Phytopathologia Mediterranea (2013) 52, 3, 517−527 RESEARCH PAPERS Pleurostomophora richardsiae, Neofusicoccum parvum and Phaeoacremonium aleophilum associated with a decline of olives in southern Italy AntoniA CARLUCCI1, MAriA LuisA RAIMONDO1, FrAncescA CIBELLI1, ALAn J.L. PHILLIPS2 and FrAncesco LOPS1 1 2 Dipartimento di Scienze Agrarie, degli Alimenti e dell’Ambiente, Università degli Studi di Foggia, Via Napoli, 25, 71121 Foggia, Italy Centro de Recursos Microbiológicos, Departamento de Ciências da Vida, Faculdade de Ciências e Tecnologia, Universidade Nova de Lisboa, Quinta da Torre, 2829-516 Caparica, Portugal Summary. In a recent survey of olive groves in the Canosa di Puglia, Cerignola and Foggia areas of southern Italy a serious decline of olive trees was seen. The symptoms comprised a general decline of the trees beginning with foliar browning and leaf drop, wilting of apical shoots, die-back of twigs and branches, and brown streaking under the bark of the trunk, branches and twigs. In more advanced stages of the disease necroses and cankers were observed on the bark. The symptoms were similar to those caused by Verticillium wilt, but morphological and molecular analyses revealed the presence of Pleurostomophora richardsiae, Phaeoacremonium aleophilum and various genera and species in the Botryosphaeriaceae. Pathogenicity tests carried out on young shoots showed that Pl. richardsiae, Pm. aleophilum and Neofusicoccum parvum were pathogenic and capable of causing brown wood streaking. Since Pl. richardsiae was the most aggressive of these three fungi and frequently it was the only one isolated from diseased trees it was considered to be a primary cause of the decline. To our knowledge, this is the irst report of Pl. richardsiae as a pathogen of olive trees. Key words: Olea europaea, olive disease, pathogen. Introduction The practice of olive cultivation spread rapidly from Asia Minor throughout the Mediterranean region due to its nutritional and health-promoting properties. Several European countries are now among the largest producers of olive oil and table olives. Indeed, Europe is the major olive-producing area in the world, and after Spain, Italy is the second largest producer, with a total crop area of 1,144.42 ha (FAOSTAT, 2012). Olives are susceptible to diferent bacterial, viral and fungal pathogens, which can cause severe dis- Corresponding author: A. Carlucci Fax: +39 0881 589501 E-mail: antonia.carlucci@unifg.it www.fupress.com/pm © Firenze University Press eases of the drupes, leaves, wood and roots. Wood decay is caused by several species, e.g., Fomes fulvus, Polyporus oleae, Schizophyllum commune, Phellinus punctatus and Trametes spp., while Verticillium dahliae is the main cause of vascular disease (JiménezDíaz et al., 1998; Nigro et al., 2005). Other fungal species are often associated with olives, such as weakly pathogenic, saprobic and endophytic fungi. Indeed, Carlucci et al. (2008) reported that Lecythophora lignicola, Phoma incompta, Phoma cava, Pleurostomophora richardsiae, Phaeoacremonium spp. and species of the Botryosphaeriaceae are all associated with brown streaking in olive xylem, but none was proved to be the cause of this symptom. During a recent survey of olive orchards in the Canosa di Puglia, Cerignola and Foggia areas of southern Italy, a decline of olive trees was noted. ISSN (print): 0031-9465 ISSN (online): 1593-2095 517 A. Carlucci et al. The symptoms consisted of a generalised decline of the trees starting with foliar browning and leaf drop, wilting of apical shoots, die-back of twigs and branches, and brown streaking under the bark of the trunk, branches and twigs. In more