Phytophthora Acerina Sp. Nov., a New Species Causing Bleeding Cankers and Dieback of Acer Pseudoplatanus Trees in Planted Forests in Northern Italy

Plant Pathology (2013) Doi: 10.1111/ppa.12153

Phytophthora acerina sp. nov., a new species causing bleeding cankers and dieback of Acer pseudoplatanus trees in planted forests in northern Italy

B. Ginettia, S. Moriccaa*, J. N. Squiresb, D. E. L. Cookeb, A. Ragazzia and T. Jungcd aDepartment of Agri-Food Production and Environmental Sciences, Plant Pathology and Entomology Division, University of Florence, Piazzale delle Cascine 28, 50144 Florence, Italy; bThe James Hutton Institute, Invergowrie, Dundee DD2 5DA, UK; cPhytophthora Research and Consultancy, Thomastrasse 75, D-83098 Brannenburg, Germany; and dInstitute for Biotechnology and Bioengineering, Centre of Genomics and Biotechnology, (IBB/CGB), Plant and Animal Genomic Group, Laboratorio de Biotecnologia Molecular e Fitopatologia, University of Algarve, Campus de Gambelas, 8005-139 Faro, Portugal

A severe dieback of Acer pseudoplatanus trees was noticed in planted forest stands in northern Italy in 2010. Affected trees showed collar rot and aerial bleeding cankers along the stems, leading to crown dieback and eventually death. An unknown Phytophthora species was consistently isolated from necrotic bark and xylem tissue and from rhizosphere soil. Based on its unique combination of morphological and physiological characters and phylogenetic analysis, this new taxon is here described as Phytophthora acerina sp. nov. Phylogenetic analysis of ITS, cox1 and b-tubulin gene regions demonstrated that P. acerina is unique and forms a separate cluster within the ‘P. citricola complex’, closely related to P. plurivora. Phytophthora acerina is homothallic with smooth-walled oogonia, thick-walled, mostly aplerotic oospores with a high abortion rate, paragynous antheridia, and persistent, morphologically variable semipapillate sporangia. Four to 5-week-old cultures produced globose to subglobose, appressoria-like and coralloid hyphal swellings and characteristic stromata-like hyphal aggregations. Optimum and maximum temperatures for growth are 25°C and 32°C, respectively. Genetic uniformity of all 15 studied isolates and the apparent absence of this species in the extensive surveys of nurseries, forests and seminatural ecosystems conducted in the previous two decades across Europe indicate a recent clonal introduction to northern Italy. Under-bark inoculation tests demonstrated high aggressiveness of P. acerina to A. pseudoplatanus indicating that this pathogen might be a serious risk to maple plantations and forests in Europe.

Keywords: biosecurity, Clade 2, invasive, nursery pathway, phylogeny, Phytophthora citricola

Introduction declines of oak and beech forests (Brasier & Scott, 1994; Jung et al., 1996, 2013b; Jung, 2009). In the past 15 years the involvement of Phytophthora In Europe, in the previous two decades, an array of 25 species in widespread declines of Acer spp., Aesculus new Phytophthora taxa were detected from forests, semi- hippocastanum, Alnus spp., Betula spp., Fagus sylvatica, natural ecosystems and plantations, of which 17 have Juglans regia, Quercus spp. and Tilia spp. across Europe been formally described (Jung & Burgess, 2009; Jung has been demonstrated (Jung et al., 1996, 2000, 2013b; et al., 2013b). This dramatic development has been Vettraino et al., 2002). The high aggressiveness of the caused by: (i) the exponential increase in the interna- most frequently involved P. cactorum, P. cambivora, tional trade of rooted nursery stock; (ii) the rapid devel- P. cinnamomi, P. plurivora, P. quercina and P. ramorum opment and application of new molecular detection and to a range of common European tree species including identiﬁcation tools; and (iii) the increased interest in Acer platanoides, Acer pseudoplatanus, F. sylvatica and environmental surveys for Phytophthora species. several Quercus spp. (Jung et al., 1996, 2003; Vettraino Novel molecular methods increased the understanding et al., 2002; Brasier & Jung, 2003; Jung & Burgess, of the true relationships between the different Phyto- 2009) strongly indicates that these Phytophthora species phthora species and between Phytophthora and other have not co-evolved with these tree species and were oomycete genera and enabled the development of natural introduced to Europe. Synergistic interactions between phylogenies for the genus and related oomycetes (Cooke root losses and bark infections caused by introduced soil- et al., 2000; Kroon et al., 2004; Blair et al., 2008). Sev- borne Phytophthora species and the increasing frequency eral well-known morphospecies such as P. citricola, of climatic extremes are a major cause for the severe P. gonapodyides, P. megasperma, P. porri or P. syringae have been shown to be complexes of morphologically and physiologically almost indistinguishable species (Brasier et al., 2003; Jung et al., 2003; Jung & Burgess, *E-mail: salvatore.moricca@uniﬁ.it

ª 2013 British Society for Plant Pathology 1 2 B. Ginetti et al.

À 2009). The morphospecies P. citricola sensu lato was 50 lgmL 1 hymexazol (Erwin & Ribeiro, 1996). The plates were divided into P. citricola sensu stricto and seven new incubated at 20°C in the dark and examined daily under the ste- closely related taxa that together form the ‘Phytophthora reomicroscope for Phytophthora-like hyphae, which were trans- citricola complex’, i.e. P. capensis, P. multivora, P. pini, ferred to V8A for initial confirmation as Phytophthora species. P. plurivora, P. citricola III, P. citricola E and P. taxon Aliquots of 250 mL soil were taken from the same five A. pseudoplatanus trees at 5–20 cm depth at a distance of 1–1Á5m ‘emzansi’, and the two new species P. mengei and P. from the stem base at four points around each tree. In addition, elongata which also belong to ITS Clade 2 but are more one sample of soil was taken from each of four sites along the distantly related to the other taxa (Jung & Burgess, canal that crosses the park at three different distances (1, 5 and 2009; Scott et al., 2009; Bezuidenhout et al., 2010; 10 m) from the canal at each site. For the isolation of Phytoph- Hong et al., 2011; Kroon et al., 2012). thora from the soil samples, green apple fruits were used as bait In 2008, a severe decline and dieback of A. pseudoplat- (Erwin & Ribeiro, 1996; Jung et al., 1996). After surface anus trees was observed in several planted forest stands in disinfection with 95% ethanol, four equidistant holes of c. 1cm northern Italy. Affected trees showed thinning and dieback diameter and c. 2 cm depth were cut into each apple with a of the crown and bleeding cankers at the stem base and sterilized scalpel. Each hole was completely filled with subsam- along the stem, with tongue-shaped, orange-brown lesions ples of soil from one sample and subsequently wetted with sterile deionized water in order to induce the germination of of the inner bark and brown to greenish staining of the potential Phytophthora resting structures. Each apple was finally cambium and adjacent xylem tissue. In 2010 and 2011, wrapped in a transparent film of polymer material and incu- Phytophthora isolates were consistently isolated from bated at 18°C in the dark for about 5–7 days, after which the necrotic bark and stained xylem tissue and from rhizo- apples were examined for the development of a fruit rot. Pieces sphere soil of A. pseudoplatanus trees with symptoms in were cut from the interface between the necrotic area and the planted forest stands of the Boscoincitta Park in Milan. healthy tissue and plated onto PARPNH. The plates were incu- Being homothallic, with persistent semipapillate sporan- bated at 20°C in the dark and outgrowing Phytophthora hyphae gia, the isolates resembled species from the ‘P. citricola were subsequently transferred to V8A. complex’. Detailed morphological and physiological stud- In December 2010, four apple baits were placed along the ies and a phylogenetic analysis of ITS, cox1 and b-tubulin entire course of a canal that runs through Boscoincitta Park. The apples were first inserted into jute bags, then placed in sequence data revealed that all isolates belong to an small cages as protection against animals and finally immersed unknown unique taxon of the ‘P. citricola complex’, in the water. After 7 days the apples were collected and trans- which is described here as Phytophthora acerina sp. nov. ported to the laboratory where isolations were carried out as described before. A second survey was conducted in February 2011 in four small ponds located in the park following the same Materials and methods procedure.

