Mycosphere

Two new species of from coastal wattle in Coorong National Park, South Australia

Trouillas FP 1, Sosnowski MR2 and Gubler WD1*

1Department of Plant Pathology, University of California, Davis, California 95616, USA 2South Australian Research and Development Institute, Adelaide, SA 5001, Australia

Trouillas FP, Sosnowski MR, Gubler WD 2010 – Two new species of Diatrypaceae from coastal wattle in Coorong National Park, South Australia. Mycosphere 1(2), 183–188.

In the present study, two species of Diatrypaceae were isolated from the wood of Acacia longifolia subsp. sophorae shrubs in the Coorong National Park, South Australia. Based on habitat, host, morphological observations and literature review, the isolates are described as the new species brunneospora and australiensis. These new taxa are fully described and illustrated and sequences of the internal transcribed spacer region of the nuclear ribosomal DNA are also provided.

Key words – Australia – Diatrypaceae – New species –

Article Information Received 16 June 2010 Accepted 13 July 2010 Published online 3 August 2010 *Corresponding author – Walter D. Gubler – e-mail – [email protected]

Introduction been made (Rappaz 1987). Among them, eight Species of Diatrypaceae () are genera have been recognized (Rappaz 1987); commonly found on stems of various woody these include Ces. & De Not. plants around the world. They are generally (four species), Diatrype Fr. (56 species), Dothi- considered to be saprotrophs, although some deovalsa Speg. (three species), Echinomyces F. species seem to be especially well established Rappaz (two species), Tul. & C. Tul. in wood of recently dead host plants (Tiffany & (26 species), Eutypella (Nitschke) Sacc. (76 Gilman 1965). A few species in this family species), Leptoperidia F. Rappaz (four species) constitute serious plant pathogens, among them and Rostronitschkia Fitzp. (one species). Eutypa lata (Pers.) Tul. & C. Tul. (syn.: E. In North America, the works of Glawe armeniacae Hansf. & Carter) has been parti- and Rogers (1984), and Tiffany and Gilman cularly well investigated as the causal agent of (1965), constitute important additional taxono- Eutypa dieback of several woody crops world- mic studies of Diatrypaceae. Recently, Vasil- wide (Carter 1991). Cryptosphaeria lignyota yeva and Stephenson (2004, 2005, 2006, 2009) (Fr.) Auersw. (syn.: C. populina (Pers.) Sacc.) described various species of Diatrypaceae from and R.W. Davidson & the Great Smoky Mountains National Park in R.C. Lorenz in this family represent severe the eastern United States, as well as Arkansas pathogens of forest trees in the United States and Texas. In California, 11 diatrypaceous and Europe (Davidson and Lorenz 1938, Hinds species were newly reported and described 1981, Jurc et al. 2006). from Vitis vinifera and various other woody The taxonomy of octosporous Diatry- host plants (Trouillas & Gubler 2004, Trouil- paceae has been revised and descriptions of las et al. 2010). The diversity of Diatrypaceae approximately 120 morphological species have from Argentina was also recently investigated

