Cryphonectria Naterciae: a New Species in the Cryphonectria-Endothia Complex and Diagnostic Molecular Markers Based on Microsate

fungal biology 115 (2011) 852e861

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Cryphonectria naterciae: A new species in the CryphonectriaeEndothia complex and diagnostic molecular markers based on microsatellite-primed PCR

Helena BRAGANÇ Aa,*, Daniel RIGLINGb, Eugenio DIOGOa, Jorge CAPELOa, Alan PHILLIPSd, Rogerio TENREIROc aInstituto Nacional de Recursos Biologicos, IP., Edifıcio da ex. Estaçao~ Florestal Nacional, Quinta do Marquês, 2784-505 Oeiras, Portugal bWSL Swiss Federal Research Institute, CH-8903 Birmensdorf, Switzerland cUniversidade de Lisboa, Faculdade de Ciências, Departamento de Biologia Vegetal, Campo Grande, 1749-016 Lisboa, Portugal dCentro de Recursos Microbiologicos, Departamento de Ciências da Vida, Faculdade de Ciências 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- 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 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 (Bragança 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- are given in parentheses. Differences for the conidia measure- lished between these and other isolates collected previously from ments among the three species were assessed by analysis of declining Q. suber, also in the Midwest of Portugal (Santos et al. variance (ANOVA), followed by LSD post-hoc means test. 2005). Cultural morphology of these Midwest isolates showed Colony growth rates for all 22 Cryphonectria isolates, to- a strong similarity with an isolate of E. gyrosa Sacc. in the Instituto gether with three Cryphonectria parasitica and three C. radicalis 854 H. Bragança et al.

Table 1 e Isolates used in this study. Cryphonectria Isolate no.a Host Originb Collector Year GenBank accession no. species ITS b-tubulin

C. naterciae C 0084 Quercus Portugal-A A. Macara 1960 EU442648 EU442658 suber C. naterciae C 0605 Q. suber Portugal-A H. Bragança & P. Piloto 2001 C. naterciae C 0606 Q. suber Portugal-A H. Bragança & P. Piloto 2001 C. naterciae C 0607 Q. suber Portugal-A H. Bragança & P. Piloto 2001 EU442649 C. naterciae C 0608 Q. suber Portugal-A H. Bragança 2000 EU442650 EU442659 C. naterciae C 0609 Q. suber Portugal-A H. Bragança & P. Piloto 2001 C. naterciae C 0610 Q. suber Portugal-A H. Bragança 2000 C. naterciae C 0611 Q. suber Portugal-A H. Bragança & P. Piloto 2001 EU442656 C. naterciae C 0612/CBS Q. suber Portugal-A H. Bragança & P. Piloto 2001 EU442657 129351/MEAN 950c C. naterciae C 0613 Q. suber Portugal-R H. Bragança 2005 C. naterciae C 0614 Q. suber Portugal-A L. Inacio 2005 EU442651 EU442660 C. naterciae C 0679/CBS Castanea Portugal-B H. Bragança 2001 EU442652 EU442661 129352 sativa C. naterciae C 0682 C. sativa Portugal-A H. Bragança & J. Marcelino 2001 C. naterciae C 0683 C. sativa Portugal-A H. Bragança & J. Marcelino 2001 C. naterciae C 0684 C. sativa Portugal-A H. Bragança & J. Marcelino 2001 C. naterciae C 0685 C. sativa Portugal-A H. Bragança & J. Marcelino 2001 EU442653 EU442662 C. naterciae C 0686 C. sativa Portugal-A H. Bragança & J. Marcelino 2001 C. naterciae C 0687 C. sativa Portugal-A H. Bragança & J. Marcelino 2001 C. naterciae C 0691 C. sativa Portugal-A H. Bragança & J. Marcelino 2001 EU442654 EU442663 C. naterciae C 0692 C. sativa Portugal-A H. Bragança & J. Marcelino 2001 C. naterciae C 0704 C. sativa Portugal-A H. Bragança & J. Marcelino 2001 C. naterciae C 0705/CBS C. sativa Portugal-A H. Bragança & J. Marcelino 2001 EU442655 129353/MEAN 951 C. parasitica EP155 C. sativa USA S. Anagnostakis 1977 EU442647 C. parasitica C 0720/MEAN 952 C. sativa Portugal-TM H. Bragança 2002 EU442646 C. parasitica C 0721 C. sativa Portugal-TM H. Bragança 2002 EU442645 C. parasitica C 0722/CBS 129354 C. sativa Portugal-T H. Bragança 2003 C. radicalis M2269 C. sativa Switzerland P. Hoegger 1995 AF548744 C. radicalis M2270 C. sativa Switzerland D. Rigling 1996 AF548745 C. radicalis M4733 C. sativa Switzerland D. Rigling 2005

