Surveys of 15 pistachio orchards were conducted from 2010 to 2017 in Bronte and Adrano (Catania province, eastern Sicily) and Agrigento and Caltanissetta provinces (western Sicily). Approximately 10 samples per orchard showing cankered twigs and branches from declining pistachio plants were randomly collected for analysis (Fig. 1). Sub-cortical and wood fragments (about 5 × 5 mm) were cut from the lesion margins between affected and healthy tissues. In addition, from one orchard in Bronte, twigs were also sampled from asymptomatic pistachio plants. Subsequently, tissue pieces were disinfected by soaking in 70% ethanol for 5 s, 4% sodium hypochlorite for 90 s, rinsed in sterile water for 60 s and dried on sterile filter paper in a laminar flow cabinet. The fragments were placed on to 1.5% (w/v) malt extract agar (MEA, Oxoid, Basingstoke, UK) and 2% potato dextrose agar (PDA, Oxoid), incubated at room temperature (25 ± 5 °C) and examined for fungal growth. Numerous slow-growing cultures were obtained and single-conidial isolations were performed with conidia collected from pycnidia produced on those cultures within one month of incubation at room temperature under natural light conditions. More than 80 single-spore isolates were obtained from symptomatic and asymptomatic tissue isolations. Amongst these, 71 isolates were characterised by molecular and phylogenetic analysis (Table 1) and the four isolates ISPaVe1958, ISPaVe2105, ISPaVe2106 and ISPaVe2148 were considered for morphological, taxonomic and pathogenic studies. For a summary of sampling information of these isolates, see Suppl. Material 1.

Figure 1. 

Symptoms caused by Liberomyces pistaciae on Pistacia vera in vivo. a Plant killed by canker on trunk b Twigs dieback c, d Shoots wilted on infected twig e Gum and cracking of the trunk f, g Internal tissue of trunk cankers h Gum exudation on branch i Internal dark discolouration in cross section of branch j Necrotic tissue in longitudinal section of twig k, l External and internal cankers on twigs.

For morphological investigations, cultures were grown on MEA, PDA and 2% corn meal agar (CMA, Sigma-Aldrich) supplemented with 2% w/v dextrose (CMD). Moreover, pycnidial formation was assessed on artificially inoculated sterilised pistachio twigs incubated in a moist chamber. The isolates used in this study are maintained in the culture collections of the Dipartimento di Agricoltura, Alimentazione e Ambiente, University of Catania (PV) and of the CREA-DC (ex CREA-PAV), the ex-type isolate ISPaVe1958 of the new pistachio pathogen was deposited at the Westerdijk Fungal Biodiversity Institute (CBS), Utrecht, The Netherlands and the holotype specimen in the Fungarium of the Department of Botany and Biodiversity Research, University of Vienna (WU).

For investigations of temperature-growth relationships of the new pistachio pathogen, the holotype isolate ISPaVe1958 and the more recent isolate ISPaVe2148 were used. Agar plugs (5 mm diam.) were taken from the edge of actively growing cultures on MEA and transferred on to the centre of 9 cm diam. Petri dishes containing 1.5% MEA. Three replicate plates were incubated at 10, 15, 20, 25, 30 and 35 °C in the dark and measurements were taken after 21 d at right angles along two lines intersecting the centre of the inoculum and the mean growth rates plus and minus the standard deviation were calculated.

The holotype isolate ISPaVe1958 (CBS 128196) of the new pistachio pathogen and the type specimens of Asteromella pistaciarum deposited in the Natural History Museum of Vienna (W) were morphologically examined. For light microscopy, squash mounts and hand sections of pycnidia were made using a razor blade and observed in tap water or in 3% KOH. Methods of microscopy included stereomicroscopy using a Nikon SMZ 1500 equipped with a Nikon DS-U2 digital camera and light microscopy with Nomarski differential interference contrast (DIC) using the compound microscope Zeiss Axio Imager.A1 equipped with a Zeiss Axiocam 506 colour digital camera. Images were captured and measured with NIS-Elements D v. 3.0 or with the Zeiss ZEN Blue Edition software. For certain images of pycnidia, the stacking software Zerene Stacker v. 1.04 (Zerene Systems LLC, Richland, WA, USA) was used. Measurements are reported as maximum and minimum in parentheses and the range representing the mean plus and minus the standard deviation of a number of measurements given in parentheses.

