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Nova Hedwigia 88 3—4 521—530 Stuttgart, May 2009

DOI: 10.1127/0029-5035/2009/0088-0521 0029-5035/09/0088-0521 $ 2.00© 2009 J. Cramer in der Gebrüder Borntraeger

Verlagsbuchhandlung, D-14129 Berlin · D-70176 Stuttgart

The family Diatrypaceae (Ascomycota) in Argentina:new species and new records

by

Cecilia C. Carmarán1* , M. Belén Pildain2 and Larissa N. Vasilyeva3

1PRHIDEB-CONICET, Departamento de Biodiversidad y Biología Experimental,Facultad de Ciencias Exactas y Naturales (UBA), Ciudad Universitaria,

Pabellón II, 4to. Piso, CP1428EHA Buenos Aires, Argentina2Centro Forestal CIEFAP, C.C.14. 9200 Esquel, Chubut, Argentina

3Institute of Biology & Soil Science, Far East Branch of the Russian Academy of Science,Vladivostok 690022, Russia

With 16 figures and 1 table

Carmarán, C.C., M.B. Pildain & L.N. Vasilyeva (2009): The family Diatrypaceae (Ascomycota) inArgentina: new species and new records. - Nova Hedwigia 88: 521–530.

Abstract: Six diatrypaceous species occurring in Argentina are discussed in this paper. Three newspecies (Diatrype gigantospora, D. wrightii, Eutypa iguazensii) and a new variety (Eutypellacorynostomoides var. argentinensis) are described and illustrated. To our knowledge, this is the firstrecord of Diatrype costesi and Eutypa leptoplaca in this country.

Key words: Argentina, Diatrypaceae, Eutypa leptoplaca, taxonomy.

Introduction

The family Diatrypaceae was established by Nitschke in 1867. Since then, severalauthors have reviewed both the family and the generic concepts (Tiffany & Gilman,1965; Glawe & Rogers, 1984; Rappaz, 1987; Vasilyeva & Stephenson, 2004, 2005;Carmarán et al., 2006).

Spegazzini (1898, 1910, 1925) began to study the biodiversity of the Diatrypaceaein Argentina and described 31 species and several varieties. In recent years, theknowledge of diatrypaceous fungi in this country has increased substantially(Carmarán & Romero, 1991; Carmarán, 2002; Romero & Carmarán, 2003; Pildainet al., 2005; Carmarán et al., 2006).

*corresponding author

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The main difficulty in identifying the diatrypaceous fungi is the high variability ofmany of the characters used for species discrimination. Sometimes, the character thatshould mark different entities can be observed within the same species. For example,some species have been described as having perithecia with necks emerging either ingroup or separately, asci with either J+ or J- apical ring, ascospores with overlappingsize ranges, and some characters can be very variable, for example the development ofstromata or the length of necks. Therefore, it is important to describe the specimensthoroughly and to indicate the most important characters for each species.

This paper is an additional contribution to the extensive project aiming at describingthe diatrypaceous fungi on native and cultivated woody plants in Argentina (Carmarán,2002; Carmarán & Romero, 2003; Carmarán et al., 2006). A few species of thisgroup, such as E. lata (Pers.: Fr.) Tul. (Descenzo et al., 1999; Rolshausen, 2006)and E. leptoplaca (Mont.) Rappaz (Trouillas & Gubler, 2004), which was found inArgentina for the first time, are known to be pathogens. These species have beenreported in Europe and the USA as responsible for significant economic damage.Both species are very similar, although E. leptoplaca has smaller ascospores andsulcate ostioles (Rappaz, 1987). Recently, Trouillas & Gubler (op. cit.) have investi-gated E. leptoplaca by using molecular techniques and working with isolates of thisspecies from gravepines.

In this paper, three new species and a new variety are described and illustrated andsome new records from Argentina are provided.

Materials and methods

The sampling and collecting methods have been previously explained (Carmarán, 2002). Specimenswere preserved in the Herbarium of Facultad de Ciencias Exactas y Naturales, Universidad deBuenos Aires, Argentina (BAFC). Samples for light microscopy were prepared from moistenedspecimens mounted in distilled water, 5% KOH, phloxine and Melzer’s reagent. Samples forepifluorescence light microscopy (EFM) were prepared in 0.05 % calcofluor (Romero & Minter,1988). Drawings were performed using a camera lucida and photographs were taken with an Olympusc-5060 wide zoom digital camera.

