OPEN ACCESS
Citation: D.R.S. Pereira, A.J.L. Phillips (2020) A new leaf spot disease of Chamaerops humilis caused by Palmeiromyces chamaeropicola gen. et sp. nov.. Phytopathologia Mediter-ranea 59(2): 353-363. DOI: 10.14601/Phyto-11213
Accepted: June 17, 2020
Published: August 31, 2020
Copyright: 2020 D.R.S. Pereira, A.J.L. Phillips. This is an open access, peer-reviewed article published by Firenze University Press (http://www. fupress.com/pm) and distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Data Availability Statement: All relevant data are within the paper and its Supporting Information files.
Competing Interests: The Author(s) declare(s) no conflict of interest.
Editor: Lizel Mostert, Faculty of AgriSciences, Stellenbosch, South Africa.
Summary. In September 2018, a leaf spot disease was noticed on a European fan palm (Chamaerops humilis L.) in Oeiras, Portugal. The aim of this study was to identify and characterize the causative agent of this disease symptom. Morphological characters and phylogenetic data derived from ITS and LSU sequences revealed that the leaf spot was caused by a flamentous fungus in the Mycosphaerellales, as a unique lineage within the Teratosphaeriaceae. This pathogen is introduced here as a new genus and species, Palmeiromyces chamaeropicola D.R.S. Pereira & A.J.L. Phillips, the cause of a newly reported leaf disease on Chamaerops humilis.
Keywords. Fungus, new genus, new species, palm tree, plant pathogen.
INTRODUCTION
Chamaerops humilis L. (Arecaceae), commonly known as the European fan palm or Mediterranean dwarf palm, is one of only two indigenous European palms, and the only one found in the Iberian Peninsula (Guzmán et al., 2017). This palm tree is distributed around the western Mediterranean Basin, occurring in Europe (Portugal, Spain, France and Italy), North Africa (Morocco, Algeria and Tunisia) and in Mediterranean islands (Balearic, Sicily, Sardinia and Malta) (Dransfield et al., 2008; Quattrocchi, 2017; Palmweb, 2020). These trees are used as sources of several products with commercial value, including textiles, food and seeds; they are also important ornamental trees (Guzmán et al., 2017). In Portugal, although these palms occur naturally, almost exclusively in the Algarve region (Carapeto et al., 2020), they are widely planted and used in gardening and landscaping due to their hardiness and aesthetic value.
Chamaerops humilis is generally free of diseases. No major diseases or pests have been reported for this palm, although it is a potential host for the red palm weevil and other harmful insects (Elliott, 2004). Some reports have shown that the principal fungal diseases on C. humilis are leaf spots. For example, this palm is a host for Graphiola phoenicis, a leaf spotting Basidiomycete found exclusively on palms (Piepenbring et al., 2012). Fröhlich and Hyde (1998) listed six species of Mycosphaerella from leaf spots on different palms, including M. chamaeropis on C. humilis. More recently, Fusarium wilt and dieback were reported from young and adult C. humilis in Spain (Armengol et al., 2005), and Pestalotiopsis chamaeropis was described from leaves of this host in Italy (Maharachchikumbura et al., 2 014) .
In the present study, a leaf spot disease was noticed on a C. humilis palm growing as an ornamental. On the lesions, ascomata, asci and ascospores with characteristics of the Mycosphaerellales (Abdollahzadeh et al., 2019) were found. Terefore, the purpose of this paper was to characterize the fungus in terms of its morphology and phylogeny.
MATERIALS AND METHODS
Specimen collection and examination
Diseased leaf segments with leaf spots were collected from an ornamental C. humilis palm in Oeiras, near Lisbon, Portugal. Plant material was transported to a laboratory in paper envelopes, and examined with a Leica MZ9.5 stereo microscope (Leica Microsystems GmbH) for observations of lesion morphology and associated fungi.
Fungal isolation
A small piece of a leaf spot lesion bearing ascomata was placed on a drop of sterile water in the inverted lid of a Petri dish. The dish base, containing half-strength potato dextrose agar (1/2 PDA) (BIOKAR Diagnostics) was placed on top of the inverted lid. Ascospores were discharged upwards and impinged on the agar surface. After incubating overnight, single germinating ascospores were transferred to fresh plates of 1/2 PDA. Cultures were then incubated in ambient light at room temperature (18-20°C).
Isolations were also made directly from leaf spots. Pieces of leaf spot tissue 1-2 mm2 were cut from the edge of each lesion, surface sterilized in 5% sodium hypochlorite for 1 min, rinsed in three times in sterile water, and then blotted dry on sterile flter paper. The fragments were plated onto 1/2 PDA containing 0.05% chloramphenicol, and incubated at room temperature until colonies developed.
