Alternaria induces airway epithelial cytokine expression independent of protease-activated receptor.

BACKGROUND AND OBJECTIVE: A novel fungal allergen, Alternaria (Alt), has been previously shown to associate with the pathogenesis of allergic rhinitis and bronchial asthma, particularly in arid and semi-arid regions. Airway epithelial cells are among the first to encounter Alt, and epithelial cytokine production and subsequent airway inflammation are early events in the response to Alt exposure. However, the underlying mechanism is unclear. As protease-activated receptor 2 (PAR2) has been implicated in most of the Alt-induced biological events, we investigated the regulation of airway inflammation and epithelial cytokine expression by PAR2. METHODS: Wild-type (WT) and Par2 knockout (Par2-KO) mice were used to evaluate the in vivo role of PAR2. Primary human and mouse airway epithelial cells were used to examine the mechanistic basis of epithelial cytokine regulation in vitro. RESULTS: Surprisingly, Par2 deficiency had no negative impact on the change of lung function, inflammation and cytokine production in the mouse model of Alt-induced asthma. Alt-induced cytokine production in murine airway epithelial cells from Par2-KO mice was not significantly different from the WT cells. Consistently, PAR2 knockdown in human cells also had no effect on cytokine expression. In contrast, the cytokine expressions induced by synthetic PAR2 agonist or other asthma-related allergens (e.g. cockroach extracts) were indeed mediated via a PAR2-dependent mechanism. Finally, we found that EGFR pathway was responsible for Alt-induced epithelial cytokine expression. CONCLUSION: The activation of EGFR, but not PAR2, was likely to drive the airway inflammation and epithelial cytokine production induced by Alt.

The Alternaria genomes database: a comprehensive resource for a fungal genus comprised of saprophytes, plant pathogens, and allergenic species.

BACKGROUND: Alternaria is considered one of the most common saprophytic fungal genera on the planet. It is comprised of many species that exhibit a necrotrophic phytopathogenic lifestyle. Several species are clinically associated with allergic respiratory disorders although rarely found to cause invasive infections in humans. Finally, Alternaria spp. are among the most well known producers of diverse fungal secondary metabolites, especially toxins. DESCRIPTION: We have recently sequenced and annotated the genomes of 25 Alternaria spp. including but not limited to many necrotrophic plant pathogens such as A. brassicicola (a pathogen of Brassicaceous crops like cabbage and canola) and A. solani (a major pathogen of Solanaceous plants like potato and tomato), and several saprophytes that cause allergy in human such as A. alternata isolates. These genomes were annotated and compared. Multiple genetic differences were found in the context of plant and human pathogenicity, notably the pro-inflammatory potential of A. alternata. The Alternaria genomes database was built to provide a public platform to access the whole genome sequences, genome annotations, and comparative genomics data of these species. Genome annotation and comparison were performed using a pipeline that integrated multiple computational and comparative genomics tools. Alternaria genome sequences together with their annotation and comparison data were ported to Ensembl database schemas using a self-developed tool (EnsImport). Collectively, data are currently hosted using a customized installation of the Ensembl genome browser platform. CONCLUSION: Recent efforts in fungal genome sequencing have facilitated the studies of the molecular basis of fungal pathogenicity as a whole system. The Alternaria genomes database provides a comprehensive resource of genomics and comparative data of an important saprophytic and plant/human pathogenic fungal genus. The database will be updated regularly with new genomes when they become available. The Alternaria genomes database is freely available for non-profit use at http://alternaria.vbi.vt.edu .

Assessing fungal community structure from mineral surfaces in Kartchner Caverns using multiplexed 454 pyrosequencing.