Sampling and isolation procedures Phytophthora isolates Soils in the Boscoincitta Park, Milan, Italy (45°29′9″N, 9°5′19″E) have a sandy-clay texture (70% coarse and fine sand, 15% silt The isolates used in the phylogenetic, morphological and physio- and 15% clay) and a slightly acidic to neutral reaction with pH logical studies are listed in Table 1. values ranging from 5Á5to7Á3. Between 2007 and 2010 annual precipitation varied between 912 mm (2007) and 1395 mm DNA isolation, amplification and sequencing (2008), with 1375 mm in 2010. Because of these favourable soil and climatic conditions, Acer pseudoplatanus stands were The 24 Phytophthora isolates obtained were subsequently trans- commonly planted in the park during previous decades. ferred to potato dextrose agar (39 g PDA, 5 g agar in 1 L Sampling was carried out from c. 30–35-year-old planted A. deionized water) and grown at 22°C for 1 week. The mycelium pseudoplatanus trees growing within a 2 km radius on non- was collected in sterile 1Á5 mL microfuge tubes by gently flooded sites in four forest stands of the Boscoincitta Park scratching hyphae from the surface of the colony with the aid of during both summer (June 2010) and winter (December 2010 a sterile scalpel and placed in the freezer (À20°C) for not less and February 2011). Affected trees showed symptoms character- than 12 h. DNA was then extracted following the protocol istic of infection by Phytophthora spp. such as general decline recommended by the extraction GenElute plant Genomic DNA and dieback of the crown, bleeding cankers at the collar and Miniprep kit (Sigma Aldrich) and finally stored at À20°C. along the stems with greenish-brown or reddish streaks at the The ITS (internal transcribed spacer) region of the ribosomal cambium and the outer xylem layers (Fig. 1). Severely damaged DNA was amplified using the primers ITS-6 (5′-GAAGGTGAA trees eventually died. GTCGTAACAAGG-3′) (Cooke et al., 2000) and ITS-4 (5′-TCC Bark samples including the cambium and adjacent stained TCCGCTTATTGATATGC-3′) (White et al., 1990). The pro- xylem tissue were taken from active lesions of five trees using a gramme used for amplification of the ITS region was: 95°C for hatchet, a knife and a scalpel. The samples were stored in polyeth- 3 min; 35 cycles of 95°C for 30 s, 55°C for 30 s, 72°C for 1 min. ylene envelopes for a maximum of 24 h. Small pieces The final extension was carried out at 72°C for 5 min. (c.89 3 9 3 mm) were cut from different parts and depths of Primers OomCoxILevup (5′-TCAWCWMGATGGCTTTTTT the phloem and xylem samples, blotted on filter paper, and plated CAAC-3′) and Fm85mod (5′-RRHWACKTGACTDATRATACC ′ onto selective PARPNH V8 agar (V8A; 16 g agar, 3 g CaCO3, AAA-3 ) modified by Robideau et al. (2011) were used to 100 mL Campbell’s V8 juice, 900 mL distilled water) amended amplify a 727 bp fragment of the mitochondrial cox1 gene from À À with 10 lgmL1 pimaricin, 200 lgmL1 ampicillin, 10 lg 15 isolates of P. acerina. To extend this, an overlapping 553 bp À À À mL 1 rifampicin, 25 lgmL 1 PCNB, 50 lgmL 1 nystatin and fragment was amplified from five isolates of P. acerina with

Plant Pathology (2013) Phytophthora acerina sp. nov. on Acer 3

(a) (b) (c)

(d) (e) (f)

(g) (h) (i)

Figure 1 Symptoms caused by Phytophthora acerina on 30–35-year-old Acer pseudoplatanus trees in forest stands of the Boscoincitta Park, Milan. (a,b) Thinning and dieback of crowns; (c) mortality and crown dieback; (d,e) bleeding canker on lower stem with tarry spots on the surface of the bark (d) and a tongue-shaped orange necrosis of the inner bark (e); (f) collar rot lesion with tarry spots; (g) tongue-shape brownish necrosis of the inner bark; (h,i) greenish to greyish discoloration of the xylem underneath a bark lesion. forward primer OomCoxI-Levlomod (5′-TTTGGTTTTTTGG A fragment of the b-tubulin gene was amplified and sequenced TCATCCAGAGG-3′) and reverse primer Fm83mod (5′-CCAA from 13 isolates of P. acerina using primers Btub_F1A (5′- TAAAAAATAACCAAAAATG-3′). The former primer anneals GCCAAGTTCTGGGARGTSAT-3′), Btub_R1A (5′-CCTGGTA at the same location as OomCoxI-Levlo (Robideau et al., 2011) CTGCTGGTAYTCMGA-3′), Btub_F2 (5′-CGGTAACAACTGG but in the forward orientation and based on P. infestans acces- GCCAAGG-3′) and Btub_R2 (5′-GATCCACTCAACGAAGTA sion U17009, and the latter is identical to Fm83 but shortened CG-3′) according to Blair et al. (2008). by two bases at the 5′ end to match the Tm of primer OomCox- All fragments were sequenced in both orientations in dye ter- I-Levlomod. The PCR programme used for amplification of both minator cycle sequencing reactions using the primers used for cox1 fragments was: 95°C for 2 min, followed by 35 cycles of PCR on an ABI 3730 Genetic Analyzer (Applied Biosystems) at 95°C for 1 min, 55°C for 1 min, 72°C for 1 min. The final The James Hutton Institute. ABI trace files were analysed using extension was carried out at a temperature of 72°C for 10 min. SEQUENCHER v. 4.6 (GeneCodes).

Plant Pathology (2013) 4 B. Ginetti et al.

Table 1 Isolates of the ‘Phytophthora citricola complex’ used in this study

Culture no.a Identiﬁcation Host Location, year Reference ITS cox1 b-tubulin

IMI 021173, P. citricola Citrus sinensis, fruit Taiwan, 1927 Scott et al. (2009) FJ237526 FJ237512 FJ665255 CBS 221.88 (type) CBS 295.29f P. citricola Citrus sp., leaf Japan, 1929 Jung & Burgess (2009) FJ560913 FJ665244 FJ665256 (authentic type) CH98U121C P. citricola – Japan Uddin et al. (unpubl.) AB367378 –– Citri-P0713b – Japan, Uddin et al. (unpubl.) AB367492 –– (Argentina) CBS 181.25, P. pini (ex- Pinus resinosa, Minnesota, Hong (unpubl.) FJ392322 –– IMI 077970 type) roots USA, 1925 22F3, P33 – Ohio, USA Hong (unpubl.) FJ392321 –– CIT-US1cf Fagus sylvatica, New York State, Jung & Burgess (2009) FJ665234 FJ665242 FJ665253 canker USA, 2003 CIT-US10cf F. sylvatica, canker New York State, Jung & Burgess (2009) FJ665235 FJ665243 FJ665254 USA, 2004 91-309 Thuja sp., canker Maumens, Lefort et al. (unpubl.) EU000125 –– Switzerland CBS 369Á61 P. citricola II Rhododendron sp. Germany, 1958 Hong (unpubl.) FJ392325 –– 22F2, P52 – New York State, Hong (unpubl.) FJ392324 –– USA, 1987 15C9 P. citricola III Acer saccharum Wisconsin, Hong (unpubl.) FJ392327 –– USA, 1985 1E1 Irrigation water Oklahoma, USA Hong (unpubl.) FJ392326 –– P11835.2d – Spain Moralejo (unpubl.) DQ648146 –– OH6/5 Quercus rubra, soil Ohio State, Balci et al. (2007) EF032477 –– USA, 2004 IMI 031372d P. citricola E Rubus idaeus Ireland Cooke et al. (2000) AF266788 –– 112 – Switzerland Bragante et al. (unpubl.) EU263906 –– 87-302 R. idaeus Switzerland, Lefort et al. (unpubl.) EU000100.1 Grandcours 83-41d – Switzerland, Lefort et al. (unpubl.) EU000081 –– Angers WAC 13201, P. multivora Eucalyptus Yalgorup, Scott et al. (2009) FJ237521 FJ237508 FJ665260 CBS 124094 (ex-type) marginata WA, 2007 WAC 13200c P. multivora E. gomphocephala Yalgorup, Scott et al. (2009) FJ237522 FJ237509 FJ665261 WA, 2007 WAC 13204c E. gomphocephala Yalgorup, Scott et al. (2009) FJ237518 FJ237507 FJ665259 WA, 2007 WAC 13205, E. marginata Jarrahdale, Scott et al. (2009) FJ237517 FJ237506 – CBS124095c WA, 1998 VHS 16168c Banksia grandis Pemberton, WA Scott et al. (2009) FJ237513 – FJ665257 IMI 329674c Soil Walpole, WA Scott et al. (2009) – FJ237504 – VHS 16439c B. littoralis Mandarah, WA Scott et al. (2009) FJ237516 FJ237505 FJ665258 P1817d Medicago sativa South Africa Kroon et al. (2004) AB367494 – AY564055 P10458d ––Blair et al. (2008) ––EU079582 P7902d Pinus radiata USA, 1992 Blair et al. (2008) ––EU080236 PLU-A5, P. plurivora F. sylvatica, root Irschenberg, Jung & Burgess (2009) FJ665225 FJ665236 FJ665247 CBS 124093c (ex-type) lesion Germany, 2004 PLU-A9c P. plurivora F. sylvatica, canker Irschenberg, Jung & Burgess (2009) FJ665226 –– Germany, 2004 PLU7 Q. robur, soil Pulling, Schubert et al. (1999) AJ007370 –– Germany, 1994 PLU9, Q. robur, soil Pulling, Scott et al. (2009) FJ237523 FJ237510 FJ665245 CBS 124087 Germany, 1994 PLU30, Q. robur, soil Cornuda, Jung & Burgess (2009) FJ665227 FJ665237 FJ665248 CBS 124089c Italy, 1995 PLU35, Q. petraea, soil Ljubliana, Scott et al. (2009) FJ237524 FJ237511 FJ665246 CBS 124090 Slovenia, 1995 PLU36c F. sylvatica, canker Munich, Jung & Burgess (2009) FJ665228 –– Germany, 1995 PLU41, A. saccharum, root Mount Royal, Jung & Burgess (2009) FJ665229 FJ665238 FJ665249 CBS 124091c Canada, 1996