Morphological observations and characterization
Microscopic structures of isolated fungi were mounted in 100% lactic acid and examined with differential interference contrast (DIC) microscopy. Observations of micromorphological features were made with Leica MZ9.5 and Leica DMR microscopes (Leica Microsystems GmbH), and digital images were recorded, respectively on the two microscopes, with Leica DFC300 and Leica DFC320 cameras (Leica Microsystems GmbH). Measurements were made with the measurement module of the Leica IM500 Image Management System (Lei-ca Microsystems GmbH). Mean, standard deviation (SD) and 95% confdence intervals were calculated from n = total of measured structures. Measurements are presented as minimum and maximum dimensions with mean and SD in parentheses. Photographs were prepared with Adobe Photoshop CS6 (Adobe).
DNA extraction, PCR amplification and sequencing
Genomic DNA (gDNA) was extracted from mycelium of cultures grown on 1/2 PDA, following a modi-fed and optimized version of the guanidium thiocy-anate method described by Pitcher et al. (1989). PCR reactions were carried out with Taq polymerase, nucle-otides, primers, PCR-water (ultrapure DNase/RNase-free distilled water) and buffers supplied by Invitrogen. Amplification reactions were performed in a TGradient Termocycler (Biometra). Amplifed PCR products were purifed and sequenced by Eurofns (Germany).
The primers ITS5 (White et al., 1990) and NL-4 (O'Donnell, 1993) were used to amplify part of the cluster of rRNA genes, including the nuclear 5.8S rRNA gene and its flanking ITS1 and ITS2 regions, along with the first two domains of the large-subunit rRNA gene (ITS-D1/D2 rDNA region). The PCR reaction mixture consisted of 50-100 ng of gDNA, 1x PCR buffer, 50 pmol of each primer, 200 μM of each dNTP, 2 mM MgCl2, 1 U Taq DNA polymerase, and was made up to a total volume of 50 μL with PCR water. The following cycling conditions were used: initial denaturation at 95°C for 5 min, followed by 40 cycles of denaturation at 95°C for 1 min, annealing at 52°C for 30 s and elongation at 72°C for 1.5 min, and a final elongation step at 72°C for 10 min.
The ITS region was sequenced only in the forward direction using the primer ITS5. The D1/D2 region (LSU) was sequenced only in the forward direction using the primers ITS5 and NL1 (O'Donnell, 1993). Consensus sequences were produced and edited with BioEdit version 7.0.5.3 (Hall, 1999).
Sequence alignment and phylogenetic analyses
ITS and LSU sequences of species in representative genera of Teratosphaeriaceae and Neodevriesiaceae in Mycosphaerellales were retrieved from GenBank by BLAST searches with the sequences generated in this study (Table 1). These were supplemented with taxa listed in recent literature (e.g. Quaedvlieg et al., 2014; Isola et al., 2016; Wang et al., 2017; Delgado et al., 2018). Cap-nodium cofeae Pat. was used as the outgroup taxon representative of a species in Capnodiales.
Sequences were aligned with ClustalX version 2.1 (Tompson et al., 1997) using the following parameters: pairwise alignment parameters (gap opening = 10, gap extension = 0.1), and multiple alignment parameters (gap opening = 10, gap extension = 0.2, DNA transition weight = 0.5, delay divergent sequences = 25%). Alignments were checked and manual adjustments were made where necessary with BioEdit. Terminal regions with data missing in some of the isolates were excluded from the analysis. The aligned ITS and LSU sequences were concatenated and combined in a single matrix.
Maximum Likelihood (ML) and Maximum Parsimony (MP) were used for phylogenetic inferences of single gene sequence alignments and the concatenated alignments. The individual gene trees were assessed for clade con-ficts between the individual phylogenies by visually comparing the trees generated. ML and MP inferences were implemented on the CIPRES Science Gateway portal version 3.3 (Miller et al., 2010), using, respectively, RAxML-HPC2 version 8.2.12 (Stamatakis, 2014) and PAUP version 4.0a165 (Swoford, 2002). The resulting trees were visualized with TreeView version 1.6.6 (Page, 1996).
MP analyses were performed using the heuristic search option with 1000 random taxa additions and Tree Bisection and Reconnection (TBR) as the branch-swapping algorithm. All characters were unordered and of equal weight, and alignment gaps were treated as missing data. Maxtrees was set to 1000, branches of zero length were collapsed, and all multiple, equally parsimonious trees were retained. Clade stability and robustness of the most parsimonious trees were assessed using bootstrap analysis with 1000 pseudoreplicates, each with ten replicates of random stepwise addition of taxa (Felsenstein, 1985; Hillis and Bull, 1993). Descriptive tree statistics for parsimony included tree length (TL), homoplasy index (HI), consistency index (CI), retention index (RI) and rescaled consistency index (RC).
