Isolation, identification and molecular ...

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South African Journal of Plant & Soil 2012, iFirst, xx–xx Printed in South Africa — All rights reserved

SOUTH AFRICAN JOURNAL OF PLANT & SOIL ISSN 0257-1862 EISSN 2167-034X http://dx.doi.org/ 10.1080/02571862.2012.701669

Isolation, identification and molecular characterisation of an isolate of Zucchini yellow mosaic virus occurring in KwaZulu-Natal, South Africa L Usher, B Sivparsad and A Gubba* Discipline of Plant Pathology, School of Agriculture, Earth and Environmental Sciences, College of Agriculture and Environmental Sciences, University of KwaZulu-Natal, Private Bag X01, Scottsville 3201, Pietermaritzburg, South Africa * Corresponding author, e-mail: [email protected] Zucchini yellow mosaic virus (ZYMV) is an economically important virus infecting cucurbits and has a worldwide distribution. In the Republic of South Africa, ZYMV has been reported as a major limiting factor to cucurbit production. The aim of this study was to identify, isolate and partially characterise a ZYMV isolate from KwaZulu-Natal (KZN). On the basis of host reactions, electron microscopy, serology, and size of the coat protein, a potyvirus infecting cucumber, squash, pumpkin and melon samples collected from farms around KZN was positively identified as ZYMV. Mechanical inoculation of test plants showed that the virus host range was limited to the cucurbits. Mottling, vein banding and blistering of leaves, and distortion of fruit were the main symptoms observed on field and inoculated hosts. Flexuous virus particles, 720−780 nm long, were observed under the electron microscope from purified virus samples. In ultra-thin leaf sections, infected plant cells contained pinwheel and scroll-type inclusion bodies. An enzyme-linked immunosorbent assay test using antibodies specific to ZYMV following single lesion virus isolation gave a positive reaction. The size of the potyvirus coat protein was shown to be about 35.7 kDa using sodium dodecyl sulphate–polyacrylamide gel electrophoresis (SDS-PAGE). Nucleotide sequence analysis of the partial coat protein (CP) gene of the ZYMV isolate from KZN revealed 95−98% sequence identity with isolates occurring in central Europe and the Indian subcontinent and 90−93% identity with isolates from Singapore and Taiwan. These high levels of sequence identity indicate that the KZN isolate is a variant of ZYMV. We propose the name ZYMV-KZN for the virus strain we have identified. Keywords: cucurbits, phylogenetic analysis, Zucchini yellow mosaic virus

Introduction The Cucurbitaceae family comprises many economically important crops that are grown both commercially and on a small scale by farmers in the Republic of South Africa (RSA) and on a worldwide basis. Zucchini yellow mosaic virus (ZYMV) has emerged as one of the most economically important viruses infecting cucurbits in many of the important cucurbit-growing regions of the world and is considered to be an example of an emerging plant virus (Desbiez and Lecoq 1997). The virus was first identified on squash in northern Italy (Lisa and Dellavalle 1981) and has spread to major cucurbit-producing regions around the world (Guner 2004, Harris et al. 2009) including the USA (Purcifull et al. 1984, Providenti and Gonsalves 1984, Nameth et al. 1985, Crosslin 1988, Stobbs and van Schagen 1990, Gracia and de Cuyo 2000), Asia (Lesemann et al. 1983, Al-Musa 1989, Wong and Lee 1992), and Europe (Lisa et al. 1981, Wright et al. 1984, Walkey et al. 1992). In the last few years, cucurbit plants exhibiting leaf mosaic and fruit deformation symptoms have become a common sight on farms in RSA. ZYMV was first detected in RSA in the early 1990s (von Wechmar et al. 1995). The virus, which resulted from a seed-borne infection of sweet melon (Citrullus lanatus), was found in a mixed infection causing severe disease symptoms (von Wechmar et al. 1995). ZYMV has been detected previously in commercial farms

