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
1
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
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: gsepulve@uta.cl
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.
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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
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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 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).
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
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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).
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.
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).
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).
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.
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
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.
Acknowledgments
We thank the lab of Professor Bernardo Arriaza
for the SEM image and the Dirección de Investigación y Posgrado de la U. Tarapacá (9711-15
Mayor Project) and Convenio de Desempeño
en Educación Superior Regional UTA-1401 for
financial support.
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.
VOLUME 44 Nº3 SEPTEMBER – DECEMBER 2017
311
Resumen
G. Sepúlveda, M. Arismendi, W. Huanca-Mamani, S. Cárdenas-Ninasivincha, R. Salvatierra,
y B. Latorre. 2017. Presencia del falso carbon (Graphiola phoenicis (Moug. ex Fr.) Poit.) sobre
Palma de canarias (Phoenix canariensis) en Isla de Pascua, Chile. Cien. Inv. Agr. 44(3): 307-311.
En hojas de Phoenix canarienis en Hanga Roa, Isla de Pascua, Chile, se encontró abundantes basidiomas
pulvinados, negros, amfígenos negros sobre las pinnas y en el raquis de las hojas, causando daño
foliar extensivo. Después de tres días en cámara húmeda se examinaron muestras con microscopio
compuesto. Además, para estudiar la relación hongo/hospedero, se utilizó microscopía electrónica
de barrido y cortes histológicos, complementariamente se aplicaron herramientas moleculares en la
identificación. Las características morfométricas y moleculares permitieron la identificación del hongo
como Graphiola phoenicis, agente causal del falso carbon en Palma de Canarias (Phoenix canariensis)
en Isla de Pascua, Chile. Este es el primer reporte de este fitopatógeno en territorio chileno.
Palabras clave: Exobasidial, falso carbón, fitopatógenos foliar.
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