advanced stages of the disease, necrosis and cankers were observed on the bark. Initially, these symptoms were thought to be caused by Verticillium spp., although brown subcortical streaking, necrosis and cankers are not generally attributed to Verticillium wilt (López-Escudero and Mercado-Blanco, 2011). Based on the isolation of other fungi from brown wood streaking in olive trees (Carlucci et al., 2008), attention was focused on other fungi, including Pl. richardsiae, Phaeoacremonium spp. and various species in the Botryosphaeriaceae. The aim of the present study was to identify and characterise the main fungi associated with declining olive trees, and to determine their role in the disease. Materials and methods Fungal isolations Samples of parts of the roots and collars, trunks and branches were collected from 42 symptomatic olive trees (cv. Coratina) 18–35-years old. The samples were transported to the laboratory for analysis. Following surface-sterilisation of the samples according to Fisher et al. (1992), the bark was removed and small samples (1–3 mm2) were taken from the immediate sub-cortical tissues with a scalpel. These samples were placed on malt extract agar [2% malt extract (Oxoid Ltd., Basingstoke, UK); 2% agar (Difco, USA)] with 500 mg L-1 streptomycin sulphate (Oxoid Ltd.), and incubated at 25°C (±3°C) in the dark. All fungal colonies morphologically similar to Pleurostomophora species were grown until they sporulated and then a conidial suspension was spread on agar plates. After 24–36 h of incubation single germinating conidia were transferred to fresh plates of potato dextrose agar (PDA, Oxoid Ltd.). Genera and species in the Botryosphaeriaceae were identiied by reference to the keys, descriptions and sequence data provided in Phillips et al. (2013). Phaeoacremonium species were identiied according to Mostert et al. 2005. Other fungi were identiied based on their micromorphology and cultural characters. Reference strains are maintained in the culture collection of the Department of Science of Agriculture, Food and 518 Phytopathologia Mediterranea Environment, of the University of Foggia, Italy. The isolation frequencies per olive plant were calculated as the number of tissue portions infected by a given fungus, divided by the total number of tissue segments incubated, and expressed as percentages. DNA extraction and microsatellite-primed polymerase chain reaction Genomic DNA of all Pleurostomophora isolates was extracted from 200 μg fresh fungal mycelium that was grown on potato dextrose agar plates for 7 days, according to a new protocol that we optimised here for Pleurostomophora strains. The mycelium was scraped of the plates and ground in liquid nitrogen to a ine powder with a pestle and mortar. Then, 250 mg of the ground mycelium was collected in 2 mL microcentrifuge tubes (Eppendorf AG, Hamburg, Germany) in 600 μL DNA extraction bufer [200 mM Tris-HCl, pH 8.0, 200 mM EDTA, pH 8.0, 0.5% (w/v) SDS, pH 7.2, 1.2% (v/v) β-mercaptoethanol], supplemented with 5 μL proteinase K (stock, 20 mg L-1; Oxoid Ltd.), and kept at 65°C for 1 h. Chloroform (600 μL; Oxoid Ltd.) was then added, and the samples were vortexed for 15 s. The samples were centrifuged (10,000 rpm, 18°C, 15 min), and the aqueous phase was transferred to new 2 mL microcentrifuge tubes, with the addition of 3 μL RNAase (stock, 40 mg L-1; Oxoid Ltd.). The samples were left at 37°C for 30 min, and then an equal volume of chloroform was added to each sample, vortexed for 15 s, followed by centrifugation (10,000 rpm, 18°C, 10 min). The aqueous phases were transferred into new 1.5 mL centrifuge tubes (Eppendorf AG), with the addition of 0.5 