Eur J Plant Pathol DOI 10.1007/s10658-013-0334-0 Ilyonectria palmarum sp. nov. causing dry basal stem rot of Arecaceae Dalia Aiello & Vladimiro Guarnaccia & Alessandro Vitale & Gabriella Cirvilleri & Giovanni Granata & Filomena Epifani & Giancarlo Perrone & Giancarlo Polizzi & Johannes Z. Groenewald & Pedro W. Crous Accepted: 28 October 2013 # KNPV 2013 Abstract During surveys conducted in 2010–2013, a complete breakage or bending of the trunk and a dry basal stem rot were observed on containerised Brahea armata, B. edulis, Howea forsteriana and Trachycarpus princeps plants in different nurseries located in eastern Sicily (southern Italy). A cylindrocarpon-like species was consistently obtained from diseased palm tissues, while known pathogens of these hosts such as Ganoderma, Phytophthora and Thielaviopsis were not found associated with symptomatic tissues or isolated on standard or selective media. A total of 40 cylindrocarpon-like isolates were collected and characterised based on morphology and DNA phylogeny. Multigene analyses based on the βtubulin, histone H3, translation elongation factor 1-α, and the internal transcribed spacers (ITS1, 5.8S, ITS2) genes facilitated the identification of a new species, described here as Ilyonectria palmarum. The pathogenicity of one representative isolate collected from each palm species was tested on plants cultivated under nursery conditions and in a growth chamber. All isolates were pathogenic to B. armata, B. edulis, H. forsteriana, and T. princeps and symptoms identical to that observed in nurseries were reproduced. Dry basal stem rot and stem bending caused by Ilyonectria palmarum represents a potentially serious problem for nurseries cultivating containerised palms. Keywords Cylindrocarpon-like asexual morph . Multi-gene analysis . Pathogenicity . Trunk bending . Trunk breakage Electronic supplementary material The online version of this article (doi:10.1007/s10658-013-0334-0) contains supplementary material, which is available to authorized users. D. Aiello : V. Guarnaccia : A. Vitale : G. Cirvilleri : G. Polizzi (*) Dipartimento di Gestione dei Sistemi Agroalimentari e Ambientali, Sezione Patologia Vegetale, University of Catania, Via S. Sofia 100, 95123 Catania, Italy e-mail: gpolizzi@unict.it G. Granata Formerly, Dipartimento di Gestione dei Sistemi Agroalimentari e Ambientali, Sezione Patologia Vegetale, University of Catania, Via S. Sofia 100, 95123 Catania, Italy F. Epifani : G. Perrone Istituto di Scienze delle Produzioni Alimentari (ISPA), Via Amendola 122/O, 70126 Bari, Italy J. Z. Groenewald : P. W. Crous CBS-KNAW Fungal Biodiversity Centre, Uppsalalaan 8, 3584 CT Utrecht, The Netherlands P. W. Crous Laboratory of Phytopathology, Wageningen University and Research Centre (WUR), Droevendaalsesteeg 1, 6708 PB Wageningen, The Netherlands P. W. Crous Microbiology, Department of Biology, Utrecht University, Padualaan 8, 3584 CH Utrecht, The Netherlands Eur J Plant Pathol Introduction Basal stem or butt rot is one of the major diseases causing losses in oil and ornamental palms, and has been reported from the USA, Malaysia and South East Asia (Elliott and Broschat 2001; Wong et al. 2012). This disease is caused by several species of Ganoderma including G. zonatum Murrill, G. boninense Pat., and G. miniatocinctum Steyaert (Miller et al. 1999; Elliott 2004; Chong et al. 2011; Wong et al. 2012). Ganoderma butt rot caused by G. zonatum is the most common and lethal disease of ornamental palms throughout Florida and it has been observed on all palm species in both natural and landscaped settings (Downer et al. 2009). Ganoderma zonatum degrades or rots the lower 4–5 ft of the trunk and other symptoms may include wilting or a general decline. Cross sections of infected trunks show a central area that is markedly different in colour (Elliott 2004). Diagnostic basidiomata are not always produced on diseased palms. The disease can therefore often only be confirmed when the palm is removed and the lower trunk sections are examined (Downer et al. 2009). Other lethal palm diseases producing trunk or crown rot are caused by species of Phytophthora and Thielaviopsis (Uchida 2004; Elliott 2006; Downer et al. 2009). Phytophthora species cause several disease symptoms including seedling blight and damping-off, root, stem, collar and bud rots. Phytophthora bud (heart) rot and root rot caused by P. palmivora (E.J. Butler) E.J. Butler and P. nicotianae Breda de Haan is widespread in Italian nurseries on Trachycarpus fortunei (Hooker) H. Wendl. (Cacciola et al. 2011; Polizzi unpublished). Thielaviopsis trunk infections frequently initiate in the upper part of the trunk and transverse sections show symptoms very similar to those induced by Ganoderma spp. (Simone 2004; Polizzi et al. 2006). On some species such as coconut and kentia palms a reddish stem bleeding may also occur (Simone 2004; Polizzi et al. 2007). During 2010, a new basal stem rot was detected on approximately 25 % of 2,000 5-year-old containerised Brahea armata S. Watson palms in a commercial nursery located in Praiola, Giarre (Catania province, eastern Sicily, Italy). The symptomatic plants showed a complete breakage or bending of the trunk as a consequence of a partial decay of one side of the basal stem (Fig. 1a–c). The root system appeared healthy, with no significant symptoms of decay (Fig. 1g). Sometimes, a new leaf started from the basal stem below the decayed tissues. Transverse sections through infected trunks showed a brown central area resembling Ganoderma infection, but the decay started from the external part of the trunk (Fig. 1e–f). In addition, no symptoms of general chlorosis, foliage necrosis, and fruiting bodies or conks were detected on infected plants. In relation to this newly discovered disease, the aims of the present study were to: determine the distribution of this disease in Sicilian nurseries; to identify the pathogen(s) associated with the disease via morphological and molecular characterisation; and to verify the pathogenicity of the microorganism(s) associated with the disease. Materials and methods Field survey and isolation During 2010–2013, surveys were conducted on different Arecaceae ornamental palms in 15 nurseries located in eastern Sicily. A total of approximately 300.000 2- to 8-year-old potted plants of B. armata (20.000 plants), B. edulis H. Wendl. (5.000 plants), Howea forsteriana C. Moore & F. v. Muell. (20.000 plants), T. fortunei (50.000 plants), T. princeps Gibbons, Spanner & San Y. Chen (1.000 plants), Chamaerops humilis L. (50.000 plants), Phoenix canariensis hort. ex Chabaud (90.000 plants), P. roebelenii O’ Brien (10.000 plants), Arecastrum romanzoffianum (Cham.) Becc. (15.000 plants), and Washingtonia robusta (Lindl.) H. Wendl. (40.000 plants) were surveyed for disease symptoms. Plants showing a complete breakage or bending of the trunk were randomly collected for analysis. The disease incidence on each host species from different nurseries was recorded. Infected basal stem tissues collected from symptomatic plants were surface disinfected with 1.5 % sodium hypochlorite for 1 min, rinsed in distilled water, and plated on Ganoderma selective medium (Ariffin and Idris 1991), carrot agar amended with 500 μl of streptomycin sulphate, acidified (lactic acid; pH=3.6) potato dextrose agar (PDA, Oxoid) and PDA amended with 100 μl of streptomycin sulphate and Phytophthora selective medium PARPH (Jeffers and Martin 1986), and incubated at 25 °C. Morphological characterisation A total of 40 cylindrocarpon-like isolates were derived from single conidia and maintained on PDA slants (Table 1). For morphological identification all isolates Eur J Plant Pathol Fig. 1 Dry basal stem rot caused by Ilyonectria palmarum sp. nov. on containerised palms. a Trunk bending on Brahea armata plants. b Trunk bending and death of B. armata plant. c Breakage of trunk on Howea forsteriana plant. d Dry basal stem rot with external tissue proliferation on B. armata plant. e–f Discolouration due to degrading tissue remains externalized within the trunk. g Root system appears healthy b a c d f were grown for 1 week at 25 °C in the dark on Petri dishes containing PDA, and synthetic nutrient-poor agar (SNA) (Crous et al. 2009). Observations were done by mounting fungal structures in clear lactic