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Liberomyces pistaciae sp. nov., the causal agent of pistachio cankers and decline in Italy
expand article infoSalvatore Vitale, Dalia Aiello§, Vladimiro Guarnaccia|, Laura Luongo, Massimo Galli, Pedro W. Crous|, Giancarlo Polizzi§, Alessandra Belisario, Hermann Voglmayr#
‡ Centro di Ricerca Difesa e Certificazione, Roma, Italy
§ University of Catania, Catania, Italy
| University of Stellenbosch, Matieland, South Africa
¶ Westerdijk Fungal Biodiversity Institute, Utrecht, Netherlands
# University of Vienna, Vienna, Austria
Open Access

Abstract

A new canker and decline disease of pistachio (Pistacia vera) is described from Sicily (Italy). Observations of the disease and sampling of the causal agent started in spring 2010, in the area where this crop is typically cultivated, Bronte and Adrano (Catania province) and later extended to the Agrigento and Caltanissetta provinces. Isolations from the margins of twig, branch and stem cankers of declining plants resulted in fungal colonies with the same morphology. Pathogenicity tests on 5-year-old potted plants of Pistacia vera grafted on P. terebinthus reproduced similar symptoms to those observed in nature and the pathogen was confirmed to be a coloniser of woody plant tissue. Comparison of our isolates with the type of the apparently similar Asteromella pistaciarum showed that our isolates are morphologically and ecologically different from A. pistaciarum, the latter being a typical member of Mycosphaerellaceae. Asteromella pistaciarum is lectotypified, described and illustrated and it is considered to represent a spermatial morph of Septoria pistaciarum. Multi-locus phylogenies based on two (ITS and LSU rDNA) and three (ITS, rpb2 and tub2) genomic loci revealed isolates of the canker pathogen to represent a new species of Liberomyces within the Delonicicolaceae (Xylariales), which is here described as Liberomyces pistaciae sp. nov. (Delonicicolaceae, Xylariales). The presence of this fungus in asymptomatic plants with apparently healthy woody tissues indicates that it also has a latent growth phase. This study improves the understanding of pistachio decline, but further studies are needed for planning effective disease management strategies and ensuring that the pathogen is not introduced into new areas with apparently healthy, but infected plants.

Keywords

Delonicicolaceae, nut disease, pathogenicity, Pistacia vera, Xylariales, 1 new species

Introduction

Cases of pistachio tree decline with gummosis, leaf canopy thinning and fruit losses have been observed for several years in the area of Bronte (Catania province, Sicily, Italy), which is considered the most typical area where high-quality pistachios are produced in Italy (http://www.dibartolosrl.it/bronte-pistachios/). Although pistachio is characterised by good rusticity, it is subject to several fungal diseases known to afflict pistachio trees in the Mediterranean area. Of these, the most commonly reported are phylloptosis, leaf spots mainly caused by Septoria pistaciae, S. pistaciarum and Pseudocercospora pistacina, gum cankers by Cytospora terebenthi and branch and twig cankers by Botryosphaeria dothidea (Chitzanidis 1995, Teviotdale et al. 2002, Vitale et al. 2007, Crous et al. 2013). The latter is widespread and already present as a latent pathogen in numerous plant communities in various parts of the world (Marsberg et al. 2017). Amongst soil-borne pathogens, Verticillium dahliae and Phytophthora spp. are reported to be particularly damaging in California (Holtz 2008). Moreover, recently a new blight was reported on pistachio fruit caused by Arthrinium xenocordella in the Agrigento province, southern Italy (Aiello et al. 2018).

From spring 2010 onwards, surveys have been carried out in 15 pistachio orchards of Catania, Agrigento and Caltanissetta provinces, Sicily, where declining trees were present. Declining plants showed twig, branch and stem cankers associated with vascular necrosis and tree decline. Abundant gummosis often occurred in association with cankered lesions. The cankered area resulted in localised, sunken lesions with several central cracks. After removing the bark, discolouration and necrotic tissue were evident and lesions deepened into the woody tissue. A coelomycetous fungus with pycnidial conidiomata was consistently isolated from these lesions.

