Soil Microbial Communities in Diverse Agroecosystems Exposed to the Herbicide Glyphosate.

Despite glyphosate's wide use for weed control in agriculture, questions remain about the herbicide's effect on soil microbial communities. The existing scientific literature contains conflicting results, from no observable effect of glyphosate to the enrichment of agricultural pathogens such as Fusarium spp. We conducted a comprehensive field-based study to compare the microbial communities on the roots of plants that received a foliar application of glyphosate to adjacent plants that did not. The 2-year study was conducted in Beltsville, MD, and Stoneville, MS, with corn and soybean crops grown in a variety of organic and conventional farming systems. By sequencing environmental metabarcode amplicons, the prokaryotic and fungal communities were described, along with chemical and physical properties of the soil. Sections of corn and soybean roots were plated to screen for the presence of plant pathogens. Geography, farming system, and season were significant factors determining the composition of fungal and prokaryotic communities. Plots treated with glyphosate did not differ from untreated plots in overall microbial community composition after controlling for other factors. We did not detect an effect of glyphosate treatment on the relative abundance of organisms such as Fusarium spp.IMPORTANCE Increasing the efficiency of food production systems while reducing negative environmental effects remains a key societal challenge to successfully meet the needs of a growing global population. The herbicide glyphosate has become a nearly ubiquitous component of agricultural production across the globe, enabling an increasing adoption of no-till agriculture. Despite this widespread use, there remains considerable debate on the consequences of glyphosate exposure. In this paper, we examine the effect of glyphosate on soil microbial communities associated with the roots of glyphosate-resistant crops. Using metabarcoding techniques, we evaluated prokaryotic and fungal communities from agricultural soil samples (n = 768). No effects of glyphosate were found on soil microbial communities associated with glyphosate-resistant corn and soybean varieties across diverse farming systems.

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.

Evidence for convergent evolution of host parasitic manipulation in response to environmental conditions.

Environmental conditions exert strong selection on animal behavior. We tested the hypothesis that the altered behavior of hosts due to parasitic manipulation is also subject to selection imposed by changes in environmental conditions over time. Our model system is ants manipulated by parasitic fungi to bite onto vegetation. We analyzed the correlation between forest type (tropical vs. temperate) and the substrate where the host bites (biting substrate: leaf vs. twigs), the time required for the fungi to reach reproductive maturity, and the phylogenetic relationship among specimens from tropical and temperate forests from different parts of the globe. We show that fungal development in temperate forests is longer than the period of time leaves are present and the ants are manipulated to bite twigs. When biting twigs, 90% of the dead ants we examined had their legs wrapped around twigs, which appears to provide better attachment to the plant. Ancestral state character reconstruction suggests that leaf biting is the ancestral trait and that twig biting is a convergent trait in temperate regions of the globe. These three lines of evidence suggest that changes in environmental conditions have shaped the manipulative behavior of the host by its parasite.

Characterization of soil nematode communities in three cropping systems through morphological and DNA metabarcoding approaches.

We used complementary morphological and DNA metabarcoding approaches to characterize soil nematode communities in three cropping systems, conventional till (CT), no-till (NT) and organic (ORG), from a long-term field experiment. We hypothesized that organic inputs to the ORG system would promote a more abundant nematode community, and that the NT system would show a more structured trophic system (higher Bongers MI) than CT due to decreased soil disturbance. The abundance of Tylenchidae and Cephalobidae both showed positive correlations to soil organic carbon and nitrogen, which were highest in the ORG system. The density of omnivore-predator and bacterial-feeding nematodes was reduced in NT soils compared to CT, while some plant-parasitic taxa increased. NT soils had similar Bongers MI values to CT, suggesting they contained nematode communities associated with soils experiencing comparable levels of disturbance. Metabarcoding revealed within-family differences in nematode diversity. Shannon and Simpson's index values for the Tylenchidae and Rhabditidae were higher in the ORG system than CT. Compared to morphological analysis, metabarcoding over- or underestimated the prevalence of several nematode families and detected some families not observed based on morphology. Discrepancies between the techniques require further investigation to establish the accuracy of metabarcoding for characterization of soil nematode communities.

A phylogenetically-based nomenclature for Cordycipitaceae (Hypocreales).