aCe collections of the INRB, I.P., Oeiras (isolates under investigation are listed in bold), CBS e culture collection of the CBS, Utrecht, The Neth- erlands; MEAN e culture collection of INRB (Ex Estaçao~ Agronomica Naciona), M e culture collection of the Swiss Federal Research Institute (WSL), Switzerland. bAe Alentejo, B e Beira Interior, R e Ribatejo e Oeste, TM e Tras-os-Montes e Alto Douro, T e Terceira. c Ex-type culture. reference isolates (Table 1), were assessed by the methods de- (2 cm 2 cm) was stripped from the cellophane overlays, scribed by Hoegger et al. (2002) and Gryzenhout et al. (2004). transferred to a 2 ml Eppendorf tube and DNA extracted fol- Cultures were grown on PDA in 90 mm diam. Petri dishes in lowing the procedures of Raeder & Broda (1985) with modifica- the dark at temperatures ranging from 10 Cto35Cat5C in- tions. First, the mycelium was suspended in 500 ml lysing tervals. Five replicate plates per isolate and temperature were buffer (300 mM TriseHCl, pH 8.5; 250 mM NaCl; 25 mM inoculated at the centre with 5 mm diam. agar discs taken EDTA, 0.5 % w v 1 SDS) and the equivalent of 200 mlof from the edge of actively growing cultures. After 4 d, two per- 450e600 mm glass beads (Sigma) was added. After vortexing pendicular diameters of each culture, minus the 5 mm diam. for 2 min, the tubes were centrifuged (max V.) for 10 min at of the agar disk, were measured and the average taken. The 4 C (adapted from Sampaio et al. 2001). The supernatant NewmaneKeuls Test of multiple mean comparison for nonin- was mixed with 350 ml of phenol and 150 ml chloroform and dependent samples and uneven number of observations centrifuged for 15 min (max V.). Subsequent steps were done (Newman 1939; Keuls 1952) was used to test for significant dif- exactly as described by Raeder & Broda (1985). The DNA was ferences ( p < 0.05) in growth between species for each tem- resuspended in 50 ml of TE (10 mM TriseHCl, 2 mM EDTA, pH perature. Statistical analyses were made using Statistica 6.1 8.0) and stored at 4 C until use. software (StatSoft, Tulsa, OK, USA). Amplification of the internal transcribed spacer (ITS) region DNA extractions and RFLP analysis

