First, second, and fourth authors: Estación Experimental “La Mayora,” Consejo Superior de Investigaciones Científicas, 29750 Algarrobo-
Costa, Málaga, Spain; third author: Seminis Vegetable Seeds Ibérica, S.A., Paraje San Nicolás, 04740 La Mojonera, Almería, Spain; and
fifth author: Centro de Edafología y Biología Aplicada del Segura (CEBAS), Consejo Superior de Investigaciones Científicas, 30100
Campus Universitario de Espinardo, Murcia, Spain.
Accepted for publication 10 February 2003.
ABSTRACT
Marco, C. F., Aguilar, J. M., Abad, J., Gómez-Guillamón, M. L., and very low CYSDV levels were detected by RT-PCR in whitefly-inoculated
Aranda, M. A. 2003. Melon resistance to Cucurbit yellow stunting leaves, and therefore, multiplication or spread of CYSDV in C-105 plants
disorder virus is characterized by reduced virus accumulation. Phyto- appeared to remain restricted to the inoculated leaves. When C-105
pathology 93:844-852. plants were graft inoculated, CYSDV RNA could be detected in phloem
tissues, but the systemic colonization of C-105 by CYSDV upon graft
The pattern of accumulation of Cucurbit yellow stunting disorder virus inoculation seemed to be seriously impeded. Additionally, in situ hybridi-
(CYSDV; genus Crinivirus, family Closteroviridae) RNA has been ana- zation experiments showed that, after C-105 graft inoculation, only a
lyzed in several cucurbit accessions. In susceptible accessions of melon portion of the vascular bundles in petioles and stems were colonized by
(Cucumis melo), cucumber (Cucumis sativus), marrow (Cucurbita maxi- CYSDV and virus could not be found in leaf veins. RT-PCR experiments
ma), and squash (Cucurbita pepo), CYSDV RNA accumulation peaked using primers to specifically detect negative-sense CYSDV RNA were
during the first to second week postinoculation in the first to third leaf carried out and showed that CYSDV replication took place in graft-
above the inoculated one; younger leaves showed very low or undetect- inoculated C-105 scions. Therefore, the resistance mechanism may in-
able levels of CYSDV. Three melon accessions previously shown to volve a restriction of the virus movement in the vascular system of the
remain asymptomatic after CYSDV inoculation under natural conditions plants and/or prevention of high levels of virus accumulation.
were also assayed for their susceptibility to CYSDV. Hybridization and
reverse transcription-polymerase chain reaction (RT-PCR) analysis of Additional keywords: Bemisia tabaci, closterovirus, plant–virus interac-
noninoculated leaves showed that only one of these, C-105, remained tion, systemic movement, tolerance.
virus-free for up to 6 weeks after whitefly inoculation. In this accession,
Cucurbit yellow stunting disorder virus (CYSDV) is a clostero- heat shock protein 70 homologue (HSP70h) and the coat pro-
virus transmitted in nature by the whitefly Bemisia tabaci tein genes of RNA 2. The genetic diversity of CYSDV isolates
(Gennadius). CYSDV was first detected in 1982 in the United is unusually low compared with other members of the family
Arab Emirates (9), and since then, it has been found in Spain, Closteroviridae, although it is still possible to differentiate two
Portugal, Morocco, Lebanon, and North America extensively genetic groups, the so-called eastern and western CYSDV sub-
affecting cucurbit crops (1,3,4,12,20,37). CYSDV induces inter- populations (30,31).