Pathogenicity tests with four fungal strains of the undescribed pistachio pathogen were performed to satisfy Koch’s postulates. Trials were carried out outdoors and in a growth chamber at 25 ± 1 °C. Potted 5-yr-old plants of Pistacia vera grafted on to P. terebinthus were used for artificial inoculations. Three plants for each isolate and six inoculation sites for each plant were considered.

Inoculations were made on stems and twigs after removing a 5 mm diam. bark disc with a cork borer, replacing it with a 5 mm plug from a 14-d-old PDA culture and covering it with sterile wet cotton, wrapped with parafilm (Pechney Plastic Packaging Inc., Chicago, USA) and aluminium foil to prevent contamination and desiccation. An equivalent number of plants and inoculation sites were inoculated with sterile PDA plugs as controls. The inoculated plants were observed every week. Symptom typology and the length of lesions were assessed after 12 months. To fulfil Koch’s postulates, re-isolation was conducted following the same procedure as described above for isolations. Tissue fragments were plated on MEA or PDA and morphological and molecular identifications by sequencing the ITS rDNA were performed.

The extraction of genomic DNA from pure cultures was performed as reported in previous studies (Voglmayr and Jaklitsch 2011, Jaklitsch et al. 2012, Guarnaccia and Crous 2017) by using the DNeasy Plant Mini Kit (QIAgen GmbH, Hilden, Germany) or the Wizard Genomic DNA Purification Kit (Promega Corporation, WI, USA). For the ex-type strain of the new species, the complete internal transcribed spacer region (ITS1-5.8S-ITS2) and a ca. 0.9 kb fragment of the large subunit nuclear ribosomal DNA (nLSU rDNA) were amplified and sequenced as a single fragment with primers V9G (de Hoog and Gerrits van den Ende 1998) and LR5 (Vilgalys and Hester 1990); the complete ITS region of the other strains was amplified with primers ITS5 and ITS4 (White et al. 1990); the RNA polymerase II subunit 2 (rpb2) gene was amplified with primers fRPB2-5F2 and fRPB2-7cR (Liu et al. 1999, Sung et al. 2007) or dRPB2-5f and dRPB2-7r (Voglmayr et al. 2016); and the beta-tubulin (tub2) gene with primer pairs T1 and T22 or Tub2Fd and Bt-2b (O’Donnell and Cigelnik 1997, Aveskamp et al. 2009). The PCR product was purified using an enzymatic PCR cleanup (Werle et al. 1994) as described in Voglmayr and Jaklitsch (2008). DNA was cycle-sequenced using the ABI PRISM Big Dye Terminator Cycle Sequencing Ready Reaction Kit v. 3.1 (Applied Biosystems, Warrington, UK) with the same primers as in PCR; in addition, primers ITS4, LR2R-A (Voglmayr et al. 2012) and LR3 (Vilgalys and Hester 1990) were used for the ITS-LSU fragment. Sequencing was performed on an automated DNA sequencer (3730xl Genetic Analyser, Applied Biosystems).

NCBI BLASTn searches of the ITS and LSU of the undescribed pistachio pathogen revealed members of Xylariales as closest matches. For phylogenetic analyses, two combined matrices were produced; GenBank accession numbers of the sequences used in the phylogenetic analyses are given in Table 1. A combined ITS-LSU matrix was generated to reveal the phylogenetic position of the undescribed pistachio pathogen within Xylariales. For this, representative GenBank sequences of Xylariales were selected from Jaklitsch et al. (2016) and supplemented with some additional GenBank sequences; six taxa of Sordariomycetes were added as outgroup. The second combined matrix contained three loci (ITS, rpb2, tub2) sequenced for 68 isolates of the undescribed pistachio pathogen; in addition, GenBank sequences of four accessions of Delonicicolaceae and of six additional members of Xylariales were added and two species of Diaporthales were used as outgroup (Guarnaccia and Crous 2017, Voglmayr et al. 2017).

