Mycologia, 96(3), 2004, pp. 598–613. q 2004 by The Mycological Society of America, Lawrence, KS 66044-8897 Botryosphaeria corticola, sp. nov. on Quercus species, with notes and description of Botryosphaeria stevensii and its anamorph, Diplodia mutila Artur Alves António Correia Mediterranean basin (Brasier 1992, 1996, Brasier et al 1993, Muñoz et al 1996, Robin et al 1998, Ragazzi et al 2000). Several factors, including drought, severe summer flooding, changes in traditional agronomic practices, wood-boring insects and fungal diseases (Brasier 1996), have been implicated in the decline. While root rot caused by Phytophthora cinnamomi Rands has been regarded as a major factor in the decline of Iberian oaks (Brasier 1992, Brasier et al 1993, Gallego et al 1999), several fungi that cause cankers and diebacks also are considered to be important contributing factors (Luque et al 2001). The most common and important canker and dieback pathogen is considered to be Botryosphaeria stevensii Shoemaker (anamorph: Diplodia mutila Fr.). Thus, Oliva and Molinas (1986) reported a Diplodia species associated with diseased cork oaks (Quercus suber L.) in northeastern Spain, while Vajna (1986) reported that D. mutila caused a branch canker and dieback of sessile oak (Q. petraea [Matt.] Liebl.) in Hungary. Later, Luque and Girbal (1989) reported that after cork removal B. stevensii invades the exposed trunks and causes wilting and death of Q. suber in northeastern Spain. As with most Botryosphaeria species, B. stevensii normally is encountered as its anamorph. The taxonomic history of D. mutila was explained by Stevens (1933) and by Sutton (1980), but some controversy surrounds the characters that define this fungus. In the original description, Montagne (1834) described the conidia as ‘‘Asci [conidia] elliptico-oblongi, didymi, sporidiis binis referti.’’ Stevens (1933) studied slides of Montagne’s exsiccatus in STR and described the conidia as hyaline and aseptate with a thick smooth, glassy wall, although pale brown, one-septate conidia sometimes were present. Both Shoemaker (1964) and Laundon (1973) agreed with Stevens’ concept. Sutton (1980), however, described the conidia as hyaline at first but becoming dark brown and one-septate when mature. In his illustration of this species he depicts a predominance of dark conidia. The only species of Botryosphaeria with mostly hyaline, aseptate, thick-walled conidia of the Diplodia type are B. stevensii and B. quercuum (Schwein.) Sacc. Shoemaker (1964) separated the two species on the basis of conidium shape as defined by their length/ width (L/W) ratios. Thus, in B. quercuum, the conid- Centro de Biologia Celular, Departamento de Biologia, Universidade de Aveiro, Campus Universitário de Santiago, 3810-193 Aveiro, Portugal Jordi Luque Departamento Protecció Vegetal, Institut de Recerca i Tecnologia Agroalimentàries, Centre de Cabrils, Ctra. de Cabrils s.n., E-08348 Cabrils, Barcelona, Spain Alan Phillips1 Centro de Recursos Microbiológicos, Faculdade de Ciências e Tecnologia, Universidade Nova de Lisboa, Quinta da Torre, 2829-516 Caparica, Portugal Abstract: Botr yosphaeria stevensii frequently has been associated with dieback and canker diseases of oak, mainly in the western Mediterranean area but more rarely in other regions. The species concept of B. stevensii has been unclear, and it is possible that some collections were identified incorrectly. A collection of fungal strains isolated from diseased oak trees and initially identified as B. stevensii was characterized on the basis of morphology and ITS nucleotide sequences. Morphology was compared with the type specimens of Physalospora mutila (5 B. stevensii) and its anamorph, Diplodia mutila. It was concluded that the isolates from oak differed from B. stevensii in having larger ascospores and conidia as well as different spore shapes and represented an as yet undescribed species, which is described here as B. corticola. Moreover, ITS sequence data separated B. corticola from all other known species of Botryosphaeria. Amended descriptions of B. stevensii and its anamorph are provided to differentiate B. stevensii from B. corticola and to clarify some of the earlier taxonomic uncertainties. Key words: Ascomycetes, Botryosphaeriaceae, ITS, oak decline, phylogeny, ribosomal DNA, systematics INTRODUCTION Since the early 1980s a serious decline of oak trees has been recognized in the Iberian Peninsula and the Accepted for publication August 27, 2003. 1 Corresponding author. E-mail: alp@mail.fct.unl.pt 598 ALVES ET AL: BOTRYOSPHAERIA CORTICOLA ia are subglobose with a L/W ratio of about 1.5 while in B. stevensii they are oblong with a L/W ratio of about 2.3. The consensus is that conidia of B. stevensii are (20–)25–27 3 10–12 mm (Stevens 1933, Shoemaker 1964, Laundon 1973, Sivanesan 1984), but Sutton (1980) considered they can be up to 31 mm long. The history of B. stevensii also is confused somewhat. Stevens (1936) discovered a Physalospora species, single ascospore cultures of which produced conidia typical of D. mutila. Because no described species had been referred to the teleomorph of D. mutila, he made a new combination in Physalospora as P. mutila (Fr.) N.E. Stevens. However, because of the lack of ascomycete elements in the type of D. mutila, he in fact described a new species. Because this was published after 1 Jan 1935 without a Latin diagnosis, the name was not valid. Shoemaker (1964) provided a Latin description for P. mutila, thus validating the name, but considered it to be better accommodated in Botryosphaeria. Because of the earlier name, Botryosphaeria mutila (Schwein.) Cooke, Grevillea 13: 101 (1885), Shoemaker proposed a new one, Botryosphaeria stevensii Shoemaker, typified by the type of P. mutila N.E. Stevens ex Shoemaker. Because the concepts of B. stevensii and D. mutila are not entirely clear, it is possible that some collections have been misidentified. When Luque and Girbal (1989) reported B. stevensii from Q. suber they mentioned that conidia of the strains they examined were larger than normal for this species but they regarded this as natural variation in the fungus. Zhou and Stanosz (2001a, b) more recently suggested that the name B. stevensii might have been applied to more than one species, thus raising the possibility that the fungus reported on oak in fact is not B. stevensii. Analysis of nucleotide sequences of ribosomal genes has contributed greatly to resolving phylogenetic relationships of fungi (Bruns et al 1991, Berbee and Taylor 1992, O’Donnell et al 1997). Comparison of the nucleotide sequences of the internal-transcribed spacer (ITS) regions of the nuclear ribosomal DNA has been used to clarify the taxonomy and phylogenetic relationships of Botryosphaeria species ( Jacobs and Rehner 1998, Denman et al 2000, Zhou and Stanosz 2001b). Analysis of ITS sequence data also has been used to help distinguish morphologically similar species in this genus (Smith et al 2001, Phillips et al 2002). Since 1989, several Botryosphaeria isolates morphologically similar to B. stevensii were obtained from oak in Spain, Portugal and Italy. The aim of the present study, therefore, was to determine whether these strains were distinguishable from B. stevensii on the ON QUERCUS 599 basis of morphological characters and the nucleotide sequence of the 5.8S ribosomal gene and its flanking ITS regions. These data were compared with strains previously identified as B. stevensii and also morphologically with the type specimens of B. stevensii and D. mutila. MATERIALS AND METHODS Isolates.