mycological research 113 (2009) 73–81 journal homepage: www.elsevier.com/locate/mycres Characterisation and phylogenetic relationships of Anisogramma virgultorum and A. anomala in the Diaporthales (Ascomycota) Heike DE SILVAa, Lisa A. CASTLEBURYb,*, Sarah GREENa, Jeffrey K. STONEc aForest Research, Northern Research Station, Roslin, Midlothian, EH25 9SY, Scotland, United Kingdom bSystematic Mycology and Microbiology Laboratory, USDA ARS, Beltsville, MD 20705, USA cDepartment of Botany and Plant Pathology, Oregon State University, Corvallis OR 97331-2902, USA article info abstract Article history: The two diaporthalean fungi Anisogramma virgultorum and A. anomala are biotrophic Received 28 November 2007 parasites. A. virgultorum causes stromatal cankers on young shoots of birch whereas Received in revised form A. anomala infects young branches of Corylus avellana. Although previous classifications 1 July 2008 based on morphological characteristics placed both species in the Gnomoniaceae, Diapor- Accepted 12 August 2008 thales, their taxonomic position within the order and their relationship to each other re- Corresponding Editor: quired further clarification. We determined the nucleotide sequences of the ITS and Brenda Wingfield partial LSU nu-rDNA regions of both species. A putative second teleomorph form of A. vir- gultorum, described in the literature as the ‘single perithecial form’, was also included in Keywords: the analysis. Based on phylogenetic analyses of LSU sequences, the stromatal forms of Betula A. virgultorum and A. anomala were part of a well-supported monophyletic sister clade to Corylus the Gnomoniaceae. The single perithecial form was placed within a clade containing repre- Eastern filbert blight sentative members of the Gnomoniaceae, separate from species of Anisogramma. These re- Gnomoniaceae sults indicate that the single perithecial form of A. virgultorum actually represents an unrelated and as yet unidentified species of Gnomoniaceae. A morphological description of asci and ascospores of the three species is given. A Wilcoxon two sample test revealed that asci of the stromatal form of A. virgultorum were significantly shorter than those of the single perithecial species. Ascospores of the stromatal form of A. virgultorum were signifi- cantly shorter and wider than those of the single perithecial species. Published by Elsevier Ltd on behalf of The British Mycological Society. Introduction eastern filbert blight on commercially grown European hazel- nut (Corylus avellana) in the eastern United States and more The genus Anisogramma comprises two well-known species: recently in Oregon and Washington, and British Columbia A. virgultorum and A. anomala. A. virgultorum produces dark (Johnson et al. 1996). A third species, originally described as brown to black erumpent stromatal cankers on young shoots Diaporthe apiospora has been placed in Anisogramma as A. apio- of Betula pendula and B. pubescens and has been observed to spora, but Barr (1978) did not include it in Anisogramma and cause shoot dieback on young trees in Scotland (H.D.S. & later placed it in the genus Apioporthella (Barr 1991). A. apio- S.G. unpubl.). A. anomala causes the devastating disease spora has a valsoid arrangement of perithecia that does not * Corresponding author. E-mail address: [email protected] 0953-7562/$ – see front matter Published by Elsevier Ltd on behalf of The British Mycological Society. doi:10.1016/j.mycres.2008.08.008 74 H. De Silva et al. suggest a relationship with Anisogramma, and therefore, is not branches in severely infected trees leading to branch dieback considered in this paper. within a few years (Gottwald & Cameron 1979; Gottwald & A. virgultorum has been reported from birch in Germany, Cameron 1980a; Johnson et al. 1994). Disease incidence may Italy, Finland, Switzerland, UK, Sweden (Massee 1914; Thei- be as high as 100 % at some sites and in some cases entire or- ßen & Sydow 1916; Vleugel 1917; Froidevaux & Mu¨ ller 1972; chards have been killed by the disease (Gottwald & Cameron Witzell & Karlsson 2002) and New Hampshire (Barr 1978). Al- 1980b; Johnson et al. 1996). though widely distributed, it has been considered a minor Historically, morphological characteristics of the stromata, pathogen of birch in the UK because of its rare occurrence perithecia, and ascospores have been used to distinguish gen- (Dennis 1968; Ellis & Ellis 1985). However, in a recent survey, era within the Diaporthales (Barr 1978; Monod 1983). However, this species was found abundantly at nine planted birch emphasis on different morphological characters by different stands across Scotland and was associated with crown die- authors and the great variation in phenotypic features has back of affected trees (De Silva et al. 2008). Ascospores formed lead to much confusion in the determination of families and within flask-shaped perithecia are released in spring from genera. For example, A. virgultorum was listed in the family stromatal cankers on infected birch shoots of the previous Valsaceae by Eriksson (1992) while Dennis (1968) and Froide- season’s growth. Spore