Species of Diatrypaceae associated with grapevine trunk diseases in eastern Spain

528 ISSN (print): 0031-9465 www.fupress.com/pm ISSN (online): 1593-2095 © Firenze University Press Phytopathologia Mediterranea (2012) 51, 3, 528−540 Corresponding author: J. Luque Fax: +34 937533954 E-mail: [email protected] Research Papers Species of Diatrypaceae associated with grapevine trunk diseases in Eastern Spain Jordi LUQUE1, Francesc GARCIA-FIGUERES2, Francisco Javier LEGORBURU3, argiñe MURUAMENDIARAZ3, Josep ARMENGOL4 and Florent p. TROUILLAS5 1 Institut de Recerca i Tecnologia Agroalimentàries (IRTA), Ctra. de Cabrils km 2, E-08348 Cabrils, Spain 2 Laboratori de Sanitat Vegetal, Via Circulació Nord, Tram VI, Carrer 3, Zona Franca, E-08040 Barcelona, Spain 3 NEIKER-Tecnalia, Basque Institute for Agriculture Research and Development, Apdo. 46, E-01080 Vitoria/Gasteiz, Spain 4 Instituto Agroforestal Mediterráneo, Universidad Politécnica de Valencia, Camino de Vera s/n, E-46022 Valencia, Spain 5 Department of Plant Pathology, University of California, Davis, California 95616, USA Summary. The presence and diversity of Diatrypaceae species occurring on grapevines in Eastern Spain were investigated. Several species were identified on the basis of morphological characters and phylogenetic analyses of the complete sequence of the internal transcribed spacers of the ribosomal DNA and part of the β-tubulin gene. Five species of Diatrypaceae isolated from the wood of diseased grapevines, pruning debris and/or perithecia were identified, including Anthostoma decipiens, Cryptovalsa ampelina, Eutypa lata, Eutypella citricola and Eutypella microtheca. Additionally, four taxa could not be identified to the species level but were closely related to Eutypa tetragona based on phylogenetic analyses. Eutypa lata was the most prevalent species and showed the greatest degree of genetic diversity. Cryptovalsa ampelina and E. microtheca ranked second in the frequency of isolations, while all the remaining species were less frequently isolated. Eutypella citricola and E. microtheca are reported for the first time as occurring on grapevine in Spain and this is the first report of A. decipiens occurring on grapevine. Key words: Anthostoma, Cryptovalsa, Eutypa, Eutypella, Vitis vinifera, Bayesian inference. Introduction Several fungi in the Diatrypaceae are known to occur on grapevines (Vitis vinifera L.) in many grapegrowing countries, including species in the genera Cryptosphaeria Ces. & De Not., Cryptovalsa Ces. & De Not. ex Fuckel, Diatrype Fr., Diatrypella (Ces. & De Not.) De Not., Eutypa Tul. & C. Tul. and Eutypella (Nitschke) Sacc. (Carter, 1988, 1991; Mostert et al., 2004; Trouillas and Gubler, 2004; Catal et al., 2007; Pitt et al., 2009; Úrbez-Torres et al., 2009; Trouillas and Gubler, 2010a, 2010b; Trouillas et al., 2010, 2011; Úrbez-Torres et al., 2011). To date, 14 known species of this family have been reported from grapevine, while about five taxa are still not assigned to any known species (Farr and Rossman, 2011). Eutypa lata (Pers.) Tul. & C. Tul., the causal agent of Eutypa dieback, is the most important grapevine pathogen known within this family (Carter, 1988, 1991). While identification, pathogenicity, epidemiology and control of E. lata have been thoroughly studied worldwide, the same subjects concerning other diatrypaceous fungi still require further investigation. However, recent reports on the identification and pathogenicity of other diatrypaceous taxa, including Cryptovalsa ampelina (Nitschke) Fuckel (Mostert et al., 2004; Luque et al., 2006; Martín et al., 2009; Trouillas and Gubler, 2010b), Eutypa leptoplaca (Mont.) Rappaz (Trouillas and Gubler, 2004, 2010b), Eutypella vitis (Schwein.) Ellis & Everh. (Catal et al., 2007; Jordan and Schilder, 2007; Úrbez-Torres et al., 2009, 2011), and Cryptosphaeria pullmanensis Glawe (Trouillas and Gubler, 529 Vol. 51, No. 3, December, 2012 Diatrypaceae species on grapevine in Spain 2010b) are contributing to a better knowledge about these fungi