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Journal of Plant Pathology (2011), 93 (3), 569-576 Edizioni ETS Pisa, 2011 569

EGYPTIAN ISOLATES OF RINGSPOT FORM A MOLECULARLY DISTINCT CLADE

A.F. Omar, S.A. El-Kewey, S.A. Sidaros and A.K. Shimaa

Department of Plant Pathology, Faculty of Agriculture, Kafrelsheikh University, 33516 Kafrelsheikh, Egypt

SUMMARY , family , have a worldwide distribu- tion and are responsible for significant yield losses to A survey to determine the incidence in open field cucurbit crops (Quiot-Douine et al., 1990). squash ( pepo) of (PRSV) are single-stranded RNA , and represent one of in Kafrelsheikh governorate regions (Balteem, Shabah, the economically most important and largest genus of Kafrelsheikh and Sheno) was carried out in 2009 and plant viruses (Shukla et al., 1994). 2010. The most commonly observed symptoms were PRSV is an important pathogen of papaya and cucur- mosaic, malformation such as blisters and narrow leaf bits (Riechmann et al., 1992). Two main PRSV strains blades and malformed fruits. The identification of have been described (Quiot-Douine et al., 1990), that PRSV by ELISA showed that the virus was highly dis- can be distinguished by their host range. PRSV-W, for- tributed in squash fields with an incidence higher than merly mosaic virus 1, which naturally in- 50%. Egyptian isolates of PRSV were easily transmitted fects cucurbit crops but is unable to infect papaya (Cari- by mechanical inoculation and by . Flex- ca papaya) (Bateson et al., 1994), and PRSV-P, which in- uous particles were observed in leaf dip preparations fects papaya naturally and cucurbits experimentally. and pinwheels and scrolls were seen in thin sections of PRSV particles are flexuous filaments (760-800x12 nm) diseased squash tissue. The RT-PCR amplified partial made of monomers of a single polypeptide species of coat protein (CP) gene was sequenced from eight about 36 kDa encapsidating a single-stranded positive- Egyptian PRSV isolates. Sequence comparisons and sense RNA molecule 10,300 nucleotide in size (Yeh et phylogenetic analysis revealed that the Egyptian isolates al., 1992; Wang and Yeh, 1997). PRSV induces variable grouped together in a distinct clade. Comparison with symptoms in papaya and cucurbit cultivars, including PRSV sequences retrieved from GenBank presented nu- vein clearing, mottling, malformed leaves, filimorphism, cleotide identities in the range of 87.5-97.1% and close ringspots and streaks on fruits, stem and petioles, and relationships of the Egyptian isolates with the two stunting (Purcifull et al., 1984). PRSV is naturally trans- Venezuelan isolates of the so-called type-P, Sucre-ElMu- mitted by several species of in a non-persistent co and Merida6, and to the Mexican and USA isolates. manner. The virus can also be transmitted mechanically, This is the first report on the incidence and characteri- and is typically not seed-transmitted (Tripathi et al., zation of PRSV in Egypt. 2008). In Egypt, squash is infected by several viruses in- cluding Squash leaf curl virus (SqLCV) (Abdel-salam et Key words: survey, potyvirus, squash, ELISA, RT- al., 2006), ZYMV (Desbiez and Lecoq, 1997; Shehata et PCR. al., 2008), (CMV) (Fattouh, 2003) and WMV (Salem et al., 2007). To the best of our knowledge, there is no prior report about PRSV infect- INTRODUCTION ing squash or other crops in Egypt, so this work focuses on identification and the first molecular characteriza- Cucurbits are very important vegetable crops in tion of the coat protein (CP) gene of Egyptian PRSV Egypt, their estimated production in 2008 being isolates and sequence comparison with other reported 3,491,207 tons (FAO, 2010). Zucchini yellow mosaic PRSV sequences. virus (ZYMV), Papaya ringspot virus (PRSV) and Water- melon mosaic virus (WMV), all belonging to the genus MATERIALS AND METHODS

Surveys, virus detection and virus isolates. The sur- Corresponding author: A.F. Omar Fax: +2.047.9102930 vey screened only for PRSV because the most prevalent E-mail: [email protected] symptoms observed on squash fruits in open-field pro- 003_JPP599RP(Omar)_569 15-11-2011 17:39 Pagina 570

