Abstract: Virus infection of Lagenaria breviflora and Coccinia barteri were observed in Calabar, Nigeria during the 2005/2006 growing season. The viruses causing the diseases were characterized in this study. Diagnostic tools were host range, transmission studies, cytopathology, electron microscopy, Immunosorbent Electron Microscopy (ISEM), serology and coat protein gene sequencing. Evidence from biological, serological and sequence data confirmed that both viruses belong to the genus Potyvirus, family Potyviridae. Both were mechanically transmissible and also transmitted by Myzus persicae and Aphis gossyppi in a fore-gut manner. They also induced cytoplasmic inclusions in infected leaf tissues. The Lagenaria virus isolate has a coat protein molecular weight of 32.5 kDa and 35.0 kDa for Coccinia isolate. Of the two viruses only Lagenaria virus isolate showed cross reactivity with MWMV in DAS-ELISA. Both viruses reacted negatively with antisera to some notable cucurbit viruses in the same test, showed weak to moderate decorations with antisera to PRSV, TuMV and TeMV in immunosorbent electron microscopy (ISEM) tests. The Coccinia virus was, however, strongly decorated by antiserum to MWMV but no decoration with the Lagenaria virus. Comparison of the amino acid sequence data of the N-terminal regions of the coat proteins to that of MWMV reported from Sudan indicated 92% and 93% identities for the Coccinia and Lagenaria viruses, respectively. It is suggested that the virus isolates reported in the study be considered strains of the MWMV Sudan isolate. This is the first report of the occurrence of MWMV in Nigeria.
INTRODUCTION
Lagenaria bleviflora (Benth.) Roberty (Adenopus breviflorus) is a member of the Cucurbitaceae family. It is characterized by glabrous stems and leaves which are distinctly 5-lobed. Arising from the axils of the leaves are branched tendrils. The plant is monoecious but the male and female flowers are borne separately on the same plant. The fruits are roundish, streaked and flattened at both ends (Burkill, 2004). Economically, the seeds are considered a good source of most essential amino acids comparable to soybean (Oshodi, 1996) and oil (85.1% unsaturated fatty acid and 65.3% linoleic acid) suggesting potential uses in soap making, shoe polish, shampoo and edible purposes (Akintayo and Bayer, 2002).
Coccinia barteri (Hork.f) Kay is also a member of the Cucurbitaceae family. It is herb characterized by unbranched tendrils which arise from the axils of leaves by which it attaches itself to supports. The leaves are variable in shape, more or less deeply 3-5 lobed, shining, glossy and dark green in colour often with white blotches. The male and female flowers are borne in a raceme and the fruits are streaked and ellipsoidal (Holstein and Renner, 2010). It is a pot-herb and of medicinal importance in Cross River State of Nigeria.
Globally, Zucchini Yellow Mosaic Virus (ZYMV), Papaya Ringspot Virus (PRSV) and Watermelon mosaic virus-2 and Cucumber Mosaic Virus (CMV) are considered among the most economically important viruses infecting cucurbits (Yardimci and Korkmaz, 2004; Lecoq et al., 2001; Fattouh, 2003; Salem et al., 2007; Massumi et al., 2007; Gholamalizadeh et al., 2008; Safaeizadeh, 2008). Others, though of limited distribution but of no less importance, are Zucchini Lethal Chlorotic Virus (ZLCV) (Yuki et al., 2000), Watermelon leaf mottle virus (De Sa et al., 2000), Zucchini green mottle mosaic virus (Choi, 2001), Melon Necrotic Spot Virus (MNSV) (Choi et al., 2003), Kyuri green mottle mosaic virus (Ko et al., 2006), Zucchini yellow fleck virus (Desbiez et al., 2007), Cucurbit Yellow Disorder Virus (CYSDV) (Wintermantel et al., 2009), Cucumber Green Mottle Mosaic Virus (CGMMV) (Shim et al., 2005; Ko et al., 2007; Liu et al., 2009), Cucurbit Aphid-borne Mosaic Yellows Virus (CABYV), (Kneirim et al., 2010) and Cucumber Green Mottle Virus (CGMV) (Moradi and Jafarpour, 2011).