ML analyses were performed using a General Time Reversible (GTR) nucleotide substitution model including a discrete gamma distribution and estimation of proportion of invariable sites (GTR+G+I) to accommodate variable rates across sites. Clade stability and robustness of the branches of the best-scoring ML tree were estimated by conducting rapid bootstrap analyses with iterations halted automatically by RAxML.
RESULTS
Symptoms and isolations
Symptoms of the disease were found on a single C. humilis palm. These were leaf spots randomly distributed on segments of several leaves, which were frequently accompanied by yellowing of the leaf tips and generalized blight (Figure 1). The leaf spots were discrete, circular to ellipsoidal, amphigenous, initially yellowish to brown-grey, each with a wide dark-brown border. The leaf spot centre became progressively greyish and brittle. Each spot was surrounded by a conspicuous yellow or brown to red-brown halo. The discrete spots frequently coalesced, giving rise to blotches of grey necrotic plant tissue. In mature or older spots, the leaf epidermis often flaked off exposing dark ascomata immersed in the necrotic tissue. The bitunicate, obovate to pyriform asci contained eight hyaline, 1-septate ascospores (Figure 1). The lack of pseudoparaphyses suggested that the fungus was closely related to genera in Teratosphaeriaceae or Mycosphaerellaceae.
Ascospores ejected from ascomata germinated slowly on 1/2 PDA. After 6-12 h 1-2 additional septa formed in each ascospore (Figure 1k and l). After 24-48 h, 1-2 swellings developed on the middle cells and the ascospores became markedly curved (Figure 1m). Further septa developed and germ tubes emerged at right angles to the long axis of each ascospore, all from one side of the ascospore (Figure 1n). Ascospores and germ tubes remained hyaline. The fungus grew slowly forming a black, amorphous mass of mycelium on 1/2 PDA that attained a diameter of ca. 3 mm after 3 months of incubation (Figure 1o). No signs of sporulation were visible even after extended periods of incubation of up to 3 months. No other fungi were isolated, even from surface sterilized tissues taken from within the leaf lesions and plated on 1/2 PDA.
Phylogenetic analyses
Results of the BLAST search with ITS and LSU of the fungus isolated from C. humilis revealed that it was closely related to species in Teratosphaeriaceae and Neodevriesiaceae, and only distantly related to Mycosphaerellaceae. The available ITS and LSU sequences of 81 strains of Teratosphaeriaceae and Neodevriesi-aceae, either sequenced in this study or retrieved from GenBank, were included in the phylogenetic analysis (Table 1). The concatenated ITS and LSU alignment of 80 ingroup taxa and one outgroup taxon comprised 1152 characters (including alignment gaps), with 599 characters for ITS and 553 for LSU, after alignment. Tree topologies resulting from maximum parsimony and maximum likelihood analyses were similar, and both presented well-resolved clades for each genus included in the analyses, mostly supported by high bootstrap values (= 70%). The ML tree is shown in Figure 2.
Of the 1152 characters, 653 were constant and 139 variable characters were parsimony-uninformative. MP analysis of the remaining 364 parsimony-informative characters resulted in 140 equally parsimonious trees of 2181 steps and a moderately high level of homoplasy as indicated by a CI of 0.381, an RI of 0.693, an HI of 0.619 and an RC of 0.264. Topology of the trees difered from one another only in the position of the isolates within the terminal groupings of the Teratosphaeria clade. All other clades were consistent in their phylogenetic positions.
The final likelihood score for the ML tree was -11854.411424. The matrix had 560 distinct alignment patterns, with 15.85% undetermined characters or gaps. Estimated base frequencies were as follows: A = 0.222286, C = 0.271515, G = 0.286947 and T = 0.219253; substitution rates AC = 1.523961, AG = 1.983358, AT = 1.573616, CG = 1.073620, CT = 5.812546 and GT = 1.000000; gamma distribution shape parameter a = 0.554479; proportion of invariable sites = 0.384063.
Although well-resolved, internal nodes within Te r-atosphaeriaceae received low bootstrap support (= 50%). Nevertheless, Teratosphaeriaceae and Neodevriesiaceae were well-separated, insomuch that represented a clade with 100% bootstrap support, which confirms the phylo-genetic diference that supports these two families.
