Fungal trunk pathogens associated with wood decay of pistachio trees in Iran

Over the growing seasons of 2011–2013, various pistachio (Pistacia vera L.) cv. Fandoghi, and wild pistachio (P. atlantica Desf. subsp. mutica) trees were inspected in Iran to determine the aetiology of trunk diseases with specific reference to species of Phaeoacremonium and Botryosphaeriaceae spp. Samples were collected from branches of trees exhibiting yellowing, defoliation, canker and dieback, as well as wood discoloration in cross sections. Fungal trunk pathogens were identified using morphological and cultural characteristics as well as comparisons of DNA sequence data of the ITS and TEF-1α (for Botryosphaeriaceae species) and β-tubulin gene (for Phaeoacremonium species) regions. Phaeoacremonium parasiticum was the dominant species followed by Phaeoacremonium aleophilum, Botryosphaeria dothidea, Neofusicoccum parvum, Phaeoacremonium cinereum, Phaeoacremonium viticola and Dothiorella viticola. Pathogenicity tests were undertaken to determine the role of these species on pistachio under field conditions. Neofusicoccum parvum and Pm. aleophilum caused the longest and smallest lesions respectively. This study represents the first report on the occurrence and pathogenicity of Phaeoacremonium species on P. vera cv. Fandoghi. This also represents the first report of Pleurostomophora sp. on pistachio and Pm. parasiticum and D. viticola on wild pistachio. Additional key words: β-tubulin gene; fungal trunk pathogens; internal transcribed spacer; pistachio decline; Pistacia vera. Abbreviations used: EF (elongation factor); ITS (internal transcribed spacer); MEA (malt extract agar); OA (oatmeal agar); PDA (potato-dextrose agar); SDW (sterile distilled water); WA (water agar). Citation: Mohammadi, H.; Sarcheshmehpour, M.; Mafi, E. (2015). Fungal trunk pathogens associated with wood decay of pistachio trees in Iran. Spanish Journal of Agricultural Research, Volume 13, Issue 2, e1007, 10 pages. http://dx.doi.org/10.5424/sjar/2015132-6560. Received: 20 Jul 2014. Accepted: 5 May 2015 Copyright © 2015 INIA. This is an open access article distributed under the Creative Commons Attribution License (CC by 3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Funding: The authors received no specific funding for this work Competing interests: The authors have declared that no competing interests exist. Correspondence should be addressed to Hamid Mohammadi: hmohammadi@uk.ac.ir.


Introduction
The genus Pistacia (Anacardiaceae) includes 11 species (Zohary, 1952). Among these, pistachio (Pistacia vera L.), wild pistachio (P. atlantica Desf. subsp. mutica (Fisch. & Mey.) Rech. F) and P. khinjuk Stocks, are the species that occur in Iran (Sheibani, 1996). Pistachio is the fifth most important commercial nut crop in the world and has been cultivated in different countries such as Iran, Turkey, other Mediterranean countries, and USA. According to the Food and Agriculture Organization (FAO, 2012), approximately 85% of the world's pistachio production currently comes from these countries. Wild pistachio is a dominant native Pistacia species in Iran. In spring of 2012 a yellowing and wilting of pistachio trees was noticed in Kerman province (south-eastern Iran). Examination of symptomatic branches revealed the presence of differ-2 wild pistachio trees. Symptomatic tissues were cut into disks, surface-disinfected by immersing in 1.5% solution of NaOCl for 30 s, and rinsed in sterile distilled water (SDW). About 10 wood pieces (3×3 mm) were taken from the margin between discolored and healthy tissue and plated onto malt extract agar (MEA, 2% malt extract, 1.5% agar; Merck, Germany) supplemented with 100 mg/L streptomycin sulphate (MEAS). Plates were incubated at 25ºC in the dark for 2 weeks, and all colonies were transferred to potato dextrose agar (PDA; Merck, Germany). Single conidial cultures were obtained from each isolates for further study. Fungal isolates that did not sporulate were purified by hyphaltipping.

