the state of Goiás; gene sequences from 63 of these were obtained and compared. Among these, one was confirmed to be M. anisopliae sensu stricto; 53 were very closely allied to M. anisopliae but require more extensive genetic characterization to determine if they might represent a new taxon in the M. anisopliae species complex. Eight of these Cerrado isolates were referable to M. robertsii, and the remaining isolate is the first South American (and Southern Hemisphere) collection of M. flavoviride var. pemphigi. These findings underline the need for better characterization of the diversity of these widely distributed fungi in Brazil. (© 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim).
Plants That Eat Animals
Apart from some spectacular exceptions, such as pitcher plants and Venus fly traps, most plants are thought to acquire nitrogen passively from microbial decomposition and the activities of nitrogen-fixing bacteria. Metarhizium species are common endophytes—fungi that live within plant tissues without causing disease. This genus is also found ubiquitously in soil, where they parasitize insects. In a series of microcosm experiments, Behie et al. (p. 1576 ) investigated whether these fungi could couple their endophytic life-styles with their parasitic modes and be a conduit by which plants could obtain nitrogen from animals. Radio-labeled moth larvae were added to the microcosms in which bean and grass plants were grown, and when the larvae were inoculated with fungi, it was only a matter of days before the nitrogen label was detected in the plants.
The anamorph genus Metarhizium is composed of arthropod pathogens, several with broad geographic and host ranges. Members of the genus, including ‘‘M. anisopliae var. frigidum’’ nomen nudum and Metarhizium flavoviride, have been used as biological insecticides. In a recent revision of the genus the variety ‘‘M. anisopliae var. frigidum’’ was suggested to be a synonym of M. flavoviride based largely on ITS sequence phylogenetic analysis. In this study we conducted morphological evaluations and multigene phylogenetic analyses with EF-1α, RPB1 and RPB2 for strains of M. flavoviride and ‘‘M. anisopliae var. frigidum.’’ Included in these evaluations were the ex-type of M. flavoviride var. flavoviride and what likely would be considered the ‘‘ex-type’ of the invalidly published taxon ‘‘M. anisopliae var. frigidum’’. Based on morphological and molecular evidence we conclude that ‘‘M. anisopliae var. frigidum’’ is distinct from M. flavoviride and the taxon M. frigidum sp. nov. is described.
The biogeography and genotype diversity of Metarhizium species in northwestern North American soils was examined; twenty ecoregions were sampled, including 58 agricultural and 80 natural habitat subsites, and areas that were glaciated during the Pleistocene epoch. One hundred and twenty nine isolates of M. brunneum, 26 isolates of M. robertsii, four isolates of M. guizhouense, one isolate of M. flavoviride, and 55 isolates of Beauveria were recovered. Metarhizium and Beauveria species were isolated in diverse ecoregions within the study area, but a trend for increased isolation of Metarhizium species in western regions of the study area was observed. Consistent with this observation, the prevalence of M. brunneum and M. robertsii decreased at higher elevations, and the opposite was true for Beauveria. Both M. brunneum and M. robertsii were more commonly isolated from agricultural and natural habitat subsites, and considerable genotypic diversity was observed in both habitats and within the same subsite. Metarhizium robertsii but not M. brunneum was more commonly isolated from non-glaciated locations; however, less diversity and richness was observed for M. brunneum recovered from glaciated versus non-glaciated locations consistent with insular biogeography. The study has implications for microbial control strategies in the region.
Tree growth at boreal/alpine treelines is generally expected to be stimulated by climate warming. However, tree growth has been shown to have changed insignificantly or reduced in numerous studies. Tree response to climate warming also varies among species in the same eco-region. The Tibetan Plateau has experienced rapid warming in the past several decades. It remains unknown how the dominant species in this region have responded to this warming, which is expected to significantly influence treeline dynamics. We obtained 288 tree cores from fir (Abies faxoniana and Abies squamata) and spruce (Picea purpurea and Picea baifouriana) at six treeline sites on the eastern edge of the Tibetan Plateau to detect their growth trends and climate responses. The results showed that the growth of fir did not change significantly at any site, whereas spruce growth increased, especially at the Songpan and Luhuo sites. The strong negative temperature or positive moisture controls of April on fir growth indicated spring drought stress. The radial growth of spruce was positively related to June and July temperature; at the Songpan and Luhuo sites, it was also positively related to the temperature from February to April. The results indicate that both pre-growing season temperature and growing season temperature accelerate spruce growth, whereas pre-growing season temperature causes drought stress to fir. The tree species composition at the alpine treeline on the eastern edge of the Tibetan Plateau will change as warming continues.