Research on the distribution and structure of fungal communities in caves is lacking. Kartchner Caverns is a wet and mineralogically diverse carbonate cave located in an escarpment of Mississippian Escabrosa limestone in the Whetstone Mountains, Arizona, USA. Fungal diversity from speleothem and rock wall surfaces was examined with 454 FLX Titanium sequencing technology using the Internal Transcribed Spacer 1 as a fungal barcode marker. Fungal diversity was estimated and compared between speleothem and rock wall surfaces, and its variation with distance from the natural entrance of the cave was quantified. Effects of environmental factors and nutrient concentrations in speleothem drip water at different sample sites on fungal diversity were also examined. Sequencing revealed 2,219 fungal operational taxonomic units (OTUs) at the 95% similarity level. Speleothems supported a higher fungal richness and diversity than rock walls. However, community membership and the taxonomic distribution of fungal OTUs at the class level did not differ significantly between speleothems and rock walls. Both OTU richness and diversity decreased significantly with increasing distance from the natural cave entrance. Community membership and taxonomic distribution of fungal OTUs also differed significantly between the sampling sites closest to the entrance and those furthest away. There was no significant effect of temperature, CO2 concentration, or drip water nutrient concentration on fungal community structure on either speleothems or rock walls. Together, these results suggest that proximity to the natural entrance is a critical factor in determining fungal community structure on mineral surfaces in Kartchner Caverns.

Discord between morphological and phylogenetic species boundaries: incomplete lineage sorting and recombination results in fuzzy species boundaries in an asexual fungal pathogen.

BACKGROUND: Traditional morphological and biological species concepts are difficult to apply to closely related, asexual taxa because of the lack of an active sexual phase and paucity of morphological characters. Phylogenetic species concepts such as genealogical concordance phylogenetic species recognition (GCPSR) have been extensively used; however, methods that incorporate gene tree uncertainty into species recognition may more accurately and objectively delineate species. Using a worldwide sample of Alternaria alternata sensu lato, causal agent of citrus brown spot, the evolutionary histories of four nuclear loci including an endo-polygalacturonase gene, two anonymous loci, and one microsatellite flanking region were estimated using the coalescent. Species boundaries were estimated using several approaches including those that incorporate uncertainty in gene genealogies when lineage sorting and non-reciprocal monophyly of gene trees is common. RESULTS: Coalescent analyses revealed three phylogenetic lineages strongly influenced by incomplete lineage sorting and recombination. Divergence of the citrus 2 lineage from the citrus 1 and citrus 3 lineages was supported at most loci. A consensus of species tree estimation methods supported two species of Alternaria causing citrus brown spot worldwide. Based on substitution rates at the endo-polygalacturonase locus, divergence of the citrus 2 and the 1 and 3 lineages was estimated to have occurred at least 5, 400 years before present, predating the human-mediated movement of citrus and associated pathogens out of SE Asia. CONCLUSIONS: The number of Alternaria species identified as causing brown spot of citrus worldwide using morphological criteria has been overestimated. Little support was found for most of these morphospecies using quantitative species recognition approaches. Correct species delimitation of plant-pathogenic fungi is critical for understanding the evolution of pathogenicity, introductions of pathogens to new areas, and for regulating the movement of pathogens to enforce quarantines. This research shows that multilocus phylogenetic methods that allow for recombination and incomplete lineage sorting can be useful for the quantitative delimitation of asexual species that are morphologically indistinguishable. Two phylogenetic species of Alternaria were identified as causing citrus brown spot worldwide. Further research is needed to determine how these species were introduced worldwide, how they differ phenotypically and how these species are maintained.

Profiling bacterial diversity and taxonomic composition on speleothem surfaces in Kartchner Caverns, AZ.

Caves are relatively accessible subterranean habitats ideal for the study of subsurface microbial dynamics and metabolisms under oligotrophic, non-photosynthetic conditions. A 454-pyrotag analysis of the V6 region of the 16S rRNA gene was used to systematically evaluate the bacterial diversity of ten cave surfaces within Kartchner Caverns, a limestone cave. Results showed an average of 1,994 operational taxonomic units (97 % cutoff) per speleothem and a broad taxonomic diversity that included 21 phyla and 12 candidate phyla. Comparative analysis of speleothems within a single room of the cave revealed three distinct bacterial taxonomic profiles dominated by either Actinobacteria, Proteobacteria, or Acidobacteria. A gradient in observed species richness along the sampling transect revealed that the communities with lower diversity corresponded to those dominated by Actinobacteria while the more diverse communities were those dominated by Proteobacteria. A 16S rRNA gene clone library from one of the Actinobacteria-dominated speleothems identified clones with 99 % identity to chemoautotrophs and previously characterized oligotrophs, providing insights into potential energy dynamics supporting these communities. The robust analysis conducted for this study demonstrated a rich bacterial diversity on speleothem surfaces. Further, it was shown that seemingly comparable speleothems supported divergent phylogenetic profiles suggesting that these communities are very sensitive to subtle variations in nutritional inputs and environmental factors typifying speleothem surfaces in Kartchner Caverns.

