Olive (Olea europaea L.), is an important oil-producing crop in Mediterranean countries such as Spain, Italy, Greece, Turkey, and many other countries such as USA (California), Argentina, Chile, Australia and New Zealand (Zohary and Hopf 1994; Noormohammadi et al. 2007). Iran has a long history with olive cultivation and there is ample evidence that regions near the Caspian Sea in northern Iran are a presumed center of origin for olive (Noormohammadi et al. 2007). The Iranian olive industry is a relatively small farming business distributed mainly in the north of the country, covering a total area of over 80,000 ha (Sadeghi 2002; Noormohammadi et al. 2007; Arzanlou et al. 2012). In recent years, there is growing interest by government and local authorities in establishing new olive orchards in different provinces of the country; thus current trends are for acreage increase and planting new varieties in different parts of the country (Noormohammadi et al. 2007). The olive plants are susceptible to numerous debilitating diseases which limit olive production in many olive-producing countries and also in Iran (Lazzizera et al. 2008; Moral et al. 2009). Fungal diseases cause significant economic losses to the olive industry through yield loss, reduction in growth, and increase in production costs (Lazzizera et al. 2008; Moral et al. 2009). Several fungal diseases such as Verticillium vascular wilt, peacock eyespot and fruit rot diseases have been reported on olive in Iran (Ershad 2009; Arzanlou et al. 2012). Various kinds of fruit rot are widespread on olive in many olive-producing countries, reducing the quality and quantity of the product through direct loss of rotten fruits, reduced commercial value of table olives and reduced quality of the oil due to fungal infections (Lazzizera et al. 2008). Diverse fungal groups have been reported to cause fruit rot on olive, including species of Botryosphaeriaceae such as Botryosphaeria, Diplodia, Lasiodiplodia, Macrophomina, Neofusicoccum and Camarosporium; Glomerellaceae including Colletotrichum; Mycosphaerellaceae incl. Pseudocercospora and several other fungal species with minor importance such as Fusicladium oleagineum, Alternaria spp., Aureobasidium pullulans, Epicoccum nigrum, Cladosporium herbarum s.l., Capnodium elaeophilum and Truncatella angustata (Avila et al. 2005; Athar 2005; Chattaoui et al. 2011; Moral et al. 2008, 2009; Sergeeva et al. 2009; Arzanlou et al. 2012). In the present study we report a new fruit rot fungal disease on olive in Iran.

In a survey on fungal agents involved in olive fruit rot in orchards of the Tarom region in the Zanjan province, fruits with rot symptoms were collected from olive orchards in four counties. In total eight orchard and seven trees per orchard were inspected. The incidence of the fruit rot was 2.4 %. Disease symptoms of fruit rot started as small, brown, water-soaked spots, increased in diameter and became wrinkled with age. The mummified fruits remained attached on the tree (Fig. 1). Isolations were made from diseased tissues by cutting small pieces from the margin of the lesions, the tissues were then surface-sterilized for 15–20 s in 70 % ethanol, rinsed with sterile water three times, dried on sterile filter paper and transferred to Potato Dextrose Agar (PDA, Fluka, Hamburg, Germany) plates supplemented with 100 mg L−1 of streptomycin sulphate. Single-spore cultures were established from sporulating colonies as described by Bakhshi et al. (2011). Briefly, under a stereomicroscope, with the tip of a wetted sterile inoculation needle a conidial mass on a conidioma was touched and the conidia suspended on PDA plates (supplemented with streptomycin sulfate (100 mg L−1)), containing 10 ml sterile water. The suspension was evenly spread on the agar surface and plates were kept in oblique position overnight. The plates then were checked under the stereomicroscope and germinated conidia were transferred to new PDA plates. Single-spore cultures are preserved on PDA in 2 ml microtube slants at 4 °C in the Culture Collection of Tabriz University and Iranian Fungal Culture Collection with accession numbers CCTU 1200 = IRAN 2183C and CCTU 12001 = IRAN 2182C, respectively.