Moroccan Watermelon Mosaic Virus (MWMV), first isolated in Morocco (Fischer and Lockhart, 1974; McKern et al., 1993) was reported to have caused severe damage to cucurbits in all commercial cucurbit producing regions. The virus has also been reported from southwest Spain (Quiot-Douine et al., 1990) and Italy (Roggero et al., 1998). In African the virus has been reported from South Africa (Van Der Meer and Garnett, 1987), Sudan (Lecoq et al., 2001), Democratic Republic of Congo (Arocha et al., 2008) and Tunisia (Yakoubi et al., 2008).
The southeastern corner of Nigeria is rich in both cultivated and wild cucurbit species. Among the cultivated ones are Cucurbita moschata (Duch ex Lam) Duch and Poir, Cucumis sativus L. Lageneria siceraria (Mol) Standl, Cucumeropsis manni (= C. edulis) and Colocynthis citrullus L. cultivated for their leaves as pot-herbs and fruits and seeds. Among the wild species are Lagenaria breviflora and Coccinia barteri which are importantly in traditional medicine. Virus infection of some of these cucurbits is widespread but largely unreported. So far, a watermelon mosaic-like virus isolated from C. edulis (= C. manni) (Igwegbe, 1983), Telfairia mosaic virus (TeMV) reported on Telfairia occidentalis (Shoyinka et al., 1987) and a strain of PRSV from Cucumis sativus (Owolabi et al., 2008) are the potyviruses that have been reported naturally infecting cucurbits in Nigeria.
In 2005-2006 growing season, virus-like induced symptoms characterized by mosaic, leaf malformation and conspicuous green-vein banding on Lagenaria breviflora while vein-clearing and sometime yellow mosaic, green vein-banding which sometimes may be masked by white blotches on the leaves, were observed on Coccinia barteri. This paper reported the biological, serological and molecular characterization of these virus isolates tentatively designated LbreV and CbarV for the L. breviflora and C. barteri virus isolates, respectively.
MATERIALS AND METHODS
Virus isolation and propagation: Symptomatic leaves obtained from both L. breviflora and C. barteria were collected from the field in sealed polyethylene bags. The infected leaf tissues were triturated in cold 0.03 mol L sodium phosphate buffer pH 8.0 in pre-cooled oven-sterilized pestle and mortar. The inocula were mechanically transferred onto healthy seedlings of a range of test plants in the greenhouse (23±2°C) and the inoculated plants were left for symptom development. Three serial local lesion transfers were made for LbreV which elicited chlorotic local lesions in Chenopodium quinoa. No local lesion hosts were identified for CbarV. However, preliminary serological tests using double antibody sandwich enzyme linked immunosorbent assay (DAS-ELISA) and sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) confirmed that symptoms observed on C. barteri was not due to mixed infection. Both viruses were propagated in C. sativus and or C. pepo by periodic sap inoculation.
Host range and symptomatology: For host range studies, inocula derived from infected leaf tissues of the propagation hosts were inoculated by rubbing on 600-mesh carborundum dusted leaves of at least 25 plant species and cultivars belonging to the Amaranthaceae, Chenopodiaceae, Solanaceae and Cucurbitaceae families. The plants were kept in the greenhouse at 23±2°C. Symptoms were recorded for a period spanning 4 weeks. All inoculated plants without visible symptoms were assayed for virus presence by back-indexing on C. manni.
Aphid transmission tests: Myzus persicae, Aphis craccivora, A gossypii and Macrosiphon euphorbiae reared on Ficia faba and A. spiraecola obtained from its natural host (Chromolaena odorata) were starved for 1 h and allowed acquisition feeding time of about 3-5 min. Ten aphids were then transferred to each of five healthy seedlings of C. pepo in insect-screened cages, kept and sprayed with an insecticide (Pirimor). The plants were then kept in the greenhouse and symptom development was monitored for about 3 weeks.
