Rodney G. Roberts

Chemical and morphological segregation of Alternaria arborescens , A. infectoria and A. tenuissima species-groups

Correct morphological identification of Alternaria is important and demands a combination of modern standardised methods and up-to-date literature. The production of secondary metabolites has previously been used as a means of identification and classification. In this study, 153 fungal isolates belonging to the genus Alternaria were examined. They were grown under standardised conditions and subjected to morphological and chemical examination. All isolates were grouped according to their three-dimensional sporulation pattern on potato carrot agar and their colony colour on dichloran rose bengal yeast extract sucrose agar (DRYES). After extraction, all isolates were analysed by a high performance liquid chromatograph equipped with a diode array detector and the resulting metabolite profiles were subjected to multivariate statistic analyses. The analyses of metabolite profiles showed that the isolates could be divided into three major species-groups that were morphologically identifiable as the A. infectoria species-group, the A. arborescens species-group and the A. tenuissima species-group. The A. infectoria species-group is chemically very different from both the A. arborescens and the A. tenuissima species-groups with only a few metabolites in common. None of the 35 A. infectoria species-group isolates produced any known metabolites and all had white or greyish white colonies on DRYES. The A. arborescens species-group and the A. tenuissima species-group, shared most of the known metabolites and had colonies of various shades of green on DRYES. One cluster of isolates belonging to the A. tenuissima species-group was able to produce tentoxin, which has not been reported previously from any A. tenuissima isolate. The results suggest that each species-group contains several taxa and these taxa need to be formally described before species specific metabolite profiles can be established.

Chemical and morphological segregation of Alternaria alternata, A. gaisen and A. longipes

Correct identification of fungi to species level is important because a specific epithet embodies a set of characters that enables us to predict, for example, the mycotoxin production of a species. Many small-spored Alternaria isolates have been misidentified due to inappropriate growth conditions and the use of spore size as the only identifying character. In this study 39 Alternaria isolates were grown under standardised conditions and subjected to chemical, morphological and physiological analyses. All isolates were extracted and analysed by HPLC-DAD. Analysis showed that both A. gaisen and A. longipes were able to produce altertoxin 1, which has not previously been reported. The resulting metabolite profiles were subjected to cluster analysis and principal component analysis. A subset of the isolates was grown at five different temperatures. Colony colour and diameter were recorded and the diameter measurements were subjected to principal component analysis. Analysis of chemical and physiological data showed that the 39 isolates segregated into the same distinct groups that are morphologically identifiable as A. alternata, A. longipes or A. gaisen. The results showed that A. longipes, A. gaisen and A. alternata are different species that can be distinguished morphologically, physiologically and chemically. Therefore, the continued use of the name Alternaria alternata for A. longipes and A. gaisen is unwarranted and pathotypes should not be used.

RAPD fragment pattern analysis and morphological segregation of small-spored Alternaria species and species groups

Accurate identification of small-spored Altemaria spp. is challenging because of morphological plasticity under non-standard conditions and the common misapplication of the name A. alternata to a variety of morphologically distinct taxa. A system used by some authors of naming phytotoxigenic alternarias as pathotypes of A. alternata has further clouded the meaning and usefulness of this specific epithet. Two hundred and sixty isolates of small-spored Alternaria , primarily from fruit substrates, were segregated into morphological groups and then subjected to RAPD-PCR analysis using total genomic DNA and three different primers. When cultured under defined conditions, the pattern of sporulation observed at 50 × magnification was predictive of genetic relatedhess as determined by cluster analysis of RAPD fragment patterns. In RAPD analyses, morphological groups or species were resolved as distinct branches of the dendrogram: Alternaria gaisen (= A. kikuchiana, A. alternata Japanese pear pathotype, group 2), A. longipes (= A. alternata tobacco pathotype, group 5), the ‘ tenuissima ’ group (group 5), the arborescens group (group 3) and the ‘ infectoria group (group 6). Isolates in groups for which there were only a few representatives clustered as a branch. Analysis of RAPD fragment patterns confirmed that when Altemaria isolates are cultured and observed under defined conditions, their phenotypic plasticity is minimized and valid taxonomic separations can be made upon morphological characteristics. We conclude that A. alternata, A. gaisen, A. longipes and other small-spored saprotrophic or perthotrophic taxa are recognizable as morphologically distinct taxa. We suggest that the ‘pathotype’ system of naming small-spored Altemaria taxa confers no predictive value relative to observable morphological and genetic characters, and should be abandoned.