veinal chlorotic spots in mature leaves which may enlarge and The control of CYSDV is currently based on chemical treat-
eventually fuse together producing yellowing of the entire leaf ments against its vector and preventive cultural practices, both
except for the veins that remain green (3,37). Yellowing symp- with limited success. The use of genetically resistant cultivars is a
toms are accompanied by substantial reductions of fruit yield good option for CYSDV control, but no resistant cultivar is com-
and quality and, therefore, this virus has a high economic impor- mercially available. Recently, we reported the existence of possi-
tance. CYSDV is a member of the genus Crinivirus (family ble sources for natural resistance or tolerance to CYSDV (19). In
Closteroviridae) (22). Its viral particles are flexible rods with this work, plants of a melon (Cucumis melo L.) accession (C-105)
lengths between 750 to 800 nm (17). CYSDV has a narrow host remained asymptomatic after natural or artificial inoculation with
range limited to species of Cucurbitaceae (3) in which it is CYSDV. Additionally, genetic tests strongly suggested that this
believed to remain confined to phloem-associated cells (13). The character was controlled by a single dominant gene named Cys
CYSDV genome consists of two molecules of single-stranded (19). Because no detection methods were used to specifically ana-
RNA of positive polarity designated RNAs 1 and 2 (3). Livieratos lyze the presence CYSDV in C-105 plants, this character could
and Coutts (18) cloned and sequenced cDNAs to the RNA 2 correspond to a tolerance (i.e., lack of symptoms) or to a resis-
of a CYSDV isolate, but the nucleotide sequence of the RNA 1 tance (i.e., reduction in, or a lack of virus accumulation). The
has not been published. The genetic diversity of CYSDV iso- work presented here was aimed at elucidating this aspect and ana-
lates from different areas of the world has been studied for the lyzing the mechanism of disease tolerance or resistance of C-105
melon plants to CYSDV. Tools for detection of CYSDV RNAs in
plant tissues were developed and the accumulation of CYSDV in
C-105 and related cultivars or species was studied. Results of our
Corresponding author: M. A. Aranda; E-mail address: m.aranda@cebas.csic.es work indicate that C-105 plants are resistant to CYSDV infection
and suggest that the resistance mechanism may involve a restric-
Publication no. P-2003-0505-02R tion of the virus movement in the vascular system of the plants
© 2003 The American Phytopathological Society and/or prevention of high levels of virus accumulation.
844 PHYTOPATHOLOGY
MATERIALS AND METHODS Establishment of the signification groups was carried out by using
the least significant difference test at P = 0.01 (SAS system; SAS
All standard DNA and RNA manipulations were carried out as Institute, Cary, NC).
described by Sambrook and Russell (32). Detection of CYSDV RNA in tissue prints. For detection of
Plant, virus, and insect cultures. Cucurbits susceptible to CYSDV positive-sense RNA in tissue prints (23), plant stems,
CYSDV used in this study were Cucumis melo cv. Amarillo (C- petioles, and major leaf veins were cut with a razor blade and, im-
250, La Mayora germplasm collection, Málaga, Spain), Cucumis mediately after cutting, the cross-sections were blotted onto posi-
sativus cv. Bellpuig, Cucurbita maxima cv. Cabello de Angel, and tively charged nylon membranes (Roche Diagnostics GmbH,
Cucurbita pepo cv. Diamant (Semillas Fitó, Spain). The melon Mannhein, Germany). CYSDV RNA on membranes was detected
accessions tested for their susceptibility to CYSDV (C-105, by molecular hybridization with an RNA digoxigenin (DIG)-
C-243, and C-300) belong to the germplasm collection of La labeled probe (see in situ hybridization) according to the manu-
Mayora. Seeds were germinated and grown in a nursery; 15-day- facturer’s recommendations (Roche Diagnostics). Briefly, prehy-
old seedlings were planted in 12-liter pots and kept in an insect- bridization and hybridization of membranes were carried out at
proof glasshouse. After 2 weeks, if required, plants were trans- 65°C in standard buffer (5× SSC [1× SSC is 0.15 M NaCl plus
planted to 18-liter pots to guarantee proper development. The 0.015 M sodium citrate], 0.1% N-Lauroylsarcosine, 0.02% sodium
CYSDV isolate used in this study (CYSDV-AlLM) was obtained dodecyl sulfate, and 2% blocking reagent [Roche Diagnostics])
from a naturally infected melon plant collected in Almeria (Spain) containing 50% formamide. Membranes were washed for 15 min,
in 1997, and maintained in La Mayora on Cucumis melo cv. once at room temperature in 2× SSC and twice at 65°C in 0.1×
Amarillo through transmission by B. tabaci (B biotype [7]). SSC. Chemiluminescent detection was carried out using the
Samples of this isolate are kept under the accession no. PV- reagents and protocols supplied by the DIG-labeling and detection
0592/EWSN_6 in the DSMZ-Deutsche Sammlung von Mikroor- kit (Roche Diagnostics).