—Material from diseased branches and trunks was collected from Quercus ilex L. or Q. suber in several regions in Spain and one region each in Portugal and Italy. Diseased material also was collected from Vitis vinifera L. and Pyrus communis L. Isolations were made from single ascospores, conidia or by directly plating out pieces of diseased tissue after surface sterilization (3 min in 70% ethanol). Ascomata or conidiomata were cut through horizontally and the contents transferred to a drop of sterile water on a flamed microscope slide. A portion of this was taken and spread over a few square centimeters of a plate of Difco potato-dextrose agar (PDA). The water on the slide was allowed to evaporate at room temperature before a drop of 100% lactic acid was applied and covered with a cover slip. The Petri dish bearing the spores was incubated at 25 C overnight. The next day individual germinating spores were transferred to fresh plates of PDA and checked by microscopy to ensure that a single spore had been transferred. In this way, single ascospore and single conidium isolates could be linked to the semipermanent preparation and their identity verified by microscopy. Strains isolated by M. E. Sánchez and A. Trapero (University of Córdoba, Spain) also were included in the study (TABLE I). Fungi isolated in our study were deposited at Centraalbureau voor Schimmelcultures (CBS, Utrecht, The Netherlands), and they are referred to throughout this paper by their CBS accession numbers (TABLE I). Specimens were lodged with the herbarium of Estação Agronómica Nacional, Oeiras, Portugal (LISE). In the phylogenetic analysis, nucleotide sequences of the 5.8S ribosomal DNA gene and the flanking ITS regions of various Botryosphaeria species were taken from GenBank (TABLE I). Morphology and cultural characters.—Cultures were maintained on half-strength PDA or oatmeal agar (Anonymous 1968). To induce sporulation, a 4 cm length of autoclaved poplar twig was added to each Petri dish. Colony characteristics were recorded from cultures grown on full-strength PDA at room temperature (ca 20–25 C) and exposed to indirect sunlight. Growth was determined on full-strength PDA incubated in the dark at 5-degree intervals between 5 C and 35 C. Observations on micromorphological features were made with a Leica DMR HC microscope with bright field or Nomarski differential interference contrast illumination. Digital images were recorded with an Olympus C-3030 camera. Measurements were made with the UTHSCSA ImageTool version 3. At least 50 ascospores or conidia of each isolate were measured on images taken with the 1003 objective lens. Data for spore measurements are presented as the low- Identitya and origin of isolates studied Teleomorph corticola corticola corticola corticola corticola corticola corticola corticola corticola corticola corticola corticola corticola corticola corticola obtusa obtusa obtusa obtusa obtusa quercuum rhodina rhodina rhodina stevensii stevensii stevensii stevensii stevensii tsugae sp. sp. Anamorph Diplodia corticola Diplodia corticola Diplodia corticola Diplodia corticola Diplodia corticola Diplodia corticola Diplodia corticola Diplodia corticola Diplodia corticola Diplodia corticola Diplodia corticola Diplodia corticola Diplodia corticola Diplodia corticola Diplodia corticola Diplodia sp. Diplodia sp. Diplodia sp. Diplodia sp. Diplodia sp. Unnamed Lasiodiplodia theobromae Lasiodiplodia theobromae Lasiodiplodia theobromae Diplodia mutila Diplodia mutila Diplodia mutila Diplodia mutila Diplodia mutila Sphaeropsis sp. Diplodia quercina Unnamed Sphaeropsis sapinea f. sp. cupressi Sphaeropsis sapinea Diplodia sp. Sphaeropsis sapinea Collection or culture numberb Host CBS112545 CBS112546 CBS112547 CBS112548 CBS112549d CBS112550 CBS112551 CBS112552 CBS112070 CBS112077 CBS112071 CBS112072 CBS112073 CBS112076 CBS 112074 K. J. 93. 56 CBS112555 CBS112556 ATCC60851 Sano 1 CBS177.89 CBS356.59 Z. S. 96-112 K. J. 93.40 CBS678.88 K. J. 93.35 ATCC60259 CBS112553 Z. S. 94-3 CBS418.64 K. J. 93.29 K. J. 93.58 Quercus suber Quercus ilex Quercus ilex Quercus suber Quercus suber Quercus suber Quercus suber Quercus suber Quercus suber Quercus suber Quercus ilex Quercus ilex Quercus suber Quercus suber Quercus suber Hardwood shrub Vitis vinifera Pyrus communis Prunus persica Malus domestica Quercus cerris Theobroma cacao Pinus radiata Pistacia sp. Quercus suber Quercus suber Malus pumila Vitis vinifera Juniperus sp. Tsuga heterophylla Quercus sp. Tsuga sp. M. E. Sánchez, A. Trapero Cádiz, Spain M. E. Sánchez, A. Trapero Huelva, Spain M. E. Sánchez, A. Trapero Córdoba, Spain A. Alves Aveiro, Portugal A. Alves Aveiro, Portugal A. Alves Aveiro, Portugal A. Alves Aveiro, Portugal A. Alves Aveiro, Portugal J. Luque Barcelona, Spain J. Luque Girona, Spain J. Luque Girona, Spain J. Luque Badajoz, Spain J. Luque Badajoz, Spain J. Luque Cádiz, Spain J. Luque Tempio Pausania, Italy G. J. Samuels New York, USA A. J. L. Phillips Montemor-o-Novo, Portugal A. J. L. Phillips Monte da Caparica, Portugal K. Britton Georgia, USA T. Sano USA A. Vannini Italy E. Müller Agalawatta, Sri Lanka W. Swart South Africa T. J. Michailides California, USA J. Luque Girona, Spain K. A. Jacobs Spain H. J. Boesewinkel Unknown A. J. L. Phillips Montemor-o-Novo, Portugal N. Tisserat USA A. Funk British Columbia, Canada E. Hecht-Poinar USA G. J. Samuels North Carolina, USA AY259089 AY259090 AY259110 AY259099 AY259100 AY259097 AY259101 AY259102 AY259105 AY259106 AY259107 AY259108 AY268420 AY259109 AY268421 AF027759 AY259094 AY259096 AF243408 AB034812 AF243399 AF243400 AF243401 AF027760 AY259104 AF027754 AF243406 AY259093 AF243403 