Download aetiology of chili pepper variegation from yurécuaro, méxico

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Journal of Plant Pathology (2011), 93 (2), 331-335
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AETIOLOGY OF CHILI PEPPER VARIEGATION
FROM YURÉCUARO, MÉXICO
M. Camacho-Tapia1, R.I. Rojas-Martínez1, E. Zavaleta-Mejía1, M.G. Hernández-Deheza1,
J.A. Carrillo-Salazar2, A. Rebollar-Alviter3 and D.L. Ochoa-Martínez1
1 Instituto
de Fitosanidad, Colegio de Postgraduados, Montecillo 56230, México
Genéticos y Productividad Genética, Colegio de Postgraduados, Montecillo 56230, México
3 Centro Regional Morelia, Universidad Autónoma de Chapingo, Morelia, Michoacán 58000, México
2 Recursos
SUMMARY
In summer 2008, symptoms resembling those of a
virus disease, i.e. chlorotic veins, deformation of the
leaves and fruits, albinism and browning, were observed
in chile pepper (Capsicum annuum L.) fields at
Yurécuaro (Michoacán, México). However, ELISA tests
were negative for the viruses most commonly associated
with this crop. Based on these results, and knowing that
in the state of Sinaloa (México), Candidatus Liberibacter solanacearum (Ca.L.s.) was detected in chile plants
showing shortened internodes, apical chlorosis and
symptoms similar to those of “yellow psillid” in potato,
it was thought that the symptoms of chile in Yurécuaro
could be induced by this bacterium. In this study the
etiology of the variegation of chile was determinated.
Symptomatic plants were collected in the Yurécuaro region in order to detect in leaves and seeds the possible
causal agent by PCR and transmission electron microscopy, and to transmit the pathogen by grafting, Bactericera cockerelli, and through seed. In leaf tissue and
seed of affected plants, Ca L.s. was detected by PCR,
using primers OA2 and OI2c. The bacterium was also
detected in thin sectioned phloem cells of affected
leaves. Disease symptoms were reproduced in plants
grafted with infected tissue and in those exposed to
adults of B. cockerelli collected from diseased plants.
The evidence obtained indicates that the variegation of
chile pepper in Yurécuaro is induced by Ca.L.s.
Key words: Candidatus Liberibacter solanacearum,
transmission electron microscopy, polymerase chain reaction, Bactericera cockerelli.
INTRODUCTION
The green chile pepper (chile, Capsicum annuum L.)
is the most widely grown vegetable crop in México,
with a cultivated area of 149,114 ha in 2007 (SIAP,
2007). The main producing Mexican states are: Sinaloa
(694,634 tons), Chihuahua (564,256 tons), Zacatecas
(209,331 tons), San Luís Potosí (133,402 tons), Tamaulipas (125,482 tons), and Michoacán (93,424 tons). The
main Michoacán municipalities growing chile are
Yurécuaro, Tanhuato, Coahuayana, Vista Hermosa and
Ecuandureo (SIAP, 2009).
During 2008, a new phytosanitary problem of chile
arose in Yurécuaro, which resembled a viral infection
because of the symptoms shown by affected plants, i.e.
variegation, deformation of the leaves and albinism, that
recalled those induced by Tobacco etch virus (TEV)
(Plant Virus Online, 2008). However, ELISA tests using
commercial kits (Agdia, USA) for the presence of TEV,
Tobacco mosaic virus (TMV), Tobacco ringspot virus
(TRSV), Cucumber mosaic virus (CMV), Alfalfa mosaic
virus (AMV), Tomato spotted wilt virus (TSWV), and
Tomato aspermy virus (TAV) were negative.