and, specifically, the role they play in the grapevine trunk diseases. Recent studies in California and Australia have suggested a possible correlation between infection of grapevines by diatrypaceous species occurring on natural and ornamental host plants in the immediate proximity of the vineyards (Trouillas and Gubler, 2010b; Trouillas et al., 2010, 2011). Grapevine infection could be partly explained by a possible opportunistic lifestyle of these fungi, normally saprobic on their natural host(s) but occasionally pathogenic to other unusual hosts (e.g. grapevine) rendered susceptible due to predisposing factors (Trouillas et al., 2010, 2011). Pathogenicity experiments on V. vinifera vines using various species of Diatrypaceae have been conducted in California vineyards. Results showed that Diatrypaceae were capable of colonizing grapevine wood. However, although some species seemed to produce vascular discolorations, the disease caused by these fungi and their virulence remained unclear (Trouillas and Gubler, 2010b). As suggested by Trouillas and Gubler (2010b), extended incubation periods while conducting pathogenicity tests should be necessary to better characterize disease expression and aggressiveness of these fungi. In vineyards, symptoms caused by E. lata early in the season (i.e. stunted shoot growth with small, cupped and chlorotic leaves) are expressed 3 to 8 years after infection (Carter, 1988). However, in greenhouse assays, symptoms have been reported to occur within 4 weeks (Péros and Berger, 1994), 6 weeks (Jung et al., 2010), 8 months (Sosnowski et al., 2007), or even during the second growing season (Muruamendiaraz and Legorburu, personal communication). The occurrence of Diatrypaceae species on grapevines in Spain is still poorly known. Studies published in the last decade confirmed the existence of only E. lata and C. ampelina. Eutypa dieback was reported for the first time in late 1970’s in Extremadura, Southwestern Spain (Arias and Moral, 1981). Symptoms of this disease were later reported in the mid 1990’s in the Rioja wine region, North Central Spain (Mateo, 1995). Several further studies have shown E. lata to be widely distributed in the grapevine growing regions in Spain (Armengol et al., 2001; Úrbez-Torres and Peláez, 2001; Péros and Berger, 2003; Santiago et al., 2005; Martín and Cobos, 2007; Luque et al., 2009; Muruamendiaraz et al., 2009). It is currently accepted that E. lata is the most common Diatrypaceae species found on grapevine in Spain. Cryptovalsa ampelina was reported for the first time in 2006 in Catalonia, Northeastern Spain, and was found mainly on pruning debris and rarely on standing vines showing symptoms of trunk diseases such as dieback and cankers (Luque et al., 2006). A moderate virulence has been suggested for this fungus (Mostert et al., 2004; Luque et al., 2006). Martín et al. (2009) also reported C. ampelina from the central region of Spain. To date, no other Diatrypaceae species have been reported from grapevines in Spain. Therefore, the present study aimed to determine the presence and diversity of other diatrypaceous fungi occurring on grapevines in this country. Morphological characters, such as colony morphology and conidial dimensions, were used in combination with phylogenetic analyses of the internal transcribed spacers of the rDNA and part of the β-tubulin gene to aid in the characterization and identification of the taxa. Materials and methods Fungal isolates and morphological characters studied The isolates used in this study were obtained from infected shoots, cordons and trunks showing dieback and/or internal wood necroses, or directly from perithecia embedded in the bark of grapevines that were surveyed in Spain during the years 2002‒2010 (Table 1). Wood chips obtained from the necrotic tissues were surface-sterilized (3‒4 min in 70% ethanol), blotted on sterile filter paper to remove excessive ethanol, and plated onto Potato Dextrose Agar (PDA, Difco Laboratories, Detroit, MI, USA) amended with streptomycin