Should Loretta Lynch be confirmed as the Attorney General of the United States?
Togninia (Calosphaeriales) is confirmed as teleomorph of ...
14
646 Mycologia, 95(4), 2003, pp. 646–659. q 2003 by The Mycological Society of America, Lawrence, KS 66044-8897 Togninia (Calosphaeriales) is confirmed as teleomorph of Phaeoacremonium by means of morphology, sexual compatibility and DNA phylogeny Lizel Mostert Department of Plant Pathology, University of Stellenbosch, P. Bag X1, Matieland 7602, South Africa Pedro W. Crous 1 Centraalbureau voor Schimmelcultures, Uppsalalaan 8, 3584 CT Utrecht, The Netherlands J. Z. (Ewald) Groenewald Department of Plant Pathology, University of Stellenbosch, P. Bag X1, Matieland 7602, South Africa Walter Gams Richard C. Summerbell Centraalbureau voor Schimmelcultures, Uppsalalaan 8, 3584 CT Utrecht, The Netherlands Abstract: Petri disease, or black goo, is a serious dis- ease of vines in most areas where grapevines are cul- tivated. The predominant associated fungus is Phaeo- moniella chlamydospora (Chaetothyriales). Several species of Phaeoacremonium (Pm.) also are associated, of which Pm. aleophilum is the most common. Al- though no teleomorph is known for Phaeoacremon- ium, the genus Togninia previously has been linked to phaeoacremonium-like anamorphs. To investigate the possible anamorph-teleomorph connection of Phaeoacremonium to Togninia, anamorphs of Togni- nia minima, T. fraxinopennsylvanica and T. novae-zeal- andiae morphologically were compared with Pm. aleophilum and some representative cultures were mated in all combinations. Although no interspecies mating proved fertile, matings between isolates of Pm. aleophilum produced a Togninia teleomorph within 3–4 weeks. Certain field isolates of Pm. aleo- philum commonly produced the teleomorph, dem- onstrating that both mating types can occur in the same vine and thus also explaining the genetic diver- sity observed for this fungus in some vineyards. To elucidate the phylogenetic relationships among these taxa, isolates were subjected to sequence analysis of the nuclear ribosomal internal transcribed spacers (ITS1, ITS2) and the 5.8S rRNA gene, as well as por- tions of the translation elongation factor 1 alpha (EF- Accepted for publication December 3, 2002. 1 Corresponding author. E-mail: [email protected] 1a) gene. The generic placement of teleomorphs within Togninia (Calosphaeriales) further was con- firmed via phylogenetic analyses of 18S small subunit (SSU) DNA. From these sequences, morphological and mating data, we conclude that T. minima is the teleomorph of Pm. aleophilum, and that it has a bial- lelic heterothallic mating system. An epitype and mat- ing type tester strains also are designated for T. min- ima. Key words: Calosphaeriales, EF-1a, ITS, 18S SSU DNA, sexual compatibility, systematics INTRODUCTION Petri disease is a well-known disease of grapevines worldwide (Mugnai et al 1999). Affected grapevines exhibit a slow dieback as well as stunted growth. The predominant associated fungus is Phaeomoniella chla- mydospora W. Gams, Crous, M.J. Wingf. & L. Mugnai (Chaetothyriales, Herpotrichiellaceae). Whether this fungus is the sole or only a contributing causal agent of the disease is uncertain. Several species of Phae- oacremonium (Pm.) (Diaporthales, Magnaportha- ceae) also commonly grow from vines affected by Pe- tri disease, including Pm. aleophilum W. Gams, Crous, M.J. Wingf. & L. Mugnai, Pm. angustius W. Gams, Crous & M.J. Wingf., Pm. inflatipes W. Gams, Crous & M.J. Wingf., Pm. mortoniae Crous & W. Gams, Pm. parasiticum (Ajello, Georg & C.J.K. Wang) W. Gams, Crous & M.J. Wingf., Pm. rubrigenum W. Gams, Crous & M.J. Wingf., and Pm. viticola Dupont. Of the Phae- oacremonium species associated with Petri disease, Pm. aleophilum consistently has been the most fre- quently isolated (Scheck et al 1998, Ari 2000, Gatica et al 2001). In various studies, Pm. aleophilum has been found to be associated with brown-streaking symptoms in Petri-diseased grapevines (Mugnai et al 1999, Ari 2000) and sectorial brown necrosis (Gatica et al 2001). The genetic variation within populations of Phaeo- moniella chlamydospora and Pm. aleophilum has been studied by various workers (Pe ´ros et al 2000, Tegli et al 2000a, b). Using RAPDs (Random Amplified Poly- morphic DNA) and RAMS (Random Amplified Mi- cro- or Mini-Satellites), Tegli et al (2000b) showed that considerable variation existed among isolates of Pm. aleophilum collected from the same field, sug-
Transcript of Togninia (Calosphaeriales) is confirmed as teleomorph of ...