Morphological identification of fungal isolates
The initial identification of fungal isolates was made based on colony morphology, colony color and growth on MEA. Phaeoacremonium isolates were identified by macroscopic characters such as colony texture, color and pigment production on PDA, MEA, and oatmeal agar (OA; 30 g oatmeal; 15 g agar; Merk, Germany). Microscopic mounts were made from aerial mycelium of the isolates on MEA. Radial growth of isolates was measured after 16 days at 25 °C (Mostert et al., 2006). Botryosphaeriaceae spp. were identified by colony and conidial morphology (Phillips, 2002). These isolates were induced to sporulate by transferring their pure cultures on 2% water agar (WA, 2% agar; Merck, Germany) containing doubleautoclaved pine needles and incubated at 25 °C under 12 h photoperiod. Isolates were checked out weekly for formation of fruiting structures and conidia. Conidial morphology from pycnidia was recorded using a compound microscope. Fifty microscopic measurements of each type of structures were made for all studied isolates.

DNA isolation and molecular identification of fungal isolates
Fungal cultures were grown on PDA, incubated at 25 °C for 10 days. Mycelium (approximately 50 mg) was scraped from the surface of cultures and ground to a fine powder in liquid nitrogen using a mortar and pestle. Total DNA was isolated using the Peq Gold Fungal DNA mini Kit (Roche, Germany) according to the manufacturer's protocols. DNA was visualized on 0.1% agarose gels stained with ethidium bromide and the DNA samples were kept at -20 °C until used for PCR amplification. For species of Phaeoacremonium, In recent years, numerous species of Phaeoacremonium W. Gams, Crous & M.J. Wingf. have been isolated and reported from different plants. Species of this genus are known to cause die-back or decline symptoms on various woody hosts worldwide (Mostert et al., 2006). Forty three species of Phaeoacremonium have been described from different plant species worldwide (Crous et al., 1996;Dupont et al., 2000;Groenewald et al., 2001;Mostert et al., 2005Mostert et al., , 2006Damm et al., 2008;Essakhi et al., 2008;Graham et al., 2009;Gramaje et al., 2009Gramaje et al., , 2012Gramaje et al., , 2014Raimondo et al., 2014;Úrbez-Torres et al., 2014). Thus far, 10 species of this genus have been reported from various woody trees in Iran (Mostert et al., 2006;Gramaje et al., 2009;Mohammadi & Banihashemi, 2012;Mohammadi, 2012Mohammadi, , 2013Mohammadi, , 2014Mohammadi et al., 2013aMohammadi et al., , 2014Soltaninejad et al., 2013;Kazemzadeh Chakusary et al., 2014;Sami et al., 2014), however the occurrence of these species and associated Botryosphaeriaceae spp. in pistachio orchards have not been investigated in this country. Although several fungal species have been isolated from pistachio trees with decline symptoms, little is known about the aetiology of these decline diseases of pistachio trees in Iran. Therefore, the goal of this study was to (i) identify the different fungal species associated with the disease by means of morphological and molecular studies, and (ii) evaluate the pathogenicity of the different fungi in one pistachio cultivar planted in Iran.

Sampling and fungal isolation
In recent years, there has been a noticeable increase in the incidence of yellowing and dieback symptoms on pistachio trees (5-25 years old) in various orchards in Kerman province, Iran. Between 2011 and 2013, 19 pistachio orchards from the main pistachio production areas (about 1,400 ha) in the Kerman province were surveyed to determine the fungi associated with trunk diseases. Affected pistachio trees showing yellowing and dieback and various symptoms in wood, including necrosis, black vascular streaking, or discolored tissues were collected in each orchard (1-4 trees per orchard). Similar symptoms associated with resin exudation were also observed on wild pistachio trees (200-400 years old, 1,300 ha) in Saadat Shahr (Fars province). In total, 32 and 19 pistachio and wild pistachio trees were evaluated, respectively. Woody samples were collected from each symptomatic tree (1-3 branches per tree) and analyzed for internal wood symptoms. In total, 89 samples were collected: 68 from pistachio and 21 from 3 Fungal trunk pathogens associated with pistachio decline lated fungi so as to fulfill Koch's postulates. Cultures were incubated at 25 °C for further identification of the isolated fungi.
