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Botryosphaeria viticola sp. nov. on grapevines: a new species with a Dothiorella anamorph

     Departament de Protecció Vegetal, Institut de Recerca i Tecnologia Agroalimentàries, Centre de Cabrils, Ctra. de Cabrils s.n., E-08348 Cabrils, Barcelona, Spain

Alan J.L. Phillips ¹

     Centro de Recursos Microbiológicos, Faculdade de Ciências e Tecnologia, Universidade Nova de Lisboa, Quinta da Torre, 2829-516 Caparica, Portugal

	ABSTRACT

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS TAXONOMY DISCUSSION LITERATURE CITED

 

Botryosphaeria viticola sp. nov., isolated from pruned canes of Vitis vinifera in NE Spain, is described and illustrated. Phylogenetic analysis based on ITS and EF1- sequences and morphological characters of both anamorph and teleomorph confirmed this taxon to be included within the group of Botryosphaeria species with Dothiorella anamorphs. It is related most closely to B. sarmentorum and B. iberica from which it differs in morphological characters of the teleomorph and DNA sequences.

Key words: Botryosphaeriaceae, Diplodia, Fusicoccum, ITS, molecular phylogenetics, translation elongation factorEF1-

	INTRODUCTION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS TAXONOMY DISCUSSION LITERATURE CITED

 
Species of Botryosphaeria Ces. & De Not. are well known as pathogens, saprophytes and endophytes on a wide range of woody angiosperm and gymnosperm hosts (Barr 1972, von Arx 1987, Denman et al 2000). While the morphology of the teleomorphic states differs little among species, a wide range of morphologies is seen in the anamorphs and it is on this basis that species are distinguished. Anamorphs of Botryosphaeria species currently are placed in the genera Fusicoccum Corda, Diplodia Fr., Lasiodiplodia Ellis & Everh., and Dothiorella Sacc. (Crous and Palm 1999, Denman et al 2000, Phillips et al 2005). These four genera clearly are separated on their morphology and their phylogenetic relationships shown by the analysis of sequence data of the internal transcribed spacer (ITS) of the rRNA repeat (Phillips et al 2005). Additional gene sequences, such as partial sequences of the ß-tubulin gene and the translation elongation factor 1-alpha (EF1-), also have been used to unambiguously differentiate species within Botryosphaeria (Slippers et al 2004, van Niekerk et al 2004).

The genus Dothiorella was resurrected to accommodate anamorphs of Botryosphaeria species with conidia that become colored and septate at an early stage of development, even before they are released from the conidiogenous cell (Phillips et al 2005). This contrasts with conidia of Diplodia species, which are hyaline and become dark and septate only some time after they are formed, normally after a period of aging once they are discharged from the pycnidia (Shoemaker 1964, Alves et al 2004, Phillips et al 2005). Another distinctive morphological feature of Botryosphaeria species with Dothiorella anamorphs is their brown, one-septate ascospores. Although ascospores of most Botryosphaeria species occasionally are known to become pale brown and one-septate with age (Shoemaker 1964, Sivanesan 1984, Alves et al 2004) others, such as B. laricis (Wehm.) von Arx & Müller and B. visci (Kalchbr.) von Arx & Müller, are reported to be dark brown (Sivanesan 1984). Ascospores of the species so far reported with Dothiorella anamorphs become intensely brown at an early stage of development (Phillips et al 2005).

During a 2003–2004 survey of fungal pathogens associated with grapevine decline in Catalonia (NE Spain), a coelomycete with characters of Dothiorella was found frequently on discarded pruned canes. An ascomycete with brown, one-septate ascospores was found rarely and the connection between the two states was confirmed by culture of single ascospores. Because no other fungal taxa with these characters have been described from Vitis vinifera L., it was thought that this fungus might represent a new species. The purpose of this study was to characterize the morphology of this fungus and to determine its phylogenetic relationship to other Botryosphaeria species through a study of ITS and EF1- nucleotide sequences.

