Epiphytic fungal community in Vitis vinifera of the Portuguese wine regions

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1 Letters in Applied Microbiology ISSN ORIGINAL ARTICLE Epiphytic fungal community in Vitis vinifera of the Portuguese wine regions M. Oliveira 1,2,3, M. Arenas 1,2,4, O. Lage 3, M. Cunha 5 and M.I. Amorim 3,6 1 i3s - Instituto de Investigacß~ao e Inovacß~ao em Saude, Universidade do Porto, Porto, Portugal 2 IPATIMUP - Institute of Molecular Pathology and Immunology, University of Porto, Porto, Portugal 3 Departamento de Biologia, Faculdade de Ci^encias, Universidade do Porto, Porto, Portugal 4 Department of Biochemistry, Genetics and Immunology, University of Vigo, Vigo, Spain 5 Departamento de Geoci^encias, Ambiente e Ordenamento do Territorio, Faculdade de Ci^encias, Universidade do Porto, Porto, Portugal 6 BioISI - Plant Functional Genomics Group, Biosystems and Integrative Sciences Institute, Porto, Portugal Significance and Impact of Study: The knowledge on the composition of the phyllosphere microbial community is still limited, especially when fungi are concerned. These micro-organisms not only play a crucial role in crop health and productivity but also interact with the winemaking process, determining the safety and quality of grape and grape-derived products. The elucidation of the micro-organisms present in the phyllosphere will have a notorious impact on plant breeding and protection programmes and disease management strategies, allowing a better control of pesticide applications. Keywords agriculture, diversity, environmental samples, fungal diseases, Vitis vinifera. Correspondence Manuela Oliveira, IPATIMUP - Institute of Molecular Pathology and Immunology, University of Porto, Rua Julio Amaral de Carvalho; Porto, Portugal /0250: received 7 February 2017, revised 7 June 2017 and accepted 2 November 2017 doi: /lam Abstract In this work, fungi present in the grapevine s phyllosphere collected from the main demarcated wine regions of Portugal were identified, and their phylogenetic relationships were analysed. A total of 46 vine samples (leaves and berries) were collected from different parts of the country, being isolated a total of 117 fungal colonies that were identified to the genus level and sequenced in the following genetic regions: internal transcribed spacer region and 18S rrna and b-tubulin gene. Next, a phylogenetic tree reconstruction for each genetic region was built. The isolates retrieved from environmental samples belonged to the genera Alternaria (31%), Cladosporium (21%), Penicillium (19%), Aspergillus (7%) and Epicoccum (3%). No genetic signatures of exchange of genetic material were detected, and consequently, the reconstructed phylogenetic trees allowed to distinguish between these different species/genera. In the fungal composition of the Vitis vinifera phyllosphere, several potential pathogens were identified that can be associated with decreases in crop productivity. Knowledge of fungi identification and genetic diversity is pivotal for the development of more adequate crop management strategies. Furthermore, this information will provide guidelines for a more specific and wiser use of fungicides. Introduction The phyllosphere is a diverse habitat for epiphytic microorganisms (Lindow and Brandl 2003). Some of these micro-organisms are deleterious whereas others are beneficial (Lindow and Brandl 2003). Although present throughout the year, these micro-organisms exhibit a seasonal succession (Blakeman 1993). Microbial populations are also unequally distributed on leaf surfaces (Allen et al. 1991; Kinkel 1997). Several sources of phyllosphere epiphytic filamentous fungi have been proposed. These micro-organisms may arrive at the plant surface from the atmosphere, water, soil, insect, seed or even from animal-borne sources (Whipps et al. 2008; Martins et al. 2013). However, not all micro-organisms that reach the phyllosphere are Letters in Applied Microbiology 66, The Society for Applied Microbiology 93

