International Journal of Virology

Year: 2015 | Volume: 11 | Issue: 1 | Page No.: 1-11
DOI: 10.3923/ijv.2015.1.11

Serological and Molecular Characterization of Cucurbit chlorotic yellows virus Affecting Cucumber Plants in Egypt

Mahmoud Ahmed Amer

Abstract: This study was aimed to identify the causal agent inducing virus-like symptoms on cucumber plants for the first report of Cucurbit chlorotic yellows virus (CCYV) in Egypt. A total of 45 symptomatic and asymptomatic cucumber samples were collected from five locations in El-Behira, government, Egypt during June, 2014. Out of 45 samples analyzed by DAS-ELISA, twenty two samples were found infected by CCYV only, two and four samples showed mixed infection with Cucurbit yellow stunting disorder virus+CCYV and Cucurbit aphid-borne yellows virus+CCYV, respectively, whereas the rest of the samples were negative to all three viruses. No RT-PCR products were obtained with all tested samples when the specific primer for Beet pseudo-yellows virus was used. Total RNA was extracted from ELISA positive samples for CCYV and a 353-bp DNA fragment of the HSP70 gene was amplified by RT-PCR using specific primer for CCYV. The synthesized cDNA probe for CCYV was used to confirm the detection of CCYV in singly and mixed infections using dot blot hybridization. The highest similarity (100%) was found with 22 isolates of CCYV isolated from melon, watermelon, cucumber and muskmelon in Japan, China, Taiwan, Lebanon and Sudan was found but the lowest similarity (94.3%) was found with two isolates from cucumber and melon in Iran. To our knowledge, this is the first report of CCYV on cucumber in Egypt.

Keywords: RT-PCR, HSP70 gene, ELISA, DNA sequencing and CCYV

INTRODUCTION

Cucurbits are of great economic importance among the major vegetable crops in Egypt, where total production of cucurbits reached 3972500 t from a cultivated area of 528588 feddan (1 feddan = 0.42 ha) in 2012 (Ministry of Agriculture and FAO reports). In the Mediterranean region, at least 28 viruses have been reported to infect cucurbit crops (Lecoq, 2003; Lecoq and Desbiez, 2012; Abrahamian et al., 2013) and several are discovered each year (Hassan and Duffus, 1990; Brown et al., 2011; Lecoq et al., 2011; Dong et al., 2012; Omar and Bagdady, 2012; El-Rahmany et al., 2014). Cucurbit yellow stunting disorder virus (CYSDV) and Cucurbit chlorotic yellows virus (CCYV) are two emergent viruses that belong to the genus Crinivirus in the family Closteroviridae. The CCYV is a novel cucurbit-infecting crinivirus which was first identified in melon crops in Kumamoto, Japan, in 2004 (Gyoutoku et al., 2008) and is now widespread throughout the country (Furuta et al., 2008; Okuda et al., 2010). Since its first report in 2004 in Japan (Gyoutoku et al., 2008) CCYV has spread to Taiwan (Huang et al., 2010), China (Gu et al., 2011; Zeng et al., 2011), Sudan (Hamed et al., 2011), Saudi Arabia (Al-Saleh et al., 2014) and Lebanon (Abrahamian et al., 2012). CCYV remains a tentative member of the genus Crinivirus since it was first fully sequenced in 2010 (Okuda et al., 2010). CCYV has a typical bipartite crinivirus genome, encoding four proteins in RNA1 and eight in RNA2 (Okuda et al., 2010). Recently, mixed infections by CYSDV and CCYV were reported in commercially cultivated cucurbits (Abrahamian et al., 2012). Both viruses cause considerable damage mainly to the Cucurbitaceae family and can infect field crops or weeds (Wintermantel et al., 2009; Okuda et al., 2010). In addition to CCYV and CYSDV, other known whitefly-transmitted yellowing viruses are Lettuce infectious yellows virus (LIYV), Tomato infectious chlorosis virus (TICV), Tomato chlorosis virus (ToCV), Sweet potato chlorotic stunt virus (SPCSV), Potato yellow vein virus (PYVV) and Lettuce chlorosis virus (LCV) (Hartono et al., 2003; Boubourakas et al., 2006; Tzanetakis et al., 2013). CCYV and CYSDV are transmitted by Bemisia tabaci Mediterranean (MED) (previously biotype Q) and MEAM1 complexes in a semi-persistent manner (Gyoutoku et al., 2008; De Barro et al., 2011). Symptoms of Criniviruses develop on older leaves and progress toward newer growth inducing nutrition deficiency-like symptoms, interveinal chlorosis, brittleness and thickening of leaves. Symptoms induced by CYSDV, CCYV and other Criniviruses are very similar and their differentiation relies on the application of laboratory-based detection techniques (Wintermantel and Wisler, 2006). CCYV causes chlorotic leaf spots and complete yellowing of leaves of cucumber (Cucumis sativus), melon (C. melo) and watermelon (Citrullus lanatus) plants, resulting in severe economic losses. For efficient control of CCYV, simple and accurate diagnosis is urgently required. Several detection methods for CCYV using Enzyme-linked Immunosorbent Assay (ELISA), Tissue-Blot Immuno Assay (TBIA), Dot-Blot Immuno Assay (DBIA), dot-blot hybridization and Reverse Transcription (RT), Polymerase Chain Reaction (PCR) (Hourani and Abou-Jawdah, 2003; Ruiz et al., 2002; Gyoutoku et al., 2008). During 2014, yellowing symptoms were frequently observed in cultivated cucumber fields. The present study aim to investigate the incidence of the CCYV was the causal agent of yellowing disease of cucumber, also, conducted a preliminary analysis to genetically characterize Egyptian CCYV isolates. To achieve this, serological and molecular methods such as DAS-ELISA, RT-PCR, dot blot hybridization and sequencing analysis were applied for identification of the CCYV.