acid. For each e g isolate, 30 measurements at × 1,000 magnification were made for each structure using a Zeiss Axio Imager 2 light microscope with differential interference contrast (DIC) illumination, and images captured via an Table 1 List of 40 cylindrocarpon-like isolates collected from containerised Arecaceae palms showing symptoms of dry basal stem rot Isolate DiGeSA numbera Host BRA1b, BRA2, BRA3, BRA4 Brahea armata Giarre, Sicily, Italy, nursery 1 BRA5b, BRA6, BRA7, BRA8, BRA9 B. edulis Riposto, Sicily, Italy, nursery 1 BRA10, BRA11, BRA12, BRA13, BRA14, BRA15, BRA16, BRA17, BRA18, BRA19, BRA20, BRA21, BRA22 TP1b, TP2, TP3, TP4,TP5,TP6, TP7, TP8, TP9 B. armata Giarre, Sicily, Italy, nursery 2 Trachycarpus princeps Riposto, Sicily, Italy, nursery 2 Howea forsteriana Aci Castello, Sicily, Italy b HF1, HF2, HF3 , HF4, HF5, HF6, HF7, HF8, HF9 a DiGeSA Dipartimento di Gestione dei Sistemi Agroalimentari e Ambientali, Catania, Italy b Isolates used for the pathogenicity trials Geographical location Eur J Plant Pathol AxioCam MRc5 camera and Zen software. Culture characteristics (texture, density, and zonation) were described on PDA after incubation at 25 °C in the dark for 2 weeks. Colony colours were determined according to Rayner (1970). Cardinal temperatures for growth were determined by inoculating 90-mm diam PDA dishes with a 3-mm diam plug cut from the edge of an actively growing colony. Colony diameters were determined after 1 week in two orthogonal directions. The strains were incubated at 4, 10, 15, 20, 25, 30, 35 °C with three replicate plates per strain at each temperature. DNA isolation, PCR and phylogeny Species level identification was obtained by DNA sequencing and phylogenetic analyses of partial β-tubulin (benA), histone H3 (HIS3) and translation elongation factor 1-α (TEF-1a) gene sequences of the 40 strains tested in this study. Each fungal strain was grown in shake culture (150 rpm, 25 °C, 2 days) on Wickerham medium, containing 40 g glucose, 5 g peptone, 3 g yeast extract, 3 g malt extract and up to 1 l water; the mycelium was subsequently filtered and lyophilised for total DNA isolation. The fungal DNA extraction was done with the Wizard Magnetic DNA Purification System for Food (Promega) with some modifications by halving the volume of the reaction, starting from 10 mg of lyophilised mycelium. The quality of genomic DNA was determined by agarose gel electrophoresis and quantification by means of a NanoDrop ND-1000 Spectrophotometer. Amplification of part of the βtubulin gene (benA) was performed using the primers T1 (O’Donnell and Cigelnik 1997) and CYLTUB1R (Crous et al. 2004), for the Histone H3 region (HIS3) primers CYLH3F and CYLH3R (Crous et al. 2004) were used and for the translation elongation factor 1-α (TEF-1a) the primers EF1-728F (Carbone and Kohn 1999) and CylEF-R2 (Crous et al. 2004) were used. Amplification reactions and cycle conditions followed Crous et al. (2004), except that an annealing temperature of 58 °C was used for all reactions. In addition, the ITS region of the six strains used in morphological and phytopathological studies was sequenced using standard conditions and primers ITS1/ITS4 (White et al. 1990). Although this locus could not successfully distinguish all species of Ilyonectria, this region was recently accepted as the universal fungal barcode region (Schoch et al. 2012). Blast searches using the NCBI GenBank nucleotide database confirmed that the isolated fungus belonged to Ilyonectria, but without a 100 % match to any known species in the database. Representative sequences (Table 2) from previous publications (Schroers et al. 2008; Cabral et al. 2012a, b) were downloaded from the NCBI GenBank nucleotide database to act as a reference backbone for the phylogenetic analyses. The preliminary alignment of the three sequenced loci (benA, HIS3, TEF-1a) was performed using BioNumerics v. 5.1 (Applied Maths), with manual adjustment by eye where necessary. Based on previous publications (e.g. Cabral et al. 2012a, b), Campylocarpon fasciculare (CBS 112613) was used as outgroup and some representative strains of the Ilyonectria radicicola species complex were