646
Mycologia, 95(4), 2003, pp. 646–659.q 2003 by The Mycological Society of America, Lawrence, KS 66044-8897
Togninia (Calosphaeriales) is confirmed as teleomorph of Phaeoacremoniumby means of morphology, sexual compatibility and DNA phylogeny
Lizel MostertDepartment of Plant Pathology, University ofStellenbosch, P. Bag X1, Matieland 7602, SouthAfrica
Pedro W. Crous1
Centraalbureau voor Schimmelcultures, Uppsalalaan8, 3584 CT Utrecht, The Netherlands
J. Z. (Ewald) GroenewaldDepartment of Plant Pathology, University ofStellenbosch, P. Bag X1, Matieland 7602, SouthAfrica
Walter GamsRichard C. Summerbell
Centraalbureau voor Schimmelcultures, Uppsalalaan8, 3584 CT Utrecht, The Netherlands
Abstract: Petri disease, or black goo, is a serious dis-ease of vines in most areas where grapevines are cul-tivated. The predominant associated fungus is Phaeo-moniella chlamydospora (Chaetothyriales). Severalspecies of Phaeoacremonium (Pm.) also are associated,of which Pm. aleophilum is the most common. Al-though no teleomorph is known for Phaeoacremon-ium, the genus Togninia previously has been linkedto phaeoacremonium-like anamorphs. To investigatethe possible anamorph-teleomorph connection ofPhaeoacremonium to Togninia, anamorphs of Togni-nia minima, T. fraxinopennsylvanica and T. novae-zeal-andiae morphologically were compared with Pm.aleophilum and some representative cultures weremated in all combinations. Although no interspeciesmating proved fertile, matings between isolates ofPm. aleophilum produced a Togninia teleomorphwithin 3–4 weeks. Certain field isolates of Pm. aleo-philum commonly produced the teleomorph, dem-onstrating that both mating types can occur in thesame vine and thus also explaining the genetic diver-sity observed for this fungus in some vineyards. Toelucidate the phylogenetic relationships among thesetaxa, isolates were subjected to sequence analysis ofthe nuclear ribosomal internal transcribed spacers(ITS1, ITS2) and the 5.8S rRNA gene, as well as por-tions of the translation elongation factor 1 alpha (EF-
Accepted for publication December 3, 2002.1 Corresponding author. E-mail: [email protected]
1a) gene. The generic placement of teleomorphswithin Togninia (Calosphaeriales) further was con-firmed via phylogenetic analyses of 18S small subunit(SSU) DNA. From these sequences, morphologicaland mating data, we conclude that T. minima is theteleomorph of Pm. aleophilum, and that it has a bial-lelic heterothallic mating system. An epitype and mat-ing type tester strains also are designated for T. min-ima.
Key words: Calosphaeriales, EF-1a, ITS, 18S SSUDNA, sexual compatibility, systematics
INTRODUCTION
Petri disease is a well-known disease of grapevinesworldwide (Mugnai et al 1999). Affected grapevinesexhibit a slow dieback as well as stunted growth. Thepredominant associated fungus is Phaeomoniella chla-mydospora W. Gams, Crous, M.J. Wingf. & L. Mugnai(Chaetothyriales, Herpotrichiellaceae). Whether thisfungus is the sole or only a contributing causal agentof the disease is uncertain. Several species of Phae-oacremonium (Pm.) (Diaporthales, Magnaportha-ceae) also commonly grow from vines affected by Pe-tri disease, including Pm. aleophilum W. Gams, Crous,M.J. Wingf. & L. Mugnai, Pm. angustius W. Gams,Crous & M.J. Wingf., Pm. inflatipes W. Gams, Crous& M.J. Wingf., Pm. mortoniae Crous & W. Gams, Pm.parasiticum (Ajello, Georg & C.J.K. Wang) W. Gams,Crous & M.J. Wingf., Pm. rubrigenum W. Gams, Crous& M.J. Wingf., and Pm. viticola Dupont. Of the Phae-oacremonium species associated with Petri disease,Pm. aleophilum consistently has been the most fre-quently isolated (Scheck et al 1998, Ari 2000, Gaticaet al 2001). In various studies, Pm. aleophilum hasbeen found to be associated with brown-streakingsymptoms in Petri-diseased grapevines (Mugnai et al1999, Ari 2000) and sectorial brown necrosis (Gaticaet al 2001).
The genetic variation within populations of Phaeo-moniella chlamydospora and Pm. aleophilum has beenstudied by various workers (Peros et al 2000, Tegli etal 2000a, b). Using RAPDs (Random Amplified Poly-morphic DNA) and RAMS (Random Amplified Mi-cro- or Mini-Satellites), Tegli et al (2000b) showedthat considerable variation existed among isolates ofPm. aleophilum collected from the same field, sug-
647MOSTERT ET AL: TOGNINIA AND PHAEOACREMONIUM ANAMORPHS
gesting that sexual reproduction might occur (Tegli2000). Considerable genetic variation suggestive ofongoing recombination also was found in UniversallyPrimed-PCR studies done with Pm. aleophilum iso-lates from Australia (Cottral et al 2001). Rooney et al(2002) subsequently reported inducing teleomorphsfor Pm. inflatipes and Pm. aleophilum under labora-tory conditions.
