Quantitation of cucurbit yellow stunting disorder virus in Bemisia tabaci (Genn.) using digoxigenin-labelled hybridisation probes

Journal of Virological Methods 101 (2002) 95 – 103 www.elsevier.com/locate/jviromet

Quantitation of cucurbit yellow stunting disorder virus in Bemisia tabaci (Genn.) using digoxigenin-labelled hybridisation probes Leticia Ruiz, Dirk Janssen, Leonardo Velasco, Eduardo Segundo, Isabel M. Cuadrado * Centro de In6estigacio´n y Formacio´n Agraria, Unidad de Virologı´a, Apartado de Correos 91, 04700 -El Ejido, Almeria, Spain Received 21 May 2001; received in revised form 5 November 2001; accepted 5 November 2001

Abstract A cost-efficient hybridisation assay was developed to estimate the amount of cucurbit yellow stunting disorder virus (CYSDV) in Bemisia tabaci (Gennadius) whiteflies infesting protected cucumber crops. cDNA from the coat protein (cp) gene and the hsp70 homologue protein gene from CYSDV were obtained by reverse transcriptase-PCR from viruliferous whiteflies and cloned into plasmids. Digoxigenin (DIG)-labelled cDNA probes reacted with extracts from these whiteflies applied on nylon membranes. Precision and linear ranges were established in a hybridisation analysis using known concentrations of unlabelled homologue cDNA. Extracts from non-viruliferous B. tabaci showed a concentration-dependent effect on the assay with cp-specific probes but not with hsp70 -specific probes. The hsp70 probe was used to evaluate natural B. tabaci populations in commercial cucumber crops, and the estimated amounts of CYSDV per whitefly were found ranging from 5.6 fg to approximately 2.5 pg of corresponding hsp70 -cDNA. © 2002 Elsevier Science B.V. All rights reserved. Keywords: Hybridisation; Bemisia tabaci; CYSDV

1. Introduction Since the last decade, cucurbit yellow stunting disorder virus (CYSDV) has constituted a major constraint on protected cucumber and melon crops in Spain and other Mediterranean countries (Sese et al., 1994; Celix et al., 1996; Louro et al., 2000). This virus is a filamentous virus that has a * Corresponding author. Tel.: + 34-950-558030; fax: +34950-558055.

bipartite single stranded RNA genome and is a member of the genus Crini6irus (family Clostero6iridae). It is transmitted semi-persistently by Bemisia tabaci (Gennadius) and causes phloemlimited infections in plants (Wisler et al., 1998). In order to develop a strategy that would manage vector-transmitted diseases, such as, yellowing disease in greenhouse crops, epidemiological information on vector population densities, virus transmission efficiency and quantitative estimations of viruliferous insect vectors is required (Flanders et al., 1991).

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A method is described to quantitate CYSDV in B. tabaci extracts by means of hybridisation with DIG-labelled probes against two CYSDV-specific genes, encoding the coat protein (cp) and a heat shock 70 homologue protein (hsp70 ), respectively. The latter gene is typical of the clostero6iridae (Tian et al., 1996), and the protein is believed to play a role as a virus movement protein (Agranovsky et al., 1998). The following features of the method were determined for both hsp70 and cp probes: precision, linearity and working range, and limit of detection and quantitation. The CYSDV-specific hsp70 homologue probe was used to estimate the viral load of whiteflies in commercial cucumber crops in greenhouses during two consecutive campaigns.

2. Materials and methods

2.1. Whiteflies A nonviruliferous B. tabaci population was obtained by collecting emerging adults from pupae infested naturally on Euphorbia pulcherima plants in Almeria. These were reared subsequently on E. pulcherima plants grown in whitefly-proof cages within a controlled temperature room at 279 1 °C with a photoperiod of L16:D8 h and 609 2% relative humidity. The nonviruliferous populations were regularly checked for the presence of CYSDV by RT-PCR (see below). Both the nonviruliferous B. tabaci population as well as populations collected from greenhouses in Almerı´a were identified as the Q biotype by RAPDPCR analysis (results not shown) as described in Ruiz et al. (1999). B. tabaci infected with CYSDV were either maintained on infected cucumber plants or were collected from commercial greenhouse crops.