2 Grapevine phyllosphere community M. Oliveira et al. capable of establishment and colonization. The ecosystem equilibrium depends on several environmental, physicochemical and genetic characteristics of the plant, and specific properties displayed by the phyllosphere microbiome, that may alter the structure and diversity of the microbial community (Whipps et al. 2008). For instance, fungal migration behaviour is influenced by its atmospheric composition that is in constant modification due to seasonal and diurnal patterns, which are conditioned by meteorological events, such as temperature, rainfall and relative humidity (Oliveira et al. 2009b,c). The accurate identification of fungal species is critical to understand the ecological roles, distribution and economic impact of the fungal communities present in many major crops, such as vineyards. Less than 10% of an estimated 15 million fungal species have been described (Hawsworth 2001; Mueller and Schmit 2007), which constitutes a major constraint to the understanding of both fungal diversity and ecology. Morphological identification of closely related fungal species requires time to grow the fungal culture and expertise to distinguish the limited and ambiguous diagnostic characters. Moreover, morphologybased identifications are not possible for several species that cannot be cultured. Furthermore, the taxonomy of some filamentous fungal genera is frequently subjected to revisions and modifications (Sim~oes et al. 2013). To overcome these limitations, the use of molecular tools is becoming increasingly popular for fungal species identification, allowing the application of genealogical concordance through phylogenetic species recognition. These tools are frequently characterized as being accurate and culture-independent (Robideau et al. 2011). In vineyards, leaves and berries are colonized by a broad range of epiphytic micro-organisms that play a significant role in crop health and productivity. Also, these micro-organisms interact with the winemaking process and determine the safety and quality of grape or grapederived products for human consumption (Whipps et al. 2008; Fernandez-Gonzalez et al. 2012; Martins et al. 2014). Vine plants are colonized by a broad range of fungal pathogens that negatively affect grapevines, causing blighting, shrivelling, vine decay and tissue damage. Among these pathogens are Plamospara viticola (downy mildew), Erysiphe necator (powdery mildew) and Botrytis cinerea (grey rot) (Fernandez-Gonzales et al. 2009; Oliveira et al. 2009a, 2015; Rodrıguez-Rajo et al. 2010; Fernandez-Gonzalez et al. 2012). Moreover, some moulds produce toxic secondary metabolites, such as mycotoxins (e.g. ochratoxin A produced by Aspergillus spp. and Penicillium spp.) that persist in wine. Oppositely, other micro-organisms are able to activate the plant defense pathways, inducing the accumulation of pathogenesisrelated (PR) proteins of grapevine acting as protection against fungal pathogen attack or other biological stresses. Some of these PR proteins, such as chitinases and taumatin-like proteins, are known to alter wine clarity and stability (Pinto et al. 2014). In the last years, high-throughput sequencing allowed to gain a deeper knowledge of the microbial composition of the phyllosphere (Bokulich et al. 2014, 2016; Gilbert et al. 2014; Pinto et al. 2014; del Carmen et al. 2016; Kecskemeti et al. 2016; Yang et al. 2016). Epiphytic fungi and bacteria play an important role in both grape health and quality. The presence of these micro-organisms is determined by a wide range of factors, such as the winegrowing region (biogeography), grape variety, and the interannual vineyard and climate variation (vintage) across a given viticultural region. All these aspects truly constitute a nonrandom microbial terroir with impacts on both grape and wine quality (Bokulich et al. 2014; Gilbert et al. 2014). Elucidation of the microbial composition of the phyllosphere will have applications in plant breeding and plant protection programmes, coordinating the development, selection and application of effective