J Phytopathol SHORT COMMUNICATION Arthrinium phaeospermum, Phoma cladoniicola and Ulocladium consortiale, New Olive Pathogens in Italy Sandra Lo Piccolo, Vincenzo Mondello, Selene Giambra, Gaetano Conigliaro, Livio Torta and Santella Burruano Dipartimento di Scienze Agrarie e Forestali, University of Palermo, Viale delle Scienze 4, 90128 Palermo, Italy Keywords decline syndrome, Olea europaea, Sicily, weak pathogens Correspondence S. Burruano, Dipartimento di Scienze Agrarie e Forestali, University of Palermo, Italy. E-mail: santella.burruano@unipa.it Received: May 7, 2013; accepted: August 13, 2013. doi: 10.1111/jph.12179 Abstract In recent years, leaf necrosis and twig dieback in the olive crop have been detected in Sicily (Italy). In this article, we identify the predominant fungal species associated with symptomatic leaves and twigs, using morphological features and DNA sequencing of the internal transcribed spacer (ITS) region, as Alternaria alternata, Arthrinium phaeospermum, Phoma cladoniicola and Ulocladium consortiale. The pathogenicity of these four species was tested on olive plants cv. Biancolilla. All species were pathogenic on leaves, but only U. consortiale produced cortical lesions on twigs, thus suggesting its main role in the Olea europaea twig dieback. To our knowledge, this is the first report of A. phaeospermum, P. cladoniicola and U. consortiale as olive pathogens. Introduction Materials and Methods Olive (Olea europaea L. var. sativa) is historically grown in most of the Mediterranean basin countries, where it represents an economically important tree crop. Recent investigations have highlighted the incidence of common O. europea diseases worldwide, such as cercosporiosis, anthracnose, peacock spot, sooty mould, fruit rot, verticillium wilt and phoma dieback (Tosi and Zazzerini 2005; Moral et al. 2008; Trapero et al. 2009). Additionally, a new decline syndrome in olive trees has been recently observed in Sicily (Italy). The syndrome consists of foliar chlorosis, associated with necrotic irregular marginal or apical spots, which then flowed resulting in withering of leaf tips. Symptomatic twigs show apical defoliations, cortical necrosis and withering (Fig. 1). A preliminary aetiological study conducted in two Sicilian olive orchards allowed us to ascertain the constant association between fungal microorganisms and symptomatic organs of declining olive trees (Ferraro et al. 2010). Thus, the aim of this study was to identify the most prevalent fungal taxa associated with symptoms and to test their pathogenicity. Samplings were conducted during spring, summer and autumn of 2007 and 2008 in two Sicilian olive orchards, located on a hill (Racalmuto, Agrigento, south-western Sicily) and on plane (San Cipirello, Palermo, north-western Sicily). For each survey, samples of symptomatic leaves and twigs were collected and subjected to isolation tests. The plant material was surface-sterilized by sequential washing in 5% NaOCl for 5 min, 95% EtOH for 1 min and 5% H2O2 for 3 min and then rinsed three times in distilled sterile water. Fragments at the margin between the healthy and affected tissue were aseptically excised and placed on 2% malt extract agar (MEA; Oxoid, Milano, Italy). Petri dishes were incubated at 25  2°C in the dark and daily observed for fungal development during two weeks. Hyphal tips or spores of representative fungal isolates were transferred to 2% MEA for a preliminary morphological identification at genus level on the basis of their macroscopic and