Untargeted Metabolomic Investigation of Wheat Infected with Stinking Smut Tilletia caries.

Tilletia caries infection of wheat (Triticum aestivum) has become an increasing problem in organic wheat agriculture throughout the world. Little is known about how this pathogen alters host metabolism to ensure a successful infection. We investigated how T. caries allocates resources from wheat for its growth over the life cycle of the pathogen. An untargeted metabolomics approach that combined gas chromatography time-of-flight mass spectrometry and ultraperformance liquid chromatography tandem mass spectrometry platforms was used to determine which primary or specialized metabolite pathways are targeted and altered during T. caries infection. We found that T. caries does not dramatically alter the global metabolome of wheat but instead alters key metabolites for its own nutrient uptake and to antagonize host defenses by reducing wheat's sweet immunity response and other related pathways. Our results highlight metabolic characteristics needed for selecting wheat varieties that are resistant to T. caries infection for organic agriculture. In addition, several wheat metabolites were identified that could be used in developing a diagnostic tool for early detection of T. caries infection.

Directed evolution of Metarhizium fungus improves its biocontrol efficacy against Varroa mites in honey bee colonies.

Entomopathogenic fungi show great promise as pesticides in terms of their relatively high target specificity, low non-target toxicity, and low residual effects in agricultural fields and the environment. However, they also frequently have characteristics that limit their use, especially concerning tolerances to temperature, ultraviolet radiation, or other abiotic factors. The devastating ectoparasite of honey bees, Varroa destructor, is susceptible to entomopathogenic fungi, but the relatively warm temperatures inside honey bee hives have prevented these fungi from becoming effective control measures. Using a combination of traditional selection and directed evolution techniques developed for this system, new strains of Metarhizium brunneum were created that survived, germinated, and grew better at bee hive temperatures (35 °C). Field tests with full-sized honey bee colonies confirmed that the new strain JH1078 is more virulent against Varroa mites and controls the pest comparable to current treatments. These results indicate that entomopathogenic fungi are evolutionarily labile and capable of playing a larger role in modern pest management practices.

Extracts of Polypore Mushroom Mycelia Reduce Viruses in Honey Bees.

Waves of highly infectious viruses sweeping through global honey bee populations have contributed to recent declines in honey bee health. Bees have been observed foraging on mushroom mycelium, suggesting that they may be deriving medicinal or nutritional value from fungi. Fungi are known to produce a wide array of chemicals with antimicrobial activity, including compounds active against bacteria, other fungi, or viruses. We tested extracts from the mycelium of multiple polypore fungal species known to have antiviral properties. Extracts from amadou (Fomes) and reishi (Ganoderma) fungi reduced the levels of honey bee deformed wing virus (DWV) and Lake Sinai virus (LSV) in a dose-dependent manner. In field trials, colonies fed Ganoderma resinaceum extract exhibited a 79-fold reduction in DWV and a 45,000-fold reduction in LSV compared to control colonies. These findings indicate honey bees may gain health benefits from fungi and their antimicrobial compounds.

The Genera of Fungi - fixing the application of the type species of generic names - G 2: Allantophomopsis, Latorua, Macrodiplodiopsis, Macrohilum, Milospium, Protostegia, Pyricularia, Robillarda, Rotula, Septoriella, Torula, and Wojnowicia.

The present paper represents the second contribution in the Genera of Fungi series, linking type species of fungal genera to their morphology and DNA sequence data, and where possible, ecology. This paper focuses on 12 genera of microfungi, 11 of which the type species are neo- or epitypified here: Allantophomopsis (A. cytisporea, Phacidiaceae, Phacidiales, Leotiomycetes), Latorua gen. nov. (Latorua caligans, Latoruaceae, Pleosporales, Dothideomycetes), Macrodiplodiopsis (M. desmazieri, Macrodiplodiopsidaceae, Pleosporales, Dothideomycetes), Macrohilum (M. eucalypti, Macrohilaceae, Diaporthales, Sordariomycetes), Milospium (M. graphideorum, incertae sedis, Pezizomycotina), Protostegia (P. eucleae, Mycosphaerellaceae, Capnodiales, Dothideomycetes), Pyricularia (P. grisea, Pyriculariaceae, Magnaporthales, Sordariomycetes), Robillarda (R. sessilis, Robillardaceae, Xylariales, Sordariomycetes), Rutola (R. graminis, incertae sedis, Pleosporales, Dothideomycetes), Septoriella (S. phragmitis, Phaeosphaeriaceae, Pleosporales, Dothideomycetes), Torula (T. herbarum, Torulaceae, Pleosporales, Dothideomycetes) and Wojnowicia (syn. of Septoriella, S. hirta, Phaeosphaeriaceae, Pleosporales, Dothideomycetes). Novel species include Latorua grootfonteinensis, Robillarda africana, R. roystoneae, R. terrae, Torula ficus, T. hollandica, and T. masonii spp. nov., and three new families: Macrodiplodiopsisceae, Macrohilaceae, and Robillardaceae. Authors interested in contributing accounts of individual genera to larger multi-authored papers to be published in IMA Fungus, should contact the associate editors listed for the major groups of fungi on the List of Protected Generic Names for Fungi (www.generaoffungi.org).

