Project Abstract

<p></p>               <strong>ABSTRACT</strong><a target="_blank" rel="nofollow"></a><div><p>Virus infection of <i>Lagenaria breviflora</i>&nbsp;and <i>Coccinia barteri</i>&nbsp;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 microcopy, 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 <i>Potyvirus</i>, family <i>Potyviridae</i>. Both were mechanically transmissible and also transmitted by <i>Myzus persicae</i>&nbsp;and <i>Aphis gossyppi</i>&nbsp;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&nbsp;&nbsp;(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&nbsp;&nbsp;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.</p></div> <p> <br></p>

Project Overview

<p></p> <p><b>1.1 INTRODUCTION</b></p><p><i>Lagenaria bleviflora </i>(Benth.) Roberty (<i>Adenopus breviflorus</i>) 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 (Burkil, 2004). Economically, the seeds are considered a good source of most essential amino acids comparable to soybean (Oshodi, 1996)&nbsp;and oil (85.1% unsaturated fatty acid&nbsp;and 65.3% linoleic acid) suggesting potential uses in soap making, shoe polish, shampoo and edible purposes (Akintayo and Bayer, 2002).</p><p><i>Coccinia barteri</i>&nbsp;(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.</p><p>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 Korkmz, 2004; Lecoq <i>et al., </i>2001; Fattouh, 2003; Salem&nbsp;<i>et al., 2007)</i><i>&nbsp;</i>).&nbsp;</p><p>Moroccan Watermelon Mosaic Virus (MWMV), first isolated in Morocco (Fischer and Lockhart,. 1974; Mckern <i>et al.</i>, 1993)&nbsp;&nbsp;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&nbsp;<i>et al., 1990)</i>&nbsp;and Italy (Roggero&nbsp;<i>et al., 1998).</i>&nbsp;In African the virus has been reported from South Africa,&nbsp;Sudan (Lecoq&nbsp;<i>et al., 2001)</i>, Democratic Republic of Congo (Arocha&nbsp;<i>et al., 2008)&nbsp;</i>and Tunisia (Yakoubi&nbsp;<i>et al., 2008).</i></p><p>The southeastern corner of Nigeria is rich in both cultivated and wild cucurbit species. Among the cultivated ones are <i>Cucurbita moschata </i>(Duch ex Lam) Duch and Poir, <i>Cucumis sativus </i>L. <i>Lageneria siceraria </i>(Mol) Standl, <i>Cucumeropsis manni</i>&nbsp;(= <i>C. edulis</i>) and <i>Colocynthis citrullus </i>L. cultivated for their leaves as pot-herbs and fruits and seeds. Among the wild species are <i>Lagenaria breviflora </i>and <i>Coccinia barteri</i>&nbsp;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 <i>C. edulis</i>&nbsp;(= <i>C. manni</i>) (Igwegbe, 1983), Telfairia mosaic virus (TeMV) reported on <i>Telfairia occidentalis (</i>Shoyinka&nbsp;<i>et al., 1987)</i>&nbsp;and a strain of PRSV from <i>Cucumis sativus (Owolabi et al., 2008)</i>&nbsp;&nbsp;are the potyviruses that have been reported naturally infecting cucurbits in Nigeria.</p><p>In 2005-2006 growing season, virus-like induced symptoms characterized by mosaic, leaf malformation and conspicuous green-vein banding on <i>Lagenaria breviflora</i>&nbsp;while vein-clearing and sometime yellow mosaic, green vein-banding which sometimes may be masked by white blotches on the leaves, were observed on <i>Coccinia barteri. </i>This paper reported the biological, serological and molecular characterization of these virus isolates tentatively designated LbreV and CbarV for the <i>L. breviflora</i>&nbsp;and <i>C</i>. <i>barteri</i>&nbsp;virus isolates, respectively.