and communal gardens in the province of KwaZulu-Natal (KZN), where it was identified as the most common virus infecting cucurbits (Cradock 1998, Cradock et al. 2001). The other viruses found to infect cucurbits in the province were Watermelon mosaic virus-M (WMV-M), Watermelon mosaic virus-2 (WMV-2) and Cucumber mosaic virus (CMV) (Cradock et al. 2001). ZYMV is a member of the genus Potyvirus in the Potyviridae family (Hollings and Brunt 1981, Murphy et al. 1995). Like other potyviruses, ZYMV has a monopartitepositive strand RNA genome (≈9 600 nucleotides) encapsidated in flexuous filamentous particles 750 nm in length (Lisa et al. 1981). Infected plants produce a range of symptoms while individual cells contain cylindrical inclusions (pinwheels) as described by Edwardson and Christie (1978). As yet, none of the identified viruses infecting cucurbits in KZN have been characterised (von Wechmar et al. 1995, Cradock et al. 2001). The aim of this study therefore was to isolate, identify and partially characterise, at the molecular level, the most important virus infecting cucurbits in KZN. In this paper, we present evidence, based on host-range studies, serology, electron microscopy and sequence data of the coat protein gene, that the most important virus infecting cucurbits in the province of KZN in RSA is ZYMV. It is intended that the information generated in this study will form

South African Journal of Plant & Soil is co-published by Taylor & Francis and NISC (Pty) Ltd

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the basis of sustainable management strategies against the virus and the diseases it causes on cucurbits in RSA. Materials and methods Sample collection and ELISA A total of 52 leaf samples showing typical virus-like symptoms were collected from cucurbit plants growing on farms and gardens in KZN province, RSA, and stored at −80 °C until analysed for virus presence. After three single-lesion transfers on Chenopodium quinoa Wild., virus samples were maintained on Zucchini squash (Cucurbita pepo) plants. These source plants provided the same virus isolate that was used in further assays. Virus presence was determined using double-antibody sandwich (DAS) ELISA as described by Clark and Adams (1977). Bio-Rad Phyto-Diagnostics commercial ELISA kits were used to test for the presence of ZYMV, WMV-M, WMV-2, CMV, Papaya ringspot virus-W (PRSV-W) and Squash mosaic virus (SqMV). Samples were ground in a small volume of extraction buffer (PBS-Tween + 2% polyvinyl pyrrolidone-40) and incubated overnight at 4 °C. Antibodies were diluted and incubated according to the ELISA kit manufacturer’s instructions. A p-nitrophenyl phosphate (pNPP) substrate solution (1 mg ml−1) was added and plates were measured at A405 in an Anthos 2001 photometer (Anthos Labtech Instruments, Austria). Samples were considered positive when A 405 values were three-fold higher than the negative controls. Host range studies Plants used in the host range studies were grown in pots and maintained at 18–25 °C in an insect-proof greenhouse. Host plants were first tested by ELISA for the presence of ZYMV and latent infections. The hosts that were inoculated included: Cucurbita pepo L. (squash and pumpkin), Cucumis sativus L. (cucumber), Citrullus lanatus (Thunb.) Mansf. (watermelon), Phaseolus vulgaris L., Nicotiana benthamiana Domin., N. tabacum L., N. rustica L., N. occidentalis Wheeler and Solanum lycopersicum L. The cotyledons of plants with fully expanded first true leaves (five for each species) were dusted with carborundum powder and then mechanically inoculated using crude extracts of ZYMV ELISA-positive leaf material in 0.01 M phosphate buffer (pH 7.2). Plants were observed for symptoms over a five-week period. Virus purification ZYMV was purified using the methods described by Prieto et al. (2001) and Berger and Shiel (1998). Infected fresh C. pepo leaf material from three- to four-week-old plants showing typical ZYMV symptoms (vein banding and mosaic) was used as the virus source. Electron microscopy Formvar-coated 300-mesh copper grids were placed for 2 min on a drop of purified virus preparation placed on parafilm. After rinsing with dH2O, the grids were negatively stained with 2% uranyl acetate (pH 7.2). For thin sectioning, symptomatic C. pepo leaves were fixed in 3% glutaraldehyde + 2% osmium tetroxide, dehydrated in a graded ethanol series and embedded in Epon 812 (Narayanasamy 2001). Ultra-thin sections cut with an ultramicrotome were