In a study of the genus Togninia Berl. (Calosphaer-iales), Hausner et al (1992) treated Togninia minima(Tul. & C. Tul.) Berl. (lectotype of Togninia) and atthe same time they described two new species, T. frax-inopennsylvanica (Hinds) Hausner, Eyjolfsdottir & J.Reid and T. novae-zealandiae Hausner, Eyjolfsdottir &J. Reid. Although no cultures of T. minima were avail-able, the two newly described species were culturedand were shown to produce anamorphs that were in-termediate between Acremonium Link : Fr. and Phia-lophora Medlar. Several anamorph genera in recentyears have been described with this approximate ap-pearance (Gams 2000). Of these, the genus Phae-oacremonium W. Gams et al closely fits the descriptionof the Togninia anamorphs illustrated by Hausner etal (1992).
The first aim of the current study was to investigatethe possible link between Phaeoacremonium and Tog-ninia. Phaeoacremonium aleophilum, a species similarin appearance to the anamorphs illustrated by Haus-ner et al (1992), was chosen for morphological com-parison with the anamorphs of T. minima, T. fraxi-nopennsylvanica and T. novae-zealandiae. A furtheraim was to determine if the formation of a teleo-morph could be elicited in Pm. aleophilum. This wasdone by mating a selection of vine isolates in culture.A final aim was to elucidate the genetic diversity with-in and among these species and to clarify the higher-order phylogenetic placement of Togninia. To ad-dress this, a phylogenetic analysis was conducted us-ing sequences of the nuclear ribosomal DNA regionencompassing the internal transcribed spacers (ITS1,5.8S and ITS2), as well as translation elongation fac-tor 1 alpha (EF-1a), and 18S ribosomal small subunit(SSU).
MATERIALS AND METHODS
Morphology. Field isolations were made from rooted nurs-ery plants and older, diseased grapevines, from which sin-gle-conidial isolates were obtained (TABLE I). Anamorphmorphology was studied on 2% malt-extract agar (MEA;Biolab, Midrand, South Africa), while perithecia were in-duced on twice-autoclaved pieces of grapevine cane placedon 2% water agar (Biolab) (GWA). Cultures were incubatedat 22 C under a 12 h fluorescent white light/dark regime.For microscopy, material was mounted in lactic acid. Thirty
measurements were taken of each type of morphologicalstructure, and averages and 95% confidence intervals weredetermined for spore dimensions. Measurements are givenwith minimum and maximum ranges in parentheses.
Vertical sections (10 mm) of fruiting bodies were cut witha Leica CM1100 freezing microtome. Colony colors weredetermined according to Rayner (1970). Cultures are main-tained in the collection of the Department of Plant Pathol-ogy at the University of Stellenbosch, and representativestrains have been deposited at the Centraalbureau voorSchimmelcultures (CBS, Utrecht, the Netherlands). Refer-ence strains of T. fraxinopennsylvanica (CBS 110212, ex-type), T. novae-zealandiae (UAMH 9589, UAMH 9590, ex-type) and T. minima (CBS 213.31, ex-type of Longoa pani-culata Curzi) also were studied.
Matings. Twenty-one Pm. aleophilum isolates were grownon MEA plates for 2 wk, using 10 plates per isolate. Conidiawere dislodged from the agar surface by means of a glassrod, and suspensions were prepared in 5 mL sterile distilledwater. Two aliquots of 100 mL each, representing two dif-ferent isolates, were pipetted onto the canes of GWA plates.Isolates were mated in all possible combinations. Controlsconsisted of a 200 mL aliquot of one isolate only. Plates wereincubated at 22 C under continuous white light. Successfulcrosses were noted 3–4 wk after mating. For a mating to beconsidered successful, perithecia had to produce largequantities of ascospores that germinated readily in culture.One such mating was chosen (LM 54 3 LM 463), and 20single ascospore isolates obtained (LM 227–LM 240, LM243–LM 247, LM 249). Further crosses were made withthese ascospore isolates using the procedure describedabove. Two strains found to be of opposite mating type ar-bitrarily were designated as MAT1-1 (LM 463) and MAT1-2(LM 54). Inter-species matings were done to investigate thebiological species boundaries of Pm. aleophilum, T. minima,T. novae-zealandiae and T. fraxinopennsylvanica.
DNA isolation and amplification. Twenty-seven Pm. aleo-philum isolates were selected for sequence comparisons(TABLE I). Sequences of the ITS, EF-1a and SSU of T. frax-inopennsylvanica (CBS 110212), T. novae-zealandiae(UAMH 9589 and UAMH 9590), T. minima (CBS 213.31)and an unknown Phaeoacremonium sp. (STE-U 3394) alsowere included. Genomic DNA was extracted using the iso-lation protocol of Lee and Taylor (1990). In studies intend-ed to determine the degree of genetic diversity within Pm.aleophilum, the 5.8S nuclear ribosomal RNA gene and theflanking internal transcribed spacers (ITS1 and ITS2) wereamplified with primers ITS1 and ITS4 (White et al 1990)and translation elongation factor 1 alpha (EF-1a) was am-plified with primers EF1-728F and EF1-986R (Carbone andKohn 1999). These PCR amplification cycles run on aGeneAmp PCR System 2700 (Perkin-Elmer, Norwalk, Con-necticut) were for both regions: 96 C for 5 min, followedby 36 cycles of (1) denaturation (94 C for 30 s), (2) an-nealing (50 C for 30 s) and (3) elongation (72 C for 90 s),and a final 7 min extension step at 72 C.