Signatures of recombination in clonal lineages of the citrus brown spot pathogen, Alternaria alternata sensu lato.

Most Alternaria spp. are considered asexual but recent molecular evolution analyses of Alternaria mating-type genes show that the mating locus is under strong purifying selection, indicating a possible role in sexual reproduction. The objective of this study was to determine the mode of reproduction of an Alternaria alternata sensu lato population causing citrus brown spot in central Florida. Mating type of each isolate was determined, and isolates were sequenced at six putatively unlinked loci. Three genetically distinct subpopulations (SH1, SH4A, and SH4B) were identified using network and Bayesian population structure analyses. Results demonstrate that most subpopulations of A. alternata associated with citrus are clonal but some have the ability to extensively recombine through a cryptic sexual cycle or parasexual cycle. Although isolates were sampled in close physical proximity (≈2,500-m² area), we were able to reject a random mating model using multilocus gametic disequilibrium tests for two subpopulations, SH1 and SH4B, suggesting that these subpopulations were predominantly asexual. However, three recombination events were identified in SH1 and SH4B and localized to individuals of opposite mating type, possibly indicating meiotic recombination. In contrast, in the third subpopulation (SH4A), where only one mating type was present, extensive reticulation was evident in network analyses, and multilocus gametic disequilibrium tests were consistent with recombination. Recombination among isolates of the same mating type suggests that a nonmeiotic mechanism of recombination such as the parasexual cycle may be operating in this subpopulation. The level of gene flow detected among subpopulations does not appear to be sufficient to prevent differentiation, and perhaps future speciation, of these A. alternata subpopulations.

The sections of Alternaria: formalizing species-group concepts.

The systematics of Alternaria and allied genera traditionally has been based on the characteristics of conidia and the sporulation apparatus. This emphasis on morphology in the reconstruction of organismal relationships has resulted in taxonomic uncertainty and flux for a number of taxa in Alternaria and the related genera Stemphylium, Embellisia, Nimbya and Ulocladium. The present study used a molecular phylogenetic approach for systematic resolution and incorporated extensive taxon sampling (n = 176 species) representing 10 genera and analyses of 10 protein-coding loci. Phylogenetic analyses based on five of these genes revealed eight distinct asexual lineages of Alternaria that cluster as the sister group to the asexual paraphyletic genus Ulocladium, while taxa with known teleomorphs currently circumscribed as Alternaria (the infectoria species-group) cluster among genera that also have representatives with known teleomorphs. This work proposes to elevate the eight well supported asexual lineages of Alternaria to the taxonomic rank of section. Evolutionary relationships among Alternaria and closely related genera are discussed.

Novel Mouse Models of Fungal Asthma.

Alternaria alternata is a ubiquitous fungus and a major allergen associated with the development of asthma. Inhalation of intact spores is the primary cause of human exposure to fungal allergen. However, allergen-rich cultured fungal filtrates are oftentimes used in the current models of fungal sensitization that do not fully reflect real-life exposures. Thus, establishing novel spore exposure models is imperative. In this study, we established novel fungal exposure models of both adult and neonate to live spores. We examined pathophysiological changes in the spore models as compared to the non-exposure controls and also to the conventional filtrate models. While both Alternaria filtrate- and spore-exposed adult BALB/c mice developed elevated airway hyperresponsiveness (AHR), filtrates induced a greater IgE mediated response and higher broncholavage eosinophils than spores. In contrast, the mice exposed to Alternaria spores had higher numbers of neutrophils. Both exposures induced comparable levels of lung tissue inflammation and mucous cell metaplasia (MCM). In the neonatal model, exposure to Alternaria spores resulted in a significant increase of AHR in both adult and neonatal mice. Increased levels of IgE in both neonatal and adult mice exposed to spores was associated with increased eosinophilia in the treatment groups. Adult demonstrated increased numbers of lymphocytes that was paralleled by increased IgG1 production. Both adults and neonates demonstrated similarly increased eosinophilia, IgE, tissue inflammation and MCM.

A PCR-Based Assay for Detection of Fusarium oxysporum f. sp. lactucae in Lettuce Seed.