Usher, Sivparsad and Gubba

placed on Formvar-coated 300-mesh copper grids and stained with 2% uranyl acetate (pH 7.2). Both preparations were viewed using a Phillips CM120 BioTWIN electron microscope and images were captured on a digital MegaViewBIII camera. Sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) SDS-PAGE was carried out as described previously with modifications (Laemmli 1970, Ausubel et al. 1997). The running gel (12.5%) comprised 3.13 ml 30% acrylamide solution, 2.38 ml dH2O, 1.88 ml of 1.5 M Tris-HCl (pH 8.8), 0.075 ml 10% (w/v) SDS, 0.038 ml of 10% (w/v) ammonium persulfate and 0.0038 ml TEMED. The stacking gel (4.0%) consisted of 0.47 ml of 30% acrylamide solution, 2.15 ml dH 2O, 0.88 ml of 0.5 M Tris-HCl (pH 6.8), 0.035 ml of 10% (w/v) SDS, 0.018 ml of 10% (w/v) ammonium persulfate and 0.008 ml TEMED. Purified Tobacco mosaic virus (TMV) was included as a positive control. Electrophoresis was performed at 100 V for 90 min. The gels were stained using 1% and 1.25% Coomassie blue and destained using methanol and acetic acid. Isolation of viral RNA, RT-PCR amplification, cloning and sequencing Total plant RNA was isolated from symptomatic C. pepo plants using the SV Total RNA isolation kit (Promega, USA). Plants had been inoculated previously with purified ZYMV. RNA preparations were then subjected to single tube RT-PCR using the RobusT I kit (Finnzymes, Finland). Specific primers, designed from all available ZYMV coat protein (CP) gene sequences in the GenBank database were used to amplify a genomic region corresponding to approximately 850 bp of the ZYMV partial CP and 3′ nontranslated region (NTR). The primers used were: ZYMV–forward (ZYMVfor; 5′-CTCATGGGAAAATTGTGCCGCGTC-3′) and ZYMV–reverse (ZYMVrev; 5′-CTTGCAAACGGAGTCTAAT CTCGAGC-3′). The resultant RT-PCR product was then cloned using a TA Cloning ® Kit (Invitrogen TM Life Technologies, USA) with the PCR cloning vector pCR®2.1 following the manufacturer’s specifications (Invitrogen™ Life Technologies, USA). The vector with the ligated insert was used to transform TOP10F′ cells according to the manufacturer’s specifications. Plasmid DNA from positive recombinant clones was purified using a QIAprep® Spin Miniprep kit (Qiagen, USA). Sequencing of plasmid DNA from 10 clones was done using a Genetic Analyser 3100 machine using standard M13F/M13R primers. Analysis of the amplified sequence A consensus sequence obtained from the 10 representative clone sequences was used in the phylogenetic study. The nucleotide sequence reported in this paper has been deposited into GenBank as accession number DQ978272. The BLAST program was used to obtain related ZYMV CP gene sequences from GenBank (Altschul et al. 1990, Benson et al. 1996). Sequences were manually verified and edited to ensure optimal alignment using BioEdit Sequence Alignment Editor version 6.0 (Hall 1999). Phylogenetic analysis using the neighbour-joining method was done using the MEGA5 software package (Tamura et al. 2011).

