Journal of Plant Pathology (2014), 96 (1), 77-84 Edizioni ETS Pisa, 2014 Mohammed et al. 77

IDENTIFICATION AND PHYLOGENETIC ANALYSIS OF COMMON PUMPKIN IN SUDAN

H.S. Mohammed1, S. Zicca2, A. Manglli2,3, M.E. Mohamed4, M.A. El Siddig1, L. Tomassoli2 and A.A. El Hussein1

1Department of Botany, Faculty of Science, University of Khartoum, P.O. Box 321, 11115 Khartoum, Sudan 2 Consiglio per la Ricerca e la Sperimentazione in Agricoltura, Plant Pathology Research Centre, Via C.G. Bertero 22, Roma, Italy 3Dipartimento di gestione dei sistemi agricoli e forestali, “Mediterranea”University of Reggio Calabria, Reggio Calabria, Italy 4Agricultural Research Corporation, Shambat Agricultural Research Station, Khartoum North, Sudan

SUMMARY Pumpkin (Cucurbita maxima Duchesne, family Cucur- bitaceae) is an important vegetable crop in Sudan where The objective of this study was to identify viral species it is grown in an area of around 2,000 ha with an average infecting pumpkin in the Khartoum and Gezira states of annual yield of 24 tonnes/ha (Mirghani and Mohammed, Sudan, and to evaluate the genetic variability among mem- 1997). bers of the same viral species. A total of 60 symptomatic Several viruses are known, that cause mosaic symptoms pumpkin leaf samples were collected from three different in cucurbits [melon, snake cucumber, tibish (Cucumis melo locations throughout the Khartoum state, and examined var. agrestis), squash and watermelon], the most common for viral incidence. Symptoms observed varied from mosa- of which are the Zucchini yellow mosaic ic, malformation, blistering of the leaves and stunting. De- (ZYMV) (Ahmed et al., 1996; Mahgoub et al., 1997) and generate genus-specific and species-specific primers were Moroccan watermelon (MWMV), the bego- used for the detection of common cucurbit viruses. Results movirus Watermelon chlorotic stunt virus (WmCSV), the showed that the highest incidence (21.7%) was for Cucur- Cucurbit -borne yellows virus (CABYV), bit chlorotic yellows virus (CCYV) and Cucurbit yellow the Squash mosaic virus (SqMV) (Lecoq et al., stunting disorder virus (CYSDV), followed by (18.3%) Zuc- 2003, 2011) and the Cucurbit chlorotic yellow chini yellow mosaic virus (ZYMV), and (15%) Watermelon virus (CCYV) (Hamed et al., 2011). chlorotic stunt virus (WmCSV). Single and multiple infec- Pumpkin is affected by many viruses, responsible for tions with more than one virus were detected. The partial most of the occurring damage worldwide, but its sanitary nucleotide sequences of the regions encoding CI and CP, status in Sudan has not been investigated except for a re- CP, HSP70h, and AV2 proteins of ZYMV, CYSDV, CCYV cent short note reporting the occurrence of Papaya ringspot and WmCSV, respectively, have been deposited in NCBI virus (PRSV) (Mohammed et al., 2012). GenBank. Phylogenetic analysis based on nucleotide se- The aim of this study was to identify viral species in- quences showed no or little variation among the studied fecting pumpkin grown in the Khartoum and Gezira viral isolates since each of them grouped well with its cor- states and to evaluate the genetic variability. responding reference species.

Key words: pumpkin, cucurbit viruses, RT-PCR, phylo- MATERIALS AND METHODS genetic analysis, Sudan. Samples collection and RNA extraction. A total of 60 symptomatic pumpkin leaf samples were collected INTRODUCTION from three different locations in Sudan, i.e. Wad-Ram- ly and Shambat areas (Khartoum), the former is one of Viral diseases are the main problem in the production the most important areas for pumpkin production, and of cucurbit plants compared to diseases caused by other Elnuba (Gezira). For viral detection 100 mg leaf tissue agents. Viruses causing significant yield losses to cucur- were ground in 1.0 ml of phosphate buffer (pH 7.2). Total bits worldwide are found within several families including RNA was extracted from 100 µl of this suspension using , , , Luteoviridae a RNeasy plant mini kit (Qiagen, Germany) according to and (Lecoq et al., 2003). At least 59 viral spe- the manufacturer’s protocol. cies within different genera are globally characterized and reported on cucurbits (Lecoq et al., 2003; King et al., 2011). RT-PCR amplification and sequencing. Based on the type of cucurbit viruses usually reported from tropical Af- Corresponding author: H.S. Mohammed Fax: +249.11.771203 rica, eight primer sets (Table 1) were selected for detection E-mail: [email protected] and identification of the corresponding virus. One-step 78 Phylogenetic analysis of pumpkin viruses in Sudan Journal of Plant Pathology (2014), 96 (1), 77-84

Table 1. Designed and published primers sets used for detection of viral species.