For Eutypa leptoplaca identification, wood with typical stromata of diatrypaceous fungi was collectedfrom a peach orchard in the town of San Pedro, Buenos Aires province, Argentina, in November2006. The culture was obtained from hamathecium elements. The isolates obtained were deposited inthe BAFC culture collection (Facultad de Ciencias Exactas y Naturales, Universidad de BuenosAires, Argentina). For genotypic analyses, the cultures used for the molecular studies were grownon malt peptone (MP) broth using 10 % (v/v) of malt extract (Brix 10) and 0.1 % (w/v) Bacto peptone(Difco), 2 mL of medium in 15 mL tubes. The cultures were incubated at 25°C for 21 d in light/darkness. DNA was extracted from the cells by using the UltraCleanTM Microbial DNA IsolationKit (MO BIO laboratories inc., Solana Beach, USA), according to the manufacturer’s instructions.The ITS region of the isolates was amplified using the universal primers ITS1 and ITS4 (White et al.,1990) while a fragment of the β-tubulin gene was amplified using the primers T1 and Bt2b (Glass &Donaldson, 1995; O’Donnell & Cigelnik, 1997). In some cases, best amplification results wereachieved by adding 6% bovine serum albumin (BSA, Promega Corp.) to the PCR reaction mix. PCRproducts were purified using a QIAGEN Gel Extraction kit (QIAGEN Inc.). Both strands of eachfragment were sequenced at the Molecular Biology Facility of the Facultad de Ciencias Exactas yNaturales, Universidad de Buenos Aires (Buenos Aires, Argentina). Forty six sequences obtainedfrom GenBank were included in the analysis, 25 from ITS and 21 from betatubulin gene, representingtwenty species of the family Diatrypaceae. The outgroup containing Xylaria curta, Xylaria berteri

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and Ganoderma lucidum was chosen according to Touillas & Gubler (2004), who generated thesequences. DNA sequences were edited with the software program BioEdit sequence alignmenteditor, version 7.0.5.3 (Hall, 1999). To determine the support for each clade, a bootstrap analysiswas performed with 1000 replications. The phylogenetic analysis of the sequence data was alsoperformed using the parsimony method and NONA version 2.0 (Goloboff, 1997) with all thecharacters equally weighted and gaps scored as missing data. Overall, 3.5% of the data matrix cellswere scored as gaps.

Taxonomy

Diatrype gigantospora Carmarán & L.N.Vassiljeva, sp. nov. Figs 1–5

ETYMOLOGY: Referring to the size of the ascospores.

Stromata sparsa. Perithecia monostycha. Asci 50–97 × 8–10 µm. Ascosporae brunneae, 12–22 ×3–4.5 µm.

Stromata discrete, with strongly developed interior, upper part yellowish, white tothe basal section, when mature almost white. Necks short, emerging separately, sulcate.Perithecia 4–10 in a stroma, sometimes compressed. Asci fusiform, 50–97 × 8–10 µm.Ascospores cylindrical to allantoid, brown to dark brown, 12–22 × 3–4.5 µm

ANAMORPH: Not seen.

HABITAT: On dead branch of an unidentified tree.

KNOWN DISTRIBUTION: Argentina.

MATERIAL EXAMINED: ARGENTINA, Jujuy, Dique La Cienaga, on decaying branch of unidentifiedtree, July 1994, C.C.Carmarán (BAFC 34610; HOLOTYPE).

