1 Journal of Plant Pathology (2014), 96 (1), Edizioni ETS Pisa, 2014 Mohammed et al. 77 IDENTIFICATION AND PHYLOGENETIC ANALYSIS OF COMMON PUMPKIN VIRUSES IN SUDAN H.S. Mohammed 1, S. Zicca 2, A. Manglli 2,3, M.E. Mohamed 4, M.A. El Siddig 1, L. Tomassoli 2 and A.A. El Hussein 1 1 Department of Botany, Faculty of Science, University of Khartoum, P.O. Box 321, Khartoum, Sudan 2 Consiglio per la Ricerca e la Sperimentazione in Agricoltura, Plant Pathology Research Centre, Via C.G. Bertero 22, Roma, Italy 3 Dipartimento di gestione dei sistemi agricoli e forestali, Mediterranea University of Reggio Calabria, Reggio Calabria, Italy 4 Agricultural Research Corporation, Shambat Agricultural Research Station, Khartoum North, Sudan SUMMARY The objective of this study was to identify viral species infecting pumpkin in the Khartoum and Gezira states of Sudan, and to evaluate the genetic variability among members of the same viral species. A total of 60 symptomatic pumpkin leaf samples were collected from three different locations throughout the Khartoum state, and examined for viral incidence. Symptoms observed varied from mosaic, malformation, blistering of the leaves and stunting. Degenerate genus-specific and species-specific primers were used for the detection of common cucurbit viruses. Results showed that the highest incidence (21.7%) was for Cucurbit chlorotic yellows virus (CCYV) and Cucurbit yellow stunting disorder virus (CYSDV), followed by (18.3%) Zucchini yellow mosaic virus (ZYMV), and (15%) Watermelon chlorotic stunt virus (WmCSV). Single and multiple infections with more than one virus were detected. The partial nucleotide sequences of the regions encoding CI and CP, CP, HSP70h, and AV2 proteins of ZYMV, CYSDV, CCYV and WmCSV, respectively, have been deposited in NCBI GenBank. Phylogenetic analysis based on nucleotide sequences showed no or little variation among the studied viral isolates since each of them grouped well with its corresponding reference species. Key words: pumpkin, cucurbit viruses, RT-PCR, phylogenetic analysis, Sudan. INTRODUCTION Viral diseases are the main problem in the production of cucurbit plants compared to diseases caused by other agents. Viruses causing significant yield losses to cucurbits worldwide are found within several families including Geminiviridae, Closteroviridae, Bromoviridae, Luteoviridae and Potyviridae (Lecoq et al., 2003). At least 59 viral species within different genera are globally characterized and reported on cucurbits (Lecoq et al., 2003; King et al., 2011). Corresponding author: H.S. Mohammed Fax: Pumpkin (Cucurbita maxima Duchesne, family Cucurbitaceae) is an important vegetable crop in Sudan where it is grown in an area of around 2,000 ha with an average annual yield of 24 tonnes/ha (Mirghani and Mohammed, 1997). Several viruses are known, that cause mosaic symptoms in cucurbits [melon, snake cucumber, tibish (Cucumis melo var. agrestis), squash and watermelon], the most common of which are the potyviruses Zucchini yellow mosaic virus (ZYMV) (Ahmed et al., 1996; Mahgoub et al., 1997) and Moroccan watermelon mosaic virus (MWMV), the begomovirus Watermelon chlorotic