control measures against important vine pathogens (Vorholt 2012; Kecskemeti et al. 2016). The majority of the available studies are focused on the bacterial component of these communities. However, fungi are also important because they play crucial roles in the decomposition of structural components, such as lignin and cellulose, and in nutrient cycling (Santamarıa and Bayman 2005; Osono 2006). Moreover, fungi could deeply affect plant function, health, and development, through a wide range of interactions acting as pathogens or as biocontrol agents to shape the microbial community dynamics of the bacteria present on the leaves (Peay et al. 2008). In the present work, the epiphytic fungal communities present in vineyards phyllosphere from different demarcated wine regions of Portugal is described, using a culture-dependent approach with subsequent morphological and molecular characterization of the isolates obtained. Results and discussion The composition of Vitis vinifera phyllosphere was assessed using leaves and berries from different cultivars collected from the main Portuguese Demarcated Wine Regions (Table 1, Figs S1 and S2), from September to December 2013, which corresponds to the season when spores start to germinate and form colonies (Blakeman 1993). Due to the highest area of vineyards and the diversity of cultivars, the Douro region contributed to the majority (49%) of the samples analysed. A total of 117 fungal isolates were obtained in pure culture and identified using their distinctive macroscopic 94 Letters in Applied Microbiology 66, The Society for Applied Microbiology

3 M. Oliveira et al. Grapevine phyllosphere community morphological characteristics. These isolates were Alternaria spp. (31%), Cladosporium spp. (21%), Penicillium spp. (19%), Aspergillus spp. (7%), Epicoccum spp. (3%) and nonidentified vegetative mycelia (20%) (Figure S3). Using molecular markers on a selected subset of the fungal isolates (72 isolates), a total of 20 different operational taxonomic units (OTUs) were identified. Penicillium accounted for a total of eight OTUs (Penicillium brevicompactum, Penicillium chrysogenum, Penicillium commune, Penicillium crustosum, Penicillium expansum, Penicillium glabrum, Penicillium oxalicum and Penicillium spinolossum). Alternaria genus included Alternaria alternata and Alternaria tenuissima. All fungal isolates belonging to the Cladosporium genus were identified as Cladosporium cladosporioides. Additionally, Epicoccum nigrum, Fusarium chlamydosporum, Paecilomyces variotii, Phoma herbarum, Rhizopus stolonifer and Ulocladium consortiale were also identified (Figs 1 3, Table S1). No signatures of genetic recombination were detected in the studied genetic regions, suggesting that the observed genetic diversity was generated through mutation events. Table 1 Location of samples analysed in this study Type of sample Wine region Location Cultivars Berries and Leaves Alentejo Cuba Alicante Bouschet Evora Aragonez Mertola Chardonnay Pias Trincadeira Moura Not identified Serpa Chardonnay Bairrada Anadia Fern~ao Pires D~ao Lous~a Cerceal Tondela Cord~oes Vila Nova Not identified de Poiares Douro Murcßa Cerceal Pinh~ao Codega de Olarinho Regua Gouveio Tinta Roeiz Touriga Franca Touriga Nacional Viosinho Penınsula Azeit~ao Aragonez de Setubal Vinhos Verdes Amarante Alvarinho Barcelos Arinto Fafe Azal Branco Felgueiras Borracßal Guimar~aes Loureiro Melgacßo Malvasia Moncß~ao Pedren~a Nogueira Trajadura Roriz Vinh~ao The 18S rdna sequences were undoubtedly useful for the identification at the genus level, but amplification of the ITS region allowed the identification at the species level. In Penicillium spp., the use of the ITS markers provided a finer species definition, allowing the separation between P. spinolossum and P. glabrum as well as the separation between P. expansum, P. crustosum and P. commune, and P. chrysogenum. The discrimination between P. crustosum and P. commune was only achieved using b-tubulin sequences. Moreover, in Aspergillus spp. the ITS markers allowed the separation