Chemical, Bioactivity, and Biosynthetic Screening of Epiphytic Fungus Zasmidium pseudotsugae.

We report the first secondary metabolite, 8,8'-bijuglone, obtained from pure cultures of the slow growing Douglas fir- (Pseudotsuga menziesii var. menziesii) foliage-associated fungus Zasmidium pseudotsugae. The quinone was characterized using extensive LC/MS and NMR-based spectroscopic methods. 8,8'-Bijuglone exhibited moderate antibiotic activity against Gram-positive pathogens and weak cytotoxic activity in the NCI-60 cell line panel and in our in-house human colon carcinoma (HCT-116) cell line. An analysis of the fungal genome sequence to assess its metabolic potential was implemented using the bioinformatic tool antiSMASH. In total, 36 putative biosynthetic gene clusters were found with a majority encoding for polyketides (17), followed by non-ribosomal peptides (14), terpenes (2), ribosomal peptides (1), and compounds with mixed biosynthetic origin (2). This study demonstrates that foliage associated fungi of conifers produce antimicrobial metabolites and suggests this guild of fungi may present a rich source of novel molecules.

Environmental variables associated with Nothophaeocryptopus gaeumannii population structure and Swiss needle cast severity in Western Oregon and Washington.

The environment has a strong influence on the abundance and distribution of plant pathogenic organisms and plays a major role in plant disease. Climatological factors may also alter the dynamics of the interactions between plant pathogens and their hosts. Nothophaeocryptopus (=Phaeocryptopus) gaeumannii, the causal agent of Swiss needle cast (SNC) of Douglas-fir, is endemic to western North America where it exists as two sympatric, reproductively isolated lineages. The abundance of this fungus and the severity of SNC are strongly influenced by climate. We used statistical and population genetic analyses to examine relationships between environment, pathogen population structure, and SNC severity. Although N. gaeumannii Lineage 2 in western Oregon and Washington was most abundant where SNC symptoms were most severe, we did not detect a significant relationship between Lineage 2 and disease severity. Warmer winter temperatures were inversely correlated with foliage retention (AFR) and positively correlated with the relative abundance of Lineage 2 (PL2). However when distance inland, which was strongly correlated with both AFR and PL2, was included in the model, there was no significant relationship between Lineage 2 and AFR. Spring/early summer dew point temperatures also were positively associated with total N. gaeumannii abundance (colonization index (CI)) and inversely correlated with AFR. Warmer summer mean temperatures were associated with lower CI and higher AFR. Our results suggest that the two lineages have overlapping environmental optima, but slightly different tolerance ranges. Lineage 2 was absent from more inland sites where winters are colder and summers are warm and dry, while Lineage 1 occurred at most sites across an environmental gradient suggesting broader environmental tolerance. These relationships suggest that climate influences the abundance and distribution of this ecologically important plant pathogen and may have played a role in the evolutionary divergence of these two cryptic fungal lineages.

Culture-based identification to examine spatiotemporal patterns of fungal communities colonizing wood in ground contact.

Timber durability is often assessed using small wood stakes exposed in direct soil contact, and the assessment generally emphasizes effects on wood rather than organisms involved. Understanding fungal colonization patterns can help identify key decay agents under varying conditions and use these patterns to improve wood protection strategies. Fungal colonization of red alder (Alnus rubra), Douglas-fir (Pseudotsuga menziesii) heartwood/sapwood, and western redcedar (Thuja plicata) field stakes was assessed over 2 y in western Oregon. Spatiotemporal fungal community variations were identified via culturing and DNA sequencing, where 814 isolates were identified from 84 stakes. Forty-six ascomycete genera were identified, with Phialophora, Trichoderma, and Epicoccum species occurring most frequently. Twenty-three basidiomycete genera were identified, with Trametes and Phanerochaete being the most common. Douglas-fir and western redcedar stakes contained the highest and lowest diversity levels, respectively, reflecting natural durability differences of these species. Fungal species abundance was higher below ground than in the above ground and groundline zones, likely reflecting more stable moisture regimes, proximity to soil-based fungi, and potential nutrient migration into wood beneath the soil surface. Ascomycetes were proportionally more abundant early in the exposure period, but basidiomycetes were also observed early in the process, and there appeared to be no consistent colonization pattern.