One-way analysis of variance (ANOVA) using the SAS Software v 9.1 (SAS Inst., Cary, NC, USA) was performed in order to evaluate differences in the extent of wood discoloration induced by fungal isolates. The LSD test was used for comparison of treatment means at p<0.05. Dunnett's t test was used to assess significant differences at p<0.05 in the extent of upward and downward wood discoloration between the control mean and the treatment means on pistachio branches.

Field symptoms and sampling
A total of 32 symptomatic pistachio trees were sampled from different orchards (Table 1). Fungal trunk pathogens were isolated from 28 trees (87.50%). Common symptoms observed in the sampled pistachio orchards included: yellowing, die-back, shoot canker, and plant death. Internal symptoms included: central necrosis, watery necrosis, brown to black internal wood discoloration, brown to black streaking and wedgeshaped necrosis when branches were cut transversely, and dark brown to black streaking when affected parts were cut longitudinally (Fig. 1). In this study, 16 wild pistachio trees (P. atlantica subsp. mutica) showing yellowing, dieback and canker symptoms were also sampled from Fars province from which several fungal species were isolated in nine trees (60%). One of the most common external symptoms on wild pistachio trees was dieback of the branches associated with resin exudation. In branches with dieback symptoms, a circular necrosis and wedge-shaped wood discoloration was generally observed when symptomatic parts were cross-sectioned. During this study some wild pistachio trees showed severe decline symptoms and eventually died.

Fungal isolation and identification
A total of 103 fungal isolates were recovered from pistachio trees showing decline symptoms and wood discoloration in cross section. Based on morphological and cultural characteristics, 32 isolates of Phaeoacremonium (31.1% of total isolates) were obtained and identified from diseased pistachio trees. The most common species isolated from orchards were Pm. parasiticum and Paecilomyces variotii ± 600 bp of the 5' end of the β-tubulin gene (BT) was amplified as described by Mostert et al. (2006) using the primer sets T1 (O'Donnell & Cigelnik, 1997) and Bt2b (Glass & Donaldson, 1995). Morphological identifications of Botryosphaeriaceae spp. were confirmed by sequence analysis of the internal transcribed spacer (ITS) nrDNA region and a section of the translation elongation factor 1-alpha (EF1-α) gene using the primers ITS1 and ITS4 (White et al., 1990), and EF1-728F and EF1-986R (Carbone & Kohn, 1999), respectively. Amplification of the ITS region and EF1-α gene were performed as described by Úrbez-Torres et al. (2008). PCR products were purified and sequenced in both directions by Macrogen Inc. Sequencing Center (Seoul, South Korea).

Pathogenicity tests
Twelve isolates representing the following species: Pm. parasiticum (isolates PRPIS1 and KER-U-PRPISM1), Pm. aleophilum (isolates PLPIS1 and PLPIS2), Pm. cinereum (isolates PCPIS1 and PCPIS2), Pm. viticola (isolates IRNHM-PV103), N. parvum (isolates NPPIS1 and NPPIS2), B. dothidea (isolates BDPIS1 and BDPIS2), and Dothiorella viticola (isolate Ker-U-SV1) were used. Inoculum was prepared for each isolate from PDA cultures incubated at 25 °C in the dark for 3 weeks, except for isolates of Botryosphaeriaceae that were incubated for 10 days. Inoculation of pistachio trees was carried out in one pistachio orchard (cv. Fandoghi, 20-year-old) located in Kerman. Thirteen plants showing neither any external symptoms nor any wood discoloration were selected and inoculated in April 2013. For each isolate, four branches of pistachio trees were chosen randomly and the outer bark at the inoculation area cleaned and sprayed with 70% ethanol. A mycelial plug (4 mm in diameter and 3 mm thick) taken from the margin of an actively growing colony on PDA was put into a 1 cm deep hole drilled radially with an ethanol-disinfected borer into a branch. The inoculated areas were protected by moist cotton and wrapped with Parafilm® (Pechiney Plastic Packaging, Menasha, USA) to prevent desiccation. Four additional branches were inoculated with sterile PDA plugs, which served as controls. Four months after inoculation, the branches