Electron microscopy: The particle morphology of the virus isolates was determined by leaf-dip serology. Virus particles were trapped onto grids pre-coated with polyclonal antiserum (TuMV-314) or monoclonal antibody (MoAb) P-3-3H8, followed by negative staining with 2% phosphotungstic acid, pH 6.0. The grids were then examined under the Upson-902 electron microscope.
Cytopathology: Small pieces from symptomatic leaf tissues of Lagenaria breviflora and Coccinia barteri virus-infected leaf tissues were taken and fixed with 3% (v/v) glutaraldehyde after four changes (x4) of 10 min duration in 0.1 M cacodylate buffer, pH 7.0 overnight. The samples were then post-fixed for 2 h with 0.66% osmium tetraoxide in two changes (x2) of 45 min duration in 0.1 veronate acetate buffer, pH 7.25. This was followed by two times (x2) dehydration of the samples through graded series of alcohol (30, 50, 70 and 90 absolute alcohol) in 1% aqueous uranyl acetate. Thereafter, the samples were embedded in gelatin capsule for 24 h at 40°C and later for 48 h at 60°C. Ultra-thin sections were made using Reichert-Jung ultramicrotome and examined under the Upson-902 electron microscope.
Gel electrophoresis and immunoblotting: Symptomatic leaf tissues of C. pepo for both viruses were homogenized (1:3 w/v) in distilled water. One hundred microlitres were treated with equal volume of Sodium Dodecyl Sulphate (SDS)s dissociation buffer (2% SDS, 0.62 mM Tris-Hcl buffer pH 6.8, 5% (v/v) 2-mercaptoethanol, 5% (v/v) glycerol and 0.001% (v/v) bromocresol blue) by boiling for 5 min. Proteins were separated by SDS-polyacrylamide gel electrophoresis (PAGE) in a Vertical Minigel-Twin electrophoresis apparatus (Biometra, Gottingen, Germany) as described by Laemmli (1970). Western blotting was carried out as described by Richter et al. (1994).
Virus purification and antisera production: The viruses were purified from infected zucchini squash (Cucurbita pepo) leaf tissues 2-3 weeks post inoculation by differential centrifugation as described by Owolabi et al. (1998). Briefly, the procedure involved grinding tissues in high salt buffer, precipitating with polyethylene glycol and isolating viral zones after sucrose density gradient centrifugation.
Antisera to the viruses were produced by injecting rabbit intramuscularly with 1 mg/1 mL purified viral suspensions emulsified with (1/1) Freund’s incomplete adjuvant followed by two booster injections with Freund’s incomplete adjuvant at weekly interval. The animals were bled starting 2 weeks after injection. Immunoglobulins G (IgGs) and alkaline phosphate conjugates were prepared using standard procedures and used for serological tests.
Serology tests: Serological relationships between the virus isolates used in this study and some definitive potyviruses were investigated in plate-trapped antigen enzyme-linked immunosorbent assay (PTA-ELISA) as described by Converse and Martin (1990) and DAS-ELISA (Clark and Adams, 1977). Polysterene microtitre plates were used and the reaction volumes consisted of 100 μL per well. Appropriate dilutions of a panel of 15 antisera mostly immunoglobulins (IgGs) (obtained from the antiserum bank, Institute of Pathogen Diagnostics, Aschersbelen, Germany) and prepared as recommended were used in the ELISA tests. Symptomatic leaf samples were ground in extraction buffer at 1:10 (w/v). ELISA readings were considered positive when they exceeded twice the readings of the corresponding controls (Walkey et al., 1994).
Immune specific electron microscopy: Antisera (IgG) to (MWMV), PRSV, Beet Yellow Mosaic Virus (BYMV), CIYVV, TuMV, (TeMV), WMV (Katabase), WMV-2, Zucchini Yellow Fleck Virus (ZYFV) and ZYMV obtained from Biologische Bundesanstalt, Brauschweig, Germany, were used in Immunospecific Electron Microscopy (ISEM) decoration tests carried out as described by Richter et al. (1994).