included in the analysis (Table 2). The phylogenetic analysis was conducted firstly on these individual locus alignments and subsequently the combined alignment of the three loci was analysed for inferring the organismal phylogeny. The combined alignment was created using the Muscle algorithm in MEGA v. 5 (Tamura et al. 2011). Each locus was first aligned separately and then concatenated in a super-gene alignment used to generate the phylogenetic tree. Phylogenetic analyses were performed in MEGA v. 5 using both Neighbour-Joining (NJ) (Saitou and Nei 1987) and Maximum Likelihood (ML) methods and the Tamura-Nei model (Tamura and Nei 1993). The best model substitution was calculated and the best option was found to be the Tamura 3parameter model with Gamma distribution. A parsimony analysis of the aligned sequence data was performed with PAUP (Phylogenetic Analysis Using Parsimony) v. 4.0b10 (Swofford 2003). Alignment gaps were treated as a fifth character state and all characters were unordered and of equal weight. Maximum parsimony analysis was performed using the heuristic search option with 100 random simple taxa additions and tree bisection and reconstruction (TBR) as the branch swapping algorithm. Branches of zero length were collapsed and all multiple, equally parsimonious trees (limited to 1,000 trees) were saved. The robustness of the trees obtained was evaluated by 1000 bootstrap replications (Hillis and Bull 1993). Tree length (TL), consistency index (CI), retention index (RI) and rescaled consistency index (RC) were calculated. MrModeltest v. 2.2 (Nylander 2004) was used to determine the best nucleotide substitution model settings for each data partition. Based on the results of the MrModeltest, a model-optimised phylogenetic reconstruction was performed for the aligned combined Eur J Plant Pathol Table 2 “Cylindrocarpon”, Ilyonectria and Neonectria isolates used in the phylogenetic analyses Species Strain number and status GenBank accession no. ITS TUB H3 EF Host/substrate “Cylindrocarpon” cylindroides “Cylindrocarpon” pauciseptatum Ilyonectria liriodendri CBS 324.61, representative strain CBS 120171a JF735312 DQ789875 JF735599 JF735788 Abies concolor EF607089 EF607066 JF735587 JF735776 Vitis sp. CBS 110.81a of C. liriodendri DQ178163 DQ178170 JF735507 JF735696 Liriodendron tulipifera, root Ilyonectria lusitanica CBS 129080 JF735296 JF735423 JF735570 JF735759 Vitis vinifera AY677290 AY677233 JF735647 JF735836 Vitis vinifera JF735288 JF735414 JF735557 JF735746 Panax quinquefolium JF735290 JF735416 JF735559 JF735748 Panax ginseng AY 677281 AY677236 JF735631 JF735820 Vitis vinifera CBS 112615, holotype of C. macrodidymum Ilyonectria mors-panacis CBS 306.35a of Ramularia mors-panacis Ilyonectria mors-panacis CBS 124662a of C. destructans f.sp. panacis Ilyonectria novozelandica CBS 112593 Ilyonectria macrodidyma BRA1 HF937429 HF922606 HF922618 HF922612 Brahea armata BRA2 HF937430 HF922607 HF922619 HF922613 Brahea armata HF3 = CBS 135754a HF937431 HF922608 HF922620 HF922614 Howea fosteriana HF7 HF937432 HF922609 HF922621 HF922615 Howea fosteriana TP1 HF937433 HF922610 HF922622 HF922616 Trachycarpus princeps TP3 HF937434 HF922611 HF922623 HF922617 Trachycarpus princeps Ilyonectria radicicola CBS 264.65a AY677273 AY677256 JF735506 JF735695 Cyclamen persicum Ilyonectria robusta CBS 308.35a of Ramularia JF735264 JF735377 JF735518 JF735707 Panax quinquefolium Ilyonectria rufa CBS 137.37, authentic strain of Coleomyces rufus CBS 113555 AY677271 AY677251 JF735540 JF735729 Dune sand JF735350 AY677234 JF735661 JF735850 Vitis sp. JF735309 DQ789869 JF735594 JF735783 Fagus sylvatica JF735310 DQ789880 JF735595 JF735784 Salix cinerea JF735308 DQ789872 JF735593 JF735782 Alnus incana JF735311 DQ789882 JF735598 JF735787 Arceuthobium tsugense JF735313 JF735438 JF735791 Malus sylvestris Ilyonectria palmarum sp. nov. Ilyonectria torresensis Neonectria ditissima Neonectria ditissima Neonectria major Neonectria neomacrospora Neonectria ramularia a CBS 226.31, authentic strain of C. wilkommii CBS 835.97, representative strain of N. galligena CBS 240.29a CBS 118984, representative strain CBS 151.29, authentic strain of