The ending of dual nomenclatural systems for pleomorphic fungi in 2011 requires the reconciliation of competing names, ideally linked through culture based or molecular methods. The phylogenetic systematics of Hypocreales and its many genera have received extensive study in the last two decades, however resolution of competing names in Cordycipitaceae has not yet been addressed. Here we present a molecular phylogenetic investigation of Cordycipitaceae that enables identification of competing names in this family, and provides the basis upon which these names can be maintained or suppressed. The taxonomy presented here seeks to harmonize competing names by principles of priority, recognition of monophyletic groups, and the practical usage of affected taxa. In total, we propose maintaining nine generic names, Akanthomyces, Ascopolyporus, Beauveria, Cordyceps, Engyodontium, Gibellula, Hyperdermium, Parengyodontium, and Simplicillium and the rejection of eight generic names, Evlachovaea, Granulomanus, Isaria, Lecanicillium, Microhilum, Phytocordyceps, Synsterigmatocystis, and Torrubiella. Two new generic names, Hevansia and Blackwellomyces, and a new species, Beauveria blattidicola, are described. New combinations are also proposed in the genera Akanthomyces, Beauveria, Blackwellomyces, and Hevansia.

Species limits, phylogeography and reproductive mode in the Metarhizium anisopliae complex.

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.

Managing the plant microbiome for biocontrol fungi: examples from Hypocreales.

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.

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.

Five new species of entomopathogenic fungi from the Amazon and evolution of neotropical Ophiocordyceps.

The neotropical biogeographic zone is a 'hot spot' of global biodiversity, especially for insects. Fungal pathogens of insects appear to track this diversity. However, the integration of this unique component of fungal diversity into molecular phylogenetic analyses remains sparse. The entomopathogenic fungal genus Ophiocordyceps is species rich in this region with the first descriptions dating to the early nineteenth century. In this study, material from various ecosystems throughout Colombia and Ecuador was examined. Molecular phylogenetic analyses of five nuclear loci including SSU, LSU, TEF, RPB1, and RPB2 were conducted alongside a morphological evaluation. Thirty-five specimens were examined representing fifteen different species of Ophiocordyceps, and five new species, Ophiocordyceps blattarioides, Ophiocordyceps tiputini, Ophiocordyceps araracuarensis, Ophiocordyceps fulgoromorphila, and Ophiocordyceps evansii, were described. An accurate identification of the host allowed us to conclude that host identity and host habitat are positively correlated with phylogenetic species of Ophiocordyceps and are probably strong drivers for speciation of neotropical entomopathogenic fungi.

Multilocus genotyping of Amylostereum spp. associated with Sirex noctilio and other woodwasps from Europe reveal clonal lineage introduced to the US.

Sirex noctilio is a woodwasp of Eurasian origin that was inadvertently introduced to the southern hemisphere in the 1900s and to North America over a decade ago. Its larvae bore in Pinus spp. and can cause significant mortality in pine plantations. S noctilio is associated with a symbiotic white rot fungus, Amylostereum areolatum, which females inject into trees when they oviposit and which is required for survival of developing larvae. We compared the genetic diversity of A. areolatum isolated from S. noctilio and other woodwasps collected from Europe and from northeastern North America to determine the origin of introduction(s) into the United States. Multilocus genotyping of nuclear ribosomal regions and protein coding genes revealed two widespread multilocus genotypes (MLGs) among the European samples, one of which is present in the US. The other two MLGs associated with S. noctilio in the US represented unique haplotypes. These latter two haplotypes were likely from unrepresented source populations, and together with the introduced widespread haplotype reveal multiple A. areolatum MLGs introduced by S. noctilio and indicate possible multiple S. noctilio introductions to North America from Europe. Our results also showed a lack of fidelity between woodwasp hosts and Amylostereum species.

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

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

Phylogeny of Hirsutella species (Ophiocordycipitaceae) from the USA: remedying the paucity of Hirsutella sequence data.