A nested polymerase chain reaction-based (nPCR) assay was developed and evaluated for the rapid detection of Fusarium oxysporum f. sp. lactucae in seed of lettuce. Three primers were designed from sequences of the intergenic spacer region of the rDNA and were used in the PCR amplifications. The first amplification employed the primer pair GYCF1 and GYCR4C and produced a product of 2,270 bp. The second amplification employed the forward primer GYCF1 and the nested primer R943 and produced a single 936-bp PCR product. The nPCR protocol developed successfully detected the target sequence in genomic DNA at 1 fg/µl. A seed assay was tested that included a 4-day incubation step in which seed were maintained under high humidity conditions to increase fungal biomass for DNA extraction. In seed lots prepared by mixing known amounts of F. oxysporum f. sp. lactucae-infested seed with noninfested seed, this assay permitted the detection of the pathogen from lots with infestation rates as low as 0.1%. Samples of lettuce seed obtained from 88 commercial lettuce seed lots were assayed for the pathogen by direct plating and by using the nPCR assay. The pathogen was not detected by either diagnostic method, suggesting the seed lots were pathogen free or the level was below detection limits.

A re-examination of the phylogenetic relationship between the causal agents of carrot black rot, Alternaria radicina and A. carotiincultae.

The phylogenetic relationship between Alternaria radicina and A. carotiincultae was reexamined based on morphology, sequence analysis of rDNA (ITS and mitochondrial small subunit [mtSSU]), protein coding genes (actin [ACT], beta-tubulin, chitin synthase [CHS], translation elongation factor [EF-1a], Alternaria allergen a1 [Alt a1], and glyceraldehyde-3-phosphate dehydrogenase [gpd]), and RAPD and ISSR analysis of total genomic DNA. Although some morphological characters overlapped to a degree, with A. radicina isolates expressing moderate variation and A. carotiincultae isolates being highly uniform, A. carotiincultae could be differentiated from A. radicina based on significantly greater growth rate on potato dextrose agar (PDA) or acidified PDA (APDA) and average number of transverse septa per conidium. Sequence of rDNA and two protein coding genes, ACT and CHS, were invariant between species. However polymorphism with the EF-1a, beta-tubulin, and Alt a1 gene strictly separated the population of A. radicina and A. carotiincultae as distinct lineages, as did RAPD and ISSR analysis. The polymorphic gpd gene did not strictly separate the two species. However isolates of A. radicina encompassed several haplotypes, one of which was the exclusive haplotype possessed by A. carotiincultae isolates, suggesting evidence of incomplete lineage sorting. The results suggest that A. carotiincultae is closely related to A. radicina but is a recently divergent and distinct lineage, which supports its status as a separate species.

Molecular diversity and allergenic profiles of Alternaria spp. from desert environments in Arizona.

This study examined the genetic diversity of small-spored Alternaria species in the southwest desert of the USA by sampling 552 isolates from different habitats (soil and plant debris) in different locations (urban and an undisturbed desert). To estimate the genetic diversity, Amplified Fragment Length Polymorphism (AFLP) fingerprinting analysis was performed for all isolates. Strains representative of the sampled genotypic diversity (n = 125) were further characterized according their sporulation pattern and the capability to produce allergens. Morphological characterization assigned the majority of the strains to the Alternaria alternata and Alternaria tenuissima morpho-groups with only two isolates assigned to the Alternaria arborescens morpho-group. AFLP fingerprinting differentiated the A. arborescens morpho-groups, but could not distinguish between the A. alternata and A. tenuissima morpho-groups. Western blot analysis showed that a large number of allergenic proteins were produced by strains. These proteins were not specific for any morpho-group nor source of isolation. A hierarchical analysis of molecular variance was performed on the AFLP data to quantify molecular variation and partition this variation among sampled locations and habitat. No statistically significant differentiation among locations and habitat was detected indicating a lack of population structure across environments.

Phylogeny of Fusarium oxysporum f. sp. lactucae Inferred from Mitochondrial Small Subunit, Elongation Factor 1-alpha, and Nuclear Ribosomal Intergenic Spacer Sequence Data.