2.2. Virus isolates CYSDV was obtained from commercial cucumber crops in Almeria, and was maintained on Cucumis sati6us cv. Marianna RZ grown in whitefly-proof cages by B. tabaci transmission. The species-identity of the isolate was confirmed

by sequencing the RT-PCR amplification product for hsp70 described below and comparison with the published sequence (Genebank Acc. No. U67170).

2.3. Nucleic acid extraction for RT-PCR and spot hybridisation assay Five to 20 individuals of B. tabaci were frozen and placed in a microtube containing 20 ml of STE (0.1 M NaCl, 10 mM Tris–HCl (pH 8.0), 1 mM EDTA (pH 8.0)) and were ground using a plastic pestle. Immediately after grinding, the tube was centrifuged (10 min per 10 000 rpm per 4 °C). The supernatant was collected and either heated at 68 °C (5 min) and put on ice or subjected to one of the following two procedures: (a) extraction of total nucleic acids with an equal volume of TE-saturated phenol: chloroform (1:1; v/v), re-extraction of the aqueous phase with an equal volume of chloroform; (b) total RNA extraction from the homogenate supernatant using the SV Total RNA Isolation System from Promega (Madison, USA). Double-stranded RNA (dsRNA) from healthy and crini6irus-infected plants was purified from the total nucleic acid extract by non-ionic cellulose (CF-11, Whatman) chromatography in the presence of 16% ethanol as described by Valverde et al. (1990).

2.4. RT-PCR All RT-PCR amplifications used the oligonucleotide primers described to amplify two separate regions of the CYSDV genomic RNA. The first region comprised the entire coat protein (cp) gene and was amplified with primers CP1 (5%-ATGGCGAGTTCGAGTGAGAA-3%) and CP2 (5%TCAATTACCACAGCCACCTG-3%) as described by Livieratos et al. (1999). The second region contained part of the hsp70 homologous gene, amplified with primers 410U (5%-AGAGACGGTAAGTAT-3%) and 410L (5%-TTGGGCATGTGACAT-3%) as described by Celix et al. (1996). First strand cDNA synthesis was carried out as follows: five ml of template was added to 100 ng of downstream primer (CP2 or 410L) and the mix-

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ture was heated at 95 °C (dsRNA template) or 68 °C (B. tabaci extract) for 5 min and then chilled in ice. Primed RNA was used for firststrand cDNA synthesis with 100 units of Superscript II™ RNase H- Reverse Transcriptase (Life Technologies, Barcelona, Spain) following the manufacturers instructions. Two microliter aliquots of the reaction mixtures were used for PCR amplification by adding 100 ng of the respective upstream primer (CP1 or 410U). Template cDNA was denatured at 96 °C for 5 min, and amplification was performed using forward and reverse primers (100 ng each of CP1/CP2 or 410U/410L) and Taq polymerase (Roche, Barcelona, Spain) in a total volume of 50 ml. Thermocycling conditions were: 2 min at 94 °C, 35 cycles of 30 s at 95 °C, 30 s at 55 °C and 1 min at 72 °C, and a final extension step at 72 °C for 5 min.

2.5. Preparation of DIG-labelled probes and hybridisation PCR-amplification products were ligated into pGEM-T Easy vector (Promega, Madison, WI) according to the manufacturers instructions and transformed into DH5a Eschericha coli cells. The plasmid inserts were sequenced and the identity was confirmed by comparison with published sequence information (Genebank Acc. Number AJ243000 for cp). Digoxigenin-labelled probes were prepared by PCR reaction containing 200 ng of linearised pGEM-T-hsp70 or pGEM-T-cp plasmid, using the PCR DIG Labeling Mix (Roche) and following the manufacturers instructions. Thermocycling conditions were identical to those described above. For hybridisation, samples were denatured at 68 °C for 5 min, snap-cooled on ice and blotted as 2 ml samples on a positively charged nylon membrane. The nucleic acids were bound to the nylon by UV crosslinking. Prehybridisation was carried out at 1 h at 42 °C followed by overnight hybridisation with 25 ng/ml of DIG-labelled probes at 42 °C. The hybridisation solution contained 5× SSC (20 × SSC = 3 M NaCl, 0.3 M sodium citrate, pH 7), 0.02% sodium dodecyl sulphate (SDS), 0.1% sodium N-lauroylsarcosine, 2% Blocking Reagent and 50% deion-