Product Annealing Primer Sequence (5’ - 3’) Target gene Target virus Reference size (bp) Temperature (°C)

Van der CP9502/ TGAGGATCCTGGTGYATHGARAAYGG Coat protein Variable 53 potyviruses Vlugt et al. CPUP GCGGATCCTTTTTTTTTTTTTTTTT gene (CP) (1999) GemA/ TAATATTACCKGWKGVCCSC Deng et al. AV2-AV1 530 55 GemB TGGACYTTRCAWGGBCCTTCACA (1994) MWMV-183/ CAGGGTTCCAAAGGTCAAAA Coat protein 241 57 MWMV This study MWMV-423 ATTTCAATGCACCACACCA gene (CP) Cylindrical ZYMV-1246/ CTTCCAGTCACCACACATGG inclusion 444 57 ZYMV This study ZYMV-1689 TAATGCTGCTGGATCAGTGC gene (CI) WCPd/ GACCTAGTGAYGGKTGCTGTGAATCAG Coat protein 950 61 WMV This study WCPr GCACTAGTCGACCCGAAATGCTAACTG gene (CP) Heat shock Crini-s2/ CATTCCTACCTGTTTAGCCA Hamed et protein gene 353 58 CCYV TGCACTTATAATCTGCTGGTAC al. (2011) Crini-as2 (HSP70h) TTGGGCATGTGACAT Coat protein Célix et al. 410L/410U 460 55 CYSDV AGAGACGGTAAGTAT gene (CP) (1996) CMV5CP/ CTCGAATTCGGATCCGCTTCTCCGCGAG Coat protein Anonymous 870 60 CMV 3CP GGCGAATTCGAGCTCGCCGTTAAGCTGGATGGAC gene (CP) (1998)

Table 2. Number of pumpkin samples infected with each of the detected viral species. specific annealing temperature (Table 1) and 1 min at 72°C with final extension for 10 min at 72°C. PCR products Virus present were visualized under UV after electrophoresis in 1.2% agarose gel and staining with 0.02% ethidium bromide. Location Crinivirus The amplified DNA fragments were purified using Ami- ZYMV WmCSV CCYV CYSDV con Ultra-0.5 Centrifugal Filter Devices (Millipore, USA). Wad-Ramly 5/20 5/20 6/20 6/20 Purified products were bi-directionally sequenced (Bio- Elnuba 2/15 0/15 3/15 3/15 Fab Research, Italy). Shambat 4/25 4/25 4/25 2/25 Total (infected 11 9 13 11 Sequence analysis. Nucleotide sequences, were assem- samples) bled and analyzed using the MEGA 5.05 program, and subjected to sequence similarity searches against GenBank Table 3. Accession numbers of partial nucleotide sequences database using the BLAST program. Deduced amino ac- of different genomic regions of pumpkin viruses. id sequences were obtained using an online translation tool (www.expasy.ch/tool/translate). Phylogenetic trees Virus Genomic GenBank Isolate code identified region accession Nos. were constructed after multiple sequence alignments us- ing ClustalW embedded in the MEGA 5.05 program and CP KC695817 ZYMV-Sud.C.20 ZYMV Neighbor-joining method with 1000 bootstrap replicates CI KC677622 (Tamura et al., 2011). CYSDV-Sud.C.22 KC677625 CYSDV CP CYSDV-Sud.C.32 KC677626 CCYV-Sud.C.35 KC677623 CCYV HSP70h RESULTS CCYV-Sud.C.37 KC677624 WmCSV-Sud.C.23 KC677627 WmCSV AV2-AV1 Notable differences were observed in the symptoms WmCSV-Sud.C.37 KC677628 shown by leaf samples collected from different locations. Yellow mosaic was dominant in plants from Shambat RT-PCR protocol was performed in a total volume of 25 whereas malformation, blistering and/or stunting associ- µl containing 2 µl of total RNA extract, 5× PCR buffer ated with yellow mosaic were frequently observed in plants (Promega, USA), 2.5 mM of each dNTP, 4 µM of each from Wad-Ramly and Elnuba. primer, 1.2 U of AMV RT (Promega, USA), 0.75 U of Go-Taq polymerase (Promega, USA), 20 U of RNase-OUT RT-PCR based detection of pumpkin viruses. Four vi- (Invitrogen, USA). Amplification was done according to ral species belonging to three viral genera namely Potyvi- the following conditions: reverse transcription at 46°C for rus, Crinivirus and Begomovirus were detected in 25% of 30 min, followed by denaturation at 95°C for 7 min, and the collected samples by RT-PCR. When genus-specific by 35 cycles of the following steps: 1 min at 94°C, 1 min at and/or species-specific primers were used, the highest Journal of Plant Pathology (2014), 96 (1), 77-84 Mohammed et al. 79