ganismen und Zellkulturen GmbH collection (Braunschweig, CYSDV RNA quantification in dot blots. The amount of
Germany). CYSDV-AlLM RNA was prepared by in vitro tran- CYSDV positive-sense RNA present in leaf samples was esti-
scription using T7 RNA polymerase (Promega, Madison, WI) in a mated from dot blot hybridization signals. For this, two to three
standard reaction (32) in which the plasmid pLM15, linearized disks (1.5 cm in diameter) were taken from different parts of each
with KpnI, was used as template. leaf and total RNA was extracted from them following the proto-
For whitefly transmission experiments, virus-free B. tabaci col described by Célix et al. (3). Aliquots of each extract were
colonies were maintained on healthy melon plants (Cucumis melo analyzed by electrophoresis in agarose gels to check RNA integ-
cv. Amarillo) in an insect-proof glasshouse (20 to 32°C, 45 to rity and to quantify total RNA. Melon total RNA extracts of
85% relative humidity [RH]) with artificial light supplemented known RNA concentration were included as standards. For each
when needed. analyzed sample, 1 µl from the total RNA extract and 1 µl of a
Whitefly and graft inoculations. Whitefly inoculation of test 1:10 dilution were spotted onto a nylon membrane. Additionally,
plants with CYSDV was performed essentially as described by 1 µl of CYSDV-transcribed RNA at a concentration of 40 µg/ml
López-Sesé and Gómez-Guillamón (19). Briefly, groups of adult plus a dilution series were spotted onto the membrane. Blots were
whiteflies were given a 24 h acquisition access period by feeding then subjected to UV RNA cross linking, prehybridized, hybri-
on virus source melon plants (Cucumis melo cv. Amarillo). Fol- dized, washed, and developed to detect CYSDV RNA as de-
lowing the acquisition access period, whiteflies were given a 48 h scribed previously. Membranes were exposed to X-ray films and
inoculation access period on test plants using 60 whiteflies per the resulting autoradiographs were transiluminated and their image
plant. Plants for inoculation were about 4 weeks old, with the first acquired by a digital camera. Digitized images were computer
true leaf fully expanded. Acquisition and inoculation were per- analyzed to quantify the intensity of the signal on each dot. Dots
formed using home-made clip-on cages. After the inoculation corresponding to the samples of CYSDV-transcribed RNA pro-
access period, whiteflies were carefully removed and plants were vided the values to calibrate the relationship between signal inten-
treated with insecticide. Fifteen days after inoculation, the leaves sity and amount of viral RNA. This calibration served to interpo-
on which the whiteflies had fed and laid their eggs were removed. late the values for the amounts of viral RNA in the test samples.
Control plants were obtained following the same scheme but using Eventually, the amount of CYSDV RNA was referred to as the
virus-free whiteflies. amount of total RNA for each extract.
For graft inoculation, Cucumis melo cv. Amarillo plants were Detection of CYSDV RNAs by reverse transcription-poly-
whitefly inoculated as described to produce the stocks needed. Ten merase chain reaction. For reverse transcription-polymerase chain
days after inoculation, the leaves where the whiteflies had fed reaction (RT-PCR) detection of positive-sense CYSDV RNA, two
were removed and analyzed for CYSDV infection by hybridiza- primers that would produce a DNA fragment of about 540 bp were
tion on tissue prints of petiole cross-sections (described below). designed based on the nucleotide sequence of the 3-terminal
Only plants that showed an intense hybridization signal were used region of CYSDV RNA 1 (J. M. Aguilar, M. Franco, C. F. Marco,
as virus sources. Infected plants were cut at the internodal stem B. Berdiales, E. Rodriguez-Cerezo, V. Truniger, and M. A.
