Tropical Plant Pathology 36 (6) November - December 2011
400
Tropical Plant Pathology , vol. 36, 6, 400-403 (2011)Copyright by the Brazilian Phytopathological Society. Printed in Brazil
www.sbfito.com.br
SHORT COMMUNICATION / COMUNICAÇÃO
Confirmation of the presence of the Citrus leprosis virus C
(CiLV-C) in Southern Mexico
Isidro Izquierdo Castillo 1, Luis Felipe Zermeño Diaz 1, Walter Mendez 2, Gabriel Otero-Colina 3, Juliana
Freitas-Astúa 4, Eliane Cristina Locali-Fabris 4, Gilberto José de Moraes 5, Renata Faier Calegario 5, Aline
Daniele Tassi 5 & Elliot W. Kitajima 5
1Comité Estatal de Sanidad Vegetal, Villahermosa, Tabasco, Mexico; 2Gobierno del Estado de Tabasco, Villahermosa, Tabasco,
Mexico; 3Instituto de Fitosanidad, Colegio de Postgraduados, Carretera México-Texcoco km 36.5, Montecillo, Texcoco
56230, Mexico; 4Embrapa Mandioca e Fruticultura Tropical, 44300-000, Cruz das Almas, BA, Brazil and Centro APTA
Citros Sylvio Moreira, 13490-970, Cordeirópolis, SP, Brazil; 5Departamento de Fitopatologia e Nematologia, Universidade
de São Paulo, ESALQ, 13418-900, Piracicaba, SP, Brazil
Author for correspondence: Elliot W. Kitajima, e-mail: ewkitaji@esalq.usp.br
ABSTRACT Citrus leprosis was detected in sweet oranges in Chiapas, Mexico, in 2005 based on symptoms. The disease was soon after observed
in sweet orange orchards at Huimanguillo and Cunduacan, state of Tabasco. Leaf samples of leprosis-affected Valencia or Hamlin sweet
oranges were collected and subjected to ultrastructural examination and molecular detection of CiLV-C by RT-PCR. Cytopathic effects
typical of CiLV-C infection as presence of short, baciliform particles in the endoplasmic reticulum cisternae and electron dense, vacuolated
viroplasma in the cytoplasm, as well as hypertrophy of some spongy parenchyma cells were observed in the tissues of the leaf lesions. RT-
PCR using specific primers for CiLV-C produced DNA fragments of the expected size. Brevipalpus mites were present in these orchards
and identified as B. phoenicis, the known vector for CiLV-C. Transmission tests with these mites confirmed their role as leprosis vectors
to sweet orange but not to lemon, Volkamerian lemon, C35 citrange and Carrizo citrange. These data confirm the presence of CiLV-C in
Tabasco, Mexico and that the citrus leprosis found in Chiapas was caused by CiLV-C.
Key words: Brevipalpus phoenicis , Chiapas, citrus leprosis, electron microscopy, molecular detection, Tabasco.
RESUMO
Confirmação da ocorrência da leprose dos citros tipo citoplasmático no sul do México Em 2005 houve um relato da presença da leprose dos citros no Estado de Chiapas, ao sul do México, baseado em sintomatologia.
Subsequentemente a doença foi constatada em pomares de laranjeiras doces no Estado de Tabasco, nas localidades de Huimanguillo e
Cunduacan. Amostras de folhas com lesões lepróticas de laranjeiras Valencia ou Hamlin foram coletadas e posteriormente examinadas ao
microscópio eletrônico e submetidas a ensaios de detecção molecular por RT-PCR usando primers específicos. Estudos ultraestruturais
revelaram alterações citopatológicas típicas causadas pelo CiLV-C (partículas baciliformes curtas no lúmen do retículo endoplasmático e
a presença de viroplasmas vacuolados e elétron-densos no citoplasma) e hipertrofia em algumas células do parênquima lacunoso. Ensaios
de RT-PCR resultaram na amplificação, na maioria das amostras de folhas dessecadas, de fragmento de DNA de tamanho esperado. Ácaros
Brevipalpus coletados nos pomares foram identificados como B. phoenicis. Ensaios de transmissão com este ácaro confirmaram seu papel
como vetor do CiLV-C para laranja doce, mas não houve transmissão para limão, limão Volkameriana, citrange C35 e citrange Carrizo.