Mycelium was grown on PDA overlayed with cellophane for The ITS region, which includes ITS1, 5.8S rRNA gene and ITS2, 7 d at 25 C in the dark. A square of fresh cultures was ampliﬁed by PCR using forward primer ITS5 and reverse C. naterciae: A new species in the CryphonectriaeEndothia complex 855

primer ITS4 described by White et al. (1990). The PCR reaction 1995). The amplification reaction mixture and reaction condi- mixture (50 mL) included 400 ng of template DNA, 2 U of Taq tions were as described by Myburg et al. (2002). DNA polymerase (Life Technologies, England), 50 pmol of each primer (Life Technologies, England), 1 PCR buffer sup- Sequence analysis m plied with the enzyme, 4 mM MgCl2 and 200 M of each m dNTP (Life Technologies, England). To each PCR tube 50 Lof All sequences were obtained in an automated DNA capillary se- mineral oil were added before amplification in a RoboCycler quencer CEQ 2000-XL (Beckman Coulter, USA, in ICAT-Lisbon 96 (Stratagene, La Jolla, CA, USA), according to the following Faculty of Sciences Sequencing Services) by a dye-labelled program: 4 min at 95 C; 30 cycles of 1 min at 95 C, 1 min at dideoxy termination method (Dye Terminator Cycle 56 C and 1 min at 72 C; 5 min at 72 C. sequencer (DTCS) start kit, Beckman Coulter). For sequencing, Based on Myburg et al. (1999) a restriction analysis of the ITS the PCR products were purified using Jet Quick-PCR Purification m region was performed. From each PCR product 5 L samples Kit (Genomed) as described by the manufacturer. Two were digested with 3 U of restriction endonuclease AluI (New sequencing reactions, one in each direction, were performed England Biolabs, Beverly, MA, USA), in a final volume of for each PCR product using the same primers as for PCR ampli- m 10 L, according to manufacturer’s instructions. The digested fication. The two sequences were assembled using the CEQ 1 PCR products were separated on 2 % w v agarose gel (Invitro- Investigator program (software CEQ 8000, Beckman Coulter). gen) in 0.5 TBE (50 mM Tris, 45 mM boric acid, 1 mM EDTA) at Sequences were edited and used in a phylogenetic analysis 90 V for 3 h, using 100 bp DNA Ladder (Gibco-BRL) as a molecu- following the protocol of Santos & Phillips (2009) with minor lar size marker. After ethidium bromide staining, the gels were changes. Phylogenetic tree were inferred in Phylogenetic Anal- analysed with KODAK 1D 3.5.2 software. ysis Using Parsimony (PAUP) v. 4.0b10 (Swofford 2002) by Max- imum Parsimony (MP) and gaps were regarded as missing data. b-tubulin gene amplification Additional sequence data used in the phylogenetic analyses were obtained from Myburg et al. (2004a, b) and Gryzenhout Two regions within the b-tubulin gene were amplified using et al. (2006a) and listed by their taxon name and accession num- primer pairs Bt1a, Bt1b (amplifying b-tubulin region 1) and ber in the tree. Sequences from Diaporthe ambigua were used as Bt2a, Bt2b (amplifying b-tubulin region 2) (Glass & Donaldson outgroup to root the phylogenetic trees. Newly generated

Fig 1 e Culture morphology on PDA (top row), and colouration of corn meal medium (bottom row): (A) and (D) e Cryphonectria naterciae; (B) and (E) e C. radicalis; (C) and (F) e C. parasitica. 856 H. Braganc¸a et al.

Table 2 e Dimension of conidia produced by C. naterciae, C. radicalis, and C. parasitica. Mean ± SD and range for each species (listed in bold) and isolate are given with 95 % conﬁdence intervals (minimum and maximum dimensions are given in parenthesis). Isolate no. n Mean SD (mm) Range (mm)

C. naterciae 150 3.7 ± 0.4 3 1.3 ± 0.1 (2.9L)3.6e3.8(L4.9) 3 (1.0L)1.2e1.3(L1.6) C 0612 50 3.4 0.2 1.2 0.1 (3.0)3.4e3.5(3.9) (1.0)1.2e1.3(1.5) C 0679 50 4.0 0.5 1.3 0.1 (2.9)3.8e4.1(4.9) (1.0)1.2e1.3(1.6) C 0705 50 3.7 0.3 1.3 0.1 (3.0)3.6e3.8(4.3) (1.0)1.3e1.3(1.5)