vol. cold 7.5 M ammonium acetate (Oxoid Ltd.). Following gentle mixing, the samples were left on ice for 30 min, and then centrifuged (13,000 rpm, 2°C, 10 min). The aqueous phase was collected and transferred to 2.0 mL centrifuge tubes, with the addition of 0.7 vol. cold isopropanol (Oxoid Ltd.) and gentle mixing; these were then left at –25°C overnight. After this, the samples were centrifuged (13,000 rpm, 2°C, 25 min), the supernatant discarded, and the pellets washed twice with 700 μL cold 70% ethanol, with centrifugation (13,000 rpm, 2°C, 15 min) and discarding of the supernatants. After the inal centrifugation, the pellets were left to dry at room temperature with the tubes open, for 10–15 min. The pellets were then dissolved in 100 μL hot (65°C) TE bufer (10 mM Tris, pH 8.0, 1 mM EDTA), Pleurostomophora richardsiae and olive decline in Apulia and left at 55°C for 10 min. Methods for DNA isolation from Botryosphaeriaceae and Phaeoacremonium are as described by Phillips et al. (2013) and Mostert et al. (2005), respectively. Microsatellite-primed polymerase chain reaction (PCR) proiles were generated for 38 Pleurostomophora isolates with M13 primers (Meyer et al., 1993) according to Santos and Phillips (2009). The isolates were clustered into a consensus dendrogram on the basis of their proiles, with the dendrogram built using the BioNumerics software, version 5.1 (Applied Maths, Kortrijk, Belgium), using Pearson’s correlation coeicients and the unweighted pair group method with arithmetic mean. The reproducibility levels were calculated as the means of the reproducibilities obtained for the M13 primer. For this purpose, 10% of the isolates were chosen at random and their proiles were analysed again. ordered and of equal weight. Maximum-parsimony analysis was performed using the heuristic search option, with random addition of sequences (1000 replications), and tree bisection–reconnection as the branch-swapping algorithm, with the MULTREES options on. Branches of zero length were collapsed and all multiple, equally parsimonious trees were saved. The robustness of the trees obtained was evaluated with 1000 bootstrap replications (Hillis and Bull, 1993). Tree length, consistency index, retention index and rescaled consistency index were calculated, and the resulting trees were visualised with TreeView, version 1.6.6 (Page, 1996). New sequences were lodged in GenBank, and the alignment and phylogenetic tree were deposited in TreeBASE (S15002). The tree was rooted to Diaporthe ambigua (AJ458389). Morphology ITS Ampliication, sequencing and phylogenetic analysis The 5.8S rDNA gene and lanking internal transcribed spacers 1 and 2 of 15 Pleurostomophora isolates as representative were ampliied with the primers ITS1 and ITS4 (White et al., 1990). PCR reactions were performed according to Carlucci et al. (2012). The ampliied PCR fragments were puriied with NucleoSpin extract II puriication kits (Macherey-Nagel) before both strands were sequenced by PRIMM srl. In addition, part of the translation elongation factor 1-α (EF 1-α) was ampliied and sequenced for the Botryosphaeriaceae according to Phillips et al. (2013) and partial sequences of the β-tubulin and actin gene were generated for Phaeoacremonium as detailed by Mostert et al. (2005). Nucleotide sequences were edited with BioEdit, version 7.0.9 (http://www.mbio.ncsu.edu/BioEdit) and aligned with additional sequences retrieved from GenBank (www.ncbi.nlm.gov), using ClustalX, v. 1.83 (Thompson et al., 1997). Phylogenetic analyses were performed using PAUP version 4.0b10 (Swofford, 2003) for neighbour joining analysis and for maximum parsimony. The Kimura 2-parameter substitution