between A. niger and A. carbonarius (being this separation confirmed using b-tubulin sequences). Finally, in Alternaria spp. the ITS markers allowed the separation between A. alternata and A. tenuissima. Isolate Fungus_29 was attributed to both Phoma spp. by the 18S rrna gene, and to Epicoccum spp. by ITS marker, highlighting the close phylogenetic relation between these two genera. In fact, it has been suggested that these fungal types belong to the same biological species (Arenal et al. 2000), corresponding to two different sexual states (anamorph and teleomorph). The use of the 18S rrna gene, the ITS region and the b-tubulin sequences allowed a much better and in-depth analysis of the phylogenetic relationship of the isolates. The obtained morphological diversity did not correspond to an elevated number of identified OUTs. This apparent lack of molecular diversity might be due to the presence of species complexes or species groups. Species complexes correspond to groups of organisms that belong to different species but exhibit similar characteristics, such as morphology, physiology and/or other phenotypic features (Almeida and Araujo 2013; Chen et al. 2016). Some authors suggested that A. alternata (Roberts et al. 2000; De Hoog and Horre 2002; Andrew et al. 2009), C. cladosporioides (Bensch et al. 2010), A. niger (Howard et al. 2011) and Penicillium subgenus Penicillium (Skouboe et al. 1999) are representative of species complexes and that consist of several heterogeneous species. Considerable controversy exists about which variants represent distinct species and which represent population variation within species (Andrew et al. 2009). To date, only an endopolygalacturonase gene and two nonidentified loci have proven to be sufficiently variable to distinguish between members of the A. alternata species group (Peever et al. 1999) while in the case of Penicillium the b-tubulin, the calmodulin and elongation factor 1-a genes were used (Peterson 2004; Wang and Zhuang 2007)). According to the Worldwide Bioclimatic Classification System (WBCS), the Portuguese territory is divided into two bioclimatic regions: the Eurosiberian subregion including the hyperhumid temperate mountains in the north-west and the Mediterranean subregion which Letters in Applied Microbiology 66, The Society for Applied Microbiology 95

4 Grapevine phyllosphere community M. Oliveira et al. L_giganteum* 18S_Fungus_069 P_commune* 18S_Fungus_072C 18S_Fungus_022 18S_Fungus_011 18S_Fungus_071B P_crustosum* 18S_Fungus_046 P_chrysogenum* 18S_Fungus_045 18S_Fungus_048 18S_Fungus_042 18S_Fungus_069B 18S_Fungus_001 P_brevicompactum* 18S_Fungus_074 P_oxalicum* 18S_Fungus_096 18S_Fungus_087 18S_Fungus_092 P_glabrum* P_spinolosum* 18S_Fungus_080 18S_Fungus_082 18S_Fungus_081 A_niger* A_tamarii* 18S_Fungus_067 18S_Fungus_076 18S_Fungus_033 P_variotii* 18S_Fungus_023 18S_Fungus_056 18S_Fungus_078 18S_Fungus_079 C_oxysporum* 18S_Fungus_024 18S_Fungus_070 C_bruhnei* 18S_Fungus_009 18S_Fungus_038 18S_Fungus_095 18S_Fungus_090 18S_Fungus_015 18S_Fungus_039 F_equiseti* 18S_Fungus_016 18S_Fungus_089 18S_Fungus_099 P_herbarum* 18S_Fungus_100 18S_Fungus_098 18S_Fungus_101 E_nigrum 18S_Fungus_029 18S_Fungus_055 18S_Fungus_032 18S_Fungus_034 18S_Fungus_040 18S_Fungus_013 18S_Fungus_054B 18S_Fungus_041 18S_Fungus_061B A_alternata* 18S_Fungus_026 18S_Fungus_071C 18S_Fungus_085 18S_Fungus_017 18S_Fungus_058 18S_Fungus_052 18S_Fungus_005 U_botrytis* 18S_Fungus_065 18S_Fungus_068 18S_Fungus_012 18S_Fungus_019 18S_Fungus_072 18S_Fungus_072B R_stolonifer* 0.03 Figure 1 Evolutionary relationships of taxa (representative isolate of each taxon), using 18S rrna gene in DNA samples extracted from leaves of diverse Vitis vinifera cultivars collected in the main Portuguese demarcated wine regions. The evolutionary history was inferred with NJ. [Colour figure can be viewed at wileyonlinelibrary.com] includes summer-dry areas (Mesquita and Sousa 2009). In our work, in the northern region of Portugal, two different patterns of fungal genera distribution were observed. In the