MOLECULAR CHARACTERISTICS OF ZUCCHINI YELLOW MOSAIC VIRUS (ZYMV) ON CUCUMBER PLANTS AND COMPARISON BETWEEN MOLECULAR CHARACTERISTICS OF TWO ISOLATES OF VIRUS (ZYMV) THAT INFECTS CUCURBITS IN RIYADH REGION, SAUDI ARABIA الخصائص الجزيئية لفيروس موزاييک وإصفرار الکوسة الخضراء (ZYMV) على الخيار ومقارنة الخصائص الجزيئية والحيوية لعزلتيـن من الفيروس على القرعيات في منطقة الري

MOLECULAR CHARACTERISTICS OF ZUCCHINI YELLOW MOSAIC VIRUS (ZYMV) ON CUCUMBER PLANTS AND COMPARISON BETWEEN MOLECULAR CHARACTERISTICS OF TWO ISOLATES OF VIRUS (ZYMV) THAT INFECTS CUCURBITS IN RIYADH REGION, SAUDI ARABIA

Jehan Saud Al Abrahaim

Princess Norah Bent Abdulrahman - Associated Professor in Microbiology (Virology –Bacteriology)

Jsaa242@hotmail.com

INTRODUCTION:

                Cucurbits are among the main vegetable crops grown in Saudi Arabia. Cucurbit are infected by several viruses such as Zucchini yellow mosaic virus (ZYMV), Cucumber mosaic virus (CMV) that cause severe diseases and serious yield reduction in cucurbit crops. From the economic point of view, ZYMV considers the most important cucurbit virus in KSA in general and in Riyadh in particular (Al Shahwain, 1995). One of the reasons makes it very dangerous; ZYMV induces different types of symptom including yellow mosaic, mottle, blisters, stunting, leaf and fruit deformation (Desbiez & Lecoq, 1997). ZYMV, a species of the genus Potyvirusin the family Potyviridae, with a positive sense-single stranded RNA of about 9.5 kb (Gal-On, 2007). Strains of from distinct geographic origins exhibit biological diversity, particularly concerning their host, symptomatology and aphid transmission (Desbiez et al., 2002). The virus was first reported in Italy and France by Lisa et al. (1981), and within a few years the virus had been reported from almost all cucurbit producing countries worldwide including those of Europe, Asia, Africa, North and South America, and Oceania (Desbiez & Lecoq, 1997 and Gal-On, 2007).

                The positive sense single-stranded RNA genome of ZYMV, like other potyviruses, was found to be translated as one polyprotein precursor containing the activity of viral RNA-dependent RNA polymerase (Hong et al., 1995).

This action has multifunctional activities involved in aphid transmission, cell-to-cell movement, systemic movement encapsidation of the viral RNA, and regulation of viral RNA amplification (Gal-On et al., 1992; Dolja et al., 1993;Varrelamann and Maiss, 2000 and Hong et al., 1995).

Since the 1970s, serological methods especially enzyme-linked immunosorbent assay (ELISA) have been used widely and successfully for detection of plant viruses and diagnosis of plant viral diseases (Clark and Adams, 1977). In the 1990s, nucleic acid-based methods such as reverse transcription (RT) and the polymerase chain reaction (PCR) began to be used in plant virus detection (Rowhani et al., 1995 and Thomson et al., 1995). Accordingly, several degenerate primers have been designed to recognize the conserved regions of viral genomes of many virus species or the whole virus genus or family(Tian et al., 1996 and Chen et al., 2001).

In this study, the biological and molecular methods were used to characterize the variability of ZYMV isolates from cucumber (Cucumis sativus) and pumpkin (cucurbita moschata) plants grown in Riyadh, Saudi Arabia.

MATERIALS AND METHODS:

Sample Collection:

Samples were collected during the 18-May-2009 growing seasons from field-grown cucumber (Cucumis sativus)and pumpkin (cucurbita moschata) plants grown in Riyadh, Saudi Arabia. Show symptoms such as: mosaic, yellowing, leaf distortion, hoestring, fruit deformation and yield reduction. Young leaves from some symptomatic plants were collected at random. All samples were kept in ice chests for transportation to the laboratory. Each plant sample was kept separately in a plastic bag at 4^oC until analyzed.