sulphate (Sigma-Aldrich Co., St. Louis, MO, USA) at 100 units per ml (PDA-str) as described by Johnston and Booth (1983). Pure cultures of fungi were obtained by isolation of single hyphal tips. Monosporic isolations from perithecia were carried out as follows. Stromata were cut with a sterile blade to reveal the perithecial contents and a drop of sterile water (about 100 μL) was placed on the cut surface of the fruiting bodies. A water drop containing masses of ascospores was then collected with a pipette, plated onto PDA-str and spores were dispersed with a Digralsky spreader. Twenty four hours after plating, a single germinating spore was selected under the microscope and transferred to a fresh PDA Petri dish. Isolates were maintained at 4°C in sterile distilled water for long-term storage. Phytopathologia Mediterranea 530 J. Luque et al. Table 1. Isolates of several Diatrypaceae species obtained from grapevine in different locations of Spain, used in this study. Species Isolate Location Province Isolation year Grapevine variety Source details Anthostoma decipiens JL567 Falset Tarragona 2004 Tempranillo Wood necrosis, arm Cryptovalsa ampelina JL413 Vimbodí Tarragona 2003 Red Grenache Perithecia, on pruning debris Cryptovalsa ampelina JL424 Capçanes Tarragona 2003 Carignane Perithecia, on pruning debris Cryptovalsa ampelina JL476 Bot Tarragona 2003 Macabeo Canker, arm Cryptovalsa ampelina JL717 El Pla del Penedès Barcelona 2009 Cabernet Sauvignon Wood necrosis, living shoot Eutypa lata JL355 Caldes de Montbui Barcelona 2002 Chardonnay V-shaped necrosis, arm Eutypa lata JL399 Pacs del Penedès Barcelona 2003 Cabernet Sauvignon Canker, arm Eutypa lata JL407 Mediona Barcelona 2003 Tempranillo V-shaped necrosis, arm Eutypa lata JL411 Vimbodí Tarragona 2003 Red Grenache V-shaped necrosis, arm Eutypa lata JL427 La Vilella Baixa Tarragona 2003 Cabernet Sauvignon Canker, arm Eutypa lata JL431 Capçanes Tarragona 2003 Carignane V-shaped necrosis, arm Eutypa lata JL432 Òdena Barcelona 2003 Macabeo V-shaped necrosis, arm Eutypa lata JL479 Batea Tarragona 2003 Red Grenache Canker, arm Eutypa lata JL600 Vilajuïga Girona 2005 Tempranillo V-shaped necrosis, arm Eutypa lata JL677 Barbastro Huesca 2007 Cabernet Sauvignon Canker, arm Eutypa lata JL720 Barriobusto Álava 2006 Tempranillo Wood necrosis, arm Eutypa lata JL721 Lanciego Álava 2006 Tempranillo V-shaped necrosis, arm Eutypa lata JL723 Laguardia Álava 2008 Tempranillo V-shaped necrosis, arm Eutypa lata JL725 Bargota Navarra 2008 Tempranillo V-shaped necrosis, arm Eutypa lata JL726 Labastida Álava 2008 Tempranillo V-shaped necrosis, arm Eutypa lata JL727 Navaridas Álava 2007 Tempranillo Perithecia, on arm Eutypa lata JL731 La Morra Burgos 2002 Tempranillo Wood necrosis, unknown part Eutypa lata JL732 Malagón Ciudad Real Unknown Red Grenache Wood necrosis, unknown part Eutypa lata JL739 Albacete Albacete 2010 Unknown Wood necrosis, unknown part Eutypa lata JL740 Yecla Murcia 2009 Unknown Wood necrosis, unknown part Eutypa lata JL741 Villena Alicante 2009 Unknown Wood necrosis, unknown part Eutypa lata JL743 La Cañada La Rioja 2010 Tempranillo Wood necrosis, unknown part Eutypa lata JL744 Azofra La Rioja 2010 Tempranillo Wood necrosis, unknown part (Continued) 531 Vol. 51, No. 3, December, 2012 Diatrypaceae species on grapevine in Spain Sporulation was enhanced by culturing isolates on PDA at 25°C with a 12/12 hour photoperiod, under near UV (Philips TLD 18W/08; Philips Electronics N.V., Amsterdam, Netherlands) and white fluorescent light (Osram L 18W/840; Osram GmbH, Munich, Germany). Isolates that did not sporulate within 3 months of incubation in the above conditions were not used for the measurements of conidia. The mean, standard deviation, 95% confidence intervals and minimum and maximum values were calculated from the measurements made with the ×100 microscope objective of 50 conidia mounted in water. Colony characters of isolates were recorded after growing the fungi on PDA at the above cited conditions for 4 weeks. When