In several surveys in the tropical forests in Thailand, specimens that looked morphologically similar to Metarhizium martiale and Cordyceps variegata, as well as other Metarhizium species were collected and cultured in vitro. A combined phylogeny of several genes including the small (18S) and large (28S) subunits of the ribosomal DNA, elongation factor 1-α (TEF), RNA polymerase II subunits 1 and 2 (RPB1, RPB2) genes has shown these to be new taxa in the Clavicipitaceae. Nigelia is described as a new genus closely related to Metarhizium, to the scale insect pathogens Aschersonia (Hypocrella), Samuelsia and Moelleriella, and to plant pathogens in Claviceps and Balansia, and other relatives. Nigelia comprises M. martiale and a new species Nigelia aurantiaca, which has been found infecting lepidopteran larvae and which produces pseudoimmersed, obliquely arranged, obpyriform perithecia with curved or bent ostioles and with whole (non-separating) cylindric ascospores. Metarhizium chaiyaphumense, M. kalasinense, M. prachinense, M. samlanense, and M. takense are described as new species of Metarhizium. Metarhizium martiale is transferred to Nigelia, and Paecilomyces reniformis is transferred to Metarhizium.
An entomogenous fungus, Metarhizium anisopliae (METSCHNIKOFF) SOROKIN is well known as the pathogen of “green muscardine” in the muscardine diseases of the silkworm. Bombyx mori and other wild insects. It has been believed that the main source of the fungus infection of the silkworm are wild insects attacked by muscardine fungi spontaneously. Therefore, in order to improve the control methods of silkworm muscardine, it is necessary to know whether there are any other sources of the fungus infection to the silkworm in fields. The present paper deals with Metarhizium anisopliae distributed in the soil of mulberry fields as the primary source of the fungus infection to the silkworm. 1. An entomopathogenic fungus, Metarhizium anisopliae was easily detected in the mulberry field soil by the dilution plate method using 0.02% Tween 40 sterile solution and the selective agar medium, Czapek Dox-rose bengal (60mg/Z)-chloramphenicol (0.1mg/ml)-cycloheximide (1.0 mg/ml)-agar (CRCC agar), or by the baiting (or trapping) method using living silkworm pupae. 2. Many isolates of M. anisopliae from the soil of the mulberry fields showed high pathogenicity to silkworm larvae. 3. Isolates of M. anisopliae obtained from the muberry field soil were divided into three forms in their conidial size and in color of colony: a short spared form with greenish black colony (the most common form, M. anisopliae (METSCHNIKOFF) SOROKIN var. ainsopliae), a short spored form with pale green colony (M. anisopliae var. anisopliae), and a long spored form with greenish black colony (M. anisopliae (METSCHNIKOFF) SOROKIN var. major TULLOCH). Foregoing results suggest that M. anisopliae is distributed in the soil as a soil-born fungus: not only overwinter in the soil of mulberry fields as reservoir, but also grow in the soil. © 1979, The Japanese Society of Sericultural Science. All rights reserved.
Establishment, persistence and local dispersal of the entomopathogenic fungi Metarhizium anisopliae (ESALQ1037) and M. robertsii (ESALQ1426) (Ascomycota: Hypocreales) were investigated in the soil and rhizosphere following soil drench application in strawberries between 2012 and 2013 at a single location in Inconfidentes, Minas Gerais, Brazil. Metarhizium spp. isolates (n = 108) were collected using selective agar media and insect bait methods, and characterized by sequence analyses of the 5’–end of the translation elongation factor 1-α and the MzFG543igs intergenic region and by multilocus simple sequence repeat analysis. Both applied fungal isolates were frequently recovered from bulk soil and rhizosphere samples of the treated plots, suggesting that they were able to establish and disperse within the soil. Persistence within the soil and strawberry rhizosphere for both fungal isolates was observed up to 12 months after application with frequencies of 25% of haplotypes similar to isolate ESALQ1037 and 87.5% of haplotypes similar to isolate ESALQ1426, respectively. Overall, M. robertsii was the most abundant species in the agroecosystem studied representing 77.8% of the isolates recovered across all sample dates.