	MATERIALS AND METHODS

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Isolates and morphology.— – Isolations were made from single conidia, single ascospores or by directly plating out pieces of diseased tissue after surface sterilization in 70% ethanol for 4 min. Isolates were cultured on Difco potato-dextrose agar (PDA) at 25 C. Sporulation was enhanced by culturing the isolates on 2% water agar bearing pieces of autoclaved grapevine canes at 25 C with a 12/12 h photoperiod. Growth rates were determined on PDA plates incubated in the dark at 5–35 C in 5 C intervals. Isolates were deposited at the Centraalbureau voor Schimmelcultures (CBS), Utrecht, The Netherlands. Specimens were lodged with the herbarium of Estação Agronómica Nacional (LISE), Oeiras, Portugal.

Examinations were made with a Leica DMR HC microscope fitted with Nomarski DIC optics. Digital images were recorded with a Leica DFC320 digital camera, and measurements were made with the Leica IM500 measurement module. The mean, standard deviation and 95% confidence intervals were calculated from measurements of 50 conidia and ascospores. Data for spore measurements are presented as the lower 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 sequencing.— – DNA was extracted from fungal mycelium as described by Alves et al (2004). Amplification of the ITS1 and ITS2 regions flanking the 5.8S ribosomal RNA gene was carried out with universal primers ITS1 and ITS4 (White et al 1990). Part of the translation elongation factor 1-alpha gene (EF1-) was amplified with primers EF1-728F and EF1-986R (Carbone and Kohn 1999). Reactions were performed on a GeneAmp^® PCR System 9700 thermal cycler (PE Applied Biosystems, Foster City, California). Each reaction contained 0.5 µL template DNA (about 100 ng genomic DNA), 20 µL Eppendorf Mastermix 2.5x (Eppendorf AG, Hamburg, Germany), 0.4 µM of each primer and made up to a final volume of 50 µL. These temperature profiles were used: one cycle of initial denaturation at 95 C for 3 min, followed by 35 cycles of denaturation (20 s at 95 C), annealing (30 s at 55 C for ITS or 58 C for EF1-) and extension (40 s at 72 C), with a final extension at 72 C for 5 min. PCR products were separated by electrophoresis in 2% agarose gels and viewed under UV light after ethidium bromide staining.

PCR products were purified with the High Pure PCR Product Purification Kit (Roche Diagnostics GmbH, Mannheim, Germany). Purified amplicons were sequenced in both directions with the PCR primers and the BigDye^TM Terminator v1.1 Cycle Sequencing Kit (Applied Biosystems, Foster City, California). The resulting fragments were analyzed on an ABI Prism 377 automated DNA sequencer (Perkin Elmer, Norwalk, Connecticut).

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 with sequences from both strands. Nucleotide sequences were deposited in GenBank (TABLE I). Additional sequences for both DNA regions corresponding to other Botryosphaeria species were obtained from GenBank (TABLE I).

	RESULTS

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Phylogenetic analysis.— – The sequence alignment of 41 isolates (TABLE I), including the two outgroup isolates, consisted of 568 characters for the ITS region and 347 characters for the EF1- gene, including the alignment gaps. Indels were coded separately and added to the end of the alignment as characters 916–1055. Alignment gaps were treated as missing data. Part of the ITS1 region (characters 86–140) could not be aligned unambiguously and was excluded from the analysis. Similarly, characters 608–654, close to the 5' end of the EF1- gene also could not be aligned and were excluded. The coded indels associated with these regions (characters 929–952, 991–1001) also were excluded. Thus 918 characters were included. New ITS and EF1- sequences were deposited in GenBank (TABLE I) and the alignments in TreeBase (S 1351). The result of the partition homogeneity test (P=0.4800) indicated that the ITS and EF1- data reflected the same underlying phylogeny, therefore the two datasets were combined in a single analysis.