region above the third leaf and the basal part of the plant was kept Aranda, unpublished data). The upstream primer was 5-GAAG-
to be used as a virus-infected stock. To produce the scions, Cucu- AATTCCAGGCAAGG-3 (MA156) and the downstream primer
mis melo plants (accession C-105 and cv. Amarillo as susceptible was 5-TCACATCATCAATCCAAAAG-3 (MA129). Reverse
control) were grown in the greenhouse for 6 weeks and pruned transcription was performed in a reaction mixture (20 µl) con-
twice during this period to obtain as many side shoots as possible. taining first strand buffer (Invitrogen BV/Novex, Groningen, the
The apical parts of the stems of these plants (approximately 7 to Netherlands), 10 µg of total RNA (3), 75 µM MA129, 0.5 mM
8 cm long) were collected, given a V shape at the cut end, and dNTPs, 20 units of ribonuclease inhibitor (Invitrogen), and
grafted onto the stock. The graft region was wrapped with Para- 200 units of Superscript RNase H reverse transcriptase (Invitro-
film M (American National Can, Chicago, IL) and a high RH gen). The mixture was incubated at 37°C for 1 h. For PCR
(60 to 80%) was maintained in the greenhouse during the amplification, 1 µl of the synthesized cDNA was added to a tube
following 2 days to avoid dehydration of the scion. Following this containing 15 µM each primer, 1 mM dNTPs, 3 mM MgCl2,
procedure, around 80% of the scions survived and the rate of reaction buffer (Bioline, London, UK), and 2.5 units of Taq DNA
successful infections of susceptible scions was 100%. polymerase (Bioline) in a final volume of 25 µl. Cycling condi-
The results of the grafting experiments were compared by tions were as follows: 30 cycles at 94°C for 30 s, 55°C for 30 s,
means of an analysis of variance after arcsine transformation (34). and 72°C for 30 s, preceded by an initial incubation of 94°C for
Vol. 93, No. 7, 2003 845
5 min, and followed by a final incubation at 72°C for 7 min. For
RT-PCR detection of negative-sense CYSDV RNA, primers that
would produce a DNA fragment of about 560 bp were designed
based on the nucleotide sequence of the 3-terminal region of
CYSDV RNA 1 (J. M. Aguilar, M. Franco, C. F. Marco, B.
Berdiales, E. Rodriguez-Cerezo, V. Truniger, and M. A. Aranda,
unpublished data). The primer for first strand cDNA synthesis
was 5-CGAAGTTTGTGCAATGCCTGACCGAAGAATTCCA-
GGCAAGG-3 (MA230), which included a 3-end portion identi-
cal to a nucleotide sequence of the positive-sense CYSDV RNA 1
and a nonviral 5-end portion (underlined). Twenty micrograms of
total nucleic acids (TNA) extracts was used as template for re-
verse transcription; TNA extracts were obtained as described by
Célix et al. (3), except that LiCl precipitation was substituted by
an ethanol precipitation. Other reverse transcription conditions
were as described for the detection of positive-sense CYSDV
RNA. After reverse transcription, 100 units of RNase A (Roche
Diagnostics) was added to the mixture and incubation proceeded
at 95°C for 5 min and then at 37°C for 20 min. The resulting
products were purified in a Microcon YM-100 (Millipore Corp.,
Bedford, MA). One microliter of the prepared cDNA was used in
PCR amplification reactions in which primers MA129 and 5-
CGAAGTTTGTGCAATGCCTGACC-3 (MA225) were used, the
latter being the nonviral nucleotide sequence included in MA230.
PCR conditions were as described for detection of positive-sense
CYSDV RNA. RT-PCR products were fractionated by electro-
phoresis in 1.2% agarose gels and visualized after ethidium bro-
mide staining.
Cytological detection of CYSDV RNA by in situ hybridiza-
tion. A DIG-11-UTP-labeled RNA probe to detect the positive-
sense CYSDV RNA was synthesized by in vitro transcription
from the plasmid pLM15. Plasmid pLM15 was linearized with
XbaI for transcription of negative-sense RNA using SP6 RNA
polymerase (Promega). Transcripts were sheared to fragments of
about 150 bp by alkaline hydrolysis to improve the in situ hy-
bridization signal. This probe was hybridized to tissue sections
and detected using an alkaline phosphatase-conjugated anti-DIG
antibody as described previously; 5-bromo-4-chloro-3-indoylphos-
phate and nitro-blue tetrazolium were used as chromogenic sub-
strates (2,36). The probe was also used for dot blot and tissue print
hybridizations (described previously).
Melon tissues were fixed in 4% formaldehyde and prepared for
in situ hybridization according to Jackson (11). Processed sections
were mounted in Shandon Mount Toluene Base (Shandon, Pitts-
burgh, PA) with a prior counterstaining in 0.1% Calcofluor (Sigma
Chemical, St. Louis) and were photographed using transmitted
light microscopy or combined epifluorescence (UV; Calcofluor
staining of cell walls) and transmitted light microscopy.