RNA Extraction:

Total RNA was extracted from dried (20 mg ZYMV-infected leaves using TRI-Reagent (Invitrogen, Carlsbad, USA) according to the manufacturer's instructions) resulting pellets were dissolved in 125 µl RNase-free water.

Reverse Transcription Polymerase Chain Reaction (RT-PCR):

Total RNA from ZYM-infected plants was extracted using a phenol/chloroform protocol (Wadsworth et al., 1988). Three μl of RNA were submitted to reverse transcription in a final volume of 20 μl, using 2μl PCR buffer 10x(0.5 M Tris-HCl, 0.7 M KCl, 0.1 M MgCl₂, pH 8), 1μl DTT (100 mmol/μl), 1μl dNTPs (10 mmol/ μl), 0.5 μl RNase inhibitors enzymes (10 mmol/μl) and 2 μl Reverse-DAG primer (100 pmol/μl) (5 /- GCT CCA TAC ATA GCT GAG ACA GC - 3/: nucleotide position 091206P203 on sequence L 3098 for one hour at 42^oC with 0.5 μl ZYMV reverse transcriptase (200 mmol/μl). 5 μl of the RT reactions were used for PCR using a 5 μl PCR buffer 10x, 2 μl MgCl₂, 1 μl dNTPs (10mmol/μl), 0.5 μl Taq polymerase (5 unit/μl), 1 μl Reverse-DAG (100 pmol), and 1 μl Forward-DAG (100 pmol) (5/-TAG GCT TGC AAA CGG AGT CTA ATC/: 091206P204 on sequence L 7457 oligonucleotides encompassing the N-terminal part of the coat protein coding region and the C-terminal part of the polymerase (NIb) (primers designed by (Desbiez et al., 2002). PCR reactions were performed by a first denaturation of the samples at 94^oC for 3 minutes followed by 35 cycles at 94^oC for 30 seconds, 43^oC for 30 seconds and 72^oC for 30 seconds and a final elongation step at 72^oC for 7 minutes. PCR products were controlled by electrophoresis on 1% agarose gel (Desbiez et al., 2002).

RESULTS:

RT-PCR:

Bands of about 1202 bp obtained for ZYMV1 (cucumber isolate)and ZYMV2 (pumpkin isolate) in PCR assay (Fig. 1). Accordingly, the molecular characteristics revealed that both isolates (ZYMV1 and ZYMV2) were closely related to ZYMV. The molecular characterization of the two Riyadh ZMYV isolates ZYMV1 and ZYMV2 is presented in Table 1. The isolates in Gen bank revealed that accession numbers was GU808330 and GU058058 for ZYMV1 and ZYMV2, respectively. The nucleotides locus were 804bp RNA and 98 bp RNA, for ZYMV1 and ZYMV2 respectively. The nucleotides sequence of the two isolates ZYMV1 and ZYMV2 presented molecular diversity that suggests that they did not share a common origin and they have adapted to their different hosts.

Fig. 1 : Poty primer for ZYMV1 in cucumber isolate and ZYMV2 in cucumber mosschata (pumpkin)"

RT for RNA, using random primer then PCR for poty (primer :10Mm 4+ 10n poty).

Table 1: Molecular characterization of two ZYMV isolates ZYMV1 and ZYMV2.

DISCUSSION:

ZYMV causes marked yellowing of infected cucurbit plants. According to the molecular characteristics of ZYMV1 isolated from cucumber and ZYMV2 isolated pumpkin were closely related to ZYMV. The similarity of the CP nucleotide sequences of ZYMV isolates from other countries such as Germany suggests a common origin. In Saudi, seeds of many vegetables including cucumber are regularly imported from Europe which suggests that infected seeds may have been a pathway for introducing ZYMV. Although seed transmission of ZYMV was reported at a very low rate, it could cause epidemics (Desbiez and Lecoq, 1997). Although Saudi isolates of ZYMV were closely related regarding to the CP gene identity, they showed different phenotypes in host plants tested. Moreover, Saudi isolates of ZYMV had a different host range (Al Shahwain, 1995, Al Abrahaim, 2012, under publication data). Ali et al., 2009, point out that two Syrian isolates of ZYMV were closely related to the CP gene identity, they showed different phenotypes in host plants tested. Moreover, Syrian isolates of ZYMV had a different host range. However, Safaeizadeh (2008) compared the biological and molecular variability of ZYMV in Iran and reported that, the limited variability of ZYMV strains from Iran was neither correlated with their biological variability, nor with their geographical origin in the country.