Todos estes dados considerados em conjunto confirmam que CiLV-C está presente em Tabasco, México e que a leprose dos citros descrita
em Chiapas é causada pelo CiLV-C.
Palavras-chave: Brevipalpus phoenicis , Chiapas, detecção molecular, leprose dos citros, microscopia eletrônica, Tabasco.
Citrus leprosis was first reported in the early 1900’s
in Florida, US (Fawcett, 1911). It was afterwards reported
in many South American countries and in the last 10 years it
has been spreading through Central America (Rodrigues et
al., 2003; Bastianel et al., 2010). Sanchez Anguiano (2005)
reported the occurrence of leprosis in southern Mexico based
on symptoms on sweet oranges. This caused serious concern
in the Mexican citrus industry, which is going through a
considerable expansion, with important production areas
in several Mexican states (Chiapas, Tabasco, Veracruz, Tamaulipas, Nuevo Leon, Chihuahua, Sonora). Mexico has
526,000 ha of citrus plantation of which ca. 70% are sweet
orange, with a net yield of 6.7 million tons with a market
value of US$ 700 million (SIAP, 2011).
In 2006, typical leprosis symptoms (lesions on the
leaves, twigs and fruits, precocious fall of fruits) (Figure 1)
were observed in many commercial orchards of Valencia
and Hamlin sweet orange (C. sinensis [L.]) at Huimanguillo
and Cunduacan, state of Tabasco. Incidence of symptomatic
trees was not high but they were found in most of visited
401
Tropical Plant Pathology 36 (6) November - December
2011
Confirmation of the presence of the Citrus leprosis virus C (CiLV-C) in Southern Mexico
grooves with clear indication of the dissemination of the
disease. Leaf lesions were essentially similar to that caused
by Citrus leprosis virus C (CiLV-C), the prevalent and
more aggressive form of leprosis than that caused by the
nuclear form (CiLV-N). Fruits had depressed brownish
lesions, usually surrounded by a chlorotic halo (Figure 1A),
and many of them were dropped on the soil. Leaf lesions
were usually large with pale green color with concentric
brownish ring of gummy tissues (Figure 1C-D). There were
many chlorotic and necrotic lesions in the twigs, and some
stems exhibited large necrotic lesions (Figure 1B). From the
size of the leaf and stem lesions, it seems that the initial
infection may have occurred at least two to three years
before. To confirm that the causal agent of these symptoms
was CiLV-C, symptomatic leaf samples were collected
randomly, placed in plastic bags and taken to the laboratory
of the Comité Estatal de Sanidad Vegetal, Villahermosa, to be processed. Twigs were sampled to verify the presence of
Brevipalpus
mites, which when present were preserved in
ethanol 70%. Part of the collected mites was mounted for
light microscopy on Hoyer’s mounting medium (Moraes &
Fletchmann, 2008) and examined under light microscopy
at the Laboratório de Acarologia of the Escola Superior de
Agricultura Luiz de Queiroz, Universidade de São Paulo
(ESALQ/USP), at Piracicaba, SP, Brazil. The remaining
mites were air-dried, mounted either dorsally or ventrally
on a double side carbon tape, covered by a thin layer of
gold in a Baltec SD050 sputter, and examined in a LEO 435
VP scanning electron microscope at the Núcleo de Apoio
à Pesquisa em Microscopia Eletrônica aplicada à Pesquisa
Agropecuária (NAP/MEPA) of ESALQ/USP, Piracicaba,
SP, Brazil. Small pieces of lesions from still fresh leaves were
cut with a sharp razor blade and immersed in a mixture of
FIGURE 1 - Leprosis symptoms found in sweet oranges in the state of Tabasco. A. Leaf and fruit lesions of Valencia
sweet orange with leprosis (Cunduacan); B. Necrotic lesions on a stem of Valencia sweet orange (Huimanguillo); C.D.
Different aspects of lesions on the leaves of Valencia sweet orange (Huimanguillo).
Tropical Plant Pathology 36 (6) November - December 2011
402
I.I. Castillo et al.
FIGURE 2 - Transmission
electron micrographs of thin
sections of leaf lesions of Valencia
sweet orange caused by CiLV-C.