Knowing that Candidatus Liberibacter solanacearum
(Ca.L.s.) was found in New Zealand (MAF, 2008) in
chile plants with mottling, yellowing, leaf deformation
and sudden death and with chile plants with symptoms
similar to those of “yellow psillid” of potato in Sinaloa,
(Munyaneza et al., 2009), it was hypothesized that this
bacterium could be involved in the aetiology of the disease occurring in Yurécuaro. Ca.L.s. is also the casual
agent of Zebra chips of potato in the USA (Abad et al.,
2009) and New Zealand (Liefting et al., 2008), affects
other solanaceous crops, such as tomato (Solanum lycopersicum) (Liefting et al., 2008, 2009) and is transmitted
by the psyllid Bactericera cockerelli (Hansen et al., 2008).
As, mentioned, Munyaneza et al. (2009) have reported the presence of Ca.L.s. in symptomatic chile but did
not determine whether this bacterium is the actual
agent causal of the disease. This investigation was therefore carried out with the aim of establishing if there
were an aetiological relationship between Ca.L.s. and
the chile variegation disease present in Yurécuaro.
MATERIAL AND METHODS
Corresponding author: R.I. Rojas-Martinez
Fax: 595.95.20200 ext.1623
E-mail: [email protected]
Symptomatic chile plants of cvs Centella and Tajin
were collected at Yurécuaro (northwest of the Mi-
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choacán state at 20º20’ north and 102º17’ west) including 10 fruits for seed extraction and taken to the laboratory for testing. Seed samples comprised also seeds harvested by the growers and from commercial lots of cvs
Centella, Tajin, Big-Brother and Camino Real.
DNA extraction. DNA was extracted from 0.3 g tissue from symptomatic and symptomless leaves according to Ahrens and Seemüller (1992) and from 0.2 g of
seeds with the Qiagen extraction kit (Quiagen, USA).
The quantity and quality of the extracted DNA was determined spectrophotometrically (Lambda Bio model
10, Perkin Elmer) at 260 nm and with electrophoresis in
0.8% agarose gel, respectively.
PCR detection of Ca. Liberibacter solanacearum.
PCR was conducted using Ca.L.s.-specific primers OA2
and OI2c, designed on the 16S rDNA region, that amplify a 1160 bp fragment (Liefting et al., 2008, 2009).
Amplification took place in a BIO-RAD thermocycler,
model i-Cycler, with an initial denaturation at 94ºC for 5
min, followed by 35 cycles at 95ºC for 30 sec for denaturation, 58ºC for 30 sec for annealing and 72ºC for 1
min for extension, and 10 min at 72ºC for final extension. Five µl of the PCR product were loaded onto a
1% agarose gel and run by electrophoresis at 90 V. Gels
were stained with ethidium bromide (0.5 µg ml-1 for 10
min) and acquired with a BIO-RAD photodocumenter
(Gel-Doc model 2000). For molecular weight reference,
a 1 Kb marker (Gibco BRL, USA) was used.
PCR products were cleaned with a Wizard kit
(Promega, USA) then cloned in Escherichia coli using a
TOPO-TA cloning kit (Invitrogen, USA) in accordance
with manufacturer’s instructions. Sequencing of 16S rDNA clones was by the Automatic Sequencer 3700xl DNA
Analyzer (Applied Biosystems, USA). Sequences were
aligned with those available in GenBank, utilizing the
BLAST tool of NCBI (http://www.ncbi.nlm.nih.gov/
BLAST/)
Seeds in which Ca.L.s. was detected, were germinated and the seedlings transplanted into eight unicel containers with three seedlings each, that were placed in a
cage with insect-proof netting to prevent contact with
insect vectors. The seedlings were kept under observation for 2 months for symptom appearance.
Transmission by grafting. Tissue from symptomatic
chile plants were grafted onto 15 healthy plants. The
cuts were made diagonally to ensure the greatest contact
area between graft and host and the grafting area was
covered with parafilm. The plants were covered with
plastic bags for a week to maintain humidity and were
kept in the greenhouse until symptoms appeared.
Transmission by Bactericera cockerelli. Four symptomatic plants that carried a large number of B. cockerelli
Journal of Plant Pathology (2011), 93 (2), 331-335
nymphs were collected from the field and taken to a
glasshouse where they were placed inside entomology
quality cages to avoid contact with other insects. Once
two generations of the insects had been obtained, DNA
was extracted from 10 samples, each consisting of three
insects, and subjected to PCR analysis.