Hirsutella (Ophiocordycipitaceae: Hypocreales) is a genus of insect, mite, and nematode pathogens with an asexual morph, which generally produce a mucilaginous cluster of one or several conidia on phialides that are basally subulate and taper to a fine neck. The generic name Hirsutella has been proposed for suppression in favour of Ophiocordyceps as a consequence of the ending of dual nomenclature for different morphs of pleomorphic fungi in 2011. Though the generic name is well established, geographically dispersed, and speciose, exceptionally few sequences are available in online databases. We examined 46 isolates of 23 Hirsutella species from the USA, curated by the USDA-ARS Collection of Entomopathogenic Fungal Cultures (ARSEF Culture Collection), that previously had not been molecularly characterized and produced a phylogeny of these organisms; we included previously published Hirsutella and Ophiocordyceps taxa. In producing the largest phylogeny of Hirsutella isolates so far, we provide: (1) context for discussing previously-hypothesized relationships; (2) evidence for revisions as taxonomic transitions move forward; and (3) available molecular data to be incorporated into further evolutionary studies of Ophiocordycipitaceae.

New 1F1N Species Combinations in Ophiocordycipitaceae (Hypocreales).

Based on the taxonomic and nomenclatural recommendations of Quandt et al. (2014) new species combinations are made for Ophiocordycipitaceae. These new combinations are compliant with recent changes in the International Code of Nomenclature for algae, fungi, and plants (ICN) and the abolition of the dual system of nomenclature for fungi. These changes include 10 new combinations into Drechmeria, four new combinations into Harposporium, 23 new combinations and 15 synonymies in Ophiocordyceps, and one new combination into Purpureocillium.

Phylogenetic-based nomenclatural proposals for Ophiocordycipitaceae (Hypocreales) with new combinations in Tolypocladium.

Ophiocordycipitaceae is a diverse family comprising ecologically, economically, medicinally, and culturally important fungi. The family was recognized due to the polyphyly of the genus Cordyceps and the broad diversity of the mostly arthropod-pathogenic lineages of Hypocreales. The other two cordyceps-like families, Cordycipitaceae and Clavicipitaceae, will be revised taxonomically elsewhere. Historically, many species were placed in Cordyceps, but other genera have been described in this family as well, including several based on anamorphic features. Currently there are 24 generic names in use across both asexual and sexual life stages for species of Ophiocordycipitaceae. To reflect changes in Art. 59 in the International Code of Nomenclature for algae, fungi, and plants (ICN), we propose to protect and to suppress names within Ophiocordycipitaceae, and to present taxonomic revisions in the genus Tolypocladium, based on rigorous and extensively sampled molecular phylogenetic analyses. When approaching this task, we considered the principles of priority, monophyly, minimizing taxonomic revisions, and the practical utility of these fungi within the wider biological research community.

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

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

Phylogenetic analysis of Deladenus nematodes parasitizing northeastern North American Sirex species.

The parasitic nematode Deladenus siricidicola is a biological control agent of the invasive woodwasp, Sirex noctilio. Since the discovery of S. noctilio in pine forests of northeastern North America in 2005, a biological control program involving the Kamona strain of D. siricidicola has been under consideration. However, North American pine forests have indigenous Sirex spp. and likely harbor a unique assemblage of associated nematodes. We assessed phylogenetic relationships among native Deladenus spp. in the northeastern United States and the Kamona strain of D. siricidicola. We sequenced three genes (mtCO1, LSU, and ITS) from nematodes extracted from parasitized Sirex spp. collected inside and outside of the range of S. noctilio. Our analyses suggest cospeciation between four North American Sirex spp. and their associated nematode parasites. Within two S. noctilio individuals we found nematodes that we hypothesize are normally associated with Sirex nigricornis. One individual of the native S. nigricornis contained Deladenus normally associated with S. noctilio. We discuss nematode-host fidelity in this system and the potential for non-target impacts of a biological control program using D. siricidicola against S. noctilio.

Fidelity among Sirex woodwasps and their fungal symbionts.