MATERIALS AND METHODSM

Sample collection: Seven cucumber open fields were surveyed in different locations 48 symptomatic and two asymptomatic cucumber samples from seven open fields distributed in a geographical area in El-Behira government, Egypt during June, 2014 were collected for virus identification. Viral symptoms included chlorotic spots followed by yellowing of whole leaves and thickening of older leaves were observed. A maximum of ten samples per field from symptomatic plants were collected and processed in Plant Virology Lab, Plant Protection Department, College of Food and Agricultural Sciences, King Saud University.

Serological detection: The presence of CCYV and other common cucurbit viruses (Cucurbit chlorotic yellows virus (CCYV), Cucurbit yellow stunting disorder virus (CYSDV), Cucumber mosaic virus (CMV), Watermelon mosaic virus (WMV), Zucchini yellow mosaic virus (ZYMV) and Cucurbit aphid-borne yellows virus (CABYV) in samples was ascertained by using Double-Antibody Sandwich (DAS) ELISA as described by Clark and Adams (1977) with polyclonal antibodies obtained from LOEWE CO, Germany) and polyclonal antibodies of CABYV was kindly provided by Dr. H. Lecoq (INRA Montfavet, France were used to serologically identify the viruses infecting cucumber). Leaf samples were ground in a pre-cooled mortar and pestle with an extraction buffer (PBST: 0.13 M NaCl, 0.003 M KCl, 0.008 M Na₂HPO₄, 0.001 M KH₂PO₄, pH 7.4) containing 0.05% Tween 20 and 0.1% nonfat dry milk and were placed in wells that had been pre-coated with specific polyclonal antisera diluted in a carbonate buffer (pH 9.6). Plates were incubated at 4°C overnight and washed three times with PBST-Tween 20 buffer. Plates were then coated with alkaline phosphatase conjugated antibody diluted in extraction buffer and incubated for 2 h at 37°C. After washing, p-nitrophenyl phosphate in diethanolamine substrate buffer (0.5 mg mL^-1, pH 9.8) was added to each well and incubated at room temperature for 30-120 min. The reaction was detected colorimetrically at A405 nm using an ELISA reader (BioTek, Instruments, Inc., USA). Two wells were used per sample. Virus-free cucurbit species grown in insect- proof cages were used as negative controls. Positive and healthy controls were included in all tests. Samples were considered to be positive if the A405 nm values were more than three times those of the healthy control.