A. Part of spongy parenchyma
cell showing a viroplasm (*)
and a group of short, bacilliform
particles (V) within a cisternae of
the endoplasmic reticulum. Sample
from Huimanguillo; B. Similar to
A, showing a large viroplasm (*).
Sample from Cunduacan; C.D.
Scanning electron micrographs,
using backscattered detector,
of Brevipalpus phoenicis
collected at sweet orange orchard
at Huimanguillo, Tabasco.
Identification was based on the
presence of five latero-dorsal setae
(arrows) in the opistosoma (C) and
two solenidia (arrows) at the end of
the tarsus of the leg II ( D).
2.5% glutaraldehyde and 2% paraformaldehyde in 0.05M,
pH 7.2 cacodylate buffer for fixation. These samples were
used for transmission electron microscopy (TEM). Several
leaves with lesions were air dried for RT-PCR analysis to
detect CiLV-C by molecular techniques.
Fixed leaf samples were further processed
(Maunsbach & Afzelius, 1999) at the NAP/MEPA, for TEM.
Tissues were washed in cacodylate buffer, post fixed in 1%
OsO
4, dehydrated in acetone and embedded in low viscosity
Spurr’s epoxy resin. Blocks were trimmed and sectioned in a
Leica UC6 ultramicrotome equipped with a diamond knife.
Sections were collected on a copper grid, stained with 3%
uranyl acetate and Reynold’s lead citrate and examined in a
Zeiss EM900 transmission electron microscopy. Dried leaves were processed for RT-PCR according
to the protocol and specific primers for CiLV-C as described
by Locali et al. (2003) at the Centro APTA Citros Sylvio
Moreira (CCSM), Cordeirópolis, SP, Brazil and Instituto de
Fitosanidad- Colegio de Postgraduados, Texcoco, México. Half of the 12 samples of leprotic leaf lesions
collected from Huimanguillo and Cunduacan examined
by TEM revealed the presence of short, bacilliform
particles within cisternae of the endoplasmic reticulum
and/or electron dense viroplasm in the cytoplasm of either
palisade and/or spongy parenchyma cells (Figure 2 A and
B), a characteristic cytopathic effect of the infection by
CiLV-C (Rodrigues et al., 2003; Kitajima et al., 2003). Also,
hypertrophied cells were commonly seen in the spongy
parenchyma as previously observed in leaf tissues infected
by CiLV-C (Marques et al., 2007). RT-PCR assays of dried leaf samples, using specific
primers for CiLV-C (Locali et al., 2003) promoted the
amplification of DNA fragments of expected size, in most
of the analyzed samples (data not shown), whose sequence
(GenBank accession HQ292778) exhibited similarity with
that of CiLV-C (GenBank accession YP_654542). Further
RT-PCR assays carried out at the Instituto de Fitosanidad,
Colegio de Postgraduados in additional samples from
Tabasco and Chiapas, resulted in positive results. Light microscope examination indicated that the
mite species is Brevipalpus phoenicis Geijskes, based on the
number (5) of the laterodorsal setae and the number of the
solenidia (2) at the end of the tarsus of the leg II (Welbourn
et al., 2003). Similar findings were made when these mites
were examined under the SEM (Figure 2 C-D). Mite transmission assays were carried out with
adult B. phoenicis from a colony maintained on sweet
orange fruits. They were transferred onto sweet orange
leaves with typical leprosis lesions, collected from leprosis-
infected orchard in Huimanguillo and left feeding for 24
h. Then they were transferred onto five healthy seedlings
of Valencia sweet orange, lemon (C. limon [L.] Burm. f.),
Carrizo citrange (Poncirus trifoliate x C. sinensis), C35
citrange and Volkamerian lemon (C. volkameriana Tan. and
Pasq.), 12 mites on a single leaf per plant, under greenhouse
conditions. Chlorotic spots appeared on infested leaves of sweet
orange 5-6 weeks after infestation in all infested sweet orange
plants, but not in four control plants, without infestation. RT-
PCR of the tissues of the leaf lesions confirmed the presence of
CiLV-C. Leprosis symptoms did not appear on lemon, Carrizo
403
Tropical Plant Pathology 36 (6) November - December
2011
Confirmation of the presence of the Citrus leprosis virus C (CiLV-C) in Southern Mexico
citrange, C35 citrange and Volkamerian lemon which are
considered highly resistant or immune to CiLV-C (Bastianel
et al., 2010).