microscopic features (Domsch et al. 1980; Barnett and Hunter 1998; Pitt and Hocking 1999). Microscopic characteristics were examined using a Ó 2013 Blackwell Verlag GmbH 1 New fungal pathogens on olive (a) (b) Fig. 1 Symptomatic olive trees in Sicily: withering of twigs (a); chlorosis and necrosis on leaves (b). S. Lo Piccolo et al. The 40-ll reaction volume contained 50–100 ng of DNA template, 2 mM MgCl2, 0.2 mM dNTP, 0.3 lM of each primer, 0.5 U of Taq DNA polymerase (Dream Taq, Fermentas, Italy) and 1 9 Dream Taq buffer (Fermentas). The amplification reaction was carried out as follows: initial denaturation at 95°C for 2 min; followed by 35 cycles of denaturation at 95°C for 30 s, annealing at 55°C for 20 s and extension at 72°C for 1 min; and a final extension at 72°C for 5 min. PCR products were sequenced in both directions using the same primers that were used for PCR. The nucleotide sequences were compared to those found in GenBank using the BLAST program (Altschul et al. 1997). The same isolates used for DNA extraction were tested for pathogenicity on five 3-year-old healthy olive plants (cv. Biancolilla). For each plant, the inoculations were performed on three twigs (in three different points) and on five leaves for each twig. In detail, a 6-mm-diameter mycelium plug derived from a 7-day-old colony grown on malt extract agar (MEA, Oxoid, UK) was placed both on leaves wounded in three points with sterile needles and on properly peeled twigs. Three control plants were inoculated with non-colonized plugs of MEA. Plants were then kept in greenhouse under controlled conditions (T = 22  1°C; R.H. = 70–80%), watered fortnightly and checked for symptoms. The length of leaf lesions was measured 2 weeks after inoculation, while the length of cortical lesions, after 20 weeks. The data were subjected to ANOVA and the Tukey’s test. To fulfil the Koch’s postulates, re-isolation assays from symptomatic tissues were carried out on MEA. Results and Discussion light microscope (Axioskop; Zeiss, Jena, Germany) coupled to an AxioCam MRc5 (Zeiss) digital camera, and images were captured using the software AxioVision 4.6 (Zeiss). For each sampling, the isolation frequency (IF) of each fungal genus in both olive groves was calculated. The species-level identification of the most prevalent fungal genera was performed on the basis of criteria given by Simmons (1998, 2007), Samson et al. (2000) and Diederich et al. (2007). One representative isolate for each fungal species identified morphologically was used for genomic DNA extraction using the standard cetyltrimethylammonium bromide (CTAB)-based protocol (O’Donnell et al. 1998). The internal transcribed spacer (ITS) regions (ITS1, 5.8S gene, ITS2) of the ribosomal DNA operon were amplified by polymerase chain reaction (PCR) using the primers ITS1F (fungal specific; Gardes and Bruns 1993) and ITS4 (universal; White et al. 1990). 2 The most prevalent fungal genera associated with leaf necrosis and twig dieback in both olive orchards were Alternaria, Arthrinium, Phoma and Ulocladium (Table 1). Other genera isolated in only one of the two olive groves were Aureobasidium, Camarosporium and Septoria. Moreover, ubiquitous genera such as Cladosporium and Penicillium were isolated with low IF in both locations. The specific identification of the isolates referable to the four predominant genera showed the presence of only one species for each genus. The isolates of Alternaria genus produced colonies that grew rapidly (5–7 cm diameter at 25°C for 7 days on MEA) and