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ized formamide. The membranes were washed under stringent condition: 2× SSC, 0.1% SDS for 10 min at room temperature followed by two washes of 15 min each in 0.1× SSC, 0.1% SDS at 68 °C. Next, the membranes were incubated with 1% blocking reagent buffer, then with anti-digoxigenin antibody conjugated with alkaline phosphatase (anti-digoxigenin-AP Fab fragments) and finally washed twice with maleate buffer plus 0.3% Tween 20. Detection by chemiluminescence was carried out with CSPD following the manufacturers instructions and overnight exposure at room temperature to a Lumi-film. All reagents were from Roche (Barcelona, Spain), unless otherwise noted.

2.6. Quantitati6e hybridisation method 6alidation The exposed chemiluminescent detection films were scanned and analysed using KODAK 1D Image Analysis Software Version 3.5. Fixed squares (or Regions of Interest, ROI’s) of 600 pixels were drawn enclosing each spot and the optical density (OD) of each blotted sample was obtained as the sum of the background-subtracted pixel values within the ROI, that is, the net intensity. The following parameters were determined. (i) Working and Linear Ranges. Positively charged nylon membranes were loaded with 2 ml samples of independently prepared dilutions of pGEM-T-cp and pGEM-T-hsp70 plasmid in TE. Considering the molecular ratio of vector versus virus specific insert fragment, the amounts of virus-specific ssDNA in the samples ranged from 10 to 54 000 fg in the membranes loaded with pGEM-cp, and from 20 to 40 000 fg in membranes loaded with pGEM-hsp70. The amounts of DNA blotted were transformed to a logarithm base 10 (log) so that the dose-response curve refers to log (dose). Linear regression coefficients and parameters including 0.95 confidence intervals were calculated, as well as, the residual values (difference between actual measurement response and the response predicted by the straight line for each concentration value).

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(ii) Limits of detection (LoD) and quantification (LoQ). Two ml samples of 10 independent blanks (B. tabaci extract in TE at concentrations corresponding to 1 adult per ml) were each measured once. LoD and LoQ were expressed as the analyte concentration corresponding to the mean sample blank value + 3s and +10s, respectively (Euroachem, 1999). (iii) Precision. The OD of 20 prepared independently samples of TE containing 1.0 pg of cp and hsp70 inserts in plasmid vectors were measured and the distribution of values obtained was calculated as (standard deviation× 100)/mean (% CV). (iv) Comparison of buffer and B. tabaci extracts as dilution media. In order to evaluate the matrix effects when measuring CYSDV-specific sequences in B. tabaci, a series of fixed concentrations of pGEM-T-cp and pGEMT-hsp70 were added to extracts of CYSDVfree B. tabaci (corresponding to 1 adult per ml prepared in TE). Considering the molecular ratio of vector versus insert, the amounts of virus-specific ssDNA loaded ranged from 2.2 fg to 2.2 pg in the membranes loaded with pGEM-cp, and from 20 fg to 20 pg in membranes loaded with pGEM-T-hsp70. The slopes of the regression lines obtained for the measurements in TE and in B. tabaci extracts were compared. Alternatively, pGEM-T-cp (0.8 pg) and pGEM-T-hsp70 (1.0 pg) were added to CYSDV-free B. tabaci extracts prepared in TE (ranging from 1 whitefly/1 ml to 1 whitefly/20 ml TE) and spotted as 2 ml aliquots on membranes, followed by hybridisation with CYSDV-cp and hsp70 DIG-PCR probes.

and autumn of 2000. The greenhouses had similar structural features (conventional polyethylene film over the roof and conventional 16/10 net over the lateral windows) and environmental conditions. In each greenhouse, 24 plants were randomly selected from which 20 B. tabaci adults were removed, introduced into a sealed glass pipette and stored at − 20 °C until further processing (usually within 1 week). Each group of 20 whiteflies was put into a microtube containing 20 ml of TE and homogenised with a pestle. After centrifugation (10 min at 12 000 rpm) the supernant was removed, heated at 68 °C for 5 min, cooled on ice and applied to membranes as 2 ml aliquots (equivalent to the amount of 2 B. tabaci specimen). Dilutions ranging from 20 to 40 000 fg of linearised and denatured pGEM-hsp70 plasmid, were applied to the membranes in 2 ml aliquots and used as standards. Hybridisation with DIG-labelled hsp70 PCR fragments and quantitation was done as described above.

