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A New Leaf Spot Disease of Chamaerops Humilis Caused by Palmeiromyces Chamaeropicola Gen

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A New Leaf Spot Disease of Chamaerops Humilis Caused by Palmeiromyces Chamaeropicola Gen Phytopathologia Mediterranea Firenze University Press The international journal of the www.fupress.com/pm Mediterranean Phytopathological Union New or Unusual Disease Reports A new leaf spot disease of Chamaerops humilis caused by Palmeiromyces chamaeropicola gen. et Citation: D.R.S. Pereira, A.J.L. Phil- lips (2020) A new leaf spot disease sp. nov. of Chamaerops humilis caused by Palmeiromyces chamaeropicola gen. et sp. nov.. Phytopathologia Mediter- ranea 59(2): 353-363. DOI: 10.14601/ Diogo R.S. PEREIRA, Alan J.L. PHILLIPS* Phyto-11213 Universidade de Lisboa, Faculdade de Ciências, Biosystems and Integrative Sciences Insti- Accepted: June 17, 2020 tute (BioISI), Campo Grande, 1749-016 Lisbon, Portugal *Corresponding author: [email protected] Published: August 31, 2020 Copyright: © 2020 D.R.S. Pereira, Summary. In September 2018, a leaf spot disease was noticed on a European fan palm A.J.L. Phillips. This is an open access, (Chamaerops humilis L.) in Oeiras, Portugal. The aim of this study was to identify and peer-reviewed article published by characterize the causative agent of this disease symptom. Morphological characters and Firenze University Press (http://www. fupress.com/pm) and distributed phylogenetic data derived from ITS and LSU sequences revealed that the leaf spot was under the terms of the Creative Com- caused by a filamentous fungus in the Mycosphaerellales, as a unique lineage within mons Attribution License, which per- the Teratosphaeriaceae. This pathogen is introduced here as a new genus and species, mits unrestricted use, distribution, and Palmeiromyces chamaeropicola D.R.S. Pereira & A.J.L. Phillips, the cause of a newly reproduction in any medium, provided reported leaf disease on Chamaerops humilis. the original author and source are credited. Keywords. Fungus, new genus, new species, palm tree, plant pathogen. Data Availability Statement: All rel- evant data are within the paper and its Supporting Information files. Competing Interests: The Author(s) INTRODUCTION declare(s) no conflict of interest. Chamaerops humilis L. (Arecaceae), commonly known as the European Editor: Lizel Mostert, Faculty of AgriSciences, Stellenbosch, South fan palm or Mediterranean dwarf palm, is one of only two indigenous Euro- Africa. pean 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, Sic- ily, 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 natural- ly, 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 Phytopathologia Mediterranea 59(2): 353-363, 2020 ISSN 0031-9465 (print) | ISSN 1593-2095 (online) | DOI: 10.14601/Phyto-11213 354 Diogo R.S. Pereira, Alan J.L. Phillips Basidiomycete found exclusively on palms (Piepenbring Morphological observations and characterization et al., 2012). Fröhlich and Hyde (1998) listed six species of Mycosphaerella from leaf spots on different palms, Microscopic structures of isolated fungi were mount- including M. chamaeropis on C. humilis. More recently, ed in 100% lactic acid and examined with differential Fusarium wilt and dieback were reported from young interference contrast (DIC) microscopy. Observations and adult C. humilis in Spain (Armengol et al., 2005), of micromorphological features were made with Leica and Pestalotiopsis chamaeropis was described from MZ9.5 and Leica DMR microscopes (Leica Microsys- leaves of this host in Italy (Maharachchikumbura et al., tems GmbH), and digital images were recorded, respec- 2014). tively on the two microscopes, with Leica DFC300 and In the present study, a leaf spot disease was noticed Leica DFC320 cameras (Leica Microsystems GmbH). on a C. humilis palm growing as an ornamental. On the Measurements were made with the measurement mod- lesions, ascomata, asci and ascospores with characteris- ule of the Leica IM500 Image Management System (Lei- tics of the Mycosphaerellales (Abdollahzadeh et al., 2019) ca Microsystems GmbH). Mean, standard deviation (SD) were found. Therefore, the purpose of this paper was to and 95% confidence intervals were calculated from n = characterize the fungus in terms of its morphology and total of measured structures. Measurements are present- phylogeny. ed as minimum and maximum dimensions with mean and SD in parentheses. Photographs were prepared with Adobe Photoshop CS6 (Adobe). MATERIALS AND METHODS Specimen collection and examination DNA extraction, PCR amplification and sequencing Diseased leaf segments with leaf spots were collected Genomic DNA (gDNA) was extracted from myce- from