Overlooked competing asexual and sexually typified generic names of Ascomycota with recommendations for their use or protection.

With the change to one scientific name for fungal species, numerous papers have been published with recommendations for use or protection of competing generic names in major groups of ascomycetes. Although genera in each group of fungi were carefully considered, some competing generic names were overlooked. This paper makes recommendations for additional competing genera not considered in previous papers. Chairs of relevant Working Groups of the ICTF were consulted in the development of these recommendations. A number of generic names need protection, specifically Amarenographium over Amarenomyces, Amniculicola over Anguillospora, Balansia over Ephelis, Claviceps over Sphacelia, Drepanopeziza over Gloeosporidiella and Gloeosporium, Golovinomyces over Euoidium, Holwaya over Crinium, Hypocrella over Aschersonia, Labridella over Griphosphaerioma, Metacapnodium over Antennularia, and Neonectria over Cylindrocarpon and Heliscus. The following new combinations are made: Amniculicola longissima, Atichia maunauluana, Diaporthe columnaris, D. liquidambaris, D. longiparaphysata, D. palmicola, D. tersa, Elsinoë bucidae, E.caricae, E. choisyae, E. paeoniae, E. psidii, E. zorniae, Eupelte shoemakeri, Godronia myrtilli, G. raduloides, Sarcinella mirabilis, S. pulchra, Schizothyrium jamaicense, and Trichothallus niger. Finally, one new species name, Diaporthe azadirachte, is introduced to validate an earlier name, and the conservation of Discula with a new type, D. destructiva, is recommended.

Recommendations on generic names competing for use in Leotiomycetes (Ascomycota).

In advancing to one scientific name for fungi, this paper treats genera competing for use in the phylogenetically defined class Leotiomycetes except for genera of Erysiphales. Two groups traditionally included in the so-called "inoperculate discomycetes" have been excluded from this class and are also not included here, specifically Geoglossomycetes and Orbiliomycetes. A recommendation is made about the generic name to use in cases in which two or more generic names are synonyms or taxonomically congruent along with the rationale for the recommendation. In some cases the recommended generic name does not have priority or is based on an asexual type species, thus needs to be protected and ultimately approved according to Art. 57.2 of the International Code of Nomenclature for algae, fungi and plants (ICN). A table is presented listing all competing generic names and their type species noting the recommended generic name. New combinations are introduced for the oldest epithet in the recommended genus including Ascocalyx berenice, Ascoconidium purpurascens, Ascocoryne albida, A. trichophora, Blumeriella filipendulae, B. ceanothi, Botrytis arachidis, B. fritillariae-pallidoflori, Calloria urticae, Calycellina aspera, Dematioscypha delicata, Dermea abietinum, D. boycei, D. stellata, Diplocarpon alpestre, D. fragariae, Godroniopsis peckii, Grovesinia moricola, Heterosphaera sublineolata, Hyphodiscus brachyconium, H. brevicollaris, H. luxurians, Leptotrochila campanulae, Monilinia polystroma, Neofabraea actinidae, N. citricarpa, N. vagabunda, Oculimacula aestiva, O. anguioides, Pezicula brunnea, P. californiae, P. cornina, P. diversispora, P. ericae, P. melanogena, P. querciphila, P. radicicola, P. rhizophila, Phialocephala piceae, Pilidium lythri, Rhabdocline laricis, Streptotinia streptothrix, Symphyosirinia parasitica, S. rosea, Unguiculariopsis caespitosa, and Vibrissea laxa.

The EDLL motif: a potent plant transcriptional activation domain from AP2/ERF transcription factors.