The aims of this study were thus to investigate the aetiology of the decline syndrome observed in Bronte and to provide morphological, taxonomic, phylogenetic and pathogenic evidence of the causal organism which proved to be an undescribed species of Liberomyces, which was initially misidentified as Asteromella pistaciarum.

Materials and methods

Field survey and isolation

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.

Morphological characterisation

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

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.

DNA extraction, PCR amplification and sequencing

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).

Phylogenetic analyses

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.

Results

Field survey and isolation

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.

Isolates and accession numbers used in the phylogenetic analyses.

Taxon Strain1,2,3 ITS3 LSU3 tub2 3 rpb2 3
Acrocordiella occulta CBS 140500ET KT949893 KT949893
Alnecium auctum CBS 124263ET KF570154 KF570154
Amphibambusa bambusicola MFLUCC 11-0617HT KP744433 KP744474
Amphisphaeria umbrina HKUCC 994 AF009805 AF452029
Anthostoma decipiens CBS 133221 KC774565 KC774565
Arthrinium phragmites CBS 135458HT KF144909 KF144956
Arthrinium saccharicola CBS 831.71 KF144922 KF144969
Barrmaelia macrospora CBS 142768ET KC774566 KC774566
Bartalinia robillardoides CBS 122705ET KJ710460 KJ710438 LT853252 LT853152
Basiseptospora fallax CBS 129020ET JF440983 JF440983
Beltrania rhombica CBS 141507 KX306749 KX306778
Beltraniella odinae NBRC 6774 006774014 006774014
Beltraniopsis neolitseae CBS 137974HT KJ869126 KJ869183
Biscogniauxia nummularia MUCL 51395ET JX658444 KT281894
Broomella vitalbae CBS 140412 KT949895 KT949895
Cainia graminis CBS 136.62 KR092793 AF431949
Calosphaeria pulchella CCTU 316 JX876610 JX876611
Camillea obularia ATCC 28093 KY610384 KY610429
Chaetosphaeria innumera MR 1175 AF178551 AF178551
Coniocessia maxima CBS 593.74HT GU553332 GU553344
Coniocessia nodulisporioides CBS 281.77IT GU553333 GU553352
Creosphaeria sassafras CBS 127876 KT949900 KT949900
Cryptovalsa rabenhorstii CBS 125574 KC774567 KC774567
Daldinia concentrica CBS 113277 AY616683 KT281895 KC977274 KY624243
Delonicicola siamense MFLUCC 15-0670HT MF167586 MF158345 MF158346
Diaporthe eres CBS 109767 KC343075 AF408350
Diaporthe limonicola CBS 142549HT MF418422 MF418582 MH797629
Diatrype disciformis CBS 197.49 - DQ470964
Discosia artocreas NBRC 8975 AB594773 AB593705
Eutypa lata CBS 208.87NT DQ006927 DQ836903
Graphostroma platystoma CBS 270.87 JX658535 AY083827
Hymenopleella hippophaeicola CBS 140410ET KT949901 KT949901
Hyponectria buxi UME 31430 - AY083834
Hypoxylon fragiforme MUCL 51264ET KC477229 KM186295
Idriella lunata MUCL 7551 KC775735 KC775710
Immersidiscosia eucalypti MAFF 242781 AB594793 AB593725
Juglanconis juglandina CBS 133343 KY427149 KY427234 KY427199
Kretzschmaria deusta CBS 163.93 KC477237 KT281896
Leiosphaerella praeclara CBS 125586ET JF440976 JF440976