A) JN192409 USA JN192428 USA 64 JN192408 USA

65 JN192405 USA JN192407 USA 99 JN192406 USA 58 JQ716413 USA I JN183062 Iran ZYMV-Sud.C.20 A 91 47 26 EF062583 Israel 86 21 DQ124239 Slovakia AB188116 Japan: Kyoto

100 AJ429071 South Korea:Seoul AJ316229 China:Zhejiang: Hangzhou - Banshan

100 AJ515911 China:Zhejiang: Hangzhou - Banshan 61 AB369279 South Korea AM422386 Taiwan:Taichung II 45 97 AJ307036 China:Zhejiang: Hangzhou - Banshan 100 AJ316228 China:Zhejiang: Hangzhou - Banshan 91 NC 003224 Taiwan: Tainan 99 AF127929 Taiwan: Tainan L29569 Reunion Island B NC 009995 MWMV Tunisia

0.05 B) AJ420016 Austria AJ420015 Austria AJ420017 Austria Fig. 1A AJ251527 Hungary

50 DQ124239 Slovakia JN315861 Serbia JN315862 Serbia 70 JN315863 Serbia AJ459955 Hungary

14 AJ459956 Hungary AJ420018 Slovenia JX310111 Venezuela 04 AB127936 Pakistan 24 JX310108 Venezuela

85 EF062583 Israel EF062582 Israel

2 JX310105 Venezuela

18 ZYMV-Sud.C.20 0 65 HM641799 Sudan JF317296 Turkey FJ705272 Iran 0 EU999760 Jordan EU999757 Syria FJ705259 Iran 46 FJ705257 Iran 6 AJ420019 Germany

89 JF792447 Australia JF792446 Australia

68 HQ529776 India 164 GQ482976 India A 40 AB458595 Syria 51 AB458596 Syria 48 JQ899266 Saudi Arabia

92 JQ899265 Saudi Arabia 42 96 JQ899263 Saudi Arabia JF317297 Turkey JX502677 Brazil JX502673 Brazil 29 14 HM450303 Cote d Ivoire 5 12 JN861004 France 40 27 HM641798 France AJ307036 China 18 AY611022 China AB188115 Japan JX028592 USA 34 36 AJ420020 Italy 79 97 HM005307 Mali 100 96 HM005312 Mali NC 003224 Taiwan 93 D13914 Florida DQ925447 Viet Nam

84 L29569 Reunion Island 56 AF014811 Singapore B AY074808 China

95 DQ925449 Viet Nam 75 AJ889243 China C NC 009995 MWMV Tunisia

0.05 Fig. 1. A. Phylogenetic tree constructed by the NJ method based on partial CI gene nucleotide sequences of 21 isolates of ZYMV from GenBank and the isolate ZYMV-Sud.C.20. MWMV was used as an out-group. Bootstrap values along the branches are sup- ported by 1000 replicates.Fig. B. Phylogenetic 1B tree constructed by the NJ method based on partial CP gene nucleotide sequences of 55 isolates of ZYMV from GenBank and the isolate ZYMV-Sud.C.20. MWMV was used as an out-group. Bootstrap values along the branches are supported by 1000 replicates.