Reverse transcription-polymerase chain reaction (RT-PCR) detection: The original field samples were used to RNA extraction using Isolate II RNA Kit (Bioline) according to the manufacturer’s protocol. RT-PCR was carried out using One-step RT-PCR reagent (PrimeScript One-step RT-PCR kit ver 2; Takara BIO. Inc. Japan). Amplifications were carried out in a Mastercycler gradient (Eppendorf, Germany). A pair of primers, BPYV I: 5’-tcg aaa gtc caa caa gac gt-3’ and BPYV II: 5’-ctg atg gtg cgc gag tg-3’), were used to detect genera Beet pseudo-yellows virus (BPYV) according to Boubourakas et al. (2006). The HSP70h gene region of CCYV was amplified using Crini-s2 (5’-cat tcc tac ctg ttt agc ca-3’) and Crini-as2 (5’-tgc act tat aat ctg ctg gta c-3’) primer designed according to Hamed et al. (2011) from CCYV sequences available at GenBank to confirm virus infection under the following reaction conditions: RT, 42°C for 30 min and 94°C for 2 min; PCR, 35 cycles of 94°C for 30 sec, 55°C for 30 sec and 72°C for 30 sec, followed by 72°C for 5 min. The PCR amplification products were then examined by agarose gel electrophoresis (1.5% agarose, TAE), followed by ethidium bromide staining according to Sambrook and Russell (2001).

Dot blot hybridization assay: The obtained RT-PCR product (353 bp) of partial Hsp70h gene of these CCYV was purified using Matrix Gel Extraction System, Life Technologies and directly labeled with digoxigenin (DIG) labeling system according to Feinberg and Vogelstein (1983) and Holtke and Kessler (1990). Sap extractions were prepared from all collected samples from cucumber (45 samples) by grinding according to Laulhere and Rozier (1976) and Podleckis et al. (1993). The DNA was fixed on the membranes by Ultraviolet cross-linked for 30 sec. Prehybridization, hybridization and immunological detection were carried out using the DIG DNA labeling and Detection kit (Roche Diagnostics) according to the manufacturer’s recommendation.

Partial nucleotide sequence cloning and analysis of CCYV HSP70h gene: Amplified RT-PCR products subsequently were ligated into pGEM-T Easy vector (Promega, Madison, WI) according to the manufacturer’s instructions and transformed into DH5a Escherichia coli cells. CCYV DNA products from RT-PCR were used directly for sequence analysis. Ethidium bromide strained DNA bands of interest were purified (Matrix Gel Extraction System, Life Technologies).

The nucleotide sequence of the partial Hsp70h gene of these CCYV isolates detected in cucumber (EG-B1, EG-B2 and EG-B3) were determined with the same primers as those used for amplification using an ABI 310 DNA Analyzer (Perkin Elmer Applied Biosystems, HITACHI). Alignment analysis for calculation of percentage identity was carried out using the DNASTAR program. Analysis of the obtained sequences was carried out using the DNASTAR software trial. To achieve a valid comparison, 30 CCYV isolate sequences belonging to different countries obtained from GenBank database (Table 1) were reduced to the longitude of the isolated sequences in this study. Sequences used for comparisons were retrieved from GenBank (www.ncbi.nlm.nih.gov). The accession numbers of international CCYV isolates, along with the country of origin, are shown in Table 1. The partial of Hsp70h gene sequence of three Egyptian CCYV isolates; EG-B1, EG-B2 and EG-B3 have been deposited in the EMBL/GenBank/DDBJ databases.

RESULTS

Detection and identification of CCYV in infected field samples by DAS-ELISA: Symptoms on infected cucumber plants included yellowing and thickening of the basal and older leaves which occasionally also showed slight epinasty, or resulted in flower abortion and a reduction in the number of fruits per plant. Frequently, the chlorotic basal leaves of older plants developed large interveinal necrotic areas (Fig. 1). Forty eight samples of symptomatic plants taken from seven cucumber fields in El-Behira, government, Egypt, were analyzed. Twenty samples were found infected by CCYV only, two samples showed mixed infection with CYSDV and CCYV, four samples showed mixed infection with CABYV and CCYV whereas the rest of the samples were negative to all three viruses when DAS-ELISA against CYSDV, CABYV and CCYV using specific polyclonal antiserum.

RT-PCR: The obtained results reveled that no RT-PCR products (251 bp) were obtained with all tested samples when the specific primers for BPYV (BPYV-CP-5' and BPYV-CP-3') was used. Six out of twenty six samples that were positive using DAS-ELISA for CCYV infection were checked by RT-PCR to confirm CCYV identification. DNA amplification product of approximately 353 bp was observed in samples that were positive to CCYV in DAS-ELISA. No DNA product was amplified in healthy plant extracts (Fig. 2).