</p><p><b>1.2 MATERIALS AND METHODS</b></p><p><b>Virus isolation and propagation:</b>&nbsp;Symptomatic leaves obtained from both <i>L. breviflora </i>and<i>&nbsp;C. barteria</i>&nbsp;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 <i>Chenopodium quinoa</i>. 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)&nbsp;confirmed that symptoms observed on <i>C. barteri</i>&nbsp;was not due to mixed infection. Both viruses were propagated in <i>C. sativus</i>&nbsp;and or <i>C. pepo</i>&nbsp;by periodic sap inoculation.</p><p><b>Host range and symptomatology:</b>&nbsp;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 <i>C. manni</i>.</p><p><b>Aphid transmission tests:</b>&nbsp;Myzus persicae, Aphis craccivora, A gossypii and <i>Macrosiphon euphorbiae</i>&nbsp;reared on <i>Ficia faba</i>&nbsp;and <i>A. spiraecola</i>&nbsp;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 <i>C. pepo</i>&nbsp;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.</p><p><b>Electron microscopy:</b>&nbsp;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.</p><p><b>Cytopathology:</b>&nbsp;Small pieces from symptomatic leaf tissues of <i>Lagenaria breviflora </i>and<i>&nbsp;Coccinia barteri</i>&nbsp;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.</p> <p> <b>Immune specific electron microscopy:&nbsp;</b>&nbsp;Antisera (IgG) to (MWMV), PRSV, Beet Yellow Mosaic Virus (BYMV), CIYVV, TuMV, (TeMV), WMV (<i>Katabase</i>), 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&nbsp;<i>et al., 1994).</i></p><p><i> </i></p><p><i><b>1.3 RESULTS</b></i></p><p><i><b>Host range and symptomatology:</b>&nbsp;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 <i>Chenopodium amaranticolor (Fig.1)&nbsp;</i>&nbsp;and <i>C. quinoa</i>&nbsp;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 <i>C. pepo, C. moschata</i>&nbsp;and <i>L. siceraria</i>, while infection of these cucurbits species by CbarV was characterized by mild mottle except in <i>L. siceraria</i>&nbsp;that showed severe leaf malformation. The following plant species were not susceptible to both viruses and there was no evidence of latent infection. Amaranthaceae: <i>Celosia trigyna</i>&nbsp;Linn., <i>Gomphra globosa</i>&nbsp;Linn.; Chenopodiaceae:<i>&nbsp;Chenopodium foetidum</i>,<i>&nbsp;C. foliosus C. murale </i>Linn<i>. C. rubrum</i>,<i>&nbsp;C. urbicum </i>and <i>C. capitatum</i>.; Cucurbitaceae:<i>&nbsp;Colocynthis citrullus </i>Mill. Gard.<i>&nbsp;Luffa aegyptica </i>Mill.; Cruciferae:<i>&nbsp;Brassica oleracea V. capitata </i>Linn.; Solanaceae:<i>&nbsp;Nicotiana benthamiana </i>Domim<i>. N. occidentalis </i>Wheeler<i>, N. tabacum V. kamsum </i>Linn<i>., Solanum melogena Linn., S. nigrum </i>Linn<i>. </i>and <i>Physalis angulata </i>Linn.</i></p><p><i><b>Aphid transmission tests:</b>&nbsp;Both viruses were transmitted in the fore-gut manner (non-persistent) by <i>M. persicae</i>&nbsp;and <i>A. gossyppii</i>&nbsp;but not by <i>A. craccivora</i>. CbarV was also transmitted by <i>M. euphorbiae</i>&nbsp;but failed to transmit LbreV.</i></p><p><i><b>Electron microscopy and cytopathology:</b>&nbsp;Leaf dip preparations of <i>C. edulis</i>&nbsp;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 <i>C. edulis (Fig. 2c.d).</i>&nbsp;</i></p><p><i><b>Electrophoresis and western blotting:</b>&nbsp;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).</i></p><i> <br></i><p></p>