183 and new species as well as new records were Coorong is a wetland ecosystem and estuary, proposed (Carmaran et al. 2009). particularly valuable for its unique ecology and The current generic delineation and clas- cultural significance. Acacia longifolia subsp. sification in the Diatrypaceae as suggested by sophorae is a common shrub indigenous to Rappaz (1987) is based primarily on characters south eastern Australia and restricted mainly to of the teleomorphic states, including stroma coastal dunes. The taxa isolated for this study morphology and organization of perithecia. differed from known species in various aspects However, much overlap of taxonomic charac- of ascospores and ascomata morphology, in ters exists between the current diatrypaceous habitat and host, and deserve description as genera. Vasilyeva and Stephenson (2004) new species. acknowledged that the concept of Diatrype as delimited by Rappaz (1987) is somewhat Methods problematic, as there is, in some instances, no Pieces of dead stems of Acacia longifolia clear separation between this and either subsp. sophorae with black and effuse stromata Eutypa or Eutypella. Moreover, phylogenetic typical of Diatrypaceae were collected in classifications of Diatrypaceae were attempted December 2008 from the Coorong National using molecular data as well as morphological Park in South Australia. Microscopic analyses characters but evolutionary relationships in the and photography were conducted using stan- family remained uncertain (Acero et al. 2004, dard light microscopy with a Leica DMLB Carmaran et al. 2006, Trouillas et al. 2010). microscope mounted with a Leica DFC480 The diatrypaceous mycota of Australia is digital camera. Stroma and perithecial charac- remained largely unexplored. A few species teristics were examined using a Leica MZ95 have been studied, mainly because of their stereo microscope. Examinations and measure- occurrence on Vitis vinifera grapevines (Carter ments of microscopic characters were carried 1991, Mostert et al. 2004, Pitt et al. 2009). The out in water. Melzer’s reagent also was used to occurrence of Diatrypaceae on native assess amyloid reaction of the apical ascus ring. Australian host plants has been given little Thirty ascospores, ten asci (spore bearing consideration. In his account of Australian portion only) and ten perithecia of each species fungi, Cooke (1892) reported nine putative isolate were measured. Pure fungal cultures species of Diatrypaceae. An additional three were obtained from hyphal tips and grown on species were described from intertidal host potato dextrose agar (PDA) (Difco Labora- plants in north Queensland: Cryptovalsa tories, Detroit) dishes incubated under 24-hours halosarceicola K.D. Hyde on Halosarcia fluorescent lighting. Thirty conidia per isolate halocnemoides in a mangrove at Cairns Airport were measured in water as conidial masses (Hyde 1993a), Eutypa bathurstensis K.D. Hyde became visible from colonies. Sequences of the & Rappaz (Hyde 1993b) and Eutypella naqsii nuclear internal transcribed spacer (ITS) region K.D. Hyde (Hyde 1995) on Avicennia sp. at of the ribosomal DNA (rDNA) were obtained Bathurst Heads. In a study of fungi associated following standard DNA extraction procedure with Acacia and Eucalyptus plants in Melville and amplification by polymerase chain reaction Island in the Northern Territory of Australia, (PCR) using primers ITS1 and ITS4 (White et Yuan (1996) reported Cryptovalsa protracta al. 1990). Sequences have been deposited into (Pers.) De Not., Diatrype stigma (Hoffm.) Fr. GenBank. Holotypes and ex-type cultures were and (Schwein.) Ellis & deposited at the Australian Scientific Collec- Everh. To our knowledge, the above references tions Unit, NSW Industry & Investment, constitute the only studies that illustrate the Orange, NSW, Australia (DAR). diatrypaceous biota in Australia. During the course of surveys for Taxonomy Diatrypaceae associated with Vitis vinifera in South Australia, two original diatrypaceous Diatrype brunneospora Trouillas, Sosnowski species were collected on native Acacia & Gubler, sp. nov. Figs 1–6 longifolia subsp. sophorae (coastal wattle) MycoBank 518611 Fabaceae) from Coorong National Park. The

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Figs – 1–6 – Morphology of Diatrype brunneospora. 1. Asci and paraphyses. 2. Mature (brown) and immature (light brown) ascospores showing typical oil drops. 3. Cylindrical to clavate ascus, with flat apex. 4. Filiform conidia. 5. Colony after 6 days on PDA dishes (85 mm diam) incubated under 24-hours fluorescent lighting. 6. Perithecial cavities and emerging perithecial ostioles in the wood of Acacia longifolia subsp. sophorae. Bars = 10 µm in 1–4; 1 mm in 6.

Etymology – referring to the brown Stromata effuse, embedded in decor- ascospores. ticated wood and incorporating host tissues, Stromata lata effusa, in ligno decorticato. rather eutypoid, blackening the host surface. Perithecia immersa, monosticha, globosa vel Perithecia in one layer, globose to ovoid, ovoidea, 0.4−0.5 mm diam, ostiolis quadri- irregularly scattered, 0.4−0.5 mm diam, sulcatis, 0.3−0.4 mm diam. Asci octospori, ostioles commonly 4-sulcate, occasionally 5- clavulato-cylindracei, parte sporifera 65−85 × sulcate, generally emerging separately, 0.3−0.4 6−7.5 µm, annulis apicalibus haud amyloideis mm diam. Asci cylindrical to clavate with flat ornati. Ascoporae oblongae, brunneae, oleosae, apex and easily breaking stipes, without (6.5−)8−10(−12.5) × 3.5−4.5 µm. Conidia amyloid reaction in Melzer’s reagent, 8-spored, filiforma, subhyalina, (20−)22−32(−34) × p. sp. 65−85 × 6−7.5 µm. Ascospores oblong, 1−1.5 µm. Hab. in ramis emortuis Acacia dark brown at maturity, usually with two oil longifolia subsp. sophorae, in South Australia. droplets, (6.5−)8−10(−12.5) × 3.5−4.5 µm.

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Figs – 7–12 – Morphology of Eutypella australiensis. 7. Allantoid subolivaceous ascospores. 8. Filiform conidia. 9. Knobbed apex of an ascus. 10. Clavate and long-stipitate ascus. 11. Colony after 6 days on PDA dishes (85 mm diam) incubated under 24-hours fluorescent lighting. 12. Emerging perithecial ostioles in the wood of Acacia longifolia subsp. sophorae. Bars = 10 µm in 7– 10; 1 mm in 12.