model (Kimura, 1980) was used for the distance analysis. All of the characters were unordered and of equal weight. Bootstrap values were obtained from 1000 neighbour-joining bootstrap replicates. For the parsimony analyses, alignment gaps were treated as missing data, and all characters were un- Colony morphology of the Pleurostomophora isolates was determined in cultures incubated on malt extract agar at 25°C (±3°C) in the dark for 3 weeks, according to Vijaykrishna et al. (2004). Microscopic characteristics of these strains were determined according to Carlucci et al. (2012). Conidial shape and size were measured from 100% lactic acid mounts, using a Leica application suite measurement module (Leica Microsystem GmbH, Wetzlar, Germany). Photomicrographs were recorded with a Leica DFC320 digital camera on a Leica DMR microscope itted with Nomarski diferential interference contrast optics. Conidial dimensions were measured with the ×100 objective and at least 30 conidia were measured for each isolate. The means, standard deviation (SD) and 95% conidence intervals were calculated. Pathogenicity tests Two isolates of Pm. aleophilum (Pal4, Pal9), two isolates of Neofusicoccum parvum (Bot84, Bot88), and two isolates of Pl. richardsiae (PLEU9, PLEU27) were included in the pathogenicity tests. The pathogenicity tests were carried out in June 2013 on green shoots (0.5–1.5 cm diam., 30–50 cm long) of 18-year-old olive trees cv. Coratina grown in an open ield in an olive grove in Canosa di Puglia. The shoots were inoculated on wounds made at the internodes. Agar plugs (0.3–0.5 cm diam.) were taken from 10-day-old cultures grown on potato dextrose agar at 23°C (±2°C), Vol. 52, No. 3, December, 2013 519 A. Carlucci et al. and inserted under the bark. After inoculation, the wounds were wrapped with wet sterile cottonwool and sealed with Parailm. The controls were inoculated with sterile agar plugs. Each experiment included 20 replicates per treatment. The shoots were examined at 25 days after inoculation when the length of wood discoloration that occurred both under the bark and in the inner wood were measured. The mean, standard deviation, and maximum and minimum lengths of wood discoloration were determined. Isolations were made from all of the inoculated shoots, the isolates were identiied as described initially, to fulill Koch’s postulates. One-way analysis of variance was performed using Statistica, version 6 (StatSoft, Hamburg, Germany), to evaluate the diferences in the extension of sub-cortical discoloration induced by the fungal isolates. Duncan’s tests were used for comparisons of treatment means, at P<0.01. Table 1. Fungi isolated from the cankers and sub-cortical brown streaking of wilted olive plants. Fungal isolation frequency (%) Fungus 20.5 11.8 8.4 14.2 2.1 4.8 3.0 3.3 14.1 6.1 5.4 8.9 Aureobasidium oleae 0.0 1.3 5.4 1.9 Botryosphaeria dothidea 0,6 2,4 1,9 1.6 Cylindrocarpon destructans 5.1 0.0 0.0 1.9 Cytospora oleina 0.0 1.3 0.6 0.6 Diaporthe spp. 0.0 3.9 1.2 1.7 Diplodia mutila 0.0 0.7 0.5 0.4 Diplodia seriata 1.1 3.3 3.1 2.4 Epicoccum nigrum 8.1 0.9 0.0 3.3 Isolations Fomitiporia mediterranea 0.0 1.7 0.0 0.3 Cankers and longitudinal brown streaking were seen under the bark of the trunk and branches of declining trees (Figure 1). The mycolora isolated from the olive plants that showed the disease symptoms was very variable and is reported in Table 1. Acremonium spp., Aspergillus spp. and Penicillium spp. were isolated more frequently than other fungi (Table 1), although they are considered to be saprobic. Phaeoacremonium spp. were isolated from the trunks and branches, with isolation frequencies that ranged