Dot blot hybridization: A cDNA probe for CCYV was synthesized using the Crini-s2 and Crini-as2 primer. Figure 3 illustrate dot blot hybridization of total RNA extraction from all naturally symptomatic and asymptomatic cucumber plant samples collected from El-behira government. Positive hybridization reaction indicated by formation of purple color on the nitrocellulose membrane was observed in RNA extracts of 26 out of 45 cucumber samples but no hybridization was observed between the cDNA probe and nucleic acid extraction from the rest collected samples (Fig. 3). These results were confirming the obtained results from DAS- ELISA and RT-PCR analysis.

Partial nucleotide sequence of CCYV HSP70h gene: The RT-PCR products obtained with the leaf samples were cloned and sequenced to identify the suspected CCYV isolates. The HSP70h which was amplified with the primers Crini-s2 and Crini-as2 primer (353 bp) were sequenced and submitted in GenBank under accession numbers (KP204871, KP204872 and KP204873). Phylogenetic analysis was carried out for the partial sequences of the HSP70h of the obtained sequences for Egyption isolates (EG-B1, EG-B2 and EG-B3) and representative sequences of CCYV isolates from Japan, China, Taiwan, Iran, Lebanon, Sudan and Saudi Arabia as shown in Table 1. The phylogenetic tree showed that all Egyptian CCYV isolates in this study are in one clusters with them.

The analysis also showed that the highest nucleotide identities were 100% obtained with two isolates (AB457591 and AB523789) of CCYV from Japan isolated from melon, seven isolates (GU721108, GU721109, GU721110, GU721111, HM581659, HQ148667 and JQ904629) isolated from melon, watermelon, cucumber and whitefly from China, three isolates (JF502222, JN126045 and JN126046) isolated from melon and muskmelon from Taiwan, eight isolates (KC633822, KC633823, KC633825, KC633826, KC633827, KC990509, KC990510 and KC990511) isolated from cucumber from Lebanon and two isolates isolated from pumpkin from Sudan (KC677623 and KC677624). While it was 95.5% with CCYV isolate from watermelon and melon (GU721106 and GU721107) from China and 4 isolates isolated from cucumber from Greece, Lebanon and Saudi Arabia (HG939521, KC633821, KC633824 and LM653107). The lowest similarity (94.3%) also was found with the Sem6 (KC559449) and Sem26 (KC559450) isolates from Iran isolated from cucumber and melon respectively when compared with the five isolates of CCYV from Egypt (Fig. 4 and Table 1).

DISCUSSION

Cucurbit viruses have always caused major losses in the quantity and quality of cucurbit crops in many countries of the world and they represent one of the most important limiting factors for growers (Provvidenti, 1996). In recent decades, approximately 39 different virus species have been reported to infect cucurbits naturally (Lecoq, 2003). Yellowing virus diseases (BPYV, CYSDV, CCYV and CABYV) are emerging as a major agricultural threat causing significant yield losses in cucumber and cucurbits. Several cucumber-growing regions of cucumber plants in open-field crops in El-Behira, government, Egypt were collected with symptoms similar to those induced by CCYV (interveinal chlorosis of middle leaves and yellowing of older leaves). Similar symptoms were observed in 2012 in open field watermelon (Citrullus lanatus) plants in Rhodes and in November 2013 in a cucumber greenhouse in Tympaki, Crete (Orfanidou et al., 2014). The yellowing symptoms differed considerably from three endemic viral diseases of cucurbits, Watermelon mosaic virus, Zucchini yellow mosaic virus and Cucumber mosaic virus (Avgelis, 1983; Boubourakas et al., 2006). Symptoms of affected cucurbit plants appeared to be more similar to those caused by whitefly transmitted yellowing criniviruses, Cucurbit yellow stunting disorder virus and Beet pseudo-yellows virus, as well as Cucurbit chlorotic yellows virus (Okuda et al., 2010), or even similar to the aphid transmitted polerovirus, CABYV which also induces yellowing symptoms in cucurbits (Omar and Bagdady, 2012). Mixed infection was found in approximately 12.5% of infected cucumber samples, whereas out of 45 samples, two samples showed mixed infection with CYSDV+ CCYV and four samples showed mixed infection with CABYV+CCYV when DAS-ELISA was used. In Lebanon, some 13% of melon and 30% of squash plants showed mixed infections of ZYMV+CABYV (Abou-Jawdah et al., 2000). The highest incidence (15%) was found in cucurbits of Turkey infected with ZYMV+WMV (Sevik and Arli-Sokmen, 2003). Mixed infections are associated with enhanced symptom expression and synergistic effects are reported for viruses in the family Luteovirridae (Savenkov and Valkonen, 2001). The high incidence of CCYV found in this study, together with the less number of mixed infections, suggests that CCYV is an important threat for cucurbit crops in Egypt. CCYV has been reported only in Japan, China, Taiwan, Lebanon and Sudan (Gu et al., 2011; Zeng et al., 2011; Hamed et al., 2011; Huang et al., 2010; Abrahamian et al., 2013; Bananej et al., 2013; Orfanidou et al., 2014). Symptoms and the presence of CCYV were confirmed by RT-PCR (Hamed et al., 2011). The cDNAs for the Hsp70h gene of CCYV genome from three CCYV isolates chosen randomly from the previous survey were produced by RT-PCR. For all samples, the expected RT-PCR products were obtained and were directly sequenced with previous cloning. Thus, fragments of 353 pb, corresponding to the partial sequences of Hsp70h gene was sequenced. The alignment of partial Hsp70h gene nucleotide sequences of three CCYV Egyptian isolates showed 100% identity, when compared with other CCYV isolates from Japan, China, Taiwan, Lebanon and Sudan while the lowest similarity (94.3%) was found with the Sem6 and Sem26 isolates from Iran isolated from cucumber and melon, respectively when compared with the five isolates of CCYV from Egypt. These isolates did not only show high similarity among themselves but they also showed significant similarity with isolates of CCYV in some neigh-boring countries, as well as in distantly located Asia and European countries too, suggesting probable transmission of this virus between these countries through transmission methods such as seeds and whiteflies.

