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The omnipresent dematiaceous hyphomycete genus Alternaria is associated with a wide variety of substrates including seeds, plants, agricultural products, humans, soil and even the atmosphere. It includes saprophytic, endophytic and pathogenic species, among which multiple plant pathogens, post-harvest pathogens, and human pathogens (causative agents of phaeohyphomycosis and hypersensitivity reactions). Molecular studies reveal that the Alternaria complex comprises nine genera. Within this complex several genera are non-monophyletic and Alternaria species cluster into multiple distinct species clades, which are not always correlated with species-groups based on morphological characteristics. The most commonly reported species in literature and type species of the genus Alternaria, A. alternata, also comprises one such species-group. The small-spored Alternaria species within this group are mainly described based on morphology and / or host-specificity, but are difficult to distinguish based on molecular techniques alone. As A. alternata is considered as one of the most prolific producers of fungal allergens and is reported as pathogen on over 100 host plants, correct species identification is of utmost importance. The research presented in this thesis discusses the taxonomic status of Alternaria and its related genera, with a further focus on the two biggest and most important species complexes; the large-spored A. porri and small-spored A. alternata species complexes. With the phylogenies and classifications presented in this thesis, more robust and understandable taxonomy and nomenclature in Alternaria and allied genera within the Alternaria complex are created.
Chapter 1 gives a general introduction to the genus Alternaria and related genera. The history of the genus and its economic importance as plant pathogen, post-harvest pathogen, causative agent of phaeohyphomycosis and common allergen causing hypersensitivity reactions are summarized. The introduction of the morphological species complexes, based on characters of the conidia, the pattern of chain formation, and the nature of the apical extensions of conidia are treated. Molecular studies recognise seven Alternaria species-groups within the Alternaria complex. Besides Alternaria, eight other genera are assigned to the Alternaria complex based on molecular and morphological studies.
Chapter 2 focusses on the relationship of Alternaria and its closely related genera within the broader Alternaria complex. The phylogenetic lineages within the Alternaria complex are delineated based on nucleotide sequence data of parts of the 18S nrDNA (SSU), 28S nrDNA (LSU), the internal transcribed spacer regions 1 and 2 and intervening 5.8S nrDNA (ITS), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), RNA polymerase second largest subunit (RPB2) and translation elongation factor 1-alpha (TEF1) gene regions. The phylogenetic data reveal a Stemphylium clade sister to Embellisia annulata and a big Alternaria clade. The Alternaria clade contains six monotypic lineages and 24 internal clades, which are treated as sections of Alternaria. In order to create a stable phylogenetic taxonomy, and supported by i) a well-supported phylogenetic node in multiple analyses, ii) a high-similarity of clades within Alternaria based on SSU, LSU and ITS data, and iii) variation in the clade order between the different gene phylogenies, 13 genera are placed into synonymy with Alternaria. Embellisia annulata is synonymized with Dendryphiella salina, and together with D. arenariae placed in the new genus Paradendryphiella. The sexual genera Clathrospora and Comoclathris, with asexual forms linked to Alternaria, cluster within the Pleosporaceae, as does Alternaria, but outside Alternaria s. str. The genus Alternariaster, described to accommodate Alternaria helianthi, clusters within the Leptosphaeriaceae.
Chapter 3 describes the reappraisal of the genus Alternariaster. Alternaria helianthi, the causal agent of leaf spot on Helianthus annuus (sunflower) was segregated from Alternaria based on conidial morphology, and placed in the new genus Alternariaster. A multi-gene phylogeny of parts of the ITS, LSU, RPB2 and GAPDH gene regions placed a fungal pathogen associated with leaf spot on Bidens sulphurea (yellow cosmos) in Brazil in close relation with Al. helianthi. Based on the close phylogenetic relation to Al. helianthi, but distinct morphological and pathogenicity characters, the fungal pathogen associated with leaf spot on B. sulphurea is newly described as Al. bidentis.