Lepteutypa fuckelii CBS 140409NT KT949902 KT949902
Liberomyces macrosporus CCF 4028HT FR715522 FR715522 FR715498 FR715509
Liberomyces pistaciae CPC 31292 = CBS 144225 MH797562 MH797697 MH797630
Liberomyces pistaciae CPC 31293 MH797563 MH797698 MH797631
Liberomyces pistaciae CPC 31294 MH797564 MH797699 MH797632
Liberomyces pistaciae CPC 31295 MH797565 MH797700 MH797633
Liberomyces pistaciae CPC 31296 MH797566 MH797701 MH797634
Liberomyces pistaciae CPC 31297 MH797567 MH797702 MH797635
Liberomyces pistaciae CPC 31298 MH797568 MH797703 MH797636
Liberomyces pistaciae CPC 31299 MH797569 MH797704 MH797637
Liberomyces pistaciae CPC 31300 MH797570 MH797705 MH797638
Liberomyces pistaciae CPC 31301 MH797571 MH797706 MH797639
Liberomyces pistaciae CPC 31302 MH797572 MH797707 MH797640
Liberomyces pistaciae CPC 31303 MH797573 MH797708 MH797641
Liberomyces pistaciae CPC 31304 MH797574 MH797709 MH797642
Liberomyces pistaciae CPC 31305 MH797575 MH797710 MH797643
Liberomyces pistaciae CPC 31315 MH797576 MH797711 MH797644
Liberomyces pistaciae CPC 31316 MH797577 MH797712 MH797645
Liberomyces pistaciae CPC 31317 MH797578 MH797713 MH797646
Liberomyces pistaciae CPC 31318 MH797579 MH797714 MH797647
Liberomyces pistaciae CPC 31319 MH797580 MH797715 MH797648
Liberomyces pistaciae CPC 31320 MH797581 MH797716 MH797649
Liberomyces pistaciae CPC 31321 MH797582 MH797717 MH797650
Liberomyces pistaciae CPC 31322 MH797583 MH797718 MH797651
Liberomyces pistaciae CPC 31323 MH797584 MH797719 MH797652
Liberomyces pistaciae CPC 31324 MH797585 MH797720 MH797653
Liberomyces pistaciae CPC 31325 MH797586 MH797721 MH797654
Liberomyces pistaciae CPC 31326 MH797587 MH797722 MH797655
Liberomyces pistaciae CPC 31327 MH797588 MH797723 MH797656
Liberomyces pistaciae CPC 31328 MH797589 MH797724 MH797657
Liberomyces pistaciae CPC 31329 MH797590 MH797725 MH797658
Liberomyces pistaciae CPC 31330 MH797591 MH797726 MH797659
Liberomyces pistaciae CPC 31332 MH797592 MH797727 MH797660
Liberomyces pistaciae CPC 31333 MH797593 MH797728 MH797661
Liberomyces pistaciae CPC 33611 MH797594 MH797729 MH797662
Liberomyces pistaciae CPC 33612 MH797595 MH797730 MH797663
Liberomyces pistaciae CPC 33613 MH797596 MH797731 MH797664
Liberomyces pistaciae CPC 33614 MH797597 MH797732 MH797665
Liberomyces pistaciae CPC 33629 MH797598 MH797733 MH797666
Liberomyces pistaciae CPC 33630 MH797599 MH797734 MH797667
Liberomyces pistaciae CPC 33848 MH797600 MH797735 MH797668
Liberomyces pistaciae CPC 33849 MH797601 MH797736 MH797669
Liberomyces pistaciae CPC 33850 MH797602 MH797737 MH797670
Liberomyces pistaciae CPC 33851 MH797603 MH797738 MH797671
Liberomyces pistaciae CPC 33852 MH797604 MH797739 MH797672
Liberomyces pistaciae CPC 33853 MH797605 MH797740 MH797673
Liberomyces pistaciae CPC 33854 MH797606 MH797741 MH797674
Liberomyces pistaciae CPC 33855 MH797607 MH797742 MH797675
Liberomyces pistaciae CPC 33856 MH797608 MH797743 MH797676
Liberomyces pistaciae CPC 33857 MH797609 MH797744 MH797677
Liberomyces pistaciae CPC 33858 MH797610 MH797745 MH797678