3. Results

3.1. Detection of CYSDV in B. tabaci by RT-PCR and hybridisation RT-PCR-amplified fragments of the predicted size for both cp (about 750 bp) and hsp70 (about 435 bp) genes were obtained from viruliferous B. tabaci, and from dsRNA preparations of CYSDV infected plants (Fig. 1). Positive amplification re-

2.7. Sampling procedures in the e6aluation of natural B. tabaci populations B. tabaci adults were sampled from commercial cucumber crops in three separate greenhouses, each spanning 600 m2 and located at an experimental farm of CIFA, Almerı´a. The crops were cultivated following the usual practices of the region during two consecutive campaigns, spring

Fig. 1. RT-PCR amplifiactions from dsRNA (1), from healthy (3) and from viruliferous (5) B. tabaci, with CYSDV-hsp70 -homologue gene specific primers. RT-PCR amplifications from dsRNA (2) from healthy (4) and from viruliferous (6) B. tabaci, with CYSDV-cp gene specific primers.

L. Ruiz et al. / Journal of Virological Methods 101 (2002) 95–103

Fig. 2. Spot hybridisation of viruliferous (number 1 – 8) and healthy (c) B. tabaci with cp-specific probes (left) and with hsp70 -specific DIG-labelled probes (right).

actions were revealed using the supernatant of B. tabaci homogenates and also in RNA extractions from the supernatant. When these crude B. tabaci samples were applied to nylon membranes, they reacted positive after hybridisation with either CYSDV — cp or hsp70 specific DIG-labelled probes (Fig. 2). Neither RT-PCR-amplified fragments of viral sequences, nor positive hybridisation results were obtained from B. tabaci that was reared on E. pulcherima plants.

3.2. Quantitation of CYSDV in B. tabaci The results of the measurements, expressed as log10 OD of different amounts of pGEM-T plasmids containing the CYSDV specific sequences, are shown in Fig. 3 and linear regression coeffi-

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cients and parameters are shown in Table 1. The graphical representation of the data suggests that the procedure yielded results proportional to the concentration and with homogeneous scattering among the linear regression line at a concentrations ranging from 20 to 8000 fg of hsp70 -cDNA when hybridised with DIG-labelled PCR fragments. A minor deviation from the regression line was observed at a concentration of 40 pg of homologous DNA. With respect to the lower end of the working range, LoQ was found to be 5.6 fg. Also the calibration of hybridising different amounts of pGEM-T-cp with DIG-labelled cpcDNA probe suggested a linear relationship between concentrations of homologous DNA and optical densities obtained (Table 1). However, a closer examination of Fig. 3B and particularly the plotted residual values, revealed a considerable concentration-dependent deviation from the slope indicative of a non-linear relationship between homologous DNA and responses. Whereas, a theoretical LoQ of 3.6 fg was obtained in case of blank samples hybridised with DIG- labelled cpcDNA, in practice, the lowest concentration (22 fg) within the linear range had to be used as LoQ. The theoretical LoD values obtained for hsp70 and cp-probes were 0.12 and 0.71 fg of homologous cDNA, respectively. Considering the equally dispersed measurements along the linear ranges obtained for both cp and hsp70 -probes, a

Fig. 3. Precision profile between hybridisation signals (optical density) and the concentration of pGEM-t-hsp70 (A) of pGEM-T-cp (B) plasmid. Right corner inserts show the respective residual graph.

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Table 1 Linear regression coefficients and parameters of the calibrations for DIG-hsp70 and cp probes. Only those for cp in B. tabaci differed at the 95% confidence interval (C.I.)

hsp70 in TE cp in TE hsp70 in TE hsp70 in B. tabaci cp in TE cp in B. tabaci

n

R

Slope

95% C.I.

Intercept

95% C.I.