We report that associations between mutualistic fungi and their economically and ecologically important woodwasp hosts are not always specific as was previously assumed. Woodwasps in the genus Sirex engage in obligate nutritional ectosymbioses with two species of Amylostereum, a homobasid\iomycete genus of white rot fungi. In the present study, the Amylostereum species and genotypes associated with three species of Sirex native to eastern North America and one relatively recent invasive Sirex from Europe were investigated by comparing intergenic spacer regions (IGS). Sirex spp. were sampled over 6 years from 23 sites in six US states, ranging from Maine in the northeast to Louisiana in the southeast, to obtain samples of Amylostereum from mycangia of adult females. Two of the native Sirex species (Sirex nigricornis and Sirex nitidus) were associated with either Amylostereum chailletii or Amylostereum areolatum, refuting the hypothesis of strict species-specific relationships. However, the invasive Sirex noctilio and the native Sirex cyaneus were each collected with only A. areolatum or A. chailletii, respectively, although S. noctilio was associated with two different IGS genotypes of A. areolatum and S. cyaneus occurs sympatrically with the other native Sirex. In Pinus, the preferred host tree of S. nigricornis and S. noctilio, these species co-occurred in 25.9 % of trees sampled, and horizontal transmission of fungal strains from S. noctilio to S. nigricornis was documented, although only in one tree. The extent that further spread and establishment of S. noctilio will alter the composition of symbionts carried by native Sirex is unknown but will depend in part on the degree of flexibility in these host-symbiont associations.

Host jumping onto close relatives and across kingdoms by Tyrannicordyceps (Clavicipitaceae) gen. nov. and Ustilaginoidea_(Clavicipitaceae).

PREMISE OF STUDY: This research seeks to advance understanding of conditions allowing movement of fungal pathogens among hosts. The family Clavicipitaceae contains fungal pathogens exploiting hosts across three kingdoms of life in a pattern that features multiple interkingdom host shifts among plants, animals, and fungi. The tribe Ustilaginoideae potentially represents a third origin of plant pathogenesis, although these species remain understudied. Fungal pathogens that cause ergot are linked morphologically with Clavicipitaceae, but are not yet included in phylogenetic studies. The placement of Ustilaginoideae and ergot pathogens will allow differentiation between the host habitat and host relatedness hypotheses as mechanisms of phylogenetic diversification of Clavicipitaceae. METHODS: A multigene data set was assembled for Clavicipitaceae to test phylogenetic placement and ancestral character-state reconstructions for Ustilaginoidea virens and U. dichromonae as well as the ergot mycoparasite Cordyceps fratricida. Microscopic morphological observations of sexual and asexual states were also performed. KEY RESULTS: Phylogenetic placement of U. virens and U. dichromonae represents a third acquisition of the plant pathogenic lifestyle in Clavicipitaceae. Cordyceps fratricida was also placed in Clavicipitaceae and recognized as a new genus Tyrannicordyceps. Ancestral character state reconstructions indicate initially infecting hemipteran insect hosts facilitates subsequent changes to a plant pathogenic lifestyle. The ancestor of T. fratricida is inferred to have jumped from grasses to pathogens of grasses. CONCLUSIONS: The host habitat hypothesis best explains the dynamic evolution of host affiliations seen in Clavicipitaceae and throughout Hypocreales. Co-occurrence in the same habitat has allowed for host shifts from animals to plants, and from plants to fungi.

Systematics and evolution of the genus Torrubiella (Hypocreales, Ascomycota).

Torrubiella is a genus of arthropod-pathogenic fungi that primarily attacks spiders and scale insects. Based on the morphology of the perithecia, asci, and ascospores, it is classified in Clavicipitaceae s. lat. (Hypocreales), and is considered a close relative of Cordyceps s. 1., which was recently reclassified into three families (Clavicipitaceae s. str., Cordycipitaceae, Ophiocordycipitaceae) and four genera (Cordyceps s. str, Elaphocordyceps, Metacordyceps, and Ophiocordyceps). Torrubiella is distinguished morphologically from Cordyceps s. lat. mainly by the production of superficial perithecia and the absence of a well-developed stipitate stroma. To test and refine evolutionary hypotheses regarding the placement of Torrubiella and its relationship to Cordyceps s. lat., a multi-gene phylogeny was constructed by conducting ML and Bayesian analyses. The monophyly of Torrubiella was rejected by these analyses with species of the genus present in Clavicipitaceae, Cordycipitaceae, and Ophiocordycipitaceae, and often intermixed among species of Cordyceps s. lat. The morphological characters traditionally used to define the genus are, therefore, not phylogenetically informative, with the stipitate stromata being gained and/or lost several times among clavicipitaceous fungi. Two new genera (Conoideocrella, Orbiocrella) are proposed to accommodate two separate lineages of torrubielloid fungi in the Clavicipitaceae s. str. In addition, one species is reclassified in Cordyceps s. str. and three are reclassified in Ophiocordyceps. The phylogenetic importance of anamorphic genera, host affiliation, and stipitate stromata is discussed.