80 Phylogenetic analysis of pumpkin viruses in Sudan Journal of Plant Pathology (2014), 96 (1), 77-84

AB523789 Japan:Kumamoto 35 JF340435 Egypt JF807055 Sudan 59 AF312801 Turkey AF312800 Turkey GU721105 China: Shanghai AF312799 Turkey GU721108 China: Ningbo AF312804 Lebanon GU721109 China: Ningbo AF312805 Jordan GU721110 China: Ningbo AF312806 USA: Texas AF312807 Mexico: Tamaulipas GU721111 China: Shouguang 33 AF312802 Lebanon HM581659 China: Ningbo 37 FJ492808 USA HQ148667 China EF210558 USA JN126045 Taiwan: Yunlin Erlun DQ903105 Jordan JN126046 Taiwan: Yilan 64 EF210561 Guatemala 15 EF210560 Guatemala JF502222 Taiwan: Yunlin 27 DQ903107 Jordan JQ904629 China: Beijing DQ903106 Jordan CCYV-Sud.C.35 DQ903108 Jordan 15 EF210559 USA AB457591 Japan:Kumamoto 31 DQ903109 Jordan JX014263 Lebanon 99 51 AF312803 Lebanon GU721107 China: Shouguang AY242078 Spain

43 65 GU721106 China: Shouguang 3164 AJ439690 Spain:Almeria AJ243000 Spain:Almeria CCYV-Sud.C.37 39 DQ903110 Jordan FJ380119 LCV USA 28 DQ903111 Jordan AY242078 CYSDV Spain 91 AF312809 Spain: Almeria NC 005210 BPYV USA: Maryland AF312810 Spain: Almeria 99 AF312808 Spain: Almeria 0.05 EF538681 Tunisia 64 CYSDV-Sud.C.22 Fig. 2. Phylogenetic tree constructed by the NJ method based 90 CYSDV-Sud.C32 on the partial HSP70h gene nucleotide sequences of CCYV AY730779 Iran: Boushehr Fig. 2. 99 JN083790 Saudi Arabia isolates from GenBank and the two Sudanese isolates CCYV AF312797 Saudi Arabia 99 (CCYV-Sud.C.35 and CCYV-Sud.C.37). CYSDV, Lettuce chlo- AF312798 Saudi Arabia rosis virus (LCV) and Beet pseudo-yellows virus (BPYV) were 99 AF312796 Saudi Arabia used as out-groups. Bootstrap values along the branches are AF312795 Saudi Arabia JQ904629 CCYV China: Beijing supported by 1000 replicates. 0.05 incidence (21.7%) was recorded for CCYV and CYSDV, Fig. 3. Phylogenetic tree constructed by the NJ method based Fig. 3. on partial CP gene nucleotide sequences of 35 isolates of followed by ZYMV (18.3%), and WmCSV (15%) (Table 2). CYSDV from GenBank and the two Sudanese isolates CYS- Multiple infections with four, three or two of these viruses DV (CYSDV-Sud.C.22 and CYSDV-Sud.C.32). CCYV was were detected in one, nine and five of the studied samples, used as an out-group. Bootstrap values along the branches are respectively. Watermelon mosaic virus (WMV) or MWMV supported by 1000 replicates. and (CMV) were not detected in any of the examined samples. (HM641799) previously identified in C. melo (Mahgoub et al., 1997) were in group A together with 48 other ZYMV Sequence analysis. The partial nucleotide sequences of isolates from different countries (Fig. 1B). the regions encoding CI and CP, CP, HSP70h, and AV2 CCY V. Partial nucleotide sequences of the HSP70h gene proteins of ZYMV, CYSDV, CCYV and WmCSV, respec- of two Sudanese pumpkin CCYV isolates (CCYV-Sud.C.35 tively were deposited in the GenBank (Table 3). and CCYV-Sud.C.37) showed high nucleotide and amino ZY MV. The studied ZYMV isolate (ZYMV-Sud.C.20) acid identities in the range 99-100% with all CCYV se- shared high nucleotide and amino acid identities (98%) quences available in the GenBank including a Sudanese in both CI and CP regions with ZYMV isolates from Is- CCYV isolate (JF807055) previously identified in C. melo rael (EF062583) and Iran (JN183062) belonging to group and C. sativus (Hamed et al., 2011). In fact, phylogenetic A (Desbiez et al., 2002). In fact, phylogenetic analysis analysis based on the HSP70h sequences revealed very low based on CI sequences available in GenBank revealed variation within CCYV population (Fig. 2). two groups, A and B, which are similar to those based CYSDV. Sequence comparison in the CP region (RNA- on CP sequence analysis (Fig. 1A). Within group A, the 2) of the two Sudanese pumpkin CYSDV isolates (CYSDV- Sudanese ZYMV-Sud.C.20 grouped in sub-cluster IA, Sud.C.32 and CYSDV-Sud.C.22) showed 99% nt identity together with ZYMV isolates from different countries, with a CYSDV isolate from Iran (AY730779) and 88.2% while the majority of the isolates from the Asiatic region and 90.8% aa identity with the same Iranian isolate. Phylo- were in sub-cluster IIA. However, a virus isolate from genetic analysis based on the alignment of CP nt sequences Réunion Island (L29569) stands alone in group B. Phylo- showed two main groups as previously reported by Rubio genetic analysis of CP sequences revealed three significant et al. (2001) and Yakoubi et al. (2007). The two studied Su- groups (A, B and C), as reported by Lecoq and Desbiez danese isolates clustered with isolates from Iran and Saudi (2012). ZYMV-Sud.C.20 and another Sudanese isolate Arabia (Fig. 3). Journal of Plant Pathology (2014), 96 (1), 77-84 Mohammed et al. 81