C. radicalis 150 3.4 ± 0.3 3 1.4 ± 0.1 (2.7L)3.4e3.5(L4.1) 3 (1.2L)1.4e1.5(L1.8) M2269 50 3.2 0.2 1.3 0.1 (2.7)3.1e3.3(3.7) (1.2)1.3e1.3(1.5) M2270 50 3.5 0.2 1.5 0.1 (3.0)3.4e3.5(4.0) (1.4)1.5e1.6(1.8) M4773 50 3.6 0.2 1.5 0.1 (3.0)3.5e3.6(4.1) (1.4)1.5e1.5(1.7)

C. parasitica 150 3.6 ± 0.4 3 1.4 ± 0.1 (2.9L)3.6e3.7(L4.5) 3 (1.1L)1.4e1.4(L1.7) C 0720 50 3.6 0.3 1.4 0.1 (3.1)3.5e3.7(4.4) (1.2)1.3e1.4(1.7) C 0722 50 3.7 0.4 1.4 0.1 (2.9)3.6e3.8(4.5) (1.1)1.4e1.4(1.6) EP155 50 3.6 0.3 1.4 0.1 (2.9)3.5e3.6(4.3) (1.2)1.4e1.5(1.7) sequences have been deposited in GenBank (Table 1) and the On corn meal medium all three C. radicalis isolates caused alignment and phylogeny in TreeBASE (TB2:S11667). a colour change from the natural beige of the medium to purple (Fig 1E). None of the C. parasitica isolates produced a purple pigmentation in this medium (Fig 1F). Isolates of C. naterciae MSP-PCR fingerprinting also produced a reddish purple pigment, although this was not as intense as the colouration produced by the C. radicalis Three different primers were tested: a core sequence of the reference cultures (Fig 1D). All isolates produced a purple col- phage M13, and two synthetic oligonucleotides, (GACA)4 and our in KOH and a yellow colour in lactic acid. (GTG)5 (Meyer et al. 1993). All PCR reactions were performed Conidia of C. naterciae (Table 2) were significantly narrower in 25 mL Eppendorf tubes containing approximately 200 ng of than C. parasitica and C. radicalis (F ¼ 96.825; p ¼ 0.0000). template DNA, 1 U of Taq DNA polymerase (Life Technologies, 2,447 Conidia of C. radicalis were significantly shorter than C. para- England), 50 pmol of each primer (Life Technologies, England), ¼ ¼ sitica and C. naterciae (F2,447 27.227; p 0.0000). 1 PCR buffer supplied with the enzyme, 2.5 mM MgCl2 and 0.1 mM of each dNTP (Life Technologies, England). To each PCR tube 50 mL of mineral oil were added and amplification was performed in a RoboCycler 96 (Stratagene, La Jolla, CA, USA), according to the following amplification program: 5 min at 94 C; 40 cycles of 1 min at 94 C, 1 min at 50 C, 2 min at 72 C; with a final step of 5 min at 72 C. Amplified DNA fragments were separated by electrophoreses in 1 % (w v 1) agarose gels (Invitrogen) at 90 V for 3 h using a 1 kb plus DNA Ladder (Invitrogen) as molecular size marker. After ethidium bromide staining, DNA banding patterns were visu- alized under UV transillumination and images were acquired using KODAK 1D 3.5.2 software.

