Highlights of the Didymellaceae: A polyphasic approach to characterise Phoma and related pleosporalean genera

Fungal taxonomists routinely encounter problems when dealing with asexual fungal species due to poly- and paraphyletic generic phylogenies, and unclear species boundaries. These problems are aptly illustrated in the genus Phoma. This phytopathologically significant fungal genus is currently subdivided into nine sections which are mainly based on a single or just a few morphological characters. However, this subdivision is ambiguous as several of the section-specific characters can occur within a single species. In addition, many teleomorph genera have been linked to Phoma, three of which are recognised here. In this study it is attempted to delineate generic boundaries, and to come to a generic circumscription which is more correct from an evolutionary point of view by means of multilocus sequence typing. Therefore, multiple analyses were conducted utilising sequences obtained from 28S nrDNA (Large Subunit - LSU), 18S nrDNA (Small Subunit - SSU), the Internal Transcribed Spacer regions 1 & 2 and 5.8S nrDNA (ITS), and part of the ß-tubulin (TUB) gene region. A total of 324 strains were included in the analyses of which most belonged to Phoma taxa, whilst 54 to related pleosporalean fungi. In total, 206 taxa were investigated, of which 159 are known to have affinities to Phoma. The phylogenetic analysis revealed that the current Boeremaean subdivision is incorrect from an evolutionary point of view, revealing the genus to be highly polyphyletic. Phoma species are retrieved in six distinct clades within the Pleosporales, and appear to reside in different families. The majority of the species, however, including the generic type, clustered in a recently established family, Didymellaceae. In the second part of this study, the phylogenetic variation of the species and varieties in this clade was further assessed. Next to the genus Didymella, which is considered to be the sole teleomorph of Phoma s. str., we also retrieved taxa belonging to the teleomorph genera Leptosphaerulina and Macroventuria in this clade. Based on the sequence data obtained, the Didymellaceae segregate into at least 18 distinct clusters, of which many can be associated with several specific taxonomic characters. Four of these clusters were defined well enough by means of phylogeny and morphology, so that the associated taxa could be transferred to separate genera. Aditionally, this study addresses the taxonomic description of eight species and two varieties that are novel to science, and the recombination of 61 additional taxa

Systematic reappraisal of species in Phoma section Paraphoma, Pyrenochaeta and Pleurophoma

Sequence data from the 18S nrDNA (SSU) and 28S nrDNA (LSU) regions of isolates of Phoma section Paraphoma were compared with those of representative isolates of the morphologically similar anamorph genera Pleurophoma and Pyrenochaeta and of the type species of Phoma sections Phoma, Pilosa and Plenodomus. Phoma section Paraphoma was found to be highly polyphyletic within the Pleosporales and only distantly related to Phoma section Phoma. The genus Paraphoma, which is based on Paraphoma radicina, is reintroduced in the Phaeosphaeriaceae with two additional taxa. The new genera Setophoma and Neosetophoma, type species Setophoma terrestris comb. nov. and Neosetophoma samarorum comb. nov., are introduced and represent species that are closely related to Paraphoma but differ based on morphological characters and molecular phylogeny. Phoma coonsii is transferred to genus Chaetosphaeronema that also belongs to the Phaeosphaeriaceae. Pyrenochaetopsis gen. nov. is introduced to accommodate the type species Pyrenochaetopsis leptospora comb. nov., as well as several other species formerly accommodated in Phoma and Pyrenochaeta. Pyrenochaetopsis is closely related to Pyrenochaeta and classified in the Cucurbitariaceae. Pleurophoma cava is transferred to genus Pyrenochaeta. The new genera elucidate the confusing taxonomy of species in genera Phoma, Pyrenochaeta and Pleurophoma and recognize monophyletic genera with distinct teleomorph affinities

Multiple Didymella teleomorphs are linked to the Phoma clematidina morphotype

The fungal pathogen Phoma clematidina is used as a biological agent to control the invasive plant species Clematis vitalba in New Zealand. Research conducted on P. clematidina as a potential biocontrol agent against C. vitalba, led to the discovery of two perithecial-forming strains. To assess the diversity of P. clematidina and to clarify the teleomorph-anamorph relationship, phylogenetic analyses of 18 P. clematidina strains, reference strains representing the Phoma sections in the Didymellaceae and strains of related species associated with Clematis were conducted. Partial sequences of the ITS1, ITS2 and 5.8S rRNA gene, the ß-tubulin gene and 28S rRNA gene were used to clarify intra- and inter-species relationships. These analyses revealed that P. clematidina resolves into three well-supported clades which appear to be linked to differences in host specificity. Based on these findings, Didymella clematidis is newly described and the descriptions of P. clematidina and D. vitalbina are amended