C. obtusiusculum (= C. magnusianum) JF735602 Ex-type strain data set to determine species relationships using MrBayes v. 3.2.1 (Huelsenbeck and Ronquist 2001; Ronquist and Huelsenbeck 2003). The heating parameter was set at 0.25 and the Markov Chain Monte Carlo (MCMC) analysis of four chains was started in parallel from a random tree topology and lasted until the average standard deviation of split frequencies came below 0.01. Trees were saved each 1,000 generations and the resulting phylogenetic tree was printed with Geneious v. 5.5.4 (Drummond et al. 2011). New sequences were lodged in GenBank and the alignment and phylogenetic tree in TreeBASE (www.treebase.org). Pathogenicity The pathogenicity of four representative isolates (HF1, BRA1, BRA5 and TP1) was evaluated on potted, 1- and 3-year-old plants of H. forsteriana, B. armata, B. edulis and T. princeps. Plants were respectively grown in a growth chamber as well as in a nursery. Trials were Eur J Plant Pathol conducted on 30 plants for each host species/age. The same number of plants was used as control. All 1-yearold plants were potted in 0.5–l containers in a 1:3:1 Canadian sphagnum peat, sand, and perlite and were inoculated by soil drench at the base of each plant (10 ml/pot) with conidial suspension (1–2.5×105 conidia ml−1) obtained from cultures grown on PDA dishes and incubated at 25 °C for 2 weeks. A mixture of conidia and mycelia as inoculum was prepared for each isolate by flooding the dishes with sterile water and rubbing the colony surface with a sterile loop. Sterile water was applied to control plants. Thirty 3-year-old plants were potted in 8-l containers in a 1:3:1 Canadian sphagnum peat, sand, and perlite and were inoculated with one mycelial plug obtained from 14-day-old colonies growing on PDA and applied to a 6 mm diam crown wound. The same number of plants was inoculated without wounding. Sterile agar plugs were used in control plants. Following inoculation, the crowns were wrapped with Parafilm and all plants were covered with plastic bags for 48 h. One-year-old plants were maintained in a growth chamber at 25±1 °C, while all 3-yearold plants were cultivated in a nursery. All plants were irrigated 2–4 times during every week and were fertilised according to farm use every 30 days with 2 or 20 g/pot of complex NPK fertilizer Nitrophoska® special (BASF). After 4 and 8 months, plants were examined for disease symptoms. Results Field survey and isolation During the survey, bending and basal stem rot were observed in all nurseries where Brahea spp. were cultivated (Fig. 1). Symptoms were detected in four nurseries on approximately 6400 of 2- to 8-year-old potted B. armata and on 1100 B. edulis plants of the same age. In these nurseries the disease incidence varied from 22.80 to 40 % on B. armata to 18.1–35.2 % on B. edulis plants. Bending and basal stem rot were also observed on 60 of 3-year-old H. forsteriana plants in one nursery and on 100 of 3-year-old T. princeps plants in another nursery (Fig. 1). On these hosts the disease incidence varied from 0.3 (H. forsteriana) to 10 % (T. princeps). No symptoms were detected on T. fortunei, C. humilis, P. canariensis, P. robelenii, A. romanzoffianum and W. robusta plants. A cylindrocarpon-like species was consistently isolated from diseased ornamental palms tissues. Species of Ganoderma, Phytophthora and Thielaviopsis were not found associated with symptomatic tissues, or recovered on standard and selective media. A total of 40 cylindrocarpon-like isolates were collected from diseased palm hosts. The majority of the isolates (22) were associated with basal stem rot of Brahea spp., nine from T. princeps, and nine from H. forsteriana (Table 1). Morphology Ilyonectria palmarum G. Polizzi, G. Perrone & Crous, sp. nov. MycoBank MB804338; Fig. 2 Perithecia formed homothallically in vitro, solitary or in groups of 2–3, developing directly on the SNA agar surface, ovoid to obpyriform, red, becoming purple-red in 3 % KOH (positive colour reaction), finely warted, 250–350 μm diam, up to 300–350 μm high; without recognisable stroma; perithecial wall consisting of two poorly distinguishable regions; outer