In plants, the ERF/EREBP family of transcriptional regulators plays a key role in adaptation to various biotic and abiotic stresses. These proteins contain a conserved AP2 DNA-binding domain and several uncharacterized motifs. Here, we describe a short motif, termed 'EDLL', that is present in AtERF98/TDR1 and other clade members from the same AP2 sub-family. We show that the EDLL motif, which has a unique arrangement of acidic amino acids and hydrophobic leucines, functions as a strong activation domain. The motif is transferable to other proteins, and is active at both proximal and distal positions of target promoters. As such, the EDLL motif is able to partly overcome the repression conferred by the AtHB2 transcription factor, which contains an ERF-associated amphiphilic repression (EAR) motif. We further examined the activation potential of EDLL by analysis of the regulation of flowering time by NF-Y (nuclear factor Y) proteins. Genetic evidence indicates that NF-Y protein complexes potentiate the action of CONSTANS in regulation of flowering in Arabidopsis; we show that the transcriptional activation function of CONSTANS can be substituted by direct fusion of the EDLL activation motif to NF-YB subunits. The EDLL motif represents a potent plant activation domain that can be used as a tool to confer transcriptional activation potential to heterologous DNA-binding proteins.

Demonstration manuscript illustrates the formatting required for submission to Mycologia.

This facsimile manuscript demonstrates the application of the formatting directions in Instructions to Authors. The abstract is written as a single paragraph.

Characterisation and phylogenetic relationships of Anisogramma virgultorum and A. anomala in the Diaporthales (Ascomycota).

The two diaporthalean fungi Anisogramma virgultorum and A. anomala are biotrophic parasites. A. virgultorum causes stromatal cankers on young shoots of birch whereas A. anomala infects young branches of Corylus avellana. Although previous classifications based on morphological characteristics placed both species in the Gnomoniaceae, Diaporthales, their taxonomic position within the order and their relationship to each other required further clarification. We determined the nucleotide sequences of the ITS and partial LSU nu-rDNA regions of both species. A putative second teleomorph form of A. virgultorum, described in the literature as the 'single perithecial form', was also included in the analysis. Based on phylogenetic analyses of LSU sequences, the stromatal forms of A. virgultorum and A. anomala were part of a well-supported monophyletic sister clade to the Gnomoniaceae. The single perithecial form was placed within a clade containing representative members of the Gnomoniaceae, separate from species of Anisogramma. These results indicate that the single perithecial form of A. virgultorum actually represents an unrelated and as yet unidentified species of Gnomoniaceae. A morphological description of asci and ascospores of the three species is given. A Wilcoxon two sample test revealed that asci of the stromatal form of A. virgultorum were significantly shorter than those of the single perithecial species. Ascospores of the stromatal form of A. virgultorum were significantly shorter and wider than those of the single perithecial species.

The histopathology of Phaeocryptopus gaeumannii on Douglas-fir needles.

Germinating ascospores of Phaeocryptopus gaeumannii produce suprastomatal appressoria from which penetration pegs enter needles. Initial infection occurs between late May and early Jul and coincides with budbreak and shoot elongation. Colonization within needles is exclusively intercellular and increases continuously during Jul-May. No intracellular hyphae or haustoria were observed, but hyphae closely appressed to mesophyll and palisade cell walls are abundant by 3-5 mo after initial infection. Pseudothecial primordia begin to form in epistomatal chambers Oct-Apr, 4-9 mo after initial infection. Pseudothecial primordia developing in the epistomatal chamber are connected to the endophytic thallus by specialized cells in the substomatal chamber that have thickened apical walls and resemble phialides but are not involved in asexual reproduction. The apical wall thickenings instead appear to function as reinforcement against the turgor pressure of the guard cells, allowing cytoplasmic continuity to be maintained between the developing pseudothecium and vegetative hyphae within the needle. Concurrent with the formation of pseudothecial primorida, epiphytic hyphae emerge from the periphery of developing pseudothecia, grow across the needle surface, form numerous anastomoses and reenter the needle by producing appressoria above unoccupied stomata. Epiphytic hyphae and their associated appressoria gradually become more abundant during Oct-Jan.