AF243405 AF027753 AF027755 Z. S. 94-158 K. J. 93.31 M. 17-3-98 Z. S. 92-43 Cupressus sempervirens Pinus sp. Pinus sylvestris Pinus sylvestris W. Swart M. de Kam S. Schroeder G. R. Stanosz AF243402 AF027756 AJ292761 AF243409 Collector Location Israel Netherlands Lower Saxony, Germany Wisconsin, USA GenBankc MYCOLOGIA Botryosphaeria Botryosphaeria Botryosphaeria Botryosphaeria Botryosphaeria Botryosphaeria Botryosphaeria Botryosphaeria Botryosphaeria Botryosphaeria Botryosphaeria Botryosphaeria Botryosphaeria Botryosphaeria Botryosphaeria Botryosphaeria Botryosphaeria Botryosphaeria Botryosphaeria Botryosphaeria Botryosphaeria Botryosphaeria Botryosphaeria Botryosphaeria Botryosphaeria Botryosphaeria Botryosphaeria Botryosphaeria Botryosphaeria Botryosphaeria Botryosphaeria Botryosphaeria Unknown Unknown Unknown Unknown 600 TABLE I. TABLE I. Continued ATCC22927 K. J. 93.095 Z. S. 97-23 K. J. 94.23 K. J. 94.26 CBS110302 CBS110299 ATCC58194 Z. S. 97-58 CBS 110301 ATCC58189 K. J. 94.09 Vaccinium sp. Cercis canadensis Liquidambar styraciflua Malus sylvestris Prunus persica Vitis vinifera Vitis vinifera Actinidia deliciosa Sophora chrysophylla Vitis vinifera Malus sylvestris Melaleuca quinquenervia Vitis sp. Eucalyptus viminalis Collector R. D. Millholland K. A. Jacobs K. Britton P. L. Pusey P. L. Pusey A. J. L. Phillips A. J. L. Phillips G. J. Samuels D. Gardner A. J. L. Phillips G. J. Samuels M. B. Rayachhetry A. B. Baudoin P. W. Crous Location GenBankc North Carolina, USA District of Columbia, USA South Carolina, USA Georgia, USA Japan Montemor-o-Novo, Portugal Oeiras, Portugal New Zealand Hawaii Palmela, Portugal New Zealand Florida, USA New York, USA Stellenbosch, South Africa AF243397 AF027752 AF241174 AF027747 AF027749 AY259092 AY259091 AF243396 AF246929 AY259098 AF243395 AF027743 AF216533 AF283690 ON QUERCUS Fungus names are those used by the collector in the original publication in which they were mentioned, or in GenBank. Designation of isolates and collection numbers: CBS 5 Centraalbureau voor Schimmelcultures; ATCC 5 American Type Culture Collection; K. J. 5 Jacobs and Rehner (1998); Z. S. 5 Zhou and Stanosz (2001a). c ITS sequences represented in italics were obtained from GenBank, the other sequences were determined in the present study. d Isolates in bold are ex-type. e KJ93.09 was referred to as B. obtusa by Jacobs and Rehner (1998) but as B. dothidea in the GenBank accession data. In the present paper, ITS sequence data identified it as B. corticola. b BOTRYOSPHAERIA CORTICOLA a Fusicoccum sp. Fusicoccum aesculi Fusicoccum aesculi Fusicoccum aesculi Fusicoccum aesculi Fusicoccum aesculi Fusicoccum luteum Fusicoccum luteum Fusicoccum sp. Fusicoccum parvum Fusicoccum parvum Fusicoccum sp. Phyllosticta sp. Unknown Host ET AL: Botryosphaeria corticis Botryosphaeria dothidea Botryosphaeria dothidea Botryosphaeria dothidea Botryosphaeria dothidea Botryosphaeria dothidea Botryosphaeria lutea Botryosphaeria lutea Botryosphaeria mamane Botryosphaeria parva Botryosphaeria parva Botryosphaeria ribis Guignardia bidwellii Mycosphaerella africana Anamorph ALVES Teleomorph Collection or culture numberb 601 602 MYCOLOGIA er and upper 95% confidence limits, with the minimum and maximum dimensions in parentheses. Dimensions of other fungal structures are given as the range of at least 20 measurements where possible. DNA extraction and PCR amplification.—Fungal isolates were grown in Czapek Dox broth or potato-dextrose broth for 5 d at approximately 23 C. Genomic DNA was isolated from fresh mycelium following an adaptation of the method of Pitcher et al (1989). The mycelium was ground in liquid nitrogen, transferred to a 2 mL Eppendorf tube and mixed with 500 mL of TE buffer (10 mM Tris-HCl, 1 mM EDTA, pH 8.0), and the contents were divided into two Eppendorf tubes. To each tube 500 mL of GES buffer (5 M guanidine thiocyanate, 100 mM EDTA, pH 8.0; 0.5% Sarkosyl) were added, mixed by inversion and placed on ice for 5 min. Then 250 mL of cold 10 M ammonium acetate was added and the tubes were placed on ice another 5 min. The aqueous phase was extracted with 1 volume of CIA (chloroform: isoamyl alcohol; 24:1 v:v) and nucleic acids were precipitated with 1 volume of cold isopropanol. After centrifugation the pellet was washed with 70% ethanol and dissolved in 500 mL of TE buffer containing RNase (50 mg/mL) followed by incubation at 37 C for 30 min. The aqueous phase again was extracted with CIA and nucleic acids were precipitated with 1/10 volume of cold 3 M sodium acetate, pH 5.2 and 2.5 volumes of cold 100% ethanol. DNA was recovered by centrifugation and the pellet washed with 70% ethanol. DNA was dissolved in 100 mL TE buffer and stored at 220 C. DNA concentrations were estimated by spectrophotometry (Cary 50 Bio, Varian, Mulgrave, Australia). PCR amplification of the nuclear 5.8S ribosomal RNA gene and its flanking ITS regions was performed on a BioRad iCycler Thermal Cycler (Hercules, California). The PCR primers ITS1 and ITS4 (White et al 1990) were supplied by MWG Biotech AG (Ebersberg, Germany). PCR reactions were carried out with Taq polymerase, nucleotides and buffers supplied by MBI Fermentas (Vilnius, Lithuania). The PCR reaction mixtures contained 13 PCR buffer (PCR buffer without MgCl2: PCR buffer with (NH4)2SO4; 1: 1 v:v), 3 mM MgCl2, 200 mM of each nucleotide, 15 pmol of each primer, 1 U of Taq polymerase, and 50–100 ng of template DNA. Each reaction volume was made up to 50 mL with sterile HPLC-grade water. Negative controls with sterile water instead of the template DNA were used in every PCR reaction. The amplification conditions were: initial denaturation of 7 min at 95 C, followed by 25 cycles of 1 min at 94 C, 1 min at 50 C, and 1 min at 72 C, and a final extension of 10 min at 72 C. After amplification, 5 mL of each PCR product were separated by electrophoresis in 1% agarose gels in 13 TAE buffer (40 mM Tris, 40 mM acetate, 2 mM EDTA, pH 8.0). Gels were stained with ethidium bromide and visualized on a UV transilluminator to assess PCR amplification. The amplified PCR fragments were purified with the Concert Rapid PCR Purification System (Gibco BRL, Eggenstein, Germany) before DNA sequencing. DNA sequencing.