Liberomyces pistaciae CPC 33859 MH797611 MH797746 MH797679
Liberomyces pistaciae CPC 33860 MH797612 MH797747 MH797680
Liberomyces pistaciae CPC 33861 MH797613 MH797748 MH797681
Liberomyces pistaciae CPC 33862 MH797614 MH797749 MH797682
Liberomyces pistaciae CPC 33863 MH797615 MH797750 MH797683
Liberomyces pistaciae CPC 33866 MH797616 MH797751 MH797684
Liberomyces pistaciae CPC 33867 MH797617 MH797752 MH797685
Liberomyces pistaciae CPC 33868 MH797618 MH797753 MH797686
Liberomyces pistaciae CPC 33869 MH797619 MH797754 MH797687
Liberomyces pistaciae CPC 33870 MH797620 MH797755 MH797688
Liberomyces pistaciae CPC 33871 MH797621 MH797756 MH797689
Liberomyces pistaciae CPC 33872 MH797622 MH797757 MH797690
Liberomyces pistaciae CPC 33873 MH797623 MH797758 MH797691
Liberomyces pistaciae CPC 33874 MH797624 MH797759 MH797692
Liberomyces pistaciae CPC 34204 MH797625 MH797760 MH797693
Liberomyces pistaciae CPC 34205 MH797626 MH797761 MH797694
Liberomyces pistaciae CPC 34206 MH797627 MH797762 MH797695
Liberomyces pistaciae CPC 34207 MH797628 MH797763 MH797696
Liberomyces pistaciae ISPaVe1958HT = CBS 128196 MH798901 MH798901 MH791335 MH791334
Liberomyces pistaciae ISPaVe2105 FR681904
Liberomyces pistaciae ISPaVe2106 FR681905
Liberomyces pistaciae ISPaVe2148 MH798902
Liberomyces saliciphilus H041 FR715510 FR715496 FR715507
Liberomyces saliciphilus H077 FR715511 FR715497 FR715508
Liberomyces saliciphilus CCF 4020HT FR715515 FR715515
Lopadostoma turgidum CBS 133207ET KC774618 KC774618
Melanconis stilbostoma CBS 121894 JQ926229 JQ926229
Melogramma campylosporum CBS 141086 JF440978 JF440978
Microdochium lycopodinum CBS 122885HT JF440979 JF440979
Microdochium phragmitis CBS 285.71ET AJ279449 EU926218 KP859076 KP859122
Nectria cinnabarina CBS 125165ET HM484548 HM484562
Neopestalotiopsis protearum CBS 114178HT LT853103 LT853251 LT853151
Pestalotiopsis knightiae CBS 114138HT KM199310 KM116227
Phlogicylindrium eucalyptorum CBS 111689 KF251205 KF251708
Phlogicylindrium uniforme CBS 131312HT JQ044426 JQ044445
Polyancora globosa CBS 118182HT DQ396469 DQ396466
Poronia punctata CBS 656.78 KT281904 KY610496
Pseudapiospora corni CBS 140736NT KT949907 KT949907
Pseudomassaria chondrospora CBS 125600 JF440981 JF440981
Pseudomassariella vexata CBS 129022ET JF440977 JF440977
Requienella fraxini CBS 140475HT KT949910 KT949910
Requienella seminuda CBS 140502ET KT949912 KT949912
Robillarda sessilis CBS 114312ET KR873256 KR873284
Sarcostroma restionis CBS 118154HT DQ278922 DQ278924
Seimatosporium cupressi CBS 224.55ET LT853083 LT853230 LT853131
Seimatosporium rosae CBS 139823ET KT198726 KT198727 LT853253 LT853153
Seiridium marginatum CBS 140403ET KT949914 KT949914
Seynesia erumpens SMH 1291 - AF279410
Strickeria kochii CBS 140411ET KT949918 KT949918
Truncatella angustata ICMP 7062 AF405306 AF382383
Vialaea insculpta DAOM 240257 JX139726 JX139726
Vialaea minutella BRIP 56959 KC181926 KC181924
Xylaria hypoxylon CBS 122620ET KY610407 KY610495
Zetiasplozna acaciae CBS 137994HT KJ869149 KJ869206