A morpholino oligomer targeting highly conserved internal ribosome entry site sequence is able to inhibit multiple species of picornavirus.

Members of the genera Enterovirus and Rhinovirus (family Picornaviridae) cause a wide range of human diseases. An established vaccine is available only for poliovirus, and no effective therapy is available for the treatment of infections caused by any pathogenic picornavirus. Peptide-conjugated phosphorodiamidate morpholino oligomers (PPMO) are single-stranded DNA-like antisense agents that readily enter cells. A panel of PPMO was tested for their antiviral activities against various picornaviruses. PPMO targeting conserved internal ribosome entry site (IRES) sequence were highly active against human rhinovirus type 14, coxsackievirus type B2, and poliovirus type 1 (PV1), reducing PV1 titers by up to 6 log(10) in cell cultures. Comparative sequence analysis led us to design a PPMO (EnteroX) targeting 22 nucleotides of IRES sequence that are perfectly conserved across greater than 99% of all human enteroviruses and rhinoviruses. EnteroX reduced PV1 replication in cell culture to an extent similar to that of other IRES-specific PPMO. Resistant PV1 arose in cell cultures after 12 passages in the presence of EnteroX and were found to have two mutations within the EnteroX target sequence. Nevertheless, cPVR transgenic mice treated once daily by intraperitoneal (i.p.) injection with EnteroX before and/or after i.p. infection with 3 x 10(8) PFU (three times the 50% lethal dose) of PV1 had an approximately 80% higher rate of survival than the controls. The viral titer in tissues taken at day 5 postinfection showed that animals in the EnteroX-treated group averaged over 3, 4, and 5 log(10) less virus in the small intestine, spinal cord, and brain, respectively, than the amount in the control animals. These results suggest that EnteroX may have broad therapeutic potential against entero- and rhinoviruses.

The systematic position of Phaeocryptopus gaeumannii.

Phaeocryptopus gaeumannii, causal agent of the Douglas-fir foliar disease Swiss needle cast, is the only known pathogenic species of the genus. Current classifications place Phaeocryptopus in the Venturiaceae (Pleosporales), typified by the apple-scab pathogen Venturia inaequalis. All core members of this family have hyphomycetous anamorphs. We sought to confirm these relationships by means of phylogenetic analyses of the small (SSU) and large (LSU) subunits and internal transcribed spacer (ITS) region of nuclear ribosomal gene sequences (nrDNA). Analyses indicated that both the genus Phaeocryptopus and the family Venturiaceae, as currently defined, are unnatural groups. Phaeocryptopus nudus, type of the genus, is aligned in the Dothioraceae (Dothideales) and P. gaeumannii in the Mycosphaerellaceae (Capnodiales) near species of Mycosphaerella and Rasutoria. Core representatives of Venturiaceae formed an unambiguous clade but ordinal placement was unresolved. The family apparently is not included in the Pleosporales, Dothideales, Myriangiales or Capnodiales. Coelomycetous Rhizosphaera form-species are accepted generally as anamorphic states of Phaeocryptopus, however the relationship never has been established conclusively. Species of Rhizosphaera are closely related to P. nudus but not to P. gaeumannii, supporting an anamorph-teleomorph connection between Rhizosphaera and Phaeocryptopus and providing further evidence that P. gaeumanii is not congeneric with P. nudus.

Comparison of taxonomic, colony morphotype and PCR-RFLP methods to characterize microfungal diversity.