(continued)

Plant Pathology (2013) Phytophthora acerina sp. nov. on Acer 5

Table 1 (continued)

Culture no.a Identiﬁcation Host Location, year Reference ITS cox1 b-tubulin

PLU77c Q. robur, nursery Nettetal, Jung & Burgess (2009) FJ665230 FJ665239 FJ665250 soil Germany, 1999 PLU92c Quercus sp., soil Turkey, 2000 Jung & Burgess (2009) FJ665231 FJ665240 FJ665251 PLU255c F. sylvatica, canker Sumuva, Czech Jung & Burgess (2009) FJ665232 –– Republic, 2007 PLU276, Carpinus betulus, Snagov, Jung & Burgess (2009) FJ665233 FJ665241 FJ665252 CBS 124092c soil Romania, 2008 P10338d Blair et al. (2008) ––EU079526 MN21HHd Rhododendron sp. USA Schwingle et al. (2007) DQ486661 –– InfGaule Gaultheria shalon Scotland Schlenzig (2005) AY879291 AY894685 – IMI 342898e Syringa vulgaris UK Cooke et al. (2000) - ITS, AF266789 AY564187 – Kroon et al. (2004) - cox1 P1822 P. capensis Stream water South Africa Bezuidenhout et al. (2010) GU191219 GU191277 GU191325 P1823 Olea capensis South Africa Bezuidenhout et al. (2010) GU191231 GU191298 GU191327 P1819 (ex-type) Curtisia dentate South Africa Bezuidenhout et al. (2010) GU191232 GU191275 GU191328 STE-U 6269 P. taxon Agathosma betulina South Africa Bezuidenhout et al. (2010) GU191228 GU191270 GU191317 ‘emzansi’ STE-U 6272 A. betulina South Africa Bezuidenhout et al. (2010) GU191220 GU191269 GU191316 B057, P. acerina A. pseudoplatanus, Milan, Italy, 2010 This study JX951285 KC156134 KC201283 CBS 133931cfg (ex-type) canker B080cf A. pseudoplatanus, Milan, Italy, 2010 This study JX951291 KC156140 KC201289 soil B035cfg A. pseudoplatanus, Milan, Italy, 2010 This study JX951282 KC156131 KC201281 canker B053cf A. pseudoplatanus, Milan, Italy, 2010 This study JX951283 KC156132 KC201282 canker B054c,f A. pseudoplatanus, Milan, Italy, 2010 This study JX951292 KC156141 KC201290 canker B055cf A. pseudoplatanus, Milan, Italy, 2010 This study JX951293 KC156142 KC201291 canker B056c,f A. pseudoplatanus, Milan, Italy, 2010 This study JX951284 KC156133 – canker B058cf A. pseudoplatanus, Milan, Italy, 2010 This study JX951286 KC156135 KC201284 canker B060cfg A. pseudoplatanus, Milan, Italy, 2010 This study JX951287 KC156136 KC201285 canker B062cf A. pseudoplatanus, Milan, Italy, 2010 This study JX951294 KC156143 KC201292 canker B063cf A. pseudoplatanus, Milan, Italy, 2010 This study JX951295 KC156144 KC201293 canker B064cfg A. pseudoplatanus, Milan, Italy, 2010 This study JX951288 KC156137 KC201286 canker B071cfg A. pseudoplatanus, Milan, Italy, 2010 This study JX951289 KC156138 KC201287 canker B077cfg A. pseudoplatanus, Milan, Italy, 2010 This study JX951290 KC156139 KC201288 canker B081cf A. pseudoplatanus, Milan, Italy, 2010 This study JX951296 KC156145 KC201294 soil CH95PHE28d Eustoma Japan Villa et al. (2006) AB217676 –– grandiflorum CH95PHEd E. grandiflorum Japan Villa et al. (2006) AB217676Á1 TARI23044d Prunus persica Taiwan, 2009 Ann et al. (unpubl.) GU111596 –– P10366d – – Coffey et al. (unpubl.) GU259257 –– aAbbreviations of isolates and culture collections: CBS, Centraalbureau voor Schimmelcultures Utrecht, Netherlands; IMI, CABI Bioscience, UK; WAC, Department of Agriculture and Food Western Australia Plant Pathogen Collection, Perth, Australia; VHS, Vegetation Health Service of the Department of Environment and Conservation, Perth, Australia; other isolate names and numbers are as given in GenBank. bSame code as isolate of Oudemans et al. (1994) which was collected in Argentina. cIsolates used in statistical analysis. dSubmitted to GenBank as P. citricola. eSubmitted to GenBank as P. inflata. fIsolates used in the morphological studies. gIsolates used in the growth temperature studies.

Plant Pathology (2013) 6 B. Ginetti et al.

Phylogenetic analysis dimensions and characteristic features of 50 mature oogonia, oospores and antheridia chosen at random were measured at Sequences of related taxa were downloaded from manually 9400 magnification at the surface of 15 9 15 mm square agar curated lists of GenBank BLAST hits and lists in relevant publi- plug cut from the centre of 14–21-day-old V8A cultures grown cations on ITS Clade 2 taxa. These were combined with in the dark at 20°C. The oospore wall index was calculated as sequences of outgroup taxa from GenBank. In the case of the the ratio between the volume of the oospore wall and the ITS region, disruption of the Clade 2 taxa alignment was pre- volume of the entire oospore (Dick, 1990). vented by a prior alignment of only the Clade 2 taxa sequences and subsequent alignment against sequences of the outgroup taxa using the CLUSTALW ‘Profile’ option. Mitochondrial cox1 Under-bark inoculation test and b-tubulin sequences were aligned using CLUSTALW under One-year-old twigs (diameter c.5–10 mm) were collected in the default settings. All alignments were inspected and adjusted field from a single mature tree of A. pseudoplatanus and Fagus manually if required. Bayesian phylogenetic analysis was con- sylvatica in March and May, respectively, shortly after budburst; ducted using a stand-alone version of MRBAYES v. 3.1.1 (Ron- leaves were removed and the twigs were cut into sections of c. quist & Heulsenbeck, 2003) in combination with tools 12 cm length. available in TOPALI v. 2.5 (Milne et al., 2008). Two duplicate In the centre of each cutting a c. 0Á5 cm diameter piece of MRBAYES analyses were run simultaneously for 2 million gener- bark was removed aseptically with a razor blade. An even-sized ations with a sampling frequency of every tenth generation and V8A disc cut from the margin of freshly growing cultures of the first 20% of samples discarded as burn-in. The general time P. acerina isolates was placed on the wound, covered with the reversible model with gamma-distributed rate variation and a removed bark piece and autoclaved wet cotton, and sealed with proportion of invariable sites was selected on the basis of the Parafilm and aluminium foil. Five isolates were tested. Controls model selection in TOPALI. The default settings of a flat Di- received only sterile V8A discs. Ten twigs were inoculated per iso- richlet prior using the six-rate parameter of the GTR substitu- late or control and placed in autoclaved glass Petri dishes containing tion model with three heated chains (temperature = 0Á2) and two layers of moist filter paper. The plates were sealed with Parafilm one cold chain were used. A 4 9 4 model of nucleotide evolu- and incubated at 20°C in the dark. After 21 days, lesion length was tion was used for the ITS analysis and in the case of the cox1 measured after removal of the outer bark. Random reisolations gene data a codon position model was applied. At the end of were made using PARPNH to fulfill Koch’s postulates. each run the convergence diagnostic potential scale reduction factor (PSRF) values for the taxon bipartitions were examined to ensure they were at or approaching a value of 1. Statistical analysis