ABSTRACT Fusarium oxysporum f. sp. lactucae, causal agent of Fusarium wilt of lettuce, is a serious pathogen recently reported in Arizona. Sequence analysis of the mitochondrial small subunit (mtSSU), translation elongation factor 1-alpha (EF-1alpha) gene, and the nuclear ribosomal DNA intergenic spacer (IGS) region was conducted to resolve relationships among f. sp. lactucae isolates, F. oxysporum isolates from other hosts, and local non-pathogenic isolates. Analysis of mtSSU sequences provided limited phylogenetic resolution and did not differentiate the lactucae isolates from 13 other F. oxysporum isolates. Analysis of EF-1alpha sequences resulted in moderate resolution, grouping seven formae speciales with the lactucae isolates. Analysis of the IGS region revealed numerous sequence polymorphisms among F. oxysporum formae speciales consisting of insertions, deletions, and single nucleotide transitions and substitutions. Repeat sequence analysis revealed several duplicated subrepeat units that were distributed across much of the region. Based on analysis of the IGS sequence data, lactucae race 1 isolates resolved as a monophyletic group with three other formae speciales of F. oxysporum. In all analyses, lactucae race 2 isolates composed a separate lineage that was phylo-genetically distinct and distantly related to the lactucae race 1 isolates.

Degradation of aflatoxin B1 from naturally contaminated maize using the edible fungus Pleurotus ostreatus.

Aflatoxins are highly carcinogenic secondary metabolites that can contaminate approximately 25% of crops and that cause or exacerbate multiple adverse health conditions, especially in Sub-Saharan Africa and South and Southeast Asia. Regulation and decontamination of aflatoxins in high exposure areas is lacking. Biological detoxification methods are promising because they are assumed to be cheaper and more environmentally friendly compared to chemical alternatives. White-rot fungi produce non-specific enzymes that are known to degrade aflatoxin in in situ and ex situ experiments. The aims of this study were to (1) decontaminate aflatoxin B1 (AFB1) in naturally contaminated maize with the edible, white-rot fungus Pleurotus ostreatus (oyster mushroom) using a solid-state fermentation system that followed standard cultivation techniques, and to (2) and to assess the risk of mutagenicity in the resulting breakdown products and mushrooms. Vegetative growth and yield characteristics of P. ostreatus were not inhibited by the presence of AFB1. AFB1 was degraded by up to 94% by the Blue strain. No aflatoxin could be detected in P. ostreatus mushrooms produced from AFB1-contaminated maize. Moreover, the mutagenicity of breakdown products from the maize substrate, and reversion of breakdown products to the parent compound, were minimal. These results suggest that P. ostreatus significantly degrades AFB1 in naturally contaminated maize under standard cultivation techniques to levels that are acceptable for some livestock fodder, and that using P. ostreatus to bioconvert crops into mushrooms can reduce AFB1-related losses.

Morphological, pathogenic, and molecular characterization of alternaria isolates associated with alternaria late blight of pistachio.

ABSTRACT Alternaria isolates were obtained from various pistachio tissues collected in five orchards in California. For all isolates, morphological characteristics of the colony and sporulation apparatus were determined and compared with those of representative isolates of A. alternata, A. tenuissima, A. arborescens, and A. infectoria. A selection of the pistachio isolates and the representative Alternaria isolates were evaluated for pathogenicity to pistachio. Molecular characteristics of these isolates were determined using random amplified polymorphism DNA (RAPD) analysis, polymerase chain reaction restriction fragment length polymorphism (PCR-RFLP) analysis of nuclear intergenic spacer rDNA, and sequence analysis of nuclear internal transcribed spacer (ITS) rDNA. Based on morphological characteristics, the pistachio isolates were grouped as identical or very similar to either A. alternata, A. tenuissima, A. arborescens, or A. infectoria. Isolates from the alternata, tenuissima, and arborescens species-groups were pathogenic to pistachio and no significant differences in pathogenicity were observed. Isolates from the infectoria species-group were only weakly pathogenic to pistachio. Based on cluster analysis of RAPD and PCR-RFLP data, three distinct clusters were evident; the infectoria cluster, the arborescens cluster, and a combined alternata/tenuissima cluster. Based on analysis of ITS sequence data, the infectoria species-group was phylogenetically distinct from the other species-groups. Isolates of the alternata, tenuissima, and arborescens species-groups comprised a monophyletic clade in which the three species-groups could not be further resolved.

