Revised taxonomy of Phoma and allied genera

The anamorphic genus Phoma includes many important plant pathogens. The identification of Phoma species based on studies in pure culture is difficult and time consuming and the in vitro characters are often variable. Moreover, the present classification of Phoma species into sections is ambiguous and morphological characters are shared with related genera. In the present study the molecular phylogeny of species of Phoma and allied genera was examined and the results obtained were used to revise the taxonomy. The DNA sequence data obtained provide tools for the development of detection and identification methods. Chapter 1 provides a general introduction of the anamorph genus Phoma and the research that has been performed in The Netherlands during the last decades is described. Phoma is characterised by producing hyaline conidia in fruiting bodies called pycnidia. The genus includes many important plant pathogens. The taxonomy of Phoma has been studied intensively at the Plant Protection Service in the Netherlands for more than 50 years, resulting in the development of a generic concept in 1997 as an outline for identification of Phoma species. In this concept species of the genus Phoma are classified based on their morphological characters into the nine sections Phoma, Heterospora, Macrospora, Paraphoma, Peyronellaea, Phyllostictoides, Pilosa, Plenodomus and Sclerophomella. The species placed in each of the sections were systematically described culminating in the publication of the “Phoma Identification Manual” in 2004, with the descriptions of 223 specific and infra-specific taxa of Phoma, and more than 1000 synonyms in other coelomycetous genera. In the Netherlands the late Gerhard Boerema, former head of the Mycology Department at the Plant Protection Service, has been the driving force behind this Phoma research for decades. The Phoma Identification Manual is a valuable tool for the morphological identification of isolates, but in vitro studies are very time consuming and need a high level of expertise. Moreover, the classification of Phoma species in sections based on morphological characters appeared artificial and several species can be classified in more than one section because of their multiple “section-specific” characters. In addition, distinctive characters of Phoma sections are shared among morphologically related coelomycetous genera including Ascochyta, Asteromella, Microsphaeropsis, Phomopsis, Phyllosticta, Pleurophoma, Pyrenochaeta and Stagonospora. Phoma sections are related to diverse teleomorph genera including Didymella, Leptosphaeria, Mycosphaerella and Pleospora. Synanamorphs of Phoma species have been recognised amongst the genera Phaeomoniella, Stagonosporopsis, Epicoccum, Phialophora and Sclerotium illustrating their heterogeneity. A large, well-studied Phoma culture collection established at the Plant Protection Service and the “Centraalbureau voor Schimmelcultures” includes more than 1100 strains of Phoma species. This collection formed the basis of an intensive molecular phylogenetic study of the genus Phoma and morphologically similar genera, which commenced in 2006. Furthermore, a literature study identified sequences of genes that are suitable for phylogenic studies and elucidation of the evolutionary history of the genus Phoma. Several potentially informative regions of the genome were sequenced in the first phase of the project as has been described in chapters 2–4. The phylogeny and DNA sequence data obtained have provided tools for the development of fast and reliable molecular detection and identification methods. The development of Real-time TaqMan PCR methods for the detection and identification of two important plant pathogenic (quarantine) species formerly described in Phoma, Stagonosporopsis andigena and S. crystalliniformis, is described in Chapter 5. In chapter 2 several genes were studied to elucidate the molecular phylogeny of Phoma and allied genera. Sequence data of the 18S nrDNA (SSU) and 28S nrDNA (LSU) regions of the type species of the Phoma sections and morphologically similar coelomycetes and related teleomorphs were compared. The results justified the introduction of the new family Didymellaceae to accommodate the generic type species Didymella exigua and Phoma herbarum. The type species of the Phoma sections Phyllostictoides, Sclerophomella, Macrospora and Peyronellaea also grouped in Didymellaceae. The generic type species Ascochyta pisi and Microsphaeropsis olivacea also grouped in Didymellaceae and it shows that these genera are closely allied to Phoma. The type species of Phoma sections Heterospora, Paraphoma, Pilosa and Plenodomus grouped in various families outside Didymellaceae and were subject of following studies. Chapter 3 provides a molecular phylogenetic re-evaluation on Phoma-like species that appeared only distantly related to the generic type species Phoma herbarum and its related Didymella teleomorph (Didymellaceae). Phoma section Paraphoma, characterised by setose pycnidia, resembles species of Pyrenochaeta and Pleurophoma. Sequence data from the SSU and LSU regions of the species classified in Phoma section Paraphoma were compared with those of representative isolates of Pyrenochaeta and Pleurophoma, and with those of the type species of the Phoma sections Phoma, Pilosa and Plenodomus. Unnamed, often sterile Phomalike strains in the collections were included. The molecular phylogeny of species that were classified in Phoma section Paraphoma appeared to be highly polyphyletic and a thorough reclassification of the species is provided. Paraphoma was reinstalled and grouped with the new genera Neosetophoma and Setophoma in Phaeosphaeriaceae. Pyrenochaeta and the new genus Pyrenochaetopsis, including mainly taxa formerly described in Phoma section Paraphoma, were closely allied in Cucurbitariaceae. In chapter 4 the molecular phylogeny of species of Phoma sections Plenodomus, Pleospora and Heterospora was determined using LSU, SSU and ITS. In a “one species = one name” approach, the species described in Phoma section Plenodomus and its teleomorph Leptosphaeria were reclassified in Leptosphaeria, Plenodomus and the new genera Paraleptosphaeria and Subplenodomus in Leptosphaeriaceae. Two species of Phoma section Heterospora, the type species Phoma heteromorphospora and its allied species Ph. dimorphospora, were transferred to the new genus Heterospora that also grouped in Leptosphaeriaceae. Leptosphaeria doliolum comprises a species complex that was revised based on multilocus sequence data of LSU, ITS, SSU, s-tubulin, and chitin synthase 1. The molecular phylogeny of species classified in Ascochyta and Phoma, section Pilosa in Pleosporaceae that produce morphologically similar pilose pycnidia, was determined based on analysis of actin sequence data. Several Phoma-like species grouped outside the suborder Pleosporineae in a LSU sequence analysis and were transferred to the genera Aposphaeria (Melanommataceae), Paraconiothyrium (Montagnulaceae) and Westerdykella (Sporormiaceae). Coniothyrium palmarum and related species were described in Coniothyriaceae. The new genera Medicopsis (Trematosphaeriaceae) and Nigrograna, of which the family is still unknown, are introduced to accommodate two medically important species formerly classified in Pyrenochaeta. In chapter 5 specific real-time (TaqMan) PCR assays were developed for the detection of the pathogens Stagonosporopsis andigena and S. crystalliniformis in leaves of potato and tomato. The molecular phylogeny with related species of Stagonosporopsis, Boeremia and Phoma based on sequence polymorphisms in the actine gene, was determined. The reliability of the DNA extraction and TaqMan PCRs for the detection of S. andigena and S. crystalliniformis in leaf material was tested in performance studies and demonstrated the specificity, analytical sensitivity, reproducibility, repeatability and robustness of both assays.