Lori M. Carris

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).

Karnal bunt of wheat newly reported from the African continent.

In December 2000 seed harvested from wheat (Triticum aestivum L.) cultivars SST 876 and SST 825 produced under sprinkler irrigation near Douglas, Northern Cape Province, South Africa, contained a substantial amount of partially bunted kernels. Kernel embryos contained black masses of teliospores, and in many instances the endosperm was partially degraded. Teliospores were brown to dark brown, densely echinulate, 25 to 45 μm in diameter with a short mycelial fragment on some of the spores. Hyaline, smooth-walled sterile cells were also present. Teliospores were soaked in sterile distilled water for 2 days, streaked on 2% water agar plates and incubated at 22°C in the dark. Teliospores germinated after 5 days, producing 50 to 250 filiform, nonconjugating, primary basidiospores and forcibly discharged allantoid, secondary basidiospores. Based on kernel appearance, a rotten fish odor in infected grain, teliospore morphology, and germination characteristics, the pathogen was identified as Tilletia indica Mitra, the cause of Karnal bunt (1). This morphological identification was confirmed at the USDA-ARS Systematic Botany and Mycology Laboratory, Beltsville, MD. Molecular verification of 12 South African isolates was provided by the Foreign Disease-Weed Science Research Unit at Fort Detrick, MD, using real-time polymerase chain reaction with the Tin3/Tin10 T. indica-specific primer set (2). Four additional isolates were confirmed as T. indica using the same primer set as well as ITS rDNA sequencing at the Beltsville laboratory. Reference specimens were deposited at the National Fungal Collection in Pretoria, South Africa (PREM 57214), and at Beltsville (BPI 748170). At present, the mode of introduction of T. indica into South Africa, as well as its precise distribution, is not known. It appears, however, that the pathogen is restricted to the Douglas production area in the Northern Cape where quarantine measures have been taken to contain and possibly eradicate the disease. References: (1) L. A. Castlebury and L. M. Carris. Mycologia 91:121, 1999. (2) R. D. Frederick et al. Phytopathology 90:951, 2000.

Internal Transcribed Spacer Sequence-Based Phylogeny and Polymerase Chain Reaction-Restriction Fragment Length Polymorphism Differentiation of Tilletia walkeri and T. indica.

ABSTRACT A polymerase chain reaction-restriction fragment length polymorphism assay to distinguish Tilleita walkeri, a rye grass bunt fungus that occurs in the southeastern United States and Oregon, from T. indica, the Karnal bunt fungus, is described. The internal transcribed spacer (ITS) region of the ribosomal DNA repeat unit was amplified and sequenced for isolates of T. indica, T. walkeri, T. horrida, and a number of other taxa in the genus Tilletia. A unique restriction digest site in the ITS1 region of T. walkeri was identified that distinguishes it from the other taxa in the genus. Phylogenetic analysis of the taxa based on ITS sequence data revealed a close relationship between T. indica and T. walkeri, but more distant relationships between these two species and other morphologically similar taxa.

Genetic variability among isolates of Tilletia barclayana, T. indica and allied species.

Tilletia barclayana, T. indica, T. rugispora and T. boutelouae are a group of floret-infecting smuts characterized by densely tuberculate teliospores. A morphologically similar smut infecting Lolium mul- tiflorum was discovered in Oregon and the south- eastern USA in 1996, although herbarium records show a similar smut on L. perenne has been present in Australia for at least thirty years. Based on PCR- RFLP analysis of the rDNA and RAPD analysis, the U.S. ryegrass bunt is most similar to T. indica, but the relatively low similarity (ca 25%) in the RAPD analysis does not support conspecificity of the two taxa. Both RAPD and PCR-RFLP analyses suggest that T barclayana comprises at least two distinct taxa. One cluster corresponds to the rice-infecting isolates, and the other to isolates infecting species of Panicum and Paspalum. Tilletia boutelouae and T. rugispora are sup- ported as species distinct from T barclayana and T indica based on both RAPD and PCR-RFLP analyses.

Molecular relationships among varieties of the Tilletia fusca ( T. bromi ) complex and related species

Twenty-six isolates representing six morphologically similar taxa of Tilletia occurring in the western U.S.A. were compared using randomly amplified polymorphic DNA (RAPD) markers and restriction fragment length polymorphisms (RFLP) of the 5·8 s rDNA and flanking ITS regions. Two distinct clusters were separated in the dendrogram based on the RAPD analysis. One cluster contained isolates of T. controversa, Apera interrupta-infecting isolates of a potentially new species of Tilletia , and isolates of the Bromus -infecting varieties bromi-tectorum and guyotiana of the T. fusca (= T. bromi ) complex. The second cluster contained Vulpia -infecting isolates of T. fusca var. fusca . The separation of the Bromus -infecting varieties from the Vulpia -infecting variety of T. fusca was supported by distinct restriction digest phenotypes. The RAPD analysis was more sensitive than the RFLP analysis, and allowed separation of the Bromus -infecting varieties bromi-tectorum and guyotiana corresponding to host specificity shown in previous studies. The presence of genetically distinct populations of T. fusca var. fusca occurring on V. microstachys and V. octoflora is indicated by RAPD and RFLP analyses. Species status of T. togwatii , a bunt infecting Poa reflexa that was previously removed from the T. fusca complex, is also supported by RAPD and RFLP analyses.