were collected, and brought to the laboratory and split lengthwise through the inoculation site. The length of wood necroses was measured upwards and downwards from the point of inoculation, and total necrosis was calculated. Surface-sterilized (1 min in 5% NaOCl) woody pieces taken from the necrotic tissues were plated on PDA to re-isolate the inocu-4 lowing and dieback symptoms. Eight isolates of Pm. aleophilum were also isolated from three pistachio trees (10.7% of positive samples) showing dieback and brown to black streaking and brown to black wood discoloration symptoms in cross section. Two isolates of Pm. cinereum were isolated from a pistachio tree (3.6% of positive samples) showing leaf yellowing and dieback and brown to black streaking in cross section. One isolate of Pm. viticola was also obtained from one pistachio tree showing leaf yellowing and brown to black streaking in cross section. In the current study, five isolates of N. parvum were obtained from two trees (7.1% of positive samples) showing dieback and wedge-shaped necrosis in cross section. Four isolates of B. dothidea were also isolated from one pistachio tree (3.6% of positive samples) showing shoot canker and wedge-shaped necrosis in cross section. In our work, eight isolates of a Pleurostomophora sp. were also obtained from pistachio trees showing yellowing and dieback symptoms. In this study only one isolate of D. viticola also was obtained from a wild pistachio tree showing dieback, resin exudation and wedge shape necrosis in cross section. The highest incidence of Phaeoacremonium isolation was from brown to black necrosis (62.50%), followed by central necrosis (18.8%), with frequencies of 20.4% and 9.7% of total fungal isolates, respectively. Pm. aleophilum, N. parvum, B. dothidea, Pm. cinereum, and Pm. viticola were identified in 7.8, 4.9, 3.9, 1.9 and 1.0% of all isolates, respectively. Numerous isolates of Aspergillus spp., Penicillium spp., Pleurostomophora sp., Nattrassia mangiferae, Trichoderma spp., and other phialidic fungi, were always associated with diseased pistachio trees in different areas. BLASTn searches in GenBank showed that β-tubulin sequences of Phaeoacremonium isolates had 100% identity with isolates of Pm. aleophilum PAL2-A (GenBank GQ903709), Pm. parasiticum CBS 109665 (Gen-Bank AY579312), Pm. cinereum Pm7 (GenBank FJ517163) and Pm. viticola (GenBank EU128094

Pathogenicity tests
Mean lengths of the extent of wood discolorations caused by Botryosphaeriaceae and Phaeocremonium species on inoculated pistachio branches are shown in Table 2. Results of the pathogenicity tests showed that all fungal species were pathogenic on inoculated pistachio trees (F = 188.68 and p<0.001). All the isolates produced brown to dark wood streaking or discoloration upward and downward from the point of inoculation as shown after removing the superficial bark (Fig. 2). N. parvum was the most virulent showing the longest (p<0.05) lesion length of 73.8 ± 1.1 mm followed by Pm. parasiticum and B. dothidea that did not differ significantly (p<0.05) between them. Pm. cinereum, Pm. viticola and D. viticola showed similar (p<0.05) lesion lengths, ranging from 48.3 to 50.8 mm. Pm. aleophilum induced the shortest (p<0.05) lesion length. The length of lesions produced by all the fungi used in the inoculation tests were longer (p<0.05) than that reached at the control (6.8 ± 0.9 mm). After cross sectioning the inoculated branches, it was determined that Phaeoacremonium ssp. produced necrotic wood tissues while the Botryosphaeriaceae spp. produced wedge-shaped necrosis (Fig. 2). Inoculated species were re-isolated at frequencies ranged between 40.0% (Pm. cinereum) and 83.4% (N. parvum) on pistachio. As showed on Fig. 3 all inoculated species caused longer basipetal than acropetal lesions in all treatments. The Dunnett's t test showed significant differences between treatments with Phaeoacremonium and Botryosphaeriaceae spp. compared to the control treatment (Fig. 3).

Discussion
In the present study we report for the first time the isolation and pathogenicity of Phaeoacremonium and brown to black wood discoloration (12.50%) and watery necrosis (6.25%), while N. parvum, B. dothidea, and D. viticola were isolated only from wedge-shaped necrosis.