The combined dataset consisted of 918 characters, of which 435 were constant and 192 variable characters were parsimony uninformative. Maximum parsimony analysis of the remaining 291 parsimony-informative characters resulted in four most parsimonious trees, one of which is shown (FIG. 1). MP trees differed only in the position of the isolates in the terminal clades, while the overall topology was the same in all trees. NJ analysis resulted in trees with the same topology as the MP trees.

View larger version (44K): [in this window] [in a new window]   FIG. 1. One of four most parsimonious trees obtained from the combined analysis of ITS and EF1- sequence data (TL = 868 steps, CI = 0.736, RI = 0.891, RC = 0.656, HI = 0.264). Bootstrap support values exceeding 50% from 1000 replications for the neighbor joining followed by the maximum parsimony analyses are shown at the nodes. The tree was rooted to Cercospora apii CBS 119.25 and Hypocreales sp. CMW7063. The origins of the B. viticola isolates are represented by SA (South Africa) or SP (Spain) after each isolate. Anamorph genera are given on the right with illustrations depicting salient features of each genus. Bar = 10 changes.

Morphology.— – Discarded pruned canes collected from five vineyards in NE Spain (TABLE I) bore pycnidia of a coelomycete with characters corresponding to Dothiorella, and one of them bore an ascomycete with bitunicate asci containing eight brown, one-septate ascospores (LISE 95177). The culture obtained from a single ascospore of this latter collection (CBS 117009) produced pycnidia and conidia that were indistinguishable from the coelomycete cited above, thus confirming the connection between the anamorph and teleomorph. Another collection (CBS 117007) was obtained from a fragment of necrotic wood.

Ascomata (FIGS. 4–6) were found rarely. Clavate asci (FIGS. 2, 8), formed among septate pseudoparaphyses (FIGS. 2, 7), contained eight, brown-walled, one-septate ascospores (FIGS. 2, 8–11). The ascospores often bore a small projection at one or both ends (FIGS. 2, 10, 11).

View larger version (181K): [in this window] [in a new window]   FIGS. 4–11. Botryosphaeria viticola. 4. Ascomata partially erumpent through the host bark. 5. Ascoma cut through horizontally showing the white contents with dark spots corresponding to asci with ascospores. 6. Vertical section through an ascoma. 7. Septate pseudoparaphyses. 8. Clavate ascus containing eight biseriate, dark-brown, one-septate ascospores. 9–11. Ascospores. The ascospores in FIGS. 10 and 11 bear small, rounded projections at either end. Bars: 4 = 0.5 mm, 5 = 0.25 mm, 6 = 50 µm, 7–11 = 10 µm.

View larger version (81K): [in this window] [in a new window]   FIGS. 2–3. Botryosphaeria viticola and its anamorph Dothiorella viticola. 2. Asci, pseudoparaphyses and ascospores of LISE 95177. 3. Conidiogenous cells and conidia of LISE 95178. Bar = 10 µm.

View larger version (184K): [in this window] [in a new window]   FIGS. 12–19. Dothiorella viticola. 12. Conidiomata partially erumpent through the host bark. 13–17. Conidiogenous cells and conidia. 16. Conidiogenous cell with periclinal thickenings. 17. Conidiogenous cells with annellations. The cell on the right has a dark-brown, one-septate conidium attached at the tip. 18, 19. Conidia taken at two different levels of focus showing the roughening on the inner surface of the wall. Bars 12 = 0.5 mm, 13–19 = 10 µm.

	TAXONOMY

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS TAXONOMY DISCUSSION LITERATURE CITED

Botryosphaeria viticola A.J.L. Phillips & Luque, sp. nov. FIGS. 2–19

Anamorph. – Dothiorella viticola A.J.L. Phillips & Luque, sp. nov.