Relationships and taxonomic status of Alternaria radicina, A. carotiincultae, and A. petroselini based upon morphological, biochemical, and molecular characteristics.

Alternaria radicina, A. carotiincultae, and A. petroselini are closely related pathogens of umbelliferous crops. Relationships among these fungi were determined based on growth rate, spore morphology, cultural characteristics, toxin production, and host range. Random amplified polymorphic DNA (RAPD) analysis of these species, other species of Alternaria, and closely related fungi was also performed. A. petroselini was readily differentiated from A. radicina and A. carotiincultae on the basis of spore morphology, production of microsclerotia, host range, and RAPD analysis. Alternaria radicina and A. carotiincultae were considerably more similar to each other than to A. petroselini, but could be differentiated on the basis of growth rate, spore morphology, colony morphology, and, to a limited extent, RAPD analysis. When grown on media having a high nutritional content, A. radicina produced a diffusible yellow pigment and crystals of the fungal metabolite radicinin. In contrast, A. carotiincultae produced little or no radicinin. However, when A. carotiincultae was grown on the same medium amended with radicinin, growth rate and colony and conidial morphology were more similar to those of A. radicina. These results suggest that the morphological differences between A. radicina and A. carotiincultae are due, at least in part, to radicinin production, and that these fungi are conspecific. Therefore, we propose that A. carotiincultae be considered a synonym of A. radicina.

Molecular characterization of Embellisia and Nimbya species and their relationship to Alternaria, Ulocladium and Stemphylium.

DNA sequences from rDNA and protein-coding regions were determined for six Embellisia and two Nimbya spp. and were compared to those from Alternaria, Ulocladium and Stemphylium spp. Sequences determined included rDNA from the nuclear internal transcribed-spacer region (ITS1/5.8S/ITS2) and the mitochondrial small-subunit (mt SSU) and a portion of the glyceraldehyde-3-phosphate dehydrogenase (gpd) gene. Phylogenetic analyses were performed on each dataset separately and then combined for total evidence analysis using methods of maximum parsimony and maximum likelihood. Results revealed that Embellisia and Nimbya clustered within a large monophyletic Alternaria-Nimbya-Embellisia-Ulocladium clade with Stemphylium as the sister taxon. Members of the infectoria species-group were the most basal group in this large polygeneric clade. Embellisia and Nimbya were sister taxa of the remaining Alternaria and Ulocladium spp. and were related more closely to Alternaria than was Stemphylium. Four Embellisia spp. formed a monophyletic clade. However, E. allii clustered with the two Nimbya spp. and E. indefessa clustered with Alternaria and Ulocladium spp., revealing that Embellisia, as currently circumscribed, is polyphyletic. Potential revisions of taxonomy for all genera are discussed.

Making a living while starving in the dark: metagenomic insights into the energy dynamics of a carbonate cave.

Carbonate caves represent subterranean ecosystems that are largely devoid of phototrophic primary production. In semiarid and arid regions, allochthonous organic carbon inputs entering caves with vadose-zone drip water are minimal, creating highly oligotrophic conditions; however, past research indicates that carbonate speleothem surfaces in these caves support diverse, predominantly heterotrophic prokaryotic communities. The current study applied a metagenomic approach to elucidate the community structure and potential energy dynamics of microbial communities, colonizing speleothem surfaces in Kartchner Caverns, a carbonate cave in semiarid, southeastern Arizona, USA. Manual inspection of a speleothem metagenome revealed a community genetically adapted to low-nutrient conditions with indications that a nitrogen-based primary production strategy is probable, including contributions from both Archaea and Bacteria. Genes for all six known CO2-fixation pathways were detected in the metagenome and RuBisCo genes representative of the Calvin-Benson-Bassham cycle were over-represented in Kartchner speleothem metagenomes relative to bulk soil, rhizosphere soil and deep-ocean communities. Intriguingly, quantitative PCR found Archaea to be significantly more abundant in the cave communities than in soils above the cave. MEtaGenome ANalyzer (MEGAN) analysis of speleothem metagenome sequence reads found Thaumarchaeota to be the third most abundant phylum in the community, and identified taxonomic associations to this phylum for indicator genes representative of multiple CO2-fixation pathways. The results revealed that this oligotrophic subterranean environment supports a unique chemoautotrophic microbial community with potentially novel nutrient cycling strategies. These strategies may provide key insights into other ecosystems dominated by oligotrophy, including aphotic subsurface soils or aquifers and photic systems such as arid deserts.

