Revised taxonomy of Phoma and allied genera Johannes (Hans) de Gruyter Thesis committee Promoters Prof. dr. P.W. Crous Professor of Evolutionary Phytopathology, Wageningen University Prof. dr. ir. P.J.G.M. de Wit Professor of Phytopathology, Wageningen University Other members Dr. R.T.A. Cook, Consultant Plant Pathologist, York, UK Prof. dr. T.W.M. Kuyper, Wageningen University Dr. F.T. Bakker, Wageningen University Dr. ir. A.J. Termorshuizen, BLGG AgroXpertus, Wageningen This research was conducted under the auspices of the Research School Biodiversity Revised taxonomy of Phoma and allied genera Johannes (Hans) de Gruyter Thesis submitted in fulfilment of the requirements for the degree of doctor at Wageningen University by the authority of the Rector Magnificus Prof. dr. M.J. Kropff, in the presence of the Thesis Committee appointed by the Academic Board to be defended in public on Monday 12 November 2012 at 4 p.m. in the Aula. Johannes (Hans) de Gruyter Revised taxonomy of Phoma and allied genera, 181 pages. PhD thesis Wageningen University, Wageningen, NL (2012) With references, with summaries in English and Dutch ISBN 978-94-6173-388-7 Dedicated to Gerhard Boerema † CONTENTS Chapter 1 Introduction 9 Chapter 2 Molecular phylogeny of Phoma and allied anamorph 17 genera: towards a reclassification of thePhoma complex Chapter 3 Systematic reappraisal of species in Phoma section 37 Paraphoma, Pyrenochaeta and Pleurophoma Chapter 4 Redisposition of Phoma-like anamorphs in Pleosporales 61 Chapter 5 The development of a validated real-time (TaqMan) 127 PCR for detection of Stagonosporopsis andigena and S. crystalliniformis in infected leaves of tomato and potato Chapter 6 General discussion 145 Appendix References 154 Glossary 167 Summary 170 Samenvatting 173 Dankwoord 176 Curriculum vitae 178 Education statement 179 CHAPTER 1 Introduction 9 Chapter 1 Chapter 1. This chapter gives a historic overview of the events leading to the initiative to write this dissertation. It discusses the changes in the taxonomic definition of Phoma, and highlights some of the important plant pathogenic species in the genus. The scope and outline of the thesis have been provided. Phoma studies in the Netherlands The anamorphic genus Phoma and morphologically similar coelomycetous genera have been studied intensively in the Netherlands during the past 50 years. Phoma became an important topic at the Plant Protection Service (Plantenziektenkundige Dienst, PD) in the sixties of the last century. A new fungal disease was found on seed potatoes, characterised by a dry rot that primarily developed during storage, leading to potentially severe economic losses. Fusarium species are commonly known as the cause of dry rot on potatoes (Secor & Salas 2001, Peters et al. 2008), but isolations made on artificial media at the Mycology Department of the PD revealed an unknown Phoma species as causal agent. Several Phoma species were described as soil inhabitants in literature and recorded on seed potatoes at that time, such as the plurivorous Phoma exigua, Ph. glomerata, Ph. eupyrena and Ph. anserina, but clear taxonomic characters for a proper identification ofPhoma species were lacking. Detailed in vitro studies that were undertaken at the PD by Boerema and co-workers clarified the taxonomy of these plurivorous Phoma species (Boerema & van Kesteren 1962, Dorenbosch 1970), which in turn revealed the disease symptoms to be caused by Ph. foveata, a pathogen first reported from Scotland (Foister 1940). Phoma foveata proved to be morphologically very similar to the plurivorous Ph. exigua (Boerema & Höweler 1967), which often appeared as a secondary invader on dry rot symptoms. Phoma foveata was considered as a pathogenic variety of the latter and reclassified as Phoma exigua var. foveata, the causal agent of potato gangrene (Boerema 1967). Phoma exigua var. foveata is identified in pure culture by the production of anthraquinones that crystallise into fine yellow-green needles. Application of a droplet of 1N NaOH on a culture on oatmeal agar, malt extract agar or cherry-decoction agar shows a violet to red discolouration of these pigments, whereas application of NaOH on a culture of Ph. exigua var. exigua, discolouration from initially green into red occurs, due to the presence of the metabolite ‘substance E’; E+ reaction (Boerema & Höweler 1967). Phoma exigua var. foveata was considered to pose a high risk to the potato industry and the pathogen was subsequently added to the list of quarantine organisms in Europe. A chromatographic detection method was developed to demonstrate the presence of anthraquinones (Mosch & Mooi 1975). To prevent the spread of the pathogen, all lots of seed potatoes were sampled and tested for several years for presence of the