South African Journal of Plant and Soil 2012, iFirst, xx–xx

A bootstrap test was conducted using 1 000 replicates and evolutionary distances were computed using the Jukes-Cantor method. Actual percentage sequence identities were generated through pairwise comparisons using CLC Main Workbench 6.6.2 (CLC Bio, Aarhus, Denmark). Results Sample collection and ELISA ZYMV was detected in 55% of the samples collected. Mixed infections of ZYMV and WMV-2 were detected in 12% of samples. CMV and WMV-M were also detected as single infections (15% and 5%, respectively) and at times as mixed infections (6%). All samples were negative for SqMV and PRSV-W. Propagation and host range studies The host range of ZYMV was found to be restricted to the cucurbit family (Table 1). Vein banding and shoe-stringing was the most common symptom observed on zucchini squash (Figure 1a and b). A yellow mottle was observed on pumpkin, cucumber and watermelon plants (Figure 1c). Gem squash plants showed chlorosis (Figure 1d). Symptoms generally started appearing four weeks post-inoculation. Hybrid squash and zucchini squash fruits showed a yellow mottle and distortion (Figure 2). Virus purification and characterisation Zucchini squash leaves mechanically inoculated with the ZYMV virus isolate under study were chosen for purification after being confirmed to be positive for ZYMV using ELISA. Electron microscopic examination of ZYMV-infected plant tissue and purified ZYMV preparations demonstrated the presence of elongated flexuous virus particles, 720–780 nm in length (Figure 3a). In ultra-thin sections of cells from infected plants, pinwheel and scroll type inclusions, typical of potyvirus infections, were readily detected in preparations stained with uranyl acetate (Figure 3b). Purified ZYMV virion proteins, when denatured with SDS and analysed by PAGE, yielded two bands with the major band being approximately 35.7 kDa in size. Purified TMV proteins were used as a standard and yielded a band of approximately 17 kDa (Figure 4). The identity of the virus was also confirmed by partially characterising its CP gene. The amplified cDNA fragment of the partial CP gene and 3′ NTR, 850 bp in length, was cloned and sequenced. BLAST analysis of the fragment confirmed the identity of the isolated virus as ZYMV based on high sequence homologies to different ZYMV sequences. Phylogenetic analysis resulted in the construction of a rooted cluster phylogram (Figure 5). The resultant tree showed that the KZN isolate of ZYMV (ZYMV-KZN) is part of a group that is composed of the isolates from Florida, Connecticut, Italy, Hungary, Austria, Slovenia and Pakistan. Within this group, the isolates from Florida, Connecticut and Italy formed a specific cluster and had 96–98% identity within themselves and were 95–96% similar to ZYMV-KZN. Isolates from Hungary, Austria, Slovenia, Pakistan and ZYMV-KZN separated into another branch of the cluster. The isolates from Hungary, Austria, Slovenia and Pakistan isolates were 98–99% identical to each other and were c. 97% similar to ZYMV-KZN. Isolates

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Table 1: Reaction of different plant species when inoculated with sap extracted from gem squash infected with ZYMV Family, species and cultivar Cucurbitaceae Cucurbita pepo Gem squash ‘Rolet’ Hybrid squash ‘Sensor’ Zucchini squash ‘Partenon’ Cucumis sativus ‘Ashely’ Citrillus lanatus ‘Charleston Grey’ Curcubita maxima Leguminosae Phaseolus vulgaris Bean Dwarf ‘Espanda’ Solanaceae Nicotiana benthamiana N. tabacum N. rustica N. occidentalis

Symptomsa

CL, M, BL of young leaves M, VB M, VB, SS M YM, St M 0

0 0 0 0

Explanation of symbols: CL = chlorosis, M = mottling, BL = blistering, VB = vein banding, SS = shoestringing, YM = yellow mottle, St = stunting, 0 = no symptoms a