region 10– 15 μm thick, composed of 3–4 layers of angular to subglobose cells, 7–20×5–8 μm; cell walls up to 1 μm thick; inner region around 5 μm thick, composed of cells that are flat in transverse optical section and angular to oval in subsurface optical face view; perithecial warts consisting of globose to subglobose cells, 15–25× 10–20 μm, that have walls up to 1.5 μm thick. Asci subcylindrical to narrowly clavate, 40–70×5–8 μm, 8spored; apex truncate to bluntly rounded, with a visible ring. Ascospores divided into two cells of equal size, ellipsoidal, tapering towards both ends, smooth to finely warted, guttulate, (9–)10(−11) × 3(−3.5) μm. Conidiophores simple or complex, sporodochial. Simple conidiophores arising laterally or terminally from the aerial mycelium or erect, arising from the agar surface, solitary to loosely aggregated, unbranched or sparsely branched, 1–4-septate, rarely consisting only of the phialide, 50–170 μm long; phialides monophialidic, cylindrical, slightly tapering toward the base, 50–70×2– 3 μm, 2.5–3 μm near aperture. Complex, sporodochial conidiophores aggregated in pionnote sporodochia, repeatedly, irregularly branched; phialides cylindrical but slightly tapering towards the tip, 25–50×2–3 μm, 1.5– 2 μm near the aperture. Micro- and macroconidia present on both types of conidiophores. Macroconidia (2–)3-septate, straight or sometimes slightly curved, Eur J Plant Pathol Fig. 2 Ilyonectria palmarum (CPC 22087). a Perithecia developing on synthetic nutrient-poor agar. b–c Asci. d Ascospores. e Conidiophores with phialides. f Three-septate macroconidia. g Microconidia. h Chlamydospores. Scale bars: A=300 μm, all others = 10 μm cylindrical or typically minutely widening towards the tip, therefore appearing somewhat clavate, mostly with a visible, slightly laterally displaced hilum, (25–)32– 37(−39)×(4–)5(−6) μm. Microconidia common on SNA, 0–1-septate, ellipsoidal to ovoidal or subcylindrical, more or less straight, with a clearly laterally displaced hilum; aseptate microconidia (6–)8– 9(−10) × 2.5–3(−4) μm; 1-septate microconidia (10–)11–13(−15)×(3–)3.5(−4) μm. Conidia formed in heads on simple conidiophores or as unpigmented masses on simple as well as complex conidiophores. Chlamydospores mostly in short, intercalary chains, golden-brown, globose to globose-ellipsoid, 8–15×9– 12 μm. Additional specimens examined Italy, Sicily, Catania province, Praiola, Giarre, Praiola, Giarre, basal stem rot on Brahea armata, Sept. 2010, G. Polizzi, DiGeSABRA1 = CPC 22085 = CBS 135755; DiGeSA-BRA2 = CPC 22086 = CBS 135228; Aci Castello, basal stem rot on Howea forsteriana, July 2011, G. Polizzi, DiGeSAHF7 = CPC 22088 = CBS 135753; Carruba, Giarre, basal stem rot on Trachycarpus princeps, Feb. 2012, G. Polizzi, DiGeSA-TP1 = CPC 22089 = CBS 135229; Carruba, Giarre, basal stem rot on Trachycarpus princeps, Feb. 2012, G. Polizzi, DiGeSA-TP3 = CPC 22090 = CBS 135756. Culture characteristics Covering surface of PDA dish after 2 week at 25 °C; surface was appressed, dense, lacking zonation, and with sparse aerial mycelium; centre dark brick, outer zone cinnamon, reverse dark brick in centre, brick in outer zone (Rayner 1970). We obtained amplicons of approximately 560 bases for benA, 510 for HIS3 and 460 for TEF-1α for all isolates. The consensus sequences from these amplicons were used in the combined alignment. The manually adjusted combined (benA, TEF-1α and HIS3) alignment contained 58 taxa (including the outgroup sequence) and, of the 1,550 characters (516, 498 and 536 for benA, TEF-1α and HIS3, respectively) used in the phylogenetic analysis, 511 (152, 189 and 170 for benA, TEF-1α and HIS3, respectively) were parsimony-informative, 218 (75, 96 and 47 for benA, TEF-1α and HIS3, respectively) were variable and parsimony-uninformative, and 821 (289, 213 and 319 for benA, TEF-1α and HIS3, respectively) were constant. Cardinal temperatures for growth After 7 days at 4 °C all isolates only grew on the agar plug, while no growth was observed at 35 °C. Optimal growth occurred at 25 °C, with colonies reaching 30–62 mm diam. Typus: Italy, Sicily, Catania province, Aci Castello, basal stem rot on Howea