fungal biology 115 (2011) 852e861 journal homepage: www.elsevier.com/locate/funbio Cryphonectria naterciae: A new species in the CryphonectriaeEndothia complex and diagnostic molecular markers based on microsatellite-primed PCR Helena BRAGANC¸ Aa,*, Daniel RIGLINGb, Eugenio DIOGOa, Jorge CAPELOa, Alan PHILLIPSd, Rogerio TENREIROc aInstituto Nacional de Recursos Biologicos, IP., Edifıcio da ex. Estac¸ao~ Florestal Nacional, Quinta do Marqu^es, 2784-505 Oeiras, Portugal bWSL Swiss Federal Research Institute, CH-8903 Birmensdorf, Switzerland cUniversidade de Lisboa, Faculdade de Ci^encias, Departamento de Biologia Vegetal, Campo Grande, 1749-016 Lisboa, Portugal dCentro de Recursos Microbiologicos, Departamento de Ci^encias da Vida, Faculdade de Ci^encias e Tecnologia, Universidade Nova de Lisboa, 2829-516 Caparica, Portugal article info abstract Article history: In a recent study intended to assess the distribution of Cryphonectria parasitica in Portugal, Received 28 May 2010 22 morphologically atypical orange isolates were collected in the Midwestern regions. Received in revised form Eleven isolates were recovered from Castanea sativa, in areas severely affected by chestnut 16 June 2011 blight and eleven isolates from Quercus suber in areas with cork oak decline. These isolates Accepted 21 June 2011 were compared with known C. parasitica and Cryphonectria radicalis isolates using an inte- Available online 8 July 2011 grated approach comprising morphological and molecular methods. Morphologically the Corresponding Editor: atypical isolates were more similar to C. radicalis than to C. parasitica. Phylogenetic analyses Andrew N. Miller based on internal transcribed spacer (ITS) and b-tubulin sequence data grouped the isolates in a well-supported clade separate from C. radicalis. Combining morphological, cultural, Keywords: and molecular data Cryphonectria naterciae is newly described in the CryphonectriaeEndothia Chestnut tree complex. Microsatellite-primed PCR fingerprinting with (GACA)4 primer discriminated Cork oak tree between C. naterciae, C. radicalis, and C. parasitica. Cryphonectria parasitica ª 2011 British Mycological Society. Published by Elsevier Ltd. All rights reserved. Cryphonectria radicalis (GACA)4 Endothiella gyrosa MSP-PCR RFLPePCR Introduction undescribed groups in the Diaporthales, by the development of orange stromatic tissue at some stage of their life cycle, a purple The Cryphonectriaceae was introduced by Gryzenhout et al. reaction in KOH and a yellow reaction in lactic acid associated (2006a) to accommodate genera from the CryphonectriaeEndothia with pigments in the stromatic tissue or in culture. There is complex (Castlebury et al. 2002). Genera in the Cryphonectriaceae also strong phylogenetic support in phylogenies based on can be distinguished from those in other families, or ribosomal and b-tubulin gene sequences (Castlebury et al. 2002). * Corresponding author. E-mail addresses: [email protected], [email protected], [email protected] 1878-6146/$ e see front matter ª 2011 British Mycological Society. Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.funbio.2011.06.014 C. naterciae: A new species in the CryphonectriaeEndothia complex 853 Cryphonectria was considered to be a synonym of Endothia Nacional de Recursos Biologicos (INRB) working culture until Barr (1978) separated the two genera based on the config- collection that was collected from Q. suber in 1960. This fungus uration and texture of the stromata and the septation and was first reported in Portugal by Camara^ (1929) causing the ‘ferru- shape of the ascospores (Roane et al. 1986; Myburg et al. gem alaranjada’ (‘orange rust’) disease on cork oak stems. 2004b). Nevertheless, species of Endothia continue to be mis- The aim of this work was to use morphological and molec- taken with Cryphonectria spp. due to their similar orange fruit- ular techniques to determine the identity of the isolates from ing structures and a shared anamorph genus, Endothiella (Barr Portugal. In addition, MSP-PCR fingerprinting was used to pro- 1978; Gryzenhout et al. 2006b). Moreover, these species share vide a fast and reliable methodology to discriminate the spe- the same hosts (Castanea spp. and Quercus spp) and geograph- cies within the CryphonectriaeEndothia complexes. ical distributions (Gryzenhout et al. 2006b). Recent taxonomic revisions restrict the name Cryphonectria (sensu stricto)toCryphonectria macrospora (Tak. Kobay. & Kaz. Material and methods Ito) M.E. Barr, Cryphonectria nitschkei (G.H. Otth) M.E. Barr Crypho- nectria parasitica and Cryphonectria radicalis (Schwein.: Fr.) M. E. Fungal isolates Barr, (Gryzenhout et al. 2006a, b, c). Of these C. parasitica is the most studied species since it is the causal agent of chestnut In this study, 22 orange Cryphonectria isolates from Castanea blight disease worldwide, and the only species of this genus sativa and Quercus suber collected in the Midwest of Portugal that is considered to be a primary plant pathogen. This fungus (Beira Interior, Ribatejo, and Alentejo) were characterized has destroyed nearly all native stands of American chestnuts (Table 1). All isolates were maintained in a culture collection [Castanea dentata (Marsh.) Borkh.]