—Both strands of the PCR products were sequenced with the ABI PRISMt BigDyey Terminator Cycle Sequencing Ready Reaction Kit with AmpliTAQ DNA Polymerase (PE Applied Biosystems, Foster City, California) in a Bio-Rad iCycler Thermal Cycler. Cycle sequencing reactions (20 mL) contained 30–90 ng DNA template, 3.2 pmol of each primer, 4 mL BigDyey Terminator and sterile HPLC-grade water. The cycle sequencing program was: 25 cycles of 96 C for 10 s, 50 C for 5 s, and 60 C for 4 min. To remove excess dye terminator the entire content of each extension reaction was mixed thoroughly with 2 mL of 3 M sodium acetate (pH 4.6) and 50 mL of 100% ethanol. Extension products were precipitated for 20 min at room temperature and centrifuged 20 min at 16 000 3 g. The supernatant was discarded and 250 mL of 70% ethanol was added. The tubes were vortexed, centrifuged 5 min at 16 000 3 g and the supernatant discarded. The tubes were dried in a heat block at 90 C for 1 min. The sequences were obtained with the ABI PRISMt 310 Genetic Analyzer (PE Applied Biosystems, Foster City, California). The complete sequences of the ITS region (partial 18S, ITS1, 5.8S gene, ITS2, and partial 28S sequences) were read and edited with Chromas 1.45 (http://www. technelysium.com.au/chromas.html). All sequences were checked manually, and nucleotide arrangements at ambiguous positions were clarified using both primer direction sequences. Sequences were deposited in the GenBank public database. Phylogenetic analysis.—DNA sequences from this study and those retrieved from GenBank were aligned with MegAlign (DNASTAR Inc., Madison, Wisconsin). The alignments were checked visually and improved manually where necessary. In the analyses, alignment gaps were treated as missing data. Sequences of Guignardia bidwellii (Ellis) Viala & Ravaz and Mycosphaerella africana Crous & M. J. Wingf. were used as outgroup. Phylogenetic analyses were performed with PAUP* (Phylogenetic Analysis Using Parsimony) version 4.0b10 (Swofford 2000). Trees were produced using both neighbor-joining (NJ) and maximum-parsimony (MP) analyses for the ITS sequence dataset. The Kimura two-parameter distance calculation was used for NJ analysis. For MP analysis, the heuristic search option with 1000 random addition sequences and TBR branch-swapping options were used. Stability of clades was assessed with 1000 bootstrap replications in a heuristic search. Other measures used were tree length, consistency index (CI), retention index (RI), rescaled consistency index (RC) and homoplasy index (HI). RESULTS Morphology and cultural characteristics.—All isolates from oak, irrespective of whether they were derived from single ascospores or conidia, were morphologically similar. Colonies on PDA formed abundant aerial mycelium that was initially white but turned darkolivaceous after 5–6 d at 25 C. The reverse side was almost black in older cultures. Pycnidia usually were aggregated and appeared after 20–25 d in cultures incubated under black light with a 12 h photoperiod. Cultures of the isolate from Vitis (CBS112553) that ALVES ET AL: BOTRYOSPHAERIA CORTICOLA was tentatively identified as D. mutila were similar, except that pycnidia were produced after 14–21 d under the same conditions. Ascospores of the strains from oak were (28.2–) 33.6–34.7(–40.6) 3 (12.0–)14.6–15.3(–18.8) mm. In contrast, ascospores of the specimen of B. stevensii we studied (BPI599153) were (24.8–)30.8–32.1(–36.2) 3 (9.5–)11.2–11.7(–13.4) mm. Conidia of the isolates from oak were (23.7–)29.6–30.3(–46.1) 3 (9.1–) 13.4–13.8(–20.5) mm, which is considerably larger than was found in D. mutila K(M)99664, which has conidia (23.5–)25.1–25.7(–27.4) 3 (12.4–)13.2– 13.5(–14.3) mm. Conidium dimensions of the strain of D. mutila isolated in this work (CBS112553) were similar to K(M)99664. Conidiomata and ascomata of the collections from oak were mostly multilocular but conidiomata of B. stevensii were mostly unilocular. The teleomorph of B. stevensii was not found in this work. Phylogenetic analysis.—ITS sequences of 37 strains of Botryosphaeria species, either sequenced in this study or retrieved from GenBank, were included in the phylogenetic analysis. The sequence alignment is available from TreeBase (S949). Of the 549 characters in the ITS dataset, 338 were constant, 53 variable characters were parsimony uninformative and 158 were parsimony informative. The 26 equally parsimonious trees generated from the heuristic search exhibited low levels of homoplasy as indicated by a consistency index (CI) of 0.848, a retention index (RI) of 0.940 and a homoplasy index (HI) of 0.152. The topology of the trees differed from one another only in the positions of isolates within terminal groupings. Tree topologies resulting from maximumparsimony and neighbor-joining analyses were similar, and only the former is shown (FIG. 1). Two major clades were resolved corresponding to Botryosphaeria species with Fusicoccum or Diplodia anamorphs. Within the Fusicoccum clade, five groups were resolved and these corresponded to five Botryosphaeria species that generally are accepted to have Fusicoccum anamorphs. Five groups also were resolved within the Diplodia clade, and four of these corresponded to known species of Botryosphaeria. The strains that were isolated from oak, which we describe here as B. corticola, clustered with isolates from oak retrieved from GenBank, namely AF027754 (KJ93.35 as B. stevensii), AF027753 (KJ93.29 as Diplodia quercina Westend.) and AF243399 (CBS177.89 as B. quercuum). Two isolates from hosts other than Quercus also lay within this subclade, namely, AF027755 (KJ93.58 as Botryosphaeria sp. from Tsuga sp.) and AF027752 (KJ93.09 as B. dothidea [Moug. : Fr.] Ces. et De Not. from Cercis canadensis L.). This sub- ON QUERCUS 603 clade was supported by a high bootstrap value of 100% and was distinct from the subclade containing isolates considered to be B. stevensii. Isolates previously identified as B. stevensii were distributed in two separate subclades, with the isolates from apple and grapevine (AF243406 and CBS112553) grouping together while an isolate from Tsuga sp. grouped in a separate subclade with Sphaeropsis sapinea f. sp cupressi from Cupressus sempervirens L. These groups were supported by high bootstrap values (.98%). Other major subclades in the Diplodia clade corresponded to B. obtusa (Schwein.) Shoemaker, B. rhodina (Cooke) Arx, and B. tsugae Funk. The B. obtusa clade contained isolates that had been identified as B. obtusa and others identified as Diplodia pinea (Desm.) J. Kickx f. (5 Sphaeropsis sapinea [Fr. : Fr.] Dyko & Sutton). Based on morphological features as well as DNA phylogeny, we consider the isolates from oak to represent a new species. We describe it here as Botryosphaeria corticola. TAXONOMY Botryosphaeria corticola A. J. L. Phillips, Alves et Luque, sp. nov. FIGS. 2–18 Anamorph: Diplodia corticola A. J. L. Phillips, Alves et Luque, sp. nov. Ascostromata in contextu hospitis inclusa, usque 1 mm