Results

Culture morphology and growth

On PDA after 1 week, the pinkish colouration due to the development of small droplets of purple exudates on the hyphae, visible under the dissecting microscope, was observed in only the three Cryphonectria radicalis isolates from Switzerland. Cryphonectria naterciae sp. nov showed, with some variation, an orange pigmentation in the centre of the culture, whereas the three C. radicalis remained white. After 4 weeks under ar- tiﬁcial light, three different types of culture morphologies were associated with C. naterciae, C. radicalis, and Cryphonectria Fig 2 e Colony growth of Cryphonectria naterciae (22 isolates), parasitica. As shown in Fig 1, C. radicalis and C. naterciae cul- C. radicalis (three isolates) and C. parasitica (three isolates) tures had similar morphologies. However, the ﬂat centre on PDA medium (mean colony growth after 4 d ± SD, area was present only in C. radicalis isolates (Fig 1B). 5 replications per isolate and temperature). C. naterciae: A new species in the CryphonectriaeEndothia complex 857

The mean colony diameters of the three taxonomic groups 600 bp, respectively. Digestion of the ITS region with the enzyme after 4 d of growth in relation to temperature are showed in Alu I produced the same RFLP profile for all isolates (two frag- Fig 2. All isolates grew best at 25 C. Isolates of C. radicalis showed ments with approximately 475 and 240 bp), which is in accor- the fastest growth at all temperatures tested, with the exception dance with the Cryphonectria parasitica profile presented by of30 C where C. parasitica grew the fastest. However, despite the Myburg et al. (1999). separation of the three taxonomic groups at 30 C, no statistical significant association could be found between growth and tax- Phylogeny onomic groups when all data were analysed. The phylogenetic tree obtained from the ribosomal ITS se- ITS and b-tubulin gene amplification and RFLP analysis quence data is shown in Fig 3. The dataset consisted of 28 ingroup taxa and two outgroups and the alignment con- Amplification of the ITS (ITS1-5.8S-ITS2) and b-tubulin gene re- tained 594 characters including alignment gaps. Of the gions resulted in single fragments of approximately 700 and 594 characters, 167 were parsimony informative and

C0608 Portugal

AF452117 Portugal

AF368327 Italy

C0685 Portugal

C0084 Portugal

100 C0691 Portugal Cryphonectria naterciae C0607 Portugal

C0612 Portugal §

C0611 Portugal

100 C0705 Portugal

C0679 Portugal

C0614 Portugal 67 C. radicalis AF548745 Switzerland

C. radicalis AF548744 Switzerland 59 100 C. radicalis AF452113 Italy

C. radicalis AF368328 Italy

C. parasitica C0720 Portugal

C. parasitica AY697927 Japan 100 C. parasitica AY194100 USA

C. parasitica C0721 Portugal

C. parasitica EP155 USA

100 100 C. nitschkei AY697937 Japan

67 C. nitschkei AY697936 Japan C. macrospora AY697942Japan 100

C. macrospora AF368331Japan

100 Endothia gyrosa AF368326 USA 71 Endothia gyrosa AF046905 USA

Endothia singularis AF368323 USA

Diaporthe ambigua AF543817

Diaporthe ambigua AF543818 10

Fig 3 e The most parsimonious tree resulting from the alignment of 576 characters of ITS rDNA region. Length [ 359; consistency index (CI) [ 0.850; retention index (RI) [ 0.944; homoplasy index (HI) [ 0.150. Bootstrap values with 1000 replications for MP are shown above the branches. Bar represents 10 changes. Diaporthe ambigua (AF543817 and AF543817) were included as outgroups. Phylogeny deposited in TreeBASE (TB2:S11667). Newly generated sequences are listed in bold together with their isolate number. Isolate followed by x is ex-type isolate and additional sequence data used in the phylogenetic analyses were obtained from Myburg et al. (2004a,b) and Gryzenhout et al. (2006a). 858 H. Braganc¸a et al.