stunt virus (WmCSV), the polerovirus Cucurbit aphid-borne yellows virus (CABYV), the comovirus Squash mosaic virus (SqMV) (Lecoq et al., 2003, 2011) and the crinivirus Cucurbit chlorotic yellow virus (CCYV) (Hamed et al., 2011). Pumpkin is affected by many viruses, responsible for most of the occurring damage worldwide, but its sanitary status in Sudan has not been investigated except for a recent short note reporting the occurrence of Papaya ringspot virus (PRSV) (Mohammed et al., 2012). The aim of this study was to identify viral species infecting pumpkin grown in the Khartoum and Gezira states and to evaluate the genetic variability. MATERIALS AND METHODS Samples collection and RNA extraction. A total of 60 symptomatic pumpkin leaf samples were collected from three different locations in Sudan, i.e. Wad-Ramly and Shambat areas (Khartoum), the former is one of the most important areas for pumpkin production, and Elnuba (Gezira). For viral detection 100 mg leaf tissue were ground in 1.0 ml of phosphate buffer (ph 7.2). Total RNA was extracted from 100 µl of this suspension using a RNeasy plant mini kit (Qiagen, Germany) according to the manufacturer s protocol. RT-PCR amplification and sequencing. Based on the type of cucurbit viruses usually reported from tropical Africa, eight primer sets (Table 1) were selected for detection and identification of the corresponding virus. One-step
2 78 Phylogenetic analysis of pumpkin viruses in Sudan Journal of Plant Pathology (2014), 96 (1), Table 1. Designed and published primers sets used for detection of viral species. Primer Sequence (5-3 ) Target gene Product size (bp) Annealing Temperature ( C) Target virus Reference CP9502/ CPUP GemA/ GemB MWMV-183/ MWMV-423 ZYMV-1246/ ZYMV-1689 WCPd/ WCPr Crini-s2/ Crini-as2 410L/410U CMV5CP/ 3CP TGAGGATCCTGGTGYATHGARAAYGG GCGGATCCTTTTTTTTTTTTTTTTT TAATATTACCKGWKGVCCSC TGGACYTTRCAWGGBCCTTCACA CAGGGTTCCAAAGGTCAAAA ATTTCAATGCACCACACCA CTTCCAGTCACCACACATGG TAATGCTGCTGGATCAGTGC GACCTAGTGAYGGKTGCTGTGAATCAG GCACTAGTCGACCCGAAATGCTAACTG CATTCCTACCTGTTTAGCCA TGCACTTATAATCTGCTGGTAC TTGGGCATGTGACAT AGAGACGGTAAGTAT CTCGAATTCGGATCCGCTTCTCCGCGAG GGCGAATTCGAGCTCGCCGTTAAGCTGGATGGAC Variable 53 potyviruses AV2-AV begomoviruses Cylindrical inclusion gene (CI) Heat shock protein gene (HSP70h) Van der Vlugt et al. (1999) Deng et al. (1994) MWMV This study ZYMV This study WMV This study CCYV CYSDV CMV Hamed et al. (2011) Célix et al. (1996) Anonymous (1998) Table 2. Number of pumpkin samples infected with each of the detected viral species. Location Potyvirus ZYMV Virus present Begomovirus Crinivirus WmCSV CCYV CYSDV Wad-Ramly 5/20 5/20 6/20 6/20 Elnuba 2/15 0/15 3/15 3/15 Shambat 4/25 4/25 4/25 2/25 Total (infected samples) Table 3. Accession numbers of partial nucleotide sequences of different genomic regions of pumpkin viruses. Isolate code ZYMV-Sud.C.20 CYSDV-Sud.C.22 CYSDV-Sud.C.32 CCYV-Sud.C.35 CCYV-Sud.C.37 WmCSV-Sud.C.23 WmCSV-Sud.C.37 Virus identified ZYMV CYSDV CCYV WmCSV Genomic region CP CI GenBank accession Nos. KC KC KC KC KC KC KC KC RT-PCR protocol was performed in a total volume of 25 µl containing 2 µl of total RNA extract, 5 PCR buffer (Promega, USA), 2.5 mm of each dntp, 4 µm of each primer, 1.2 U of AMV RT (Promega, USA), 0.75 U of Go-Taq polymerase (Promega, USA), 20 U of RNase-OUT (Invitrogen, USA). Amplification was done according to the following conditions: reverse transcription at 46 C for 30 min, followed by denaturation at 95 C for 7 min, and by 35 cycles of the following steps: 1 min at 94 C, 1 min at CP HSP70h AV2-AV1 specific annealing temperature (Table 1) and 1 min at 72 C with final extension for 10 min at 72 C. PCR products were visualized under UV after electrophoresis in 1.2% agarose gel and staining with 0.02% ethidium bromide. The amplified DNA fragments were purified using Amicon Ultra-0.5 Centrifugal Filter Devices (Millipore, USA). Purified products were bi-directionally sequenced (Bio- Fab Research, Italy). Sequence analysis. Nucleotide sequences, were assembled and analyzed using the MEGA 5.05 program, and subjected to sequence similarity searches against GenBank database using the BLAST program. Deduced amino acid sequences were obtained using an online translation tool ( Phylogenetic trees were constructed after multiple sequence alignments using ClustalW embedded in the MEGA 5.05 program and Neighbor-joining method with 1000 bootstrap replicates (Tamura et al., 2011). RESULTS Notable differences were observed in the symptoms shown by leaf samples collected from different locations. Yellow mosaic was dominant in plants from Shambat whereas malformation, blistering and/or stunting associated with yellow mosaic were frequently observed in plants from Wad-Ramly and Elnuba. RT-PCR based detection of pumpkin viruses. Four viral species belonging to three viral genera namely Potyvirus, Crinivirus and Begomovirus were detected in 25% of the collected samples by RT-PCR. When genus-specific and/or species-specific primers were used, the highest
3 Journal of Plant Pathology (2014), 96 (1), Mohammed et al. 79 A) JN USA JN USA 64 JN USA 65 JN USA JN USA 99 JN USA 58 JQ USA JN Iran ZYMV-Sud.C EF Israel DQ Slovakia AB Japan: Kyoto 100 AJ South Korea:Seoul AJ China:Zhejiang: Hangzhou - Banshan 100 AJ China:Zhejiang: Hangzhou - Banshan 61 AB South Korea AM Taiwan:Taichung AJ China:Zhejiang: Hangzhou - Banshan AJ China:Zhejiang: Hangzhou - Banshan NC Taiwan: Tainan 99 AF Taiwan: Tainan L29569 Reunion Island NC MWMV Tunisia I II B A B) 0.05 AJ Austria AJ Austria AJ Austria AJ Hungary 50 DQ Slovakia JN Serbia JN Serbia 70 JN Serbia AJ Hungary 14 AJ Hungary AJ Slovenia JX Venezuela AB Pakistan JX Venezuela EF Israel EF Israel JX Venezuela ZYMV-Sud.C.20 HM Sudan JF Turkey FJ Iran 0 EU Jordan EU Syria FJ Iran 46 FJ Iran 6 AJ Germany 89 JF Australia JF Australia 68 HQ India 164 GQ India 40 AB Syria A AB Syria JQ Saudi Arabia JQ Saudi Arabia 96 JQ Saudi Arabia JF Turkey JX Brazil JX Brazil 14 HM Cote d Ivoire 5 12 JN France 27 HM France AJ China AY China AB Japan JX USA AJ Italy HM Mali HM Mali 93 NC Taiwan D13914 Florida DQ Viet Nam L29569 Reunion Island AF Singapore AY China DQ Viet Nam AJ China B C NC 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 supported by 1000 replicates. B. Phylogenetic 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.