from Taiwan and Singapore were 92–93% and 90% similar to ZYMV-KZN, respectively. These isolates separated into a different cluster, whereas the isolate from China appeared as an outlier (Figure 5). Discussion Based on results from this study, a virus infecting cucurbits in KZN has been identified and characterised. Previous studies have based the identification of viruses infecting cucurbits in KZN solely on ELISA reactions (von Wechmar et al. 1995, Cradock et al. 2001). This is the first study to fully characterize a ZYMV-infecting cucurbit virus in KZN through molecular sequencing. This virus has been shown to be a typical potyvirus. Flexuous virus particles 720–780 nm in length, typical of potyviruses, were observed under an electron microscope. Pinwheel and scroll type inclusion bodies, which are induced in plant cells following infection by potyviruses, were also observed. SDS-PAGE analysis showed the migration of two bands. The major band corresponds to the CP of the virus under study and was about 35.7 kDa in size, which is close to the expected size of 36 kDa for most potyviruses (Büchen-Osmond and Purcifull 1996). The occurrence of the second band has been reported previously (Hill and Benner 1990) and is thought to be the result of partial degradation of the polypeptide by host or microbial proteolytic enzymes (Shukla et al. 1994). Occurrence of the second band may also result from cleavage from two different sites in the polypeptide at the junction between the nuclear inclusionb protein and the coat protein (Niblett et al. 1991, Yeh et al. 1992). The positive reaction of the virus to antibodies specific to ZYMV in ELISA confirmed the identity of the potyvirus to be ZYMV. We propose the name ZYMV-KZN for this virus isolate. ZYMV is an important virus of cucurbits in KZN, where it causes severe disease and economic losses (Cradock

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(a)

(b)

(c)

(d)

Figure 1: A range of symptoms seen on mechanically inoculated cucurbit plants 2 weeks after inoculation with crude extracts of Zucchini yellow mosaic virus ZYMV. Leaves showed (a) vein banding, (b) shoe-stringing, (c) mottling and (d) chlorosis. Inoculated plants were maintained in a greenhouse

(a)

(b)

Figure 2: Fruit deformation because of Zucchini yellow mosaic virus (ZYMV) infection on zucchini squash plants under greenhouse conditions. Symptoms were recorded 10–12 weeks after inoculation of plants with crude ZYMV extracts


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(a)

(b)

200 nm

200 nm

Figure 3: Electron micrographs of Zucchini yellow mosaic virus (ZYMV) particles. (a) Filamentous particles of ZYMV from a purified preparation and negatively stained with uranyl acetate, (b) ultra-thin sections of ZYMV-infected gem squash leaf tissue showing pinwheel (PW) and scroll (SC) type inclusion bodies

1

2

3

kDa

61.3 49.0 36.4

24.7 19.2

13.1

9.3

Figure 4: Sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) protein pattern of the KwaZulu-Natal isolate of Zucchini yellow mosaic virus ZYMV (lane 3). Lane 1, wide-range molecular mass standard (172.6, 111.4, 79.6, 61.3, 49.0, 36.4, 24.7, 19.2, 13.1 and 9.3 kDa; lane 2, Tobacco mosaic virus control coat protein

1998). The results of our host range studies for ZYMV-KZN were consistent with reactions previously reported for other ZYMV isolates (Lisa et al. 1981, Davis 1986, Gal-On 2007). Some isolates have been reported to produce systemic infection of P. vulgaris and N. benthamiana (Desbiez and Lecoq 1997) but the KZN isolate did not. This suggests a difference in the infectivity and virulence of ZYMV-KZN with other ZYMV isolates. Results from this study show that the ZYMV-KZN isolate has a 95–98% sequence identity with isolates occurring in central Europe and the Indian subcontinent. These high levels of sequence identity (>95%) indicate that the KZN isolate is a variant of ZYMV. The isolates from North America (Florida and Connecticut) form a different cluster. We speculate that the seed that was the source of the original infection in RSA might have originated from these parts of the world, specifically from the Indian subcontinent. Given the active trade between the Indian subcontinent and RSA, this is a plausible explanation of the origin of the ZYMV-KZN isolate. Previous studies have shown that there are other viruses besides ZYMV that infect cucurbits in KZN (Cradock 1998). Any strategy to control virus diseases in cucurbits must therefore take the multiple infections under field conditions into consideration if the control is to be effective and sustainable. Tolerant and/or resistant cultivars have been commonly used to control/manage virus diseases. Often, the tolerant/resistant cultivars are not available. In cases

