Seedborne transmission of Zucchini yellow mosaic virus and Cucumber mosaic virus in Styrian Hulless group of Cucurbita pepo1 I. Tóbiás1*, B. Szabó2, K. Salánki3, L. Sári4, H. Kuhlmann5, and L. Palkovics2 1
Plant Protection Institute, Hungarian Academy of Sciences, P.O. Box 102, 1525 Budapest, Hungary 2 Corvinus University of Budapest, Department of Plant Pathology, 1118 Budapest, Hungary 3 Agricultural Biotechnology Center, P.O.Box 411, 2100 Gödöllő, Hungary 4 Tessedik Sámuel College, 5540 Szarvas, Hungary 5 MQB Dr. Kuhlmann, 71083 Herrenberg, Germany * Corresponding author e-mail:
[email protected]
Keywords: Hulless pumpkin, seedborne virus transmission, ZYMV, CMV, sequences of coat protein Abstract A total of 62 seed lots originating from hulless pumpkin fruit exhibiting typical symptoms of ZYMV infection were tested in insect-free greenhouses in three consecutive years. Of those, 25 seed lots showed seedborne virus transmission that ranged from 0.29 to 15.34 % of the seeds. In total 19997 seeds were sowed, 16039 plants were monitored and 225 plants were seedborne virus infected. Of those, 166 had ZYMV and 59 had mixed infection of ZYMV and CMV as identified by ELISA serological tests, by indicator plants or by RT-PCR. The average rate of seedborne transmission of ZYMV was 1.4 %. Coat protein gene of CMV and ZYMV were characterized and compared. INTRODUCTION Zucchini yellow mosaic virus (ZYMV) was first identified in northern Italy. Soon after, it was identified in areas throughout the world where cucurbits are cultivated, including Mediterranean countries, Japan, Germany, Central Europe, China, Chile, Australia, Mexico, Mauritius, Canada and the USA (Desbiez and Lecoq 1997; Greber et al. 1987; Nameth et al. 1986; Prieto et al. 2001; Robinson et al. 1993; Sammons et al. 1989; Tóbiás et al. 1996). This relatively new, but aggressive virus (member of the potyvirus group) has spread rapidly throughout the world, suggesting an efficient transmission from plant to plant by several aphid species in a nonpersistent manner (Lisa and Lecoq 1984) and long distance distribution via infected seeds (Davis and Mizuki 1986; Schrinjwerkers et al. 1991; Fletcher et al. 2000). The efficient intercontinental spread of the virus can be explained by international trading of infected seeds. However, the rate of disease transmission via seed is low and difficult to prove (Provvidenti and Robinson 1987; Gleason and Provvidenti 1990; Robinson et al. 1993). There is some evidence for seed transmission in the case of zucchini (Davis and Mizuki 1986; Schrinjwerkers et al. 1991), buttercup squash (Fletcher et al. 2000) and hulless oilseed pumpkin (Tóbiás and Kovács 2001). This work focused on studying the seedborne virus transmission on hulless oilseed 1
Cucurbitaceae 2008, Proceedings of the IXth EUCARPIA meeting on genetics and breeding of Cucurbitaceae (Pitrat M, ed), INRA, Avignon (France), May 21-24th, 2008 189
pumpkin, the effect of storage time on the rate of virus transmission via infected seeds, and characterization of the virus isolates transmitted by seeds. Since coat protein (CP) analysis has become a primary method for taxonomic assignment of potyviruses and cucumoviruses, our aim was to characterize this genomic region of ZYMV and CMV originating from virus-infected pumpkin seedlings. MATERIALS AND METHODS Seed material and experimental conditions In 2001 in the vicinity of Szarvas (Hungary) field grown hulless oilseed pumpkin (Cucurbita pepo subsp. pepo var. pepo Styrian Hulless Group) plants were naturally infected and exhibited typical symptoms of ZYMV infection. All fruit with mild, severe and very severe distortions were harvested and weighed. The seeds were taken out of the fruit, dried, weighed and stored at 5°C in a cooling chamber. Seed lots