We compared three methods for estimating fungal species diversity in soil samples. A rapid screening method based on gross colony morphological features and color reference standards was compared with traditional fungal taxonomic methods and PCR-RFLP for estimation of ecological indices of soil microfungal community composition. Normalized counts of colony morphotypes on dichloran rose bengal medium were used to estimate species richness (S) and evenness (J) and to calculate Shannon's diversity (H) and Simpson's (SI) dominance indices. Isolates were obtained by dilution plating techniques from litter and soil layer samples taken from Douglas-fir forest and clear-cut areas at two locations in the Cascade Mountains. The highest correspondence (97%) was observed between taxonomic identification and RFLP patterns (32:33). Cladistic analyses of PCR-RFLP patterns indicated an 81% correspondence between RFLP patterns:colony morphotypes (33:41). A correspondence of 78% was observed between traditional taxonomic identification:colony morphotypes (32:41). Statistical analyses of ecological indices based on quantitative application of the colony morphotyping method indicated significant differences (P < 0.05) in fungal community composition between forested and clear-cut areas at the Toad Road site but not at the Falls Creek site. Comparisons of ecological indices based on traditional identification of taxa by microscopic characterization on defined culture media resulted in identical findings of statistical significance. The colony morphotyping approach is proposed as a screening method to identify potential effects of land management practices, edaphic factors and pollutants on microfungal diversity.

Equine cutaneous fungal granuloma: a study of 44 lesions from 34 horses.

Forty-four nodular and noninvasive cutaneous fungal granulomas were identified in 34 horses over a 14.5-year period. Cutaneous fungal granulomas were most common in young horses (mean age 6.1 +/- 4.2 years; range 1-19 years). There was no apparent breed or sex bias. Granulomas were either single or multiple, and most often occurred in the skin of the head and neck. The characteristic histological finding was a nodular dermal mass with a mean diameter of 7.3 mm (range 2.5-20 mm) and an intact overlying epithelium. Lesions most often exhibited intense lymphocytic inflammation, with admixed pyogranulomatous inflammation associated with a small to moderate number of fungal elements. Causative fungi were both pigmented and nonpigmented organisms of variable morphology. Penetrating plant material was identified in three cases. Granulomas caused by nonpigmented fungi were most common in horses from wet regions. Both pigmented and nonpigmented fungi were found in granulomas from horses in dry regions. Cutaneous fungal granulomas occurred in February through November, with peaks in April and July. No correlation of yearly incidence with annual average temperature or rainfall was detected. This study confirms that equine cutaneous fungal granuloma is relatively common in horses in the Pacific Northwest. Morphology of causative fungi was variable, but the signalment, history, and clinical and overall histological features were very similar. Surgical excision was curative.

Quasispecies diversity determines pathogenesis through cooperative interactions in a viral population.

An RNA virus population does not consist of a single genotype; rather, it is an ensemble of related sequences, termed quasispecies. Quasispecies arise from rapid genomic evolution powered by the high mutation rate of RNA viral replication. Although a high mutation rate is dangerous for a virus because it results in nonviable individuals, it has been hypothesized that high mutation rates create a 'cloud' of potentially beneficial mutations at the population level, which afford the viral quasispecies a greater probability to evolve and adapt to new environments and challenges during infection. Mathematical models predict that viral quasispecies are not simply a collection of diverse mutants but a group of interactive variants, which together contribute to the characteristics of the population. According to this view, viral populations, rather than individual variants, are the target of evolutionary selection. Here we test this hypothesis by examining the consequences of limiting genomic diversity on viral populations. We find that poliovirus carrying a high-fidelity polymerase replicates at wild-type levels but generates less genomic diversity and is unable to adapt to adverse growth conditions. In infected animals, the reduced viral diversity leads to loss of neurotropism and an attenuated pathogenic phenotype. Notably, using chemical mutagenesis to expand quasispecies diversity of the high-fidelity virus before infection restores neurotropism and pathogenesis. Analysis of viruses isolated from brain provides direct evidence for complementation between members in the quasispecies, indicating that selection indeed occurs at the population level rather than on individual variants. Our study provides direct evidence for a fundamental prediction of the quasispecies theory and establishes a link between mutation rate, population dynamics and pathogenesis.

Population structure suggests reproductively isolated lineages of Phaeocryptopus gaeumannii.