included in the analysis resulting in one most parsimonious 744 7 bp, 669 4 bp, and 457 4 bp); three with Cryphonectria tree. radicalis isolates (974 16 bp, 783 7 bp, 673 2 bp) and three Since not all isolates used for the ITS region could be se- with Cryphonectria parasitica isolates (762 14 bp, 638 8 bp, quenced for ß-tubulin gene (Table 1), only the tree obtained and 389 2 bp). Since the Portuguese isolates had the same from ribosomal ITS sequence data are presented taking into banding patterns, this method confirmed the C. naterciae iden- account that ITS and ß-tubulin phylogenies are congruent tification for the nonsequenced isolates. (data not shown). The phylogenetic relations found amongst Cryphonectria Taxonomy spp. and Endothia spp. are compatible with the results presented by Myburg et al. (2004a, b) and Gryzenhout et al. Cryphonectria naterciae M.H. Bragança, E. Diogo, & A.J.L. Phillips (2006a, b). These data confirm the existence of two separate sp. nov. Fig 5. clades within Cryphonectria radicalis isolates considered by Cryphonectria radicalis affinis sed coloniis in agaro solano tuberoso Gryzenhout et al. (2006b) as C. radicalis sensu stricto that in- (PDA) uniformibus, laevibus, aurantiacis-ferrugineis non luteis- cludes two isolates from Switzerland and two from Italy. aurantiacis granulosibus, circulo centrali albido translucido abest et gut- tae parvae purpureae hyphae abest. A C. radicalis differt coloniis in The new species, Cryphonectria naterciae groups our Portuguese farina frumento luteo-purpurascentibus non purpureis. Conidia (2.9) isolates with another Portuguese isolate and an Italian isolate 3.6e3.8(4.9) (1.0e)1.2e1.3(1.6) mm. (100 % bootstrap support). As expected, Portuguese Cryphonec- tria parasitica isolates are in the same clade with the other Conidiomata pseudostromatic, globose, pulvinate, occur- C. parasitica (99 % bootstrap support). The final identification ring separately, superficial to slightly immersed, erumpent of isolates is presented in Table 1. through the host epidermis, yellow to orange, obpyriform, multilocular, variable in size up to 390 mm wide and 420 mm MSP-PCR fingerprinting high, with a single ostiole (Fig 5(AeB)), basal tissues pseudo- parenchymatous (Fig 5D). Conidiophores cylindrical, septate, Distinct electrophoretic band patterns were obtained with branched, hyaline, smooth, up to 33 mm long, 1.9 mm wide e M13, (GTG)5, and (GACA)4 primers, with each primer enabling (Fig 5(E G)). Conidiogenous cells phialidic, apical or lateral differentiation of all three species, (Fig 4). Electrophoretic on branches beneath the septum, cylindrical tapering towards e e m banding patterns obtained by (GACA)4 were more evident the end, (6.9 )7.2 9.8( 10.9) long and (1.3 )1.5 2.0( 2.3) m than M13 and (GTG)5 patterns. The (GACA)4 PCR fingerprints wide, collarete and periclinal thickening inconspicuous (Fig showed well-separated main DNA fragments (bp SD), four 5(EeG)). Conidia (2.9)3.6e3.8(4.9) (1.0)1.2e1.3(1.6) mm, associated with Cryphonectria naterciae isolates; (971 10 bp, hyaline, one celled (or aseptate), egutulate, cylindrical to

e Fig 4 MSP-PCR proﬁles of Cryphonectria naterciae, C. radicalis, and C. parasitica isolates using (GACA)4, M13, and (GTG)5 primers: 1 e C 0084, 2 e C 0605, 3 e C 0607, 4 e C 0608, 5 e C 0611, 6 e C 0612, 7 e C 0614, 8 e C 0679, 9 e C 0685, 10 e C 0691, 11 e C 0705, 12 e CM2269, 13 e CM2270, 14 e CM4733, 15 e CEP155, 16 e C 0720, 17 e C 0721, 18 e C 0722. M e molecular size marker (1 kb plus DNA Ladder). Diagnostic bands are indicated by arrows: 457 bp for C. naterciae and 389 bp for C. parasitica. C. naterciae: A new species in the CryphonectriaeEndothia complex 859