28 32 12 12 12 12

0.989 0.982 0.995 0.997 0.981 0.969

0.575 0.353 0.573 0.629 0.353 0.587

0.540–0.610 0.327–0.378 0.532–0.613 0.596–0.661 0.303–0.402 0.482–0.692

4.662 5.957 4.670 4.460 5.957 5.318

4.553–4.772 5.878–6.037 4.548–4.793 4.362–4.558 5.851–6.063 5.093–5.544

single concentration of homologous cDNA was used to determine the precision of the method which was found to be 5.35 and 3.36%, respectively. The responses of cp and hsp70 -probes hybridised against aliquots containing a different amounts of plasmid containing cp or hsp70, diluted either in TE or a B. tabaci extract, were compared. As shown in Fig. 4 and from the calculated linear regression parameters (Table 1) no differing signals were obtained with TE and with B. tabaci extracts in the case of hsp70 probes. However, when using cp-probes, B. tabaci extracts reduced considerably the responses at the lowest concentrations used. The different matrix effects on hsp70 and cp probes were also obvious using a fixed amount of plasmid containing cp or hsp70 sequences in a series of B. tabaci extract dilutions in TE. Vector extract reduced responses of cp-probes significantly in a concentration-dependent manner until a dilution of 1 whitefly per 10 ml (Fig. 5).

3.3. CYSDV quantitation in commercial greenhouse B. tabaci populations The amount of CYSDV hsp70 -copies in B. tabaci collected from commercial greenhouses ranged from the established quantitation limits (5.6 fg) up to almost 2500 fg per whitefly (Table 2). The mean viral load from whiteflies sampled had similar values at the initial phase of each crop season and probably reflects the existence of a homogeneous vector population initially present in the environment, and which entered subsequently into the greenhouses.

4. Discussion Generally, crini6iruses generate only low titres in infected plants (Wisler et al., 1998), and due to the lack of specific and sensitive antisera, diagnosis of these pathogens in plants has to recur to either dsRNA purifications or RT-PCR amplification (Celix et al., 1996). RT-PCR detection of CYSDV in plants has been achieved successfully by amplification of viral cp (Livieratos et al., 1999) and hsp70 genes (Celix et al., 1996). In the present paper we showed that both diagnostic sequences are also detectable by RT-PCR in viruliferous B. tabaci (Fig. 1). Amplifications were obtained using either supernants of homogenised whiteflies, or total RNA purifications. This permits the development of a highly sensitive and quantitative RT-PCR assay of CYSDV as has been achieved for viruses in aphids (Canning et al., 1996) and in nematodes (Van de Wilk et al., 1994). However, the application of a quantitative assay for CYSDV for management strategy decisions would depend on the cost-efficient analysis of many samples to be carried out by skilled extension service technicians. Within this perspective, the introduction of techniques similar to ELISA or hybridisation, labelled non-isotopically would be less demanding. Hybridisation with DIG-labelled probes has become a widely used method for plant virus detection (Saldarelli et al., 1996) and with chemiluminiscent substrates revealing comparable sensitivities to probes labelled non-isotopically (Musiani et al., 1991; Narva´ ez, et al., 2000). Since DIG-labelled probes detected CYSDV in viruliferous B. tabaci (Fig. 2) a quantitative hybridisation assay might be an alternative

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Fig. 4. Analyte-concentration dependant interferance of B. tabaci extract on hybridisation. Different concentrations of hsp70 -cDNA (A) and cp-cDNA (B) were diluted either in TE (open circles) or in an extract of non-viruliferous B. tabaci (1 whitefly per ml) (filled circles).

for RT-PCR-based techniques. Analysis of CYSDV in B. tabaci was carried out routinely using the supernatant of 20 whiteflies homogenised in 20 ml of extraction buffer, thus minimising the errors of recovery caused by the use of extractions with organic solvents or by other purification procedures. In our experience, the number of whiteflies and the volume of extraction buffer constituted a compromise between the minimum volume to efficiently homogenise whiteflies in a microtube and the volume of aliquots applied to a nylon membrane (2 ml). As a consequence, the chemiluminescent signal of each spot reflects the amount of homologous oligonucleotide in two whiteflies. Calibration of the quantitative assay requires the use of pure virus preparations. However, dilutions of plasmid containing virus-specific sequences have been successfully used to calibrate a quantitative hybridisation assay of TYLCV in B. tabaci (Caciagli and Bosco, 1996). Since crini6iruses generally are extremely difficult to purify (Wisler et al., 1998) we preferred to use cloned cDNA for quantitative hybridisation in which it is assumed that the binding kinetics of the DIG-labelled probes are similar for the single stranded RNA genome as for the cDNA. For both, cp and hsp70 probe, the concentration response relationship was linear until about