from 1.8% to 4.4%. Fungi belonging to the Botryosphaeriaceae (i.e., Botryosphaeria dothidea, Diplodia mutila, Diplodia seriata, Lasiodiplodia theobromae, Neofusicoccum luteum, Neofusicoccum parvum) were isolated with isolation frequencies ranging from 0.4% to 5.8%. Pleurostomophora richardsiae was isolated more frequently from trunks and branches, with isolation frequency ranging from 18.3% to 23.4%, respectively. This fungus was also isolated from the roots, although with a relatively low isolation frequency of 1.7%. Phaeoacremonium and Botryosphaeriaceae spp. were mainly isolated from older trees (more than 25 years old), while Pl. richardsiae was isolated mainly from younger trees (18–22 years old) (data not shown). Other fungi, identiied from their micromorphological and cultural features, were infrequently iso- Lasiodipodia theobromae 0.7 2.0 2.0 1.5 Lecythophora lignicola 0.4 0.4 1.2 1.1 Microsphaeropsis olivacea 0.0 0.4 0.0 0.2 Neofusicoccum luteum 0.0 0.8 1.0 0.5 Neofusicoccum parvum 1.9 7.8 8.3 5.8 Penicillium spp. 1.7 4.8 3.6 5.4 Phaeoacremonium aleophilum 0.0 4.4 1.8 2.1 Phoma incompta 0.0 0.4 0.6 0.3 Pleurostomophora richardsiae 1.7 18.3 23.4 13.5 Verticillium spp. 0.9 0.0 0.0 0.3 Bacteria 28.6 12.2 21.6 20.9 No fungi 6.8 9.6 17.2 7.9 Results 520 Phytopathologia Mediterranea Acremonium spp. Root/ Trunk Branches Total collar Alternaria alternata Aspergillus spp. Total Number of plants analyzed 100.0 100.0 100.0 100.0 42 lated from a few olive plants, such as Alternaria alternata, Aureobasidium oleae, Cylindrocarpon destructans, Cytospora oleina, Diaporthe spp., Epicoccum nigrum, Phoma incompta, Fomitiporia mediterranea, Lecythopho- Pleurostomophora richardsiae and olive decline in Apulia Figure 1. Disease symptoms observed on olive trees. (a) Wilt and die-back. (b–d) Sub-cortical browning as longitudinal streaking from young (12 years old) and adult (26 years old) trees. (e) Cankers and brown streaking on branches. White arrow, region from which Pl. richardsiae was frequently isolated. (f, g) Microscopic features of Pl. richardsiae. (f) Conidiophores and conidiogenous cells. (g) Brown, globose, and hyaline, cylindrical conidia. Scale bar in f, g = 10 μm. Vol. 52, No. 3, December, 2013 521 A. Carlucci et al. Table 2. Fungal isolates used in the phylogenetic study Species a Isolate numbera Host Locality GenBank ITS Collector Pl. ochracea CBS 131321 Human yellowgrain mycetoma Khartoum, Sudan N.A. Mhmoud and A. Fahal JX073270 Pl. ootheca CBS 115329 Degrading wood British Columbia L. Mostert HQ878590 Pl. repens CBS 294.39 Pine lumber Florida, USA R.W. Davidson AF083195 Pl. richardsiae PLEU3 Olive cv. Coratina Foggia, Italy A. Carlucci KF751176 PLEU4 Olive cv. Coratina Canosa di Puglia, Italy F. Lops - PLEU5 Olive cv. Coratina Cerignola, Italy A. Carlucci KF751177 PLEU6 Olive cv. Coratina Cerignola, Italy A. Carlucci - PLEU7 Olive cv. Coratina Canosa di Puglia, Italy F. Lops KF751178 PLEU8 Olive cv. Coratina San Severo, Italy M.L. Raimondo KF751179 PLEU9 Olive cv. Coratina Canosa di Puglia, Italy F. Lops - PLEU11 Olive cv. Coratina Foggia, Italy F. Lops - PLEU21 Olive cv. Coratina Cerignola, Italy A. Carlucci - PLEU22 Olive cv. Coratina Cerignola, Italy A. Carlucci KF751180 PLEU24 Olive cv. Coratina Cerignola, Italy A. Carlucci - PLEU25 Olive cv. Coratina Canosa di Puglia, Italy F. Lops KF751181 PLEU26 Olive cv. Coratina Foggia, Italy F. Lops KF751182 PLEU27 Olive cv. Coratina Cerignola, Italy A. Carlucci - PLEU29 Olive cv. Coratina Torremaggiore, Italy M.L. Raimondo KF751183 CBS 270.33 - Sweden E. Melin AY729811 Pm. aleophilum CBS 246.91 Root and stem Yugoslavia M. Muntañola-Cvetkovic AF017651 Pm. angustius CBS 