RNA purification, cDNA synthesis and sequence analysis: RNA was extracted from purified virions of both isolates by incubation for 30 min in 10 mM Tris-HCl pH 7.6, 100 mM NaCl. 0.5% SDS and 200 μg mL-1 proteinase K at 30°C followed by two phenol extraction and ethanol preparation as described by Maiss et al. (1988). Complementary DNA (cDNA) synthesis was performed both by oligo (dT) and random priming according to Gubler and Hoffman (1983). Double stranded cDNA was dC-tailed and annealed to Pst I cut dG-tailed pBR 322. Cells of Escherichia coli strain DH 1 were rendered competent by the dimethylsulfoxide/dithiothreitol procedure according to Hanahan (1983) and transformed with 10 ng of recombinant DNA. Colonies were transferred to nitrocellulose filters place on LM agar and grown overnight. Lysis of bacteria, DNA denaturation and fixation were carried out as described by Grunstein and Hogness, (1975). Clones containing plasmids with cDNA insert were identified and isolated by a modified alkaline lysis method (Birnboim and Doly, 1979). Sequencing was carried using the method of Sanger et al. (1977) and sequence data were analyzed by the use of computer program from Schwindinger and Warner (1984).
Sequence alignment was obtained using EMBOSS procedure and distance matrix by EMBLOSUM62. The deduced amino acid sequence data obtained were compared to that of MWMV.
RESULTS
Host range and symptomatology: Both virus isolates somewhat had narrow host ranges. The host range of CbarV was restricted to the cucurbitaceous plants as non-cucurbits tested were not susceptible. Besides infecting a good number of the cucurbit test plants, the LbreV induced chlorotic local lesions in Chenopodium amaranticolor (Fig. 1) and C. quinoa though inconspicuous (Table 1). The susceptible cucurbits reacted differently to the two virus isolates. For example, LbreV induced conspicuous and severe mosaic leaf malformation and green vein-banding in C. pepo, C. moschata and L. siceraria, while infection of these cucurbits species by CbarV was characterized by mild mottle except in L. siceraria that showed severe leaf malformation. The following plant species were not susceptible to both viruses and there was no evidence of latent infection. Amaranthaceae: Celosia trigyna Linn., Gomphra globosa Linn.; Chenopodiaceae: Chenopodium foetidum, C. foliosus C. murale Linn. C. rubrum, C. urbicum and C. capitatum.; Cucurbitaceae: Colocynthis citrullus Mill. Gard. Luffa aegyptica Mill.; Cruciferae: Brassica oleracea V. capitata Linn.; Solanaceae: Nicotiana benthamiana Domim. N. occidentalis Wheeler, N. tabacum V. kamsum Linn., Solanum melogena Linn., S. nigrum Linn. and Physalis angulata Linn.
Aphid transmission tests: Both viruses were transmitted in the fore-gut manner (non-persistent) by M. persicae and A. gossyppii but not by A. craccivora. CbarV was also transmitted by M. euphorbiae but failed to transmit LbreV.
Electron microscopy and cytopathology: Leaf dip preparations of C. edulis infected by the CbarV and LbreV isolates revealed flexuous rod shaped particles resembling those of potyviruses (Fig. 2a, b). LbreV induced laminated aggregates while CbarV produced laminated aggregates and tubes in thin sections made from C. edulis (Fig. 2c, d).
Electrophoresis and western blotting: The results showed that the molecular weight (Mr) of LbreV was about 32.5 kDa while that of the CbarV was approximately 35.0 kDa (Fig. 3).
| Table 1: | Reactions of some host range plants to Lagenaria and Coccinia virus isolates |
| Fig. 1(a-d): | Chlorotic local lesions induced by the Lagenaria virus isolate in C. Amaranticolor (a) and mosaic and dark green-vein banding in Cucurbita pepo (b). Leaf malformation induced by the Coccinia virus isolate in Cucumeropsis mannii (c and d) |
Serological tests: Both LbleV and CbarV showed strong homologous reactions with their, respectively antisera with values of 2.00 and 1.47 compared to 0.02 and 0.01 when reacted with antiserum to PRSV. LbreV reacted positively with antibody to CbarV (0.41) but there was no reciprocal reaction between CbarV and antiserum to LbreV (Table 2). Both virus isolates reacted positively with potyvirus specific MoAb P-3-3H8 and polyclonal antibody TuMV-314 in PTA-ELISA. Of the two viruses only LbreV showed cross reaction with MWMV in DAS-ELISA. Both failed to react with antisera to BCMV, ClYMV, PRSV, TuMV-326, Soybean Mosaic Virus (SoyMV) and WMV-2 in the same test. However, in ISEM decoration tests, the LbleV was moderately decorated by anitisera to PRSV, TuMV and TeMV, very weakly decorated by antisera to WMV (kalabase), WMV-2 and ZYFV but no decorations were observed with MWMV and ZYMV antisera.