10 pg of target sequence diluted in TE was reached. When diluted in an extract of non-viruliferous B. tabaci, DIG-cp probes showed a significant loss of sensitivity, and therefore of

Fig. 5. Interferance of B. tabaci extract on hybridisation. Fixed amounts of hsp70 -cDNA (1.0 pg (A)) and cp-cDNA (0.8 pg (B)) were resuspended in different concentrations of non-verifolous B. tabaci extracts diluted in TE. The results are expressed as the percentage of log optical density (O.D) of controls, which was cDNA diluted in TE only and which is represented as full (mean) and dashed (95% C.I.) horizontal lines.

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Table 2 Quantative determination of CYSDV in naturally infected B. tabaci Campaign

Spring Autumn

Commercial cucumber greenhouses I

II

III

597 (2468) 110 (720)

371 (2252) 222 (942)

305 (2323) 83 (307)

Mean (maximum) amounts of CYSDV-hsp70 copies, expressed in fg, measured per whitefly from a group of 20 individuals and per cucumber plant (n= 24). The whitefly populations were collected at the first-leaf stage during two consecutive campaigns in the year 2000 and in three separate greenhouses.

linearity, at plasmid-insert concentrations below 22 fg. This was in contrast with DIG-hsp70 probes, that did not show significantly different responses when comparing plasmid containing a hsp70 insert diluted in TE and in B. tabaci extract (Figs. 4 and 5). These features of the cp and hsp70 probes were also prominent when hybridised against approximately 960 B. tabaci populations collected from the field (results not shown), making the hsp70 probes the tool of first choice for quantitation of CYSDV in B. tabaci. As such we were able to provide the first estimates of the viral load of a crini6irus in natural B. tabaci populations. Most of the populations evaluated yielded CYSDV concentrations corresponding to amounts of hsp70 cDNA ranging from 5.6 to about 2500 fg per whitefly (Table 2), which are values that fitted well within the established linear range of concentration versus response. Assuming that a single copy of the hsp70 gene fragment used (450 nt) represents about 1/40 of the total genome of CYSDV (17 000 nt), one can conclude that the infected whiteflies contain between 0.8 and 80 pg (weight of the entire genome) of the crini6irus. These concentrations are in line with those found for other RNA viruses such as luteovirus in aphids (Canning et al., 1996) but are also well beyond those found for DNA viruses such as TYLCV that are detected at concentrations of about 1 ng in a single whitefly (Caciagli and Bosco, 1996).

In general, whitefly screening yields number of whiteflies present in a crop, and these data can be used to establish action thresholds, which are levels of pest populations at which control should be implemented to avoid significant damage to the crop. Such thresholds are significant where pests, such as, thrips (Shipp et al., 2000), or whiteflies (Toscano et al., 1999) cause direct damage to crops. However, for virus susceptible crops, realistic thresholds would be based on infective arthropod vectors, like thrips transmitting tomato spotted wilt virus, or leafhoppers transmitting phytoplasma-borne aster yellows disease (Goodwin et al., 1999), and would consider transmission efficiencies and viral loads of vectors. So far, setting up action thresholds for whitefly transmitted viruses has been unattainable because of the lack of a fast and reliable method to assess the viral load of whiteflies or even the numbers of viruliferous whiteflies present in a crop. However, the results of the present paper show that the CYSDV load of B. tabaci populations from cucumber greenhouses can be estimated in a costefficient manner using DIG-labelled hsp70 specific probes. This suggests that the method described can yield data that may be considered for the development of management strategies of yellowing disease.

Acknowledgements The first and fourth authors had fellowships from the Direccı´on General de Investigacio´ n (Consejerı´a de Agricultura y Pesca) of the Junta de Andalucı´a. The second and third author had fellowships from INIA. This work was supported by project SC99-050 (INIA). We also thank Antonia Belmonte for technical assistance and Vince Reid for careful revision of the manuscript.

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