570 Papaya ringspot virus in Egypt Journal of Plant Pathology (2011), 93 (3), 569-576

duction areas were ringspots, similar to the reported as an abrasive. The extract was rubbed onto squash symptoms induced by PRSV on cucurbit and papaya cotyledons and true leaves. Ten squash plants were in- fruits. The occurrence of ringspot symptoms, distinct oculated for each isolate. Test and control plants from those typically induced on squash in Egypt by (rubbed with buffer only) were kept in a growth cham- WMV, ZYMV, or CMV led us to examine the causal ber at 28-30ºC. Test plants were observed for symptom virus. After mechanical transmission to squash under development. greenhouse conditions, the virus was first identified as a potyvirus using the potyvirus group-specific ELISA kit transmission. Aphid transmission was carried (Agdia, USA). Subsequently we utilized a potyvirus out according to Pinto et al. (2008) by using a colony group-specific primer pair (Van der Vlugt et al., 1999) Myzus persicae. Aphids were collected from zucchini to amplify a portion of the 3’ terminal region of the squash plants and, after identification by the Entomolo- genome. Cloning and sequencing of the ca. 650 bp PCR gy Department, Faculty of Agriculture, Kafrelsheikh product resulted in the identification of the virus as University, were reared on sweet pepper under an PRSV. We therefore carried out a survey to determine proof cage to establish virus-free colonies. For transmis- the distribution and variability of PRSV in the primary sion trials, groups of apterous aphids were starved in squash production areas of Kafrelsheikh governorate. plastic boxes for 30 min, then placed on infected plants The virus isolates characterized in this work were ob- for an acquisition access period of 60 sec. Groups of 5 tained from naturally infected squash () aphids were transferred to test plants for an inoculation plants with symptoms of vein clearing, vein banding and access period of 1 h. Zucchini squash plants were inocu- mosaic on the leaves. The fruits were malformed and lated at the cotyledonary stage, and then sprayed with showed the already mentioned ringspots. Viral isolates insecticide. Virus infections were determined by symp- were collected in 2009 and 2010 from 20 fields random- tom appearance and ELISA. ly distributed in a geographical area of roughly 80 km2 of different regions of Kafrelsheikh governorate (Bal- Electron microscopy. A partially purified suspension teem, Shabah, Kafrelsheikh and Sheno), where squash is of isolate Sheno 1 was prepared according to Black et grown in spring and summer. Incidence of virus symp- al. (1963). A 20 µl aliquot of the preparation was pipet- toms was visually evaluated in each field by examining ted onto a formvar-coated copper grid, the excess liquid 500 plants following a W-shaped itinerary, and ex- was removed by blotting with filter paper and the grid pressed as the percentage of the total plants (Kassem et al., 2007). Eight hundred samples were collected from squash plants with ringspots on the fruits for PRSV de- tection and from plants with different symptoms such as mosaic, yellowing and symptomless fruits that could be infected by other viruses. Each field was visited and sampled twice, before and at the beginning of harvest- ing, collecting 10 to 15 leaf samples per field, which were then assayed for determining their sanitary status. PRSV was detected by ELISA using a commercial kit (Loewe Biochemica GmbH, Germany) according to the manufacturer’s instructions. Optical densities (OD) were recorded at 405 nm with a microplate reader (Stat Fax 4200, USA) and samples showing OD values higher than twice the average of the negative control were con- sidered positive (Mnari-Hattab et al., 2009).

Virus isolation and mechanical transmission. The eight Egyptian PRSV isolates characterized in this study (Sheno 1, Sheno 2, Balteem 1, Balteem 2, Balteem 3, Shabah, Kafr 1, and Kafr 2) were singled out by succes- sive local lesion transfers to Chenopodium amaranticolor (Fig. 1) (Yeh et al., 1984). A single local lesion was used to inoculate squash as propagative host plant. The local lesion-purified isolates were used for all further charac- terization studies. Young symptomatic squash leaves were ground in 0.2 M potassium phosphate buffer pH Fig. 1. Necrotic local lesions induced in Chenopodium ama- 7, containing 0.02 M sodium sulfite and carborundum ranticolor by Egyptian PRSV isolates. 003_JPP599RP(Omar)_569 15-11-2011 17:39 Pagina 571