All alignments were produced with the server version of MAFFT (www.ebi.ac.uk/Tools/mafft), checked and refined using BioEdit v. 7.0.9.0 (Hall 1999). After exclusion of ambiguously aligned regions and long gaps, the final ITS-LSU matrix contained 1340 nucleotide characters and the three loci matrix 1941 nucleotide characters (660 from ITS, 781 from rpb2 and 500 from tub2). The alignment and phylogenetic trees were deposited in TreeBASE (http://purl.org/phylo/treebase/phylows/study/TB2:S23059).

Maximum Likelihood (ML) analyses were performed with RAxML (Stamatakis 2006) as implemented in raxmlGUI v. 1.3 (Silvestro and Michalak 2012), using the ML + rapid bootstrap setting and the GTRGAMMAI substitution model with 1000 bootstrap replicates.

Maximum Parsimony (MP) analyses were performed with PAUP v. 4.0a161 (Swofford 2002), using 1000 replicates of heuristic search with random addition of sequences and subsequent TBR branch swapping (MULTREES option in effect, steepest descent option not in effect, COLLAPSE command set to MINBRLEN). Molecular characters were unordered and given equal weight; gaps were treated as missing data. Bootstrap analyses with 1000 replicates were performed in the same way, with 5 rounds of replicates of heuristic search with random addition of sequences and subsequent TBR branch swapping during each bootstrap replicate, with each replicate limited to 1 million rearrangements in the analysis of the three-loci matrix.

Cankers and decline symptoms caused by the undescribed pistachio pathogen were detected in 10 orchards amongst the 15 investigated. The disease was primarily observed in the winter period and during late spring.

In the Bronte and Adrano areas (Catania province), symptoms were observed during the dormant season. Symptomatic plants showed gum exudation and often bark scaling on trunk and/or branches. When bark scaling occurred, it appeared as cracking and peeling of the bark. On trunks and large branches, cankers first appeared as visible dead circular areas that developed in the bark, which subsequently became dark and sunken. From that point onwards, infected areas expanded in all directions but much faster along the main axis of the stem, branch or twig. Under some environmental conditions, the host produced callus tissue around dead areas limiting the canker. Under the bark, cankers were characterised by discolouration and necrotic tissues and, in some cases, these extended to the vascular tissues and pith (Figs 1, 2).

During the active growing season, the symptomatic plants also showed canopy decline. Inflorescences and shoots, originating from infected branches or twigs, wilted and died. When the trunk was girdled by a canker, a collapse of the entire plant occurred (Fig. 1).

More than 80 single-spore isolates were obtained from symptomatic and a few also from asymptomatic pistachio plants. Amongst these, 71 isolates were characterised by molecular phylogenetic analyses and 68 deposited at the Westerdijk Fungal Biodiversity Institute, Utrecht, Netherlands (Table 1).

Figure 2. 

Symptoms reproduced from mycelial plug inoculation with Liberomyces pistaciae on 5-year-old potted plants of Pistacia vera. Stem symptoms after a, b 3 wks c 6 months d, e 12 months f, g Cankers on twigs.