Shared ITS DNA substitutions in isolates of opposite mating type reveal a recombining history for three presumed asexual species in the filamentous ascomycete genus Alternaria

About 15,000 species of ascomycete fungi lack a known sexual state. For fungi with asexual states in the anamorph genera Embellisia, Ulocladium, and Alternaria, six species have known sexual states but more than 50 species do not. In sexual filamentous ascomycetes, opposite mating type information at the MAT1 locus regulates mating and the opposite mating type genes each have a clonal, non-recombining phylogenetic history. We used PCR to amplify and sequence fragments of the opposite mating type genes from three supposedly asexual species, A. brassicae, A. brassicicola and A. tenuissima. Each haploid fungal isolate had just one mating type, but both mating types were present in all the three species. We sequenced the ribosomal ITS regions for isolates of opposite mating type, for the three asexual species and four known related sexual species. In a phylogenetic analysis including other ITS sequences from GenBank, the three asexual species were not closely related to any of the known sexual species. Isolates of opposite mating type but the same species had identical ITS sequences. During any period of asexual evolutionary history, lineages of each mating type would have had a separate evolutionary history and any ITS substitutions shared between isolates of opposite mating type would have had to accumulate by convergence. Allowing for varying substitution rates and assuming a Poisson distribution of substitutions, the probability that isolates of opposite mating type shared an ITS substitution through convergence was low. This suggests that isolates of opposite mating type of A. brassicae, A. brassicicola and A. tenuissima were exchanging substitutions through sexual or parasexual reproduction while the ITS was evolving. If sexuality was lost, it was lost after the period of evolutionary history represented by the shared substitutions.

Extracellular Lipase Production by Fungi from Sunflower Seed

ABSTRACTTwo hundred and twenty-three lyophilized isolates of fungi representing 91 species from sunflower seed were revived and grown in lipase medium with and without emulsified sunflower oil. Lip...

New and rare fungi from cherry fruits.

Capronia hystrioides sp. nov. (anamorph Phaeoramularia hachijoensis) was isolated from cherry fruit. Also isolated were Sporormiella subticinensis comb. nov., Leptodiscella africana, and Cladosporium malorum, a synonym of C. porophorum.

COMPARATIVE STUDIES OF CRYPTOSPORIOPSIS CURVISPORA AND C. PERENNANS. II. CYTOLOGY AND VEGETATIVE COMPATIBILITY

Cryptosporiopsis curvispora, cause of apple anthracnose, and C. perennans, cause of perennial canker, were indistinguishable in gross cytology; macroconidia, microconidia, phialides and hyphal cells of each species were mostly uninucleate. Complementation of nitrate-nonutilizing mutants occurred within and between isolates of each species but not between species. Such complementations were common within and between isolates of C. perennans but rare within and between isolates of C. curvispora. The two fungi are considered to be distinct species. The teleomorph of C. perennans is transferred to Pezicula as P. perennans comb. nov.

Ascotricha xylina: its occurrence, morphology, and typification

The isolation of Ascotricha xylina from sunflower (Helianthus annuus) oilseed grown in the U.S. is the first record of this fungus from sunflower seed and from the Western hemisphere. Examination of specimens of A. xylina from world herbaria confirmed the determination as A. xylina, and led to the discovery ofthe holotype that was lost for 33 years and the recognition of an isotype. The sizes of ascospores and widths of perithecial hairs from these specimens showed greater variation than described by Ames. Undescribed conidiogenous areas on perithecial hair apices were observed in culture. Centrum tissues appeared similar to other Ascotricha species, with broad, septate paraphyses interspersed among asci with non-amyloid apices.

Alternaria cerasidanica sp nov., isolated in Denmark from drupes of Prunus avium

The ex-type strain of Alternaria cerasidanica was isolated in 2001 from an immature, asymptomatic drupe of Prunus avium collected at a commercial cherry orchard near Sklskør, Denmark. Cultural morphology, sporulation pattern and cluster analyses of combined RAPD, RAMS (microsatellite), and AFLP fingerprints of A. cerasidanica and 167 strains of Alternaria spp. support the placement of A. cerasidanica within the A. infectoria species-group sensu Simmons and its segregation from other members of this group. A. cerasidanica is currently monotypic and known only from preharvest sweet cherry fruit in Denmark.