Vinhos Verdes Demarcated Wine Regions, explicitly included in the Eurosiberian subregion, a prevalence of wet weather spores, such as Aspergillus and Penicillium, was observed (Oliveira et al. 2009b) over the dry weather spores, such as Alternaria, Cladosporium, and Epicoccum (Oliveira et al. 2009b). Contrary, in the adjacent Douro region and the southern part of the country (comprising the Penınsula de Setubal and the Alentejo Demarcated Wine Regions), clearly included in the Mediterranean subregion, a prevalence of dry weather spores was registered. Finally, the central region of Portugal, including the Bairrada and D~ao Demarcated Wine Regions, considered as the transition between the two bioclimatic subregions (Costa et al. 1999), an almost similar presence of both spore types was observed (Table S2). Studies on V. vinifera phyllosphere fungal composition, using both culture-dependent and culture-independent 96 Letters in Applied Microbiology 66, The Society for Applied Microbiology

5 M. Oliveira et al. Grapevine phyllosphere community ITS_Fungus_042 ITS_Fungus_049 P_expansum* P_crustosum* P_commune* ITS_Fungus_072D ITS_Fungus_069 ITS_Fungus_046 ITS_Fungus_023 ITS_Fungus_022 ITS_Fungus_021 ITS_Fungus_071B ITS_Fungus_069B ITS_Fungus_011 ITS_Fungus_048 P_chrysogenum* P_brevicompactum* ITS_Fungus_074 P_oxalicum* P_spinolossum* ITS_Fungus_096 ITS_Fungus_087 ITS_Fungus_092 P_glabrum* ITS_Fungus_080 A_niger* ITS_Fungus_077 A_carbonarius* ITS_Fungus_081 ITS_Fungus_082 ITS_Fungus_033 P_variotii* ITS_Fungus_009 ITS_Fungus_079 ITS_Fungus_015 C_oxysporum* ITS_Fungus_002 ITS_FungusS_007 ITS_Fungus_016 ITS_Fungus_051B ITS_Fungus_090 ITS_Fungus_095 C_cladosporioides* ITS_Fungus_024 ITS_Fungus_078 ITS_Fungus_051 ITS_Fungus_059B ITS_Fungus_056 ITS_Fungus_070 ITS_Fungus_100 ITS_Fungus_101 ITS_Fungus_099 ITS_Fungus_098 ITS_Fungus_029 E_nigrum* ITS_Fungus_089 P_herbarum* ITS_Fungus_005 U_consortiale* ITS_Fungus_013 ITS_Fungus_034 ITS_Fungus_040 ITS_Fungus_003 ITS_Fungus_052 ITS_Fungus_071C A_alternata* ITS_Fungus_072 ITS_Fungus_017 ITS_Fungus_068 ITS_Fungus_085 ITS_Fungus_032 ITS_Fungus_012 A_tenuissima* ITS_Fungus_065 ITS_Fungus_057B ITS_Fungus_055 ITS_Fungus_018 ITS_Fungus_037 ITS_Fungus_019 ITS_Fungus_041 ITS_Fungus_058 ITS_Fungus_061B ITS_Fungus_054B ITS_Fungus_093B ITS_Fungus_075 R_srolonifer* L_giganteum* ITS_Fungus_001 F_chlamydosporum* ITS_Fungus_086 ITS_Fungus_092B 0.2 Figure 2 Evolutionary relationships of taxa (representative isolate of each taxon), using ITS region in DNA samples extracted from leaves of diverse Vitis vinifera cultivars collected in the main Portuguese demarcated wine regions. The evolutionary history was inferred with NJ. [Colour figure can be viewed at wileyonlinelibrary.com] techniques, also reported the Ascomycota phylum as the primary component of this particular microenvironment (Serra et al. 2005; Pinto et al. 2014). Furthermore, previous works that analysed grapes from four Portuguese demarcated wine regions at three different phenological phases identified fungal strains, being Cladosporium (25%), Alternaria (24%), Botrytis (15%), Penicillium (9%), and Aspergillus (8%) the most frequent genera (Serra et al. 2005). Among these, 920% of the strains were nonmycotoxigenic nor produced mycotoxins of known relevance. The most common mycotoxigenic species isolated were ochratoxin A potential producers (54% A. niger aggregate and 06% A. carbonarius) and trichothecenes (04% Fusarium spp. and 08% Trichothecium roseum) (Serra et al. 2005). Another study that analysed 199 strains of filamentous fungi isolated from various vineyards in Burgundy (France) identified Penicillium (585%) as the most frequently isolated strain, being P. spinolusum the most commonly found (227%), whereas no strains of Aspergillus were isolated (Diguta et al. 2011). The same authors also isolated C. cladosporioides, B. cinerea, E. nigrum, A. alternata, Trichoderma koningiopsis, P. diplodiella, C. herbarum, Acremonium alternatum, Thanatephorus cucumeris, and F. oxysporum. The same work reports that a single PCR is unable to distinguish between some species within a genus, as in the Letters in Applied Microbiology 66, The Society for Applied Microbiology 97