A survey of the genetic diversity and population structure of the Douglas-fir Swiss needle cast pathogen Phaeocryptopus gaeumannii was conducted with single-strand conformational polymorphisms (SSCP) to screen for variability in mitochondrial and nuclear housekeeping genes. Thirty host populations representing the natural range of Douglas-fir as well as locations where the tree was planted as an exotic were sampled. Sequencing of SSCP variants revealed that the method accurately detected both single nucleotide and indel polymorphisms. Sequence information was used to construct multilocus gene genealogies and to test various hypotheses of recombination (outcrossing) and clonality (selfing). We found that P. gaeumannii in the region of Oregon's Swiss needle cast epidemic exhibits strong multilocus gametic phase disequilibrium and is subdivided into two reproductively isolated sympatric lineages. Low genotypic diversity together with the presence of overrepresented genotypes in both lineages suggests a predominantly selfing reproductive mode. Genotypes of one lineage were found in isolates from a widespread geographic distribution, occurring throughout much of the Pacific Northwest as well as nonindigenous populations abroad that have historical reports of disease. Genotypes of the second lineage were detected only in isolates from Oregon's coastal region. Within the main epidemic area, abundance of this second lineage in young plantations appeared to be correlated with disease severity.

Remote site control of an active site fidelity checkpoint in a viral RNA-dependent RNA polymerase.

The kinetic, thermodynamic, and structural basis for fidelity of nucleic acid polymerases remains controversial. An understanding of viral RNA-dependent RNA polymerase (RdRp) fidelity has become a topic of considerable interest as a result of recent experiments that show that a 2-fold increase in fidelity attenuates viral pathogenesis and a 2-fold decrease in fidelity reduces viral fitness. Here we show that a conformational change step preceding phosphoryl transfer is a key fidelity checkpoint for the poliovirus RdRp (3Dpol). We provide evidence that this conformational change step is orientation of the triphosphate into a conformation suitable for catalysis, suggesting a kinetic and structural model for RdRp fidelity that can be extrapolated to other classes of nucleic acid polymerases. Finally, we show that a site remote from the catalytic center can control this checkpoint, which occurs at the active site. Importantly, similar connections between a remote site and the active site exist in a wide variety of viral RdRps. The capacity for sites remote from the catalytic center to alter fidelity suggests new possibilities for targeting the viral RdRp for antiviral drug development.

Ribavirin and lethal mutagenesis of poliovirus: molecular mechanisms, resistance and biological implications.

Positive strand RNA virus populations are a collection of similar but genetically different viruses. They exist as viral quasispecies due to the high mutation rates of the low fidelity viral RNA-dependent RNA polymerase (RdRp). It is thought that this genomic heterogeneity is advantageous to the population, allowing for adaptation to rapidly changing environments that present varying types and degrees of selective pressure. However, one consequence of this extensive diversity is the susceptibility to mutagens that further increase sequence variation. Since RNA viruses live at the edge of maximal variability, an increase in the mutation rate is likely to force the virus beyond the tolerable mutation frequency into 'error catastrophe'. One such mutagen, ribavirin, is an antiviral nucleoside analog that is mutagenic to several RNA viruses. Ribavirin is incorporated into the viral genome causing lethal mutagenesis and a subsequent decrease in the specific infectivity. Even so, passaging poliovirus in the presence of low to intermediate concentrations of the drug leads to the emergence of a viral population resistant to the effects of ribavirin. These viruses have a point mutation in the RdRp that increases the overall polymerase fidelity. Interestingly, as predicted by the quasispecies theory, ribavirin resistant viruses are less adaptable, as they are more susceptible to other non-mutagenic antiviral drugs and are highly attenuated in vivo. Here, we review the mechanism of action of ribavirin on poliovirus and other RNA viruses, the possibility for escape via increased fidelity of the viral polymerase, the consequences of this response on viral population dynamics, and the biological implications for the therapeutic use of mutagenic antiviral agents.

A climate-based model for predicting geographic variation in swiss needle cast severity in the Oregon coast range.