forsteriana, July 2011, G. Polizzi, holotype CBS H-135754, culture ex-type DiGeSA-HF3 = CPC 22087 = CBS 135754. Molecular identification Eur J Plant Pathol Eur J Plant Pathol The first of five equally most parsimonious trees obtained from a heuristic search with 100 random taxon additions of the combined sequence alignment using PAUP v. 4.0b10. Strains from the novel species are included in the coloured box. The scale bar shows 30 changes, and bootstrap support values from 1000 replicates and Bayesian posterior probability values are shown at the nodes. An asterisk (“*”) indicates nodes with bootstrap support values of 100 % and Bayesian posterior probability values of 1. Thickened lines indicate the strict consensus branches and the tree was rooted to Campylocarpon fasciculare CBS 112613 (benA = AY677221, TEF-1a = JF735691, HIS3 = JF735502) ƒFig. 3 The percentage of variable sites and parsimony informative sites for each locus differ, the TEF-1α sequences have the highest percentage (57.23 %) of variable and parsimony informative sites, and HIS3 have the lowest variability (40.49 %). Five equally most parsimonious tree were saved from the heuristic search and the first of these is shown in Fig. 3 (TL = 1,632, CI = 0.718, RI = 0.829, RC = 0.595). Based on the results of MrModeltest, the following priors were set in MrBayes for the different partitions: all partitions had dirichlet base frequencies a b c d e f Fig. 4 Symptoms induced on containerised palms by Ilyonectria palmarum sp. nov. isolates 8 months after plugs inoculations. a–c. Dry basal stem rot and breakage of trunk on Brahea armata plants. g d. Trunk bending and emission of new leaves below the decayed tissues on B. edulis plants. e–g. Basal stem rot, trunk bending and tissue proliferation on Howea forsteriana plants Eur J Plant Pathol and, per partition, the HKY85 model with gammadistributed rates (for benA), the GTR model with gamma-distributed rates (for TEF-1α), or the GTR model with inverse gamma-distributed rates (for HIS3). The alignment contained 622 unique site patterns (188, 231, and 203 for benA, TEF-1α and HIS3, respectively). The Bayesian analysis lasted 1,995,000 generations and the consensus trees and posterior probabilities were calculated from the 2,994 trees left after discarding the first 25 % for burn-in. The phylogenetic tree obtained from the Bayesian analysis has the same overall topology as that obtained from the parsimony analysis (data not shown; posterior probability values superimposed on Fig. 3). In addition, the Maximum Likelihood phylogenetic tree obtained in MEGA 5 software had the highest log likelihood of (−6807.4661) with a total of 1,360 positions in the final dataset (see Supplementary Fig S3). Phylogenetic analyses were also conducted on the individual data partitions (data not shown) and the resulting trees contained a similar overall topology, fulfilling the requirements of genealogical concordance phylogenetic species recognition (GCPSR, Taylor et al. 2000). Pathogenicity All Ilyonectria isolates tested were pathogenic on the host species inoculated, and produced identical symptoms to those observed in the nurseries (Fig. 4). After 4 months, trunk bending was only detected on the 3year-old plants with wounds. After 8 months, plants showed basal stem rot and trunk bending. The root rot was observed occasionally in 1-year-old plants of H. forsteriana. Plant death was however observed on Table 3 Pathogenicity of Ilyonectria palmarum isolates on ornamental palms 4 and 8 months after inoculation Palm species/age Howea forsteriana/1 year Control Brahea armata/1 year Control Brahea edulis/1 year Control B. armata/3 years Isolate Control B. armata/3 years Control B. edulis/3 years Control Trachycarpus princeps/3 years Control After 4 monthsb After 8 monthsb TB BSR DP TB DP HF3 CS/NW 0 1 2 0 2 SW/NW 0 0 0 0 0 BRA1 CS/NW 0 1 1 0 2 – SW/NW 0 0 0 0 0 BRA5 CS/NW 0 2 1 0 2 – SW/NW 0 0 0 0 0 BRA1 BRA5 Control H. forsteriana/3 years Number of symptomatic plants – Control B. edulis/3 years Inoculation methoda AFP/NW 0 0 0 0 0 AP/NW 0 0 0 0 0 AFP/NW 0 0 0 0 0 AP/NW 0 0 0 0 0 HF3 AFP/W 2 0 28 5 3 – AP/W 0 0 0 0 0 BRA1 AFP/W 