diametro, erumpescentia, solitaria, stromatiformis, multilocularis, atrobrunnea vel nigra, cum ostiolis centralibus. Asci clavati, inter pseudoparaphyses filiformes interspersi, 160– 250 3 30–33 mm, octosporati, bitunicati cum loculo apicali bene evoluto. Ascosporae irregulariter biseriatae, hyalinae, unicellulares, (28.2–)33.6–34.7(– 40.6) 3 (12.0 –)14.6– 15.3(–18.8) mm, fusoides vel rhomboides, medio latissimae, fundis obtusis, apicibus obtusis vel subobtusis. Conidiomata in contextu hospitis inclusa, solitaria, stromatiformia, globosa, usque 1 mm diametro. Cellulae conidiogenae holoblasticae, hyalinae, subcylindricae, 12–19(–24) 3 4–6 mm, percurrenter cum 2–3 proliferationibus prolificentes, vel in plano eodem periclinaliter incrassatae. Conidia hyalinae, unicellulares, parietibus crassis, ovoidea, apicibus obtuse rotundato, in fundo obtuse rotundato, (23.7–)29.6–30.3 (–46.1) 3 (9.1–)13.4–13.8(–20.5) mm. Pseudothecia stromatic, immersed, partially erumpent when mature, dark brown to black, more or less circular, up to 1 mm diam, multiloculate, individual locules 200–300 mm diam, thick-walled, wall composed of outer layers of thick-walled, dark brown textura angularis, inner layers of thin-walled, hyaline textura angularis. Ostiole circular, central, papillate, periphysate. Pseudoparaphyses hyaline, branched, septate, 2–3 mm wide. Asci 160–250 3 30–33 mm (including stipe), clavate, stipitate, bitunicate, containing eight, biseriate ascospores. Ascospores (28.2–) 604 MYCOLOGIA FIG. 1. Phylogenetic relationships among Botryosphaeria corticola and other Botryosphaeria species based on maximumparsimony analysis of combined ITS1, 5.8S and ITS2 sequence data. Bootstrap percentages are given below the branches (1000 replicates) and branch lengths proportional to the number of changes are given above the branches. GenBank accession numbers are listed for isolates not sequenced in this study. 33.6–34.7(– 40.6) 3 (12.0 –)14.6–15.3(–18.8) mm (mean 6 S.D. of 90 ascospores 5 34.1 6 2.5 3 14.9 6 1.6 mm), L/W ratio of 2.3 6 0.2, broadly fusiform to rhomboid, widest in the middle, both ends obtuse, hyaline, moderately thick-walled (ca 1 mm), smoothwalled, aseptate, rarely becoming light brown and 1or 2-septate with age. Conidiomata eustromatic, immersed, partially erumpent when mature, dark brown to black, more or less circular, up to 1 mm diam, multiloculate, individual locules 200–300 mm diam, wall composed of three layers, an outer of dark brown, thick-walled textura angularis, a middle layer of dark brown thin-walled cells and an inner layer of thin-walled hyaline cells. Ostiole central, circular, papillate. Conidiophores absent. Conidiogenous cells 12–19(–24) 3 4–6 mm, holoblastic, discrete, cylindrical, hyaline, smooth, indeterminate, proliferating at the same level giving rise to periclinal thickenings, or ALVES ET AL: BOTRYOSPHAERIA CORTICOLA ON QUERCUS 605 near Aveiro, on dead branches of Quercus suber L., Feb 2002, A. Alves, (LISE 94839, culture ex type CBS112549). Additional cultures examined. ITALY. SARDINIA: Tempio Pausania, on trunk of Q. suber, Feb 2001, J. Luque & A. Franceschini, (CBS112074). PORTUGAL. BEIRA LITORAL. Requeixo near Aveiro, on dead branch of Quercus suber L., Feb 2002, A. Alves, (CBS112550, CBS112551, CBS112548, CBS112552). SPAIN. ANDALUSIA: Cádiz, cankered branch of Q. ilex, Jul 2000, M.A. Sanchez & A. Trapero, (CBS112545); Huelva, branch of Q. ilex, Aug 2000, M.A. Sanchez & A. Trapero, (CBS112546); Córdoba, branch of Q. ilex, Aug 2000, M.A. Sanchez & A. Trapero, (CBS112547). CATALONIA: Girona, Vidreres, trunk bark of Q. suber, Mar 1989, J. Luque (CBS678.88), May 1994, J. Luque (CBS112077), Girona, Sant Feliu De Buixalleu, dead branch of Quercus ilex L., May 1995, J. Luque (CBS112071); Barcelona, Vallgorguina, trunk bark of Quercus suber, Jan 1992, J. Luque (CBS112070), trunk canker of Q. suber, Dec 2000, J. Luque (CBS112076). EXTREMADURA: Badajoz, Valdebotoa, dead branch of Q. ilex, Aug 2000, J. Luque, (CBS112072), Villar Del Rey, trunk canker of Q. suber, Aug 2000, J. Luque (CBS112073). FIGS. 2–4. Botryosphaeria corticola and its anamorph Diplodia corticola. 2. Ascus and pseudoparaphyses of LISE 94840. 3. Ascospores of LISE 94840. 4. Conidia and conidigenous cells of CBS112549 on oatmeal agar. Scale bars 5 10 mm. proliferating percurrently to form one or two indistinct annelations. Conidia (23.7–)29.6–30.3(–46.1) 3 (9.1–)13.4–13.8(–20.5) mm, mean and standard deviation of 250 conidia 5 29.9 6 2.6 3 13.6 6 1.4 mm, length/width ratio (average of 250 conidia) 5 2.2 6 0.3, hyaline, aseptate, eguttulate or sometimes with a large central guttule, contents granular, smooth, thick-walled, oblong to cylindrical, straight, both ends broadly rounded, rarely becoming brown and septate with age. Colonies 36–44 mm diam on PDA after 4 d in the dark at 25 C. Cardinal temperatures for growth were min 5 C, max below 35 C, opt 20– 25 C. Etymology. Derived from the Latin root for the word for bark in reference to its habitat on its main host, the cork oak (Quercus suber). Hosts. Quercus species and possibly other hosts. Distribution. Iberian Peninsula, Italy, N. America. HOLOTY PE of B. corticola. PORTUGAL. BEIRA LITORAL: Requeixo near Aveiro, on dead branches of Quercus suber L., Feb 2002, A. Alves (LISE 94840, culture ex type CBS112549). HOLOTY PE of D. corticola. PORTUGAL. BEIRA LITTORAL: Requeixo Because of the similarity of B. corticola to B. stevensii, and to clarify some taxonomic uncertainties in the latter species and its anamorph (D. mutila), we studied material filed under B. stevensii in BPI, and D. mutila in STR and K. Fries (1823) described Sphaeria mutila and distributed two exsiccati under that name as Scler. Suec. 164 and 385. We examined material of these two exsiccati in STR and found both to be devoid of spores. Stevens (1933) and Sutton (1980) also reported that these two exsiccati in BPI and K had no spores. Sutton (1980) reported that 164 was an ascomycete of the Botryosphaeria type and pointed out that Sphaeria mutila should be adopted for the ascomycetous element it represents. Montagne sent Fries a fungus that was identified as S. mutila. The record was listed under S. mutila Fr. by Montagne (1834) with the note that this species would become the type of a new genus, Diplodia, later characterized by Fries (1849). Therefore, the name of the pycnidial fungus dates from Montagne (1834); it is typified by his material and the correct citation is Diplodia mutila Fr. in Montagne (1834). Montagne distributed this fungus in his exsiccatus No. 498. No material of this could be found in STR, according to Françoise Deluzarche of the Institut de Botanique, Strasbourg, France. However, Montagne’s specimen of D. mutila in Kew, K(M)99664 (isotype), was examined and agreed in all aspects with Stevens’ (1933) account of Montagne’s exs. 498 but differed from the description given by Sutton (1980). While Sutton (1980) referred to the conidia as initially hyaline with a large central guttule, later becoming 606 MYCOLOGIA FIGS. 5–11. Botryosphaeria corticola LISE 94840. 