101737 Vitis vinifera France P. Larignon AF197976 Pm. inlatipes CBS 391.71 Quercus virginiana Texas R.S. Halliwell AF197990 Pm. mortoniae CBS 101585 Root stock California L. Morton AF295328 CBS 211.97 Fraxinus excelsior Sweden J. Stenlid AF295329 Pm. parasiticum CBS 860.73 - USA Z. Yan U31841 Pm. rubrigenum CBS 498.97 Decorticated wood Puerto Rico S. Huhndorf AF197988 Pm. viticola LCP 933886 Vitis vinifera France P. Larignon AF118137 D. ambigua CMW5287 Malus domestica Pretoria, South Africa N. Moleleki AJ458389 Ex-type isolates are shown in bold. ra lignicola, Microsphaeropsis olivacea, and Verticillium spp. Because of their low frequency and inconsistent isolation frequency these were not considered to be the cause of the disease symptoms observed. 522 Phytopathologia Mediterranea Molecular identiication and phylogenetic analysis Identities of the Botryosphaeriaceae and Phaeoacremonium species (Table 1) was conirmed by BLAST searches in GenBank. Since the association of Pl. Pleurostomophora richardsiae and olive decline in Apulia PLEU7 CBS 270.33 PLEU29 PLEU27 PLEU26 PLEU3 PLEU4 100 PLEU5 Pleurostomophora richardsiae PLEU25 PLEU24 PLEU22 PLEU21 PLEU11 100 PLEU9 PLEU6 PLEU8 CBS 115329 Pleurostomophora ootheca 93 CBS 294.39 Pleurostomophora repens 100 CBS 131321 Pleurostomophora ochracea CBS 101585 99 60 91 Phaeoacremonium mortoniae AF295329 CBS246.91 Phaeoacremonium aleophilum AF118137 Phaeoacremonium viticola 91 AF197976 Phaeoacremonium angustius 100 99 94 CBS 498.97 Phaeoacremonium rubrigenum CBS 860.73 Phaeoacremonium parasiticum CBS 391.71 Phaeoacremonium inflatipes AJ458389 Diaporthe ambigua 1 Figure 2. One of four most parsimonious trees obtained from heuristic searches of ITS sequence data. Bootstrap support values from 1000 replicates are shown at the nodes. The tree is rooted to Diaporthe ambigua. Vol. 52, No. 3, December, 2013 523 A. Carlucci et al. phology agreed in all ways with the descriptions provided by Schol-Schwarz (1970), Domsch et al. (1980), De Hoog et al. (2000) and Vijaykrishma et al. (2004). richardsiae with declining olive trees was unusual, a more intensive phylogenetic study was undertaken. The microsatellite-primed PCR dendrogram of 38 isolates generated a single clade (data not shown), from which 15 isolates were chosen for sequencing the ITS (Table 2). The ITS dataset consisted of 27 ingroup isolates and 1 outgroup isolate (Table 2). After alignment, and following the exclusion of incomplete portions at each end, the dataset consisted of 498 characters. Of these 498 characters 305 were constant, while 54 were variable and parsimonyuninformative. Maximum parsimony analysis of the remaining 139 parsimony-informative characters resulted in four most parsimonious trees, one of which is shown in Figure 2 (tree length = 325; consistency index = 0.825, retention index = 0.930 and homoplasy index = 0.175). The neighbour joining analysis produced a tree with similar topology to the maximum parsimony tree. The 15 Pleurostomophora isolates sequenced in this study clustered in the same clade with the ex-type isolate of Pl. richardsiae (CBS 270.33, GenBank AY729811). Pathogenicity tests The results of pathogenicity tests were all determined at 25 days from inoculation. All three species tested in the inoculation experiment produced brown streaking in the wood. The most aggressive fungus was Pl. richardsiae, at 25 days after inoculation the two isolates caused signiicantly longer regions of brown streaking (averages of 3.3 and 3.7 cm) than the other two species tested, and resulted in death of all young shoots (Table 3). Although inoculation with the two N. parvum isolates resulted in death of all the