possible, fungi were tentatively identified from their colony and conidial morphology by comparing with previous studies (Trouillas and Gubler, 2010b; Trouillas et al., 2010; Trouillas et al., 2011). DNA extraction and sequencing DNA was extracted from the 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 using the universal primers ITS1 and ITS4 (White et al., 1990). Part of the β-tubulin gene was amplified by using the primers Bt2a and Bt2b (Mostert et al., 2006). All reactions were performed on a GeneAmp® PCR System 9700 thermal cycler (PE Applied Biosystems, Foster City, CA, USA), following the temperature profiles described in Luque et al. (2005) for the ITS region, and Mostert et al. (2006) for the β-tubulin gene. Purification of PCR products was according to the methods described in Luque et al. (2005). The purified amplicons were sequenced in both directions using the aforementioned primers and the BigDye™ Terminator v1.1 Cycle Sequencing Kit (Applied Biosystems, Foster City, CA, USA). The resulting fragments were analyzed on an ABI Prism 377 automated DNA sequencer (Perkin Elmer, Norwalk, CT, USA). Sequences were read and edited with BioEdit Sequence Alignment Editor Version 7.0.8 (Hall, 1999). All sequences were checked manually, and nucleotide arrangements at ambiguous positions were clarified using sequences from both strands. Nucleotide sequences obtained in this study were deposited in GenBank (Table 2). Identification of isolates was confirmed by comparing the DNA sequences of the above mentioned regions Species Isolate Location Province Isolation year Grapevine variety Source details Eutypa sp. JL488 Batea Tarragona 2003 Red Grenache V-shaped necrosis, arm Eutypa sp. JL688 Barbastro Huesca 2007 Merlot V-shaped necrosis, arm Eutypa sp.JL690 Barbastro Huesca 2007 Chardonnay V-shaped necrosis, arm Eutypa sp. JL742 Albacete Albacete 2010 Unknown Wood necrosis, unknown part Eutypella citricola JL583 Olèrdola Barcelona 2004 Chenin Blanc Wood necrosis, trunk Eutypella citricola JL734 Murcia Murcia 2009 Unknown Wood necrosis, unknown part Eutypella microtheca JL609 Vilajuïga Girona 2005 Tempranillo Perithecia, on pruning debris Eutypella microtheca JL625 Peralada Girona 2005 Don Mariano Wood necrosis, living shoot Eutypella microtheca JL735 Villanueva de Alcolea Castellón 2009 Unknown Wood necrosis, unknown part Eutypella microtheca JL738 Novelda Alicante 2009 Unknown Wood necrosis, unknown part All isolates available through IRTA (J. Luque). All isolates obtained from infected wood except for Cryptovalsa ampelina JL413 (monosporic), C. ampelina JL424 (monosporic), Eutypa lata JL727 (multisporic), and Eutypella microtheca JL609 (monosporic), which were all obtained from perithecia. Other accession number for selected isolates deposited at Centraalbureau voor Schimmelcultures (CBS), Utrecht, The Netherlands are: JL411, CBS 121487; JL413, CBS 117484; JL424,CBS 117485; JL476, CBS 117486. Table 1. Continues. Phytopathologia Mediterranea 532 J. Luque et al. with those deposited at GenBank, and by combining these sequences in the phylogenetic analyses. Phylogenetic analyses Thirty seven isolates obtained in this study were used in the phylogenetic analyses. Additional sequences corresponding to other Diatrypaceae (26 for the ITS and 25 for the β-tubulin datasets) were obtained from GenBank (Figures 1 and 2) to be included in the analyses. Daldinia concentrica (Bolton) Ces. & De Not. (Xylariaceae) was used as the outgroup (with GenBank accession numbers FJ185300 for ITS and FJ185285 for β-tubulin). Taxa from GenBank were selected to include diatrypaceous species previously reported from grapevine as well as species showing high similarity with our query sequences. DNA sequences of both ITS and β-tubulin datasets were aligned using ClustalW (Thompson et al., 1994). Alignments were verified and adjusted manually using BioEdit. ITS and b-tubulin datasets were combined into a single dataset, which did not include Anthostoma decipiens (DC.) Nitschke because no β-tubulin sequence