Statistical analyses were carried out using STATA11 (Stata Statisti- Colony morphology, growth rates and cardinal cal software) to determine if morphometric and growth rate dif- temperatures ferences between P. acerina and other taxa of the ‘P. citricola complex’ were statistically significant. As the 50 measurements Morphology of hyphae and colony growth patterns were for every character in each isolate showed a normal distribution, described from 7-day-old cultures grown at 20°C in the dark on parametric tests were used. Data were tested for homogeneity of V8A, malt extract agar (MEA) and half-strength PDA (19Á5g variances between independent data. In case of homogeneity, a two- PDA, 7Á5 g agar, 1 L distilled water; Difco). Colony morpholog- sided t-test was applied. If the hypothesis of homoskedasticity was ies were described according to Erwin & Ribeiro (1996) and violated, a modified t-test for heterogeneous variances was used. Jung & Burgess (2009). For temperature–growth relationships, three replicate V8A plates per isolate were incubated at 10, 15, 20, 25, 30, 32 and Results 35°C. Radial growth rate was recorded after 5–7 days along two lines intersecting the centre of the inoculum at right angles. Isolation results In December 2010, P. acerina was isolated from necrotic Morphology of sporangia and gametangia bark and underlying stained xylem of all five mature A. pseudoplatanus trees sampled in four planted forest Sporangia and gametangia of 15 isolates of P. acerina were measured on V8A as described by Jung et al. (2003). Because stands of the BoscoincittaPark.Phytophthora acerina was morphometric data of the same isolates may differ considerably also isolated from rhizosphere soil of one of these five trees. between different studies, the authentic type of P. citricola s. str. No other Phytophthora species was recovered from bark (CBS295.29), the ex-type of P. plurivora (CBS124093) and cankers and rhizosphere soil of declining maple trees. P. pini isolates CIT US1 and CIT US10 (all used by Jung & Bur- In contrast, P. acerina could not be isolated from water gess, 2009) were included to verify whether the measurements bodies or from soil sampled near the canal. However, five of the present study could be compared with those published by other Phytophthora spp. were found, i.e. P. lacustris, P. Jung & Burgess (2009). Sporangia were produced by immersing taxon ‘PgChlamydo’, P. gonapodyides, P. inundata and P. 9 15 15 mm square agar plugs taken from growing margins of taxon ‘Walnut’. 3–5-day-old colonies in 90 mm Petri dishes filled with deionized water, and young leaves of Quercus ilex floating at the surface. After 6 h the water was replaced by nonsterile pond water. The Phylogenetic analysis plates were incubated at 20–25°C. After 24–36 h, dimensions and characteristic features of 50 mature sporangia per isolate The 760 bp ITS sequences of 15 isolates of P. acerina chosen at random were determined at 9400 magnification were identical to each other but were not an exact match (Axiophot dissecting microscope; Carl Zeiss). For each isolate, to any sequence in GenBank. They were most closely

Plant Pathology (2013) Phytophthora acerina sp. nov. on Acer 7

P10650 HQ261589 P. infestans

0·99 P0255 HQ261635 P. palmivora

PSEU 6 AY230190 P. pseudosyringae

Cg.2.3.3 AY241924 P. bisheria P1319 FJ801727 P. capsici 0·99 0·99 P10219 FJ801320 P. tropicalis

STE-U6269 GU191228 P. taxon ‘emzansi’ 1 1 P1823 GU191231 P. capensis

WAC13204 FJ237518 VHS16439 FJ237516 Phytophthora multivora 1 CBS 124095 FJ237521 0·98 CH98U121C AB367378 P0713 AB367492 Phytophthora citricola sensu stricto IMI021173 FJ237526 CBS 295.29 FJ560913 IMI 031372 AF266788 112 EU263906 Phytophthora citricola E 83-141 EU000081 CBS 181.25 FJ392322 CIT-US1 FJ665234 Phytophthora pini CIT-US10 FJ665235 22F3 FJ392321 0·77 91-309 EU000125 1E1 FJ392326 15C9 FJ392327 Phytophthora citricola III OH6/5 EF032477 P11835.2 DQ648146 NX05a JQ730714 P. taxon a NX05d JQ730716 P. taxon a 0·72 191 JQ730711 P. taxon a IMI342898 AF266789 CBS 124087 FJ237523 0·89 PLU7 AJ007370 CBS 124090 FJ237524 CBS 124093 FJ665225 0·91 CBS 124089 FJ665227 PLU92 FJ665231 PLU-A9 FJ665226 Phytophthora plurivora PLU36 FJ665228 CBS 124091 FJ665229 PLU77 FJ665230 CBS 124092 FJ665233 PLU255 FJ665232 MN21HH DQ486661 CBS 379.61 FJ392325 22F2 FJ392324 B035 JX951282 B063 JX951295 B081 JX951296 B062 JX951294 B058 JX951286 B055 JX951293 B054 JX951292 0·8 B053 JX951283 B080 JX951291 Phytophthora acerina B056 JX951284 CBS 133931 JX951285 B060 JX951287 B071 JX951289 B064 JX951288 B077 JX951290 CH95PHE28 AB217676 0·02 TARI 23044 GU111596 P10366 GU259257

Figure 2 Bayesian inference tree based on the rDNA ITS sequences of isolates of Phytophthora in ITS Clade 2. Numbers below the branches indicate the posterior probability values. aSubmitted to GenBank as P. plurivora.

Plant Pathology (2013) Table 2 Polymorphic nucleotides (numbers to be read vertically) from aligned sequence data of ITS, cox1 and b-tubulin gene regions showing the variation between isolates of Phytophthora acerina, 8 P. citricola s. str., P. plurivora, P. pini, P. citricola III and P. citricola E. Blue shading denotes polymorphisms found in P. citricola s. str., green shows polymorphisms found in P. plurivora, orange shading is for polymorphisms found in P. citricola I, III and E and yellow shading highlights polymorphisms only found in P. acerina. Grey shading denotes no data available

ITS cox1 β-tubulin 1 1 1 1 1 1 1 1 3 3 5 5 5 6 6 7 1 2 2 3 3 3 4 5 5 5 6 6 6 7 7 7 7 8 9 9 0 0 1 1 2 2 3 3 4 8 0 1 2 6 5 5 9 4 4 6 5 9 5 9 5 0 1 7 9 9 2 4 6 6 0 2 6 0 1 5 7 6 1 6 3 9 1 2 0 4 0 8 1 2 7 Isolate no. 9 5 0 6 4 1 7 1 2 4 0 8 2 9 3 4 0 2 3 9 0 9 1 4 3 1 0 2 1 3 4 4 2 9 6 2 9 8 0 8 8 2 4 9 6 Phytophthora acerina a CBS 133931 A – T –T A T T G R G G– GACT AGT T ACCAC C A C GCATT B054 A – T – T A T T G R G G – G A C T A G T T A C C A C C A C G A A C T T G A T A A G C A T T B055 A – T – T A T T G R G G – G A C T A G T T A C C A C C A C G A A C T T G A T A A G C A T T – A T – T A T T G R G G – G A C T A G T T A C C A C C A C G A A C T T G A T A A G C A T T B062 – B063 A – T – T A T T G R G G – G A C T A G T T A C C A C C A C G A A C T T G A T A A G C A T T B081 A – T – T A T T G R G G – G A C T A GT T A CCA C C A C G A A C T T G A T A A G C A T T Phytophthora acerina b TARI 23044 A – T – T A T T G G G G– P10366 A – T –T A T T G G G G – CH95PHE28 A – T – T A T T G A G G C TT Phytophthora taxonc 191 A – T – T A C C G G G G – NX05a A –T – T A T C G G G G –

NX05d A –T – T A T C G G GG– Ginetti B. Phytophthora citricola s.str. IMI 021173 A – C T T A T C G G G G – T T A C A T A A A T C A T T G A A T T T A C G T C G T G C A C T CBS 295.29 A – C T T A T C G G G G – A T C A T T G A A T T T A C G T C G T G C A C T P0713 A – C T T A T C G G G G–