Ascomata pseudothecia, plerumque solitaria, inter-dum aggregata, in hospitis inclusa, erumpescentia, globosa vel pyriformis, cum ostioli centrali, papillata, nigra, usque 240 µm diam, paries pseudothecii usque 60 µm crassis, stratis textura angularis, extus e cellulis atrobrunneis vel brunneis composita, intus e cellulis hyalinus revestimentum loculi facientibus. Asci bitunicati, clavati, 100–110 x 25–30 µm, stipitati, octospori, pseudoparaphysibus multis filiformibus, septatis 3.5–4.5 µm latis interspersi. Ascosporae fusiformis oblongis vel subclavatis, apicibus obtusis, in fundo subacutis, (19–)22.6–23.5(–27) x (8.4–)10.6–11.2(–14.5) µm. Conidiomata in contextu hospitis inclusa, solitaria vel aggregata, stromatiformia, globosa, 200–360 µm diametro. Cellulae conidiogenae holoblasticae, hyalinae, subcylindricae, 5–14 x 3–7 µm, proliferatione percurrenti limitata, ut videtur 1–2 annellationibus paucis, vel inplano eodem periclinaliter incrassate. Conidia brunnea, uniseptata, parietibus crassis, ovoidea, apicibus obtuse rotundato, in fundo obtuse rotundato vel truncata (16–)20.2–20.6(–26) x (7–)9.2–9.4(–12) µm.

Ascomata dark brown to black, stromatic, pyriform, pseudothecial, isolated or in botryose clusters up to 2 mm diam, initially immersed in host, partially erumpent at maturity, up to 240 µm diam, ostiole circular, central, papillate; wall up to 60 µm thick, of dark brown thick-walled textura angularis, and lined with thin-walled, hyaline cells. Pseudoparaphyses thin-walled, hyaline, frequently septate, slightly constricted at septum, 3.5–4.5(–5.0) µm wide. Asci 100–110 x 25–30 µm, arising from base of ascoma, stipitate, clavate, thick-walled, bitunicate with a thick endotunica and a well-developed apical chamber, 8-spored, irregularly biseriate. Ascospores oblong, ovate to subclavate, (19–)22.6–23.5(–27) x (8.4–)10.6–11.2(–14.5) µm (mean ± S.D. = 23.1 ± 0.2 x 10.9 ± 0.1 µm n = 62) mostly 1-septate, slightly constricted at septum, dark brown, moderately thick-walled, finely verruculose on inner surface, often inequilateral, widest in lower one-third to middle of apical cell, often with a small rounded projection at tip and base of spore, basal cell tapering toward obtuse base. Conidiomata pycnidial, separate or aggregated into botryose clusters up to 2 mm diam, individual pycnidia spherical to globose, black, immersed, partially erumpent when mature, unilocular, 200–360 µm diam (mean: 295 µm; n = 25), thick-walled, wall of three layers: an outer layer of dark brown, thick-walled textura angularis, a median layer of dark brown thin-walled cells textura angularis, and an inner layer of thin-walled, hyaline cells. Ostiole single, central, circular, papillate. Conidiophores hyaline, cylindrical, branched. Conidiogenous cells discrete or integrated, cylindrical to broad lageniform, (5–)8.6–9.8(–14) x (3–)4.4–4.9(–7) µm, hyaline, smooth, holoblastic, indeterminate, proliferating at same level to form periclinal thickenings or rarely proliferating percurrently giving rise to 1–2 annellations. Conidia brown, oblong to subcylindrical, septate, occasionally slightly constricted at septum, moderately thick-walled, externally smooth, internally finely verruculose, ends rounded, often with a truncate base, (16–)20.2–20.6(–26) x (7–)9.2–9.4(–12) µm (mean ± SD = 20.4 ± 0.1 x 9.3 ± 0.1 µm, n = 200) length/width ratio 2.2 ± 0.02, n = 200. Colonies on PDA reaching 40 mm radius after 3 d at 25 C. Aerial mycelium present, colonies cottony, dark olive to grayish, darkening from the center of the colony after 3 d; colony fully darkened after 6–10 d. Pycnidia produced after 20–30 d in culture at 23 C under near UV black light (12/12 h photoperiod). Cardinal temperatures for growth: minimum 5 C, optimum 20–25 C, maximum below 35 C.