In conclusion, Serological and molecular data tend to indicate that the two Saudi ZYMV isolates were biologically variable and presented a low molecular diversity. This suggests that they share a common origin but that they have subsequently adapted to their different hosts.

The present study reports comparative biological and molecular variability of ZYMV in Saudi Arabia for the first time. The information obtained in this study will be helpful to improve control strategies for such destructive virus in KSA.

REFERENCES:

Ali, M. C., T. Maoka and Natsuaki K. T. (2009): Biological and Molecular characterize-ation of two Zucchini yellow mosaic virus isolates from Syria. Bulletin OEPP/EPPO Bulletin, 39, 99-104.

Al-Shanwan, I. M., O. A. Abdalla and Al-Saleh, M. A. (1995): Response of greenhouse- grown cucumber cultivars to an isolate of ZYMV. Plant Dis., 79 (9): 898-901.

Chen, J., Chen, J., and Adams, M. J., (2001): A universal PCR primer to detect members of the Potyviridae and its use to examine the taxonomic status of several members of the family. Arch. Virol. 146, 757–766.

Clark, M. F. and Adams, S.A.N. (1977): Characteristics of Micro Plates Method of Enzyme-linked-immunosorbent Assay for Detection of Plant Viruses. J. Gen. Virol., 34: 475-483.

Desbiez, C. and Lecoq, H. (1997): Zucchini yellow mosaic virus. Plant Pathology 46, 809–829.

Desbiez, C., Wipf-Scheibel, C. and Lecoq, H. (2002): Biological and Serological Variability, Evolution and Molecular Epidemiology of Zucchini yellow mosaic virus with Special Reference to Caribbean Islands. Virus Res., 85: 5-16.

Dolja, V. V., Herndon, K. L., Pirone, T. P. and Carrington, J. C. (1993): Spontaneous mutagenesis of a plant potyvirus genome after insertion of a foreign gene. J. Virol. 67: 5968-5975.

Gal-On A. (2007): Zucchini yellow mosaic virus: insect transmission and pathogenicity– the tails of two proteins. Molecular Plant Pathology 8, 139–150.

Gal-On, A., Antignus, Y., Rosner, A. and Raccah, B. (1992): A zucchini yellow mosaic virus coat protein gene mutation restores aphid transmissibility but has no effect on multiplication. J. Gen. Virol. 73: 2183-2187.

Hong, Y., Levay, K., Murphy, J. F., Klein, P. G., Shaw, J. G. and Hunt, A. G. (1995): A potyvirus polymerase interacts with the viral coat protein and VPg in yeast cells. Virology. 214:159-166.

Lisa, V., Baccardo, G., D., Agostino, G., Dellavalle, G., and D., Aquilio, M. (1981): Characterization of a Potyvirus that Causes Zucchini yellow mosaic. Phytopathology., 71:667-672

Rowhani, A., Maningas, M. A., Lile, L. S., Daubert, S. D., Golino, D. A., (1995): Development of a detection system for viruses of woody plants based on PCR analysis of immobilized virions. Phytopathology, 85: 347–352.

Safaeizadeh, M. (2008): Comparative biological and Molecular variability of zucchini yellow mosaic virus in Iran. Asian Journal of Plant Pathology, 2(1): 30-39.

Thomson, K. G., Dietzgen, R. G., Gibbs, A. J., Tang, Y.C., Liesack, W., Teakle, D.S. and Stackebrandt, E. (1995): Identification of zucchini yellow mosaic potyvirus by RT-PCR and analysis of sequence variability. J. Virol. Methods, 55:83-96.

Tian T., Klaassen V.A., Soong J., Wisler G., Duffus J. E. and Falk B. W., (1996): Generation of cDNAs specific to Lettuce infectious yellows closterovirus and other whitefly-transmitted viruses by RT-PCR and degenerate oligonucleotide primers corresponding to the closterovirus gene encoding the heat shock protein 70 homolog. Phytopathology, 86: 1167-1173.

Varrelamann, M. and Maiss, E. (2000): Mutations in the coat protein gene of plum pox virus suppress particle assembly, heterologous encapsidation and complementation in transgenic plants of Nicotiana benthamiana. J. Gen. Virol. 81: 567-576.