Green muscardine caused by Metarhizium rileyi affects sericulture, and is typically enzootic and occurs frequently at low incidence. We collected 152 M. rileyi isolates from silkworm cadavers in eight sericulture areas in seven provinces of China, and four strains from other Lepidoptera larvae in Qianshan(QS) County, Anhui province. Nine microsatellite inter-simple sequence repeat (ISSR) primers produced 91 distinct and reproducible bands, revealing a high level (90.11%) of DNA polymorphism. Unweighted Pair Group Method with Arithmetic Mean (UPGMA) cluster analysis divided the populations into four groups, with isolates from Qianshan County forming a single branch. All the 156 M. rileyi isolates were heterogenic and polyphyletic and didn’t displayed typical regional distribution except strains from Qianshan country. PCA analysis of the nine populations of M. rileyi revealed similar phylogenies among accessions. Genetic differentiation index (Gst) among eight enzootic populations was 0.3789 and gene flow (Nm) was 0.4098, suggesting the low gene flow maintained a high degree of differentiation. Gst between the enzootic population of Qianshan County and other seven populations exceeded the threshold of severe differentiation, with moderate differentiation between the remaining seven enzootic populations. Analysis of molecular variance (AMOVA) showed most ISSR variation (61%) among isolates occurred within populations. No significant correlation was observed between geographical and genetic distance. According to cluser analysis based on single enzootic population, every enzootic population showed dominance, namely mainly constituted of strains with high genetic similarity. These data indicated that the green muscardine in each local silkworm population was predominantly caused by a native group of M. rileyi. Furthermore, Gst and Nm of M. rileyi from silkworm and other Lepidoptera larvae in Qianshan County were 0.1174 and 1.8791, respectively, suggesting strains isolated from different hosts in Qianshan County do not show obvious host specificity. This demonstrated that host transfer may take place in silkworm and other insects.
A wild, forest-dwelling cockroach from the subfamily Ectobiidae (order Blattodea) in a nature reserve in Cavalcante, in the state of Goiás, Brazil, was found to be infected by a new, genetically distinct species in the Metarhizium flavoviride species complex that we describe here as Metarhizium blattodeae. The status of this fungus as a new species is supported by both multigenic sequence comparisons and protein profiles generated by MALDI-TOF (matrix-assisted laser desorption/ionization time-of-flight) mass spectrometry. This is one of the first reports of a naturally occurring fungal pathogen affecting any sylvatic (forest-dwelling) cockroach from any part of the world. M. blattodeae caused up to 96% mortality of Periplaneta americana nymphs (a serious peridomestic cockroach species) after 10 days.
The occurrence of deuteromycetous entomopathogenic fungi was examined in 266 soil samples representing 86 locations across temperate and near northern habitats in Ontario, Canada. Entomopathogenic fungi were isolated by baiting the soil with waxworm larvae, Galleria mellonella L., and incubating at 8, 15, or 25 degrees C. Entomopathogenic fungi were isolated from 91% of the locations sampled across Ontario. The most abundant species were Metarhizium anisopliae (Metschn.) Sorok. (357 isolates) and Beauveria bassiana (Bals.) Vuill. (187 isolates). Thirteen isolates of Paecilomyces spp. were also found. Beauveria bassiana was isolated more frequently in soils from near northern locations, relative to M. anisopliae. Beauveria bassiana was isolated more frequently from larvae baited in soils incubated at 8 and 15 degrees C, while M. anisopliae was isolated most frequently at 25 degrees C. Thus, B. bassiana is more psychrophilic than M. anisopliae. From 47 of the locations in a temperate area (southern Ontario and the Kawartha Lakes region), two sites, one from an agricultural habitat and one from a natural habitat, were sampled within 1 km of each other. In these locations, B. bassiana was predominantly recovered more often from soils of natural habitats, while M. anisopliae was recovered more often in agricultural habitats. The occurrence of M. anisopliae and B. bassiana was not related to soil type or pH.