In studies intended to determine the higher order phy-logeny of Togninia, primers NS1 and NS4 (White et al1990) were used to amplify the 59 end of the 18S ribosomal
648 MYCOLOGIAT
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I.P
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Togn
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isol
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Spec
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Cul
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No.
Gen
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Hos
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77c
LM
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LM
467c
LM
468
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1107
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0708
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7993
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649MOSTERT ET AL: TOGNINIA AND PHAEOACREMONIUM ANAMORPHS
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926
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445
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921
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DNA (SSU) gene for a subset of four isolates representingthe different Togninia species and Pm. aleophilum. The cy-cling conditions consisted of an initial denaturation step of94 C for 7 min, followed by 36 cycles of (1) denaturation(95 C for 45 s), (2) annealing (55 C for 60 s) and (3)elongation (72 C for 120 s), and finally a 2 min extensionstep at 72 C.
PCR products were analyzed by electrophoresis at 85 Vfor 30 min in a 0.8% (w/v) agarose gel in 0.5 3 TAE buffer(0.4 M Tris, 0.05 M NaAc, and 0.01 M ethylene diaminetetraacetic acid [EDTA], pH 7.85) and visualized under UVlight with a GeneGenius Gel Documentation and AnalysisSystem (Syngene, Cambridge, United Kingdom) followingethidium bromide staining.
PCR products were purified according to the manufac-turer’s instructions using a commercial kit (Nucleospin Ex-tract 2 in 1 Purification Kit, Machery-Nagel GmbH & Co.,Germany). Sequencing reactions were carried out with ABIPRISM Big Dye Terminator version 3.0 Cycle SequencingReady Reaction Kit (PE Biosystems, Foster City, California),according to the manufacturer’s recommendations, andwere analyzed on an ABI Prism 3100 DNA Sequencer (Per-kin-Elmer, Norwalk, Connecticut). Sequences were depos-ited at GenBank (TABLE I), and the alignment was depos-ited in TreeBase (ITS and EF-1a: SN1269–3614; SSU:SN1269–3617).
Phylogenetic analysis. Raw sequence data were analyzed us-ing EditView 1.0.1 (http://www.appliedbiosystems.com),and sequences were manually aligned by inserting gaps.Phylogenetic analyses were conducted using PAUP (Phylo-genetic Analysis Using Parsimony) version 4.0b10 (Swofford2000). Gaps were treated as a fifth character, and all char-acters were unordered and of equal weight. Phialophora ri-chardsiae (Nannf.) Conant (CBS 270.33, GenBank ITS 5AY179948, EF-1a 5 AY179914) and Cercospora apii Fresen.(CBS 119.25, GenBank ITS 5 AY179949, EF-1a 5AY179915) were used as outgroups for both the EF-1a andITS analyses. Six Pm. aleophilum isolates (LM 44, LM 52,LM 75, LM 83, LM 113, and LM 115) were excluded fromthe combined analyses. Their respective EF and ITS se-quences were 100% similar to the sequences of LM 24, LM441, LM 466, LM 443, LM 440, LM 463, LM 460, LM 34and LM 5. Maximum-parsimony analysis was performed us-ing the heuristic search option with a 1000 random-taxonadditions and tree bisection and reconstruction (TBR) asthe branch-swapping algorithm. Bootstrap support for theITS and EF-1a analysis for internal branches was evaluatedfrom 1000 heuristic search replicates and 1000 random tax-on additions. Tree length, consistency index (CI), retentionindex (RI) and the rescaled consistency index (RC) valuesalso were calculated. A partition homogeneity test in PAUP(Swofford 2000) was conducted to test the congruence be-tween the ITS and EF-1a sequence datasets. Small subunitsequences were added to an alignment obtained fromTreeBase (M911). Sequences representative of the differentorders within the class Sordariomycetes, as well as the orderChaetothyriales (Chaetothyriomycetes), were retrievedfrom GenBank and added to the alignment. Neighbor-join-ing analyses of the SSU alignment (using uncorrected ‘‘p’’,
650 MYCOLOGIA
Kimura-2-parameter and Jukes-Cantor substitution models)were done with PAUP version 4.0b10 (Swofford 2000). Rho-dosporidium toruloides Banno and Athelia bombacina Pers.were used as outgroups for the neighbor-joining analysis.