release studied over a two-year period vaux & Mu¨ ller (1972) included this species in the Diaporthaceae. coincided with shoot elongation of birch seedlings, a period More recently, A. virgultorum was placed in the Gnomoniaceae when the host appears to be most susceptible to infection (Barr 1978) while in The Dictionary of the Fungi, the genus Ani- (H.D.S. & S.G. unpubl.). Ascospores are released in spring to in- sogramma is listed in the Valsaceae (Hawksworth et al. 1995). fect young expanding shoots, and the first symptoms, such as The aim of this present study is to determine whether the dark brown staining of the phloem and splitting of the epider- two forms of A. virgultorum are actually conspecific, to charac- mis, are observed on infected current season shoots during terise both forms in comparison with A. anomala, and to infer mid to late July, approximately eight to ten weeks after infec- the phylogenetic relationships of these species to other taxa in tion. Ascostromata then develop on infected birch shoots by the Diaporthales using nucleotide sequences of the complete mid-August (H.D.S. & S.G. unpubl.). ITS region and LSU nu-rDNA gene. A species with similar ascospores that has been identified as a second teleomorph form of A. virgultorum was found in Switzerland and described by Froidevaux & Mu¨ ller (1972) as Materials and methods having small individual fruiting bodies, each containing a sin- gle perithecium, that are scattered around the infected birch Fungal material shoot. In the more common stromatal form numerous peri- thecia occur densely packed in rows within each stroma. Eighteen isolates of Anisogramma virgultorum, one isolate of Whereas the stromatal form was commonly found on birch the single perithecial species and one isolate of A. anomala trees infected with A. virgultorum across Scotland (De Silva were included in this study. Isolates of both birch pathogens et al. 2008), the rare single perithecial species has, to date, were collected from planted and site-natural birch stands only been observed on two B. pubescens trees at one site in across Scotland and A. anomala on infected shoots of Corylus avellana were obtained from one location in Oregon (Table 1). the north of Scotland. Froidevaux & Mu¨ ller (1972) do not men- tion how frequently the single perithecial form was encoun- All infected birch shoots were stored at À20 C until use. Cul- tered, but note that it was not as common as the stromatal turing of A. virgultorum and the single perithecial species was form. attempted by streaking ascospores onto malt agar (2 % malt In North America, A. anomala is indigenous on native hazel agar, 2 % sucrose, 2 % bacteriological agar and 0.2 % yeast ex- (C. americana) and causes a disease of the commercial crop tract) amended with 0.5 % activated charcoal following auto- species, C. avellana, known as eastern filbert blight that pre- claving. While ascospores of all isolates of A. virgultorum vents establishment of this crop in the northeastern USA germinated, their germ tubes lysed after a few days on this (Barss 1930). In 1970 this disease was first discovered on com- medium. Monoascospore cultures could only be isolated for mercially grown European hazelnut trees in the state of the single perithecial species. These were maintained in an Washington (Davison & Davidson 1973) and has since spread incubator at 20/15 C day/night temperatures with a 12 h to Oregon. In Oregon and Washington, ascospores mature photoperiod. Light was supplied as cool white fluorescent and are released from cankers during periods of rain from and near-UV light. Colonies were subcultured to obtain actively early winter to late spring. However, new infections by asco- growing mycelium for DNA extraction. As A. virgultorum could spores occur only on young, developing shoots over a period not be cultured, molecular analyses were conducted by DNA of several weeks in spring. Infection occurs on succulent, extraction using perithecial contents taken directly from newly emerged leaves and shoots soon after breaking of the a freshly cut stroma or by direct PCR methods. Similarly, vegetative buds. The disease cycle requires one or more years, DNA of A. anomala was extracted from perithecial contents including a 12–24 month latent period from the time of infec- taken from infected C. avellana shoots. tion to expression of first symptoms on affected branches (Stone et al. 1992; Johnson et al. 1994). The disease spreads by Voucher material new infections from ascospores and by perennial expansion of existing cankers along and around infected branches. Pe- Herbarium specimens of Anisogramma virgultorum and rennial cankers can measure from a few centimetres to over A. anomala are stored at the USDA ARS Systematic Mycology 2 m in length. Larger cankers often girdle the scaffolding Laboratory, in Beltsville MD, USA. Shoot material infected Phylogenetics of Anisogramma 75 Table 1 – Fungal isolates used for ITS and LSU analyses Isolate Fungal species Host species Origin Collection date Source AV01 Anisogramma virgultorum Betula pubescens Carroch, Scotland 06.09.2006 H.