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HUNGARY

70

0.01

89

AUSTRIA

99

SLOVENIA

99

PAKISTAN ZYMV–KZN

79

FLORIDA CONNETICUT

72 99

94

ITALY TW–TN1

27

TW–TNML1 TW–TC1

100 44 59

TW–PT5 TW–CY2

SING–1 100

SING–2 CHINA

Figure 5: Phylogram based on coat protein gene sequences of Zucchini yellow mosaic virus (ZYMV) isolates. The ZYMV isolate from KwaZulu-Natal (KZN) sequenced in this study was compared with ZYMV isolates reported from other parts of the world: TW-TNML1 (GenBank accession no. AF127932), TW-TC1 (AF127931), TW-PT5 (AF127934), TW-CY2 (AF127930), TW-TN1 (AF127933), Florida (D13914), Connecticut (D00692), Italy (AJ420020), Hungary (AJ251527), Austria (AJ420017), Slovenia (AJ420018), Pakistan (AB127936), ZYMV-KZN (South Africa) (DQ978272), Singapore-1 (AF014811), Singapore-2 (X62662), China (AF513552)

where they are available, they are overcome by new virus isolates after a few years. All this poses serious challenges to the farmer, the breeder and virologist. Recently, the use of transgenic plants to control virus diseases of plants has gained currency (Beachy 1997, Gonsalves 1998, Ferreira et al. 2002). To this end, transgenic cucurbits with resistance to multiple virus infections under field conditions have been developed and have performed well under field conditions (Fuchs and Gonsalves 1995, Fuchs et al. 1997, Tricoli et al. 1995, Yu et al. 2011). The information generated in this study can be a first step in a programme to develop transgenic cucurbits with resistance to multiple virus infections for KZN and South African farmers. This is of particular importance in terms of genetically variable virus strains being able to overcome strain-specific resistance in an introduced transgenic cultivar, hence nullifying considerable time consuming and costly efforts in resistance development. The data of low genetic variability provided by this study will be pertinent to the design of a cucurbit cultivar with transgenic virus resistance that will be effective in all locations in KZN and possibly in South Africa. Our efforts in this direction have begun.

Acknowledgements — We thank Celeste Hunter for all her assistance in the laboratory and Vijay Bandu and the rest of the staff at the Centre for Electron Microscopy, University of KwaZuluNatal, for all their assistance and contributions throughout the year. LU and BS were supported by scholarships from the National Research Foundation.

References Al-Musa AM. 1989. Severe mosaic caused by Zucchini yellow mosaic virus in cucurbits from Jordan. Plant Pathology 38: 541–546. Altschul SF, Gish W, Miller W, Myers EW, Lipman DJ. 1990. Basic local alignment search tool. Journal of Molecular Biology 215: 403–410. Ausubel F, Brent R, Kingston RE, Moore DD, Seidman JG, Smith JA, Struhl K (eds). 1997. Short protocols in molecular biology: a compendium of methods from current protocols in molecular biology (3rd edn). New York: Wiley. Beachy RN. 1997. Mechanisms and applications of pathogenderived resistance in transgenic plants. Current Opinion in Biotechnology 8: 215–220. Benson DA, Boguski M, Lipman DJ, Ostell J. 1996. GenBank. Nucleic Acids Research 24: 1–5.