Wadsworth G. J., Redibaugh, M. G. and Scandalios, J. G. (1998): A Procedure for the Small-scale Isolation of Plant RNA Suitable for RNA Blot Analysis. Anal. Biochem., 172: 279-283.

MOLECULAR CHARACTERISTICS OF ZUCCHINI YELLOW MOSAIC VIRUS (ZYMV) ON CUCUMBER PLANTS AND COMPARISON BETWEEN MOLECULAR CHARACTERISTICS OF TWO ISOLATES OF VIRUS (ZYMV) THAT INFECTS CUCURBITS IN RIYADH REGION, SAUDI ARABIA

Jehan Saud Al Abrahaim

Princess Norah Bent Abdulrahman - Associated Professor in Microbiology (Virology –Bacteriology)

Jsaa242@hotmail.com

INTRODUCTION:

                Cucurbits are among the main vegetable crops grown in Saudi Arabia. Cucurbit are infected by several viruses such as Zucchini yellow mosaic virus (ZYMV), Cucumber mosaic virus (CMV) that cause severe diseases and serious yield reduction in cucurbit crops. From the economic point of view, ZYMV considers the most important cucurbit virus in KSA in general and in Riyadh in particular (Al Shahwain, 1995). One of the reasons makes it very dangerous; ZYMV induces different types of symptom including yellow mosaic, mottle, blisters, stunting, leaf and fruit deformation (Desbiez & Lecoq, 1997). ZYMV, a species of the genus Potyvirusin the family Potyviridae, with a positive sense-single stranded RNA of about 9.5 kb (Gal-On, 2007). Strains of from distinct geographic origins exhibit biological diversity, particularly concerning their host, symptomatology and aphid transmission (Desbiez et al., 2002). The virus was first reported in Italy and France by Lisa et al. (1981), and within a few years the virus had been reported from almost all cucurbit producing countries worldwide including those of Europe, Asia, Africa, North and South America, and Oceania (Desbiez & Lecoq, 1997 and Gal-On, 2007).

                The positive sense single-stranded RNA genome of ZYMV, like other potyviruses, was found to be translated as one polyprotein precursor containing the activity of viral RNA-dependent RNA polymerase (Hong et al., 1995).

This action has multifunctional activities involved in aphid transmission, cell-to-cell movement, systemic movement encapsidation of the viral RNA, and regulation of viral RNA amplification (Gal-On et al., 1992; Dolja et al., 1993;Varrelamann and Maiss, 2000 and Hong et al., 1995).

Since the 1970s, serological methods especially enzyme-linked immunosorbent assay (ELISA) have been used widely and successfully for detection of plant viruses and diagnosis of plant viral diseases (Clark and Adams, 1977). In the 1990s, nucleic acid-based methods such as reverse transcription (RT) and the polymerase chain reaction (PCR) began to be used in plant virus detection (Rowhani et al., 1995 and Thomson et al., 1995). Accordingly, several degenerate primers have been designed to recognize the conserved regions of viral genomes of many virus species or the whole virus genus or family(Tian et al., 1996 and Chen et al., 2001).

In this study, the biological and molecular methods were used to characterize the variability of ZYMV isolates from cucumber (Cucumis sativus) and pumpkin (cucurbita moschata) plants grown in Riyadh, Saudi Arabia.

MATERIALS AND METHODS:

Sample Collection:

Samples were collected during the 18-May-2009 growing seasons from field-grown cucumber (Cucumis sativus)and pumpkin (cucurbita moschata) plants grown in Riyadh, Saudi Arabia. Show symptoms such as: mosaic, yellowing, leaf distortion, hoestring, fruit deformation and yield reduction. Young leaves from some symptomatic plants were collected at random. All samples were kept in ice chests for transportation to the laboratory. Each plant sample was kept separately in a plastic bag at 4^oC until analyzed.

RNA Extraction:

Total RNA was extracted from dried (20 mg ZYMV-infected leaves using TRI-Reagent (Invitrogen, Carlsbad, USA) according to the manufacturer's instructions) resulting pellets were dissolved in 125 µl RNase-free water.