Mitospore stages of Disciotis, Gyromitra and Morchella in the inland Pacific Northwest USA.

Colonies of Costantinella species growing on soil, moss and woody debris in the autumn in the inland Pacific Northwest USA were established in culture. Five different mitospore taxa were distinguished based on colony color, presence or absence of setae and internal transcribed spacer region (ITS) rDNA amplicon size. Sequence data from the largest and second largest subunits of RNA polymerase II, translation elongation factor 1-α, D1 and D2 domains of nuclear large subunit rDNA and ITS were used to connect each of the distinct mitospore taxa to corresponding vernal-fruiting Pezizales, including Disciotis cf. venosa, Gyromitra cf. esculenta and three species of Morchella. Both meiospore and mitospore stages of Morchella brunnea and M. populiphila collected in spring and autumn within a meter of each other at two urban sites had identical multilocus haplotypes, providing evidence connecting the two stages of the life cycle. Among other Morchella mitospore stages collected, some had identical haplotypes to previously sampled meiospore stages, while others were distinct, possibly representing undescribed species. Mitospore isolates with sequences assigning them to Disciotis or Gyromitra had different haplotypes from meiospore stages occurring in the same area. Meiospore stages of Disciotis and Gyromitra sampled as part of the study were also genetically distinct from European collections of D. venosa and G. esculenta, indicating more diversity is present in these taxa than is reflected in the current taxonomy. The widespread occurrence of mitospore stages of these fungi suggests that the life cycles of morels, false morels and allied taxa are more complex than previously recognized.

Revision of Entyloma (Entylomatales, Exobasidiomycetes) on Eryngium.

The genus Entyloma consists of more than 160 species of smut fungi distributed worldwide on dicots, with Apiaceae being one of the main host families. This study aims to clarify the systematics and phylogeny of Entyloma on Eryngium (Apiaceae) with molecular and morphological data. Eleven species from Eryngium are discussed herein. Four of them are described as new taxa: E. carmeli sp. nov. on Eryngium falcatum, E. eryngii-cretici sp. nov. on Eryngium creticum, E. eryngii-maritimi sp. nov. on Eryngium maritimum and E. ho-chunkii sp. nov. on Eryngium yuccifolium. Analysis of the internal transcribed spacer (ITS) region of rDNA is presented and supports the polyphyly of Entyloma on Eryngium.

Development of sequence characterized amplified genomic regions (SCAR) for fungal systematics: proof of principle using Alternaria, Ascochyta and Tilletia.

SCARs were developed by cloning RAPD-PCR amplicons into commercially available vectors, sequencing them and designing specific primers for PCR, direct sequencing and phylogenetic analysis. Eighteen to seventy percent of cloned RAPD-PCR amplicons were phylogenetically informative among closely related small-spored Alternaria spp., Ascochyta spp. and Tilletia spp., taxa that have been resistant to phylogenetic analysis with universally primed, protein-coding sequence data. Selected SCARs were sequenced for larger, population-scale samples of each taxon and demonstrated to be useful for phylogenetic inference. Variation observed in the cloned SCARs generally was higher than variation in nuclear ribosomal internal transcribed spacer (ITS) and several protein-coding sequences commonly used in lower level fungal systematics. Sequence data derived from SCARs will provide sufficient resolution to address lower level phylogenetic hypotheses in Alternaria, Ascochyta, Tilletia and possibly many other fungal groups and organisms.

Development of molecular markers and preliminary investigation of the population structure and mating system in one lineage of black morel (Morchella elata) in the Pacific Northwestern USA.

Phylogenetic analysis of LSU/ITS sequence data revealed two distinct lineages among 44 morphologically similar fruiting bodies of natural black morels (Morchella elata group) sampled at three non-burn locations in the St Joe and Kanisku National Forests in northern Idaho. Most of the sampled isolates (n = 34) represented a dominant LSU/ITS haplotype present at all three sites and identical to the Mel-12 phylogenetic lineage (GU551425) identified in a previous study. Variation at 1-3 nucleotide sites was detected among a small number of isolates (n = 6) within this well supported clade (94%). Four isolates sampled from a single location were in a well supported clade (97%) distinct from the dominant haplotypes and may represent a previously un-sampled, cryptic phylogenetic species. Species-specific SNP and SCAR markers were developed for Mel-12 lineage isolates by cloning and sequencing AFLP amplicons, and segregation of AFLP markers were studied from single ascospore isolates from individual fruiting bodies. Based on the segregation of AFLP markers within single fruiting bodies, split decomposition analyses of two SCAR markers, and population genetic analyses of SNP, SCAR, and AFLP markers, it appears that members of the Morchella sp. Mel-12 phylogenetic lineage are heterothallic and outcross in nature similar to yellow morels. This is the first set of locus-specific molecular markers that has been developed for any Morchella species, to our knowledge. These markers will prove to be valuable tools to study mating system, gene flow and genetic structure of black morels at various spatial scales with field-collected fruiting bodies and eliminate the need to culture samples in vitro.