originating from different fruit were kept separately. Seed transmission experiments were conducted in a greenhouse under strict sanitary conditions from mid January to the end of March of 2002, 2003 and 2004 with randomly chosen seed lots. The seedlings were visually observed, and all plants showing any sign of abnormality (mosaic symptoms, leaf deformation, vein clearing etc.) were marked and examined. ELISA experiments Leaf material from the marked plants was tested for infection of ZYMV (Bioreba Art No 161222) using DAS-ELISA according to the manufacturer's protocol. In case of no reaction with ZYMV kit in ELISA, samples were assayed with WMV-2 (Bioreba Art No. 161122) and CMV (Bioreba Art. No. 160622) ELISA kits, RT-PCR and test plants. Test plant experiments The following indicator plants were used: Cucurbita pepo ‘Black Beauty’, Nicotiana benthamiana, Chenopodium quinoa and C. amaranticolor. The sample leaves showing typical symptoms of virus infection were triturated in 0.06 M phosphate buffer (pH 7.2) and inoculated onto carborundum dusted test plant leaves. Nucleic acid isolation, RT-PCR, cloning and sequence analysis Total RNA was extracted from leaves having obvious signs or symptoms using the method of White and Kaper (1989). The primers were designed to amplify the coat protein gene of ZYMV and CMV. The following primers were used: ZYMV CP1 (sense) (5’-GTA ATG CTA ACC ATG GGG CAC TCA G-3’) and ZYMV CP3 (antisense) (5’-GGG GAT CCG ACC TAC CCT TTA CTG-3’) for ZYMV, and CMV 43 (antisense): 5’-GCG GAT CCT GGT CTC CTT-3’, CMV 58 sense): 5’GGC TGC AGT CCG CGA GAT TGC GGT-3’ for CMV detection. The first strand of cDNA was synthesized from total RNA using MBI Fermentas Revert Aid First Strand cDNA Synthesis Kit and ZYMV-CP3 and CMV43 primers respectively. Two µl from this reaction mixture were used for PCR experiments. Amplification was performed in a volume of 50 µl of PCR buffer (10mM Tris-HCl pH 9.5, 1.5 mM MgCl2, 50 mM KCl, 0.1 % Triton X100, containing100 ng dATP, dCTP, dGTP, and dTTP-t, 0.1 nM primers and 5 U Taq polymerase (Fermentas). Forty reaction cycles 190
were performed (Eppendorf Mastercycler Gradient) with the following parameters: 1/ZYMV - template denaturation 94°C 15 s, primer annealing 60°C for 30 s and DNA synthesis at 72°C for 120 s, 2/ CMV - template denaturation 94°C 30 s, primer annealing 50°C for 30 s, DNA synthesis at 72°C for 90 s. After electrophoresis in 1 % agarose gels, the amplified DNA fragments were observed or excised and isolated with the MBI Fermentas DNA Extraction Kit. Three isolates from each virus (ZYMV-15/1, ZYMV-80/1, ZYMV-59/2, CMV-69/38, CMV-15/6 and CMV-80/1) were chosen to determine the nucleotide sequences of the coat protein. The PCR products of ZYMV-15/1, ZYMV-80/1, ZYMV-59/2 and CMV-80/1 isolates were cloned in Promega vector (pGEM-T Easy Vector). CMV-69/38 and CMV-15/6 PCR products were sequenced directly. DNA sequences were obtained with a DyeDeoxy Terminator Kit (Applied Biosystems, Foster City, CA. USA) using reverse and universal (-20) or CMV primers. Sequence analysis was performed employing University of Wisconsin Genetics Computer Group (GCG) sequence analysis software package version 8.1 using DISTANCE, GAP, SEQED, TRANSLATE and BOXSHADE programs. RESULTS Seedborne transmission of viruses In the tests for seedborne transmission of viruses, seeds of Styrian Hulless Group of C. pepo were used. Seed lots were originated from 62 fruit showing typical symptoms of ZYMV infection; 28 fruit were severely distorted, 28 distorted and 6 showed mild distortion. The weight of fruit varied between 2.1 kg and 4.9 kg and the seed numbers per fruit from 60 to 580. There was a correlation between fruit weight and seed number. In the tests of 2002 as a first step 22 seed lots were assayed, 