A non-radioactive dot-blot hybridization technique have been used to detect several plant viruses and have been shown to be more sensitive and more specific than serology (Eiras et al., 2001; Sanchez-Navarro et al., 1996, 1998; James et al., 1999; Kataya et al., 2008). In dot-blot hybridization experiments, the virus could be detected using a DIG-labelled probe. Non-isotopic dot-blot hybridization becomes more accessible to non-specialized or less well equipped laboratories, since leaf samples can be homogenized even in the field and sap extracts applied immediately to nylon membranes. The necessity to fix the RNA onto the membrane using a UV light source is a disadvantage but the technique has several advantages: speed, the probe can be re-used up to three times and hundreds of samples can be processed simultaneously and samples can be stored until required. This becomes essential when early detection is required, particularly with the known phloem tropism and low virus concentration of criniviruses in plants (Kataya et al., 2008).

Phylogenetic tree constructed by DNASTAR (Fig. 4) divided CCYV isolates into five clusters: Group I include isolates from Japan, Sudan, China and two isolates isolated from muskmelon from Taiwan. Group II included the two isolates isolated from cucumber from Iran. Group III included the five isolates isolated from cucumber from Lebanon. Group VI included the two isolates isolated from cucumber from Lebanon. Group VII included the two isolates isolated from cucumber from Greece and Saudi Arabia, whereas the Group VIII include the three Egyptian isolates of CCYV from Egypt. The data presented here show that it was one of the most prevalent and wide-spread viruses in open field crops of the El-behira region. In agreement with the present findings, field-grown cucurbit crops (cucumber, melon, squash and watermelon) from Iran, China, Greece, Lebanon, also showed high CCYV incidence (Gu et al., 2011; Zeng et al., 2011; Abrahamian et al., 2012; Bananej et al., 2013; Orfanidou et al., 2014). In conclusion, symptoms, RT-PCR and sequencing results confirm the presence and establishment of CCYV in cucumber crops in Egypt. It is remarkable that the sequences obtained from the Egyptian samples show only negligible sequence differences from some Iran isolates. Because of the large whitefly vector populations, the spread of CCYV to neighboring countries in Africa and potentially southern Europe, or wherever cucurbits are grown, can be expected. To our knowledge, this is the first report of CCYV in Egypt.

ACKNOWLEDGMENTS

The author gratefully acknowledges the financial support by the Agricultural Research Center, Deanship of Scientific Research of, King Saud University, Riyadh, King Saud University. A special thank also to Dr. M.A. Al-Saleh at Plant Protection Department, College of Food and Agricultural Sciences, King Saud University, for useful advice, suggestion and provision of the necessary facilities valuable for this research.

REFERENCES