CH98U121C A – C T T A T C G G G G – al. et Phytophthora plurivora CBS 124087 A –T T C A C C G G GG– T A ATGT T TTCTCC T A A A AAT T C A A T G AGC ATT CBS 124090 A – T T C A C C G G G G – T A A T G T A T TCTCC T A AAAATTC AATGA GTATT CBS 124089 A – T T C T C C G G G G – T C T C C T A A A A A T T C A A T G A G C A T T CBS 124091 A – T T C A C C G G G G– T CT C C T A A A A A T TCAATGA GTAT T CBS 124092 A – T T C A C C G G G G – T C T C C T A A A A A T T C A A T G A GT A T T CBS 124093 A – T T C A C C G G G G – T C T C C T A AAAATTC AATGA GTATT PLU77 A – T T C A C C G G G G– TCTCC T A AAAATTCA ATGA GTATT PLU92 A – T T C T C C G G G G – TCTCC T A AAAATTCAATGA GCATT InfGaul A – T T C A C C G G G G – T C T C C T A A A A A T T C A A T G A PLU7 A – T T C A C C G G G G – PLU-A9 A – T T C A C C G G G G– PLU36 A – T T C A C C G G G G– PLU255 A – T T C A C C G G GG– IMI 342898 A – T T C A C C G G G G – MN21HH A – T T C A C C G G G G – CBS 379.61 A – T T C A C C G G G G – ln Pathology Plant 22F2 A – T T C A C C G G G G – Phytophthora pini CIT-US1 A A T – T A T C G G G A – A C C A T T A A A T A T T C A A T G A A C A T C CIT-US10 A A T – T A T C G G G A – A C C A T T A A A T A T T C A A T G A A C A T C

(2013) CBS 181.25 A A T – T A T C G G A A – A CCA T T A T A T A T T C A A T A A A C A T C 91-309 A A T – T A T C G G G A – 22F3 A A T – T A T C G G G A – Phytophthora acerina sp. nov. on Acer 9

related to four GenBank accessions, three of which are 0 7 1 6 shown in Figure 2. Each of the four accessions shared T C 8 2 9 the base change at position 541 with all isolates of 1 4 4 P. acerina and two accessions had a single discrete -tubulin -tubulin β 8 2 3 change at position 752 not shared with any isolate in the 3 8 0 clade (Table 2). At position 564 all isolates of P. acerina had a clear heterozygous base (R = A/G) that was not reported in the GenBank accessions. However, two 2 4 1 8 accessions had an A and two had a G at this location. GA A CA T C T CA A GA 2 0 0 1 All these isolates formed a distinct clade in the ITS tree T 2 1 1 8 within a closely related cluster of six taxa that also 1 1 9 1 includes P. plurivora, P. citricola III, P. pini and P. citri- 2 1 0 9 cola E, with P. citricola s. str. at a position slightly basal 0 3 1 6 to the cluster (Fig. 2). Twelve ITS sequence differences T C A T A G A C A A 6 9 9 have been shown to discriminate amongst these six taxa (Table 2). T 9 1 2 Mitochondrial cox1 sequence was generated for 14 8 6 4 isolates of P. acerina, with 1194 bp sequenced for ﬁve T A 7 7 4 isolates and 694 bp for the remaining nine isolates. No A 7 5 3 sequence variation was observed amongst the P. acerina 7 1 1 isolates. There were no exact matches on GenBank. 7 0 2 Database coverage at the cox1 locus is not as compre- 1

x hensive as for the ITS region but sequences are available 6 6 0 o c for isolates of the closely related P. plurivora, P. citricola T T G A G T 2 6 1 III, P. pini and P. citricola s. str. Across these ﬁve taxa, A 6 0 3 27 single base changes were observed over the 1194 bp

5 6 4 of cox1 sequence, eight of which were unique to isolates CC 6 1 5 of P. acerina (Table 2). The phylogenetic analysis of A 4 9 5 cox1 sequences indicated that isolates of P. acerina 0 2 4 formed a distinct well-supported clade alongside the ﬁve other recognized taxa (Fig. 3). 3 9 9 Beta-tubulin gene sequences of 1100 bp in length were 9 3 3 generated for 13 isolates of P. acerina and once again no 7 2 3 intraspeciﬁc variation was observed. Five single base pair

2 1 0 changes were observed amongst the closely related 2 0 4 P. acerina, P. plurivora, P. citricola III, P. pini and 1 5 3 P. citricola s. str. (Table 2) but there was insufﬁcient phylogenetic signal (data not shown) in this gene to 9 9 resolve these taxa. Two bases speciﬁc to P. pini and P. citricola III were noted but other differences were 2 – 7 5 – – shared amongst P. plurivora, P. citricola s. str. and 6 9 A 8 P. acerina. 6 5 0 GA GA G A G – 5 6 4 G GG A – A C C A T T A T A T T C A A G A G A G – G A G A G A G – Taxonomy 2 4 G 5 G A G A G A G 4 1 C 5 C Phytophthora acerina B. Ginetti, T. Jung, D. E. L. — S Cooke, S. Moricca sp. nov. MycoBank MB 804019; 9 3 T C 7 T C T C T C T I

. – .

I Figs 4 6. I 1 5 A 3 A E I a a l l Sporangia abundantia in cultura liquida, persistentia, o o 5 4 1 T A T C T A T T c c i i r r terminalia, interdum lateralia aut intercalaria, semi-papil- t t i i c c – T A T A T – 6 6 – T A T A T – – T A – T – – – – T A – T P. citricola a a P. plurivora lata vel rare bipapillata, ovoidea, limoniformia vel r r o o 2 0 T T T T h h t t obpyriformia, rare ellipsoidea, obovoidea vel distorta, h h p p 1 5 – T – – T – T o o saepe cum obturamento conspicuo basale, apex interdum t t y y h h arcuatus, 50Á2 Æ 11Á2 9 33Á2 Æ 7Á3 lm, ratio longitudo P A P A A – A – A ad altitudinem 1Á5 Æ 0Á2. Sporangiophora simplicia vel ramosa sympodiis laxis irregularibus; interdum inserta (Continued) lateraliter ad sporangia, inﬂationes ad nodos rarae. Systema sexus homothallicum, solum partim functionale; 112 A 1 E 1 1 E A – P11835.2 A – – A P11835.2 OH6/5 15C9 Isolate no. Isolate 9 83-141 IMI 031372 Submitted to GenBank as Isolates produced during this study. Submitted to GenBank as

Table 2 a b c oogonia in medio 50% abortiva, terminalia, lateralia

Plant Pathology (2013) 10 B. Ginetti et al.

H270 AY129167 Phytophthora pseudotsugae

1 15/99 AY898627 Phytophthora infestans

ATCC56193 AY129173 Phytophthora colocasiae

302 AY129166 Phytophthora capsici

0·89 PD97/11132 AY564161 Phytophthora tropicalis 0·68 DDS1450 FJ237504

WAC13204 FJ237507 0·81 WAC13201 FJ237508 1 Phytophthora multivora 0·98 WAC13200 FJ237509

WAC13205 FJ237506

0·99 VHS16439 FJ237505 STE-U 6272 GU191269 Phytophthora taxon ‘emzansi’ STE-U 6269 GU191270

1 P1823 GU191298

0·83 P1822 GU191277 Phytophthora capensis 1 0·69 P1819 GU191275

IMI 021173 FJ237512 Phytophthora citricola sensu stricto 1 CBS 295.29 FJ665244

0·78 CIT-US1 FJ665242 CIT-US10 FJ665243 Phytophthora pini 0·8 CBS 181.25 GQ247650

1E1 GU071239 0·8 Phytophthora citricola III 15C9 GU071240 0·74 CBS 124091 FJ665238 0·75 PLU77 FJ665239

CBS 124092 FJ665241

0·98 InfGaul AY894685 IMI 342898 AY564187 Phytophthora plurivora CBS 124090 FJ237511

CBS 124087 FJ237510

CMW37938 JX272328 0·73 CBS 124089 FJ665237

PLU92 FJ665240

B054 KC156141 B055 KC156142

B062 KC156143 Phytophthora acerina 1 B063 KC156144 0·2 B081 KC156145

Figure 3 Bayesian inference tree based on sequences of the mitochondrial gene cox1 of isolates of Phytophthora in ITS Clade 2. Numbers below the branches indicate the posterior probability values.