The initial symptom, observed 3 weeks after artificial fungal inoculation, was gum exudation produced around the point inoculated. After removing the bark, a dark discolouration and necrotic tissue were visible (Figs 2a, b). After 6 months, external cankers were observed in correspondence with the inoculated sites and small cracks were present in the sunken central area (Fig. 2c). After 12 months, symptoms were very obvious and similar to the cracked cankers observed in nature. Long and deep cracks were evident on the sunken area that defined the cankered lesion. After removing the bark, it was evident that the pathogen was able to colonise the wood and long discolourations were present (Figs 2d, e). After 12 months from inoculation, the length of lesions ranged from 12 to 45 mm. For ISPaVe1958 and ISPaVe2105, length lesions averaged 16.7 ± 3.0 and 31 ± 1.0 mm, respectively, while for ISPaVe2106 and ISPaVe2148, 18 ± 0.0 mm and 29.7 ± 2.0 mm. Controls measured 4.0 ±1.0 mm in average. Cultures, morphologically identical with the new pistachio pathogen, were re-isolated from these cankers, fulfilling Koch’s postulates. Moreover, ITS sequence comparison of these re-isolated cultures confirmed the species identity.

The growth rate experiments revealed 30 °C as optimal temperature for both isolates with an evidently better growth of the holotype ISPaVe1958 at this temperature in comparison to ISPaVe2148 (Fig. 3).

Figure 3. 

Temperature-growth relationships of the holotype isolate ISPaVe1958 compared to the more recent isolate ISPaVe2148 of Liberomyces pistaciae on 1.5% MEA. Mean growth rates (mm) plus and minus the standard deviation, calculated on three replicates after 21 d of incubation, are shown.

Of the 1340 nucleotide characters of the ITS-LSU matrix, 519 are parsimony informative. The best ML tree (-lnL = 19486.775), revealed by RAxML, is shown as a phylogram in Fig. 4. Maximum parsimony analyses revealed 14 MP trees 4008 steps long (not shown). The backbone of the MP trees differs in several deeper unsupported nodes from the ML tree (not shown); notably in the MP tree, the Liberomyces clade was not the most basal node of Xylariales, although without support (not shown).

In the ML and MP analyses of the ITS-LSU matrix (Fig. 4), the Xylariales received maximum support, but backbone support within Xylariales was low to absent. The new species clustered within the Liberomyces clade, which was sister to Delonicicola siamense (Delonicicolaceae, Xylariales). The Delonicicolaceae received high support (100% ML and 99% MP bootstrap support), but their closest relatives remained unclear due to lack of significant backbone support in all deeper nodes (Fig. 4). The sister-group relationship of L. saliciphilus and L. macrosporus received moderate support (81% ML and 89% MP bootstrap support).

Figure 4. 

Phylogram of the best ML tree (-lnL = 19486.775) revealed by RAxML from an analysis of the combined ITS-LSU matrix of selected Xylariales, showing the phylogenetic position of Liberomyces pistaciae (bold) within Delonicicolaceae. ML and MP bootstrap support above 50% are given above or below the branches.

Of the 1941 nucleotide characters of the ITS-rpb2-tub2 matrix, 743 are parsimony informative (201 from ITS, 343 from rpb2, 199 from tub2). The best ML tree (-lnL = 12820.324), revealed by RAxML, is shown as a phylogram in Fig. 5. Maximum parsimony analyses revealed 6 MP trees 2669 steps long, with a tree backbone identical to that of the ML tree (not shown).

The analyses of the ITS-rpb2-tub2 matrix (Fig. 5) revealed similar topologies to the analyses of the ITS-LSU matrix. The Xylariales and Delonicicolaceae received high support in ML and MP analyses. The new pistachio pathogen formed a genetically homogeneous clade with high to maximum support, confirming that all isolates sequenced belong to the same species. As in the ITS-LSU analyses, it was placed as sister to the highly supported Liberomyces saliciphilus-L. macrosporus clade with moderate support.

Figure 5. 

Phylogram of the best ML tree (-lnL = 12820.324) revealed by RAxML from an analysis of the combined ITS-rpb2-tub2 matrix of selected Xylariales, showing the phylogenetic position of Liberomyces pistaciae (bold) within Delonicicolaceae. The tree was rooted with two species of Diaporthales (Diaporthe limonicola, Juglanconis juglandina). ML and MP bootstrap support above 50% are given above the branches.