5. Ascomata partially erumpent through the host bark. 6. Multilocular ascoma cut through horizontally showing the white contents typical of Botryosphaeria of six loculi. 7. Vertical section through an ascostroma showing the thick wall and three loculi opening through periphysate ostioles. 8. A mature ascus containing ascospores, several immature asci and pseudoparaphyses. 9, 10. Ascospores. 11. A brown, 2-septate ascospore. Scale bars: 5 5 1 mm, 6 5 0.5 mm, 7 5 100 mm, 8 5 20 mm, 9–11 5 10 mm. ALVES ET AL: BOTRYOSPHAERIA CORTICOLA ON QUERCUS 607 FIGS. 12–18. Diplodia corticola. 12. Sectioned conidiomata of LISE 94839. 13, 14. Percurrently proliferating conidiogenous cell (CBS112549) in surface view (13) and optical section (14) with annelations arrowed. 15. Phialide of CBS112549. 16. Conidia (CBS112549). 17. Conidia (CBS 112077). 18. Brown and septate conidia (CBS 112077). Scale bars: 12 5 100 mm, 13–18 5 10 mm. dark brown and medianly 1 eusepate, we found that the vast majority of conidia in K(M)99664 were hyaline and aseptate, although pale brown and one- or two-septate conidia were seen rarely. The conidia usually had a large central guttule. Furthermore, the dimensions that Sutton (1980) reported (27–31 3 12– 13.5) are somewhat larger than we found (23.5–27.5 3 12–14). Stevens (1933) gave the conidia as (20–) 25–27 3 10–12(–16) mm. In these respects, our findings from this specimen correspond more closely to Stevens’ (1933) description of this species than to Sutton’s (1980) account. 608 MYCOLOGIA FIGS. 19–22. Botryosphaeria stevensii and its anamorph Diplodia mutila. 19. Ascus and pseudoparaphyses (BPI 599153). 20. Ascospores (BPI 599153). 21. Conidia and conidiogenous cells (BPI 599153). 22. Conidia and conidiogenous cells (K(M)99664). Scale bars 5 10 mm. When Stevens (1936) described Physalospora mutila he referred to a specimen on a cut twig of Fraxinus excelsior L. collected from Saltash, Cornwall, England, as the type specimen. A specimen fitting this description (BPI 599151) was examined. Unfortunately, no ascomycete could be found, but the anamorphic fungus on this specimen corresponded in all ways with Diplodia mutila K(M)99664. There was, however, ample material of the teleomorph on BPI 599153, which is a specimen on apple collected by Stevens from the same locality at the same time he collected BPI 599151. This specimen (BPI 599153) is designated here as lectotype of P. mutila. Botryosphaeria stevensii Shoemaker, Can. J. Bot. 42: 1299. 1964. FIGS. 19–38 5 Physalospora mutila N.E Stevens, Mycologia 28:333. 1936, as Physalospora mutila (Fries) N. E. Stevens comb. nov. Anamorph: Diplodia mutila Fries in Montagne, Ann. Sci. nat., sér. 2, 1:302 (1834). [ Sphaeria mutila Fries, Syst. Mycol. 2:424–425. 1823. Synonyms of D. mutila are given by Stevens (1933). The description is based on BPI 599153 (P. mutila) and K(M)99664 (D. mutila). Ascomata unilocular, solitary or clustered, immersed, partially erumpent when mature, globose, up to 300 mm diam, dark brown to black, thickwalled, wall composed of outer layers of thick-walled, dark brown textura angularis, inner layers of thinwalled, hyaline textura angularis. Ostiole central, circular, papillate, periphysate. Pseudoparaphyses hyaline, branched, septate, 2–3 mm wide. Asci clavate, stipitate, bitunicate, 100–160 3 14–22 mm (including stipe), containing eight, biseriate ascospores. Ascospores (24.8–)30.8–32.1(–36.2) 3 (9.5–)11.2–11.7 (–13.4) mm (mean 6 S.D. of 50 ascospores 5 31.5 6 2.3 3 11.4 6 0.9 mm) with length width ratio of 2.8 6 0.3, fusiform, widest in the middle, both ends obtuse, hyaline, thin-walled, smooth, aseptate, rarely becoming light brown and 1- or 2-septate with age. Conidiomata solitary or aggregated in clusters of up to five or more, immersed, partially erumpent when mature, dark brown to black, more or less globose, up to 600 mm diam, wall composed of three layers, an outer of dark brown, thick-walled textura angularis, a middle layer of dark brown thin-walled cells, an inner layer of thin-walled hyaline cells. Ostiole central, circular, papillate. Conidiophores absent. Conidiogenous cells (11–)12–15 3 4–5 mm, holoblastic, discrete, cylindrical, hyaline, smooth, indeterminate, proliferating at the same level giving rise to periclinal thickenings, or proliferating percurrently to form one or two indistinct annelations. Conidia hyaline, aseptate, smooth, thick-walled, oblong to ovoid, straight, both ends broadly rounded, (23.5– )25.1–25.7(–27.4) 3 (12.4–)13.2–13.5(–14.3) mm, mean and standard deviation of 50 conidia 5 25.4 6 1.0 3 13.4 6 0.5 mm, length/width ratio (average of 50 conidia) 5 1.9 6 0.1, rarely becoming pale brown and septate with age. Culture examined. PORTUGAL. ALENTEJO. Montemor-o-Novo, Vitis vinifera, 1996, A.J.L. Phillips (CBS 112553). Specimens examined. Physalospora mutila. ENGLAND. CORNWALL: Saltash, on bark of Fraxinus excelsior, 22 Aug 1935, N.E. Stevens (BPI 599151); Saltash, on bark of Malus sp., 22 Aug 1935, N.E. Stevens (BPI 599153, LECTOTY PE designated herein). SURREY: Ranmore Common, on Fraxinus sp., 19 Apr 1957, C. Booth (BPI 599150 ex IMI 69064). Diplodia mutila: FRANCE. ARDENNE: Sedan, on bark of Populus nigra, date not known, Montagne (K(M)99664 ISOTY PE). Sphaeria mutila: Scler. Suec. 164 (STR); Scler. Suec. 385 (STR). DISCUSSION Based on morphological characters and nrDNA ITS sequences we conclude that the Botryosphaeria that occurs mainly on oak is different from B. stevensii ALVES ET AL: BOTRYOSPHAERIA CORTICOLA ON QUERCUS 609 FIGS. 23–28. Botryosphaeria stevensii BPI 599153. 23. Ascomata partially erumpent through the host bark. 24. Sectioned ascoma. 25. Ascus with ascospores. 