. In Europe, many European at the Unidade de Silvicultura e Recursos Florestais, INRB, chestnuts areas (Castanea sativa Mill.) have been similarly af- I.P., Oeiras, Portugal and in the Swiss Federal Research Insti- fected by the blight since the start of the 1930s. However, impact tute (WSL). For comparison, three Cryphonectria radicalis iso- and disease severity has been stalled due to natural higher resis- lates from Switzerland (culture collection of the WSL) and tance in the European chestnut and the presence of natural four Cryphonectria parasitica isolates, three from the Portuguese hypovirulence in the pathogen (Anagnostakis 1987; Heiniger & collection, and one from USA (WSL collection), were used. Ref- Rigling 1994; Bissegger et al. 1997; Milgroom & Cortesi 2004). In erence isolates were deposited in the public culture collection the United States and in some European countries, C. parasitica of the Centraalbureau voor Schimmelcultures (CBS), Utrecht, has also been found on some species of Quercus (Torsello et al. The Netherlands and the INRB culture collection (MEAN). 1994; Radocz & Tarcali 2005), although blight symptoms are notassevereasinCastanea (Radocz & Tarcali 2005). Culture morphology and growth The saprophytic C. radicalis occurs in Europe, North Amer- À ica, and Japan, being closely related to C. parasitica (Hoegger Isolates were grown on PDA (Difco 39 g L 1)in90mmdiam.Petri et al. 2002; Venter et al. 2002; Myburg et al. 2004a, b). Isolates dishes are incubated at 25 C in the dark for 1 week. The cultures of C. radicalis are difficult to detect in nature because they were then exposed to diffuse daylight at room temperature on are possibly almost displaced or their occurrence is cryptically the laboratory bench, and culture morphology was recorded masked by C. parasitica(Hoegger et al. 2002). once a week for a period of 4 weeks. All cultures were checked Gryzenhout et al. (2006b) proposed two different phyloge- for typical Cryphonectria radicalis characteristics, i.e., purple netic groups of C. radicalis. The first one (C. radicalis sensu droplets (as seen with the dissecting microscope) and a ‘flat’ stricto) defined by the North America type specimen and corre- central area (Hoegger et al. 2002). Agar pieces (5 Â 5 mm) taken sponding morphologically to a group containing isolates from from the edge of actively growing cultures were immersed in Switzerland, Greece, Italy, and Japan. The second group con- parallel into 0.2 ml of 3 % of KOH and into 0.2 ml of lactic acid sisted of two isolates, one from Italy and another from Portu- to check for purple and yellow discolouration of the mycelium gal (Myburg et al. 2004a, b). This Portuguese isolate was as described by Castlebury et al. (2002). In addition, all isolates formerly described as Endothiella gyrosa and was collected were grown on corn meal medium (10 g of corn meal autoclaved from Quercus suber L. Nevertheless, the taxonomy of C. radicalis for 20 min at 120 C with 20 ml of distilled water in 100 ml Erlen- remains unclear despite all the knowledge that have been meyer flasks) to test for the purple discolouration that is charac- gathered in recent years, mainly because the teleomorph teristic of C. radicalis in this medium (Hoegger et al. 2002). Two (required for accurate morphological species differentiation) cultures per isolate were incubated at 25 C in the dark and is not commonly found in nature and is not easily produced assessed for discolouration of the medium after 7 weeks. in vitro (Myburg et al. 2004b; Gryzenhout et al. 2006b). Observations of micromorphological features and measure- In a recent study on the distribution of C. parasitica in Portugal ment from three isolates of each group of Cryphonectria (20-d-old (Braganc¸a et al. 2007), morphologically atypical orange isolates PDA cultures) were made as described by Santos & Phillips were collected from chestnut stands that were severely affected (2009). Mean, standard deviation (SD) and 95 % confidence inter- by the blight in the Midwest of the country. The different mor- vals were calculated from measurements of 150 conidia of each phology of these isolates was noticed on potato dextrose agar group of Cryphonectria. Minimum and maximum dimensions (PDA) among C. parasitica cultures. A close relationship was estab-