NOTES: The unusual combination of characters, such as size of ascospores, pigmentationwithin the stroma and the morphology of asci, did not allow us to assign the specimento any described species of the Diatrypaceae. This specimen was included in thephylogenetic analysis carried out by Carmarán et al. (2006). The species appears tohave a strong relationship with Cryptosphaeria sulcata A.I. Romero & Carmarán andDiatrype albopruinosa (Schwein.) Cooke var salicina Rehm since both species havesimilar size and colour of ascospores, but different stromatal features. On the otherhand, this material is superficially similar to Diatrype implicata (Lév.) Rappaz andDiatrype patagonica Speg., but none of the latter has such asci and ascospores as in theArgentinean material. The ascal morphology is also peculiar for this family. Thus,with fluorescence microscopy, an apical ring and the external wall of asci were stronglyfluorescing, the cytoplasmic channel was seen in immature asci, and their clavateshape was characteristic. As pointed out by Carmarán et al. (op. cit.), the ascalmorphology could be a good predictor of the phylogenetic relationships.

Eutypa iguazensii Carmarán & L.N.Vassiljeva, sp. nov. Figs 6–9

ETYMOLOGY: Referring to the place where the species was collected.

Stromata sparsa. Asci 70–95 × 9–10 µm. Ascosporae allantoideae, hyalinae, 13–21 × 4–5 µm.

Stromata discrete, with strongly developed interior, upper part green-brown, whiteto the basal section, generally when mature only a light brown region can be seen.

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Figs 1–14. Diatrype gigantospora: 1. Stromata (holotype BAFC 34610). 2. Fascicles of asci.3–5. Detail of asci, with fluorescent microscopy, and transmission light. 6–9. Eutypa iguazensii(BAFC 51112). 6–7. Stromata. 8. Asci. 9. Asci with fluorescent microscopy. 10–14. Diatrypewrightii (BAFC 51117). 10–11. Stromata. 12. Asci. 13. Asci with fluorescent microscopy.14. Empty asci with fluorescent microscopy. Bars: 3–12, 19 = 10 µm; 1–2, 13–18 = 5 µm.

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Necks short, emerging separately or in groups, sulcate. Perithecia 4–10 in a stroma,usually not compressed. Asci fusiform, 70–95 × 9–10 µm, J+ or J-. Ascospores allan-toid, light brown, 13–21 × 3–4 µm.

ANAMORPH: Not seen.

HABITAT: On bark of angiosperm.

KNOWN DISTRIBUTION: Argentina.

MATERIAL EXAMINED: ARGENTINA, Misiones, Parque Nacional Iguazú, on decaying branch ofunidentified tree, April 1993, C.C.Carmarán (BAFC 51112; HOLOTYPE).

NOTES: Among taxa with eight-spored asci, there is a group of species includingD. macowaniana Thüm., D. dothidioides Rehm, D. microstoma Syd. & P.Syd.D. praeandina (Speg.) Rappaz and Eutypa andicola Speg. with similar stromaticcharacters. They have strongly developed interior of stromata, green-brown in upperpart and white in basal section. They do not have a black zone line in the substrate,the necks emerge separately, and they have J+ or J- asci and pale brown, 7–15 ×2–3 µm ascospores. These similarities suggest that the taxa involved comprise acomplex of species that have a very strong relationship within the group or perhapsthat those features could represent a convergent evolution.

The material of Eutypa iguazensii could be assigned to this complex. It has severalsimilar features, but differs in having larger ascospores and a very irregular stromaticsurface. It also resembles Eutypella leprosa, but unlike that species, E. iguazensiihas the coloured stromatal interior.

Diatrype wrightii Carmarán & L.N.Vassiljeva, sp. nov. Figs 10–14

ETYMOLOGY: In memory of Doctor Jorge E.Wright.

Asci 70–95 × 9–10 µm. Ascosporae allantoideae, hyalinae, 13–21 × 4–5 µm.

Stromata discrete, with strongly developed interior, white, black zone line present inthe substrate. Necks short, emerging separately, not prominent, sometimes difficultto see, sulcate. Perithecia 5–15. Asci fusiform, 30–35 × 4–5 µm, J+. Ascosporesallantoid, hyaline, 5–8 × 1.5–2.5 µm

ANAMORPH: Not seen.

HABITAT: On wood of angiosperm.

KNOWN DISTRIBUTION: Argentina.

