The presence of false smut (Graphiola phoenicis) on Canary date palm (Phoenix canariensis) in Easter Island, Chile

Abstract

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 Materials and methods Plant Sample and morphologic identificationIn 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 cm 2 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 identificationFor 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 ...

Two new species in a new genus and a critical revision of Brachybasidiaceae (Exobasidiales, Basidiomycota) in honor of Franz Oberwinkler

“…In addition, sequences of all available species of Brachybasidiaceae and Graphiolaceae were added including sequences from the public catalog of the NITE Biological Resource Center collection (NBRC), Japan. GenBank accession numbers of the sequences used (Begerow et al 2002;Boekhout et al 2003;Cao et al 2018;Crous et al 2003;Kottke et al 2010;Kruse et al 2017;Macedo et al 2016;Maier et al 2006;Matheny et al 2006;Nasr et al 2019;Piepenbring et al 2010Piepenbring et al , 2012aSchoch et al 2014;Sepúlveda et al 2017;Tanaka et al 2008;Wang et al 2015;Yasuda et al 2005) are cited in Fig. 2.…”

Graphiola phoenicis associated with false smut on Canary Island date palm (Phoenix canariensis) in Korea

“…Poit. on P. canariensis reported from Chile (Sepúlveda et al, 2017). Yellow to brown spots appeared on both sides of the leaves and black fruiting bodies (sori) formed on the leaf surfaces (Figure 1a,b).…”

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Phoenix canariensis Hort. ex Chabaud, commonly known as the Canary Island date palm, is widely planted as a highly decorative, exotic, ornamental plant all over the world in tropical and subtropical regions (Morici, 1998). In Korea, palms were first planted along the streets on Jeju Island from the late 1960 s in order to provide an exotic landscape.

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“…For molecular identification, genomic DNA was extracted from three cultures using the Quick-DNA Fungal/Bacterial Miniprep Kit (Zymo Research). Partial large subunit (LSU) rDNA regions were amplified with NL1m (5′-GCATATCAATAAGCGGAGGAAAAG-3′) and NL-4m (5′-GGTCCGTGTTTCAAGACG-3′) primers (Sepúlveda et al, 2017). The polymerase chain reaction (PCR) cycling conditions were denaturation at 94°C for 5 min; this was followed by 35 cycles at 94°C for 1 min, 55°C for 1 min, and 72°C for 1 min, then a final extension at 72°C for 2 min.…”