Our study has shown that pistachio trees represent a rich catch-crop for species of the genus Phaeoacremonium (Pm. parasiticum, Pm. aleophilum, Pm. cinereum and Pm. viticola). Phaeoacremonium species are known to cause dieback or decline symptoms on various woody hosts especially on grapevine, as the causal agents of esca and Petri disease (Mostert et al., 2006). Although, numerous species of this genus have also been associated with trunk diseases of a various woody hosts other than grapevine worldwide Damm et al., 2008;Cloete et al., 2011;Ismail et al., 2013;Gramaje et al., 2014). Of the Phaeoacremonium species, 65.6% of the isolates were identified as Pm. parasiticum, which was found in all the sampled areas during this study. This species has been isolated from Quercus virginiana in USA (Halliwell, 1966); Nectandra sp. in Costa Rica (Hawksworth et al., 1976), Prunus avium in Greece (Rumbos, 1986), Prunus armeniaca in South Africa (Damm et al., 2008) and Tunisia (Hawksworth et al., 1976); Actinidia chinensis (Di Marco et al., 2004) and Olea europea in Italy (Nigro et al., 2013); Phoenix dactylifera in Iraq (Hawksworth et al., 1976) and Iran (Mohammadi, 2014); and pome fruit trees (Sami et al., 2014) and Cupressus sempervirens  in Iran.
Botryosphaeriaceae species associated with wood decay of pistachio trees in Iran. The present survey has also shown several internal wood symptoms similar to those observed on grapevine worldwide (Luque et al., 2009;Van Niekerk et al., 2011). The most frequent external symptoms were leaf yellowing and dieback while central necrosis was recorded as the most frequent internal wood discoloration associated with pistachio decline. In our case, we found at least five different kinds of wood necrosis. Visual aspects of the different wood lesions are based on the necrotic types established by Larignon & Dubos (1997) and Van Niekerk et al. (2011) for grapevine trunk diseases. Similar wood lesions have been also reported on other woody hosts such as pome fruit trees  Fungal trunk pathogens associated with pistachio decline in this study, the lesions caused by N. parvum were longer than those caused by the other species. In a preliminary study, Mousavi et al. (2014a, b) obtained similar results when testing the pathogenicity of Botryosphaeriaceae and Phaeoacremonium spp. on detached pistachio shoots. These results were consistent with other pathogenicity studies that reported N. parvum to be one of the most pathogenic Botryosphaeriaceae spp. on grapevines in several countries including South Africa (Van Niekerk et al., 2004), Spain , Australia (Savocchia et al., 2007), USA (Úrbez-Torres &Gubler, 2009) andNew Zealand (Billones-Baaijens et al., 2013). Botryosphaeria dothidea is one of the main pathogens of pistachio (Michailides, 1991), but was previously reported to be weakly pathogenic to healthy vines, Eucalyptus and Syzygium in South Africa (Van Niekerk et al., 2004;Pavlic et al., 2007;Slippers et al., 2007). Phaeoacremonium aleophilum produced smaller lesions than the other species inoculated on pistachio. This is consistent with previous pathogenicity tests performed on pear in South Africa (Cloete et al., 2011). Phaeoacremonium parasiticum and D. viticola, which were obtained from wild pistachio, were pathogenic on pistachio trees. Recently, D. viticola has been isolated and reported from pistachio trees in Greece by Chen et al. (2014b). With the exception of D. viticola, all the other species were reported from grapevine in Iran. Therefore wild pistachio in Iran should be considered as a potential source of this species for vineyards and pistachio orchards.