2 1 28 6 3 – AP/W 0 0 0 0 0 BRA5 AFP/W 3 2 26 6 4 – AP/W 0 0 0 0 0 TP1 AFP/W 2 1 22 5 3 – AP/W 0 0 0 0 0 Thirty plants were inoculated for each species/age a CS/NW conidial suspension drench applied with no wound; SW/NW sterile water drench applied with no wound; AFP/NW agar fungal plug applied with no wound; AP/NW agar plug applied with no wound; AFP/Wagar fungal plug applied with wound; AP/Wagar plug applied with wound b BSR basal stem rot; TB trunk bending; DP death of plant Eur J Plant Pathol plants of H. forsteriana and Brahea spp., with and without wounds. Basal stem rot and trunk bending were observed on all host species when the plants were inoculated with agar fungal plug applied with wound (Table 3). Isolations from the symptomatic plants consistently yielded the test fungi. All uninoculated control plants remained healthy. Discussion Basal stem or trunk rot of palms is normally caused by different fungal species including Ganoderma and Phytophthora spp., and T. paradoxa. In our study, a new and widespread dry basal stem rot was detected on potted B. armata, B. edulis, H. forsteriana, and T. princeps ornamental palms cultivated in different nurseries in eastern Sicily. A cylindrocarpon-like asexual morph was consistently obtained from symptomatic tissues of these palms, while Ganoderma spp., Phytophthora spp., and T. paradoxa were not detected in standard or selective media. According to the morphological classification of isolates associated with trunk rot of palms, this species has microconidia and chlamydospores, and thus belongs to “Cylindrocarpon group 3” sensu Booth (1966). Species in this group, along with their neonectria-like sexual morphs were recently relocated to the genus Ilyonectria (Chaverri et al. 2011). Several important plant pathogenic species inducing black foot rot of a range of hosts belong to this genus (Seifert et al. 2003; Halleen et al. 2004, 2006; Chaverri et al. 2011; Cabral et al. 2012a, b). Based on the multigene DNA analysis conducted of known species, isolates from palm were shown to represent a novel species, described here as I. palmarum. Ilyonectria palmarum was pathogenic to the different host species inoculated, and produced identical symptoms to those observed in nurseries, thus confirming its role as causal agent of the new basal rot of containerised palms in Italy. On the basis of the disease incidence and severity observed in several nurseries we believe that this disease poses a serious threat, especially to the containerised field-grown species of Brahea. The reasons that promoted this unprecedented occurrence of I. palmarum associated with a basal stem rot are unknown. The containerised palm production could have a role in promoting infections because the palms are frequently stressed, remain containerised throughout production, and several wounds could be incurred during transplanting. The predominant potting component in field-grown palm nurseries in most of the eastern Sicily is autochtone volcanic soil mixed with peat and perlite or vermiculite to assure plant stability and proper development. Thus, the use of non-disinfected volcanic soil could represent a possible source of pathogen inoculum. Species of Ilyonectria are commonly found in soil, and cause root diseases on a range of diverse hosts worldwide (Sánchez et al. 2002; Seifert et al. 2003; Halleen et al. 2006; Alaniz et al. 2007, 2009; AgustíBrisach et al. 2011; Dart and Weeda 2011; Petit et al. 2011; Cabral et al. 2012a, b; Özben et al. 2012; ÚrbezTorres et al. 2012; Vitale et al. 2012; Erper et al. 2013). It is interesting to note, however, that Ilyonectria spp. were not previously reported as causal agents of palm diseases. Previously, Halleen et al. (2006) identified a single strain (IMI 313237) clustering with the ex-type culture of I. radicicola (CBS 264.65) and isolated from arecoid palm (Seifert et al. 2003), but no data about the pathogenicity of this isolate is reported in the literature. As far as we could establish, this is the first confirmed report of basal stem rot and stem bending caused by a Ilyonectria species on palms. 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