6 Grapevine phyllosphere community M. Oliveira et al. R._stolonifer TUB_Fungus_75 L._giganteum TUB_Fungus_07 TUB_Fungus_02 TUB_Fungus_09 TUB_Fungus_15 TUB_Fungus_24 TUB_Fungus_28 C._oxysporum TUB_Fungus_56 TUB_Fungus_41 TUB_Fungus_48 P._crustosum TUB_Fungus_71B TUB_Fungus_45 TUB_Fungus_22 TUB_Fungus_34 TUB_Fungus_46 TUB_Fungus_72C TUB_Fungus_21 TUB_Fungus_23 TUB_Fungus_40 TUB_Fungus_47 TUB_Fungus_42 TUB_Fungus_49 P._commune P._chrysogenum P._brevicompactum TUB_Fungus_01 TUB_Fungus_29 P._variotii TUB_Fungus_33 F._equiseti TUB_Fungus_86 F._incarnatum E._nigrum P._herbarum A._alternata A._arborescens U._consortiale U._botrytis TUB_Fungus_05 TUB_Fungus_76 TUB_Fungus_77 A._niger A._carbonarius TUB_Fungus_ Figure 3 Evolutionary relationships of taxa (representative isolate of each taxon), using b-tubulin gene in DNA samples extracted from leaves of diverse Vitis vinifera cultivars collected in the main Portuguese demarcated wine regions. The evolutionary history was inferred with NJ. [Colour figure can be viewed at wileyonlinelibrary.com] case of Penicillium, due to a sequence identity of the amplified region (90-99%) (Diguta et al. 2011). The results herein presented point out to the existence of regional patterns of occurrence of different fungal types present in V. vinifera phyllosphere. As such, the Vinhos Verdes Region was characterized by the presence of Aspergillus and Penicillium fungal spores. Contrarily, in the Douro, Penınsula de Setubal and Alentejo regions, Alternaria, Cladosporium and Epicoccum were the main fungal spores present. Bairrada and D~ao Regions showed a similar presence of all spore types. All these fungal types are associated with different impacts on both grapevine health and wine quality. Knowledge of the microbial composition of the phyllosphere will lead to more effective control measures. Moreover, this study permits to infer about the intricate connections between the phyllosphere and the surrounding environment, pointing out to the need of future studies for a better characterization of V. vinifera phyllosphere, using alternative culture-independent techniques, such as the combined metagenomics profiles of the atmosphere, soil and phyllosphere. Material and methods Sampling This study was conducted in the Portuguese demarcated wine regions of Alentejo (15% of the samples), Bairrada (9%), D~ao (9%), Douro (49%), Penınsula de Setubal (7%) and Vinhos Verdes (13%). In these regions, from September to December of 2013, ten leaves and two bunches of grapes from Vitis vinifera cultivars were aseptically collected from the top part of the plant (Table 1, Figure S1). Growth of fungal cultures and morphological observations For the isolation of fungal colonies, leaves and berries were placed in contact with Sabouraud dextrose agar (SDA; Diagnostici Liofilchem) medium and removed after 2 min. Cultures were grown at 25 C for 3 days (Figure S2). Colonies were isolated and identified to the 98 Letters in Applied Microbiology 66, The Society for Applied Microbiology

7 M. Oliveira et al. Grapevine phyllosphere community genus level using distinctive macroscopic and microscopic morphological features. Monospore isolates were cultured in SDA medium, at 25 C, for 5 days (Figure S3). DNA extraction, PCR and DNA sequencing Conidia harvested with a cytobrush were placed in TE buffer (10 mmol l 1 Tris-HCl, 1 mmol l 1 EDTA, ph 80) and cell concentration adjusted to 10 6 to 10 8 conidia per ml, by cell counting in a Neubauer chamber. DNA was extracted from spores as previously described (Oliveira et al. 2010, 2014). For gene amplification and sequencing, fungal universal primers were used: (1) internal transcribed spacer region 1, the 58S rrna gene and ITS region 2 were amplified using the primer pair ITS1 and ITS4 (Vilgalys et al. 1994); (2) 18S rrna gene region using the primers NS1 and NS4 (White et al. 1990); and (3) b-tubulin gene using the primers Bt2a and Bt2b (Glass and Donaldson 1995). PCR amplifications were conducted in a final volume of 50 ll, consisting of 25 ll of My Taq TM HS MIX (Bioline; London, UK), 10 ll of primer mix, 10 ll of fungal DNA and 05 ll of ultrapure water (Qiagen; Hilden, Germany). PCRs were run at the following conditions: initial denaturation at 95 C for 10 min, followed by 35 cycles of denaturation at 95 C for 1min, annealing at C for 1 min, and extension at 72 C for 1 min, followed by a final extension at 72 C for 10 min. Amplification products were purified with a mixture consisting of Exonuclease I (Thermo Scientific; Waltham, MA, USA) and FastaAP (1 : 5) (Thermo Scientific; Waltham, MA, USA), according to manufacturer s instructions. Sequencing reactions were run at the following conditions: initial denaturation at 96 C for 2 min, followed by 35 cycles of denaturation at 96 C for 15 s, annealing at 50 C for 9 s, and extension at 60 C for 2 min, followed by a final extension at 60 C for 10 min. Samples were amplified using both forward and reverse primers. Sequencing data were processed and analysed with Sequencing 5.2 analysis software (Applied Biosystems; Foster City, CA, USA). Evolutionary analyses The sequences obtained were aligned using MAFFT (Katoh and Standley 2013) and refined [noninformative positions (assumed as gaps in more than 80% of the sequences) were trimmed] with trimal (Capella-Gutierrez et al. 2009). The best-fitted substitution model was selected with jmodeltest2 (Darriba et al. 2012) under the Bayesian information criterion (BIC) (Luo et al. 2010). The lack of signature of genetic recombination was identified with GARD (Pond et al. 2006), allowing the inference of a phylogenetic tree for every genetic region under study (Schierup and Hein 2000; Arenas and Posada 2010; Mallo et al. 2015). Neighbour-joining (NJ) and maximum-likelihood (ML) phylogenetic trees were reconstructed with MEGA (Tamura et al. 2013) and PhyML (Guindon et al. 2010), including statistical support for the internal nodes by a bootstrap analysis (Felsenstein 1985). Sequences were identified by similarity searches against other sequences available from GenBank with the BLASTn search algorithm (Table S1). Acknowledgments Research supported by the Reitoria da Universidade do Porto (PP_IJUP2011_54), the European Regional Development Fund (ERDF) through the COMPETE Operational Competitiveness Programme, and national funds through the Fundacß~ao para a Ci^encia e Tecnologia (FCT) under projects PEst-C/MAR/LA0015/2013 and PEst-C/ SAU/LA0003/2013. MO (SFRH/BPD/66071/2009) and MA (IF/00955/2014) are supported by FCT fellowships funded by POPH-QREN Promotion of Scientific Employment, the European Social Fund, and National Funds of the Ministry of Education and Science. MA was also supported by the Spanish Government (RYC ). The authors kindly acknowledge A. Fernandes, C. Calheiros, C. Martins, J. Bondoso, L. Fortes, N. Nogueira, and P. Gracßa (FCUP), J. Guerner Moreira (DRAPN), J. Sofia and M. Neves (DRAPC), R. Amador, M. C. Val, and C. Carlos (ADVID), F. Mata (ATEVA), and L. Mendes (AVIP) for their assistance. Conflict of Interest The authors have no conflict of interest to declare. References Allen, E.A., Hoch, H.C., Steadman, J.R. and Stavely, R.J. (1991) Influence of leaf surface features on spore deposition and the epiphytic growth of phytopathogenic fungi. In Microbial ecology of leaves ed. Andrews, J.H. and Hirano, S.S. pp New York, NY: Springer. Almeida, L.A. and Araujo, R. (2013) Highlights on molecular identification of closely related species. Infect Genet Evol 13, Andrew, M., Peever, T. and Pryor, B. (2009) An expanded multilocus phylogeny does not resolve morphological species within the small-spored Alternaria species complex. Mycologia 101, Arenal, F., Platas, G., Monte, E. and Pelaez, F. (2000) ITS sequencing support for Epicoccum nigrum and Phoma epicoccina being the same biological species. Mycol Res 104, Letters in Applied Microbiology 66, The Society for Applied Microbiology 99

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