Hosts. – Vitis vinifera.

Geographical range. – South Africa, Spain.

Specimens examined. – SPAIN. CATALONIA: Vimbodí, near the Monastery of Poblet, on pruned canes of Vitis vinifera cv. Garnatxa Negra, 12 Aug 2004, J. Luque & S. Martos, (HOLOTYPE of Botryosphaeria viticola, LISE 95177, culture ex type CBS 117009; HOLOTYPE of Dothiorella viticola, LISE 95178), on pruned canes of Vitis vinifera cv. Garnatxa Negra, 28 May 2003, J. Luque & R. Mateu. Batea, on pruned canes of V. vinifera cv. Macabeu, 29 Jul 2004, J. Luque & S. Martos. Bot, on pruned canes of V. vinifera cv. Ull de llebre, 29 Jul 2004, J. Luque & S. Martos. Gandesa, on necrotic wood from a diseased branch of Vitis vinifera cv. Macabeu, 15 Jun 2004, J. Luque & S. Martos. Sant Esteve Sesrovires, on pruned canes of V. vinifera cv. Macabeu, 10 Aug 2004, J. Luque & S. Martos. Sant Sadurní d’Anoia, on pruned canes of V. vinifera cv. Xarel·lo, 27 May 2004, J. Luque & J. Reyes.

Notes. – The teleomorph is extremely rare compared to the abundant anamorph.

	DISCUSSION

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In this study Botryosphaeria viticola is described as a new species. It is closely related to B. sarmentorum and B. iberica, according to the morphological data and DNA sequences of the ITS and EF1- gene regions. Ascospores of these three species are brown and one-septate, which contrasts with the hyaline and aseptate ascospores commonly regarded as typical for Botryosphaeria based on B. dothidea having Fusicoccum aesculi anamorph (Denman et al 2000, Slippers et al 2004). As explained by Phillips et al (2005), these unusual characters can be accommodated in the concept of Botryosphaeria as emended by Saccardo (1877). Moreover B. laricis and B. visci are reported to have brown, one-septate ascospores (Sivanesan 1984), and septation of ascospores has been reported for other Botryosphaeria species, albeit after they have aged (Shoemaker 1964, Alves et al 2004). Nevertheless B. viticola, B. sarmentorum and B. iberica are unusual in that their ascospores become brown and septate early in their development. Such features of the ascospores could place these three species in Dothidotthia Höhn. Barr (1972) considered that the only difference between Botryosphaeria and Dothidotthia is that in Dothidotthia the ascospores are brown and septate while in Botryosphaeria they are hyaline and aseptate. However the phylogenetic analysis placed these species within Botryosphaeria and this raises the possibility that Dothidotthia may be a synonym of Botryosphaeria. On the other hand it is possible that Botryosphaeria is paraphyletic and comprises a number of separate genera. Any decisions on the status of this genus should be made on the basis of data resulting from the analysis of 18S and possibly 28S rDNA sequence data. Furthermore such a study also should take into account a thorough study of Dothidotthia to determine its status in relation to Botryosphaeria. Nevertheless, considering the data presented here on ITS and EF1- sequences, B. viticola, B. sarmentorum and B. iberica clearly fall within the species of Botryosphaeria.

In the present study isolates of B. dothidea lay within a clade separate from the one containing other species with Fusicoccum anamorphs. This can be seen in studies of phylogeny of Botryosphaeria (e.g. Zhou and Stanosz 2001, Slippers et al 2004, Alves et al 2004). However, with the inclusion of species with Dothiorella anamorphs in the present study, the separation of B. dothidea from other species with Fusicoccum anamorphs is more clearly defined. This is a further indication that Botryosphaeria may be paraphyletic.