| Fig. 2(a-d): | Flexuous rod particles of Lagenaria breviflora virus isolate (a) and Coccinia barteri virus (b) stained with uranyl acetate. Laminated aggregates (LA) (c) laminated aggregates and tubes (TU) (d) induced by the Lagenaria and Coccinia virus isolates, respectively. Bars = 38.5 nm |
| Fig. 3: | Sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) and Western blotting of some potyviruses. Pre-stained protein marker (M) (16.5-175 kDa). Lane 1: Coccinia barteri virus, Lane 2: Papaya rinspot virus (Owolabi et al., 2008); Lane 3: Lagenaria breviflora virus; Lane 4: Cucurbita mosaic virus (In press); Lane 5: Watermelon mosaic virus-2 (Hungary); Lane 6: Potato virus Y; Lane 7: healthy sap from Nicotiana benthamiana. Viruses were detected after incubation with P-3-3H8 monoclonal antibody. |
| Fig. 4: | Sequence alignment of the N-terminal regions of the coat protein of Lageneria breviflora virus (LbreV), Coccinia barteri virus (CbarV) and MWMV (Sudan isolate). Stars (*) indicate points of differences in the amino sequences of the viruses |
| Table 2: | Serological relationships between the Lagenaria (LbreV) and Coccinia virus (CbarV) isolates and papaya ringspot virus (PRSV) using polyclonal antisera (PAS) raised against the viruses |
| aAntiserum was obtained from Institute of Pathogen Diagnostics, Ashersleben. bAntiserum against Cucurbita mosaic virus (a potyvirus from Nigeria. In press). cAntiserum was obtained from Institute of Biochemistry and Plant Virology, Braunchweig, Germany, † = Readings were made at E450 nm | |
On the other hand, CbarV was strongly decorated by MWMV antiserum, moderately decorated by antisera to PRSV, very weakly decorated by antisera to TuMV and TeMV but no decoration at all with antisera to WMV (katabase), WMV-2, Zucchini Yellow Fleck Virus (ZYFV) and ZYMV.
Sequence data: Comparing the amino acid (aa) sequences of the N-terminal regions of LbreV and CbarV with the corresponding region in MWMV showed greater variability at the proximal end (Fig. 4). Sequence alignment of the amino acid sequences of the conserved regions of LbreV and CbarV indicated 92 and 93% sequence identities, respectively when compared to that of MWMV while the difference in the sequences of both viruses study differed by 7%.
DISCUSSION
The characteristics of the viruses isolated from L. breviflora and C. barteri reported in this study are consistent with those of other members of the genus Potyvirus previously reported in the literature. The viruses are transmitted in a fore-gut manner by aphids and have flexuous rod-shaped particles (Lecoq et al., 2001; Owolabi et al., 2008). They induced tubes and laminated aggregates, have coat protein molecular weights of 32.5 and 35.0 kDa which fell within the range of 30-47 kDa for potyviruses (Hull, 2002). The virus isolates also reacted serological to potyvirus specific monoclonal antibodies and decorated to varying degrees by antisera to some putative members of the genus in ISEM decoration tests. They also share high degree of sequence homologies with a putative member of the genus (Quiot-Douine et al., 1990; Shukla et al., 1994; Lecoq et al., 2001).