Reverse Transcription Polymerase Chain Reaction (RT-PCR):

Total RNA from ZYM-infected plants was extracted using a phenol/chloroform protocol (Wadsworth et al., 1988). Three μl of RNA were submitted to reverse transcription in a final volume of 20 μl, using 2μl PCR buffer 10x(0.5 M Tris-HCl, 0.7 M KCl, 0.1 M MgCl₂, pH 8), 1μl DTT (100 mmol/μl), 1μl dNTPs (10 mmol/ μl), 0.5 μl RNase inhibitors enzymes (10 mmol/μl) and 2 μl Reverse-DAG primer (100 pmol/μl) (5 /- GCT CCA TAC ATA GCT GAG ACA GC - 3/: nucleotide position 091206P203 on sequence L 3098 for one hour at 42^oC with 0.5 μl ZYMV reverse transcriptase (200 mmol/μl). 5 μl of the RT reactions were used for PCR using a 5 μl PCR buffer 10x, 2 μl MgCl₂, 1 μl dNTPs (10mmol/μl), 0.5 μl Taq polymerase (5 unit/μl), 1 μl Reverse-DAG (100 pmol), and 1 μl Forward-DAG (100 pmol) (5/-TAG GCT TGC AAA CGG AGT CTA ATC/: 091206P204 on sequence L 7457 oligonucleotides encompassing the N-terminal part of the coat protein coding region and the C-terminal part of the polymerase (NIb) (primers designed by (Desbiez et al., 2002). PCR reactions were performed by a first denaturation of the samples at 94^oC for 3 minutes followed by 35 cycles at 94^oC for 30 seconds, 43^oC for 30 seconds and 72^oC for 30 seconds and a final elongation step at 72^oC for 7 minutes. PCR products were controlled by electrophoresis on 1% agarose gel (Desbiez et al., 2002).

RESULTS:

RT-PCR:

Bands of about 1202 bp obtained for ZYMV1 (cucumber isolate)and ZYMV2 (pumpkin isolate) in PCR assay (Fig. 1). Accordingly, the molecular characteristics revealed that both isolates (ZYMV1 and ZYMV2) were closely related to ZYMV. The molecular characterization of the two Riyadh ZMYV isolates ZYMV1 and ZYMV2 is presented in Table 1. The isolates in Gen bank revealed that accession numbers was GU808330 and GU058058 for ZYMV1 and ZYMV2, respectively. The nucleotides locus were 804bp RNA and 98 bp RNA, for ZYMV1 and ZYMV2 respectively. The nucleotides sequence of the two isolates ZYMV1 and ZYMV2 presented molecular diversity that suggests that they did not share a common origin and they have adapted to their different hosts.

Fig. 1 : Poty primer for ZYMV1 in cucumber isolate and ZYMV2 in cucumber mosschata (pumpkin)"

RT for RNA, using random primer then PCR for poty (primer :10Mm 4+ 10n poty).

Table 1: Molecular characterization of two ZYMV isolates ZYMV1 and ZYMV2.

DISCUSSION:

ZYMV causes marked yellowing of infected cucurbit plants. According to the molecular characteristics of ZYMV1 isolated from cucumber and ZYMV2 isolated pumpkin were closely related to ZYMV. The similarity of the CP nucleotide sequences of ZYMV isolates from other countries such as Germany suggests a common origin. In Saudi, seeds of many vegetables including cucumber are regularly imported from Europe which suggests that infected seeds may have been a pathway for introducing ZYMV. Although seed transmission of ZYMV was reported at a very low rate, it could cause epidemics (Desbiez and Lecoq, 1997). Although Saudi isolates of ZYMV were closely related regarding to the CP gene identity, they showed different phenotypes in host plants tested. Moreover, Saudi isolates of ZYMV had a different host range (Al Shahwain, 1995, Al Abrahaim, 2012, under publication data). Ali et al., 2009, point out that two Syrian isolates of ZYMV were closely related to the CP gene identity, they showed different phenotypes in host plants tested. Moreover, Syrian isolates of ZYMV had a different host range. However, Safaeizadeh (2008) compared the biological and molecular variability of ZYMV in Iran and reported that, the limited variability of ZYMV strains from Iran was neither correlated with their biological variability, nor with their geographical origin in the country.

In conclusion, Serological and molecular data tend to indicate that the two Saudi ZYMV isolates were biologically variable and presented a low molecular diversity. This suggests that they share a common origin but that they have subsequently adapted to their different hosts.