MATERIAL EXAMINED: ARGENTINA, Buenos Aires, Isla Martín García, on decaying branch of unidentifiedtree, May 1992, C.C.Carmarán (BAFC 51117; HOLOTYPE). ARGENTINA, Buenos Aires, Chivilcoy,on decaying branch of unidentified tree, September 2002, L.Iannone (BAFC 51271).

NOTES: There is a group of taxa superficially linked to Diatrype disciformis (Hoffm.:Fr.) Fr., Diatrype bullata (Hoffm.: Fr.) Fr., D. tremellophora Ellis, D. asterostomaBerk. & M.A.Curtis and D. laurina Rehm. These taxa have a similar size of ascospores(in a range of 5–12 µm), well-developed stromata that are white inside, and a blackzone line is formed in the substrate. The differences between members of this groupare mainly amyloid reaction, “aspect in culture” (Rappaz 1987) and small differences

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in ascospores range (for example: D. disciformis 5–8.5 µm, D. asterostroma 7–11,D. laurina 9.5–12 µm). D. costesi (Speg.) Petr. & H.Syd. and our D. wrightii, bothof which share several features but do not have a black line in the substrate, appearto be very close to this group. The new species has been found only in wood ofangiosperm, never in bark.

Eutypella corynostomoides (Rehm) Rappaz var. argentinensis Carmarán &L.N.Vassiljeva, var. nov.

ETYMOLOGY: Referring to the region where the material was collected.

Stromata late effusa. Asci 8–18 × 4–6 µm. Ascosporae allantoideae, olivaceae, 3–6 × 1–1.5 µm.

Stromata effuse, interior not well-developed, frequently with a subepidermic stromaticlayer. Necks long, emerging in groups or separately, prominent, ostioles sulcate.

Fig. 15. The most parsimonious trees obtained from ITS sequence data (L = 450, Ci: 0,71, Ri:0,80).The branches with support ≤ 50 were collapsed. SP ITS = Argentinean strain.

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Fig. 16. The most parsimonious trees obtained from β-tub. sequence data (L = 802, Ci:0,93, Ri:0,96).The branches with support ≤ 50 were collapsed. SP BT = Argentinean strain.

Perithecia 1–15, immersed in the wood. Asci fusiform, 8–18 × 4–6 µm, J-. Ascosporesallantoid, brown, 3–6 × 1–1.5 µm.

ANAMORPH: Not seen.

HABITAT: On wood of angiosperm.

KNOWN DISTRIBUTION: Argentina.

MATERIAL EXAMINED: ARGENTINA, Misiones, Parque Nacional Iguazú, on decaying branch ofunidentified tree, April 1994, C.C.Carmarán (BAFC 51151; HOLOTYPE).

NOTES: Taxa without well-developed stromata, but with long necks, uniform asci,and small ascospores were assigned to Peroneutypa Berl. (Carmarán et al., 2006).The material here studied displays similar features, but the asci are fusiform. On theother hand, Eutypella corynostomoides presents similar characters as well, but differsin having amyloid asci and pale, allantoid ascospores that are different from brownishand almost straight ascospores of E. corynostomoides.

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Table 1. List of taxa used in the phylogenetic analysis.

Taxon GenBank Betatubulin GeneBank ITS

Diatrype bullata (Hoffm.: Fr.) Fr. DQ007002Diatrype flavovirens (Pers.: Fr.) Fr. DQ006959Diatrype stigma (Hoffm.: Fr.) Fr. DQ007003 AJ302439Diatrypella frossti Peck AF192322Diatrypella sp. DQ007001Diatrypella sp. AY684217Diatrypella sp. DQ006947Eutypa armeniacae Hansf. & M.V.Carter DQ006948Eutypa lata (Pers.: Fr.) Tul. & C.Tul. AY684215 AY462547

AY684213 AY684228DQ006989 AY694232AY684214 AY694233

DQ006932Eutypa lejoplaca (Fr.: Fr.) Fuckel AY684197 AY684221

AY684196 AY684238Eutypa leptoplaca (Mont.) Rappaz AY684211 AJ302453

AY684210 AY684235DQ006963 AY684236DQ006961 DQ006924EU649783* FJ267699*

Eutypa maura (Fr.: Fr.) Fuckel DQ006967 AY684224AY684198 DQ006926

Eutypa petrakii Rappaz DQ006971Eutypa sparsa Romell AY684201 AY684220

AY684214Eutypella alsophila (Mont.) Berk. AJ302467Eutypella scoparia (Schwein.) Ellis & Everh. AF373064