was available for this species in GenBank. The incongruence-length difference test (ILD) (Farris et al., 1995) or partition homogeneity test (HomPart) was performed in PAUP* ver. 4.0b10 (Swofford, 2002) to determine whether the ITS and btubulin datasets could be combined. Prior to the ILD, uninformative characters were removed and the ILD test was run using a heuristic search and simple addition of taxa for 1,000 random partitions of the data. Prior to Bayesian inference (BI) analyses, the most appropriate nucleotide substitution models were chosen using jModeltest version 0.1.1 (Posada, 2008). BI analyses were carried out using Mr. Bayes version 3.2 (Ronquist and Huelsenbeck, 2003). Four Markov chains were run simultaneously for 1×106 generations, and these were sampled every 100 generations. Data from the first 1,000 generations were discarded as the burn-in period, and after confirming that likelihood values were stabilized prior to the 1,000th generation. The 50% majority rule consensus tree and posterior probability of the tree nodes were calculated from the pooled samples. Results A total of 38 isolates of putative Diatrypaceae species from Spain were used in this study (Table 1). Most of these isolates were obtained from infected wood of diseased vines, whereas two isolates of C. ampelina and one isolate of Eutypella microtheca Trouillas, W.M. Pitt & Gubler were obtained from perithecia occurring on grapevine pruning debris. One isolate of E. lata (JL727) was also obtained from perithecia, but found on old dead wood. Isolates were obtained from typical V-shaped cankers as well as from irregular-shaped necroses. Shapes of cankers associated with the various isolates collected are summarized in Table 1. While 37 isolates were used in the phylogenetic analyses, conidial measurements were obtained for 21 isolates only. These included one isolate of A. decipiens, three of C. ampelina, 12 of E. lata, two of Eutypella citricola Speg., and three of E. microtheca. Production of conidia was not observed for Eutypa sp. isolates JL488, JL688, JL690 and JL742. PCR amplification of the ITS region gave products of approximately 0.6 kb while those of the β-tubulin were about 0.4 kb. The ITS and β-tubulin dataset contained 64 and 63 sequences, respectively, including the outgroup. The ITS matrix consisted of 604 aligned characters and the β-tubulin matrix consisted of 403 characters, including gaps. Results of the ILD test (P<0.05) showed that the ITS and β-tubulin data were incongruent and thus could not be combined. For this reason, two separated BI analyses were carried out and the resulting phylogenetic trees are presented in Figures 1 and 2. The Akaike Information Criterion (AIC) implemented in jModeltest was used to determine the best fitting models for the ITS and β-tubulin datasets, and resulted as GTR+G for the ITS dataset and HKI+I+G for the β-tubulin dataset. The phylogenetic tree of ITS showed five main clades that were supported by posterior probabilities over 95%. These groups included fungi in Cryptovalsa (group 1), Diatrype and Diatrypella (group 2), Eutypella and Anthostoma (group 3), and Eutypa (groups 4 and 5) (Figure 1). On the other hand, the phylogeny of the β-tubulin gene showed four large clades, coinciding with the genera Cryptovalsa (group 1), Diatrype and Diatrypella (group 2), Eutypella (group 3), and Eutypa (group 4), although Eutypa (group 4) only received a posterior probability value of 0.81 (Figure 2). Anthostoma decipiens JL567 was also included in this clade. In addition to C. ampelina and E. lata, already known from grapevine in Spain, phylogenetic analyses also detected E. citricola, E. microtheca and A. decipiens. Species identification of these five taxa was 533 Vol. 51, No. 3, December, 2012 Diatrypaceae species on grapevine in Spain strongly supported by the high sequence similarities with reference sequences from GenBank (99 to 100%) and by the posterior probabilities obtained in the BI analyses (0.97 to 1). The ITS and β-tubulin analyses