Plant Pathology (2013) Phytophthora acerina sp. nov. on Acer 11

(a) (b) (c) (d) (e) (f)

(g) (h) (i) (j) (k)

(l) (m) (n) (o)

Figure 4 Semipapillate sporangia of Phytophthora acerina on V8 juice agar after 24–36 h ﬂooding with nonsterile pond water. (a,b) ovoid; (c) mouse-shaped, intercalary inserted; (d) elongated-ovoid with vacuole; (e–g) obpyriform, (f,g) with vacuole; (h) limoniform, sporangiophore widening towards base of sporangium; (i) ellipsoid with vacuole; (j) mouse-shaped with vacuole; (k) distorted, intercalary inserted; (l,m) distorted with vacuoles; (n) elongated-ovoid, empty sporangium after release of zoospores, with conspicuous basal plug; (o) irregular catenulate hyphal swellings. Scale bar = 10 lm, applies to a–o.

vel sessilia, globosa, rare subglobosa vel excentrica, irregular, sometimes catenulate hyphal swellings were 31Á3 Æ 3Á8 lm. Oosporae apleroticae vel pleroticae, infrequently formed on sporangiophores (Fig. 4o). Spo- 27Á8 Æ 3Á3 lm, paries 2Á0 Æ 0Á3 lm, in maturitate fre- rangia were persistent, semipapillate, less frequently bi- quenter pigmentati lutei ad luteifusci. Antheridia paragyn- or tripapillate or bilobed (<1% over all isolates) and osa, 12Á5 Æ 2Á3 9 9Á8 Æ 1Á6 lm. Aggregationes hypharum sometimes formed a conspicuous basal plug that pro- frequenter in agaro ‘V8A’ et in cultura liquida, diameter 15– truded into the sporangium (Fig. 4n). In all isolates spo- 150 lm. Inﬂationes hypharum subglobosae. Chlamydospo- rangial shapes showed a wide variation including ovoid rae non observatae. Temperaturae crescentes in agaro (54Á9% over all isolates; Fig. 4b), limoniform (14Á1%; ‘V8A’, optima c.25°Cetmaximac.32°C. Coloniae in Fig. 4h), obpyriform (8Á7%; Fig. 4e,f,g), ellipsoid (4Á1%; agaro ‘V8A’ chrysanthemum cum mycelio aerio restricto. Fig. 4i), elongated–ovoid (3Á9%; Fig. 4d,n), obovoid Regiones ‘rDNA ITS’, ‘cox1’et‘b-tubulin’ cum unica (1Á8%), broad–ovoid (1Á5%; Fig. 4a), mouse-shaped sequentia (GenBank JX951285, FJ665236, FJ665247). (1Á7%; Fig. 4c,j) and other distorted shapes (8Á7%; Etymology: Name refers to Acer pseudoplatanus from Fig. 4l,m). Sporangia with special features such as curved which all studied isolates had been isolated. apices (13Á6% over all isolates; Fig. 4c,i,j,m), lateral Sporangia of P. acerina were rarely observed on solid attachment of the sporangiophore (10Á3%; Fig. 4c), agar but were produced abundantly in nonsterile pond intercalary insertion (7Á7%; Fig. 4k), hyphal swellings, water. Sporangia were typically borne terminally on sometimes catenulate, on the sporangiophore (2Á4%; unbranched sporangiophores. Small subglobose or Fig. 4o), a short hyphal projection (1Á2%; Fig. 4c), vacu-

Plant Pathology (2013) 12 B. Ginetti et al.

(a) (b) (c) (d)

(e) (f) (g) (h)

Figure 5 Oogonia of Phytophthora acerina formed in V8 juice agar. (a–e) Mature viable oogonia with oospores containing ooplasts, and paragynous antheridia; (a) plerotic oospore and paragynous antheridium with ﬁnger-like projection; (b) elongated oogonium with aplerotic oospore and paragynous antheridium with ﬁnger-like projection; (c) sessile oogonium with almost plerotic oospore; (d) aplerotic oospores, the left one with two pellucid bodies (nuclei), the right one with two ooplasts; (e) elongated oogonium with tapering base and oospore with several small globules instead of one large ooplast; (f) oogonium that aborted before oospore formation (top) and viable oogonium (bottom); (g) sessile oogonium with plerotic aborted oospore; (h) two oogonia that aborted before oospore formation. Scale bar = 10 lm, applies to a–h.

oles (Fig. 4b,d,f,i,j,l,m) and a widening of the sporangio- range 15Á9–39Á3 lm; range of isolate means 24Á6– phore towards the base of the sporangium (Fig. 4h) were 32Á4 lm). The mean proportion of aplerotic oospores observed in all isolates. Sporangia in older water cultures (Fig. 5b,d,e) over all P. acerina isolates was 69Á6% (40– usually germinated directly. Zoospores were discharged 96%). In most isolates of P. acerina an unusually high through exit pores of 8Á2 Æ 1Á8 lm (Fig. 4n). Sporangial proportion of oogonia aborted before (Fig. 5f,h) or dimensions of 15 isolates of P. acerina averaged 52Á0 Æ immediately after the formation of the oospore wall 13 9 32Á8 Æ 7Á7 lm (overall range 20Á3–105Á7 9 11Á1– (Fig. 5g). The mean abortion rate of 14 isolates was 51Á3 lm) with a length/breadth (l/b) ratio of 1Á6 Æ 0Á3 38Á5% with isolate means ranging from 10–99%. Oo- (overall range 1Á2–2Á6; range of isolate means 1Á4–2Á0). spores had a medium wall thickness with a mean wall Mean sporangial dimensions of the authentic type of diameter of 2Á0 Æ 0Á38 (overall range 0Á8–3Á4; range of P. citricola s. str. (CBS295.29), the ex-type of P. plurivo- isolate means 1Á8–2Á4) and a mean oospore wall index of ra (CBS124093) and P. pini isolates CIT US1 and CIT 0Á37 Æ 0Á05 (overall range 0Á16–0Á63; range of isolate US10 deviated from the data in Jung & Burgess (2009) means 0Á35–0Á42). Antheridia of P. acerina were exclu- by less than 2 lm (data not shown). sively paragynous (Fig. 5a–d,f), globose to obovoid, Phytophthora acerina is homothallic with paragynous club-shaped or irregular, sometimes with a ﬁnger-like antheridia. Oogonia of P. acerina were borne terminally projection (2% over all isolates), and usually attached or were laterally sessile (Fig. 5c,g), had smooth walls close to the oogonial stalk. They measured and were usually globose to slightly subglobose (Fig. 5a, 12Á8 Æ 3Á4 9 9Á5 Æ 1Á6 lm. Mean oogonia and oospore c,d,g,h). Elongated oogonia (8Á6% over all isolates; diameters of the authentic type of P. citricola s. str. Fig. 5e), slightly elongated oogonia (2Á7%; Fig. 5f) and (CBS295.29), the ex-type of P. plurivora (CBS124093) excentric oogonia (3Á9%; Fig. 5b) were present in all iso- and P. pini isolates CIT US1 and CIT US10 deviated lates. In some isolates oogonial walls turned golden-yel- from the data in Jung & Burgess (2009) by less than low to golden-brown during ageing. The oogonia of 14 2 lm and oospore wall diameters were almost identical isolates of P. acerina showed a mean diameter of to those reported by Jung & Burgess (2009) (data not 32Á0 Æ 4Á4 lm (overall range 19Á2–45Á5 lm; range of shown). isolate means 28Á1–36Á6 lm). Oospores were usually glo- In ageing cultures (3–6 weeks old) and in nonsterile bose, but could be subglobose in elongated oogonia. pond water, P. acerina isolates produced globose to sub- Oospore diameters averaged 28Á4 Æ 3Á9 lm (overall globose and coralloid hyphal swellings (Fig. 6a,b,c) and

Plant Pathology (2013) Phytophthora acerina sp. nov. on Acer 13

(a) (b) (c) (d)

(e) (f) (g) (h)

(i) (j) (k) (l)

Figure 6 Vegetative structures formed by Phytophthora acerina in V8 juice agar. (a,b) Swollen lateral hyphal swellings; (c) lateral globose swelling with radiating short hyphae; (d) two lateral hyphae twisting around each other; (e) multiple branching at the end of a short lateral hypha; (f) multiple branching along a lateral hypha; (g,h) hyphal aggregations resulting from multiple successive branching and thickening of lateral hyphae; (i) multiple branching at the end of a main hypha; (j–l) dense hyphal aggregations resulting from multiple successive branching, twisting, intermingling and thickening of lateral hyphae. Scale bar = 10 lm, applies to a–l. abundant hyphal aggregations (Fig. 6d–l). The hyphal Colony morphology, growth rates and cardinal aggregations started with multiple lateral branching of temperature short sections along the hyphae (Fig. 6f,k,l) or by multiple branching at the end of main hyphae (Fig. 6i) or of Isolates of P. acerina formed appressed to submerged short lateral hyphae (Fig. 6e,g,h). Subsequent twisting colonies with a uniform to faintly stellate growth pattern and dense intermingling (Fig. 6d) of these irregular, cor- on MEA and rosaceous to petaloid colonies with moder- alloid lateral hyphae and swellings resulted in dense stro- ate aerial mycelium on half-strength PDA (Fig. 7). On mata-like structures with diameters of 10–150 lm. The V8A colony morphology was more variable, ranging authentic type of P. citricola s. str. (CBS295.29), the ex- from chrysanthemum to faintly petaloid and stellate pat- type of P. plurivora (CBS124093) and P. pini isolates terns, and from limited to woolly aerial mycelium. Col- CIT US1 and CIT US10 did not form dense hyphal ony growth patterns of P. citricola s. str. (CBS295.29), aggregations. Very rarely, in nonsterile pond water loose the ex-type of P. plurivora (CBS124093) and P. pini iso- intermingling and twisting of individual hyphae could be lates CIT US1 and CIT US10 on all three agar media observed in cultures of the authentic type of P. citricola corresponded well with those reported by Jung & Bur- s. str. and the ex-type of P. plurivora. gess (2009) (data not shown). Diameters of primary

Plant Pathology (2013) 14 B. Ginetti et al.