The anamorphs of B. iberica, B. sarmentorum and B. viticola are morphologically similar; it would be difficult to separate them on morphological characters alone. On the other hand, the teleomorphs show distinctive characters that separate the species from one another. Although the dimensions of ascospores of the three species overlap, they can be distinguished on the morphology of their ascospores. Ascospores of B. viticola frequently bore a rounded projection at the tip and base (FIG. 15) and taper more strongly to the base than in B. iberica. While B. iberica has been found mainly on Quercus spp. and B. sarmentorum on several hosts other than Quercus (Phillips et al 2005), B. viticola appears to be restricted to grapevines. However it is not known if host association is a consistent character for these three species.

To our knowledge Botryosphaeria viticola is the third known species together with B. iberica and B. sarmentorum with anamorphs in Dothiorella. Dothiorella differs from Diplodia in morphological and molecular characteristics (Phillips et al 2005). Conidia of Dothiorella become brown and septate at an early stage, even before dehiscence from the conidiogenous cell, whereas conidia in Diplodia are hyaline and become dark and septate only with age (Shoemaker 1964, Alves et al 2004, Phillips et al 2005). This distinction has not always been recognized, with the result that the genus Diplodia has been used frequently for species with brown, one-septate conidia (Wollenweber 1941). It is possible that many of the species placed in Diplodia would be better accommodated in Dothiorella. Judging from the number of synonyms listed by Wollenweber (1941) for D. sarmentorum, many of these species are inseparable morphologically. However, as shown in this study and that of Phillips et al (2005), although the anamorphs of B. sarmentorum, B. iberica and B. viticola are indistinguishable morphologically, they can be separated on morphology of the teleomorphs and these differences are well supported by the ITS and EF1- sequence data.

The isolate CBS 117006 exhibited some differences in culture morphology from the other isolates studied. The reverse side of colonies of CBS 117006 became red-brown after 3–5 d on PDA at 25 C, with a progressive darkening of the pigment after 6–10 d. Such coloration was not seen in the other isolates of B. viticola. Furthermore there were some small differences in ITS and EF1- sequences between isolates of B. viticola (one substitution and one deletion in ITS and nine substitutions in EF). This was reflected in the phylogeny where two isolates from Spain clustered with two isolates from South Africa, but isolate CBS 117006 fell outside this B. viticola subclade. These differences in nucleotide sequences and colony morphology are regarded as intraspecific variation.

Van Niekerk et al (2004) studied a Diplodia sp. from pruned canes in South Africa with morphological characters that fit within the description of Dothiorella viticola. The phylogenetic analysis in our study showed clearly that two isolates of this Diplodia sp. (CBS 112869, CBS 112870) clustered with other isolates of B. viticola, which supports the identification of this fungus as B. viticola. The known occurrence of B. viticola in Spain and South Africa would suggest that this species may be widely distributed, possibly wherever grapevines are grown. Botryosphaeria viticola appears to be a saprophyte, according to the results obtained by van Niekerk et al (2004) in different pathogenicity tests with the strain CBS 112869. Furthermore B. viticola has been found mainly on old, discarded pruned canes and has not been associated with any disease symptoms, thus reinforcing the notion that it may be a saprophytic species.

	ACKNOWLEDGMENTS

 
This work was financed by the Instituto Nacional de Investigación y Tecnología Agraria y Alimentaria (INIA) under project RTA03-058-C2-1 (Spain) and by the European Regional Development Fund and Fundação para a Ciência e a Tecnologia under project POCTI/AGR/56140/2004 (Portugal). Soledad Martos was supported by a grant from DURSI-AGAUR (Regional Government of Catalonia, Spain). We thank these viticulturists and wine producers for letting us survey their vineyards (Spain): Miguel Torres, Ca n’Estella, Joaquim Aguiló, Raventós i Blanc and Rosend Esteve.

	FOOTNOTES

 
Accepted for publication July 9, 2005.