RESULTS
Fig. 3. Detection of Cucurbit yellow stunting disorder virus (CYSDV) in graft-inoculated plants. A, A drawing of a typical plant in which a C-105 scion was
grafted onto a susceptible infected stock. The positions of the plant that were analyzed to detect CYSDV by hybridization in tissue print assays are indicated
with arrows. Positions I and II represent the petiole and the stem of the stock, respectively, III represents the stock stem just below the graft union, IV and V
represent the scion stem, and VI represents a scion petiole. B, An example of an autoradiograph from a membrane containing tissue prints of cross-sections
from the specific positions within the grafted plants indicated in A and hybridized to detect CYSDV RNA. Columns 1 to 3 correspond to susceptible scions
grafted onto CYSDV-infected stocks, column 4 corresponds to a susceptible scion grafted onto a healthy stock, and columns 5 to 20 correspond to C-105 scions
grafted onto CYSDV-infected stocks. The original source of scion tissue in columns 5 to 9, 10 to 15, and 16 to 20 was multiple shoots from three single source
plants.
848 PHYTOPATHOLOGY
signal could be detected in any of the graft-inoculated C-105 leaf
veins examined (Fig. 6C).
DISCUSSION
represent the petiole and stem of the stock, respectively, III represents the
stock stem just below the graft union, IV and V represent the scion stem,
and VI represents a scion petiole (positions I to VI represent older to
younger tissue in the plant as indicated in Figure 3A).
z Values followed by different letters differ significantly (P 0.01) according
Fig. 6. Detection of Cucurbit yellow stunting disorder virus (CYSDV) RNA by in situ hybridization in cross-sections of leaves of graft-inoculated plants.
Images are from A, midrib veins from a leaf of a susceptible scion grafted onto a healthy stock, B, midrib veins from a leaf of a susceptible scion grafted onto a
CYSDV-infected stock, and C, midrib veins from a leaf of a C-105 scion grafted onto a CYSDV-infected stock. External phloem (eP), internal phloem (iP), and
xylem (X) are indicated for one of the veins on each panel. The dark spots (indicated by arrows) correspond to CYSDV-specific in situ hybridization signal.
Magnification bars are shown.
850 PHYTOPATHOLOGY
are under the same or different genetic control. It is possible that vascular transport of Potato virus A in diploid potatoes. Mol. Plant-
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populations. There are cases of partial resistances that were over- chlorotic stunt virus reveal several new features for the genus Crinivirus.
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evolutionary factors are involved in the determination of resis- phylogenetic analysis of Cucurbit yellow stunting disorder virus RNA2.
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19. López-Sesé, A. I., and Gómez-Guillamón, M. L. 2000. Resistance to
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the durability of resistance (25,26). If this were the case for plant L. Hortscience 35:110-113.
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population size, gene and genotype flow between CYSDV popu- yellowing disease of cucurbit crops in Portugal. Plant Dis. 84:1156.
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1998. Identification and characterization of a locus (RTM1) that restricts
individuals (25,26). To our knowledge, very little information is long-distance movement of tobacco etch virus in Arabidopsis thaliana.
available on these aspects of the CYSDV biology. Currently, we Plant J. 14:177-186.
are studying the genetic variability of natural CYSDV populations 22. Martelli, G. P., Agranovsky, A. A., Bar-Joseph, M., Boscia, D.,
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ACKNOWLEDGMENTS
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the International Committee on Taxonomy of Viruses. M. H. V. Van
This work was partly financed by Seminis Ibérica, S.A., through a
Regenmortel, C. M. Fauquet, D. H. L. Bishop, E. Carstens, M. K. Estes,
subsidy of the “Fomento de Tecnología Industrial” program (Ministerio S. M. Lemon, J. Maniloff, M. A. Mayo, D. J. McGeoch, C. R. Pringle,
de Industria y Energía, Spain). J. M. Aguilar was the recipient of a “For- and R. B. Wickner, eds. Academic Press, San Diego, CA.
mación y Especialización en Líneas de Interés para el Sector Industrial” 23. Mas, P., and Pallas, V. 1995. Nonisotopic tissue-printing hybridization–A
fellowship of the Consejo Superior de Investigaciones Científicas new technique to study long-distance plant-virus movement. J. Virol.
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