A) JN618982 Oman 66 (A) JN618981 Oman 98 JN618983 Oman JN624386 Oman 58 70 JN618984 Oman AJ245650 Sudan

32 74 WmCSV-Sud.C.23 98 WmCSV-Sud.C.37 HM368371 Lebanon 6 EF201809 Israel 79 44 JX444576 Jordan 4 45 JX131283 Jordan JX480489 Iran 3 AJ245652 Iran 10 4 JX480486 Iran JX480485 Iran 14 JX480484 Iran JX480483 Iran JX480482 Iran AY702650 TYLCV Morocco: Agadir

0.02

B) JX480483 Iran (B) JX480482 Iran 77 JX480484 Iran JX480485 Iran 42 JX480486 Iran JX480489 Iran 42 AJ245652 Iran JX444576 Jordan JX131283 Jordan 47 50 HM368371 Lebanon 38 EF201809 Israel 78 AJ245650 Sudan JN618984 Oman JN618983 Oman 51 JN618982 Oman 64 JN618981 Oman JN624386 Oman WmCSV-Sud.C.37 WmCSV-Sud.C.23 AY702650 TYLCV Morocco: Agadir

0.02 Fig. 4. Phylogenetic tree constructed by the NJ method based on nucleotide sequences of AV2 region (A) and partial CP region (B) of 17 WmCSV isolates from GenBank and the two Sudanese isolates WmCSV (WmCSV-Sud.C.23 and WmCSV-Sud.C.37). TomatoFig. yellow 4. leaf curl virus (TYLCV) was used as an out-group. Bootstrap values along the branches are supported by 1000 rep- licates.

WmCSV. The nucleotide sequence of AV2/AV1 genes cluster together with the previously reported watermelon of the two Sudanese pumpkin isolates (WmCSV-Sud.C.23 isolates from Sudan and Oman (Fig. 4A and B). and WmCSV-Sud.C.37) shared 97% nt identity with a watermelon isolate of WmCSV previously identified in Sudan (AJ245650). Comparison of deduced aa se- DISCUSSION quences in the complete AV2 and partial AV1 regions of WmCSV-Sud.C.23 showed 98% and 97% identity with The present investigation showed that pumpkin fields the WmCSV Sudanese isolate (AJ245650), respectively. in Sudan are severely infected with viruses such as CCYV, WmCSV-Sud.C.37 shared 97% identity in both regions CYSDV, ZYMV and WmCSV. CCYV is a newly charac- with the same reference isolate. The phylogenetic tree terized crinivirus (Gyoutoku et al., 2009) reported as the based on both AV2 and CP showed that the two pump- most cucurbit-damaging virus (Kubota et al., 2011). It kin WmCSV isolates reported here grouped in the same threatens cucurbit production in many Asiatic countries 82 Phylogenetic analysis of pumpkin viruses in Sudan Journal of Plant Pathology (2014), 96 (1), 77-84