shoots, the length of the brown wood streaking was signiicantly less (averages of 1.7 and 2.3 cm) than in shoots inoculated with Pl. richardsiae. The isolates of Pm. aleophilum caused brown streaking 0.7–1.0 cm in length, but did not kill the shoots. Furthermore, symptoms induced by the two Pm. aleophilum isolates were found in only 23% and 28% of inoculated plants, whereas Pl. richardsiae and N. parvum induced brown wood streaking in all the inoculated shoots. All fungi were re-isolated from symptomatic tissues, thus fulilling Koch’s postulates (Table 3). Morphology The Pl. richardsiae strains produced two diferent types of conidia, namely, brown globose conidia 1.5 μm diam., and hyaline, allantoid to cylindrical conidia (6–6.5 × 2 μm) (Figure 1). The micromor- Table 3. Pathogenicity assays carried out with two isolates each of Pm. aleophilum, Pl. richardsiae and N. parvum inoculated on young olive shoots (cv. Coratina). Brown streaking length (cm) after 25 days Fungal species Mean SD Max-Mina (%) Dead shoot Control H2O d 0.0 Ab - - - No Pm. aleophilum PAL4 0.7 AB 0.5 1.3–0.0 23 Yes PAL9 1.0 BC 0.7 2.2–0.0 28 Yes BOT84 1.7 CD 0.7 2.6–0.2 100 Yes BOT88 2.3 D 0.7 3.5–1.3 100 Yes PLEU9 3.3 E 1.5 6.6–0.6 100 Yes PLEU27 3.7 E 1.5 6.7–1.4 100 Yes N. parvum Pl. richardsiae a b 524 Re-isolation Isolate ID Maximum and minimum values detected on the basis of 20 observations. Values followed by a diferent capital letter in each column are signiicantly diferent according to Duncan’s test (P >0.01). Phytopathologia Mediterranea Pleurostomophora richardsiae and olive decline in Apulia Discussion Although a range of fungi were isolated from the wood of declining olive trees, most were infrequent or inconsistently associated with the symptoms. However, three species (N. parvum, Pl. richardsiae and Pm. aleophilum) were consistently isolated from diseased trees and at moderate to high frequencies. Thus, these three species were considered to be potential pathogens and possible causes of the disease, and were selected for further studies on their role in the disease. The results of the pathogenicity tests demonstrated that N. parvum, Pl. richardsiae and Pm. aleophilum are pathogenic on olives. However, only N. parvum and Pl. richardsiae induced symptoms in all inoculated shoots, and infections by both of them resulted in death of the shoots. This suggests that both species are likely causes of the decline and brown wood streaking of olive trees. Of the two, Pl. richardsiae was considered to be the most aggressive since it caused signiicantly longer regions of brown wood streaking than N. parvum. Furthermore, it was frequently the only fungus isolated from symptomatic younger trees, while N. parvum was never found alone but was always associated with Pl. richardsiae. Various species of the Botryosphaeriaceae were isolated from diseased trees. Some are known to be pathogens responsible for brown wood streaking in several hosts including olive, grapevine, apricot, peach, and oak (von Arx, 1987; Denman et al., 2000; Phillips et al. 2002; Alves et al., 2004; Niekerk et al., 2004; Damm et al., 2007; Burruano et al. 2008; Lazzizera et al. 2008; Phillips et al. 2008; Úrbez-Torres, 2011). On olives, species of the Botryosphaeriaceae are primarily known to cause fruit rot (Chattaoui et al., 2011; Lazzizera et al. 2008; Moral et al., 2008; Phillips et al., 2005), but some have been reported associated with branch dieback, cankers and blighted shoots of worldwide (Taylor et al., 2001; Moral et al., 2010; Romero et al., 2005; Kaliterna et al., 2012). However, this is the irst time that N. parvum has been associated with decline of olive trees in southern