ABSTRACT Since the early 1990s, Swiss needle cast disease caused by Phaeocryptopus gaeumannii has been increasing in Douglas-fir plantations in the Oregon Coast Range. Considerable variation in disease severity across the affected area often has been noted. We investigated the influence of site microclimate on fungal colonization as a basis for this variation with a combination of seedling inoculation and field studies. Development of P. gaeumannii ascocarps on inoculated seedlings subjected to mist, irrigation, and shading treatments was followed for 10 months. Contrary to expectations, numbers of ascocarps on foliage were negatively correlated with shade and mist and positively correlated with temperature. Numbers of ascocarps on foliage, site temperature, and leaf wetness were monitored over 5 years at nine field sites in the Oregon Coast Range. Factors most highly correlated with ascocarp abundance were winter mean daily temperature and spring cumulative leaf wetness. Predictive models for disease severity on the basis of these correlations were tested against disease and climate data measured at field sites during 2003-2004. A temperature-based disease prediction model was developed in combination with geographical information systems (GIS)-linked climate databases to estimate disease levels across a portion of the Oregon Coast Range. This model can be used for hypothesis testing and as a decision support tool for forest managers.

Poliovirus escape from RNA interference: short interfering RNA-target recognition and implications for therapeutic approaches.

Short interfering RNAs (siRNAs) directed against poliovirus and other viruses effectively inhibit viral replication. Although RNA interference (RNAi) may provide the basis for specific antiviral therapies, the limitations of RNAi antiviral strategies are ill defined. Here, we show that poliovirus readily escapes highly effective siRNAs through unique point mutations within the targeted regions. Competitive analysis of the escape mutants provides insights into the basis of siRNA recognition. The RNAi machinery can tolerate mismatches but is exquisitely sensitive to mutations within the central region and the 3' end of the target sequence. Indeed, specific mutations in the target sequence resulting in G:U mismatches are sufficient for the virus to escape siRNA inhibition. However, using a pool of siRNAs to simultaneously target multiple sites in the viral genome prevents the emergence of resistant viruses. Our study uncovers the elegant precision of target recognition by the RNAi machinery and provides the basis for the development of effective RNAi-based therapies that prevent viral escape.

Phylogenetics of Lophodermium from pine.

Lophodermium comprises ascomycetous fungi that are both needle-cast pathogens and asymptomatic endophytes on a diversity of plant hosts. It is distinguished from other genera in the family Rhytismataceae by its filiform ascospores and ascocarps that open by a longitudinal slit. Nucleotide sequences of the internal transcribed spacer (ITS) region of nuclear ribosomal DNA were used to infer phylogenetic relationships within Lophodermium. Twenty-nine sequences from approximately 11 species of Lophodermium were analyzed together with eight sequences from isolates thought to represent six other genera of Rhytismataceae: Elytroderma, Lirula, Meloderma, Terriera, Tryblidiopsis and Colpoma. Two putative Meloderma desmazieresii isolates occurred within the Lophodermium clade but separate from one another, one grouped with L. indianum and the other with L. nitens. An isolate of Elytroderma deformans also occurred within the Lophodermium clade but on a solitary branch. The occurrence of these genera within the Lophodermium clade might be due to problems in generic concepts in Rhytismataceae, such as emphasis on spore morphology to delimit genera, to difficulty of isolating Rhytismataceae needle pathogens from material that also is colonized by Lophodermium or to a combination of both factors. We also evaluated the congruence of host distribution and several morphological characters on the ITS phylogeny. Lophodermium species from pine hosts formed a monophyletic sister group to Lophodermium species from more distant hosts from the southern hemisphere, but not to L. piceae from Picea. The ITS topology indicated that Lophodermium does not show strict cospeciation with pines at deeper branches, although several closely related isolates have closely related hosts. Pathogenic species occupy derived positions in the pine clade, suggesting that pathogenicity has evolved from endophytism. A new combination is proposed, Terriera minor (Tehon) P.R. Johnst.