Fig 5 e Fruiting structures of C. naterciae e (A) and (B) pycnidia on Quercus suber and Castanea sativa stems respectively. (C) e tendrils of conidia on C. sativa stem in vitro; (D) e pycnidium; (E) e conidiogenous cells; (F) e conidia; (G) e conidiophores. Bars: (AeC) e 1 mm; (D) e 200 mm; (EeG) e 5 mm.

fusoid, seldom slightly allantoid (Fig 5F), exuded in the form of Specimens examined: Portugal, Alentejo, Pegoes,~ on bark of Q. yellow to orange twisted cirri (Fig 5C). suber, 2001, H. Bragança & P. Piloto, holotype CBS H-20572, culture Cultural characteristics: on PDA, colonies flat, felty, orange to ex-type CBS 129351 (C 0612, MEAN 950) See Table 1 for other isolates studied. ferruginous colour, uniform, margins even, with scarce sporu- lation (Fig 1A). Optimum temperature for growth 25 C(Fig 2) On corn meal producing a reddish purple pigment (Fig 1D). Notes: This species can be distinguished from C. radicalis by Etymology: After the Portuguese phytopathologist Maria the absence of small droplets of purple exudates on the hyphae Natercia Santos (b. 1940). growing on PDA (visible under the dissecting microscope) and Teleomorph: Not seen. by the absence of a ‘flat’ centre area in PDA plates. It can be dis- Habitat:OnQuercus suber and Castanea sativa. tinguished from Cryphonectria parasitica by causing a reddish Known distribution: Italy (Myburg et al. 2004a), Portugal. purple colour change in corn meal (Fig 1D). 860 H. Bragança et al.

both C. radicalis and C. naterciae found in Europe are indige- Discussion nous fungi, however, remain to be shown. The new species described here, C. naterciae, was particularly common in the Eleven isolates from Castanea sativa and eleven from Quercus province of Alentejo on both Q. suber and C. sativa. In the suber were studied. All isolates from both hosts formed a clear main chestnut growing area of Portugal (Tras-os-Montes), and well-supported clade sister to Cryphonectria radicalis, and however C. naterciae isolates were neither reported in the on account of the morphological and phylogenetic distinc- past nor detected during the recent national survey on chest- tions they were considered to represent a separate species de- nut blight, which yielded the isolates from chestnut in Alen- scribed here as Cryphonectria naterciae. Thus far only two other tejo (Braganc¸a et al. 2007). isolates of this species are known, one collected in Italy and Preliminary results from an experiment with C. sativa and another in Portugal (Myburg et al. 2004a). Q. suber showed that C. naterciae is not pathogenic on C. sativa, Morphologically all the Portuguese isolates belonged to the but no conclusive results were achieved with Q. suber (data not Cryphonectriaceae, which was recently described by shown). More intensive studies are needed to evaluate the Gryzenhout et al. (2006a). Furthermore, culture morphology pathogenicity of C. naterciae and to determine its main host on PDA as well as the reddish purple pigmentation in corn in Portugal and also in other areas in Europe. The combination meal medium suggested a close relationship with C. radicalis. of the morphological and molecular method based on MSP-

