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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 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.
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 gel-red (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 (Figure 1A-C). Sori are fruiting bodies of 0.5 to 1.2 mm in diameter with a subepidermal origin, with dark and hard outer walls (Figure 1A-C).
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.
As sori mature, white to creamy thread-like filaments (Figure 1B-C) emerge through the ostiole of each sorus. Spherical to elliptical spermacia 2.5-3.0 μm diameter, with thick hyaline walls, were produced (Figure 1D). In SEM images (Figure 1D-F), it was possible to observed abundant spermacia attached to the filament, suggesting that filaments help with dispersal. The morphometric characterization was coincident with previous descriptions of G. phoenicis (Cole, 1983; Tubaki and Yokoyama, 1971).
To confirm the morphological identification of G. phoenicis, a PCR fragment of ITS and LSU rDNA of isolates from Easter Island were successfully amplified and sequenced, obtaining 517 and 560 bp fragments for ITS and LSU, respectively, which were deposited in GenBank (Accession numbers KX344499 and KX344500, respectively).
Blast analysis performed with ITS sequences showed a 98% similarity of the Easter Island isolate with G. phoenicis from South Africa (KP730059), and LSU showed a 99% with G. phoenicis from Japan (AF009862). Phylogenetic analysis revealed a Graphiola cluster with nodal support of 86%. Between Graphiola species, the Easter strain has the highest similarity with G. phoenicis (Figure 2). This also supports the assignation of the Easter strain as G. phoenicis.
Figure 2 Phylogenetic tree based on LSU analysis of Graphiola phoenicis Pascua strain and closest species using the Kimura two-parameter model and maximum likelihood algorithm with 1,000 bootstrap replicates.
The results obtained confirm the identification of G. phoenicis as a basidiomycete fungus, the cause of false smut on Canary date palm trees on Easter Island. G. phoenicis is a plant pathogen affecting numerous species of palm trees in the world (Piepenbring, 2012). For instance, it has been reported on P. roebelenii in Argentina (Cúndom, 2009) and Florida, USA (Martinez, 1966) and on P. dactylifera in Brazil, Egypt, India, Kenya, Libya (Edongali, 1996), and Qatar (Abbas and Abdulla, 2004). The infection is favored by high humidity and high foliage density. This is the first record of G. phoenicis on Phoenix canariensis on Easter Island, Chile.
We used ITS and LSU sequences of this basidiomycete for molecular analysis and selected LSU for further analysis because this marker is widely recommended for genus- and species-level identification of all rust fungi (Hyde et al. 2014). LSU sequencing and phylogenetic analysis placed the Pascua strain isolated from Easter Island within the cluster composed by G. phoenicis and two other unidentified species. A similar cluster was also reported by Piepenbring et al. (2012).
These results lead us to assert that the “Pascua” strain isolated from Easter Island that is attacking Canary date palms in Rapa Nui belongs to G. phoenicis.
