Presence of false smut (Graphiola phoenicis (Moug. ex Fr.) Poit.) on Canary date palm (Phoenix canariensis) on Easter Island, Chile

Cien. Inv. Agr. 44(3):307-311. 2017 www.rcia.uc.cl plant pathology DOI: 10.7764/rcia.v44i3.1787 research note Presence of false smut (Graphiola phoenicis (Moug. ex Fr.) Poit.) on Canary date palm (Phoenix canariensis) on Easter Island, Chile Germán Sepúlveda1, Mabel Arismendi1, Wilson Huanca-Mamani1, Steffany Cárdenas-Ninasivincha1, Ricardo Salvatierra2, and Bernardo Latorre3 Universidad de Tarapacá, Facultad de Ciencias Agronómicas, Avda. General Velasquez 1775, Arica, Chile 2 CEAZA,Universidad de La Serena, Campus Andrés Bello, La Serena, Chile. 3 Pontificia Universidad Católica de Chile, Facultad de Agronomía e Ingeniería Forestal, Departamento de Fruticultura y Enología. Casilla 306-22, Santiago, Chile 1 Abstract G. Sepúlveda, M. Arismendi, W. Huanca-Mamani, S. Cárdenas-Ninasivincha, R. Salvatierra, and B. Latorre. 2017. Presence of false smut (Graphiola phoenicis (Moug. ex Fr.) Poit.) on Canary date palm (Phoenix canariensis) on Easter Island, Chile. Cien. Inv. Agr. 44(3): 307-311. Graphiola phoenicis was found on leaves of Phoenix canariensis in Hanga Roa, Easter Island, Chile. Amphigenous black pulvinate basidiomata were abundant on the leaf blades and rachides, causing extensive foliar damage. The samples were examined by a light microscope after three days in a humid chamber. In addition, the molecular tools scanning electron microscopy and histological sectioning were used to study the fungal/host relationship, complementing the identification. Morphometric and molecular characteristics led to the identification of the fungus as Graphiola phoenicis causing false smut on the Canary date palms (Phoenix canariensis). This is the first report of this plant pathogen in Chilean territory. Keywords: Exobasidiales, false smut, Graphiola leaf spot Introduction Easter Island, part of Chilean territory, is located in the South Pacific (27° 08’S; 109° 26’W) 3,747 km west of the South American coast. The landscape of the island is dominated by grasses, shrubs and isolated tree species such as Eucalyptus globulus and other exotic trees. Palynological, phytolithic and paleontological studies showed Received May 25, 2017. Accepted December 01, 2017. Corresponding author: that the endemic vegetation was extinct – for example, Paschalococos disperta (Dransfield et al. 1984), congeneric with Central Chile’s Jubaea chilensis, (Grau, 2005). Among valued ornamental and introduced Arecaceae species, the Canary Island date palm Phoenix canariensis Hort. ex Chaub., native to the Canary Islands, populates urban and rural places, importing a tropical look to the landscape of Easter Island. Until now, there have been no reports of foliar diseases affecting P. canariensis on Easter Island. Here, we report the presence of a false smut fungus attacking P. canariensis. 308 CIENCIA E INVESTIGACIÓN AGRARIA Materials and methods Plant Sample and morphologic identification In January 2015, a random sample of diseased leaves from palms of P. canariensis was collected in Hanga Roa, Avenue Atanu Tekena (27° 09’08.48”S; 109° 25’53.52”W; 28 masl). Symptoms were characterized initially by very small yellow lesions that turned dark brown in the center with fuzzy edges, affecting primarily the oldest leaves. Lesions appeared isolated or else grouped on both side of the leaves. Morphometric studies were conducted on additional herbarium samples of diseased leaves, and micrometric leaf sections were obtained for optical microscope observations. Sections of approximately 0.25 cm2 were obtained for environmental scanning electron microscope (SEM) observations in an EVO LS 10 microscope (Carl Zeiss, Germany), placed in aluminum sample holders with carbon-contact-bearing adhesives, and analyzed under vacuum with variable pressure mode (VP) (chamber pressure 150 Pa (under vacuum) and column 2×10-5 Torr (high vacuum)). The working distance (WD) varied depending on the sample type. The acceleration voltage was 15 KV, the tilt was 0° to 90°, and the images were taken with a resolution of 3.024 × 2.304 pixels at a scanning speed of 12 min 54 s. Molecular identification For molecular identification, dark lesions on foliar pinnae were collected, and DNA extraction was successful using an E.Z.N.A.® Insect DNA Kit (Omega Bio-Tek, Georgia, EEUU) according to the manufacturer’s instructions. Subsequently, the internal transcribed spacer region (ITS) and the D1/ D2 domain of the large subunit ribosomal DNA 28S (LSU rDNA) were amplified using primers ITS4 (5’-TCCTCCGCTTATTGATATGC-3) and ITS1 (5’-TGAACCTGCAGAAGGATCATTA-3’) (White et al., 1990; Barnes and Szabo, 2007) as well as NL1m (5’-GCATATCAATAAGCGGAGGAAAAG-3’) and NL-4m (5’-GGTCCGTGTTTCAAGACG-3’) (O’Donnell, 1993). PCR amplifications of the LSU and ITS rDNA were performed in a final volume of 20 μL. The reactions contained 1 µL of DNA extract; 5 p moles of each primers; 2.5 mM each dNTP; 2 mM MgCl2; 1X PCR buffer (KCl); 1 unit of Taq DNA polymerase (Thermo Scientific) and sterile distilled water. Cycling conditions were 5 min at 94 °C; 35 cycles of 1 min at 94 °C, 1 min at 55 °C and 1 min at 72 °C; and a final elongation step of 2 min at 72 °C. PCR blank reaction controls were incorporated. Each PCR product (3 μL) was visualized on a 1.5% agarose gel stained with gelred (Biotium). The amplified products were sent to Macrogen (South Korea) for purification and direct sequencing. The nucleotide sequences were visualized and edited using 4Peaks software (http://nucleobytes. com/4peaks/) and checked manually; nucleotides with ambiguous positions were clarified. The sequences obtained were compared with rDNA D1/D2 and ITS data sequences from strains available in GenBank (www.ncbi.nem. nih.gov) by using BLASTn, and sequences with ≥98% similarity were downloaded in FASTA format. The sequence alignment and phylogenetic analysis were conducted using MEGA version 6.0 (Tamura et al., 2013). Alignments were checked and manually adjusted when necessary. The Kimura 2-Parameter model (Kimura, 1980) was used to estimate evolutionary distance, and the gaps were treated as missing data. Phylogenetic reconstruction was performed using the maximum likelihood algorithm, and the robustness of the branches was assessed by bootstrap analysis (Felsenstein, 1985) of 1,000 replicates. Results and Discussion Foliar pinnae showed small yellow to dark lesions on both sides of the leaf blade, with brown to black globular, cylindrical or irregular sori VOLUME 44 Nº3 SEPTEMBER – DECEMBER 2017 309 Figure 1. Reproductive structures of the false smut fungus (Graphiola phoenicis) affecting Canary date palms (Phoenix canariensis) on Easter Island, Chile. A: Sori distributed on the surface of P. canariensis leaflets. Bar=2 mm. B and C: Sorus profile view, with abundant thread-like filaments containing spermacia. Bars=0.5 mm. D: A thread-like filament with spermacia. Bar=10 µm. E: SEM image of sorus. Bar=100 µm. F: Detail of a thread-like filament with spermacia. Bar=10 µm. (Figure 1 (...truncated)