AF373064.1AJ302465

Eutypella sp. EF488380Eutypella vitis (Schwein.) Ellis & Everh. DQ006999.1Xylaria berteri (Mont.) Cooke AY951763Xylaria curta Fr. DQ322144

*Sequences obtained from BAFC 51667

New records and comments about the Argentinean species

Diatrype costesi (Speg.) Petr. & H.Syd, Ann. Mycol. 32: 25 (1934).

HABITAT: On angiosperm wood.

KNOWN DISTRIBUTION: Argentina, Chile.

MATERIAL EXAMINED: ARGENTINA, Misiones, Parque Nacional Iguazú, on decaying branch ofunidentified tree, April 1994, C.C.Carmarán (BAFC 51151); CHILE, under Ectosphaeria costesi,los Perales, on Cryptocarpa sp. 1918, C.L.Spegazzini, (LPS 425 HOLOTYPE).

NOTES: This taxon was described by Spegazzini (1921) on the basis of material fromChile. This is the first report from Argentina.

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Eutypa leptoplaca (Mont.) Rappaz, Mycol. Helv. 2(3): 341 (1987).

HABITAT: On angiosperm wood.

KNOWN DISTRIBUTION: Europe, North America, Australia, Argentina.

MATERIAL EXAMINED: ARGENTINA, San Pedro, on a decaying branch of Prunus armeniaca,November 2006, C.C.Carmarán (BAFC 51667).

NOTES: The material studied fits with the description of E. leptoplaca, sensu Rappaz(1987), which is very close to E. lata, and both are known pathogens (Peroz et al.,1999, Catal et al., 2007). The Argentinean specimen was collected from fallen twigsof a Prunus species, a typical substrate of E. lata. The trees examined in the orcharddid not present symptoms characteristic of this fungus.

Trouillas & Gubler (2004) have demonstrated the pathogenicity of E. leptoplaca ongrapevine and have identified isolates from several substrates. To our knowledge,no members of the genus Prunus have been reported as substrate of this species.Taking into account that this is the first report of E. leptoplaca in our country, thatit has important implications in plant health in Argentina, and that it has a greatvariability of morphological characters, it was therefore, necessary to confirm theidentification with molecular techniques.

The results of the phylogenetic analyses of the sequences from the ITS1 andbetatubulin genes supported the morphological identification (Figs 15 and 16). TheArgentinean isolates appear to be in the same clade with other members of the species,with a 78 bootstrap value for the ITS analysis, and a 97 bootstrap value for thebetatubulin analysis.

References

CARMARÁN, C.C. (2002): Contribución al estudio del orden Diatrypales en las zonas subtropicalesde la República Argentina. - Boletín de la Sociedad Micológica de Madrid 26: 43–56.

CARMARÁN, C.C. & A.I. ROMERO (1992): Problemas taxonómicos en el orden Diatrypales.Contribución a su esclarecimiento I. - Boletín de la Sociedad Argentina de Botánica. 28: 139–150.

CARMARÁN, C.C., A.I. ROMERO & L.M. GIUSSANI (2006): An approach towards a newphylogenetic classification in Diatrypaceae. - Fungal Diversity 23: 67–87.

CATAL, M., S.A. JORDAN, S.C. BUTTERWORTH & A.M.C. SCHILDER (2007): Detection ofEutypa lata and Eutypella vitis in grapevine by nested multiplex polymerase chain reaction.- Phytopathology 97: 737–747.

DESCENZO, R.A., S.R. ENGEL, G. GOMEZ, E. JACKSON, G.P. MUNKVOLD, J. SÉLLER &N. RIELAN (1999): Genetic analysis of Eutypa strains from California supports the presence of twopathogenic species. - Phytopathology 89: 884–893.

GLASS, N.L. & G. DONALDSON (1995): Development of primer sets designed for use with thePCR to amply conserved genes from filamentous ascomycetes. - Applied & EnvironmentalMicrobiology 61: 1323–1330.