Italy or anywhere else in the world. Neofusicoccum parvum is well known as a pathogen of woody hosts and is regarded as a serious pathogen on some including grapevines, pine and Eucalyptus (Golzar and Burgess, 2011; Iturritxa et al., 2011; Úrbez-Torres, 2011; Úrbez-Torres et al., 2013). Since it was always associated with Pl. richardsiae infections in the present study, and never found on its own, N. parvum was not considered to be the primary cause of olive decline and brown wood streaking. Nevertheless, it is clearly pathogenic on olives and would likely contribute to the severity of the decline Phaeoacremonium aleophilum was also isolated from olive trunks and branches, although at relatively low frequencies. Since it caused relatively mild symptoms consisting of small regions of brown wood streaking in only some of the inoculated shoots, this species was considered to play an insigniicant role in the decline disease. Pleurostomophora richardsiae was considered to be the main agent responsible for wilt of apical foliage, brown streaking under the bark, and cankers of trunks and branches. It was always isolated from symptomatic wood tissues of all diseased plants, and frequently it was the only fungus isolated from younger (18–22 old years) diseased olive trees. Botryosphaeriaceae and Phaeoacremonium species, some of which are already known as pathogens of olives (Carlucci et al., 2008; Lazzizera et al., 2008), were isolated more frequently from symptomatic tissues of older plants (more of 25 old years). These species were never isolated alone but were always associated with other fungi including Pl. richardsiae. Pleurostomophora was introduced by Vijaykrishna et al. (2004) to accommodate the phialophora-like anamorphs of Pleurostoma species. Four species are currently recognised, namely Pl. richardsiae, Pl. repens, Pl. ootheca (Vijaykrishna et al., 2004) and Pl. ochracea (Mhmoud et al., 2012). Pleurostomophora richardsiae (syn. Phialophora richardsiae) is also known to be a human pathogen (Uberti-Foppa et al. 1995; Ikai et al. 1988; Pitrak et al. 1988; De Hoog et al. 2000) and the cause of subcutaneous phaeohyphomycotic cysts after traumatic implantation (Guého et al. 1989). More recently it was reported as a pathogen of grapevines by Eskalen et al. (2004) and Rolshausen et al. (2010) in California (USA), and by Halleen et al. (2007). Pleurostomophora ochracea is known to be the cause of human eumycetoma (Mhmoud et al., 2012), while Pl. repens and Pl. ootheca have only been associated with pine lumber and degrading wood, respectively (Vijaykrishna et al., 2004). Eskalen et al. (2004) and Rolshausen et al. (2010) demonstrated in pathogenicity tests that Pl. richardsiae can infect pruning wounds of grapevine, although other fungi such as Botryosphaeriaceae species and Phaeomoniella chlamydospora were more aggressive. Halleen et al. (2004) consider Pl. richardsiae to be a Vol. 52, No. 3, December, 2013 525 A. Carlucci et al. vascular pathogen of grapevines, since it was able to cause vascular discoloration similar to Petri disease when it was inoculated into grapevines. White et al. (2011) reported that Pl. richardsiae was not found abundantly in their studies carried out on diseased grapevines, maybe because the vines were very old. Indeed, they isolated from grapevine higher rates of Basidiomycete fungi, which are known agents of white rot, and Pa. chlamydospora, the fungus responsible for internal symptoms such as black and brown wood streaking. To our knowledge, this is the irst report of brown wood streaking and decline of olive trees caused by Pl. richardsiae. 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