However, some of these characters, such as the absence of PCR with (GACA)4 primer, as well as the phylogenetic small purple droplets in the Portuguese cultures, did not allow approach used in this study, could contribute towards the us to definitely assign the Portuguese isolates to C. radicalis development of diagnostic molecular markers to identify spe- (Hoegger et al. 2002). Conidial dimensions of the three species cies in Cryphonectria. scarcely differed but C. naterciae conidia were slightly narrower than in the other species. In a previous study that included two of these isolates (Gryzenhout et al. 2006b) it was Acknowledgements suggested that they may represent a distinct species. How- ever, Gryzenhout et al. 2006b did not introduce a new species We are grateful to Dr Cristina Lopes (INRB, Portugal) for her help name for these isolates because none of the sequences that in tracing old Portuguese publications on the subject. We also they studied could be linked to cultures or herbarium thank Lurdes Inacio (INRB, Portugal) for providing a fresh iso- specimens. late of Cryphonectria sp., Gonçalo Costa and Tania^ Tenreiro The polymorphisms found for each MSP-PCR primer, i.e., (ICAT, Portugal) for helping with sequence data, Jose Marcelino M13, (GTG)5, and (GACA)4, clearly distinguished three different (Universidade dos Açores, Portugal) and Pedro Naves (INRB, patterns among the analysed isolates, and these groups corre- Portugal) for English revision of the manuscript and Nuno sponded to the three species under study. Of these primers, Horta for helping us with photos arrangements. This work (GACA)4 produced the most distinctive banding patterns for was partly supported by PIDDAC 206 and PIDDAC 204 projects. each group of isolates. A single and well-separated PCR band of 457 bp was found to be specific for C. naterciae isolates and between the two specific bands for Cryphonectria parasitica references (389 bp and 638 bp) the smaller band can be unequivocally diagnostic for this species. Cryphonectria radicalis could be iden- tified due to the lack of the bands diagnostic for C. naterciae. Anagnostakis S, 1987. Chestnut blight: the classic problem of an Moreover, in C. parasitica the band around 970 bp is absent introduced pathogen. Mycologia 79:23e37. (this band is present in both C. naterciae and C. radicalis). These Barr M, 1978. The Diaporthales of North America with emphasis 7 e findings indicate that these bands are reliable markers to dif- on Gnomonia and its segregates. Mycologia Memoir :1 232. ferentiate rapidly and easily C. radicalis, C. naterciae, and C. par- Bissegger M, Rigling D, Heiniger U, 1997. Population structure and disease development of Cryphonectria parasitica in European asitica (diagnostic bands are indicated by arrows in Fig 4). This chestnut forests in the presence of natural hypovirulence. type of technique has previously been reported to give good Phytopathology 87:50e59. differentiation of related species in other genera (Sampaio Bragança H, Simoes~ S, Onofre N, Tenreiro R, Rigling D, 2007. et al. 2001; Gadanho et al. 2003; Roque et al. 2006). Cryphonectria parasitica in Portugal e diversity of vegetative ITS PCR-RFLP with Alu I revealed the existence of a common compatibility types, mating types, and occurrence of hypo- 37 e profile for each of the three species analysed, which is clearly virulence. Forest Pathology : 391 402. Camara^ M, 1929. Contributiones ad Mycofloram Lusitaniae, Cen- distinct from the profile for Endothia gyrosa (Myburg et al. 1999). turiae VIII et IX. In: Anais do Instituto Superior de Agronomia, Vol. Although this method did not discern the three groups in 3, Lisboa, 1930, pp. 34. study, it allowed us to reject the hypothesis that Portuguese Castlebury A, Rossman Y, Jaklitsch J, Vasilyeva L, 2002. A pre- isolates are E. gyrosa. liminary overview of the Diaporthales based on large subunit Cryphonectria radicalis was not found in Portugal during this nuclear ribosomal DNA sequences. Mycologia 94: 1017e1031. study. To date, the known distribution of C. radicalis, in Europe Elliston JE, 1982. Hypovirulence. Advances in Plant Pathology 1: e is restricted to the central and south-eastern part of the con- 1 33. Gadanho M, Almeida J, Sampaio J, 2003. Assessment of yeast diver- tinent including Switzerland, Italy, and Greece (Myburg et al. sity in a marine environment in the south of Portugal by 2004a). Cryphonectria radicalis has been reported from Europe microsatellite-primed PCR. Antonie van Leeuwonhoek 84:217e227. before the introduction of C. parasitica and was thought to Glass L, Donaldson G, 1995. Development of primer sets designed have evolved with European chestnut (Elliston 1982). Whether for use with the PCR to amplify conserved genes from C. naterciae: A new species in the CryphonectriaeEndothia complex 861

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