The results of the different analyses lead to the
conclusion that CiLV-C is present in the symptomatic leaf
samples collected at Huimanguillo and Cunduacan, state
of Tabasco, and RT-PCR assays confirmed that the disease
observed in sweet orange orchards at Chiapas (Sanchez
Anguiano, 2005) is caused by CiLV-C. It is not known exactly how CiLV-C was introduced into
Mexico, despite quarantine measurements. One speculation is
that CiLV-C may have been brought accidentally into Southern
Mexico by the flux of immigrants from Guatemala bringing in
contaminated orange fruits, discarded during the journey. Mexican authorities are taking measures to avoid the
spread of the disease to other citrus growing regions as well as
to reduce or eradicate the main focus in affected areas. Because
of the destructive nature of CiLV-C and the high cost of control
of the vector, B. phoenicis, there is also a serious concern in
the Caribbean area about the risk of dispersal of this disease
also to other countries, including southern USA, a major citrus
producer, and where the disease has not been registered after
the 1960’s (Childers et al., 2003).
ACKNOWLEDGEMENTS
This work received financial support from Fundação
de Apoio à Pesquisa do Estado de São Paulo - FAPESP (proc.
2008/52691-9) and Conselho Nacional de Desenvolvimento
Científico e Tecnológico - CNPq (47.1705/2009-8). The
authors are grateful do Dr. Hilda Gomes, USDA/Miami for
the images of leprosis symptoms depicted in Figures 1A, B
and D.
REFERENCES
Bastianel M, Novelli VM, Bassanezi R, Kitajima EW, Machado
MA, Kubo KS, Freitas-Astua J (2010) Citrus leprosis: Centennial of an unusual mite-virus pathosystem. Plant Disease 94:284-292.
Childers CC, Rodrigues JCV, Derrick KS, Achor DS, French JV,
Welbourn WC, Ochoa R, Kitajima EW (2003) Citrus leprosis and
its status in Florida and Texas: Past and present. Experimental and
Applied Acarology 30:181-202.
Fawcett HS (1911) Scaly bark or nail head rust of citrus. Florida
Agriculture Experiment Station Bulletin 106.
Kitajima EW, Chagas CM, Rodrigues JCV (2003)
Brevipalpus-
transmitted plant virus and virus-like diseases: Cytopathology and
some recent cases. Experimental and Applied Acarology 30:135-
160.
Locali EC, Freitas-Astua J, Souza AA, Takita MA, Astua-Monge
G, Antonioli R, Kitajima EW, Machado MA (2003) Development
of a molecular tool for the diagnosis of leprosis, a major threat to
citrus production in the Americas. Plant Disease 87:1317-1321.
Marques JPR, Freitas-Astua J, Kitajima EW, Apezzatto-da-Glória
B (2007) Lesões foliares e de ramos de laranjeira-doce causadas
pela leprose-dos-citros. Pesquisa Agropecuária Brasileira 42:1531-
1536.
Maunsbach AB, Afzelius BA (1999) Biomedical electron
microscopy. Illustrated methods and interpretations. San Diego
USA. Academic Press.
Moraes GJ, Fletchmann CHW (2008) Manual de acarologia.
Acarologia básica e ácaros de plantas cultivadas no Brasil 1ª ed, v.
1. Ribeirão Preto SP. Holos.
Rodrigues JCV, Kitajima EW, Childers CC, Chagas CM (2003)
Citrus leprosis virus vectored by Brevipalpus phoenicis (Acari:
Tenuipalpidae) on citrus in Brazil. Experimental and Applied
Acarology 30:161-179.
Sánchez-Anguiano H (2005) NAPPO Phytosanitary Alert System
www.pestalert.org/notifications.cfm?region=Mexico (Access on
November 7, 2011)
SIAP (Sistema de Información Agropecuaria y Pesquera). www.
siap.gob.mx (Access on November 7, 2011).
Welbourn WC, Ochoa R, Kane EC, Erbe EF (2003) Morphological
observations on Brevipalpus phoenicis (Acari: Tenuipalpidae)
including comparisons with B. californicus and B. obovatus.
Experimental and Applied Acarology:107-133.
TPP 452 - Received 25 November 2011 - Accepted 18 January 2012 Section Editor: F. Murilo Zerbini