According to pathogenicity tests, all isolates used in this study, caused longer basipetal than acropetal lesions on pistachio. Úrbez-Torres et al. (2008) obtained similar results when testing the pathogenicity of D. seriata and L. theobromae on 1-year-old and green shoots of grapevine. This study is the first to report on the occurrence and pathogenicity of Phaeoacremonium spp. on pistachio trees. Based on our knowledge, this also represents the first record of Pleurostomophora sp. on pistachio and Pm. parasiticum and D. viticola on wild pistachio. A species of Pleurostomophora, Pl. richardsiae, was reported by Eskalen et al. (2004) as being a pathogen of grapevine. Additionally, Carlucci et al. (2013) have reported Pl. richardsiae as the main agent of wilting of apical leaves and cankers in olive trees in Italy. Eskalen et al. (2004) and Rolshausen et al. (2010) showed that Pl. richardsiae can infect pruning wounds of grapevine. Although most of the Phaeoacremonium species have been isolated from grapevine worldwide, members of this genus are not host specific. The same species can occur on several different woody hosts and more than one species can occur on a single host. In this way, various woody trees In our study, 25% of Phaeoacremonium species were identified as Pm. aleophilum. This species is the most common Phaeoacremonium species found associated with esca and Petri diseases in grapevines (Mostert et al., 2006) however, it has been also isolated from other woody trees, such as A. chinensis and O. europea in Italy (Crous & Gams, 2000), Malus domestica in South Africa (Cloete et al., 2011) and USA (Úrbez-Torres et al., 2013), and Prunus spp. (Damm et al., 2008 and Pyrus communis in South Africa (Cloete et al., 2011). Other Phaeoacremonium species were isolated in low frequency and included Pm. cinereum and Pm. viticola that were isolated in two and one cases, respectively. Phaeoacremonium cinereum has been previously reported affecting grapevines in Iran and Spain (Gramaje et al., 2009). This species has been recently isolated from necrotic wood of walnut trees in Iran (Mohammadi et al., 2013c). Regarding Pm. viticola, this species was reported from grapevine in Iran, France, USA and South Africa (Moster et al., 2006), from A. chinensis in France (Hennion et al., 2001), from Prunus armeniaca, Prunus salicina (Damm et al., 2008) and Pyrus communis in South Africa (Cloete et al., 2011), and from Sorbus intermedia in Germany (Mostert et al., 2006). Two Botryosphaeriaceae species, B. dothidea and N. parvum were isolated from pistachio trees showing a wedge-shaped wood discoloration. On grapevine, symptoms of Botryosphaeria dieback are characterized by wedge-shaped cankers, wood necrosis, bud death, and plant dieback (Úrbez-Torres, 2011) and several species of Botryosphaeriaceae were isolated from grapevines with these symptom types. B. dothidea has been found on numerous woody trees, including stone and pome fruit trees in South Africa (Damm et al., 2007;Slippers et al., 2007), pistachio trees in California (Chen et al., 2014b), olive fruits (Moral et al., 2010), almond trees in Spain (Gramaje et al., 2012) and grapevine in several countries such as Spain (Armengol et al., 2001) and Portugal (Phillips, 2002). Recently, B. dothidea has been isolated and reported from pistachio trees showing panicle blight in Iran (Mohammadi et al., 2015). Neofusicoccum parvum has been isolated from pome and stone fruit trees in South Africa , from avocado in California (McDonald et al., 2009), from almond (Inderbitzin et al., 2010), English walnut (Chen et al., 2014a), and grapevine in California (Úrbez-Torres & Gubler, 2009). In Iran, both species have been isolated from cypress  and grapevine (Arabnezhad & Mohammadi, 2012;Mohammadi et al., 2013b).
The pathogenicity tests on pistachio trees showed that all inoculated species used were pathogenic on this host. Of the seven species tested on pistachio branches can act as alternative hosts for these pathogens. According to our results, pistachio and wild pistachio trees, as well as other woody hosts, namely cypress , date palm (Mohammadi, 2014), pome fruit trees (Sami et al., 2014), walnut (Mohammadi et al., 2013c), and stone fruit trees (Soltaninejad et al., 2013), should be considered as potential inoculum sources of viable inoculum for trunk disease pathogens in Iran, from which grapevines could be infected and these hosts could serve as an additional mode of pathogen survival in the absence of grapevine plants. Conversely, fungal trunk pathogens could have spread from grapevine plants to these woody hosts. Future studies should be undertaken to clarify the identities of other Phaeoacremonium and Botryosphaeriaceae species fungi on pistachio and wild pistachio trees in this country.