The isolate obtained from C. humilis clustered in a separate and previously undescribed lineage among the selected genera in Teratosphaeriaceae and Neodevrie-siaceae. Nevertheless, its placement between these two families received low bootstrap support. Considering the results from both MP and ML analyses, this fungus clusters close to genera in Teratosphaeriaceae. A total of nine unique base pair diferences in the ITS locus and five in the LSU locus among the 81 isolates included in the phylogenies confirms the novel lineage of the isolate as a new genus here introduced in Mycosphaerellales.
Taxonomy
Based on DNA phylogeny and morphology, the isolate collected from leaf spots on C. humilis was distinct from all other known species and genera in Mycosphaerellales. The data presented here indicate that this fungus resides in Teratosphaeriaceae as a new species and a new genus. Descriptions of the fungus are provided below.
Palmeiromyces D.R.S. Pereira & A.J.L. Phillips, gen. nov. MycoBank: MB 834638
Etymology: Named for the Portuguese word for palms (palmeiras), the host on which it was found.
Type species: Palmeiromyces chamaeropicola D.R.S. Pereira & A.J.L. Phillips, sp. nov.
Ascomata pseudothecial, amphigenous, subepider-mal, immersed to erumpent, scattered or clustered, globose to subglobose, dark-brown, ostiolate. Ostiole circular, aperiphysate. Peridium thin-walled, composed of cells forming a textura angularis, outer layer composed of thick-walled, dark-brown to brown cells, inner layers composed of thin-walled, hyaline cells. Pseudopara-physes absent. Asci bitunicate, fssitunicate, pyriform to obovoid, with well-developed ocular chamber, smooth-walled, hyaline, 8-spored. Ascospores biseriate, hyaline, broadly ellipsoidal to subcylindrical, with rounded ends, smooth- and thin-walled, medianly 1-septate, not constricted at the septum.
Palmeiromyces chamaeropicola D.R.S. Pereira & A.J.L. Phillips, sp. nov. (Figure 1)
MycoBank: MB 834639
Etymology: Named for Chamaerops humilis, the host genus from which it was collected.
Holotype: AVE-F-11
Leaf spots sunken, circular to broadly ellipsoidal, 3-7 x 2-3 mm (mean ± SD = 4.67 ± 1.06 x 2.52 ± 0.51 mm), identical on both leaf surfaces, brown-grey to yellowish centre, later becoming greyish and fragile, with a dark-brown border (ca. 1 mm wide), surrounded by a conspicuous yellow or brown to red-brown halo, occasionally coalescing, randomly distributed. Mature leaf spots contain several immersed ascomata. Ascomata pseudothecial, amphigenous, subepidermal, immersed to erumpent, scattered or clustered in groups of two or three, globose to subglobose, dark-brown, up to 90 μm diam., ostiolate. Ostiole circular, up to 21 μm diam., aperiphysate. Peridium thin-walled, composed of cells forming a textura angularis, outer layer composed of thick-walled, dark-brown to brown cells, inner layers composed of thin-walled, hyaline cells. Pseudo-paraphyses absent, but pseudoparenchymatous, cellular hamathecium remnants present. Asci bitunicate, outer wall up to 2 μm thick, fssitunicate, pyriform to obovoid, slightly curved, broader at the base, well-developed ocular chamber, smooth-walled, hyaline, 8-spored, 21.4-57.9 x 8.2-19.1 μm (mean ± SD = 33.22 ± 9.69 x 14.20 ± 3.75 μm, n = 11). Ascospores biseriate, broadly ellipsoidal to subcylindrical, with rounded ends, occasionally slightly curved, hyaline, smooth- and thin-walled, guttulate, medianly 1-septate, not to slightly constricted at the septa, 9.5-21.9 x 2.8-5.5 μm (mean ± SD = 17.31 ± 3.23 x 4.05 ± 0.72 μm, n = 50); mean ± SD ascospore length/ width ratio = 4.30 ± 0.58 (n = 50).
Material examined: PORTUGAL, Oeiras, National Sports Centre of Jamor, on leaf spots of Chamaerops humilis (Arecaceae), 20 September 2018, Alan J.L. Phillips (holotype AVE-F-11, ex-type living culture CDP 0 01).
GenBank Numbers: ITS: MT068628; LSU: MT076194.
Distribution: Oeiras, Portugal.
Host: Chamaerops humilis.