Journal of Plant Pathology (2011), 93 (3), 569-576 Omar et al. 571

Fig. 2. Symptoms in squash plants infected by PRSV. In the field, leaf distortions, narrow leaf blades and blisters (A), malformed and ringspots shape on fruits (B). In the greenhouse, mosaic and blisters (C), malformation of the leaves (D).

was negatively stained with 2% sodium phospho- One-step PCR. One-step RT-PCR Kit (Qiagen, USA) tungstate (PTA), pH 7.0. Particles were visualized using was used for RT-PCR in a 50 µl sample containing 1 µl a JEOL 1200 EX transmission electron microscopy. RNA, 20 µl RNase-free water, 10 µl 5X Qiagen OneStep

Leaf tissue pieces of squash plants inoculated with RT-PCR buffer (containing 12.5 mM MgCl2), 10 µl 5X PRSV-Sheno 1 were processed for thin sectioning ac- Q-Solution, 2 µl dNTP mix (10 mM each), 2 µl Qiagen cording to standard preparative techniques (fixation in OneStepRT-PCR Enzyme Mix and 2.5 µl of the forward glutaraldehyde, postfixation in osmium tetroxide, and primer Mo926 (5`- TCTAAAAATGAAGCTGTGGA-3`, embedding in Epon-araldite epoxy resin). Thin sections 10 pmol), and the reverse primer Mo1008 (5`- GTG- were stained with uranyl acetate and lead citrate prior CATGTCTCTGTTGACAT-3`, 10 pmol), corresponding to observation. to nucleotide positions 9258-9277 and 10077-10096 of the PRSV-YK sequence, respectively. These primers can RNA extraction. RNA was extracted from eight sin- be used to detect PRSV-P or PRSV-W type isolates by gle local lesion squash isolates (Sheno 1, Sheno 2, Bal- RT-PCR with no significant differences in the length of teem1, Balteem 2, Balteem 3, Shabah, Kafr 1, and Kafr the amplified N-terminal region of the CP genes (Wang 2) propagated in squash. All the samples used for RNA et al. 1994). The reaction was performed at 50°C for 30 extraction were ELISA positive for PRSV. Fresh squash min, 95°C for 15 min, followed by 40 cycles of 94°C for 1 tissue was used for RNA extraction using RNeasy kit min, 55°C for 1 min, 72°C for 1 min and a final extension (Qiagen, USA) following the manufacturer’s instruc- period of 72°C for 10 min. PCR products were run on a tions. The extracted RNA was used as a template for re- 1.5% agarose gel with 1-Kb plus DNA Ladder (Invitro- verse transcription. gen, USA) as molecular weight marker. 003_JPP599RP(Omar)_569 15-11-2011 17:39 Pagina 572

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Nucleotide sequencing analysis. Specific PCR DNA products of the eight Egyptian isolates amplified from infected squash samples were used directly for sequenc- Infection (%) ing. Nucleotide sequences were determined in both di- rections at INRA, Bordeaux (France) and raw sequence chromatograms were assembled and edited using GAP4 (Bonfield et al., 1995) to remove ambiguous data from Positive plants (No.) the sequence termini. Homologies to known sequences were detected using the BLASTX algorithms against the non-redundant GenBank database (http://www.ncbi. Tested plants (No.) Total nlm.nih.gov/blast). Multiple alignments were performed using ClustalW (Thompson et al., 1994) and the phylo- genetic analyses were conducted with MEGA4 using the maximum parsimony method (Tamura et al., 2007).