Berger PH, Shiel PJ. 1998. Potyvirus isolation and RNA extraction. In: Foster GD, Taylor SC (eds), Plant virology protocols: from virus isolation to transgenic resistance. Methods in Molecular Biology vol. 81. New Jersey: Humana Press. pp 151–159. Büchen-Osmond C, Purcifull D. 1996. Zucchini yellow mosaic virus potyvirus. In: Brunt A, Crabtree K, Dallwitz M, Gibbs A, Watson L (eds), Viruses of plants: descriptions and lists from the VIDE Database. Wallingford: CAB International. pp 1414–1417. Clark MF, Adams AN. 1977. Characteristics of the microplate method of enzyme-linked immunosorbent assay for the determination of plant viruses. Journal of General Virology 34: 475–483. Cradock KR. 1998. Control of insect-transmitted viruses in cucurbit crops in KwaZulu-Natal. MSc thesis, University of Natal, Pietermaritzburg, South Africa. Cradock KR, da Graca, JV, Laing M. 2001. Viruses infecting cucurbits in KwaZulu-Natal, South Africa. Revista Mexicana de Fitopatologia 19: 251–252. Crosslin JM. 1988. First report of Zucchini yellow mosaic virus in Cucurbita pepo in the Pacific Northwest [Abstract]. Plant Disease 73: 362. Davis RF. 1986. Partial characterization of Zucchini yellow mosaic virus isolated from squash in Turkey. Plant Disease 70: 735–738. Desbiez C, Lecoq H. 1997. Zucchini yellow mosaic virus. Plant Pathology 46: 809–829. Edwardson JR, Christie RG. 1978. Use of virus induced inclusions in classification and diagnosis. Annual Review of Phytopathology 16: 31–55. Ferreira SA, Pitz KY, Manshardt R, Zee F, Fitch M, Gonsalves D. 2002. Virus coat protein transgenic papaya provides practical control of Papaya ringspot virus in Hawaii. Plant Disease 86: 101–105. Fuchs M, Gonsalves D. 1995. Resistance of transgenic hybrid squash ZW-20 expressing the coat protein gene of Zucchini yellow mosaic virus and Watermelon mosaic virus 2 to mixed infections by both potyviruses. Bio/Technology 13: 1466–1473. Fuchs M, McFerson JR, Tricoli DM, McMaster JR, Deng RZ, Boeshore ML, Reynolds JF, Russell PF, Quemada HD, Gonsalves D. 1997. Cantaloupe line CZW-30 containing coat protein genes of Cucumber mosaic virus, Zucchini yellow mosaic virus, and Watermelon mosaic virus 2 is resistant to these three viruses in the field. Molecular Breeding 3: 279–290. Gal-On A. 2007. Zucchini yellow mosaic virus: insect transmission and pathogenicity – the tail of two proteins. Molecular Plant Pathology 8: 139–150. Gonsalves D. 1998. Control of Papaya ringspot virus in papaya: a case study. Annual Review of Phytopathology 36: 415–437. Gracia O, de Cuyo L. 2000. First report of Zucchini yellow mosaic virus in Argentina [Abstract]. Plant Disease 84: 371. Guner N. 2004. Papaya ringspot virus watermelon strain and Zucchini yellow mosaic virus resistance in watermelon. PhD thesis, North Carolina State University, Raleigh, USA. Hall TA. 1999. BioEdit: a user-friendly biological sequence alignment editor and analysis program for Windows 95/98/NT. Nucleic Acids Symposium Series 41: 95–98. Harris KR, Ling K-S, Wechter P, Levi A. 2009. Identification and utility of markers linked to Zucchini yellow mosaic virus resistance gene in watermelon. Journal of the American Society for Horticultural Science 135: 529–534. Hill JH, Benner HI. 1990. Properties of Soybean mosaic virus and its isolated protein. Phytopathologishe Zietschrift 97: 272–281. Hollings M, Brunt AA. 1981. Potyvirus group. CMI/AAB Descriptions of Plant Viruses no. 245. Kew: Commonwealth Mycological Institute. Laemmli UK. 1970. Cleavage of structural proteins during the assembly of the head of bacterial phage T4. Nature 227: 680–685. Lesemann DE, Makkouk KM, Koenig R, Samman EN. 1983.