Notes: Palmeiromyces chamaeropicola was found associated with leaf spots of C. humilis, but pathogenic-ity has not been tested. Nevertheless, there is evidence that this species represents an obligate biotroph causing a previously undescribed disease on C. humilis. Phylo-genetically, P. chamaeropicola is closely related to genera in Teratosphaeriaceae (Figure 2). Morphology of the sexual morph, ascospores with a peculiar mode of germination, lack of an asexual morph and very slow growth in culture correspond to genera in Te rat o s p h a-eriaceae (Crous et al., 2007). However, ascospores of P. chamaeropicola lack mucous sheaths, which is a characteristic of Teratosphaeriaceae (Crous et al., 2007; Quaed-vlieg et al., 2 014).
DISCUSSION
In this study, a new species in Teratosphaeriaceae, Palmeiromyces chamaeropicola, is described and a new genus is established to accommodate the fungus. Phy-logenetic analyses based on ITS and LSU sequences revealed that Palmeiromyces represents a separate lineage close to several Teratosphaeriaceae genera, as well as to Neodevriesiaceae. The evidence gained from unique nucleotide diferences among the several genera included in the phylogeny supports this novelty at genus-level. This species was associated with, and is considered to be the cause of, a leaf spot disease of the palm C. humilis.
Morphologically Palmeiromyces chamaeropicola resembles Mycosphaerellaceae and Teratosphaeriace-ae, characterized by small, inconspicuous ascomata immersed in the host tissue, which produce pyriform asci with eight hyaline, ellipsoidal and medianly 1-sep-tate ascospores. The presence of pseudoparenchymatal remnants in ascomata of Palmeiromyces and the absence of paraphyses place it within Teratosphaeriaceae, since Crous et al. (2007) used these characters to separate Teratosphaeriaceae from Mycosphaerellaceae. Nevertheless, the low bootstrap support for the internal nodes and thus for the branches between P. chamaeropicola and the remaining taxa in the phylogenetic analyses suggest that future studies may reveal a different phylo-genetic position for this taxon, possibly as a new family. Genera in Teratosphaeriaceae and Mycosphaerellaceae are often defined based on DNA sequence data, and on morphology of their asexual morphs. However, P. chamaeropicola barely grew in culture with no signs of asexual sporulation. This is common in Teratosphaeria species, which are cultivated with difculty (Crous et al., 2007, 2009c). Morphologically P. chamaeropicola resembles Mycosphaerella cocoës, which was found associated with leaf spots on various palm hosts, such as Calamus, Cocos and Mauritia (Fröhlich and Hyde, 1998). However, no cultures linked to the holotype of M. cocoës are extant and thus no DNA data are available for this species. Comparison of species within Mycosphaerellaceae and Teratosphaeriaceae solely on the basis of morphology is unreliable (Hunter et al., 2006; Crous et al., 2008, 2009c). Crous et al. (2008) noted that the morphological species concept had in the past obscured the presence of novel taxa, which have been resolved by means of molecular analyses. Terefore, it was not possible to determine the phylogenetic relationship between P. chamaeropicola and M. cocoës.
The phylogenetic position of P. chamaeropicola within Mycosphaerellales is uncertain and no accurate nearest neighbours could be indicated in the present analyses. In addition, a high level of homoplasy was detected in the MP analysis. Most genera within Teratosphaeria are polyphyletic, and within Capnodiales are paraphyl-etic (Crous et al., 2007). Convergence is observed in several genera, especially with respect to the morphology of asexual morphs (Crous et al., 2007, 2009b; Ruibal et al., 2008; Quaedvlieg et al., 2014). Capnodiales was recently sub-divided into seven orders (Abdollahzadeh et al., 2019) with Mycosphaerellaceae and Teratosphaeriaceae accommodated in Mycosphaerellales.
The phylogenetic analyses in this study suggest that Palmeiromyces could represent a new family, although it is not regarded as such in the present study, mainly due to low taxon sampling for the new genus. Besides the DNA phylogenetic data, P. chamaeropicola lacks several morphological characters that are diagnostic for Teratosphaeria, the type genus of Te r a t o s p h a -eriaceae. These characters include ascospores that turn brown and verruculose while still in the asci, as well as the presence of mucoid sheaths around the ascospores (Quaedvlieg et al., 2014), all of which are lacking in Palmeiromyces. In addition, the germination pattern of ascospores of Palmeiromyces is completely distinct from those reported in Mycosphaerellaceae, although this pattern is seen in some species in Teratospha-eriaceae. Tus, Palmeiromyces clearly represents a separate genus within Mycosphaerellales where several phy-logenetic lineages remain poorly resolved due to limited taxon sampling (Quaedvlieg et al., 2014). However, its position within Teratosphaeriaceae cannot be established and it is possible that future studies with greater taxon sampling may split Palmeiromyces from The rato -sphaeriaceae.