11 of them were from severely distorted fruit, 8 seed lots from distorted and 3 from mildly distorted fruit. Table 1 shows, that seedborne transmission of virus infection was detected in 7 seed lots. The rate of virus transmission in different seed lots varied from 0.3 % to 15.34 %. From a total of 6073 plants, 104 were virus infected (1.71 %). In 83 samples ZYMV was detected by ELISA methods, in 22 plants RT-PCR was able to detect the presence of ZYMV and in these samples in addition CMV was detected by ELISA technique and test plant reaction (local necrotic reaction on inoculated leaves of C. amaranticolor and C. quinoa plants). Seed lots no. 1, 10, 55 and 82 were carrying a mixed infection of ZYMV and CMV. Seeds originated from distorted fruit (10) and from severely distorted fruit (55) showed almost the same rate of virus infection (13.14 % and 15.34 % respectively). Seeds from severely distorted fruit (3, 17, 25, 31 and 37 seed lots) produced virus free seedlings just as seeds from mild distorted fruit (27, 28 and 46 seed lots) or distorted fruit (7, 22, 36, 72, 75 and Kákai seed lots). After one year of storage 21 seed lots were tested in 2003 (Tab. 2). Of those, 11 seed lots were from severely distorted, 8 from distorted and 2 from mildly distorted fruit. Out of these 21 seed lots, 11 showed seedborne transmission of virus infection. The rate of virus transmission varied between 0.29 % and 6.99 %. Most of the seedborne virus transmission was detected in seed lots from severely distorted fruit. Interestingly, 3.12 % virus transmission was observed in 63 seed lots originating from mildly distorted fruit. Of 5846 plants, only 58 were virus infected (0.99 %). In 40 191
samples, ZYMV was detected by ELISA serological methods, and in 18 plants, mixed infection of ZYMV and CMV was observed. In these samples the presence of ZYMV was detected by RT-PCR and CMV by ELISA method and test plants. Seedlings from seed lot no. 23 and 83 all had mixed infection by ZYMV and CMV carrying both viruses in the seeds. In other cases (seed lots no. 51, 63, 70), most of the seeds were ZYMV infected and some seeds had a mixed infection. Seed lots no. 12, 19, 34, 35, 65 and 71 showed seedborne transmission of ZYMV only.
Viral pathogen
Virus transmission rate %
Number of virus infected plants
Number of plants
Number of seeds
Weight of fruit (kg)
Severity of symptoms on fruitz
Seed Sample
Table 1. Seed transmission experiment in 2002
Stock 1 *** 2.1 240 228 1 0.44 1 ZYMV, 1 CMV 3 *** 2.9 220 195 0 0.00 7 ** 2.3 288 261 0 0.00 10 ** 2.4 156 137 18 13.14 18 ZYMV, 5 CMV 17 *** 2.6 275 266 0 0.00 18 *** 3.9 359 334 1 0.30 1 ZYMV 22 ** 3.1 220 203 0 0.00 25 *** 4.3 504 378 0 0.00 27 * 3.5 380 354 0 0.00 28 * 3.6 270 259 0 0.00 31 *** 3.3 580 557 0 0.00 33 ** 3.7 350 319 10 3.13 10 ZYMV 36 ** 3.9 330 271 0 0.00 37 *** 3.4 320 242 0 0.00 43 *** 3.3 310 283 2 0.71 2 ZYMV 45 *** 4.9 360 274 1 0.36 46 * 3.6 60 52 0 0.00 55 *** 2.8 393 378 58 15.34 58 ZYMV, 12 CMV 72 ** 3.5 427 401 0 0.00 75 ** 3.2 139 128 0 0.00 82 *** 3.0 250 238 13 5.46 13 ZYMV, 4 CMV Kákai ** 3.6 348 315 0 0.00 z *** - severely distorted fruit, ** - distorted fruit, * - mild distortion
In 2004 after 2 years of storage, 19 seed lots were tested (Tab. 3). In six seed lots (15, 49, 59, 69, 74 and 80 seed lots), seedborne transmission of virus was detected and ranged from 0.30 % (74) to 11.73 % (69). From 4120 plants, 64 were seedborne virus infected (1.55 %). Seed lots no. 15, 69, 74 and 80 were carrying both viruses, while no. 49 and 59 were infected by ZYMV only. Germination rate was almost the same in the first two 192
years (89.6 % and 87.5 %), but in the third year it was decreased to 63 %. The seedborne virus infection rate (1.71 %, 0.99 % and 1.55 %) was almost the same in these experiments.