Metarhizium spp. have recently been shown to be associated with the roots of different plants. Here we evaluated which Metarhizium species were associated with roots of oat (Avena sativa), rye (Secale cereale) and cabbage (Brassica oleracea), common crop plants in Denmark. Thirty-six root samples from each of the three crops were collected within an area of approximately 3 ha. The roots were rinsed with sterile water, homogenized and the homogenate plated onto selective media. A subset of 126 Metarhizium isolates were identified to species by sequencing of the 5' end of the gene translation elongation factor 1-alpha and characterized by simple sequence repeat (SSR) analysis of 14 different loci. Metarhizium brunneum was the most common species isolated from plant roots (84.1% of all isolates), while M. robertsii (11.1%) and M. majus (4.8%) comprised the remainder. The SSR analysis revealed that six multilocus genotypes (MLGs) were present among the M. brunneum and M. robertsii isolates, respectively. A single MLG of M. brunneum represented 66.7%, 79.1% and 79.2% of the total isolates obtained from oat, rye and cabbage, respectively. The isolation of Metarhizium spp. and their MLGs from roots revealed a comparable community composition as previously reported from the same agroecosystem when insect baiting of soil samples was used as isolating technique. No specific MLG association with a certain crop was found. This study highlights the diversity of Metarhizium spp. found in the rhizosphere of different crops within a single agroecosystem and suggests that plants either recruit fungal associates from the surrounding soil environment or even govern the composition of Metarhizium populations.
Biological control of spittlebugs with Metarhizium (Hypocreales: Clavicipitaceae) in Brazilian sugarcane is an example of effective pest management. However, little is known about the richness, distribution and ecology of Metarhizium species in Brazilian agroecosystems. We investigated Metarhizium diversity within a collection of 96 Brazilian isolates from spittlebugs and other insects, strains used for spittlebug control and soil isolates from sugarcane and other field crops and pristine habitats. A multilocus phylogenetic analysis of 5′-TEF and nuclear intergenic loci MzFG543igs and MzIGS3 yielded robust support for current species limits of the two most abundant taxa, Metarhizium
anisopliae and Metarhizium robertsii, and the resolution of two lineages that lie beyond currently recognized species limits in this complex. With a single exception, all isolates from insects belong to a single subclade of M. anisopliae. These data will serve as resources about Metarhizium biodiversity for insect biological control initiatives in South America.
The entomopathogenic fungal Metarhizium anisopliae lineage harbors cryptic diversity and was recently split into several species. Metarhizium spp. are frequently isolated from soil environments, but the abundance and distribution of the separate species in local communities is still largely unknown. Entomopathogenic isolates of Metarhizium spp. were obtained from 32 bulked soil samples of a single agroecosystem in Denmark using Tenebrio molitor as bait insect. To assess the Metarhizium community in soil from the agricultural field and surrounding hedgerow, 123 isolates were identified by sequence analysis of 5' end of elongation factor 1-α and their genotypic diversity characterized by multilocus simple sequence repeat (SSR) typing. Metarhizium brunneum was most frequent (78.8%) followed by M. robertsii (14.6%), while M. majus and M. flavoviride were infrequent (3.3% each) revealing co-occurrence of at least four Metarhizium species in the soil of the same agroecosystem. Based on SSR fragment length analysis five genotypes of M. brunneum and six genotypes of M. robertsii were identified among the isolates. A single genotype within M. brunneum predominated (72.3% of all genotypes) while the remaining genotypes of M. brunneum and M. robertsii were found at low frequencies throughout the investigated area indicating a diverse Metarhizium community. The results may indicate potentially favorable adaptations of the predominant M. brunneum genotype to the agricultural soil environment.
Metarhizium anisopliae and Beauveria bassiana sensu lato were isolated, from 7 and 41 % of soil samples from a commercial banana field, with average fungal density of 4.3 × 103 and 8.2 × 103 CFU g−1 soil, respectively. Twenty-one morphologically distinct B. bassiana and four M. anisopliae sensu lato isolates from different plots within the field were further characterized. ISSR fingerprinting revealed six different clusters for B. bassiana, whereas gene sequencing revealed three M. anisopliae sensu stricto and one unclassified Metarhizium sp. Bioassays with one or more representative isolates from each Metarhizium species and B. bassiana cluster showed that all indigenous isolates had lower virulence and significantly greater ST50s than reference (exotic) isolates. The data suggest that the low virulence of most indigenous isolates toward Cosmopolites sordidus adults and their relatively low density in soil samples, may help explain the low occurrence of epizootics caused by entomopathogenic fungi in populations of this pest, also known to burrow under the soil surface in banana plantations.