Palmeiromyces chamaeropicola was collected from diseased foliage of C. humilis and reveals a new insight into the Teratosphaeriaceae leaf diseases (TLD) and Mycosphaerellaceae leaf diseases (MLD). Although the pathogenicity of P. chamaeropicola has not been tested, the extremely slow growth rate in culture and almost complete lack of growth on agar suggests that this fungus has highly specific growth requirements and can be regarded as an obligate biotroph. The fungus barely grows in culture, attaining a colony diameter of only 1 mm after 1 month of incubation. Furthermore, this growth was a black, amorphous mass of sterile mycelium, which can hardly be regarded as a colony. This extremely slow growth rate is often reported in important leaf spotting fungi within Cap-nodiales (Crous et al., 2008), so P. chamaeropicola represents a new record of a phytopathogenic fungus. The report of a previously undescribed leaf spotting fungus in Mycosphaerellales represents a significant advance in the knowledge on TLD and MLD, since these fungi are important phytopathogens in various plant hosts, including Eucalyptus (Hunter et al., 2006; Crous et al., 2009a; Pérez et al., 2009; 2013; Taylor et al., 2012; Quaedvlieg et al., 2014). Furthermore, several species within Mycosphaerellales families, especially Te r a t o -sphaeriaceae, are of quarantine importance in many countries in Europe (Crous et al., 2009a; Quaedvlieg et al., 2012; Crous et al., 2019).
The present study has revealed a new disease of the ornamental, indigenous palm, C. humilis, caused by a fungal species in a previously unknown genus in the Mycosphaerellales. The geographical distribution of this fungus is, for now, confned to a single plant in the Lisbon district of Portugal. The survey conducted in this study can only be regarded as preliminary and it is likely that wider surveys will reveal more cases of this previously undescribed disease. Terefore, further sampling is essential to understand the geographical and ecological range of P. chamaeropicola. Future studies should also aim to elucidate the ecology and physiology of Palmeiro-myces to assess its traits as a phytopathogen.
ACKNOWLEDGEMENTS
Work supported by UIDB/04046/2020 and UIDP/04046/2020 Centre grants from FCT, Portugal (to BioISI).
LITERATURE CITED
Abdollahzadeh, J., Groenewald, J.Z., Coetzee, M.P.A., Wingfield, M.J., Crous, P.W., 2019. Evolution of lifestyles in Capnodiales. Studies in Mycology (in press). DOI: 10.1016/j.simyco.2020.02.004
Armengol A., Moretti A., Perrone G., Vicent A., Bengoe-chea J.A., García-Jiménez J., 2005. Identification, incidence and characterization of Fusarium proliferatum on ornamental palms in Spain. European Journal of Plant Pathology 112: 123-131.
Carapeto A., Clamote F., Schwarzer U., Pereira A.J., Almeida J.D., et al., 2020. Chamaerops humilis L. -mapa de distribuição. Flora-On: Flora de Portugal Interactiva, Sociedade Portuguesa de Botânica. http:// www.fora-on.pt/#wChamaerops+humilis. Accessed 25 February 2020.
Crous P.W., Braun U., Groenewald J.Z., 2007. Mycosphaerella is polyphyletic. Studies in Mycology 58: 1-32.
Crous P.W., Summerell B.A., Mostert L., Groenewald J.Z., 2008. Host specificity and speciation of Mycosphaere-lla and Teratosphaeria species associated with leaf spots of Proteaceae. Persoonia 20: 59-86.
Crous P.W., Groenewald J.Z., Summerell B.A., Wingfield B.D., Wingfield M.J., 2009a. Co-occurring species of Teratosphaeria on Eucalyptus. Persoonia 22: 38-48.
Crous P.W., Schoch C.L., Hyde K.D., Wood A.R., Guei-dan C., ... Groenewald J.Z., 2009b. Phylogenetic lineages in the Capnodiales. Studies in Mycology 64: 17-47.
Crous P.W., Summerell B.A., Carnegie A.J., Wing-field M.J., Groenewald J.Z., 2009c. Novel species of Mycosphaerellaceae and Teratosphaeriaceae. Persoonia 23: 119-146.
Crous P.W., Wingfield M.J., Cheewangkoon R., Carnegie A.J., Burgess T.I, ... Groenewald J.Z., 2019. Foliar pathogens of eucalypts. Studies in Mycology 94: 125-298.
Delgado G., Miller A.N., Piepenbring M., 2018. South Florida microfungi: Castanedospora, a new genus to accommodate Sporidesmium pachyanthicola (Cap-nodiales, Ascomycota). Cryptogamie Mycologie 39: 109-127.