Fig. 1
figure 1

Disease symptoms and pathogenicity assay. a Symptomatic fruits of Olea europaea naturally infected with Pilidium concavum. b Control (uninoculated) fruits of cultivar Zard without symptoms. c–d Symptoms developed on in vitro inoculated olive fruits, 7 and 14 days after inoculation, respectively

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Cultural, including colony color, shape and growth rate and microscopic features were studied on Potato Dextrose Agar (PDA), Malt Extract Agar (MEA) and Oatmeal Agar (OA) culture media after 7 days of incubation on 25 °C under alternating fluorescent (12 h) and near ultraviolet (12 h) light. Microscopic characters were studied using a smash mount technique with sterile distilled water as described by Arzanlou et al. (2007). Morphology and size of microscopic structures were assessed based on 30 measurements. In culture, the mycelia were hyaline to pale brown, with septate, branched; 2–4 μm wide hyphae. The sporodochia, (275–) 280–300 (–350) × (70–) 82–119 (–130) μm, were scattered, sessile, relatively spherical, thick-walled, initially light colored, turning brownish or almost black in older cultures. Conidiophores forming a dense palisade, hyaline to pale brown, tapering distally, (12–) 23–31 (–40) × (1–) 1.8–2.3 (–3) μm. Conidiogenous cells phialidic, hyaline, tapering towards the apex. Conidia 1-celled, hyaline, fusiform to slightly falcate, apex acute, base obtuse, thin-walled, formed singly on the conidiogenous cells, (3–) 5–6 (–8) × (1–) 1.4–1.7 (–2) μm.

The colony diameter was assessed based on three replicates. On MEA colonies reaching 29 mm diam in 7 day, initially white, turning brown in center with the production of conidiomata, felted, with sparse aerial mycelium. On PDA colonies reaching 37 mm diam in 7 day, white, with sparse aerial mycelium. On OA colonies reaching 44 mm diam in 7 day, initially white, turning brown in the center with production of conidiomata (Fig. 2). The identity of the causal agent was further confirmed using sequence data from ITS-rDNA region. For this purpose, DNA was extracted from 7-day-old cultures grown on PDA, using the protocol of Moller et al. (1992). The primers V9G (de Hoog and Gerrits van den Ende 1998) and LR5 (Vilgalys and Hester 1990) were used to amplify the 3′ end of the 18S rRNA gene, ITS1, 5.8S rDNA, ITS2 and the first approximately 900 bp of the 5′ end of the 28S nrRNA gene. PCR was performed on a GeneAmp PCR System 9700 (Applied Biosystems, Foster City, CA). The reaction mixture contained 1× PCR buffer, 1 mM MgCl2, 60 μl of 1 mM dNTPs, 0.2 pM of each primer, 0.5 U of Taq polymerase, 0.5 μl DMSO, and 10–15 ng of fungal genomic DNA. The final reaction volume was adjusted to 12.5 μl by adding sterile distilled water. The thermal cycling condition consisted of an initial denaturation at 95 °C for 5 min, followed by 40 cycles of 30 s at 94 °C, 30 s at 52 °C and 1 min at 72 °C, followed by a final extension cycle at 72 °C for 7 min. The amplicon was sequenced using PCR primers and two additional internal primers, ITS4 (White et al. 1990) and LR0R (Rehner and Samuels 1994) in order to obtain good quality sequence over the entire length of the amplicon, by using a BigDye Terminator Cycle Sequencing Kit v. 3.1 (Applied Biosystems, Foster City, CA) and analysed on an ABI Prism 3700 (Applied Biosystems, Foster City, CA). The sequence for the ITS region was subjected to a Megablast search at NCBI’s GenBank nucleotide database and sequences with high similarity were downloaded from GenBank and aligned together with the sequence obtained in this study. Sequence alignment was carried out by using the ClustalW algorithm implemented in MEGA 5 (Tamura et al. 2011). Phylogenetic analysis was performed using maximum likelihood method with program default settings in MEGA 5. Bootstrap analysis was performed with 1,000 replicates. The phylogenetic tree was rooted to Chaetomella raphigera (GenBank accession number AY487085.1). The sequence was deposited in GenBank and the alignments in TreeBASE (www.treebase.org).