Chapter 4 treats the Alternaria species which form the largest section of Alternaria, sect. Porri. This section contains almost all Alternaria species with medium to large conidia with long beaks, some of which are important plant pathogens. A multi-gene phylogeny on parts of the ITS, GAPDH, RPB2, TEF1 and Alternaria major allergen (Alt a 1) gene regions, supplemented with morphological and cultural studies, forms the basis for species recognition in this section. The polyphasic data reveal 63 species in sect. Porri, of which 10 are newly described, and 27 names are synonymized.
Chapter 5 treats the small-spored Alternaria species, which reside in sect. Alternaria. A lot of confusion around the naming of species within this section exists, since the naming is mostly based on morphology and host-specificity, although the molecular variation is minimal. Whole genome sequencing, combined with transcriptome profiling and multi-gene sequencing of nine gene regions, SSU, LSU, ITS, GAPDH, RPB2, TEF1, Alt a 1, endopolygalacturonase (endoPG) and an anonymous gene region (OPA10-2), is used to create a clear and stable species classification in this section. The nine sequenced Alternaria genomes range in size from 32.0 - 39.1 Mb. The number of repetitive sequences varies significantly, with a relative low percentage of repeats within sect. Alternaria. The genome identity within sect. Alternaria is high, compared to the genome identity for isolates from other sections to the A. alternata reference genome. Similarly, a relative low percentage of single nucleotide polymorphisms (SNPs) were observed in genomic and transcriptomic sequences between isolates from sect. Alternaria, compared to the percentage of SNP’s found in isolates from different sections compared to the A. alternata reference genome. A set of core proteins was extracted from the genome and transcriptome data, and primers were designed on two eukaryotic orthologous group (KOG) protein loci with a relatively low degree of conservation within section Alternaria. The phylogenies from these two gene regions, KOG1058 and KOG1077, could not distinguish the described morphospecies within sect. Alternaria better than the phylogenies based on the nine commonly used gene regions for Alternaria. Based on genome and transcriptome comparisons and molecular phylogenies, Alternaria sect. Alternaria consists of only 11 phylogenetic species and one species complex. Thirty-five morphospecies are synonymized under A. alternata. The subclades that are formed by these isolates are incongruent between the different gene regions sequenced; no two genes show the same groupings for any of the over 100 isolates. A sequence-based identification guide is provided for the species which are now recognized in sect. Alternaria. None of the genes sequenced in this study can distinguish all of the species recognized here on its own.
Chapter 6 investigates the molecular diversity of indoor Alternaria isolates in the USA, with the help of a phylogeographic / population genetic approach. Isolates collected throughout the USA were identified using ITS, GAPDH and endoPG gene sequencing, followed by genotyping and population genetic inference of the sect. Alternaria isolates and 37 reference isolates, using five microsatellite markers. Phylogenetic analyses revealed that 98 % (153 isolates) of the indoor isolates consisted of species from Alternaria sect. Alternaria. The remaining 2 % (three isolates) represented one sect. Infectoriae and two sect. Pseudoulocladium isolates. From the 153 isolates that belonged to sect. Alternaria, one could be assigned to A. burnsii, 15 to the A. arborescens species complex and the remaining 137 isolates were identified as A. alternata. Based on the microsatellite data, no specific indoor population could be distinguished. Population assignment analyses of the A. alternata isolates suggested that subpopulations did not exist within the sample, which we thus divided into four artificial subpopulations to represent four quadrants of the USA. Genotypic diversity was extremely high for all quadrants and a test for linkage disequilibrium suggested that A. alternata has a cryptic sexual cycle. The SouthWest-USA population displayed the highest level of uniqueness, based on private alleles. Intriguingly, the highest amount of gene flow, between SouthWest-USA and SouthEast-USA, correlated with the west-to-east movement of the antitrade winds. This suggests that indoor A. alternata isolates, although extremely diverse, have a continental distribution and high levels of gene flow over the continent.
Chapter 7 discusses the data presented in this thesis. The implications of the performed studies are placed in a broader context, with a focus on the relation between morphology and the new species classification based on molecular tools and the use of genome data in contrast to multi-gene data.
|Qualification||Doctor of Philosophy|
|Award date||10 Sept 2015|
|Place of Publication||Wageningen|
|Publication status||Published - 2015|
- molecular taxonomy
- plant pathogenic fungi