The present study reports comparative biological and molecular variability of ZYMV in Saudi Arabia for the first time. The information obtained in this study will be helpful to improve control strategies for such destructive virus in KSA.

REFERENCES:

Ali, M. C., T. Maoka and Natsuaki K. T. (2009): Biological and Molecular characterize-ation of two Zucchini yellow mosaic virus isolates from Syria. Bulletin OEPP/EPPO Bulletin, 39, 99-104.

Al-Shanwan, I. M., O. A. Abdalla and Al-Saleh, M. A. (1995): Response of greenhouse- grown cucumber cultivars to an isolate of ZYMV. Plant Dis., 79 (9): 898-901.

Chen, J., Chen, J., and Adams, M. J., (2001): A universal PCR primer to detect members of the Potyviridae and its use to examine the taxonomic status of several members of the family. Arch. Virol. 146, 757–766.

Clark, M. F. and Adams, S.A.N. (1977): Characteristics of Micro Plates Method of Enzyme-linked-immunosorbent Assay for Detection of Plant Viruses. J. Gen. Virol., 34: 475-483.

Desbiez, C. and Lecoq, H. (1997): Zucchini yellow mosaic virus. Plant Pathology 46, 809–829.

Desbiez, C., Wipf-Scheibel, C. and Lecoq, H. (2002): Biological and Serological Variability, Evolution and Molecular Epidemiology of Zucchini yellow mosaic virus with Special Reference to Caribbean Islands. Virus Res., 85: 5-16.

Dolja, V. V., Herndon, K. L., Pirone, T. P. and Carrington, J. C. (1993): Spontaneous mutagenesis of a plant potyvirus genome after insertion of a foreign gene. J. Virol. 67: 5968-5975.

Gal-On A. (2007): Zucchini yellow mosaic virus: insect transmission and pathogenicity– the tails of two proteins. Molecular Plant Pathology 8, 139–150.

Gal-On, A., Antignus, Y., Rosner, A. and Raccah, B. (1992): A zucchini yellow mosaic virus coat protein gene mutation restores aphid transmissibility but has no effect on multiplication. J. Gen. Virol. 73: 2183-2187.

Hong, Y., Levay, K., Murphy, J. F., Klein, P. G., Shaw, J. G. and Hunt, A. G. (1995): A potyvirus polymerase interacts with the viral coat protein and VPg in yeast cells. Virology. 214:159-166.

Lisa, V., Baccardo, G., D., Agostino, G., Dellavalle, G., and D., Aquilio, M. (1981): Characterization of a Potyvirus that Causes Zucchini yellow mosaic. Phytopathology., 71:667-672

Rowhani, A., Maningas, M. A., Lile, L. S., Daubert, S. D., Golino, D. A., (1995): Development of a detection system for viruses of woody plants based on PCR analysis of immobilized virions. Phytopathology, 85: 347–352.

Safaeizadeh, M. (2008): Comparative biological and Molecular variability of zucchini yellow mosaic virus in Iran. Asian Journal of Plant Pathology, 2(1): 30-39.

Thomson, K. G., Dietzgen, R. G., Gibbs, A. J., Tang, Y.C., Liesack, W., Teakle, D.S. and Stackebrandt, E. (1995): Identification of zucchini yellow mosaic potyvirus by RT-PCR and analysis of sequence variability. J. Virol. Methods, 55:83-96.

Tian T., Klaassen V.A., Soong J., Wisler G., Duffus J. E. and Falk B. W., (1996): Generation of cDNAs specific to Lettuce infectious yellows closterovirus and other whitefly-transmitted viruses by RT-PCR and degenerate oligonucleotide primers corresponding to the closterovirus gene encoding the heat shock protein 70 homolog. Phytopathology, 86: 1167-1173.

Varrelamann, M. and Maiss, E. (2000): Mutations in the coat protein gene of plum pox virus suppress particle assembly, heterologous encapsidation and complementation in transgenic plants of Nicotiana benthamiana. J. Gen. Virol. 81: 567-576.

Wadsworth G. J., Redibaugh, M. G. and Scandalios, J. G. (1998): A Procedure for the Small-scale Isolation of Plant RNA Suitable for RNA Blot Analysis. Anal. Biochem., 172: 279-283.