Infection (%) The coat protein (CP) gene sequence of Egyptian PRSV isolates, Sheno 1, Sheno 2, Balteem 1, Balteem 2, Baltem 3, Shabah, Kafr 1 and Kafr 2 have been deposit- ed in the EMBL/GenBank/DDBJ databases under ac- Positive plants (No.) 371619 41.10 32.00 31.70 190 90 130 77 29 41 40.53 32.22 31.54 140 70.00 390cession 254 numbers 65.13 FR696589 (805 bp), FR696590 (804 bp), FR696591 (811 bp), FR696592 (789 bp), FR696593 (812 bp), FR696594 (813 bp), FR696595 (816 bp), FR696596 (813 bp). Sequences used for com- parisons were retrieved from GenBank (www.ncbi.nlm. nih.gov). Accession numbers and countries of origin of Tested plants (No.) 43.70% 42.53% the different viral isolates used for comparison are ) l shown in Fig. 5. tota tota ati c/ ati RESULTS AND DISCUSSION tom p ym Inspected plants (s 2010 Surveys and relative importance of PRSV. A total of 800 symptomatic squash leaves were tested for the pres- ence of PRSV by ELISA (Table 1). Samples from symp- tomatic field plants did not always gave positive ELISA responses, irrespective of the regions and years of sam- Infection (%) pling where a visual estimation of possible PRSV infec- tion was carried out. This may depend on the fact that these samples contained viruses (e.g. WMV and/or

Positive plants (No.) ZYMV) which can induce symptoms resembling those caused by PRSV. In any case, of the total samples tested in 2009 and 2010, 47.25% and 53%, respectively, react- ed with the PRSV antiserum, showing that PRSV is Tested plants (No.) 1004070 40190 13 22 40.00 114 32.50 31.40 1500/2000 60.00 600/1500 1120/2500 3125/4000 90 50 60 200 41.65% 63.45% widely distributed and established in the major squash growing area in Kafrelsheikh governorate. Percentage of infection ranged from 31.4% in Kafrelsheikh to 60% in Sheno regions in 2009, and from 31.7% to 70% in 2010. PRSV incidence did not differ significantly from 2009 to 2010 in each region, except for Sheno, where it 2009 Inspected plants (symptomatic/total) went up from 60% to 70%, an increase perhaps due to the growing of squash in the same fields for two consec- utive years, or to increased aphid vector populations or other epidemiological factors. Visual estimation of the Results of Papaya ringspot virus (PRSV) detection by ELISA in squash samples collected from open fields during 2009 and 2010. proportion of field plants showing virus-like symptoms for each area, together with the results of ELISA testing Region of sampling BalteemShabahKafrelsheikh 1720/2500 Sheno 1210/2000 562/1500 2300/3500 All regions 60.97% provided an estimation of PRSV incidence in all regions Table 1. Table 003_JPP599RP(Omar)_569 15-11-2011 17:39 Pagina 573

Journal of Plant Pathology (2011), 93 (3), 569-576 Omar et al. 573

weeks of inoculation (Fig. 2C, D). Those symptoms were very similar to those seen in the field, which gave positive reaction with ELISA. Aphid transmission. Both W and P strains of PRSV are known to be vectored by aphids including Myzus persicae in a non-persistent manner (Purcifull et al., 1984). Likewise M. persicae was able to transmit the 8 Egyptian PRSV isolates from infected squash plants to healthy squash plants in a non-persistent manner with 60 sec acquisition and 1 h inoculation periods. Virus transmission efficiency was 100%. All the plants ex- pressing characteristic symptoms of PRSV were ELISA- positive. Cytopathology. Cylindrical inclusions (pinwheels and scrolls) were observed in the cytoplasm in squash cells infected with the local isolate Sheno 1 (Fig. 3A) and flexuous, filamentous particles were observed in leaf dip preparations (Fig. 3B), in agreement with known traits of members of the family Potyviridae (Purcifull and Ed- wardson, 1967). Fig. 3. A. Three electron micrographs showing cylindrical in- Genetic diversity and phylogenetic analysis of PRSV clusions, i.e. pinwheel (Pw) and scroll (Sc), in the cytoplasm isolates. All the Egyptian PRSV isolates yielded prod- of squash leaf cells infected by PRSV. Bars: 250 nm. B. Fila- ucts of the expected size (820 nt) after one-step RT-PCR mentous virus particles negatively stained with 2% PTA from amplification using Mo926/M1008 primers (Fig. 4). No a dip of a quash leaf infected by PRSV. Bar: 200 nm. amplification was obtained from the reaction mixture when RNA from healthy plants was used. When CP nu- which, in both years, amounted to more than 31% cleotide sequences of virus Egyptian isolates (789 to 816 (Table 1). PRSV is widespread in Hawaii and Florida, nts in size), were analysed and compared with those of Oceania, India, Malaysia, Philipipines, Taiwan, and other PRSV isolates representative of virus diversity and Thailand, West Africa and Europe (Quiot-Douine et al., geographic distribution, it was found the those of 1990; Yeh and Gonsalves, 1994), its incidence on cucur- Egyptian PRSV displayed a very low level of genetic di- bit plants ranging from 5 to 100% in zucchini, 4 to versity (98.2 to 100% nucleotide identity). In particular, 100% in cucumber and pumpkin and 10 to 100% in Kafr 1 was identical to Kafr 2, whereas Balteem 3 had bottlegourd, choyote and watermelon (Kassem et al., identities of 99.7% and 99.6% with Sheno 2, Balteem 1 2007; Dahal et al., 1997). Therefore, PRSV incidence in and Sheno 1, respectively, and higher than 99% with the region under study appears to be high, even when the others. Comparative sequence analyses at the nt lev- compared with other geographical areas where PRSV el of PRSV isolates from different countries and the has been reported among the viruses causing significant Egyptian ones, revealed that there is no significant cor- economic losses to cucurbits.