appeared floccose, whitish to grey brown, with the reverse brown to black. Conidiophores were septate and pale brown, simple or branched, geniculate and bored conidia in chains at the apex. Conidia (7–10 9 23–34 lm), up to nine in a chain, were brown, ovoid to ellipsoidal, often with a Ó 2013 Blackwell Verlag GmbH S. Lo Piccolo et al. Ó 2013 Blackwell Verlag GmbH Table 1 Isolation frequency (%) of fungal taxa isolated from symptomatic leaves and twigs of declining olive trees from two Sicilian olive orchards during 2007 and 2008 Racalmuto San Cipirello Leaves Twigs 2007 2008 Leaves 2007 2008 Twigs 2007 2008 2007 2008 Fungal taxa Sp Su Au Sp Su Au Sp Su Au Sp Su Au Sp Su Au Sp Su Au Sp Su Au Sp Su Au Alternaria alternata Arthrinium phaeospermum Aureobasidium Camarosporium Cladosporium Penicillium Phoma cladoniicola Septoria Ulocladium consortiale TOT 56.8 ― ― 5.6 8.2 0.8 ― ― ― 71.4 2.1 ― ― ― ― ― 6.2 ― ― 8.3 16.6 2.8 0.9 4.6 6.4 1.4 8.3 ― 2.3 43.3 1.1 0.5 2.1 1.6 0.5 1.1 11.6 ― 1.1 19.6 0.6 0.6 ― ― ― ― ― ― 0.6 1.8 6.3 24.2 2.0 0.8 2.0 0.4 1.2 ― 30.6 67.5 4.5 0.5 ― 2.9 6.0 1.5 ― ― 0.5 15.9 ― ― ― ― ― ― ― ― ― ― 2.9 5.7 3.7 2.9 0.9 0.9 0.9 ― 5.7 23.6 ― ― 1.9 ― ― ― ― ― ― 1.9 0.8 ― 0.7 ― ― ― ― ― 12.1 13.6 ― ― ― ― ― 1.7 ― ― 6.9 8.6 54.1 ― ― ― 3.5 ― ― ― ― 57.6 38.8 5.2 ― ― 4.5 ― 1.1 0.2 0.2 50.0 51.4 1.2 ― ― 7.5 ― 9.2 6.1 ― 75.4 26.7 3.6 ― ― 4.1 0.3 2.4 3.8 ― 40.9 12.5 0.8 ― ― 0.6 ― 5.3 1.4 0.3 20.9 23.1 13.4 ― ― 8.0 4.8 ― 1.1 18.2 68.6 ― ― ― ― 6.1 ― ― ― ― 6.1 2.4 ― ― ― 3.3 0.8 ― 5.3 8.6 20.4 ― ― ― ― ― ― ― ― 3.8 3.8 5.7 ― ― ― 0.9 ― ― ― 3.4 10.0 ― ― ― ― 2.6 ― ― 0.5 ― 3.1 ― 0.5 ― ― ― ― 0.9 ― 0.5 1.9 (c) (a) (f) (d) (b) New fungal pathogens on olive (e) (h) short beak, muriform with up to six transverse and 2–3 longitudinal or oblique septa, smooth or roughened. These isolates were identified as Alternaria alternata (Fr.) Keissl (Fig. 2a,b). Arthrinium isolates were characterized by rapidly growing colonies (5–7 cm diameter at 25°C for 7 days on MEA), with floccose whitish aerial mycelium and sporulating dark spots on the surface. Conidiophores were hyaline or pale brown, produced clusters of dark brown, lenticular, smooth conidia (5–8 9 9–11 lm), and provided with (g) Fig. 2 Morphology of a 7-day-old colony on MEA and conidia of Alternaria alternata (a, b), Arthrinium phaeospermum (c, d), Phoma cladoniicola (e, f) and Ulocladium consortiale (g, h). 3 New fungal pathogens on olive S. Lo Piccolo et al. an equatorial germ slit. These isolates were identified as Arthrinium phaeospermum (Corda) M.B. Ellis (Fig. 2c,d). Phoma isolates, identified as Phoma cladoniicola Diederich, Kocourk., & Etayo (Fig. 2e,f), showed fast-growing colonies (6–8 cm diameter at 25°C for 7 days on MEA) that appeared flat, powdery to velvety, initially white and later olive grey, dark brown to black in reverse. Black pycnidia with a subspherical to pyriform shape (50–100 lm) and ostiolate were observed. Conidiogenous cells lining in the inner wall of the pycnidial cavity were short, ampulliform, hyaline and smooth. Conidia (1.5–3 9 3–6 lm) (a) (b) (c) were ellipsoid, hyaline, smooth and biguttulate. The isolates of Ulocladium produced colonies growing mildly fast (4–6 cm diameter at 25°C for 7 days on MEA) that appeared effuse, powdery, olive brown to nearly black and brown in reverse. Conidiophores were simple or branched, pale brown, smooth and strongly geniculate. Conidia (20–35 9 15–20 lm), single or in short chains, were obovoid or ellipsoidal, golden brown, smooth or slightly rough and muriform with 1–5 transverse and 1–2 