Dransfield J., Uhl N.W., Asmussen C.B., Baker W.J., Har-ley M.M., Lewis C.E., 2008. Genera Palmarum - the
Evolution and Classification of Palms. 2nd ed. Royal Botanic Gardens, Kew, London, UK, 744 pp.
Elliott M.L., 2004. Compendium of Ornamental Palm Diseases and Disorders. 2nd ed. American Phytopatho-logical Society Press, Saint Paul, Minnesota, USA, 69 pp.
Felsenstein J., 1985. Confdence limits on phylogenies: an approach using the bootstrap. Evolution 39: 783-791.
Fröhlich J., Hyde K.D., 1998. Fungi from palms. XXXVI-II. The genera Mycosphaerella and Sphaerella. Sydow-ia 50: 171-181.
Guzmán B., Fedriani J.M., Delibes M., Vargas P. , 2017. The colonization history of the Mediterranean dwarf palm (Chamaerops humilis L., Palmae). Tree Genetics & Genomes 13: 24.
Hall T.A., 1999. BioEdit: a user-friendly biological sequence alignment editor and analysis program for Windows 95/98/NT. Nucleic Acids Symposium Series 41: 95-98.
Hillis D.M., Bull J.J., 1993. An empirical test of bootstrapping as a method for assessing confdence in phylo-genetic analysis. Systematic Biology 42: 182-192.
Hunter G.C., Wingfield B.D., Crous P.W., Wingfield M.J., 2006. A multi-gene phylogeny for species of Mycosphaerella occurring on Eucalyptus leaves. Studies in Mycology 55: 147-161.
Isola D., Zucconi L., Onofri S., Caneva G., de Hoog G.S., Selbmann L., 2016. Extremotolerant rock inhabiting black fungi from Italian monumental sites. Fungal Diversity 76: 75-96.
Maharachchikumbura S.S.N., Hyde K.D., Groenewald J.Z., Xu J., Crous P.W., 2014. Pestalotiopsis revisited. Studies in Mycology 79: 121-186.
Miller M.A., Pfeifer W. , Schwartz T., 2010. Creating the CIPRES Science Gateway for inference of large phy-logenetic trees. In: Proceedings of the Gateway Computing Environments Workshop (GCE), 2010, November 14, 2010, New Orleans, Los Angeles, USA, 1-8.
O'Donnell K., 1993. Fusarium and its near relatives. In: Te Fungal Holomorph: Mitotic, Meiotic and Pleomor-phic Speciation in Fungal Systematics (D.R. Reynolds, J.W. Taylor, ed.), CAB International, Wallingford, England, UK, 225-233.
Page R.D.M., 1996. TreeView: an application to display phylogenetic trees on personal computers. Bioinfor-matics 12: 357-358.
Palmweb, 2020. Palmweb: Palms of the World Online. http://www.palmweb.org/. Accessed 24 February 2020.
Pérez C.A., Wingfield M.J., Altier N.A., Blanchette R.A., 2009. Mycosphaerellaceae and Teratosphaeriaceae associated with Eucalyptus leaf diseases and stem cankers in Uruguay. Forest Pathology 39: 249-360.
Pérez C.A., Wingfield M.J., Altier N.A., Blanchette R.A., 2013. Species of Mycosphaerellaceae and Teratospha-eriaceae on native Myrtaceae in Uruguay: evidence of fungal host jumps. Fungal Biology 117: 94-102.
Piepenbring M., Nold F., Trampe T., Kirschner R., 2012. Revision of the genus Graphiola (Exobasidiales, Basidiomycota). Nova Hedwigia 94: 67-96.
Pitcher D.G., Saunders N.A., Owen R.J., 1989. Rapid extraction of bacterial genomic DNA with guanidium thiocyanate. Letters in Applied Microbiology 8: 151- 156.
Quaedvlieg W. , Groenewald J.Z., de Jesús Yáñez-Morales M., Crous P.W., 2012. DNA barcoding of Mycosphaerella species of quarantine importance to Europe. Persoonia 29: 101-115.
Quaedvlieg W. , Binder M., Groenewald J.Z., Summerell B.A., Carnegie A.J., ... Crous P.W., 2014. Introducing the consolidated species concept to resolve species in the Teratosphaeriaceae. Persoonia 33: 1-40.
Quattrocchi, F.L.S.U., 2017. The CRC World Dictionary of Palms: Common Names, Scientifc Names, Eponyms, Synonyms, and Etymology. 1st ed. CRC Press, Taylor & Francis Group, Boca Raton, Florida, USA, 2753 pp.