26. Ascus tip. 27, 28. Ascospores. Scale bars: 23 5 0.5 mm, 24 5 100 mm, 25 5 20 mm, 26–28 5 10 mm. and that it represents a previously undescribed species, namely B. corticola. It closely resembles both B. stevensii and B. quercuum, two other Botryosphaeria species with mostly hyaline, aseptate and thick-walled conidia but differs in the larger ascospores and conidia and in the shapes of the spores. In addition, ITS sequence data clearly separate B. corticola from all other known species of Botryosphaeria and place it within the group with Diplodia anamorphs. It is probable that B. corticola previously has been identified under other names. This is reflected in the GenBank sequences accessed in this study in which strains originally identified as B. dothidea, B. quercuum, B. stevensii or D. quercina (TABLE I) had ITS sequences that grouped them with B. corticola. However, we were unable to confirm the identity of the species described on Q. petraea in Hungary by Vajna (1986) or the Diplodia species that Oliva and Molinas (1986) isolated from Q. suber in northeastern Spain. Therefore it is possible that B. stevensii and B. quercuum also occur on oak. Differences in conidium dimensions between B. corticola and B. stevensii are most apparent in the 95% confidence intervals and means calculated from the corresponding measurements. Furthermore, conidia of B. stevensii rarely exceed 30 mm in length while those of B. corticola are commonly longer and can reach 40 mm. Differences between the two species also are seen in the teleomorph. Asci and ascospores of B. corticola are larger and the ascospores have a different shape from those of B. stevensii, but this feature may be less reliable for species differentiation than conidium characters. Although Stevens (1936) gave the ascospores of P. mutila (5 B. stev- 610 MYCOLOGIA FIGS. 29–38. Diplodia mutila. 29. Conidiomata partially erumpent through the host bark (K(M)99664). 30. Sectioned conidioma (K(M)99664). 31–34. Conidiogenous cells: 31. K(M) 99664, 32. BPI 599151, 33. BPI 599153, 34. CBS112553. 35– 38. Conidia: 35. K(M) 99664, 36. BPI 599151, 37. BPI 599153, 38. CBS112553. ALVES ET AL: BOTRYOSPHAERIA CORTICOLA ensii) as 30–36(–39) 3 (12–)13–14(–16) mm, we found that in BPI 599153 (which Stevens regarded as P. mutila) they were somewhat smaller, measuring (24–)28–35(–36) 3 (9.5–)10–13(–13.5) mm. Ascomata and conidiomata of B. corticola in nature are mainly multilocular, while in B. stevensii they are mostly unilocular. Again, this feature may have minor taxonomic significance because the shape and form of ascomata and conidiomata varies within a species according to the substrate on which they are formed (Stevens 1933, Witcher and Clayton 1963, Rayachhetry et al 1996, Phillips et al 2002). The fungus described here as B. corticola is clearly different from B. quercuum. Arx and Müller (1954) quoted an extensive synonymy under B. quercuum including B. stevensii (as Physalospora mutila) and some other species with Fusicoccum anamorphs. But Arx and Müller (1954) based their assessment of the genus on a study of herbarium material of the teleomorph and took no account of anamorph characters. It since has been recognized that, although teleomorphs of Botryosphaeria species are morphologically similar, they can be differentiated on characters of the anamorphs (Shoemaker 1964, Laundon 1973, Pennycook and Samuels 1985). Thus, when Shoemaker (1964) redescribed B. quercuum he distinguished it from B. stevensii on the shape of the conidia. In B. quercuum the conidia are subglobose (18–)21–24(–25) 3 (12–)15–16(–17), L/W 5 1.5, but in B. stevensii they are oblong (L/W 5 1.9). This distinction was followed by Sivanesan (1984), and it generally is regarded as the character that separates the two species. The subglobose conidia of B. quercuum also are distinctly different from the oblong conidia of B. corticola (L/W 5 2.2). Conidia of D. mutila often have been referred to as becoming brown and septate with age (Shoemaker 1964, Laundon 1973), but Sutton (1980) inferred that mature conidia are dark brown and septate. However, we consider that conidia of this species are hyaline, even when mature. Conidia of both B. stevensii and B. corticola can germinate when hyaline and aseptate, thus indicating that in this state they are fully mature. The pale brown and septate conidia that occasionally are seen should be regarded as atypical because they are seen only in old cultures or on material from the field whose age cannot be determined accurately. Conidia in the specimen of D. mutila in K(M)99664 were clearly hyaline. Sutton (1980) considered this an isotype of D. mutila, and this was accepted here. Denman et al (2000) suggested that the name Diplodia should be adopted to accommodate the darkspored anamorphs of Botryosphaeria species. Zhou and Stanosz (2001b) developed this idea and re- ON QUERCUS 611 ferred to the two main clades in the genus Botryosphaeria as sections Brunnea (species with wide, brown conidia of the Diplodia type) and section Hyala (species with narrow, hyaline, fusicoccum-type conidia). However, because B. corticola, B. stevensii and B. quercuum have mainly hyaline conidia, the distinction between the two sections on the basis of conidium coloration is tenuous. The main difference is that in sect. Brunnea the conidia are more than 10 mm wide with a relatively thick-wall while in sect. Hyala the conidia are less than 10 mm wide and thin-walled. These characters comply with the genera Diplodia and Fusicoccum that already are linked to these anamorphs, and at present there does not seem to be any reason to replace these genus names with section names. Furthermore, some strains of species in the B. dothidea complex with Fusicoccum anamorphs develop a brown pigment with age. The name Diplodia should not be restricted to anamorphs with brown conidia but also can apply to species with hyaline conidia, as reflected in the amended genus concept for Diplodia (Denman et al 2000). Percurrent proliferation in conidiogenous cells has been regarded as more typical of Sphaeropsis than of Diplodia (Sutton 1980). Although Denman et al (2000) stated that isolates of Diplodia produce percurrent proliferations, they did not refer to any published source for this information. However, Phillips (2002) showed annellate conidiogenous cells in the Diplodia anamorphs of B. stevensii and B. obtusa. In the present study we confirm that percurrent proliferation occurs in conidiogenous cells of D. mutila and D. corticola. Furthermore, proliferation at the same level resulting in periclinal thickenings was seen in both species. Therefore, the description of Diplodia given by Denman et al (2000) should be amended further to include phialides sensu Sutton (1980). The data generated from analysis of the ITS sequences of strains isolated in this study and from sequences retrieved from GenBank revealed that B. stevensii is not a homogeneous species, confirming the suggestion by Zhou and Stanosz (2001a, b) that the name has been applied to more than one species. This species appears to be a complex that