also distinguished a separate clade of another putative Eutypa sp. that remained unidentified but showed a close relatedness to Eutypa tetragona (Duby) Sacc. (Figures 1 and 2). Several isolates of each species were obtained from different geographic regions (Table 1). Eutypa lata was the diatrypaceous species mostly isolated (23 isolates) and showed the greatest phylogenetic diversity in both genes (Figures 1 and 2). However, the intraspecific diversity of E. lata did not correspond to a relationship of geographical proximity, and intraspecific groups were not consistent across the two DNA phylogenies. ITS and β-tubulin sequences of C. ampelina (4), E. citricola (2), and E. microtheca (4) showed no genetic variation among isolates of each species despite their different geographical origins. Colonies on PDA of the various fungal species were overall quite similar in morphology. However, slight differences could be observed. Colonies of C. ampelina on PDA were white to cream-white, woolly, with diffuse margins, and rapid growth (9 cm in diam. in 4 days at 25°C). The reverse of colonies was first pale-yellow, later (>20 days) developing irregular, mostly central, dark areas. The sporodochiumlike conidiomata consisted of conidiophores aggregated on blackened mycelial crusts, and produced cream coloured conidial masses within 4 weeks. Cultures of E. lata on PDA were variable in morphology: they were white when young (<1 week at 25°C), sometimes turning cream-white with age (4 weeks), and with diffuse margins. Occasionally, production of brownish exudates was detected in some isolates Table 2. Nucleotide sequences of the Spanish isolates of Diatrypaceae deposited in GenBank. Species Isolate ITS β-tubulin Anthostoma decipiens JL567 JN975370 JN975407 Cryptovalsa ampelina JL413 JN975335 JN975371 Cryptovalsa ampelina JL424 AY920391 JN975372 Cryptovalsa ampelina JL476 JN975336 JN975373 Cryptovalsa ampelina JL717 JN975337 JN975374 Eutypa lata JL355 JN975338 JN975375 Eutypa lata JL399 JN975339 JN975376 Eutypa lata JL407 JN975340 JN975377 Eutypa lata JL411 JN975341 JN975378 Eutypa lata JL427 JN975342 JN975379 Eutypa lata JL432 JN975343 JN975380 Eutypa lata JL479 JN975344 JN975381 Eutypa lata JL600 JN975345 JN975382 Eutypa lata JL677 JN975346 JN975383 Eutypa lata JL720 JN975347 JN975384 Eutypa lata JL721 JN975348 JN975385 Eutypa lata JL723 JN975349 JN975386 Eutypa lata JL725 JN975350 JN975387 Eutypa lata JL726 JN975351 JN975388 Species Isolate ITS β-tubulin Eutypa lata JL727 JN975352 JN975389 Eutypa lata JL731 JN975353 JN975390 Eutypa lata JL732 JN975354 JN975391 Eutypa lata JL739 JN975355 JN975392 Eutypa lata JL740 JN975356 JN975393 Eutypa lata JL741 JN975357 JN975394 Eutypa lata JL743 JN975358 JN975395 Eutypa lata JL744 JN975359 JN975396 Eutypa sp. JL488 JN975360 JN975397 Eutypa sp. JL688 JN975361 JN975398 Eutypa sp. JL690 JN975362 JN975399 Eutypa sp. JL742 JN975363 JN975400 Eutypella citricola JL583 JN975364 JN975401 Eutypella citricola JL734 JN975365 JN975402 Eutypella microtheca JL609 JN975366 JN975403 Eutypella microtheca JL625 JN975367 JN975404 Eutypella microtheca JL735 JN975368 JN975405 Eutypella microtheca JL738 JN975369 JN975406 Phytopathologia Mediterranea 534 J. Luque et al.  0.06 JL488Eutypa sp. DQ006923Eutypatetragona DQ006924Eutypaleptoplaca JL609Eutypellamicrotheca JL720Eutypalata JL741Eutypalata DQ006935Eutypalata JL732Eutypalata HQ692540Cryptovalsaampelina JL407Eutypalata HM164735Eutypapetrakii var.petrakii HQ692593Diatrypellavulgaris JL734Eutypellacitricola DQ006928Eutypalata JL479Eutypalata JL476Cryptovalsaampelina HM164726Eutypalata JL726Eutypalata DQ006943Eutypellavitis AY684223Eutypatetragona HQ692620Cryptovalsarabenhorstii DQ006926Eutypamaura JL583Eutypellacitricola JL740Eutypalata HM164718Eutypalata JL725Eutypalata JL739Eutypalata HQ692572Eutypellamicrotheca JL731Eutypalata HQ692568Eutypellamicrotheca FJ185300Daldiniaconcentrica JL413Cryptovalsaampelina GQ293938Diatrypeoregonensis JL399Eutypalata GQ293926Diatrypellaverruciformis JL744Eutypalata DQ006922Eutypalejoplaca