Pathogenicity

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.

Growth rates

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.

Phylogenetic analyses

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.

Taxonomy

As a result of the morphological and molecular phylogenetic investigations, the undescribed pistachio pathogen is described as a new species, Liberomyces pistaciae. In addition, for comparison, a morphological re-description and illustrations are also provided for the apparently similar, little known pistachio pathogen, Asteromella pistaciarum, based on type material and it is recognised as a synonym of Septoria pistaciarum.

Liberomyces pistaciae Voglmayr, S. Vitale, D. Aiello, Guarnaccia, Luongo & Belisario, sp. nov.

MycoBank No: 827682
Fig. 6

Diagnosis

Species with distinctly smaller conidia (3.2–5.0 × 1.0–2.0 μm) than in Liberomyces saliciphilus Pažoutová, M. Kolařík & Kubátová and L. macrosporus Pažoutová, M. Kolařík & Kubátová.

Type

ITALY. Sicily: Bronte (Catania province), on cankered twig of Pistacia vera, June 2010, A. Belisario (holotype: WU 39967; ex-type culture CBS 128196 = ISPaVe1958).

Etymology

Named after its host genus, Pistacia.

Description

Conidiomata pycnidial, superficial or immersed, single to densely aggregated, subglobose or cupular, uni- or irregularly plurilocular, first hyaline to pale brown, turning dark brown to blackish, without ostiole, irregularly rupturing at the apex and exuding a pale whitish conidial drop at maturity, (100–)170–260(–330) µm diam. (n=40). Pycnidial wall thin, of pale brown cells, (2.0–)3.5–6.3(–10.0) μm diam. (n=162) forming a textura angularis, outside darker, thicker-walled and more rounded, inside lined by a layer of angular hyaline cells giving rise to conidiophores. Conidiophores short, densely fasciculate, up to three times branched, hyaline, smooth, arising from the inner wall of the entire conidioma, 10–28 µm long. Conidiogenous cells holoblastic with sympodial proliferation, lageniform to cylindrical, (5.5–)6.5–8.5(–10.0) × 1.7–2.5(–2.7) µm (n=52), in dense intercalary or terminal whorls of 2–9. Conidia straight to allantoid, hyaline, smooth, 1-celled, (3.2–)3.8–4.5(–5.0) × (1.0–)1.2–1.5(–2.0) μm, l/w = (2.0–)2.7–3.5(–4.7) µm (n=182).

Culture characteristics

Colonies slow-growing (about 4 cm in diam. in 1 month on MEA, 4 cm in 2 weeks on CMD at 22 °C), initially white, turning pale to dark brown with age, with a whitish slightly lobed margin (Fig. 6a and b), surface mycelium sparse. Red to brown pigments diffusing in growth medium. Densely aggregated pycnidia formed after 7 d on the inoculum plug, successively also on the colony surface.

Notes

Morphologically, Liberomyces pistaciae is similar to the other two species of the genus, L. macrosporus and L. saliciphilus, but the latter have distinctly longer conidia (5–7.5 µm in L. saliciphilus, 8–13 µm in L. macrosporus).

Figure 6. 

Liberomyces pistaciae. a–d Cultures (a MEA, 6 weeks, 22 °C b CMD, 6 weeks, 22 °C c PDA, 3 weeks, 25 °C d PDA, 2 weeks, 25 °C) e Pycnidia produced on artificially inoculated sterilised pistachio twigs f–h Pycnidia in face view on MEA i Pycnidial wall in face view j–n Conidiophores and conidiogenous cells o–q Conidiogenous cells (o young p, q showing sympodial conidiation) r Conidia. All in water. Sources: a–c, f–r ex-holotype strain ISPaVe1958 = CBS 128196 d, e PV1= CPC 31292. Scale bars: 500 µm (e, f); 200 µm (g, h); 10 µm (i–l); 5 µm (m–r).

Septoria pistaciarum Caracc., Boll. Stud. Inform. R. Giard Colon Palermo 13: 10 [extr.] (1934).

Fig. 7

Asteromella pistaciarum Bremer & Petr., Sydowia 1(4–6): 253 (1947). For additional synonymy and a description of the Septoria morph, see Crous et al. (2013). Syn.

Type

of Asteromella pistaciarum. TURKEY. Ankara, on leaves of Pistacia vera, 29 Oct. 1944, H. Bremer (lectotype of Asteromella pistaciarum designated here: W 1973-15537, MBT383208; isotype: W 1979-11134).