Biological properties of PRSV isolates. Field Symp- toms. The diverse symptoms associated with PRSV in- fection in the surveyed fields were very similar to those previously reported in squash (Provvidenti, 1993). The symptoms included severe plant stunting. On the leaves, a green mosaic or mottled pattern which was usually ac- companied by malformations, including puckering, blis- ters, leaf distortions, and narrow leaf blades. The youngest leaves were often reduced to just the main veins. Fruits were often malformed, and could show ringspot patterns (Fig. 2A, B). Mechanical transmission. All the 8 Egyptian isolates Fig. 4. Agarose gel electrophoresis of PCR products from the of PRSV under this study were mechanically transmit- CP gene using primers Mo926/Mo1008 of Papaya ringspot vi- ted to greenhouse-grown squash plants with 100% effi- rus Egyptian isolates. M: 1-Kb plus DNA Ladder (Invitrogen, USA). Lane 1, Balteem 1; lane 2, Balteem 2; lane 3, Balteem ciency. Symptoms of vein clearing, mosaic, vein band- 3; lane 4, Shabah; lane 5, Kafr 1; lane 6, Kafr 2; lane 7, Sheno ing, and malformation were produced within 2 to 3 1; lane 8, Sheno 2; lane 9, healthy squash plant. 003_JPP599RP(Omar)_569 15-11-2011 17:39 Pagina 574

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Fig. 5. Phylogenetic analysis of coat protein nucleotide sequence of 8 Egyptian and 28 other PRSV isolates. Their evolutionary hi- stories were inferred using the Maximum Parsimony method. Tree #1 out of 12 most parsimonious trees (length = 651) is shown. The percentage of replicate trees in which the associated taxa clustered together in the bootstrap test (1000 replicates) is shown next to the branches. All positions containing gaps and missing data were eliminated from the dataset. There were a total of 718 positions in the final dataset, out of which 167 were parsimony informative. Phylogenetic analyses were conducted using MEGA4.

relation of CP sequence diversity with the geographical sequence variation among the CP gene from PRSV iso- origin of the isolates. Egyptian isolates were more close- lates within these countries (Bateson et al., 1994; Que- ly related to Venezuela Sucre-El Muco, Venezuela-Meri- mada et al., 1990). However, Jain et al. (1998, 2004) da6, Mexico-QrFC-3 and USA-Florida strain W (95- found substantial variation in the CP gene at the nt level 97.4% identity) but were more distant from a Sri Lanka (up to 14% divergence) among Asian PRSV isolates. isolate (85.9-86.8% identity). Nucleotide identity A phylogenetic tree generated from the CP gene nu- ranged from 87.5% to 97.1% with other isolates (type cleotide sequences showed that Egyptian PRSV isolates W and P) from different countries (not shown). North form a separate clade with 98% bootstrap support, sug- American and Australian data indicate that there is little gestive of a single introduction of PRSV in Egypt and 003_JPP599RP(Omar)_569 15-11-2011 17:39 Pagina 575