Fig. 2
figure 2

Cultural and morphological characteristics of Pilidium concavum. a–c Seven day-old colonies on MEA, PDA and OA. d Unfledged (immature) conidiomata on OA after 7 days. e Mature conidiomata on OA after 7 days. f–h Conidiophores and conidiogenous cells on OA. i Conidia on OA. Bars (d–e) = 100 μm, (f–i) = 10 μm

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Koch’s postulates were performed on surface-sterilized healthy fruits of Cultivar “Zard” as follow described. Single-spore cultures were grown on PDA plates for a week to sporulate abundantly; spore suspension with a final concentration of 1 × 107 conidia per ml distilled water was prepared. Healthy mature green olive fruits were dipped in 70 % ethanol for 20–30 s and rinsed three times in sterilized water. Fruits were then dipped in the spore suspension and placed in Petri dishes on sterile filter paper. The filter paper was kept wet during the experiment. For the controls, fruits were dipped in sterilized distilled water. The experiment was carried out by using two fungal isolates and ten fruits for each the fungal isolates (5 Petri dishes, each containing two fruits). Petri dishes were maintained on the lab bench at 22 ± 2 °C, for 14 days.

The morphological characteristics are in full agreement with the description of P. concavum (Desm.) Hِhn. (synanamorph Hainesia lythri (Desm.) Hِhn.; teleomorph Discohainesia oenotherae (Cooke and Ellis) Nannf.) (Sutton 1980; Rossman et al. 2004; Lopes et al. 2010; Geng et al. 2012), and its identify was further corroborated by phylogenetic comparison of the ITS-rDNA region of isolate CCTU 1200 with other sequences of Pilidium from GenBank which showed 100 % homology with Pilidium concavum isolates from strawberry and other plant species (Fig. 3). The sequence is available in GenBank with GenBank Accession No. KF255414. The pathogenicity assay showed that Pilidium concavum was pathogenic to olive fruits. The disease symptoms appeared as brown sunken lesions after 7 days and wrinkle of fruits after 14 days, which then expanded to other parts of the fruit. Fruits in the control set remained free of blemish (Fig. 1). The original fungus was re-isolated from inoculated fruits showing the symptoms and no fungal growth was observed in the controls.

Fig. 3
figure 3

A maximum likelihood phylogenetic tree obtained for the ITS regions and 5.8S rDNA sequences. Bootstrap support values from 1,000 replicates are indicated at the nodes. The tree was rooted to Chaetomella raphigera. The scale bar indicates 0.02 substitutions per site

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Even though there are several names published in this the coelomycetous genus Pilidium, only two species, the type P. acerinum and P. concavum are currently accepted (Rossman et al. 2004; Kirk et al. 2008). Members of this genus have phylogenetic affinity with Leotiomycetes (Rossman et al. 2004; Kirk et al. 2008). A teleomorph connection has been established for P. concavum in Discohainesia Nannf. in the family Dermateaceae, order Helotiales, while P. acerinum is only known the anamorph (Rossman et al. 2004). Pilidium species often produce two types of conidiomata: black pycnidia, the most commonly encountered in nature, and also sporodochia, commonly seen in pure cultures (Palm 1991; Rossman et al. 2004). The two conidiomatal types of P. concavum had been considered as two distinct species. Palm (1991) determined that the light-colored sporodochia of Hainesia lythri just represent another form of P. concavum. Which of the three names available for this fungus will persist, has not yet been established. In the present study only the sporodochial form was developed in pure culture; pycnidia were neither observed on naturally infected fruits nor on different culture media. Pilidium spp. are known to cause disease on crop plants and trees such as Strawberry, Eucalyptus, Elephant’s ears (Bergenia crassifolia (L.) Fritsch) and Paeonia suffruticosa Andrews (Cardin et al. 2009; Duan and Kang 2010; Debode et al. 2011; Geng et al. 2012). Tan-brown leaf spot and fruit rot on strawberry caused by P. concavum have been reported from many countries (Opgenorth and White 1991; Golebniak and Jarosz 2004; Cardin et al. 2009; Duan and Kang 2010; Geng et al. 2012). P. acerinum has been considered as a weak pathogen on Eucalyptus in South Africa (Crous 1991). In the present study, P. concavum was isolated from olive orchards in the Tarom region in Zanjan province in northwestern Iran. Although P.concavum has recently been reported to cause fruit rot disease on Prunus domestica in northern Iran (Sayari et al. 2013), to the best of our knowledge, this is the first report on the occurrence of P. concavum on olive anywhere in the world. The disease cycle, distribution, host range and impact of the disease on crop yield remains to be studied.