Figure 7 Colony morphology of Phytophthora acerina isolates B077, B063, B080 and CBS 133931 (from top to bottom) after 7 days’ growth at 20°C on V8 juice agar, potato dextrose agar and malt extract agar (from left to right). hyphae of P. acerina averaged 5Á1 Æ 1Á2 lm and varied Italy, under accession no. FI AGR 057). Ex-type culture from 2Á6to8Á7 lm. All five P. acerina isolates tested CBS 133931. For other specimens examined see Table 1. had identical cardinal temperatures and similar radial growth rates at all temperatures (Fig. 8). The maximum Under-bark inoculation test growth temperature for P. acerina was 32°C. All isolates were unable to grow at 35°C and did recommence All five isolates of P. acerina were pathogenic to 1-year- growth when plates previously incubated for 5 days at old twigs from mature trees of both A. pseudoplatanus 35°C were transferred to 18–20°C. Optimum tempera- and F. sylvatica (Fig. 9). ture for growth was 25°C with radial growth rates ran- After 22 days, mean lesion length on A. pseudoplat- ging from 7Á5to7Á9 mm per day. At 20°C P. acerina anus was 9Á9 Æ 0Á3 cm (overall range 6Á9–11Á3 cm) showed growth rates of 6Á5 Æ 0Á2 mm per day on V8A while lesion lengths on F. sylvatica averaged and 4Á3 Æ 0Á2 mm per day on PDA. 4Á6 Æ 0Á2 cm (overall range 3Á1–7Á8 cm). This difference in aggressiveness to both tree species was statistically significant (P < 0Á001). Specimens examined Typus: Italy: Boscoincitta Park, Milan, isolated from bleed- Notes ing bark canker of Acer pseudoplatanus, 2010, B. Ginetti. Holotype B057 (dried culture on V8A, herbarium of the A detailed list of morphological and physiological char- Museum of Natural History of the University of Florence, acters and data of P. acerina and other known taxa of

Plant Pathology (2013) Phytophthora acerina sp. nov. on Acer 15

Figure 8 Radial growth rates of six isolates of Phytophthora acerina on V8 juice agar at different temperatures.

Figure 9 Lesions caused by Phytophthora acerina in the under-bark inoculation trial after 21 days: greyish to greenish lesions on twigs of Acer pseudoplatanus (left side) and orange-brown lesions on twigs of Fagus sylvatica (right side). the ‘P. citricola complex’ is given in Table 3. Phyto- to stellate patterns with woolly mycelium (Fig. 7). On phthora acerina differs from the other taxa by its PDA the rosaceous to petaloid colonies clearly discrimi- abundant production of dense stromata-like hyphal nated P. acerina from P. plurivora (chrysanthemum), aggregations in both solid agar and in nonsterile liquid P. citricola s. str. (striate) and P. multivora (uniform) culture. In addition, no other taxon except for P. taxon (Jung & Burgess, 2009). Similar to P. plurivora, P. citri- ‘emzansi’ showed such a high oogonial abortion rate cola s. str., P. citricola E and P. multivora, P. acerina (Table 3). Further differences between P. acerina and had a growth optimum at 25°C whereas the optimum other taxa of the ‘P. citricola complex’ are listed below. temperature was 22Á5°C for P. capensis and P. taxon Oogonia of P. acerina were sometimes laterally ‘emzansi’ and 30°C for P. pini (Jung & Burgess, 2009; attached and could even be sessile, a feature not observed Bezuidenhout et al., 2010). At 32Á5°C the maximum in other taxa of the ‘P. citricola complex’. The oospore temperature for growth in P. acerina was markedly wall index of P. acerina was significantly lower than in higher than in P. capensis and P. taxon ‘emzansi’ P. multivora (0Á37 vs 0Á52; P < 0Á001) but significantly (27Á5°C; Bezuidenhout et al., 2010). higher than in all other taxa (P < 0Á001; Table 3). Unlike all the other taxa from the ‘P. citricola com- Discussion plex’, P. acerina isolates produced variable colonies on V8A, with some isolates forming chrysanthemum pat- In the first comprehensive phylogenetic analysis of the terns and limited aerial mycelium as in all other taxa, genus Phytophthora the major ITS Clade 2 contained while other isolates of P. acerina formed faintly petaloid seven species of which only P. citricola and P. inflata

Plant Pathology (2013) 16 B. Ginetti et al.

Table 3 Morphological characters and dimensions (µm) and temperature–growth relations of Phytophthora acerina, P. capensis, P. citricola s. str., P. multivora, P. pini, P. plurivora and P. taxon ‘emzansi’

P. citricola P. taxon P. acerina P. capensis s. str. P. multivora P. pinia P. pini P. plurivora ‘emzansi’

No. of isolates 15b 3c 2d 6e 2d 4f 7d 2c investigated Sporangia l 9 b mean 52 Æ 13 9 39Á1 Æ 6 9 52 Æ 7Á9 9 51 Æ 10Á4 9 53Á7 Æ 6Á5 9 47Á4 9 31Á547Á4 Æ 7Á7 9 46Á9 Æ 8Á6 9 32Á8 Æ 7Á7 24 Æ 3Á3 29Á9 Æ 5Á1 30 Æ 5Á1 33Á8 Æ 3Á9 33Á5 Æ 5Á9 27Á4 Æ 5Á7 Range of isolate 42Á5–61Á6 9 50Á9–52 9 44Á2–62Á1 9 51Á2–56Á2 9 39Á6–52Á3 9 means 26Á8–38Á3 29Á9 26Á2–34Á2 33Á5–34Á1 28Á9–38Á8 Total range 20Á3–105Á7 9 27Á5–50 9 36À75 9 36À58 9 39–70 9 31Á5–75Á3 9 27Á5–80Á5 9 35–67Á5 9 11Á1–51Á3 17Á5–32Á5 21À40 13À33 20–42Á1 22Á9–38Á9 16Á7–69Á6 17–47Á5 l/b ratio 1Á6 Æ 0Á26 1Á6–1Á71Á73 Æ 0Á28 1Á7 Æ 0Á22 1Á6 Æ 0Á16 1Á52 1Á43 Æ 0Á19 1Á8–1Á9 Oogonia Mean diameter 32Á0 Æ 4Á424Æ 2Á530Á0 Æ 3Á026Á5 Æ 1Á931Á2 Æ 2Á630Á328Á5 Æ 3Á330Á7 Æ 3Á1 Range of isolate 28Á1–36Á329Á7–30Á325Á5–27Á830Á9–31Á427Á5–29Á9 means Total range 19Á2–45Á520–27Á516Á7–35Á919–37 21Á3–36 22Á2–41Á415–37Á525–37Á5 Oospores Aplerotic 69Á6% 44% 45% 43% 44Á3% 47% oospores (40–96%) (32–56%) (36–52%) (38–48%) (22–62%) Mean diameter 28Á4 Æ 3Á922Á7 Æ 227Á1 Æ 2Á823Á6 Æ 1Á827Á7 Æ 2Á326 25Á9 Æ 3Á127Á9 Æ 2Á6 Total range 15Á9–39Á320–27Á515Á3–30Á917Á3–33Á118Á4–33Á219Á6–34Á214–35Á822Á5–32Á5 Wall diameter 2Á0 Æ 0Á42Á71Á68 Æ 0Á35 2Á6 Æ 0Á51Á8 Æ 0Á36 1Á71Á45 Æ 0Á35 < 2Á5 Oospore wall 0Á38 Æ 0Á09 0Á56g 0Á33 Æ 0Á05 0Á52 Æ 0Á07 0Á34 Æ 0Á05 0Á3 Æ 0Á06 0Á45h index Antheridia l 9 b mean 12Á8 Æ 3Á4 9 9 Æ 1Á8 9 12Á8 Æ 2Á7 9 12Á9 Æ 1Á9 9 12Á2 Æ 2Á1 9 12Á6 9 11Á111Á1 Æ 4Á4 9 14Á5 Æ 2Á1 9 9Á48 Æ 1Á6 9Á1 Æ 1Á7 8Á2 Æ 1Á7 8Á7 Æ 1Á3 9Á0 Æ 1Á6 8Á4 Æ 3Á1 14 Æ 1Á4 l 9 b range 5Á4–28Á5 9 5–12Á57Á5–18Á5 9 8–20 9 5–14 7Á7–16Á9 9 10Á2–15 9 7–21 9 10–20 9 5Á1–15Á2 5Á4–14Á4 6Á1–12Á6 10Á2–12Á9 5Á3–16 12Á5–17Á5 Abortion rate 38Á5% 42–46%i (10–99%) Hyphal + ––––––– aggregations Maximum 32 27Á5323232353227Á5 temperature (°C) Optimum 25 22Á5252530252520 temperature (°C) Growth rate on 7Á75 Æ 0Á19 6Á9 Æ 0Á16Á5 Æ 0Á02 9Á2 Æ 0Á74 8Á1 Æ 0Á18 V8A at optimum (mm per day) Growth rate at 20°C (mm per day) V8A 6Á52 Æ 0Á20 6Á6g 6Á2 Æ 0Á04 4Á8 Æ 0Á66Á3 Æ 0Á23 6Á3 Æ 0Á15g PDA 4Á28 Æ 0Á16 2Á0 Æ 0Á23Á3 Æ 06Á5 Æ 0Á42 3Á2 Æ 0Á2 aDesignated as P. citricola I in Jung & Burgess (2009). bSix of the 15 isolates were included in the growth tests. cData from: Bezuidenhout et al. (2010). dData from: Jung & Burgess (2009). eData from: Scott et al. (2009). fData from: Hong et al. (2011). gGrowth rate on CA at 20°C. hValues calculated from data in Bezuidenhout et al. (2010). iAborted plus immature oospores. were homothallic with paragynous antheridia (Cooke molecular re-evaluation demonstrated that the original et al., 2000). This clade has since expanded consider- P. inflata is a lost species and that isolates designated ably including, in 2012, 25 species and informally des- in recent years as P. inflata were conspecific with the ignated taxa (Jung & Burgess, 2009; Bezuidenhout newly described P. plurivora (Jung & Burgess, 2009). et al., 2010; Hong et al., 2011; Kroon et al., 2012). A The morphospecies P. citricola turned out as a