Interkingdom gene transfer of a hybrid NPS/PKS from bacteria to filamentous Ascomycota.

Nonribosomal peptides (NRPs) and polyketides (PKs) are ecologically important secondary metabolites produced by bacteria and fungi using multidomain enzymes called nonribosomal peptide synthetases (NRPSs) and polyketide synthases (PKSs), respectively. Previous phylogenetic analyses of fungal NRPSs and PKSs have suggested that a few of these genes were acquired by fungi via horizontal gene transfer (HGT) from bacteria, including a hybrid NPS/PKS found in Cochliobolus heterostrophus (Dothideomycetes, Ascomycota). Here, we identify this hybrid gene in fungi representing two additional classes of Ascomycota (Aspergillus spp., Microsporum canis, Arthroderma spp., and Trichophyton spp., Eurotiomycetes; Chaetomium spp. and Metarhizium spp., Sordariomycetes) and use phylogenetic analyses of the most highly conserved domains from NRPSs (adenylation (A) domain) and PKSs (ketoacyl synthase (KS) domain) to examine the hypothesis that the hybrid NPS7/PKS24 was acquired by fungi from bacteria via HGT relatively early in the evolution of the Pezizomycotina. Our results reveal a unique ancestry of the A domain and KS domain in the hybrid gene relative to known fungal NRPSs and PKSs, provide strong evidence for HGT of the hybrid gene from a putative bacterial donor in the Burkholderiales, and suggest the HGT event occurred early in the evolution of the filamentous Ascomycota.

Modelling and visualizing morphology in the fungus Alternaria.

Alternaria is one of the most cosmopolitan fungal genera encountered and impacts humans and human activities in areas of material degradation, phytopathology, food toxicology, and respiratory disease. Contemporary methods of taxon identification rely on assessments of morphology related to sporulation, which are critical for accurate diagnostics. However, the morphology of Alternaria is quite complex, and precise characterization can be laborious, time-consuming, and often restricted to experts in this field. To make morphology characterization easier and more broadly accessible, a generalized statistical model was developed for the three-dimensional geometric structure of the sporulation apparatus. The model is inspired by the widely used grammar-based models for plants, Lindenmayer-systems, which build structure by repeated application of rules for growth. Adjusting the parameters of the underlying probability distributions yields variations in the morphology, and thus the approach provides an excellent tool for exploring the morphology of Alternaria under different assumptions, as well as understanding how it is largely the consequence of local rules for growth. Further, different choices of parameters lead to different model groups, which can then be visually compared to published descriptions or microscopy images to validate parameters for species-specific models. The approach supports automated analysis, as the models can be fit to image data using statistical inference, and the explicit representation of the geometry allows the accurate computation of any morphological quantity. Furthermore, because the model can encode the statistical variation of geometric parameters for different species, it will allow automated species identification from microscopy images using statistical inference. In summary, the approach supports visualization of morphology, automated quantification of phenotype structure, and identification based on form.

Differentially expressed cDNAs in Alternaria alternata treated with 2-propenyl isothiocyanate.

The molecular mechanism of the fungal tolerance phenotype to fungicides is not completely understood. This knowledge would allow for the development of environmentally friendly strategies for the control of fungal infection. With the goal of determining genes induced by 2p-ITC, a forward suppressive subtractive hybridization was performed using cDNAs from ITC-treated Alternaria alternata as a "tester" and from untreated fungus as a "driver." Using this approach, a library containing 102 ESTs was generated that resulted in 50 sequences after sequence assembly (17 contigs and 33 singletons). Blastx analysis revealed that 38% and 40% of the sequences showed significant similarity with known and hypothetical proteins, respectively, whereas 18% were not similar to known genes. These last sequences could represent novel genes that play an unknown role in the molecular responses of fungi during adaptation to 2p-ITC. Clones similar to opsins, ABC transporters, calmodulin, ATPases and SNOG proteins were identified. Using real-time RT-PCR analysis, significant inductions of an ABC transporter and a Ca(++) ATPase during 2p-ITC treatment were discovered. These results suggest that the fungal resistance phenotype to 2p-ITC involves calcium ions and 2p-ITC efflux via an ABC transporter.