The occurrence of deuteromycetous entomopathogenic fungi was examined in 266 soil samples representing 86 locations across temperate and near northern habitats in Ontario, Canada. Entomopathogenic fungi were isolated by baiting the soil with waxworm larvae, Galleria mellonella L., and incubating at 8, 15, or 25°C. Entomopathogenic fungi were isolated from 91% of the locations sampled across Ontario. The most abundant species were Metarhizium anisopliae (Metschn.) Sorok. (357 isolates) and Beauveria bassiana (Bals.) Vuill. (187 isolates). Thirteen isolates of Paecilomyces spp. were also found. Beauveria bassiana was isolated more frequently in soils from near northern locations, relative to M. anisopliae. Beauveria bassiana was isolated more frequently from larvae baited in soils incubated at 8 and 15°C, while M. anisopliae was isolated most frequently at 25°C. Thus, B. bassiana is more psychrophilic than M. anisopliae. From 47 of the locations in a temperate area (southern Ontario and the Kawartha Lakes region), two sites, one from an agricultural habitat and one from a natural habitat, were sampled within 1 km of each other. In these locations, B. bassiana was predominantly recovered more often from soils of natural habitats, while M. anisopliae was recovered more often in agricultural habitats. The occurrence of M. anisopliae and B. bassiana was not related to soil type or pH.Key words: Metarhizium, Beauveria, entomopathogenic fungi, fungal population biology, soil ecology.
Metarhizium spp. are insect pathogenic fungal species, which are widely used as biological control agents for pest insects. The natural occurrence of Metarhizium spp. and their potential for natural regulation of pest insects have obtained more and more attention because of an increasing demand for sustainable agriculture. Therefore, the interest on management strategies enhancing the natural occurrence of Metarhizium spp. and consequently the success of conservation biological control has been growing. Such strategies include habitat management as well as establishment of semi-natural habitat types, which may provide a refuge for Metarhizium spp. and may allow for recolonization of adjacent arable fields. The present study provides an overview of the densities of Metarhizium clade 1 consisting of M. majus, M. guizhouense, M. pinghaense, M. anisopliae, M. robertsii, and M. brunneum in different habitat types at field, local, and regional scales in Swiss agricultural landscapes. Soil samples were analyzed for Metarhizium clade 1 densities with a cultivation-independent qPCR-based detection and quantification tool. Based on this tool, 19.3% of 176 collected soil samples were tested positive for Metarhizium clade 1. The densities of Metarhizium clade 1 were heterogeneously distributed among the soil samples and differed significantly among sampled areas as well as sites. Despite this heterogeneity significant habitat-specific associations were observed. High Metarhizium clade 1 densities were found in soil samples collected from low-input permanent grassland and improved field margins indicating that both semi-natural habitat types may provide potential refuges for Metarhizium clade 1 species. Based on these results, more detailed analyses can be performed in permanent grassland and improved field margins to identify factors, which may promote the natural occurrence of Metarhizium clade 1 species and improve their function as natural regulator of pest insect populations. (C) 2011 Elsevier B.V. All rights reserved.
In the present study, the occurrence and species diversity of insect-associated fungi in soil collected mainly from forest habitats in different regions of China were compared by using the ‘Galleria biat method’. Insect-associated fungi were defined to include known insect pathogenic fungi, opportunistic pathogens and secondary colonizers isolated from the Galleria mellonella bait insect exposed to the soil samples in question. Insect-associated fungi were detected in 55.5% of the 425 soil samples. A total of 377 fungi belonging to 46 species and 27 genera were isolated and identified. Among them, 6 species were known insect pathogenic fungi, 21 were opportunistic pathogens and 19 were secondary colonizers. Insect pathogenic fungi were most prevalent and Paecilomyces farinosus, Beauveria bassiana and Metarhizium anisopliae var. anisopliae (Hyphomycetes) were the most common species, comprising 19.6%, 14.1% and 10.6% of the total number of isolates, respectively. Opportunistic pathogens also had high occurrences in the soil with the percentage frequency added up to 36.9%. Among the opportunistic fungi, Fusarium oxysporum, F. solani, Geomyces pannorum, Clonostachys rosea f. catenulata and an unidentified Fusarium sp. resulted in the highest G. mellonella mortality in the preliminary pathogenicity test. Using principal component analysis, two components accounted for 76.5% of the total variance were extracted. Component 1 was positively correlated with species richness and species diversity and negatively correlated with the average altitude of the sampling region. Component 2 was negatively correlated with species evenness and positively correlated to the level of insect pathogenic fungi. The two-axis ordination of communities showed clear separation of the fungal community in South Central China, indicating higher occurrence of insect-associated fungi in the soil of subtropical humid region than the other regions.