Ruibal C., Platas G., Bills G.F., 2008. High diversity and morphological convergence among melanised fungi from rock formations in the Central Mountain System of Spain. Persoonia 21: 93-110.
Stamatakis A., 2014. RAxML version 8: a tool for phy-logenetic analysis and post-analysis of large phylog-enies. Bioinformatics 30: 1312-1313.
Swoford D.L., 2002. PAUP*. Phylogenetic Analysis Using Parsimony (*and other methods). Version 4. 2nd ed. Sinauer Associates, Sunderland, Massachusetts, USA.
Taylor K., Andjic V. , Barber P.A., Hardy G.E.S., Burgess T.I., 2012. New species of Teratosphaeria associated with leaf diseases on Corymbia calophylla (Marri). Mycological Progress 11: 159-169.
Tompson J.D., Gibson T.J., Plewniak F., Jeanmougin F., Higgins D.G., 1997. The ClustalX windows interface: fexible strategies for multiple sequence alignment aided by quality analysis tools. Nucleic Acids Research 25: 4876-4882.
Wang M.M., Shenoy B.D., Li W. , Cai L., 2017. Molecular phylogeny of Neodevriesia, with two new species and several new combinations. Mycologia 109: 965-974.
White T.J., Bruns T., Lee S., Taylor J., 1990. Amplifca-tion and direct sequencing of fungal ribosomal RNA genes for phylogenetics. In: PCR protocols: a Guide to Methods and Applications (M.A. Innis, D.H. Gelfand, J.J. Sninsky, T.J. White, ed.), Academic Press, San Diego, California, USA, 315-322.
You have requested "on-the-fly" machine translation of selected content from our databases. This functionality is provided solely for your convenience and is in no way intended to replace human translation. Show full disclaimer
Neither ProQuest nor its licensors make any representations or warranties with respect to the translations. The translations are automatically generated "AS IS" and "AS AVAILABLE" and are not retained in our systems. PROQUEST AND ITS LICENSORS SPECIFICALLY DISCLAIM ANY AND ALL EXPRESS OR IMPLIED WARRANTIES, INCLUDING WITHOUT LIMITATION, ANY WARRANTIES FOR AVAILABILITY, ACCURACY, TIMELINESS, COMPLETENESS, NON-INFRINGMENT, MERCHANTABILITY OR FITNESS FOR A PARTICULAR PURPOSE. Your use of the translations is subject to all use restrictions contained in your Electronic Products License Agreement and by using the translation functionality you agree to forgo any and all claims against ProQuest or its licensors for your use of the translation functionality and any output derived there from. Hide full disclaimer
© 2020. This work is published under https://creativecommons.org/licenses/by/4.0/ (the “License”). Notwithstanding the ProQuest Terms and Conditions, you may use this content in accordance with the terms of the License.
Abstract
In September 2018, a leaf spot disease was noticed on a European fan palm (Chamaerops humilis L.) in Oeiras, Portugal. The aim of this study was to identify and characterize the causative agent of this disease symptom. Morphological characters and phylogenetic data derived from ITS and LSU sequences revealed that the leaf spot was caused by a flamentous fungus in the Mycosphaerellales, as a unique lineage within the Teratosphaeriaceae. This pathogen is introduced here as a new genus and species, Palmeiromyces chamaeropicola D.R.S. Pereira & A.J.L. Phillips, the cause of a newly reported leaf disease on Chamaerops humilis.
You have requested "on-the-fly" machine translation of selected content from our databases. This functionality is provided solely for your convenience and is in no way intended to replace human translation. Show full disclaimer
Neither ProQuest nor its licensors make any representations or warranties with respect to the translations. The translations are automatically generated "AS IS" and "AS AVAILABLE" and are not retained in our systems. PROQUEST AND ITS LICENSORS SPECIFICALLY DISCLAIM ANY AND ALL EXPRESS OR IMPLIED WARRANTIES, INCLUDING WITHOUT LIMITATION, ANY WARRANTIES FOR AVAILABILITY, ACCURACY, TIMELINESS, COMPLETENESS, NON-INFRINGMENT, MERCHANTABILITY OR FITNESS FOR A PARTICULAR PURPOSE. Your use of the translations is subject to all use restrictions contained in your Electronic Products License Agreement and by using the translation functionality you agree to forgo any and all claims against ProQuest or its licensors for your use of the translation functionality and any output derived there from. Hide full disclaimer
Details
1 Universidade de Lisboa, Faculdade de Ciências, Biosystems and Integrative Sciences Institute (BioISI), Campo Grande, 1749-016 Lisbon, Portugal