4 80 Phylogenetic analysis of pumpkin viruses in Sudan Journal of Plant Pathology (2014), 96 (1), AB Japan:Kumamoto JF Sudan GU China: Shanghai GU China: Ningbo GU China: Ningbo GU China: Ningbo GU China: Shouguang 33 HM China: Ningbo HQ China JN Taiwan: Yunlin Erlun JN Taiwan: Yilan 15 JF Taiwan: Yunlin JQ China: Beijing CCYV-Sud.C AB Japan:Kumamoto JX Lebanon 99 GU China: Shouguang GU China: Shouguang CCYV-Sud.C.37 FJ LCV USA AY CYSDV Spain NC BPYV USA: Maryland 0.05 Fig. 2. Phylogenetic tree constructed by the NJ method based on the partial HSP70h gene nucleotide sequences of CCYV isolates from GenBank and the two Sudanese isolates CCYV (CCYV-Sud.C.35 and CCYV-Sud.C.37). CYSDV, Lettuce chlorosis virus (LCV) and Beet pseudo-yellows virus (BPYV) were used as out-groups. Bootstrap values along the branches are supported by 1000 replicates. incidence (21.7%) was recorded for CCYV and CYSDV, followed by ZYMV (18.3%), and WmCSV (15%) (Table 2). Multiple infections with four, three or two of these viruses were detected in one, nine and five of the studied samples, respectively. Watermelon mosaic virus (WMV) or MWMV and Cucumber mosaic virus (CMV) were not detected in any of the examined samples. Sequence analysis. The partial nucleotide sequences of the regions encoding CI and CP, CP, HSP70h, and AV2 proteins of ZYMV, CYSDV, CCYV and WmCSV, respectively were deposited in the GenBank (Table 3). ZYMV. The studied ZYMV isolate (ZYMV-Sud.C.20) shared high nucleotide and amino acid identities (98%) in both CI and CP regions with ZYMV isolates from Israel (EF062583) and Iran (JN183062) belonging to group A (Desbiez et al., 2002). In fact, phylogenetic analysis based on CI sequences available in GenBank revealed two groups, A and B, which are similar to those based on CP sequence analysis (Fig. 1A). Within group A, the Sudanese ZYMV-Sud.C.20 grouped in sub-cluster IA, together with ZYMV isolates from different countries, while the majority of the isolates from the Asiatic region were in sub-cluster IIA. However, a virus isolate from Réunion Island (L29569) stands alone in group B. Phylogenetic analysis of CP sequences revealed three significant groups (A, B and C), as reported by Lecoq and Desbiez (2012). ZYMV-Sud.C.20 and another Sudanese isolate JF Egypt 59 AF Turkey AF Turkey AF Turkey AF Lebanon AF Jordan AF USA: Texas AF Mexico: Tamaulipas AF Lebanon 37 FJ USA EF USA DQ Jordan 64 EF Guatemala EF Guatemala 27 DQ Jordan DQ Jordan DQ Jordan EF USA 31 DQ Jordan 51 AF Lebanon AY Spain AJ Spain:Almeria AJ Spain:Almeria 39 DQ Jordan 28 DQ Jordan 91 AF Spain: Almeria AF Spain: Almeria 99 AF Spain: Almeria EF Tunisia 64 CYSDV-Sud.C CYSDV-Sud.C32 AY Iran: Boushehr 99 JN Saudi Arabia AF Saudi Arabia 99 AF Saudi Arabia 99 AF Saudi Arabia AF Saudi Arabia JQ CCYV China: Beijing Fig. 3. Phylogenetic tree constructed by the NJ method based on partial CP gene nucleotide sequences of 35 isolates of CYSDV from GenBank and the two Sudanese isolates CYS- DV (CYSDV-Sud.C.22 and CYSDV-Sud.C.32). CCYV was used as an out-group. Bootstrap values along the branches are supported by 1000 replicates. (HM641799) previously identified in C. melo (Mahgoub et al., 1997) were in group A together with 48 other ZYMV isolates from different countries (Fig. 1B). CCYV. Partial nucleotide sequences of the HSP70h gene of two Sudanese pumpkin CCYV isolates (CCYV-Sud.C.35 and CCYV-Sud.C.37) showed high nucleotide and amino acid identities in the range % with all CCYV sequences available in the GenBank including a Sudanese CCYV isolate (JF807055) previously identified in C. melo and C. sativus (Hamed et al., 2011). In fact, phylogenetic analysis based on the HSP70h sequences revealed very low variation within CCYV population (Fig. 2). CYSDV. Sequence comparison in the CP region (RNA- 2) of the two Sudanese pumpkin CYSDV isolates (CYSDV- Sud.C.32 and CYSDV-Sud.C.22) showed 99% nt identity with a CYSDV isolate from Iran (AY730779) and 88.2% and 90.8% aa identity with the same Iranian isolate. Phylogenetic analysis based on the alignment of CP nt sequences showed two main groups as previously reported by Rubio et al. (2001) and Yakoubi et al. (2007). The two studied Sudanese isolates clustered with isolates from Iran and Saudi Arabia (Fig. 3).