Once the presence of Ca.L.s. in the colony of insects
was confirmed, groups of 10 healthy chile plants held in
the greenhouse inside cages with insect-proof netting
were exposed to 50 adult insects to allow transmission.
Phylogenetic relationships. A phylogenetic tree was
constructed with the nucleotide sequence of Ca L.s. present in B. cockerelli amplified with primers OA2 and OI2c
and comparable sequences retrieved from GenBank: Ca.
L. solanacearum (FJ957897), Ca. L. solanacearum
(EU834130), Ca. L. solanacearum (FJ957896), Ca. L.
solanacearum (FJ395204), Ca. L. solanacearum
(FJ395205), Ca. L. solanacearum (FJ395206), Ca. L.
solanacearum (GU373049), Ca. L. solanacearum
(EU935004), Ca. L. psyllaurous (EU812559), Ca. L.
africanus (EU921620), Ca. L. asiaticus (EU921615), and
Ca. L. americanus (EU921625). Seven distinct bacteria
from this group were included as roots: Brucella melitensis (AY513507), Synorhizobium spp. (AY500255),
Bradyrhizobium japonicum (EU481826), Rhodospirillum
rubrum (NC_007643), Wolbachia spp. (DQ412085), Escherichia coli (NC_010473), and Helicobacter pylori
(EU544200). The phylogenetic tree was produced using
DNASTAR Lasergene with alignment by Clustal V.
Electron microscopy. The main vein was excised
from leaves of symptomatic chile plants and cut to small
fragments that were fixed in 6% glutaraldehyde in 0.1
M phosphate buffer, pH 7.2. After washing in phosphate buffer, tissue fragments were post-fixed in osmium tetroxide for 1 h, dehydrated with graded alcohol
dilutions and embedded in epoxy resin. Sections 120
nm thick were cut with an ultramicrotome, stained with
1% uranyl acetate, and were observed with a JOEL
JEM-1200 EX electron microscope at 60 kV.
RESULTS AND DISCUSSION
All symptomatic but not the symptomless tissue samples from the field were PCR-positive for Ca.L.s. (Fig. 1).
Positive amplification was also obtained from DNA extracted from seeds produced by symptomatic plants and
from seeds produced by plants obtained from seeds of
diseased plants (Fig. 2A).
When nucleotide sequences of PCR amplicons were
compared by BLAST with those deposited in GenBank,
100% identity was found with the nucleotide sequence
of Ca.L.s. (FJ957897).
Chile plants obtained from seed of diseased fruit
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Fig. 1. PCR products obtained with primers OA2 and OI2c
in plants of Capsicum annuum L. Lanes 1 and 18: molecular
weight marker 1 Kb (Invitrogen); lane 2, asymptomatic plant
of Capsicum annuum L. (control); lane 3, leaf with albinism;
lane 4, variegated leaf; lane 5, variegated leaf with yellow
veins; lane 6, leaf showing browning; lane 7, leaf with blistering and deformation of the lamina; lane 13, negative control
(injected sterile water).
showed the typical variegation symptoms 2 months after
transplanting (Fig. 2B). Comparable symptoms developed also in chile plants 90 days after grafting with tissue from diseased plants. The leaves showed initial
chlorosis of the tips followed by necrosis. After 2
months, there was deformation of the leaves of new
shoots and the variegation was more evident. After 90
days, PCR tests were positive for the presence of Ca.L.s.
Fig. 2. A. PCR products obtained with primers OA2 and
O12c. Lane 1, Molecular weight markers of 100 bp; lane 2,
negative control; lanes 3-11, DNA from seed of the cultivars
evaluated: 3-4 Centella, 5-6 Tajin, 7-8 seed collected by the
grower, 9-10 Big-brother, 11 Camino Real. B. Chile pepper
plant (Capsicum annuum L.) with symptoms of variegation
obtained 2 months after germination of seed from plants with
symptoms of variegation.