Hitherto, non-cucurbit potyviruses such as Amaranthus mosaic virus (Taiwo and Owolabi, 2004), Eggplant severe mild mottle and Eggplant green mosaic viruses (Ladipo et al., 1988a, b) and a mosaic- inciting virus isolated from Senna hirsuta (Owolabi and Proll, 2001) have been described from Nigeria. Records of cucurbit viruses included a watermelon-like virus (Igwegbe, 1983), CMV (Atiri, 1985), TeMV (Shoyinka et al., 1987) and a strain of PRSV (Owolabi et al., 2008). These viruses differ widely in their host ranges compared to the virus isolates reported in this study. Besides sharing properties such as particle morphology (flexuous rods), transmission by aphids in a fore-gut manner, induction of cytoplasmic inclusions which are basic to members of the genus Potyvirus (Hull, 2002), no further comparison could be made with LbleV and CbarV for lack of antisera to these viruses.
Serological data obtained reveal lack of relationships between LbleV and CbarV and most of the potyviruses commonly associated with cucurbits whose antisera were used in this study. The weak to moderate decoration of LbleV by antisera to PRSV, TuMV and TeMV and in the case of CbarV by MWMV is, however, suggestive of some degree of relatedness to these viruses. That both viruses differ from PRSV is confirmed by lack of reactivity with the polyclonal antisera prepared against LbleV and CbarV. Also, Coat protein sequence data for TeMV (Maiss et al., 1988,) suggest that it is more related to Cowpea Aphid-borne Mosaic Virus (CABMV) than to any of the cucurbit viruses and therefore different from the viruses reported in this study.
Biological, serological and molecular characterization of both LbleV and CbarV indicate that both viruses unambiguously differ from each other. Host range studies show that the former induced chlorotic local lesions in C. amaraticolor and C. quinoa which are not susceptible to the latter. Results from this study also indicate differences in the symptoms induced even in plants susceptible to both viruses. CbarV was transmitted by M. euphorbiae but failed to transmit LbreV. The virus isolates also differ in their molecular weights. Serologically, each virus reacted specifically with it antiserum and gave no cross reactivity with antiserum of the other in DAS-ELISA. Furthermore, only LbleV shows cross reaction with MWMV in DAS-ELISA while the particles of CbarV were decoration by antiserum to MWMV but failed to decorate LbleV.
Yakoubi et al. (2008) have reported the existed of three clusters of MWMV. Isolates from Tunisia (MWMV-Tn) said to be closely related to those from Spain, Italy and France formed one cluster with the type strain from Morocco. The second cluster comprised the isolates from Cameroun, Niger Republic and Democratic Republic of Congo which showed wide divergent from others MWMV strains (75-80% identity) while the third cluster included isolates from Swaziland and South Africa. The Sudan isolate considered to be at the fringe of MWMV species (Lecoq et al., 2001) presented only 75-80% amino acid sequence identity in the CP with the other isolates. From the result of this study, sequence alignment of amino acid composition of the N-terminal regions of the CP of both LbleV and CbarV with that of MWMV Sudan isolate showed 92 and 93% sequence identity.
Shukla et al. (1994) have suggested that virus isolates with less than approximately 40% sequence identity should be regarded as belonging to different genera. Isolates with 55 to 75% sequence identity, they posited, should be considered as different species while isolates presenting 74 to 88% sequence identity should be regarded as different entities within a related subset of a species. They also suggested that viruses with 90% amino acid sequence identity should be regarded as strains of the same virus. Consequently, these virus isolates reported in this study should be considered strains of MWMV.
CONCLUSION
The differences observed in the biological characteristics (host range and insect transmission) and immunological attributes of the virus isolates in this study apparently stem from differences in their sequence data. Our results further emphasize the need to combine biological, serological and molecular characteristics in delimiting strains of virus species. To our knowledge, this is the first report of MWMV strains in Nigeria.
ACKNOWLEDGMENTS
The first author is grateful to the German Academic Exchange Service (DAAD) for the award of a fellowship to carry out part of this study in the Republic of Germany. We are equally grateful to the staff of the Virology Laboratory of the Institute of Resistance Research and Pathogen Diagnostics, Aschersleben (now Quedlinburg) for their technical assistance, Mrs. S. Ballhause for the photographs, Mrs. Zimmerman for the electron micrographs and thin sections and the Senate of the University of Calabar for partly funding the work through the Senate Research Grant.