Description

of the asteromella-like spermatial morph. Infection localised, producing distinct, brown, irregularly polyangular lesions of 0.5–1.5 mm diam., successively confluent, sharply delimited by leaf veins, visible on both sides of the leaf. Pycnidia (57–)69–101(–106) µm wide, (99–)107–134(–143) µm high (n=12), subepidermal, gregarious, solitary or in small groups, ellipsoid to pyriform, dark brown to black, with a central, circular, well-visible apical ostiole; peridium 8–19 µm wide, pseudoparenchymatous, of dark brown cells (3.0–)3.8–7.0(–10.3) µm diam. (n=50). Inner side lined with hyaline cells giving rise to phialides and short conidiophores. Conidiophores 1–3-celled, cells more or less square-shaped, bearing intercalary and terminal phialides. Conidiogenous cells enteroblastic, phialidic, hyaline, (3.7–)5.0–8.5(–10.5) × (2.5–)3.0–4.0(–4.7) µm (n=30), ampulliform to broadly lageniform, straight or curved. Conidia (3.4–)4.3–5.4(–6.6) × (0.9–)1.0–1.3(–1.5) μm, l/w = (2.8–)3.5–4.8(–6.1) (n=67), oblong, 1-celled, hyaline, with 1–2 subterminal guttules.

Notes

The classification and description of the lectotype of Asteromella pistaciarum is here added as it is morphologically similar to Liberomyces pistaciae and the latter had therefore initially been misidentified as the former (see e.g. Pažoutová et al. 2012, who included a sequence of Liberomyces pistaciae as Asteromella pistaciarum in their phylogenies). In addition, Asteromella pistaciarum has not been addressed in previous taxonomic accounts. Two isotype specimens are located in the Natural History Museum of Vienna (W) from which W 1973-15537 is here selected as the lectotype based on preservation and abundance of the specimens. In the original description, Bremer and Petrak (1947) reported a close association of Asteromella pistaciarum with Septoria pistaciarum and an immature mycosphaerella-like sexual morph, which they consider to represent the same species. We agree with this treatment and consider Asteromella pistaciarum to be the spermatial morph of Septoria pistaciarum, the former therefore becoming a synonym of the latter based on priority.

Figure 7. 

Asteromella pistaciarum W 1973-15537 (lectotype). a, b Pycnidia in leaf in face view c–e Pycnidia embedded in leaf in vertical section f Pycnidial wall with phialides and conidia in vertical section g Pycnidial wall in tangential section h–l Conidiophores and conidiogenous cells m Conidia. Scale bars: 10 mm (a); 100 µm (b); 20 µm (c–e); 10 µm (f–l); 5 µm (m).

Discussion

This study represents the first work determining the causal agent of cankers and decline of pistachio trees in Sicily, the major production area of Italy. In the field, severe symptoms of canker were observed on branches, shoots and trunks. In some cases, decline and death of host plants also occurred. The fungus almost exclusively isolated from these symptoms was Liberomyces pistaciae and the decline syndrome was strictly reproduced by artificial inoculation experiments. Seventy-one isolates recovered from different orchards over a 7-yr period were identified by molecular analysis. The molecular phylogenetic analyses (Figs 4, 5) clearly demonstrate that the genus Liberomyces is affiliated with the Xylariales, which confirms the results of previous analyses (Pažoutová et al. 2012, Perera et al. 2017). In both of our analyses, the genus Liberomyces is a sister group to Delonicicola siamense with moderate to high support, for which Perera et al. (2017) established a new family and order, Delonicicolaceae and Delonicicolales. However, if the order Delonicicolales is accepted, the Xylariales are unsupported in Perera et al. (2017) as well as in our phylogenetic analyses of the ITS-LSU matrix (Fig. 4). In the order Xylariales, insufficient backbone resolution and support of phylogenies based on ITS-LSU rDNA has been commonly observed (e.g. Jaklitsch and Voglmayr 2012, Jaklitsch et al. 2016), which often significantly increases if protein-coding genes like rpb2 and tub2 are considered (e.g. Voglmayr et al. 2018, Wendt et al. 2018). However, for most lineages of Xylariales, only ITS-LSU rDNA data are currently available. Remarkably, also in the phylogenetic analyses of Perera et al. (2017), which were inferred from a combined SSU, ITS, LSU and rpb2 matrix, internal support of Xylariales is absent if Delonicicolales are classified as a separate order. This fact may be due to lack of rpb2 sequence data for many lineages within Xylariales. Therefore, we consider the establishment of a separate order Delonicicolales premature and presently we propose the classification of Delonicicolaceae within Xylariales in which this family also fits morphologically, given its conidiomatal morphology and conidiogenesis.