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After six weeks exposure of chile plants to insects
collected from the field, typical variegation symptoms
were observed (Fig. 3B) and PCR detected the bacteria
in the insects that had fed for 2 months on the plants
(Fig. 3A).
Nucleotide sequences of the amplified product and
DNA from symptomatic plants and from insects that
had been feeding on them (accession Nos HQ379735,
HQ379737) were 98% homologous with the nucleotide
sequence of Ca.L.s. genome deposited in GenBank
(FJ957897).
The phylogenetic tree comprising the sequence of the
1,160 kb amplicon from the 16Sr region of the bacterium from Yurécuaro and those from isolates deposited
in GenBank showed a close relationship with the bacteria Ca. Liberibacter psyllaurous and Ca. Liberibacter
solanacearum (Fig. 4).
Bacilliform and circular structures 4 µm long and 0.1
µm in diameter, interpreted as longitudinal and cross
sections, respectively, of walled bacterial cells were observed in the vascular cylinder of the main veins of
symptomatic chile leaves (Fig. 5 A-B). These dimensions
are within the range reported for Ca.L.s. by Tanaka et
al. (2007), Bové (2006) and Liefting et al. (2009).
The symptoms induced by Ca.L.s. in chile fields of
Yurécuaro are different from those reported in New
Zealand in the same crop, possibly because of the different cultivars involved, but recall those described
from citrus, i.e. mottling, chlorotic veins, deformation
of the fruits, and shrunken coffee-coloured seed (Garnier et al., 2000; Khairulmazmi et al., 2008; Da Graca et
al., 2004).
Fig. 3. A. PCR products obtained with the primers OA2 and
O12c. Lane 1, Molecular weight markers of 100 bp; lane 2,
negative control; lanes 3-6, DNA from insects. B. Chile pepper plant (Capsicum annum L.) with symptoms of variegation,
yellowing and reduced leaf area obtained after 90 days exposure to the insects Bactericera cockerelli.
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Fig. 4. Phylogenetic tree of the 16S rRNA region of bacteria, made in MegAlign by the alignment method V of Clustal with the accession numbers obtained from GenBank. The scale bar indicates a substitution for each 100 nucleotides.
The date of Ca.L.s. introduction into Mexico is uncertain. The disease has been reported in 2009, whilst
the presence of B. cockerelli the vector responsible for
its dissemination dates back to 1947 (Plesch, 1947, cited
by Vega et al., 2008), long before this bacterium became
a threat for solanacous plants.
Fig. 5. Transmission electron photomicrographs of phloem
tissue of chile pepper infected by Ca. Liberibacter solanacearum; the arrows indicate oblong bodies in the phloem of the
central veins of symptomatic leaves.
In practice, control of Ca.L.s. in potato crops has focused on vector management due to the phloem-restricted localisation of Liberibacter and dissemination operated primarily by insect vectors (Bové, 2006). Results of
this investigation suggest that the most likely route
through which the bacterium arrived to Yurécuaro is the
seed, which is still contributing to its dissemination locally. Disease symptoms were observed before the insect
vector was detected in chili fields (unpublished observations), thus supporting the likelihood that seeds play a
relevant role in pathogen dissemination. According to
Schuster and Coyne (cited by Orvin and Hinkle, 1976),
bacteria may enter the seed via the vascular system, travel
to the germination tube of pollen grains and the hilum of
mature seed and invade their dorsal suture. Later, they
may migrate to the funiculus through the raphe, pass to
the seed coat, and rest there for a long periods of time.
Since the presence of bacteria in seed lots represents the
primary source of inoculation, one of the measures that
may help reducing the phytosanitary problem, is the use
of bacterium-free seeds. On the other hand, integrated
management of B. cockerelli is necessary to avoid disease
spread within a field. In the absence of chile crops, the
insect vector has other host plants, and the bacterium can
also survive as it was recorded from Solanum betaceum
and Physalis peruviana (Liefting et al., 2009).
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