5 Journal of Plant Pathology (2014), 96 (1), Mohammed et al. 81 A) JN Oman 66 JN Oman 98 JN Oman JN Oman JN Oman AJ Sudan WmCSV-Sud.C WmCSV-Sud.C.37 HM Lebanon EF Israel JX Jordan 4 45 JX Jordan JX Iran AJ Iran JX Iran JX Iran 14 JX Iran JX Iran JX Iran AY TYLCV Morocco: Agadir 0.02 B) JX Iran JX Iran JX Iran JX Iran JX Iran JX Iran 42 AJ Iran JX Jordan 47 JX Jordan 50 HM Lebanon 38 EF Israel 78 AJ Sudan JN Oman JN Oman JN Oman 64 JN Oman JN Oman WmCSV-Sud.C.37 WmCSV-Sud.C.23 AY 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). Tomato yellow leaf curl virus (TYLCV) was used as an out-group. Bootstrap values along the branches are supported by 1000 replicates. WmCSV. The nucleotide sequence of AV2/AV1 genes of the two Sudanese pumpkin isolates (WmCSV-Sud.C.23 and WmCSV-Sud.C.37) shared 97% nt identity with a watermelon isolate of WmCSV previously identified in Sudan (AJ245650). Comparison of deduced aa sequences in the complete AV2 and partial AV1 regions of WmCSV-Sud.C.23 showed 98% and 97% identity with the WmCSV Sudanese isolate (AJ245650), respectively. WmCSV-Sud.C.37 shared 97% identity in both regions with the same reference isolate. The phylogenetic tree based on both AV2 and CP showed that the two pumpkin WmCSV isolates reported here grouped in the same cluster together with the previously reported watermelon isolates from Sudan and Oman (Fig. 4A and B). DISCUSSION The present investigation showed that pumpkin fields in Sudan are severely infected with viruses such as CCYV, CYSDV, ZYMV and WmCSV. CCYV is a newly characterized crinivirus (Gyoutoku et al., 2009) reported as the most cucurbit-damaging virus (Kubota et al., 2011). It threatens cucurbit production in many Asiatic countries
6 82 Phylogenetic analysis of pumpkin viruses in Sudan Journal of Plant Pathology (2014), 96 (1), (Huang et al., 2010; Okuda et al., 2010; Abrahamian et al., 2012; Okuda et al., 2013) causing, for example, losses estimated at 10-20% in China (Gu et al., 2011). CCYV has recently been reported in Sudan from muskmelon and cucumber (Hamed et al., 2011). In this study, it was detected in 21.7% of the pumpkin samples showing yellowing symptoms. All the surveyed locations were affected and CCYV was found alone or in mixed infection with the other identified viruses. CYSDV is distributed throughout the Mediterranean (Papayiannis et al., 2005; Yakoubi et al., 2007; Lecoq and Desbiez, 2012), the Arab Emirates (Hassan and Duffus, 1991) and North America (CABI/EPPO, 2004). Its occurrence aggravates the phytosanitary status of the crop in Sudan as the virus was reported to affect cucurbit crops seriously in many production areas (Abou-Jawdah et al., 2000; Yakoubi et al., 2007). CYSDV was found in all regions investigated in the present study and with the same incidence level as CCYV. Both CCYV and CYSDV produce symptoms, such as interveinal chlorosis, yellowing and brittleness of lower leaves, which resemble those produced by nutritional deficiencies, a condition which usually leads to underrating disease incidence in the field. WmCSV was first identified in 1988 in Yemen (Jones et al., 1988) and rapidly spread across North Africa, Middle East (Iran, Jordan, Lebanon, Palestine, Oman) and Sudan (Kheyr-Pour et al., 2000; Al-Musa et al., 2011; Samsatly et al., 2012; Ali-Shtayeh et al., 2012; Khan et al., 2012), where it causes very severe damage to different cucurbits, especially in protected crops. Although, WmCSV was not detected in the Elnuba area, the prevalence of large populations of the vector (whiteflies) may cause disaster should the virus be introduced in this area. Consequently, the adoption of strict integrated control measures