¹ Corresponding author. E-mail: alp{at}mail.fct.unl.pt

	LITERATURE CITED

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS TAXONOMY DISCUSSION LITERATURE CITED

 
Alves A, Correia A, Luque J, Phillips AJL. 2004. Botryosphaeria corticola sp. nov. on Quercus species, with notes and description of Botryosphaeria stevensii and its anamorph Diplodia mutila. Mycologia 96:598–613.[Abstract/Free Full Text]

Barr ME. 1972. Preliminary studies on the Dothideales in temperate North America. Contr Univ Michigan Herb 9:523–638.

Carbone I, Kohn LM. 1999. A method for designing primer sets for speciation studies in filamentous ascomycetes. Mycologia 91:553–556.[CrossRef]

Crous PW, Palm ME. 1999. Reassessment of the anamorph genera Botryodiplodia, Dothiorella and Fusicoccum. Sydowia 51:161–175.

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 Mycol 45:129–140.

Hasegawa M, Kishino H, Yano TA. 1985. Dating of the human ape splitting by a molecular clock of mitochondrial-DNA. J Mol Evol 22:160–174.[CrossRef][Medline]

Hillis DM, Bull JJ. 1993. An empirical test of bootstrapping as a method for assessing confidence in phylogenetic analysis. Syst Biol 42:182–192.[CrossRef]

Phillips A, Alves A, Correia A, Luque J. 2005. Two new species of Botryosphaeria with brown, 1-septate ascospores and Dothiorella anamorphs. Mycologia 97:513–529.[Abstract/Free Full Text]

Saccardo PA. 1877. Fungi veneti novi vel critici vel Mycologiae Venetae addendi. Michelia 1:1–72.

Shoemaker RA. 1964. Conidial states of some Botryosphaeria species on Vitis and Quercus. Can J Bot 42:1297–1303.

Simmons MP, Ochoterena H. 2000. Gaps as characters in sequence-based phylogenetic analyses. Syst Biol 49:369–381.[CrossRef][Medline]

Sivanesan A. 1984. The bitunicate ascomycetes and their anamorphs. Vaduz, Liechtenstein: J Cramer. 701 p.

Slippers B, Crous PW, Denman S, Coutinho TA, Wingfield BD, Wingfield MJ. 2004. Combined multiple gene genealogies and phenotypic characters differentiate several species previously identified as Botryosphaeria dothidea. Mycologia 96:83–101.[Abstract/Free Full Text]

Swofford DL. 2003. PAUP*. Phylogenetic analysis using parsimony (*and other methods) version 4. Sunderland, Massachusetts: Sinauer Associates.

Thompson JD, Gibson TJ, Plewniak F, Jeanmougin F, Higgins DG. 1997. The Clustal X windows interface: flexible strategies for multiple sequence alignment aided by quality analysis tools. Nucleic Acids Res 25:4876–4882.[Abstract/Free Full Text]

Van Niekerk JM, Crous PW, Groenewald JZ, Fourie PH, Halleen F. 2004. DNA phylogeny, morphology and pathogenicity of Botryosphaeria species on grapevines. Mycologia 96:781–798.[Abstract/Free Full Text]

Von Arx JA. 1987. Plant Pathogenic Fungi. Nova Hedwigia Beih 87:288p.

White TJ, Bruns T, Lee S, Taylor J. 1990. Amplification and direct sequencing of fungal ribosomal RNA genes for phylogenetics. In: Innis MA, Gelfand DH, Sninsky J J, White TJ, eds. PCR protocols: A guide to methods and applications. San Diego, California: Academic Press. p 315–322.

Wollenweber HW. 1941. Diplodia sarmentorum Fries und ihre Verbreitung. Zentralbl Bakteriol Parasitendk 103:347–357.

Young ND, Healy J. 2003. GapCoder automates the use of indel characters in phylogenetic analysis. BMC Bioinformatics 4: art. 6.[CrossRef][Medline]

Zhou S, Stanosz GR. 2001. Relationships among Botryosphaeria species and associated anamorphic fungi inferred from the analyses of ITS and 5.8s rDNA sequences. Mycologia 93:516–527.[CrossRef]

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