Viral pathogen
Virus transmission rate %
Number of virus infected plants
Number of plants
Number of seeds
Weight of fruit (kg)
Severity of symptomsz
Seed sample
Table 2. Seed transmission experiments in 2003
11 ** 2.3 250 239 0 0.00 12 ** 2.4 255 143 10 6.99 10 ZYMV 19 *** 3.1 308 267 1 0.37 1 ZYMV 20 *** 2.2 297 268 0 0.00 23 *** 3.6 413 341 1 0.29 1 ZYMV, 1 CMV 29 * 3.1 363 309 0 0.00 34 *** 3.5 419 386 2 0.51 2 ZYMV 35 ** 3.1 336 245 1 0.40 1 ZYMV 38 ** 4.1 200 100 0 0.00 39 ** 3.1 160 137 0 0.00 51 *** 3.6 380 362 9 2.48 9 ZYMV, 2 CMV 53 ** 3.8 303 240 0 0.00 63 * 3.7 319 288 9 3.12 9 ZYMV, 7 CMV 65 *** 3.5 274 261 2 0.76 2 ZYMV 70 *** 2.9 339 333 12 3.60 12 ZYMV, 7 CMV 71 *** 3.4 328 306 10 3.26 10 ZYMV 83 *** 2.9 340 270 1 0.37 1 ZYMV, 1 CMV 84 ** 3.7 400 385 0 0.00 86 *** 4.5 380 366 0 0.00 87 ** 3.4 340 331 0 0.00 Oliva *** 2.8 275 269 0 0.00 z *** - severely distorted fruit, ** - distorted fruit, * - mild distortion
Molecular characterization of viruses transmitted by seed ZYMV and CMV infection in pumpkin seedlings were identified by DASELISA, test plants and reverse transcription PCR (RT-PCR). The PCR detection assays were based on ZYMV-specific and CMV-specific primers described earlier (Choi et al. 1999; Tóbiás and Palkovics 2003). The ZYMV primers amplify the coat protein gene from the second amino acid to the stop codon (The first amino acid of CP is constant in ZYMV because of the cleavage site Q/S). The CMV primers amplify an approximately 910-bp fragment including the complete 656-bp coat protein gene. Comparison of ZYMV isolates The coat protein of three ZYMV isolates originating from seed transmission experiments showed very high similarity both to each other and to the previously
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well-characterized Hungarian isolates of ZYMV. The nucleotide sequence identity and amino acid sequence similarity of CP region varied from 85.9 to 99.9 % and from 90.3 to 99.6 %, respectively. The N-terminus is the only region in the entire coat protein that is unique to potyvirus and contains virus-specific and virus strain-specific epitopes (Shukla et al. 1991). Figure 1 shows amino acid sequence identities of the Nterminal region of ZYMV isolates. The tripeptide sequence DAG located in the Nterminus of the coat protein is required for virus transmission by aphids. The ZYMV isolates from Austria, Slovenia and Hungary all have asparagine (N) instead of aspartic acid (D) and lysin (K) at positions 16 and 17, alanine (A) instead of valine (V) at position 27, and methionine (M) instead of valine (V) at 37 compared to other ZYMV isolates. These sequences are specific only to Central European isolates and suggest a common ancestor. From an analysis of amino acid sequence identities of the most variable N-terminal region of ZYMV, it is interesting that strains from Italy, Germany, Japan (M39), China (Hangzhou) and California have 100 % homology. We hypothesize a common origin as well as the occurrence of seed transmission of ZYMV in other Cucurbita species, not only in Styrian Hulless of C. pepo.