Plant Pathology (2013) Phytophthora acerina sp. nov. on Acer 17

complex of morphologically similar and phylogeneti- plex’. Such hyphal aggregations are not produced by any cally closely related taxa comprising P. citricola s. str., other taxon of the ‘P. citricola complex’ but are known the four newly described species P. capensis, P. multi- from P. cinnamomi, P. lateralis and P. gregata, a species vora, P. pini and P. plurivora, and the three informally with a comparably high oospore abortion rate, (Brasier designated taxa P. citricola III, P. citricola E and P. et al., 2010; Jung et al., 2011, 2013a). The hyphal aggre- taxon ‘emzansi’ forming the ‘P. citricola complex’ in gations of P. acerina resembled the stromata formed by subclade 2a and the two more distantly related new P. ramorum beneath the cuticle of infected leaves (Mora- species P. mengei and P. elongata belonging to subc- lejo et al., 2006), and it is likely that they play a role as lade 2c (Jung & Burgess, 2009; Bezuidenhout et al., survival structures in the life cycle of P. acerina. 2010; Hong et al., 2011; Kroon et al., 2012). The genetic uniformity of all 15 sequenced isolates, Phylogenetic analysis of the ITS, cox1 and b-tubulin pathogenicity to two common forest tree species of Eur- gene regions, as well as detailed morphological and phys- ope, and the fact that none of the sequences submitted to iological comparisons with all described species and des- GenBank from P. citricola-like isolates recovered during ignated taxa from the ‘P. citricola complex’, demonstrate extensive surveys of thousands of nursery stands, horti- that P. acerina is unique and forms a separate cluster cultural plantations and planted and seminatural forest within the complex, with P. plurivora being its closest stands in Europe is identical to P. acerina, strongly sup- known relative. ports the hypothesis that there has been a relatively All 15 genetically analysed isolates of P. acerina recent clonal introduction of P. acerina to northern Italy. shared identical ITS, cox1 and b-tubulin sequences. In The origin of P. citricola s. str. in Japan and Taiwan and GenBank, ITS sequences from four isolates designated as recent findings of P. acerina-like isolates in Japan P. citricola can be found that are similar (but not com- (CH95PHE28 and CH95PHE31) and Taiwan (TARI pletely homologous) to P. acerina. The ITS sequence of 23044), and of P. plurivora or P. plurivora-like isolates isolates TARI 23044 isolated in 2003 from Prunus persi- in remote, undisturbed healthy forests in Nepal (Vettrai- ca in Taiwan by Ann et al., and P10366 from the World no et al., 2011), Taiwan (T. Jung and M. Horta Jung, Phytophthora Collection (WPC:10366A631), submitted University of Algarve, Faro, Portugal, unpublished) and by Coffey et al., without information on host, geo- Yunnan (GenBank nos. JQ730711.1, JQ730714.1, graphic origin and date of isolation, are identical to each JQ730715.1 and JQ730716.1; Huai et al., 2013) provide other (GU111596.1 and GU259257.1, respectively). Phy- indirect evidence that the centre of origin of P. acerina tophthora acerina differs from these two isolates by might also lie in Eastern Asia. 1 bp. The other two strains (CH95PHE28 and With the exception of P. citricola s. str. and P. citrico- CH95PHE31), isolated from Eustoma grandiflorum in la E which seem to be relatively specific to Citrus spp. Japan by Villa et al. (2006), also share identical ITS and Rubus idaeus, respectively, most taxa from the ‘P. sequences (AB217676.1 and AB217677.1) and differ citricola complex’ are able to cause root rot, bark can- from P. acerina by 2 bp. The presence of A and G in kers and shoot dieback on multiple hosts from different these isolates at position 564 where P. acerina has an R genera and families. In particular, P. plurivora has a suggests these isolates might be ancestral to P. acerina wide host range with 45 species in 16 dicotyledonous but mtDNA data is required to verify this. In the ITS and four coniferous families, respectively, listed in the region, P. acerina differs from P. citricola s. str. by 4 bp original description including A. pseudoplatanus and (ex-type IMI021173) and 5 bp (CH98U121C), respec- several other Acer spp. (Jung & Burgess, 2009). In the tively; from P. pini by 4 bp (CIT-US1) and 5 bp (ex-type pathogenicity test of the present study, P. acerina was CBS 181.25), respectively; from both P. citricola E (83– aggressive to bark of its host species A. pseudoplatanus, 141) and P. citricola III (1E1) by 4 bp; from P. plurivora indicating that it might pose a serious threat to maple by 5 bp (ex-type CBS 124087) and 6 bp (PLU7), respec- stands in Italy and the rest of Europe if it cannot be tively; from P. capensis (P1822) and P. taxon ‘emzansi’ contained in the forest stands of the Boscoincitta Park (STE-U6269) by 8 and 11 bp, respectively; and from near Milan and if it is not immediately eradicated P. multivora (ex-type CBS 124095 and isolate Citri- wherever it may turn up in nurseries and plantations in P1817) by 10 bp. Analysis of the cox1 sequence data the future. More pathogenicity tests are required to clearly discriminated P. acerina as an evolutionarily dis- determine the potential host ranges of P. acerina, tinct lineage amongst the ‘P. citricola complex’. Differ- P. pini, P. multivora, P. citricola III and the not yet ences between P. acerina and other taxa ranged from introduced P. capensis and P. taxon ‘emzansi’ among 15–16 bp (P. plurivora)to23bp(P. citricola s. str.). As the native European tree and shrub species and the shown in other studies, the levels of variation are much most important exotic tree species used in forest and lower within the b-tubulin gene (Jung & Burgess, 2009; horticultural plantations. Hong et al., 2011). After P. plurivora, which was introduced a long time Besides many smaller differences to individual taxa ago and has become well established in seminatural eco- which are listed in the Notes, the high oospore abortion systems, plantings and nurseries across Europe, and the rate of 40–96% and the abundant production of dense recently arrived P. multivora, P. pini, P. citricola III and stromata-like hyphal aggregations clearly discriminate P. P. citricola E, P. acerina is the sixth species from the acerina from all other members of the ‘P. citricola com- ‘P. citricola complex’ that has been introduced to Europe.

Plant Pathology (2013) 18 B. Ginetti et al.

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