JL677Eutypalata JL567Anthostomadecipiens AM399021Anthostomadecipiens GQ293954Diatrypewhitmanensis HM164737 Eutypalaevata JL355Eutypalata JL721Eutypalata AJ302458Eutypaastroidea JL432Eutypalata JL600Eutypalata JL717Cryptovalsaampelina JL743Eutypalata DQ006944Eutypalata JL411Eutypalata JL723Eutypalata JL690 Eutypa sp. JL742Eutypa sp. AJ302457Diatrypeflavovirens JL424Cryptovalsaampelina AY684237Eutypaleptoplaca JL738Eutypellamicrotheca HQ692580Eutypellacitricola JL427Eutypalata JL625Eutypellamicrotheca JL735Eutypellamicrotheca JL727Eutypalata JL688Eutypa sp. 1 0.96 0.91 0.99 0.96 0.87 0.86 0.83 0.97 1 0.96 0.66 1 1 1 1 0.51 1 1 1 0.83 1 1 1 1 0.92 0.88 1 2 4 3 5 Figure 1. Majority rule consensus tree resulting from the Bayesian analysis of the ITS sequence data, with posterior probabilities reported at the nodes. Sequences obtained from GenBank are indicated by their accession numbers while isolates obtained in this study are indicated by their code numbers. Clades are numbered in circles. Bars represent expected changes per site. 535 Vol. 51, No. 3, December, 2012 Diatrypaceae species on grapevine in Spain  0.07 JL738Eutypellamicrotheca HQ692470Cryptovalsaampelina JL734Eutypellacitricola JL355Eutypalata HM164760Eutypalata JL742Eutypa sp. JL413Cryptovalsaampelina JL583Eutypellacitricola FJ185285Daldiniaconcentrica JL717Cryptovalsaampelina DQ006992Eutypalata JL424Cryptovalsaampelina HQ692520Eutypellacitricola JL688Eutypa sp. HM164769Eutypapetrakii var.petrakii GQ293991Diatrypellaverruciformis JL677Eutypalata JL740Eutypalata JL411Eutypalata HQ692526Eutypellamicrotheca DQ006999Eutypellavitis JL732Eutypalata JL739Eutypalata JL720Eutypalata JL479Eutypalata AY684202Eutypatetragona JL723Eutypalata JL731Eutypalata DQ006960Eutypatetragona DQ007000Eutypalata JL600Eutypalata AY684212Eutypaleptoplaca GQ293997Diatrypeoregonensis JL609Eutypellamicrotheca JL726Eutypalata GQ294010Diatrypewhitmanensis JL625Eutypellamicrotheca DQ006966Eutypaastroidea HM164771Eutypalaevata JL690Eutypa sp. DQ006974Eutypalejoplaca DQ006961Eutypaleptoplaca JL407Eutypalata JL399Eutypalata JL427Eutypalata JL725Eutypalata DQ006959Diatrypeflavovirens AY684198Eutypamaura JL741Eutypalata JL744Eutypalata JL743Eutypalata JL567Anthostomadecipiens HQ692504Diatrypellavulgaris JL727Eutypalata HM164752Eutypalata HQ692522Cryptovalsarabenhorstii JL432Eutypalata DQ006973Eutypalata JL721Eutypalata JL476Cryptovalsaampelina JL735Eutypellamicrotheca JL488Eutypa sp. HQ692536Eutypellamicrotheca 0.63 0.99 0.71 1 0.99 0.86 0.91 1 0.98 1 0.82 0.93 1 0.81 0.89 1 1 0.87 1 0.88 1 1 1 0.59 1 1 0.95 0.96 1 0.71 1 1 2 3 4 Figure 2. Majority rule consensus tree resulting from the Bayesian analysis of the β-tubulin sequence data, with posterior probabilities reported at the nodes. Sequences obtained from GenBank are indicated by their accession numbers while isolates obtained in this study are indicated by their code numbers. Clades are numbered in circles. Bars represents expected changes per site. Phytopathologia Mediterranea 536 J. Luque et al. (JL720, JL726, JL741). The reverse of colonies was first white, later (>20 days) pale-yellow to creamy in most isolates, and cultures producing exudates each exhibited a brown reverse after 4 weeks. Most isolates developed blackened, circular areas, which coalesced with time (over 4 weeks) to each show a blackish colony reverse for some isolates. Production of conidiomata and conidia were only observed in half of the studied isolates. Cultures of E. citricola on PDA were white, with low dense aerial mycelium and woolly mycelium aggregates, sometimes turning to pale cream (3 weeks). Blackened, circular areas bearing sporodochia were seen occasionally on the colony surfaces. Cultures of E. microtheca on PDA were similar to those of E. citricola but developing a light pinkish-orange colour in the central parts of the colonies in some isolates (JL625, JL735), as reported by Trouillas et al. (2011). The culture of A. decipiens JL567 on PDA was white to cream-white and woolly, with a moderate growth (9 cm in diam. in 7 days at 25°C). The reverse of the colony was first pale yellow, later (>2 weeks) creamy, with irregular blackened areas. Old colonies (4 weeks) turned to dark grey. Colonies of isolates JL488, JL688, JL690 and JL742 on PDA were overall similar, however slight differences of morphology could be observed. All colonies appeared white initially, with little aerial mycelium, but rapidly (about 3 days) turned into a pale-cream colour. After one week, isolates JL688 and JL690 developed dense, cottony mycelium with moderate aerial growth, while mycelium in JL488 and JL742 remained almost appressed to the culture medium. Reverse colour of the colonies were first white (<7 d), turning pale yellow in isolates JL488 and JL742 after 10 d while isolates JL688 and JL690 remained white. Nevertheless, 4-week-old cultures were similar in colour for all isolates, mostly pale-cream in both sides. Isolates JL688 and 690 (approx. 9 cm in diam. after 7 d) grew a little faster than JL488 and JL742 (approx. 9 cm in diam. after 12 d). Production of conidiomata and conidia were not observed in any of these Eutypa sp. isolates in two independent assays. Conidial dimensions of representative isolates of all species, except for the unknown Eutypa sp., which did not sporulate, are shown in Table 3. Conidia of A. decipiens were lunate, (7)‒9‒ (11) × (1) ‒1.05‒ (1.1) μm, and were the smallest conidia of the studied isolates. Conidia of the remaining species were similar in form (curved at the obtuse end and straight at the truncate base) and with overlapping lengths, which did not allow for species differentiation based on conidial form. Mean length of conidia for C. ampelina ranged from 21.5 μm to 24.2 μm, while a great phenotypic variation of mean conidium length was detected among E. lata isolates, which ranged from 21.9 μm (isolate JL723) to greater than 34 μm (JL427 and JL 431) (Table 3). Mean length of conidia for E. citricola ranged from 14.7 μm to 15.6 μm, which overlapped with the corresponding range in E. microtheca (15.2 μm to 15.9 μm). Mean widths of conidia for nearly all isolates ranged from 1.0 μm to 1.5 μm (Table 3). Discussion This study has confirmed the occurrence of five species of Diatrypaceae associated with grapevines in Eastern Spain, namely Anthostoma decipiens, Cryptovalsa ampelina, Eutypa lata, Eutypella citricola and Eutypella microtheca. Additionally, four unidentified isolates of a Eutypa closely related to E. tetragona were detected on the basis of the phylogenetic analyses. However, we did not consider introducing new species names for these isolates as none of them (JL488, JL688, JL690 and JL742) produced either conidia in vitro or sexual reproductive structures required for the description of new fungal species. Furthermore, the ITS and β-tubulin phylogenetic analyses showed incongruent branch topologies for these isolates, which did not allow us to definitely conclude if multiple Eutypa spp. occurred within this clade. Despite the incongruence found between the ITS and β-tubulin phylogenies, both trees inferred through Bayesian methods showed that species identification of our isolates was consistent among the estimated phylogenies. However, the position of A. decipiens still remains unclear. While this taxon was a sister clade to Eutypella in the ITS tree, it was included in the large clade of Eutypa in the β-tubulin tree. Regarding the new putative Eutypa sp. isolates, morphological observations were in accordance with the groupings observed in the ITS and the β-tubulin trees; thus, JL688 and JL690 showed similar characteristics (i.e. colony colour and growth) to JL488 and JL742. In addition, these isolates that were closely related to E. tetragona could in turn be distant relatives of E. leptoplaca. While C. ampelina and E. lata were already known in Spain from previous studies (Arias and Moral, 1981; Armengol et al., 2001; Santiago et al., 2005; Luque et al., 2006; Martín and Cobos, 2007; Martín et al., 2009; Muruamendiaraz et al., 2009), A. decipi-