is needed to prevent possible future epidemics. Concerning aphid-borne viruses, the examined pumpkin samples were infected by PRSV (Mohammed et al., 2012) and ZYMV, both of which cause severe damage to pumpkins (Wakman et al., 2002) leading to yield reduction in many countries (Krstic et al., 2002; Zhao et al., 2003; Jossey and Babadoost, 2008; Pachner et al., 2011). In Sudan, ZYMV is considered as one of the major components of the viral pathosystem that dramatically decreases cucurbit production in many parts of the country (Mahgoub et al., 1997, 1998). In this study, phylogenetic analysis based on sequence comparison of the amplified genomic region for each identified virus was performed. No relevant differences were detected when Sudanese CCYV isolates were compared with each other and with CCYV sequences from GenBank, which supports Lin et al. (2012) conclusion that HSP70h is a conserved gene in criniviruses. The CP coding region has been used to study the 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 Aranda, 2005; Sweiss et al., 2007; Yakoubi et al., 2007). Although all CYSDV isolates previously reported from Africa grouped with a Western subpopulation of the virus (Rubio et al., 2001), Sudanese isolates grouped with the Eastern subpopulation suggesting a common origin with isolates from Iran and Saudi Arabia. This could possibly be attributed to the wider trade links and air traffic between Sudan and other countries in this region. Phylogenetic analysis performed with Sudanese WmCSV isolates revealed some particularities when the two DNA-A genomic regions coding for AV2 protein (complete) and CP protein (partial), were separately analyzed. In fact, AV2 sequences of WmCSV isolates from GenBank grouped in two main clusters similar to those reported for the complete DNA-A component (Khan et al., 2012), and the Sudanese isolates WmCSV-Sud C.23 and WmCSV-Sud C37 were in the same cluster with a previously identified Sudanese isolate of this virus. Upon analysis of the two Sudanese isolates in the highly conserved CP region (Wyatt and Brown, 1996), the same evolutionary linkage with reference isolates including the previously identified WmCSV Sudanese isolate was evident. In addition, the sequences identity comparison indicated that there is a close relation among WmCSV isolates from different cucurbits including pumpkin, watermelon, melon and squash. This could be attributed to the endemic populations of the whitefly (Bemisia tabaci) vector in the region and indicates that this vector has a similar epidemiological behaviour regardless of the host (Kheyr-Pour et al., 2000). Two genomic regions (CI and CP) have been studied for ZYMV and the phylogenetic analysis did not reveal evidence of any evolutionary differences between the Sudanese isolate reported here and the one previously characterized (Mahgoub et al., 1997) indicating a high degree of stability in these genes through the years. This investigation provides basic information on pumpkin viruses present in three areas important for cucurbit production within Sudan. The high incidence of both aphid- and whitefly-transmitted viruses may be due to the large population of these vectors usually observed in cucurbits and other field crops in the country. Efficient measures such as the use of plastic mulches, floating covers, vector-targeting insecticides and resistant pumpkin cultivars are required to control virus spread in field crops. ACKNOWLEDGEMENTS The authors would like to acknowledge the Ministry of Higher Education and Scientific Research, Sudan for their financial support under the grant No. 95/2011.
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