(Huang et al., 2010; Okuda et al., 2010; Abrahamian et Aranda, 2005; Sweiss et al., 2007; Yakoubi et al., 2007). al., 2012; Okuda et al., 2013) causing, for example, losses Although all CYSDV isolates previously reported from estimated at 10-20% in China (Gu et al., 2011). CCYV has Africa grouped with a Western subpopulation of the virus recently been reported in Sudan from muskmelon and (Rubio et al., 2001), Sudanese isolates grouped with the cucumber (Hamed et al., 2011). In this study, it was de- Eastern subpopulation suggesting a common origin with tected in 21.7% of the pumpkin samples showing yellow- isolates from Iran and Saudi Arabia. This could possibly ing symptoms. All the surveyed locations were affected be attributed to the wider trade links and air traffic be- and CCYV was found alone or in mixed infection with tween Sudan and other countries in this region. the other identified viruses. Phylogenetic analysis performed with Sudanese CYSDV is distributed throughout the Mediterranean WmCSV isolates revealed some particularities when the (Papayiannis et al., 2005; Yakoubi et al., 2007; Lecoq and two DNA-A genomic regions coding for AV2 protein Desbiez, 2012), the Arab Emirates (Hassan and Duffus, (complete) and CP protein (partial), were separately ana- 1991) and North America (CABI/EPPO, 2004). Its oc- lyzed. In fact, AV2 sequences of WmCSV isolates from currence aggravates the phytosanitary status of the crop GenBank grouped in two main clusters similar to those in Sudan as the virus was reported to affect cucurbit reported for the complete DNA-A component (Khan et crops seriously in many production areas (Abou-Jawdah al., 2012), and the Sudanese isolates WmCSV-Sud C.23 et al., 2000; Yakoubi et al., 2007). CYSDV was found in and WmCSV-Sud C37 were in the same cluster with a all regions investigated in the present study and with the previously identified Sudanese isolate of this virus. Upon same incidence level as CCYV. Both CCYV and CYSDV analysis of the two Sudanese isolates in the highly con- produce symptoms, such as interveinal chlorosis, yellow- served CP region (Wyatt and Brown, 1996), the same ing and brittleness of lower leaves, which resemble those evolutionary linkage with reference isolates including produced by nutritional deficiencies, a condition which the previously identified WmCSV Sudanese isolate was usually leads to underrating disease incidence in the field. evident. In addition, the sequences identity comparison WmCSV was first identified in 1988 in Yemen (Jones et indicated that there is a close relation among WmCSV al., 1988) and rapidly spread across North Africa, Middle isolates from different cucurbits including pumpkin, wa- East (Iran, Jordan, Lebanon, Palestine, Oman) and Sudan termelon, melon and squash. This could be attributed to (Kheyr-Pour et al., 2000; Al-Musa et al., 2011; Samsatly the endemic populations of the (Bemisia tabaci) et al., 2012; Ali-Shtayeh et al., 2012; Khan et al., 2012), vector in the region and indicates that this vector has a where it causes very severe damage to different cucurbits, similar epidemiological behaviour regardless of the host especially in protected crops. Although, WmCSV was not (Kheyr-Pour et al., 2000). detected in the Elnuba area, the prevalence of large popu- Two genomic regions (CI and CP) have been studied lations of the vector () may cause disaster should for ZYMV and the phylogenetic analysis did not reveal the virus be introduced in this area. Consequently, the evidence of any evolutionary differences between the Su- adoption of strict integrated control measures is needed danese isolate reported here and the one previously char- to prevent possible future epidemics. acterized (Mahgoub et al., 1997) indicating a high degree Concerning aphid-borne viruses, the examined pump- of stability in these genes through the years. kin samples were infected by PRSV (Mohammed et al., This investigation provides basic information on 2012) and ZYMV, both of which cause severe damage to pumpkin viruses present in three areas important for cu- pumpkins (Wakman et al., 2002) leading to yield reduc- curbit production within Sudan. The high incidence of tion in many countries (Krstic et al., 2002; Zhao et al., both aphid- and whitefly-transmitted viruses may be due 2003; Jossey and Babadoost, 2008; Pachner et al., 2011). In to the large population of these vectors usually observed Sudan, ZYMV is considered as one of the major compo- in cucurbits and other field crops in the country. Effi- nents of the viral pathosystem that dramatically decreases cient measures such as the use of plastic mulches, floating cucurbit production in many parts of the country (Mah- covers, vector-targeting insecticides and resistant pump- goub et al., 1997, 1998). kin cultivars are required to control virus spread in field In this study, phylogenetic analysis based on sequence crops. comparison of the amplified genomic region for each identified virus was performed. No relevant differences were detected when Sudanese CCYV isolates were com- ACKNOWLEDGEMENTS pared with each other and with CCYV sequences from GenBank, which supports Lin et al. (2012) conclusion that The authors would like to acknowledge the Ministry of HSP70h is a conserved gene in criniviruses. Higher Education and Scientific Research, Sudan for their The CP coding region has been used to study the financial support under the grant No. 95/2011. variability of CYSDV populations by many authors who reported high genetic uniformity between isolates from different parts of the world (Rubio et al., 2001; Marco and Journal of Plant Pathology (2014), 96 (1), 77-84 Mohammed et al. 83

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Received April 9, 2013 Accepted July 28, 2013