RESULTS
Morphology. In culture, T. minima strain CBS 213.31produced a Phaeoacremonium anamorph similar toPm. aleophilum, but distinct from the Phaeoacremon-ium anamorphs associated with T. fraxinopennsylvan-ica and T. novae-zealandiae. A detailed description ofT. minima, based on the teleomorphs formed by iso-lates of Pm. aleophilum (TABLE I), as well as the ma-terial designated by Hausner et al (1992), is providedbelow:
Togninia minima (Tul. & C. Tul.) Berl., Icon. Fung.3: 11. 1900. FIGS. 1–24[ Calosphaeria minima Tul. & C. Tul., Sel. Fung. Carpol.
2: 112, plate XIII:23–24. 1863.[ Calosphaeria (Erostella) minima (Tul. & C. Tul.) Sacc.,
Syll. Fung. 1: 101. 1882.[ Erostella minima (Tul. & C. Tul.) Traverso, Fl. Ital.
Crypt. 1: 156. (1905) 1906.5 Calosphaeria alnicola Ellis & Everh., Proc. Acad. Nat.
Sci. Phila. 221. (1890) 1891.[ Togninia alnicola (Ellis & Everh.) Berl., Icon. Fung. 3:
10. 1900.5 Longoa paniculata Curzi, Atti Ist. Bot. R. Univ. Pavia,
Ser 3, 3: 204. 1927.
Anamorph. Phaeoacremonium aleophilum W. Gams,Crous, M.J. Wingf. & Mugnai, Mycologia 88: 791.1996.Mycelium consisting of branched, septate hyphae;
hyphae occurring singly or in strands of up to 10,tuberculate (with warts to 1 mm) to verruculose,pale brown, becoming paler toward the conidiogen-ous region, 1.5–3 mm wide. Chlamydospores absent.Perithecia heterothallic, mostly aggregated, not val-soid, sometimes solitary, mostly subepidermal alsoon the surface of the epidermis; perithecia subglo-bose, sometimes obpyriform, with a long cylindricalneck, (160–)250–285(–420) mm diam and basal part(200–)285–325(–400) mm tall. Wall consisting of tworegions of textura angularis: outer region darkbrown, cells smaller and more rounded than innerlayer, approx. 8–10 cells thick (individual cells notvisible further outward), 20–40 mm thick; inner re-gion hyaline (centrum) to pale brown, 5–7 cells and12–28 mm thick; surface covered with brown, septatehyphal appendages that become hyaline towardstheir tips (more abundant on older perithecia). Peri-thecial necks black, 1–3(–6) per perithecium, curved,verrucose, with apex often proliferating secondarilyupon aging and then appearing nodulose; nodules
(–120 mm wide) also appearing lower down on theneck; necks 800–1800 (av. 1055) mm long, 35–130(av. 69) mm wide at the base, and 20–60 (av. 41) mmwide at the apex, neck sometimes dividing into twonear the apex. Multinecked perithecia often with athin wall dividing the perithecial chamber. Paraph-yses hyaline, septate, cylindrical, narrowing towardsthe tip, 45–125 (av. 83) mm long, 2–4 mm wide atthe base and 1.5–2 mm at the apex, persistent. Asciarising in acropetal succession from sympodiallyproliferating ascogenous hyphae that appear spicatewhen mature, hyaline, clavate, with bluntly roundedapices and with sides straight or tapering towardsthe truncate or bluntly obtuse bases (17–)19–20(–27)3 4–5 mm; apical complex 0.5–1 mm thick, of indis-tinct structure, with a nonamyloid apical ring (neg-ative in Melzer’s reagent). Ascogenous hyphae hyaline,branched, smooth-walled, 2–3 mm wide (inflated ba-ses 25 mm wide). Ascospores 1-celled, hyaline, oblong-ellipsoidal to allantoid with rounded ends, sometimescontaining small guttules at the ends, (4–)4.5–5(–6.5)3 1–2 mm (av. 5 3 1.5 mm).
Conidiophores mostly micronematous, arising fromaerial or submerged hyphae, erect, simple, frequentlyreduced to conidiogenous cells, rarely 1–2-septate,subcylindrical, pale brown, paler towards the tip,smooth to verruculose, straight to gently curved, 4–40 mm tall, 2–3 mm wide. Conidiogenous cells terminalor lateral, mostly monophialidic, subcylindrical tonarrowly ellipsoidal, smooth to verruculose, subhya-line, 3–21 mm long, 1–2.5 mm wide at the base, 1–1.5mm wide at the apex, with a terminal, inconspicuous,almost convergent, 0.5–1 mm long, 1 mm wide collar-ette. Conidia aggregating in slimy heads, hyaline, ob-long-ellipsoidal to allantoid, when larger oblong toreniform, becoming 2-guttulate with age, (2.5–)3–4.5(–7) 3 1.5–2.5(–3) mm (av. 4 3 2 mm).
Substrates and geographical distribution. In vascularbundles of vines and twigs of woody plants: Vitis vi-nifera (Argentina, Australia, Chile, France, Italy, NewZealand, South Africa, Spain, Turkey, U.S.A. and Yu-goslavia), Actinidia sinensis (Italy), bark of a tropicalrain forest tree (Papua New Guinea), Olea europaea(Italy) (Crous et al 1996, Larignon et al 1997, Ari2000, Crous and Gams 2000, Armengol et al 2001,Groenewald et al 2001).