7

Natural infection of cucumbers by Zucchini yellow mosaic virus in Lebanon. Phytopathologishe Zietschrift 108: 304–313. Lisa V, Boccardo G, D’Agostino G, Dellavalle D, D’Aquilo M. 1981. Characterization of a potyvirus that causes Zucchini yellow mosaic virus. Plant Disease 71: 667–672. Lisa V, Dellavalle G. 1981. Characterization of two potyviruses from zucchini squash. Phytopathology 100: 279–286. Murphy FA, Fauquet CM, Bishop DHL, Ghabrial SA, Jarvis AW, Martelli GP, Mayo MA, Summers MD. 1995. Virus taxonomy: sixth report of the International Committee on Taxonomy of Viruses. Archives of Virology 10: 350–354. Nameth ST, Dodds IA, Paulus AO, Kishaba A. 1985. Zucchini yellow mosaic virus associated with severe diseases of melon and watermelon in southern California desert valleys. Plant Disease 69: 785–788. Narayanasamy P. 2001. Plant pathogen detection and disease diagnosis (2nd edn). Cambridge: Marcel Decker. Niblett CL, Zagula KR, Calvert LA, Kendall TL, Stark DM, Smith CE, Beachy RN, Lommel SA. 1991. cDNA cloning and nucleotide sequence of wheat streak mosaic virus capsid protein gene. Journal of General Virology 72: 499–504. Prieto H, Bruna A, Hinrichsen P, Muñoz C. 2001. Isolation and molecular characterization of a Chilean isolate of Zucchini yellow mosaic virus. Plant Disease 85: 644–648. Providenti R, Gonsalves D. 1984. Occurrence of Zucchini yellow mosaic virus in cucurbits from Connecticut, New York, Florida and California. Plant Disease 68: 443–446. Purcifull DE, Adlerz WC, Simone GW, Herbert E, Christie SR. 1984. Serological relationships and partial characterization of Zucchini yellow mosaic virus isolated from squash in Florida. Plant Disease 68: 230–233. Shukla DD, Ward CW, Brunt AA. 1994. The Potyviridae. Cambridge: Cambridge University Press. Stobbs LW, van Schagen JG. 1990. First report of ZYMV in Ontario [Abstract]. Plant Disease 74: 394. Tamura K, Dudley J, Nei M, Kumar S. 2011. MEGA5: Molecular Evolutionary Genetics Analysis using maximum likelihood, evolutionary distance, and maximum parsimony methods. Molecular Biology and Evolution 28: 2731–2739. Tricoli DM, Carney KJ, Russell PF, McCaster JR, Groff DW, Hadden KC, Himmel PT, Hubbard JP, Boeshore ML, Quemada HD. 1995. Field evaluation of transgenic squash containing single or multiple coat protein gene constructs for resistance to Cucumber mosaic virus, Watermelon mosaic virus 2, and Zucchini yellow mosaic virus. Bio/Technology 13: 1458–1465. von Wechmar MB, Jaffer MA, Purves M. 1995. Zucchini yellow mosaic virus (first report) and other cucurbit infecting viruses in South Africa: 1989–1993. South African Journal of Science 91: 11–12. Walkey DGA, Lecoq H, Collier R, Dobson S. 1992. Studies on the control of Zucchini yellow mosaic virus in courgettes by mild strain protection. Plant Pathology 41: 762–771. Wong SM, Lee SC. 1992. First report of Zucchini yellow mosaic virus in Singapore. [Abstract]. Plant Disease 76: 972. Wright DM, Le Cuirot C, Hill SA. 1984. Identification of Zucchini yellow mosaic virus from courgettes in Jersey by immune electron microscopy. Plant Pathology 33: 591–594. Yeh SD, Jan FJ, Chian CH, Doong TJ, Chen MC, Chung PH, Bau HJ. 1992. Complete nucleotide sequence and genetic organisation of Papaya ringspot virus RNA. Journal of General Virology 73: 2531–2541. Yu T-A, Chiang C-H, Wu H-W, Li C-M, Yang C-F, Chen J-H, Chen Y-W, Yeh S-D. 2011. Generation of transgenic watermelon to Zucchini yellow mosaic virus and Papaya ringspot virus type W. Plant Cell Reports 30: 359–371.

Received 30 May 2012, accepted 6 June 2012