GLAWE, D.A. & J.D. ROGERS (1984): Diatrypaceae in the Pacific Northwest. - Mycotaxon20: 401–460.

GOLOBOFF, P.A. (1997): NONA, version 2.0 for Windows. Computer program and documentationdistributed by the author, website: http://www.cladistics.com.

eschweizerbartxxx author

530

HALL, T.A. (1999): BioEdit: a user-friendly biological sequence alignment editor and analysisprogram for Windows 95/98/NT. - Nucleic Acids Symposium Series 41: 95–98.

O’DONNELL, K. & E. CIGELNIK (1997): Two divergent intragenomic rDNA ITS2 types withina monophyletic lineage of the fungus Fusarium are nonorthologous. - Molecular Phylogenetics andEvolution 7: 103–116.

PEROZ, J.P., I. JAMAUX-DESPREAUX, G. BERGER & D. GERBA (1999): The potentialimportance of diversity in Eutypa lata and co-colonising fungi in explaining variation in developmentof grapevine dieback. - Mycological Research 103: 1385–1390.

PILDAIN, M.B., M.V. NOVAS, & C.C. CARMARÁN (2005): Evaluation of anamorphic state,wood decay and productions of lignin-modifying enzymes for Diatrypaceaous fungi from Argentina.- Journal of Agricultural Technology 1(1): 81–96.

RAPPAZ, F. (1987): Taxonomie et momenclature des Diatrypacees à asques octospores. - MycologiaHelvetica 2: 285–648.

ROLSHAUSEN, P.E., N.E. MAHONEY, R.J. MOLYNEUX & W.D. GUBLER (2006):A reassessment of the species concept in Eutypa lata, the causal agent of Eutypa dieback of grapevine.- Phytopathology 96: 369–377.

ROMERO, A.I. & C.C. CARMARÁN (2003): First contribution to the study of Cryptosphaeriafrom Argentina. - Fungal Diversity 12: 161–167.

ROMERO, A.I. & D.W. MINTER (1988): Fluorescence microscopy: an aid to the elucidation ofAscomycetes structure. - Transactions of the British Mycological Society 90: 457–470.

SPEGAZZINI, C. (1898): Fungi Argentini novi v. critici. - Anales del Museo Nacional de BuenosAires 6: 1–23.

SPEGAZZINI, C. (1910): Fungi chilensis. - Revista de la Facultad de Agronomía y Veterinaria deLa Plata 6: 1–205.

SPEGAZZINI, C. (1921): Mycetes chilenses. - Boletin Acad. Nac. Cienc. Cordoba 25: 1–124.

SPEGAZZINI, C. (1925): Séptima Contribución a la micología chilena. - Revista Chilena de HistoriaNatural 29: 58–64.

TIFFANY, L.H. & J.C. GILMAN (1965): Iowa Ascomycetes IV. Diatrypaceae. - Iowa stateJournal of Science 40: 121–161.

TROUILLAS, F.P. & W.D. GUBLER (2004): Identification and characterization of Eutypa leptoplaca,a new pathogen of grapevine in Northern California. - Mycological Research 108: 1195–1204.

VASILYEVA, L.N. & S.L. STEPHENSON (2004): Pyrenomycetes of Great Smoky MountainsNational Park I. Diatrype Fr. (Diatrypaceae). - Fungal Diversity 17: 191–201.

VASILYEVA, L.N. & S.L. STEPHENSON (2005): Pyrenomycetes of Great Smoky MountainsNational Park II. Diatrypella (Ces. et De Not.) Nitschke and Cryptovalsa Ces. et De Not. Diatrypaceae.- Fungal Diversity 19: 189–200.

WHITE, T.J., T.L. BRUNS, S. LEE & J.W. TAYLOR (1990): Amplication and direct sequencingof fungal ribosomal RNA genes for phylogenetics. In: PCR Protocols: a Guide to methods andApplications (eds. M. Innis, D.H. Gelfand, J.J. Sninsky & J.T. White). Academic press, San Diego,USA.

Received 24 June 2008, accepted in revised form 8 October 2008.