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since, as stated above they are more closely related to Jain R.K., Pappu H.R., Pappu S.S., Varma A., Ram R.D., the Venezuelan isolates type-P, Sucre-El Muco and 1998. Molecular characterization of Papaya ringspot po- Merida6 and to the Mexican and USA isolates, it is tyvirus isolates from India. Annals of Applied Biology 132: tempting to hypothesize that the introduction was from 413-425. the New World. Jain R.K., Sharma J., Sivakumar A.S., Sharma P.K., Byadgi The Egyptian isolates did not appear to correlate A.S., Verma A.K., Varma A., 2004. Variability in the coat protein gene of Papaya ringspot virus isolates from multi- with the geographic regions from where they were sam- ple locations in India. Archives of Virology 149: 2435-2442. pled, especially Balteem 3, which clustered with Sheno Kassem M.A., Sempere R.N., Juárez M., Aranda M.A., isolates. This observation suggests that little or no geo- Truniger V., 2007. Cucurbit aphid-borne yellows virus is graphic barriers seem to exist to PRSV long-range dis- prevalent in field-grown cucurbit crops of southeastern persal in Egypt. All Australian isolates grouped together Spain. Plant Disease 91: 232-238. with Poland-BON isolate in one clade as well as the Mnari-Hattab M., Gauthier N., Zouba A., 2009. Biological Brazilian isolates W3, ES and PR which clustered with and molecular characterization of the Cucurbit aphid- Jamaica-Pondside-type P isolate in another clade. On borne yellows virus affecting cucurbits in Tunisia. Plant the other hand, the Indian isolates Up, Indore and Hy- Disease 93: 1065-1072. derabad composed another clade. Furthermore, the Pinto Z.V., Rezende J.A.M., Yuki V.A., Piedade S.M.S., 2008. Asian isolates (Sri Lanka, Thailand, Philippines, Taiwan Ability of and Myzus persicae to transmit Cu- and Japan) clustered together with the two Vietnamese cumber mosaic virus in single and mixed infection with two isolates, VNP-29 and VNW-50 in a separate clade, all of potyviruses to zucchini squash. Summa Phytopathologica which away from the Egyptian isolates clade (Fig. 5). 34: 183-185. Provvidenti R., 1993. Resistance to viral diseases in cucurbits. In: Kyle M.M. (ed.). Resistance to Viral Diseases of Vegeta- bles: Genetics and Breeding, pp. 8-43. Timber Press, Port- ACKNOWLEDGEMENTS land, OR, USA. Purcifull D.E., Edwardson J.R., 1967. Watermelon mosaic The authors wish to thank Dr. Xavier Foissac (UMR virus: tubular inclusion in pumpkin leaves and aggregates Genomique Diversité et Pouvoir Pathogène, INRA- in leaf extracts. Virology 32: 393-401. Université Victor Sègalen Bordeaux 2, Villenave Purcifull D., Edwardson J., Hiebert E., Gonsalves D., 1984. d’Ornon, France) for providing facilities for sequenc- Papaya ringspot virus. CMI/AAB Descriptions of Plant ing. Viruses. No. 292. Quemada H.L, Hostis B., Gonsalves D., Reardon I.M., Hein- rikson R., Hiebert E.L., Sieu L.C., Slightom J.L., 1990. REFERENCES The nucleotide sequences of the 3’-terminal regions of Pa- paya ringspot virus strains W and P. Journal of General Vi- Abdel-Salam A.M., Abdallah N.A., Soliman D.Z.R., Rezk rology 71: 203-210. A.A.S., 2006. The incidence of Squash leaf curl bego- Quiot-Douine L., Lecoq H., Quiot J. B., Pitrat M., Labonne movirus (SqLCV) in Egypt. Arab Journal of Biotechnology G., 1990. Serological and biological variability of virus iso- 9: 375-388. lates related to strains of Papaya ringspot virus. Phy- Bateson M.F., Handerson J., Chaleeprom W., Gibbs A.J., topathology 80: 256-263. Dale J.L., 1994. Papaya ringspot potyvirus: isolate variabili- Riechmann J.L., Lain S., Garcia J.A., 1992. Highlights and ty and the origin of PRSV type P (Australia). Journal of prospects of potyvirus molecular biology. Journal of Gener- General Virology 75: 3547- 3553. al Virology 73: 1-16. Black L.M., Brakke K., Vatter A.E., 1963. Purification and Salem T.Z., El-Gamal S.M., Sadik A.S., 2007. Use of helper electron microscopy of tomato spotted wilt virus. Virology component proteinase gene to identify a new Egyptian iso- 20: 120-130. late of Watermelon mosaic potyvirus. International Journal Bonfield J.K., Smith K.F., Staden R., 1995. A new DNA se- of Virology 3: 107-116. quence assembly program. Nucleic Acids Research 24: Shehata F.S., El-Borollosy A.M., 2008. Induction of resistance 4992-4999. against Zucchini yellow mosaic potyvirus and growth en- Dahal D., Lecoq H., Albrechtsen S.E., 1997. Occurrence of hancement of squash plants using some plant growth-pro- Papaya ringspot potyvirus and cucurbit viruses in Nepal. moting Rhizobacteria. Australian Journal of Basic and Ap- Annals of Applied Biology 130: 491-502. plied Sciences 2: 174-182. Desbiez C., Lecoq H., 1997. Zucchini yellow mosaic virus. Shukla D.D., Ward C.W., Brunt A.A., 1994. Genome struc- Plant Pathology 46: 809-829. ture, variation and function. In: Shukla D.D., Ward C.W., Brunt A.A. (eds). The Potyviridae, pp. 74-112. CAB Inter- FAO, 2010. Statistical Agriculture Databases. Available from: national, Wallingford, Oxon, UK. URL httm:// faostat.fao.org/. Tamura K., Dudley J., Nei M., Kumar S., 2007. MEGA4: Fattouh F.A., 2003. Double infection of a cucurbit host by Molecular Evolutionary Genetics Analysis (MEGA) soft- Zucchini yellow mosaic virus and Cucumber mosaic virus. ware version 4.0. Molecular Biology and Evolution 24: Pakistan Journal of Plant Pathology 2: 85-90. 1596-1599. 003_JPP599RP(Omar)_569 15-11-2011 17:39 Pagina 576