Due to the pycnidial conidiomata and conidia of similar sizes, the current pistachio pathogen, here described as Liberomyces pistaciae, was initially identified as Asteromella pistaciarum, the true identity of which was unclear at that time. No sequence data are available for authentic material of the latter. However, a re-investigation of the type specimen of A. pistaciarum revealed substantial differences between both species, providing a clear distinction between the two organisms. While the type of A. pistaciarum has short reduced conidiophores with intercalary and terminal ampulliform phialides (Fig. 7h–l), L. pistaciae has densely fasciculate conidiophores with verticillately arranged holoblastic, lageniform to cylindrical conidiogenous cells with sympodial conidial proliferation (Fig. 6j–q). In addition, the type of A. pistaciarum has distinctly more elongate conidia with a l/w of (2.8–)3.5–4.8(–6.1), compared to (2.0–) 2.7–3.5(–4.7) in L. pistaciae. Moreover, the disease symptoms are markedly different. The type collection of A. pistaciarum represents a foliar pathogen causing clearly delimited polyangular leaf lesions with gregarious subepidermal pycnidia on both sides of the leaf (Fig. 7a), whereas L. pistaciae causes a canker disease of stems and branches. Although no recent collections, sequence data or cultures are available for A. pistaciarum, its close association with Septoria pistaciarum and an immature mycosphaerella-like sexual morph on the holotype specimen, which was already noted in the original description (Bremer and Petrak 1947), provides strong evidence that A. pistaciarum represents the spermatial morph of Septoria pistaciarum and it is therefore here considered to be a synonym of the latter.

There are many fungal genera which can act as plant pathogens, but may behave also as latent pathogens, while closely related species are symptomless endophytes (Carroll 1988). This is apparently also the case in the pathogen Liberomyces pistaciae, which might have a latent phase within the host tissues since it was also isolated from asymptomatic pistachio plants. A latent phase represents a specific condition where the fungus can either develop symptoms or induce changes in the physiology of the host plant (Romero et al. 2001, Crous et al. 2015, 2016). Furthermore, Millar (1980) and Andrews et al. (1985) observed that certain latent pathogens become pathogenic when the host is stressed and this may be the case in L. pistaciae on pistachio trees in Bronte. In this regard, the ecology of its closest relatives, L. macrosporus and L. saliciphilus, is of interest, as they were isolated as bark and wood endophytes from several woody hosts (Pažoutová et al. 2012), indicating that the primary ecology of the genus Liberomyces may be endophytic, from which the pathogenic L. pistaciae may have evolved. However, detailed studies are necessary to evaluate the influence of stress on pathogenicity of L. pistaciae.

On the basis of the high disease incidence and the frequency of this species observed in several orchards in the last years, we believe that L. pistaciae represents a menace to pistachio production in Sicily. As no epidemiological data are yet available, it is not possible to suggest any control strategies to avoid L. pistaciae infections. Nevertheless, the use and distribution of infected propagation material taken from nurseries and mechanical injuries or pruning wounds could play an important role in promoting the infections. The recent increase in importance of this and other diseases of pistachio in Sicily has stimulated further research and studies are in progress to extend the survey to other areas and to obtain important information to formulate effective disease management strategies.

Acknowledgements

This study was financially supported by the research project “Ricerche per il miglioramento della frutticoltura meridionale” (FRUMED) financed by the National Ministry of Agriculture (MiPAAF). The financial support by the Austrian Science Fund (FWF; project P27645-B16) to H. Voglmayr is gratefully acknowledged. Cordial thanks are due to A. Igersheim (W) for the loan of specimens and to W. Till (WU) for managing the herbarium loans.

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