Redisposition of Phoma-like anamorphs in Pleosporales

The anamorphic genus Phoma was subdivided into nine sections based on morphological characters, and included teleomorphs in Didymella, Leptosphaeria, Pleospora and Mycosphaerella, suggesting the polyphyly of the genus. Recent molecular, phylogenetic studies led to the conclusion that Phoma should be restricted to Didymellaceae. The present study focuses on the taxonomy of excluded Phoma species, currently classified in Phoma sections Plenodomus, Heterospora and Pilosa. Species of Leptosphaeria and Phoma section Plenodomus are reclassified in Plenodomus, Subplenodomus gen. nov., Leptosphaeria and Paraleptosphaeria gen. nov., based on the phylogeny determined by analysis of sequence data of the large subunit 28S nrDNA (LSU) and Internal Transcribed Spacer regions 1 & 2 and 5.8S nrDNA (ITS). Phoma heteromorphospora, type species of Phoma section Heterospora, and its allied species Phoma dimorphospora, are transferred to the genus Heterospora stat. nov. The Phoma acuta complex (teleomorph Leptosphaeria doliolum), is revised based on a multilocus sequence analysis of the LSU, ITS, small subunit 18S nrDNA (SSU), ß-tubulin (TUB), and chitin synthase 1 (CHS-1) regions. Species of Phoma section Pilosa and allied Ascochyta species were determined to belong to Pleosporaceae based on analysis of actin (ACT) sequence data. Anamorphs that are similar morphologically to Phoma and described in Ascochyta, Asteromella, Coniothyrium, Plectophomella, Pleurophoma and Pyrenochaeta are included in this study. Phoma-like species, which grouped outside the Pleosporineae based on a LSU sequence analysis, are transferred to the genera Aposphaeria, Paraconiothyrium and Westerdykella. The genera Medicopsis gen. nov. and Nigrograna gen. nov. are introduced to accommodate the medically important species formerly known as Pyrenochaeta romeroi and Pyrenochaeta mackinnonii, respectively

Taxonomie van plant-pathogene schimmels als basis voor identificatie en detectie: resultaten van het Uitvoeringsconsortium Schimmels

Plant-pathogene schimmels worden traditioneel geïdentificeerd op basis van morfologische karakteristieken. Dit is over het algemeen tijdrovend, veel expertise en leidt vaak tot misidentificatie. Het doel van het werk uitgevoerd door het Uitvoeringsconsortium Schimmels was dan ook gericht op de verbetering van detectie- en identificatietechnieken van plant pathogene schimmels met de nadruk op quarantaineorganismen. Op grond van het economische, wetenschappelijke, en ecologische belang, de aanwezigheid van Q-organismen, en de wetenschappelijke startpositie binnen Nederland is in eerste instantie de keuze gevallen op de geslachten Colletotrichum, Phoma, en Phytophthora. De gegenereerde dat zijn opgenomen in de Q-bank