pathogen. In time the incidence of Ph. exigua var. foveata decreased, and the damage caused by the pathogen became less significant, possibly due to improved potato storage conditions. Phoma exigua var. foveata was eventually removed from the quarantine list. The pathogen was later accepted at species rank (Boerema, Loerakker & Hamers 1987) and has recently been accommodated in the newly established genus, Boeremia (Aveskamp et al. 2010). Boeremia foveata is still a quarantine organism in several countries outside Europe. A second phytosanitary problem involving Phoma appeared some years later, with the discovery of Phoma lingam, the causal agent of blackleg on Brassica species and their seeds. This initated research to define the in vitro characteristics of the pathogen and those of morphologically related saprobic Phoma species that were simultaneously found on the seed coat (Boerema & van Kesteren 1964). Fundamental studies of Phoma followed on the conidiogenesis of the generic type species Phoma herbarum (Boerema & Bollen 1975). The taxonomy of various Phoma species was 10 Introduction studied (Boerema & Dorenbosch 1973, Boerema 1976) by interpretation of previous in 1 vitro studies by Wollenweber & Hochapfel (1936) and Dennis (1946). Metabolites observed and identified in vitro, such as pigments or crystals, proved to be reliable characters for species identification (Dorenbosch 1970, Monte et al. 1991, Noordeloos et al. 1993). Hundreds of isolates, mainly obtained from infested plant material submitted for diagnosis to the PD, provided the foundation for a thorough revision of the genus Phoma. The culture and morphological characters of the species were combined with ecological data, phytopathological studies and data obtained from herbarium specimens. Phoma species described in old literature could be interpreted, but also many new species were described. This work was performed in collaboration with the Centraalbureau voor Schimmelcultures (CBS) where the taxonomy of the coelomycetes Ascochyta, Asteromella, Coniothyrium, Microsphaeropsis, Phyllosticta, Pleurophoma, Pyrenochaeta, and Stagonospora was studied. These genera show morphological characters that are very similar to Phoma in its broadest generic concept. A next step in Phoma taxonomy was the classification of the genus into nine sections (van der Aa et al. 1990, Boerema 1997). The Phoma sections Phoma, Heterospora, Macrospora, Paraphoma, Peyronellaea, Phyllostictoides, Pilosa, Plenodomus and Sclerophomella were recognised based on the characteristics of the type species of each section. The sections include both specific pathogens of (cultivated) plants and plurivorous, saprophytic or opportunistic species. Standardised descriptions of the Phoma species per section followed in a series of “Contributions towards a monograph of Phoma”, published in Persoonia in the period 1992– 2003 (de Gruyter & Noordeloos 1992, Boerema 1993, 2003, de Gruyter et al. 1993, 1998, 2002, Boerema et al. 1994, 1996, 1997, Boerema & de Gruyter 1998, 1999, van der Aa et al. 2000, de Gruyter 2002, de Gruyter & Boerema 2002) including various new taxa. These contributions were the precursors of the “Phoma Identification Manual” (Boerema et al. 2004). The manual provides keys and descriptions of more than 200 specific and infra-specificPhoma taxa. Our knowledge of the taxonomy of Phoma is still incomplete; the Phoma Identification Manual covers mainly Phoma species from European origin and the most common Phoma species known from other continents. Many Phoma species still remain to be discovered. There have been thousands of Phoma-like species described in literature, even including species from Europe, of which the true identity remains unknown. Most of the Phoma species have an unknown sexual state. As far as has been recorded, the teleomorph of Phoma resides in Didymella, Leptosphaeria, Mycosphaerella and Pleospora. Several Phoma sections are linked to a specific teleomorph genus (Boerema et al. 2004). Synanamorphs have been described in the genera Stagonosporopsis, Epicoccum, Phialophora, Sclerotium and Phaeomoniella (Boerema and Bollen 1975, Sutton 1977, Boerema 1997, Boerema et al. 1993, 1994, 1997, Crous & Gams 2000). It seemed that the taxonomic work on Phoma at the PD was completed with the publication of the Phoma Identification Manual in 2004. At that time, it could not be foreseen that already within one year a new project on the molecular phylogeny of Phoma would start. A joined research proposal was submitted by the PD and the Ministry of Agriculture Nature and Food Quality in the framework of the FES programme “Versterking Infrastructuur Plantgezondheid” and granted. The project comprised the setup of an online database providing biological, molecular and taxonomic