Cultural characteristics. See Crous et al (1996).Type specimens. YUGOSLAVIA. On roots and stems
of Vitis vinifera, 1990, M. Muntanola-Cvetkovic (CBS246.91 dried specimen and ex-type culture of Pm.aleophilum). SOUTH AFRICA. WESTERN CAPEPROVINCE: Wellington and Paarl, respectively, stemsof Vitis vinifera, 2001, L. Mostert, LM 463 (MAT1-15 CBS 111015) 3 LM 54 (MAT1-2 5 CBS 110703),
651MOSTERT ET AL: TOGNINIA AND PHAEOACREMONIUM ANAMORPHS
FIGS. 1–7. Togninia minima and its anamorph Phaeoacremonium aleophilum. 1. Perithecia. 2. Asci with spicate arrangementon ascogenous hyphae. 3. Paraphyses. 4. Ascospores. 5. Asci. 6. Conidiophores and conidiogenous cells. 7. Conidia. Bars 510 mm.
652 MYCOLOGIA
FIGS. 8–17. Togninia minima. 8. Perithecia on Vitis cane. 9. Perithecium with three necks. 10. Perithecium with globoidspore mass. 11. Vertical section through a perithecium. 12, 13. Asci with spicate arrangement on ascogenous hyphae. 14.Vertical section through a perithecium wall. 15. Asci and paraphyses. 16. Asci. 17. Ascospores. Bars 5 40 mm in FIGS. 8–11,10 mm in FIGS. 12–17.
653MOSTERT ET AL: TOGNINIA AND PHAEOACREMONIUM ANAMORPHS
FIGS. 18–24. Phaeoacremonium aleophilum, anamorph of Togninia minima. 18–21. Conidiophores and conidiogenous cells.22. Conidia after 3 mo. 23. Conidia aggregated in globoid masses. 23. Conidia after 7 d. Bar 5 10 mm.
[epitype of T. minima, designated here] dried speci-men, herb. CBS 6580.
Notes. No information previously has been avail-able regarding the anamorph of T. minima. The onlyculture currently available that previously has beenidentified as T. minima is CBS 213.31. It differs fromfreshly isolated strains of Pm. aleophilum in severalmorphological characters. Phaeoacremonium aleophil-um isolates in general have buff (190d) to honey(210b) colonies, while colonies of CBS 213.31 arewhite to buff (190d) with woolly mycelial tufts. Thedifference might be due to cultural degeneration.Conidiogenous cells (1.5 mm at the base) and conidia(1 mm wide) of CBS 213.31 were slightly narrowerthan those of Pm. aleophilum (2.5 and 2 mm, respec-tively).
Phaeoacremonium aleophilum differs from ana-morphs of T. fraxinopennsylvanica and T. novae-zeal-andiae. Conidia of T. fraxinopennsylvanica and T. no-vae-zealandiae were up to 9 and 10.5 mm long, re-spectively, significantly longer than those of Pm. aleo-philum, which did not exceed 7 mm. Also, thecollarettes of T. fraxinopennsylvanica and T. novae-
zealandiae (1.0–1.8 mm) were longer than those ofPm. aleophilum (0.5–1 mm).
Matings. Perithecia produced by crossing Pm. aleo-philum isolates were contrasted with those describedfor T. minima, T. fraxinopennsylvanica and T. novae-zealandiae (TABLE II). No interspecies mating was ob-served, nor were any matings obtained with CBS213.31. The teleomorph of Pm. aleophilum was iden-tical, however, to that described by Hausner et al(1992) for T. minima. Furthermore, ascospores of T.minima are longer and perithecia are wider and havelonger necks than those of T. fraxinopennsylvanicaand T. novae-zealandiae (TABLE II).
Of the 21 isolates of Pm. aleophilum that were mat-ed, 10 belonged to one mating type and 11 to theother (FIG. 25). The bi-allelic heterothallic matingsystem suggested by these results was verified withcrosses among the F1 ascospore progeny. Of 20 sin-gle-spore isolates used from one perithecium, 10grouped in one mating type and 10 in the other, thusagreeing with a 1:1 Mendelian segregation of matingtype (FIG. 26).
654 MYCOLOGIA
TA
BL
EII
.Pe
rith
ecia
lm
orph
olog
yof
the
vari
ous
Togn
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spec
ies
stud
ied
Asc
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and
dim
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ons
(mm
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ons
(mm
)Pe
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s(m
m)
Nec
kle
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m)
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ce
T.m
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6.5
31–
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)di
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and
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863)
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5.5
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215
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–25)
33.