Cultural characteristic – Colonies white Trouillas CNP01, herb. and ex-type culture = on PDA, growing slowly and covering half DAR 80711, GenBank accession no for ITS (with apparently no further increase in colony rDNA sequence: HM581946. size) of an 85 mm Petri dish after 20 days. Notes – Because of the taxonomic Conidia filiform, subhyaline, 17−28 × 1−1.5 confusion around Diatrypaceae and the µm. difficulty in segregating the various genera, the Known distribution – South Australia. assignment of this isolate to the genus Diatrype Material examined – AUSTRALIA, may require reconsideration in the future. This SOUTH AUSTRALIA, Coorong National Park, has morphological similarity with along Princes Highway, on dead wood of members of the genus Eutypa. However, based Acacia longifolia subsp. sophorae (Labill.) on preliminary phylogenies, this fungus Court, December 2008, holotype: F.P. appears best placed in Diatrype.

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Eutypella australiensis Trouillas, Sosnowski Acknowledgements & Gubler, sp. nov. Figs 7–12 We acknowledge Dr. Michael Priest MycoBank 518612 curator of the Plant Pathology Herbarium Etymology – referring to the Australian (DAR), at the Orange Agricultural Institute, origin. NSW Australia. Stromata lata effusa, in ligno decorticato. Perithecia immersa, nonnunquam exposita, References monosticha vel polysticha, globosa vel ovoidea, ostiolis trisulcatis. Asci octospori, claviti, longe Acero FJ, González V, Sánchez-Ballesteros J, stipitati, annulis apicalibus haud amyloideis Rubio V, Checa J, Bills GF, Salazar O, ornate, parte sporifera 40–50 × 7–8.5 μm. Platas G, Peláez F. 2004 − Molecular Ascoporae allantoideae, lutescentiae vel phylogenetic studies on the Diatrypaceae olivascentiae. Conidia filiforma, subhyalina, based on rDNA-ITS sequences. Mycolo- (16−)18−22(−25) × 1−1.5 µm. Hab. in ramis gia 96, 249–259. emortuis Acacia longifolia subsp. sophorae, in Carmaran CC, Romero AI, Giussani LM. 2006 South Australia. − An approach towards a new Stromata poorly developed, wide phylogenetic classification in Diatrypa- spreading, on decorticated wood surface. ceae. Fungal Diversity 23, 67–87. Perithecia in contact, in one or two layers, Carmaran CC, Pildain MB, Vasilyeva LN. globose to ovoid, raising the outer wood layer 2009 – The family Diatrypaceae and eventually becoming exposed, 0.25−0.4 () in Argentina: new species mm diam, with short perithecial necks, ostioles and new records. Nova Hedwigia 88, commonly 3-sulcate, emerging mostly sepa- 521–530. rately or in groups of two, 0.1−0.2 mm in Carter MV. 1991 – The status of Eutypa lata as diameter. Asci clavate, with apex more or less a pathogen. Monograph, Phytopatholo- knobbed, long-stipitate, without amyloid gical Paper No 32. Commonwealth reaction in Melzer’s reagent, 8-spored, p. sb. Agricultural Bureau, International Myco- 40–50 × 7–8.5 μm. Ascospores allantoid to logical Institute. U.K. suballantoid, yellowish to subolivaceous, 8–10 Cooke MC. 1892 – Handbook of Australian × 3 μm. fungi. Williams and Norgate, London, 1 Cultural characteristic – Colonies white −457. on PDA, covering 85 mm Petri dish after 5 Davidson RW, Lorenz RC. 1938 – Species of days, mycelium later turning grey, beginning Eutypella and Schizoxylon associated from the center of the colony. Conidia filiform, with cankers of . Phytopathology subhyaline, (16−)18−22(−25) × 1−1.5 µm. 28, 733–745. Known distribution – South Australia. Glawe DA, Rogers JD. 1984 – Diatrypaceae in Material examined – AUSTRALIA, the Pacific Northwest. Mycotaxon 20, SOUTH AUSTRALIA, Coorong National Park, 401–460. along Princes Hwy, on dead wood of Acacia Hyde KD. 1993a – Cryptovalsa halosarceicola longifolia subsp. sophorae (Labill.) Court, sp. nov. an intertidal saprotroph of December 2008, holotype: F.P. Trouillas Halosarceia halocnemoides. Mycological CNP03, herb. and ex-type culture = DAR Research 97, 799–800. 80712, GenBank accession no for ITS rDNA Hyde KD, Rappaz F 1993b – Eutypa sequence: HM581945. bathurstensis sp. nov. from intertidal Notes – Because of the taxonomic Avicennia. Mycological Research 97, confusion around Diatrypaceae and the 861–864. difficulty in separating the various genera, the Hyde KD. 1995 – Eutypella naqsii sp. nov. assignment of this isolate to the genus from intertidal Avicennia. Mycological Eutypella may require reconsideration in the Research 99, 1462–1464. future. Based on preliminary phylogenies, this Hinds TE. 1981 – Cryptosphaeria canker and fungus appears best placed in Eutypella. Libertella decay of aspen. Phytopa- thology 71, 1137–1145.

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