needs to be resolved by examining more isolates and collections. Thus, there appears to be a distinction between strains of B. stevensii isolated from apples and grapevines and those from Gymnosperms. This distinction suggests that these two groups in fact may represent different taxa. However, before any firm conclusions can be drawn, more strains from these and other hosts will have to be studied. ACKNOWLEDGMENTS This work was financed by the European Regional Development Fund (FEDER) and Fundação para a Ciência e a 612 MYCOLOGIA Tecnologia (FCT) under project POCTI/AGR/44348/2002 (Portugal) and Comisión Interministerial de Ciencia y Tecnologı́a (CICyT) and the European Regional Development Fund (ERDF) under the research project 1FD97-0911-C3-1 (Spain). A Alves was supported by Grant No. SFRH/BD/ 10389/2002 from FCT. The curators of BPI, K, PAD and STR kindly let us study specimens in their care. Marı́a Esperanza Sánchez and António Trapero (University of Córdoba, Spain) donated some of the strains included in this work. LITERATURE CITED Anonymous. 1968. Plant pathologists pocketbook. Kew, England: CMI. 267 p. Arx JA von, Müller E. 1954. Die Gattungen der Amerosporen Pyrenomyceten. Beitr Kryptogamenfl Schweiz 11(1):1–434. Berbee ML, Taylor JW. 1992. Convergence in ascospore discharge mechanisms among pyrenomycete fungi based on 18S ribosomal RNA gene sequences. Mol Phylogen Evol 1:59–71. Brasier CM. 1992. Oak tree mortality in Iberia. Nature 360: 539. . (1996). Phytophthora cinnamomi and oak decline in southern Europe. Environmental constraints including climate change. Ann Sci For 53:347–358. , Robredo F, Ferraz JFP. 1993. Evidence for Phytophthora cinnamomi involvement in Iberian oak decline. Plant Pathol 42:140–145. Bruns TD, White TG, Taylor JW. 1991. Fungal molecular systematics. Ann Rev Ecol Syst 22:525–564. Denman S, Crous PW, Taylor JE, Kang J-C, Pascoe I, Wingfield MJ. 2000. An overview of the taxonomic history of Botryosphaeria, and a re-evaluation of its anamorphs based on morphology and ITS rDNA phylogeny. Stud Myc 45:129–140. Fries EM. 1823. Systema mycologicum 2:276–620. . 1849. Summa veg Scand 1–572. Gallego FJ, Perez de Algaba A, Fernandez Escobar R. 1999. Etiology of oak decline in Spain. Eur J For Path 29:7– 27. Jacobs KA, Rehner SA. 1998. Comparison of cultural and morphological characters and ITS sequences in anamorphs of Botryosphaeria and related taxa. Mycologia 90:601–610. Laundon GF. 1973. Botryosphaeria obtusa, B. stevensii and Otthia spiraeae in New Zealand. Trans Brit Mycol Soc 6:369–374. Luque J, Girbal J. 1989. Dieback of cork oak (Quercus suber) in Catalonia (NE Spain) caused by Botryosphaeria stevensii. Eur J For Path 19:7–13. , Parladé J, Pera J. 2001. El decaimiento del alcornoques en Cataluña: sı́ntomas y hongos asociados. Investigación Agraria: Sistemas y Recursos Forestales 10: 271–289. Montagne JFC. 1834. Notice sur les plantes cryptogames récemment découvertes en France contenant aussi l’indication précis des localités de quelques espèces les plus rares de la flore française. Ann Sci Nat Bot, sér 2, 1:295–307. Muñoz MC, Cobos P, Martı́nez G, Soldevilla C, Dı́az M. 1996. Micoflora y patologı́a del alcornoque (Quercus suber L.) 1996 Madrid (Spain): Ministry of Agriculture, Fisheries and Food (MAPA). 328 p. O’Donnell KE, Cigelink KE, Weber NS, Trappe JM. 1997. Phylogenetic relationships among ascomycetous truffles and the true and false morels inferred from 18S and 28S ribosomal DNA sequence analysis. Mycologia 89:48–65. Oliva M, Molinas ML. 1986. Participación de Diplodia sp. en el escaldado del alcornoque. Scientia Gerundensis 12:123–130. Pennycook SR, Samuels GJ. 1985. Botryosphaeria and Fusicoccum species associated with ripe fruit rot of Actinidia deliciosa (kiwifruit) in New Zealand. Mycotaxon 24: 445–458. Phillips AJL. 2002. Botryosphaeria species associated with diseases of grapevines in Portugal. Phytopath Medit 41:3– 18. , Fonseca F, Povoa V, Castilho R, Nolasco G. 2002. A reassessment of the anamorphic fungus Fusicoccum luteum and description of its teleomorph Botryosphaeria lutea sp. nov. Sydowia 54:59–77. Pitcher D, Saunders N, Owen R. 1989. Rapid extraction of bacterial DNA with guanidium thiocyanate. Letters Appl Microbiol 8:151–156. Ragazzi A, Moricca S, Dellavalle I, Turco E. 2000. Italian expansion of oak decline. In: Ragazzi A, Dellavalle I, Moricca S, Capretti P, Raddi P, eds. Decline of oak species in Italy—problems and perspectives. Firenze, Italy: Accademia Italiana di Scienzi Forestali. p 39–75. Rayachhetry MB, Blakeslee GM, Webb RS, Kimbrough JW. 1996. Characteristics of the Fusicoccum anamorph of Botryosphaeria ribis, a potential biological control agent for Melaleuca quinquenervia in South Florida. Mycologia 88:239–248. Robin C, Desprez-Loustau ML, Capron G, Delatour C. 1998. First record of Phytophthora cinnamomi on cork and holm oaks in France and evidence of pathogenicity. Ann Sci For 55:869–883. Shoemaker RA. 1964. Conidial states of some Botryosphaeria species on Vitis and Quercus. Can J Bot 42:1297–1301. Sivanesan A. 1984. The bitunicate ascomycetes and their anamorphs. Vaduz, Liechtenstein: J Cramer. 701 p. Smith H, Crous PW, Wingfield MJ, Couthinho TA, Wingfield BD. 2001. Botryosphaeria eucalyptorum sp. nov., a new species in the B. dothidea-complex on Eucalyptus in South Africa. Mycologia 93:277–285. Stevens NE. 1933. Two apple black rot fungi in the United States. Mycologia 25:536–548. . 1936. Two species of Physalospora in England. Mycologia 28:330–336. Sutton BC. 1980. The Coelomycetes. Kew, England: CMI. 696 p. Swofford DL. 2000. PAUP*. Phylogenetic Analysis Using Parsimony (*and other methods). Version 4. Sinauer Associates, Sunderland, Massachusetts, USA. ALVES ET AL: BOTRYOSPHAERIA CORTICOLA Vajna L. 1986. Branch canker and dieback of sessile oak (Quercus petraea) in Hungary caused by Diplodia mutila I. Identification of the pathogen. Eur J For Path 16:223–229. White TJ, Bruns T, Lee S, Taylor J. 1990. Amplification and direct sequencing of fungal genes for phylogenies. In: PCR protocols: a guide to methods and applications. San Diego, California: Academic Press. p 315–322. Witcher W, Clayton CN. 1963. Blueberry stem blight caused ON QUERCUS 613 by Botryosphaeria dothidea (B. ribis). Phytopathology 53:705–712. Zhou S, Stanosz GR. 2001a. Primers for amplification of mt SSU rDNA, and a phylogenetic study of Botryosphaeria and associated anamorphic fungi. Mycol Res 105:1033–1044. , . 2001b. Relationships among Botryosphaeria species and associated anamorphic fungi inferred from the analyses of ITS and 5.8s rDNA sequences. Mycologia 93:516–527.