Richard A. Humber

Reconstructing the early evolution of Fungi using a six-gene phylogeny

The ancestors of fungi are believed to be simple aquatic forms with flagellated spores, similar to members of the extant phylum Chytridiomycota (chytrids). Current classifications assume that chytrids form an early-diverging clade within the kingdom Fungi and imply a single loss of the spore flagellum, leading to the diversification of terrestrial fungi. Here we develop phylogenetic hypotheses for Fungi using data from six gene regions and nearly 200 species. Our results indicate that there may have been at least four independent losses of the flagellum in the kingdom Fungi. These losses of swimming spores coincided with the evolution of new mechanisms of spore dispersal, such as aerial dispersal in mycelial groups and polar tube eversion in the microsporidia (unicellular forms that lack mitochondria). The enigmatic microsporidia seem to be derived from an endoparasitic chytrid ancestor similar to Rozella allomycis, on the earliest diverging branch of the fungal phylogenetic tree.

A multilocus phylogeny of the Metarhizium anisopliae lineage

Metarhizium anisopliae, the type species of the anamorph entomopathogenic genus Metarhizium, is currently composed of four varieties, including the type variety, and had been demonstrated to be closely related to M. taii, M. pingshaense and M. guizhouense. In this study we evaluate phylogenetic relationships within the M. anisopliae complex, identify monophyletic lineages and clarify the species taxonomy. To this end we have employed a multigene phylogenetic approach using near-complete sequences from nuclear encoded EF-1α, RPB1, RPB2 and β-tubulin gene regions and evaluated the morphology of these taxa, including ex-type isolates whenever possible. The phylogenetic and in some cases morphological evidence supports the monophyly of nine terminal taxa in the M. anisopliae complex that we recognize as species. We propose to recognize at species rank M. anisopliae, M. guizhouense, M. pingshaense, M. acridum stat. nov., M. lepidiotae stat. nov. and M. majus stat. nov. In addition we describe the new species M. globosum and M. robertsii, resurrect the name M. brunneum and show that M. taii is a later synonym of M. guizhouense.

Phylogeny and systematics of the anamorphic, entomopathogenic genus Beauveria

Beauveria is a cosmopolitan anamorphic genus of arthropod pathogens that includes the agronomically important species, B. bassiana and B. brongniartii, which are used as mycoinsecticides for the biological control of pest insects. Recent phylogenetic evidence demonstrates that Beauveria is monophyletic within the Cordycipitaceae (Hypocreales), and both B. bassiana and B. brongniartii have been linked developmentally and phylogenetically to Cordyceps species. Despite recent interest in the genetic diversity and molecular ecology of Beauveria, particularly as it relates to their role as pathogens of insects in natural and agricultural environments, the genus has not received critical taxonomic review for several decades. A multilocus phylogeny of Beauveria based on partial sequences of RPB1, RPB2, TEF and the nuclear intergenic region, Bloc, is presented and used to assess diversity within the genus and to evaluate species concepts and their taxonomic status. B. bassiana and B. brongniartii, both which represent species complexes and which heretofore have lacked type specimens, are redescribed and types are proposed. In addition six new species are described including B. varroae and B. kipukae, which form a biphyletic, morphologically cryptic sister lineage to B. bassiana, B. pseudobassiana, which also is morphologically similar to but phylogenetically distant from B. bassiana, B. asiatica and B. australis, which are sister lineages to B. brongniartii, and B. sungii, an Asian species that is linked to an undetermined species of Cordyceps. The combination B. amorpha is validly published and an epitype is designated.

Evolution of entomopathogenicity in fungi

The recent completions of publications presenting the results of a comprehensive study on the fungal phylogeny and a new classification reflecting that phylogeny form a new basis to examine questions about the origins and evolutionary implications of such major habits among fungi as the use of living arthropods or other invertebrates as the main source of nutrients. Because entomopathogenicity appears to have arisen or, indeed, have lost multiple times in many independent lines of fungal evolution, some of the factors that might either define or enable entomopathogenicity are examined. The constant proximity of populations of potential new hosts seem to have been a factor encouraging the acquisition or loss of entomopathogenicity by a very diverse range of fungi, particularly when involving gregarious and immobile host populations of scales, aphids, and cicadas (all in Hemiptera). An underlying theme within the vast complex of pathogenic and parasitic ascomycetes in the Clavicipitaceae (Hypocreales) affecting plants and insects seems to be for interkingdom host-jumping by these fungi from plants to arthropods and then back to the plant or on to fungal hosts. Some genera of Entomophthorales suggest that the associations between fungal pathogens and their insect hosts appear to be shifting away from pathogenicity and towards nonlethal parasitism.