an ornamental C. humilis palm in Oeiras, near Lis- lium of cultures grown on 1/2 PDA, following a modi- bon, Portugal. Plant material was transported to a lab- fied and optimized version of the guanidium thiocy- oratory in paper envelopes, and examined with a Leica anate method described by Pitcher et al. (1989). PCR MZ9.5 stereo microscope (Leica Microsystems GmbH) reactions were carried out with Taq polymerase, nucle- for observations of lesion morphology and associated otides, primers, PCR-water (ultrapure DNase/RNase- fungi. free distilled water) and buffers supplied by Invitrogen. Amplification reactions were performed in a TGradient Thermocycler (Biometra). Amplified PCR products were Fungal isolation purified and sequenced by Eurofins (Germany). The primers ITS5 (White et al., 1990) and NL-4 A small piece of a leaf spot lesion bearing ascomata (O’Donnell, 1993) were used to amplify part of the clus- was placed on a drop of sterile water in the inverted lid ter of rRNA genes, including the nuclear 5.8S rRNA of a Petri dish. The dish base, containing half-strength gene and its flanking ITS1 and ITS2 regions, along potato dextrose agar (1/2 PDA) (BIOKAR Diagnostics) with the first two domains of the large-subunit rRNA was placed on top of the inverted lid. Ascospores were gene (ITS-D1/D2 rDNA region). The PCR reaction mix- discharged upwards and impinged on the agar sur- ture consisted of 50–100 ng of gDNA, 1× PCR buffer, face. After incubating overnight, single germinating 50 pmol of each primer, 200 μM of each dNTP, 2 mM ascospores were transferred to fresh plates of 1/2 PDA. MgCl2, 1 U Taq DNA polymerase, and was made up Cultures were then incubated in ambient light at room to a total volume of 50 μL with PCR water. The follow- temperature (18–20°C). ing cycling conditions were used: initial denaturation at Isolations were also made directly from leaf spots. 95°C for 5 min, followed by 40 cycles of denaturation at Pieces of leaf spot tissue 1–2 mm2 were cut from the 95°C for 1 min, annealing at 52°C for 30 s and elonga- edge of each lesion, surface sterilized in 5% sodium tion at 72°C for 1.5 min, and a final elongation step at hypochlorite for 1 min, rinsed in three times in sterile 72°C for 10 min. water, and then blotted dry on sterile filter paper. The The ITS region was sequenced only in the forward fragments were plated onto 1/2 PDA containing 0.05% direction using the primer ITS5. The D1/D2 region chloramphenicol, and incubated at room temperature (LSU) was sequenced only in the forward direction until colonies developed. using the primers ITS5 and NL1 (O’Donnell, 1993). Consensus sequences were produced and edited with BioEdit version 7.0.5.3 (Hall, 1999). New leaf spot on European fan palm 355 Sequence alignment and phylogenetic analyses variable rates across sites. Clade stability and robustness of the branches of the best-scoring ML tree were esti- ITS and LSU sequences of species in representa- mated by conducting rapid bootstrap analyses with iter- tive genera of Teratosphaeriaceae and Neodevriesiaceae ations halted automatically by RAxML. in Mycosphaerellales were retrieved from GenBank by BLAST searches with the sequences generated in this study (Table 1). These were supplemented with taxa list- RESULTS ed in recent literature (e.g. Quaedvlieg et al., 2014; Isola et al., 2016; Wang et al., 2017; Delgado et al., 2018). Cap- Symptoms and isolations nodium coffeae Pat. was used as the outgroup taxon rep- resentative of a species in Capnodiales. Symptoms of the disease were found on a single C. Sequences were aligned with ClustalX version 2.1 humilis palm. These were leaf spots randomly distribut- (Thompson et al., 1997) using the following parameters: ed on segments of several leaves, which were frequently pairwise alignment parameters (gap opening = 10, gap accompanied by yellowing of the leaf tips and general- extension = 0.1), and multiple alignment parameters ized blight (Figure 1). The leaf spots were discrete, cir- (gap opening = 10, gap extension = 0.2, DNA transition cular to ellipsoidal, amphigenous, initially yellowish to weight = 0.5, delay divergent sequences = 25%). Align- brown-grey, each with a wide dark-brown border. The ments were checked and manual adjustments were made leaf spot centre became progressively greyish and brittle. where necessary with BioEdit. Terminal regions with Each spot was surrounded by a conspicuous yellow or data missing in some of the isolates were excluded from brown to red-brown halo. The discrete spots frequently the analysis. The aligned ITS and LSU sequences were coalesced, giving rise to blotches of grey necrotic plant concatenated and combined in a single matrix.
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