Tilletia puccinelliae, a new species of reticulate-spored bunt fungus infecting Puccinellia distans.

A shipment of Fults alkaligrass seed (Puccinellia distans) grown in Washington state containing bunted florets was intercepted by quarantine officials at China's Tianjin Entry-Exit Quarantine and Inspection Bureau. The bunted florets were filled with irregularly shaped, reticulately ornamented teliospores that germinated in a manner characteristic of systemically infecting Tilletia spp. on grass hosts in subfamily Pooideae. Based on morphological characters and a multigene phylogenetic analysis of the ITS region rDNA, eukaryotic translation elongation factor 1 alpha and a region of the second largest subunit of RNA polymerase II including a putative intein, the Puccinellia bunt is genetically distinct from known species of Tilletia and is proposed as a new species, T. puccinelliae.

Tilletia vankyi, a new species of reticulate-spored bunt fungus with non-conjugating basidiospores infecting species of Festuca and Lolium.

A bunt fungus, exhibiting a spore germination pattern unique to known reticulate-spored species of Tilletia was found infecting plants in seed production fields of Festuca rubra ssp. rubra (red fescue) and F. rubra ssp. fallax (Chewing's fescue) in Oregon, and in seed lots of Lolium perenne (perennial ryegrass) from Australia and Germany. Teliospores germinated to form 20-40 uninucleate, non-conjugating basidiospores, and colonies derived from single basidiospores produced teliospores in culture. In inoculation studies using single basidiospore colonies, perennial ryegrass and L. perenne ssp. multiflorum (Italian or annual ryegrass) were infected. A phylogenetic analysis, based on ITS region rDNA, eukaryotic translation elongation factor 1 alpha, and the second largest subunit of RNA polymerase II demonstrated that the fescue and ryegrass bunts are conspecific, and distinct from known species of Tilletia.

Nonsystemic bunt fungi--Tilletia indica and T. horrida: a review of history, systematics, and biology.

The genus Tilletia is a group of smut fungi that infects grasses either systemically or locally. Basic differences exist between the systemically infecting species, such as the common and dwarf bunt fungi, and locally infecting species. Tilletia indica, which causes Karnal bunt of wheat, and Tilletia horrida, which causes rice kernel smut, are two examples of locally infecting species on economically important crops. However, even species on noncultivated hosts can become important when occurring as contaminants in export grain and seed shipments. In this review, we focus on T. indica and the morphologically similar but distantly related T. horrida, considering history, systematics, and biology. In addition, the controversial generic placement and evolutionary relationships of these two species are discussed in light of recent molecular studies.

Phylogenetic analysis of Tilletia and allied genera in order Tilletiales (Ustilaginomycetes; Exobasidiomycetidae) based on large subunit nuclear rDNA sequences.

The order Tilletiales (Ustilaginomycetes, Basidiomycota) includes six genera (Conidiosporomyces, Erratomyces, Ingoldiomyces, Neovossia, Oberwinkleria and Tilletia) and approximately 150 species. All members of Tilletiales infect hosts in the grass family Poaceae with the exception of Erratomyces spp., which occur on hosts in the Fabaceae. Morphological features including teliospore ornamentation, number and nuclear condition of primary basidiospores and ability of primary basidiospores to conjugate and form an infective dikaryon were studied in conjunction with sequence analysis of the large subunit nuclear rDNA gene (nLSU). Analysis based on nLSU data shows that taxa infecting hosts in the grass subfamily Pooideae form one well supported lineage. This lineage comprises most of the reticulate-spored species that germinate to form a small number of rapidly conjugating basidiospores and includes the type species Tilletia tritici. Two tuberculate-spored species with a large number of nonconjugating basidiospores, T. indica and T. walkeri, and Ingoldiomyces hyalosporus are also included in this lineage. Most of the species included in the analysis with echinulate, verrucose or tuberculate teliospores that germinate to form a large number (>30) of nonconjugating basidiospores infect hosts in the subfamilies Panicoideae, Chloridoideae, Arundinoideae and Ehrhartoideae. This group of species is more diverse than the pooid-infecting taxa and in general do not form well supported clades corresponding to host subfamily. The results of this work suggest that morphological characters used to segregate Neovossia, Conidiosporomyces and Ingoldiomyces from Tilletia are not useful generic level characters and that all included species can be accommodated in the genus Tilletia.