5–5(
–6)
220–
490
diam
Ber
lese
(190
0)T.
min
ima
Obl
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soid
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4.7–
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2
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27)
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)
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4.8
195–
290
diam
400–
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sner
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2)
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Phylogenetic analysis. The combined alignment (ITSand EF-1a) had a total length of 863 characters, ofwhich 301 were constant, 329 were parsimony unin-formative and 233 were parsimony informative. Theresult of the partition homogeneity test showed thatthe ITS and EF-1a datasets were congruent (P 50.72) and therefore could be combined. Parsimonyanalysis of the combined datasets, using a heuristicsearch with 1000 random taxa additions, resulted in10 most-parsimonious trees (Tree length 5 901 steps,CI 5 0.937, RI 5 0.903, RC 5 0.846 and HI 5 0.063).Togninia minima (CBS 213.31) grouped in the cladewith all Pm. aleophilum sequences, as well as the ex-type strain of Pm. aleophilum. The analysis clearly de-limits the three Togninia species with good bootstrapsupport (FIG. 27). Some variation was observed with-in the Pm. aleophilum clade, as two isolates (CBS100400, 101357) formed a subclade with 100% boot-strap support within the Pm. aleophilum clade. Thesetwo isolates differed from the rest of the Pm. aleo-philum isolates at 11 (for EF-1a) and two (for ITS)nucleotide positions. Another group within the Pm.aleophilum clade also received significant support(87%). These sequences, however, differed from theother isolates in the Pm. aleophilum clade at only twonucleotide positions in the EF-1a dataset. A total of21 characters proved variable in the combined data-set (2.4%). The EF-1a area was found to be morevariable (15 nucleotides, 4.6%) than the ITS area (6nucleotides, 1.1%).
In the phylogram of the SSU sequence data (FIG.28), Pm. aleophilum, Togninia minima (CBS 213.31),T. fraxinopennsylvanica and T. novae-zealandiaegrouped together, forming the Calosphaeriales clade(Sordariomycetes) (100% bootstrap). Magnaporthegrisea (T.T. Hebert) M.E. Barr, as well as a teleo-morph of Harpophora W. Gams (Gaeumannomycesgraminis (Sacc.) Arx & D.L. Olivier, Magnaportha-ceae, incertae sedis), formed a well-supported subcla-de (100% bootstrap) within the class Sordariomyce-tes. Phialophora verrucosa Medlar, anamorphic Cap-ronia semiimersa (Cand. & Sulmont) Unter. & F.A.Naveau clustered with Capronia spp. (Chaetothyri-ales) with 100% bootstrap support.
DISCUSSION
Togninia is accepted as being typified by T. minima(Clements and Shear 1931). This viewpoint has beenfollowed by Hausner et al (1992), as explained inHolm (1992), and also is accepted by Barr (M. E.Barr pers comm, 20 Feb 2002), contrasting with herearlier view (Barr et al 1993) that Togninia was a syn-onym of Pleurostoma Tul. & C. Tul. A more compli-cated issue addressed by Hausner et al (1992) was
655MOSTERT ET AL: TOGNINIA AND PHAEOACREMONIUM ANAMORPHS
FIG. 25. Schematic representation of a mating study with single conidial isolates of Phaeoacremonium aleophilum. A (2)indicates that no perithecia formed, while (1) indicates formation of perithecia that exuded copious amounts of fertileascospores.
FIG. 26. Schematic representation of crosses from single ascospores of Togninia minima obtained from a fertile mating(LM 54 3 LM 463). A (2) indicates that no perithecia formed, while (1) indicates formation of perithecia that exudedcopious amounts of fertile ascospores.
whether the genus name Togninia had precedenceover Erostella. Erostella first was described as subgenusof Calosphaeria Tul. & C. Tul., and subsequently waselevated to generic level as Erostella (Sacc.) Trav.(1905) and again as Erostella (Sacc.) Sacc. (1906).Both Erostella and Togninia have Calosphaeria minimaTul. & C. Tul. as lectotype. The key to resolving thismystery lies in the precise interpretation of the Latindescription of Togninia provided by Berlese (1900),who placed 12 species and one variety in this genus.The decision of Clements and Shear (1931) to des-
ignate T. minima as lectotype of Togninia is not ob-viously supported by Berlese’s text. Berlese (1900, p.20), stated ‘‘C.[alosphaeria] herbicola E.[llis] etE.[verhart] id. C. ambigua Berl.[ese] est novi generisTogninia typus.’’ Two interpretations of this text arepossible. The first is that Berlese actually typified Tog-ninia by T. ambigua Berl.; in this case the lectotypi-fication of Clements and Shear (1931) should be re-jected and Erostella can be resurrected for the taxatreated by Hausner et al (1992), with Calosphaeriaminima as lectotype. The second interpretation,
656 MYCOLOGIA
FIG. 27. One of 10 most-parsimonious trees obtained from heuristic searches of a combined alignment of the 5.8S rRNAgene and flanking ITS1 and ITS2 regions and the translation elongation factor 1-alpha gene (length 5 901 steps, CI 50.937, RI 5 0.903, RC 5 0.846 and HI 5 0.063). Bootstrap support values (1000 repl.) above 65% are shown at the nodes.Phialophora richardsiae and Cercospora apii were used as outgroups.
which was explained by Holm (1992) and subse-quently followed by Hausner et al (1992), is that Ber-lese did not consider C. herbicola to be synonymouswith C. ambigua. Under the genus Togninia, Berlese(1900, p. 9) listed only T. ambigua, while he trans-ferred C. herbicola to Jattaea on p 8 but C. minima toTogninia on p 11. The Latin ‘‘id.’’, ‘‘idem’’, means‘‘the same as’’, but it also has been translated as ‘‘dit-to’’, which is more appropriate here, as Berlese(1900) clearly did not treat C. herbicola as synony-mous with C. ambigua but actually placed them indifferent genera. The species in this Calosphaeriacomplex are thus ‘‘novi generis Togninia typus’’,meaning that they are of the Togninia-type (Holm
1992), and consequently the lectotypification of Tog-ninia with T. minima, as proposed by Clements andShear (1931), can be accepted.