longitudinal or oblique septa. These isolates were identified as Ulocladium consortiale (Th€ um.) E.G. Simmons (Fig. 2g,h). The ITS (d) (e) (f) (g) 4 Fig. 3 Pathogenicity on 3-year-old olive plants (cv. Biancolilla) of Alternaria alternata, Arthrinium phaeospermum, Phoma cladoniicola and Ulocladium consortiale. Irregular necrotic lesions on leaves caused by A. alternata (a), A. phaeospermum (b), P. cladoniicola (c) and U. consortiale (d) 2 weeks after inoculation. Cortical lesion produced by U. consortiale (e) 20 weeks after inoculation: detail of lesion on twig (f) and brown internal necrosis visible in longitudinal section (g); white arrows indicate the point of inoculation. Ó 2013 Blackwell Verlag GmbH S. Lo Piccolo et al. New fungal pathogens on olive Table 2 Mean lesion length on olive leaves cv. Biancolilla, 2 weeks after inoculation with four fungal species Species Mean lesion length (cm)  SEa Alternaria alternata Arthrinium phaeospermum Phoma cladoniicola Ulocladium consortiale Control 2.2 3.5 2.6 3.4 0.0      0.3 bB 0.4 cC 0.4 bBC 0.2 cC 0.0 aA 1971) and decay of caraway seedlings (Mazur and Nawrocki 2004). To our knowledge, this is the first report of A. phaeospermum, P. cladoniicola and U. consortiale as O. europaea pathogens. Acknowledgements We thank Giancarlo Polizzi for his valuable suggestions and for critically reading the manuscript. a Mean and standard error based on 75 replicates per treatment. Values followed by equal letters are not significantly different. Lowercase and uppercase letters refer to P ≤ 0.05 and P ≤ 0.01, respectively. sequences of the four selected isolates, compared with those available in GenBank, confirmed the morphological identifications. Particularly, the isolates belonging to genera Alternaria (GenBank accession number KC577267), Arthrinium (KC577268), Phoma (KC577269) and Ulocladium (KC577270) showed 99% homology with A. alternata (JN673372), 100% with A. phaeospermum (KC253945), 99% with P. cladoniicola (JQ238629) and 99% with U. consortiale (HM036619), respectively. Pathogenicity tests showed that all the assayed fungi caused irregular necrotic leaf spots, which spread from the inoculum point to the leaf apex (Fig. 3a–d). Ulocladium consortiale was the only fungus able to produce cortical lesions (3–3.5 cm long) on twigs (Fig. 3e–g); no symptoms were observed in controls. The length of leaf lesions produced by four fungi differed significantly from the control (P ≤ 0.05 and P ≤ 0.01; Table 2). Moreover, A. phaeospermum and U. consortiale produced significantly larger lesions than A. alternata and P. cladoniicola. Alternaria alternata, A. phaeospermum, P. cladoniicola and U. consortiale were re-isolated from all the symptomatic organs, and the colonies were morphologically identical to those of the original isolates. In this study, we report the pathogenicity of A. alternata, A. phaeospermum, P. cladoniicola and U. consortiale on olive trees. The results indicate the four tested fungi as pathogens on leaves and the main role of U. consortiale in the twig dieback. On olive, only A. alternata has already been reported as pathogen of leafy cuttings of shoots (Bourbos et al. 1999). Arthrinium phaeospermum has been described as an endophyte in blueberry (Munitz et al. 2013) and maize seeds (Kido et al. 2012) and is associated with flower malformation in Aloe zebrina (Niaz and Dawar 2009), while P. cladoniicola has been described as a lichenicolous fungus (Urbanavichus and Urbanavichene 2011). 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