Genetic variation among natural populations of Tilletia controversa and T. bromi.

ABSTRACT Isolates of Tilletia controversa and T. bromi were sampled from wheat and two Bromus species hosts, respectively, in the Pacific Northwest, and genetic variation within and among populations was determined. Fifty-one random amplified polymorphic DNA markers from eleven primers were treated as phenotypic 1 and 0 character state data to estimate similarities and analyze molecular variance (AMOVA) among populations and as putative genetic loci to carry out analyses of gene diversity. Phenotypic analysis of T. controversa and T. bromi isolates revealed two distinct clusters that were 37% similar. The T. bromi cluster was subdivided further into two groups, corresponding to host, with 40% similarity. Cluster analysis based on allele frequencies produced similar results and also supported two T. bromi groups based on host. No evidence of natural hybridization and introgression was detected between the T. controversa and T. bromi populations. Both AMOVA and gene diversity analyses detected moderate levels of differentiation among T. controversa populations, whereas T. bromi populations were highly differentiated. The level of genetic differentiation observed between the T. bromi populations on different Bromus species hosts supports the hypothesis that a high degree of host specificity exists in the wild grass-infecting smuts. We speculate that the higher level of genetic differentiation among the T. bromi populations compared with the T. controversa populations on wheat may be due to selection by a more genetically diverse host population.

Influence of Host Resistance on the Genetic Structure of the White Pine Blister Rust Fungus in the Western United States

Cronartium ribicola, the causal agent of white pine blister rust, has been devastating to five-needled white pines in North America since its introduction nearly a century ago. However, dynamic and complex interactions occur among C. ribicola, five-needled white pines, and the environment. To examine potential evolutionary influences on genetic structure and diversity of C. ribicola in western United States, population genetic analyses of C. ribicola were conducted using amplified fragment length polymorphism (AFLP) molecular markers. The fungus was sampled at six sites. Collections for two of the six sites were from separate plantings of resistant-selected western white pine and sugar pine. Heterozygosity based on polymorphic loci among populations ranged from 0.28 to 0.40, with resistant-selected plantations at the extremes. Genetic differentiation was also highest between these two populations. Principal coordinates analysis and Bayesian assignment placed most isolates that are putative carriers of virulence to major-gene resistance into a discernable cluster, while other isolates showed no clustering by site or host species. These results indicate that C. ribicola in western North America is not genetically uniform, despite its presumed single site of introduction and relatively brief residence. Moreover, major-gene resistance appears to have imposed strong selection on the rust, resulting in reduced genetic diversity. In contrast, no evidence of selection was observed in C. ribicola from hosts that exhibit only multigenic resistance.

Characterization of interspecific hybrids between Tilletia controversa and T. bromi.

Tilletia controversa was hybridized with T bromi and T. laevis in wheat plants under greenhouse conditions. Teliospore morphology, germination rate and segregation of RAPD markers confirmed the identity of the F1 progeny as interspecific hybrids. The T. controversa X T. bromi hybrids exhibited reduced basidiospore fusion and pathogenicity, and a high rate of basidiospore mortality relative to the parental lines and T. controversa X T. laevis hybrids. Distortion observed in the segregation of RAPD markers in T. controversa X T. bromi hybrids, but not in T controversa X T. laevis hybrids, may be due to greater overall differences between the genomes of T. controversa and T bromi relative to T controversa and T. laevis. Tilletia controversa has limited sexual compatibility with T bromi but their hybrids are not likely to survive under natural conditions.

Morphological and molecular characterization of Colletotrichum nymphaeae and C. nupharicola sp. nov. on water-lilies (Nymphaea and Nuphar)

Nine isolates of a Colletotrichum sp. collected in North America on yellow water-lily (Nuphar luteum subsp. polysepalum) at five locations in the Pacific Northwest (PNW) and one isolate from Nymphaea odorata in Rhode Island were compared to three isolates of C. nymphaeae occurring on Nymphaea alba and Nuphar luteum in Europe. The appressoria of all North American isolates were significantly wider than those of C. nymphaeae. Conidia of the nine PNW isolates were significantly wider than those of both the Rhode Island isolate and C. nymphaeae isolates. All isolates were compared using random amplified polymorphic DNA (RAPD) and restriction fragment length polymorphism of the ITS region (RFLP-ITS). Two distinct clusters were differentiated in the dendrogram based on the RAPD analysis. The ten North American isolates formed one cluster and the European isolates of C. nymphaeae formed a second cluster. The separation of the North American isolates from the European isolates was supported by distinct restriction digest phenotypes of the ITS region. Based on morphological and molecular characterization the North American fungus is proposed as a new species, C. nupharicola.

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.