Because no authentic material of T. minima exists,Hausner et al (1992) designated the original illustra-tion (Tulasne and Tulasne 1863) as iconotype. Tog-ninia thus is conceived as a genus that has solitary toclustered, globose perithecia with papillate to beak-like apices that can be smooth or ornamented. Asciare unitunicate with truncate bases and thickenedapices, appearing in a spicate arrangement on asco-genous hyphae. Paraphyses are present, hyaline, sep-tate, and ascospores are hyaline, aseptate, allantoidto ellipsoidal. Anamorphs are acremonium- to phi-
657MOSTERT ET AL: TOGNINIA AND PHAEOACREMONIUM ANAMORPHS
FIG. 28. Neighbor-joining phylogenetic tree obtained from partial 18S rDNA sequences. Bootstrap support values (1000repl.) are shown above the nodes. Taphrina deformans and Rhodosporidium toruloides were used as outgroups. Sequencesmarked with an asterisk were taken from TreeBase (M911).
alophora-like (Hausner et al 1992). Hausner et al(1992) based their redescription of T. minima in parton two specimens, one collected from Alnus in theUnited States (N.A.F. 2514) and another from Pru-nus pennsylvanica collected in Canada (SSMF 725–7179). No cultures, however, were available for study.Clements and Shear (1931) treated Longoa Curzi assynonym of Calosphaeria, a genus that is heteroge-neous (M. E. Barr pers comm). In her revision of theCalosphaeriales, Barr (1985) did not treat Longoa,but based on its morphology Eriksson and Hawk-sworth (1986) considered the type species, Longoapaniculata (CBS 213.31, ex-type), to be a synonym ofT. minima. This synonymy is supported by the origi-
nal description and molecular data obtained in thisstudy.
A comparison of our fungus with the original de-scriptions of Togninia provided by Tulasne and Tu-lasne (1863) and Berlese (1900), as well as with theredescription of Hausner et al (1992), shows that ourfungus clearly belongs in Togninia. Furthermore, itsgeneric relationship with T. fraxinopennsylvanica andT. novae-zealandiae is confirmed via the tight clusterseen in 18S SSU sequence data (FIG. 28).
Before any teleomorph was known for Phaeoacre-monium, the genus was considered to have affinitiesfor the Magnaporthaceae (Dupont et al 1998). Mem-bers of this family generally are characterized by hav-
658 MYCOLOGIA
ing long, hairy necks and septate ascospores (Kirk etal 2001). The family is considered to be close to theDiaporthales (Winka and Eriksson 2000). The Tog-ninia teleomorphs of Phaeoacremonium differ strong-ly from those of other Magnaporthaceous fungi. Inaddition, our 18S rDNA sequence analysis shows thatthe Togninia and Phaeoacremonium species investigat-ed here form a distinct clade apart from the Diapor-thales (FIG. 28), supporting their placement in theorder Calosphaeriales for reasons outlined by Barr(1985). Phylogenetic analyses of DNA sequence datahave shown that T. minima forms a separate clusterwith T. fraxinopennsylvanica and T. novae-zealandiae,as well as other species presently known in Phaeoacre-monium (Groenewald et al 2001). Togninia teleo-morphs also have been induced for several otherPhaeoacremonium spp., which await further descrip-tion once their mating strategies have been resolved.
Results from mating studies clearly have shown thatT. minima has a biallelic heterothallic mating system.Such systems in fungi have been reviewed by Glassand Nelson (1994). Field isolates obtained from in-dividual diseased vines could be induced to form theteleomorph in vitro on cane sections, indicating thatcompatible mating types co-occur in nature on thesame vine. The conditions necessary for perithecialformation in the field remain unknown. The extentto which teleomorphs occur in the field also is un-known. The discovery of the teleomorph is importantfor the design and deployment of disease-controlstrategies, as well as for the overall understanding ofthe epidemiology of Petri disease.
ACKNOWLEDGMENTS
The authors acknowledge Winetech and the South AfricanNational Research Foundation (NRF) for financial support.Dr. Margaret E. Barr (B.C., Canada) is thanked for her com-ments on the status of the Calosphaeriales, while Drs. JamesReid and Georg Hausner (Univ. Manitoba, Canada) arethanked for their numerous letters, for providing copies ofall their correspondence related to the Togninia/Erostellaarguments and for taking the trouble to revive ex-type cul-tures of the species treated in their study.
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