Phylogeny and DNA-based identification in Phoma and related genera

This thesis treats the taxonomy of a generic complex presently known as PhomaSacc. emend Boerema &amp; Bollen. This group of fungi comprises more than 200 taxa at species or variety level that are characterised by the production of hyaline, non-septate conidial spores in pycnidial conidiomata. The genus is omnipresent in the environment, and exponents can be found on a wide range of host substrates. For many years the genus Phomawas the main research topic of a group of mycologists at the Dutch National Plant Protection Service. The studies conducted in the last decennia of the previous century culminated in a handbook that monographed the majority of the species in the above-mentioned generic complex. This handbook marked the end of the era in which the taxonomy of this genus mainly relied on morphological observations and cultural descriptions. However, it can also be regarded as the starting point of the present study. The aim of the present project was to integrate DNA-based identification methods into the taxonomic system established by the previously mentioned group of researchers. The major part of this study therefore deals with the validation of current generic and species concepts. An extensive literature review of the biology, taxonomy and identification methods to the species in this genus is provided in <strong>Chapter 2</strong>, with specific reference to the progress that has been made in Phomataxonomy after the publication of the abovementioned handbook. The advantages and disadvantages of the current taxonomical system are discussed. Furthermore, this chapter describes the general biology of the species in this fungal group, including their life cycles, distribution and host substrates. The importance of the genus for plant health and quarantine issues is illustrated, and the development of a rapid and robust identification technique based on DNA barcodes is advocated. <strong>Chapter 3</strong>treats species in Phomasection Peyronellaea. Species in this section are typified by the production of dictyochlamydospores, and thus have additional morphological characters to use in taxon delineation in comparison with species in the other Phomasections. All species in this group were subjected to a morphological re-examination and phylogenetic analyses employingITS, actin, and &beta;-tubulin nucleotide sequences. Based on multi-gene analyses, Phomasection Peyronellaeacould not be maintained as a taxonomic entity, due to the polyphasic nature of taxa in this section. The morphological study revealed that for five species a taxonomic revision was required. A further five species appeared to be new to science, including Ph. microchlamydospora, Ph. omnivirens, and Ph. schachtii. Also the taxonomic noveltiesPh. coffeae-arabicaeand Ph. sancta are described here, and are allocated to the genus Peyronellaea, re-erected in Chapter 5. In <strong>Chapter 4</strong>the diversity among species and varieties belonging to the Ph. exiguaspecies complex is investigated. The Ph. exiguaspecies complex includes nine taxa at varietal level and four species that have a high morphological similarity both in vivoand in vitro, whilst historical relations with plant hosts cannot be maintained. Among this group, both omnipresent saprobes as well as host-specific plant pathogens are present &ndash; including the potato pathogen Ph. foveata. The diversity in this complex is studied by means of Internal Transcribed Spacer regions 1 &amp; 2 and intervening 5.8S nrDNA (ITS) and actin nucleotidesequence analyses and a DNA fingerprinting technique rarely used to study fungal diversity. This technique, DNA Amplification Fingerprinting (DAF) employs short, arbitrary primers that form a loop, or a mini-hairpin, under specific temperature conditions and is frequently used in molecular plant breeding. The amplified DNA fragments were isolated and sequenced in order to develop taxon-specific markers and primer combinations based on the SCARs (Sequence Characterised Amplified Regions) and actin sequence data generated. These tools can aid rapid identification of this morphologically highly similar set of taxa. Two separate taxa were recognised within the type variety Ph. exiguavar. exigua. In the following chapter these taxa are described and all species and varieties in this complex are recombined into the new genus Boeremia. <strong>Chapter 5</strong>provides further details about the taxonomy and phylogeny of the species of interest, with a special focus on the taxa that are phylogenetically placed with Didymellaceae. In total 206 taxa were treated, of which 159 have affinities with Phoma. The genus is circumscribed in the first section of this chapter. The phylogeny was reconstructed using 28S nrDNA (Large Subunit) and 18S nrDNA (Small Subunit) sequence data. It was shown that the currently used Boeremaean subdivision of the phomoid taxa and the phylogeny were inconsistent, as the genus was highly polyphyletic. Species belonging to the form-genus Phomawere retrieved in as much as six distinct clades within Pleosporales. These clades even represent different families. The majority of the phomoid taxa, including the type species Ph. herbarumand most exponents of the sections Macrospora, Peyronellaea, Heterospora, and Phyllostictioides, were found in a single clade that represented the Didymellaceae. Most species that are associated with the Phomasections Plenodomusand Pilosacluster with the Leptosphaeriaceaeand Pleosporaceaeclades. Furthermore, some species were also found to cluster in the Sporormiaceaeand Cucurbitariaceaeclades. In the second part of this chapter, the phylogenetic variation of the species and varieties in Didymellaceaeis further assessed, using a phylogenetic reconstruction that is based on DNA sequences of the Large Subunit, ITS, and part of the &beta;-tubulin (TUB) gene region. Besides the teleomorph genus Didymella, members of the teleomorph genera Leptosphaerulinaand Macroventuriawere also found to cluster in this clade. Based on the reconstructed phylogeny, Didymellaceaesegregate into at least 18 distinct clusters, of which many can be associated with specific morphological characters. Furthermore, a number of taxa did not match any of these clusters, suggesting that an evolutionary correct subdivision of the Phomaspecies in Didymellaceaeis even more complex. Taxa in four of these phylogenetic clusters were also defined well enough by means of morphology to elevate these groups to new or reinstalled genera, namely Stagonosporopsis, Epicoccum, Boeremiaand Peyronellaea. A total of 61 taxa were recombined and several new species of Phomawere introduced, namely Ph. brasiliensis, Ph. bulgarica, Ph. dactylidis, Ph. dimorpha, Ph. longicolla, Ph. minor, Ph. pedeiaeand Ph. saxea. Furthermore, two new varieties were described, Boeremia exigua var. gilvescensand B. exigua var. pseudolilacis. Finally, the results presented in this dissertation are highlighted and discussed in<strong>Chapter 6</strong>. In total 13 species of Phomaand two taxa at varietal level were newly described during the course of this study. Moreover the taxonomic status of species in the form-genus Phomawere further clarified, and insight provided into the phylogenetic status of Didymellaceae, a fungal family that was recently established, comprising most species of Phoma, Ascochytaand Didymella. All macro- and micromorphological data obtained in this study, as well as the DNA sequences, were placed online in a publicly available polyphasic identification database (www.q-bank.eu). This will enable scientists and institutes involved in plant health to correctly identify phomoid species. Rapid identification of these species based on the tools and data generated in this study, can facilitate swift clearing of plant material and arable products during export and import, and prevent the spread of quarantine organisms

Biology and recent developments in the systematics of Phoma, a complex genus of major quarantine significance.

Species of the coelomycetous genus Phoma are ubiquitously present in the environment, and occupy numerous ecological niches. More than 220 species are currently recognised, but the actual number of taxa within this genus is probably much higher, as only a fraction of the thousands of species described in literature have been verified in vitro. For as long as the genus exists, identification has posed problems to taxonomists due to the asexual nature of most species, the high morphological variability in vivo, and the vague generic circumscription according to the Saccardoan system. In recent years the genus was revised in a series of papers by Gerhard Boerema and co-workers, using culturing techniques and morphological data. This resulted in an extensive handbook, the ¿Phoma Identification Manual¿ which was published in 2004. The present review discusses the taxonomic revision of Phoma and its teleomorphs, with a special focus on its molecular biology and papers published in the post-Boerema era