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.

Chapter V-1 – Fungi: Identification

Publisher Summary This chapter presents the basic skills and information needed to allow nonmycologists to identify the major genera and the most common species of fungal entomopathogens to the generic. Major species of fungal entomopathogens have basic diagnostic characters making them quickly identifiable. The identification of most entomopathogenic fungi necessarily depends on the observation of microscopic characters. Vegetative states of most fungi have little taxonomic value and are not characterized in the key. It is suggested that if no spores are seen in a collection, specimens should be incubated for a further time in room conditions of temperature, humidity and light. Outside the Entomophthorales, the only other significant associations with arthropods are found in the Trichomycetes, a diverse group of fungi that are mainly endocommensal in the guts of insects or crustaceans. Watermolds produce uni- or biflagellate zoospores that are both dispersive and infective units. Flagellate zoospores may be released from two possible sorts of sporangia, those with either thin or thick walls. It would be unusual to detect hosts infected by these fungi during their vegetative states, as these fungi are usually only detected when sporangia have been formed or are releasing zoospores.

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.

Survey of entomopathogenic nematodes and fungi endemic to pecan orchards of the Southeastern United States and their virulence to the pecan weevil (Coleoptera: Curculionidae).

Abstract The pecan weevil, Curculio caryae (Horn), is a major pest of pecans in the Southeastern United States. Entomopathogenic nematodes and fungi are potential alternatives to chemical insecticides for C. caryae control. Our objective was to survey pecan orchards in the southeastern United States for entomopathogenic nematodes and fungi and determine the virulence of the new isolates to C. caryae larvae. Soil was collected from 105 sites in 21 orchards in Arkansas, Georgia, Louisiana, and Mississippi. Entomopathogens were isolated by exposing soil to C. caryae and greater wax moth larvae, Galleria mellonella, (L.). We isolated entomopathogenic fungi and nematodes from 16 and 6 of the 21 orchards surveyed, respectively. The entomopathogenic fungi included Beauveria bassiana (Balsamo) Vuillemin and Metarhizium anisopliae (Metschnikoff) Sorokin, and nematodes included Heterorhabditis bacteriophora Poinar, Steinernema carpocapsae (Weiser), Steinernema glaseri (Steiner), and Steinernema rarum (Doucet). This is the first report of Steinernema rarum in the United States. Soil characteristics in orchards were analyzed for pH, organic matter, and nutrients; we detected a negative relationship between fungal occurrence and manganese levels in soil and a positive relationship between M. anisopliae occurrence and calcium or magnesium levels. In laboratory assays, virulence of 15 nematode and 22 fungal isolates to C. caryae larvae was tested in small plastic cups containing soil. Results indicated poor susceptibility of the C. caryae larvae to entomopathogenic nematodes. Several fungal isolates that caused significantly higher mortality in C. caryae larvae than other strains (including a commercial strain of B. bassiana) should be investigated further as potential control agents of C. caryae.

Chapter V-5 – Fungi: Preservation of cultures

Publisher Summary This chapter discusses preservation of microbial cultures. Storing fungi, at ambient temperatures rather than in refrigeration, may require fewer resources than any other preservation strategy. Storing culture, slants under a layer of sterile mineral oil, is one of the oldest, simplest and least expensive methods for long-term culture preservation. Cultures kept under mineral oil may remain viable for decades. Pathogenicity of entomopathogenic fungi may be undiminished after several months of storage. Storage of metabolically inactive fungi under sterile distilled water may be least technologically demanding of any preservation techniques. Lyophilization may be one of the most widely used technologically sophisticated approaches to preserve fungal germplasm and is the primary technique used in most general service culture collections. Standard domestic freezers might seem to be an ideal and economical tool for keeping frozen cultures. The use of anhydrous silica gel crystals as a carrier for culture propagules is limited to aerobic bacteria and fungi that grow on solid culture media. The choice of cryoprotectant determines the temperature, the heat of fusion, at which the cryoprotectant freezes with a strongly exothermic reaction. It is observed that the laboratory that relies on nitrogen storage facilities has made a long-term and expensive commitment to maintain cultures perceived as having a very high intrinsic value.

Metarhizium frigidum sp. nov.: a cryptic species of M. anisopliae and a member of the M. flavoviride complex

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.