Viral pathogen
Virus transmission rate %
Number of virus infected plants
Number of plants
Number of seeds
Weight of fruit (kg)
Severity of symptomsz
Seed sample
Table 3. Seed transmission experiment in 2004
5 ** 2.2 326 315 0 0.00 8 ** 2.5 232 219 0 0.00 14 ** 3.6 327 276 0 0.00 15 ** 2.8 298 189 17 8.99 17 ZYMV, 3 CMV 21 *** 2.4 282 261 0 0.00 41 ** 3.6 240 167 0 0.00 44 *** 4.5 513 169 0 0.00 49 *** 2.5 360 106 1 0.94 1 ZYMV 57 ** 3.2 344 143 0 0.00 58 ** 4.9 480 41 0 0.00 59 *** 4.1 398 26 2 7.69 2 ZYMV 61 ** 3.6 397 368 0 0.00 68 ** 3.4 305 212 0 0.00 69 *** 3.8 389 358 42 11.73 42 ZYMV, 14 CMV 74 ** 2.8 335 327 1 0.30 1 ZYMV, 1 CMV 79 ** 3.7 363 348 0 0.00 80 ** 2.8 300 175 1 0.57 1 ZYMV, 1 CMV 81 *** 2.9 400 302 0 0.00 85 * 3.8 250 118 0 0.00 *** - severely distorted fruit, ** - distorted fruit, * - mild distortion
Comparison of CMV isolates The coat protein of two CMV isolates originated from seed lots 69/38 and CMV-15/6 showed 100 % homology both in nucleic acid and deduced amino acid 194
sequence. The CMV-80/1 isolate was different at nucleic acid sequence (98.32 %), but deduced amino acid shows 100 % homology with them. The seedborne CMV isolates were compared to other CMV strains originating from cucurbit plants isolated in Japan and USA (CMV-Km, CMV-Pepo, CMV-C and CMV-C7-2), seedborne CMV in spinach (CMV-Sp104) and CMV strains previously characterized in Hungary (CMV-Ns, CMV-Rs and CMV-Trk7). Nucleotide and deduced amino acid sequence analysis revealed that CMV-69/38 and CMV-80/1 isolates were closest to CMV-C, CMV-Ns, CMV-Rs and were placed to CMV subgroup I.A. It is interesting that previously characterized CMV-Ns and CMV-Rs strains originating from different host (tobacco and radish) and different part of Hungary showed 100 % homology with CMV-80/1 and CMV-69/38 isolates in deduced amino acid sequence data. CONCLUSIONS In this study, seeds originating from 62 hulless pumpkin fruit showing typical symptoms of virus infection were tested in 3 consecutive years. In total, 19997 seeds were sown, 16039 plants were monitored and 225 plants were virus infected. Seed lots from 25 fruit revealed seedborne transmission of viruses in a range of 0.29 % to 15.34 % in individual fruit. Seedborne virus transmission occurred more often in severely distorted fruit, but not all of this type of fruit produced virus infected seedlings. In our observations, seed transmission of virus cannot be predicted from the symptoms of individual fruit. There was no correlation of virus transmission rate and the symptoms observed on the fruit. The average rate of seedborne transmission of ZYMV was 1.4 %. This is lower than the 18.0 % rate reported by Davis and Mizuki (1986), and higher than the 0.05 % seed transmission rate reported by Schrijnwerkers et al. (1991). Both assayed C. pepo ‘Black Beauty’ plants, but correspond with the results of Burgsmans and Fletcher (2000). After 2.5 years of storage, germination of the seeds decreased from 89.6 % to 63 %, but the seedborne transmission of the ZYMV remained constant at 1.71 % and 1.55 %, respectively. In total, 225 virus (ZYMV) infected plants were detected. The molecular properties of selected ZYMV isolates were similar to previously characterized Hungarian ZYMV isolates. In 59 plants, the presence of CMV was detected in addition to the presence of ZYMV. The molecular characterization of the selected CMV isolates showed that they belong to the CMV subgroup 1A and showed 100 % homology with the previously characterized CMV-Ns and CMV-Rs strains belonging to the same subgroup.