576 Papaya ringspot virus in Egypt Journal of Plant Pathology (2011), 93 (3), 569-576

Thompson J.D., Higgins D.G., Gibson T.J., 1994. CLUSTAL ringspot potyvirus. Archives of Virology 142: 271-285. W: improving the sensitivity of progressive multiple se- Wang C.H., Ban H.J. Yeh S.D., 1994. Comparison of the nu- quence alignment through sequence weighting, position- clear inclusion protein and coat protein genes of five pa- specific gap penalties and weight matrix choice. Nucleic paya ringspot virus strains distinct in geographic origin Acids Research 22: 4673-4680. and pathogenecity. Phytopathology 84: 1205-1210. Tripathi S., Suzuki J.Y., Ferreira S.A., Gonsalves D., 2008. Pa- Yeh S.D., Gonsalves D., Provvidenti R., 1984. Comparative paya ringspot virus-P: characteristics, pathogenicity, se- studies on hosts and serology of Papaya ringspot virus and quence variability and control. Molecular Plant Pathology 1. Phytopathology 74: 1081- 9: 269-280. 1085. Van der Vlugt R.A.A., Steffens P., Cuperus C., Brag E., Lese- Yeh S.D., Jan F.J., Chiang C.H., Doong T.J., Chen M.C., mann D.E., Bos L., Vetten H.J., 1999. Further evidence Chung P.H., Bau H.J., 1992. Complete nucleotide se- that Shallot yellow stripe virus (SYSV) is a distinct po- quence and genetic organization of Papaya ringspot virus tyvirus and reidentification of Welsh onion yellow stripe RNA. Journal of General Virology 73: 2531-2541. virus as a SYSV strain. Phytopathology 89: 148-155. Yeh S.D., Gonsalves D., 1994. Practices and perspective of Wang C.H, Yeh S.D., 1997. Divergence and conservation of control of Papaya ringspot virus by cross protection. Ad- the genomic RNAs of Taiwan and Hawaii strains of papaya vances in Virus Research 10: 237-257.

Received January 8, 2011 Accepted April 1st, 2011