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Austria-10 Austria-11 Austria-12 Austria-2 Austria-5 Austria-6 Austria-Slovenia-1 Hungary-2 Hungary-f1 Hungary-sz6 15/1 59/2 80/1 Hungary-5 Hungary-10 Hungary-8 Hungary-sz3 Taiwan-CY2 Taiwan-PT5 Korea-A Taiwan-TN3 Austria-Berlin-1 Austria-Italy China-Hangzhou Japan-M39 USA-California USA-Connecticut Japan-M Taiwan-TNML1 China-Beijing Taiwan-TC1 Israel Taiwan-NT1 China-Hainan China-Ningbo Japan-169 USA-Florida Korea-cu China-Shanxi Singapore
AJ420015 AJ420016 AJ420017 AJ420012 AJ420013 AJ420014 AJ420027 AJ459954
AJ459955 AJ251527 AJ45956 AF127930 AF127934 AJ429071 AF127929 AJ420028 AJ420020 AF308732 AB063251 L31350 D00692 AB004641 AF127932 AY074809 AF127931 M35095 AF127933 AF486823 AY074810 AB004640 D00593 AF062518 AY074808 X62662
SGTQPTVADAGATKKNNEDDKGKNKDATGSGSGEKTMAAVTKD SGTQPTVADAGATKKNNEDDKGKNKDATGSGSGEKTMAAVTKD SGTQPTVADAGATKKNNEDDKGKNKDATGSGSGEKTMAAVTKD SGTQPTVADAGATKKNNEDDKGKNKDATGSGSGEKTMAAVTKD SGTQPTVADAGATKKNNEDDKGKNKDATGSGSGEKTMAAVTKD SGTQPTVADAGATKKNNEDDKGKNKDATGSGSGEKTMAAVTKD SGTQPTVADAGATKKNNEDDKGKNKDATGSGSGEKTMAAVTKD SGTQPTVADAGATKKNNEDDKGKNKDATGSGSGEKTMAAVTKD SGTQPTVADAGATKKNNEDDKGKNKDATGSGSGEKTMAAVTKD SGTQPTVADAGATKKNNEDDKGKNKDATGSGSGEKTMAAVTKD SGTQPTVADAGATKKNNEDDKGKNKDATGSGSGEKTMAAVTKD SGTQPTVADAGATKKNNEDDKGKNKDATGSGSGEKTMAAVTKD SGTQPTVADAGATKKNNEDDKGKNKDATGSGSGEKTMAAVTKD SGTQPTVADAGATKKNNEDDKGKNRDATGSGSGEKTMAAVTKD SGTQPTVADAGTTKKNNEDDKGKNKDATGSGSGEKTMAAVTKD SGTQPTVADAGATKKNNEDDKGKNKDATGPGSGEKTMAAVTKD SGTQPTVADAGATKKNNEDDKGKNRDATGSGSGEKTMATVTKD SGTQPTVADAGATKKDKEDDKGKNKDVTSSGSGEKTVAAATKD SGTQPTVADAGATKKDKEDDKGKNKDVASSGSGEKTVAAATKD SGTQPTAADAGATKKDKEDDKGKNKDVTGSGSGEKTVAAVTKD SGTQPTAADAGATKKDKEDDKGKNKDVTGSGSGEKTVAAVTKD SGTQPTVADAGATKKDKEDDKGKNKDVTGSGSGEKTVAAVTKD SGTQPTVADAGATKKDKEDDKGKNKDVTGSGSGEKTVAAVTKD SGTQPTVADAGATKKDKEDDKGKNKDVTGSGSGEKTVAAVTKD SGTQPTVADAGATKKDKEDDKGKNKDVTGSGSGEKTVAAVTKD SGTQPTVADAGATKKDKEDDKGKNKDVTGSGSGEKTVAAVTKD SGTQPTVSDAGATKKDKEDDKGKNKDVTGSGSGEKTVAAVTKD SGTQPTVADAGATKKDKEDDKGKNKDVTGSGSGEKTVTAVTKD SGTQPTVADAGATKKDKEDDKGKNKNVTGSGSGEKTVAAVTKD SGTQPTVADAGATKKDKEDDKGRNKDVTGSGSGEKTVAAATKD SGTQPTVADAGATKKDKEDDKGKNKDVTSSGSGEKTMAAVTKD SGTQPTVADTGATKKDKEDDKGKNKDVTGSGSSEKTVAAVTKD SGTQPTVAD..ATKKDKEDDKGKNKDVTGSGSGEKTVAAVTKD SGTQPTVADAGATKKDKEDDKAKNRDATSSGSGEKTVAAVTKD SGTQTTVADAGATKKDKEDDKGRNKDVTGSSSGEKTVAAATKD SDTKTTVADAGATKKDKEDDKGKNKDVTSSSSGEKAIAAATKD SGTQPTVADARVTKKDKEDDKGENKDFTGSGSGEKTVVAAKKD SGTQQTVADAGATKKDKEDDKAKNKDATSSGSGAKTVAAVTKA SGTQPTVADAGATKKEKEEDKGKNKDATSSSGNDKTITPAKKD SDTQTREAGAGASKKDKDEDKDKKKDVASSSASEKAVATATKD
Figure 1. Amino acid sequence alignment for the N-terminal region of coat protein of ZYMV isolates.
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