Arroyave, José A.

Lozano, I

Morales, Francisco José

Velasco, Ana Cecilia

Lozano, I.

CGSpace (CGIAR)

• .' l' ~t'aJt Jou; na; 'tJ/P!aizt Pmt'ioh: Detection of a foveavirus associated with the lethal 'ringspot' disease of African oil palm (Elaeis guineensis Jacq.) in South America ,í.' . cy \ -' \ ,f-v ( ....... \J \¡..I ¡¡) , • \ ., ~ "\ I ~y t'. '" ,. 'U/' F.l:sMorales,1. Lozano, A,e. Velasco and J.A. Arroyave lnternational Cenler for Tropical Agriculture (CL4T), Virology Research Unil, AA. 67/3, Cali, Colombia (Phone: 57-2·4450000; Fax: 57-2-4450073; E-mail: f.morales@cgiar,º.rn Key words: Mancha anular, Bactris sp., West Africa Abstrae! A filamentous virus ca. 800 nm in length was consístently associated with a lethal disease of young African oil palms, referred to as "ringspot" in Colombia, Ecuador and Peru, in South America. This disease has also becn reported from Ivory Coast in West Africa. Partíal molecular characterizatíon showed this virus 10 be distinct but related lo tentative species of lhe Foveavirus genus, namely Cherry green ring ¡¡¡ottle virus and Cherry necrotic rusty maule virus, and more distantly lo Apple stem pittíng virus, lhe type foveavirus species. 1 I 2 lntroduction The 'ringspot' disease of African oil palm (Elaeis guineensis Jacq.) was first observed in 1969 (Dzido et al., 1978; Arévalo, 1988), in the north-western foothills of the Amazon region of San Martin, Peru. The disease affected mainly one year-old palms, which developed a systemic yellowing particularly noticeable in the youngcr leaves. Ringspots and elongated rings were observed on leaflets and rachis of affectcd leaves, which cventually turncd brown and died wíthín three months aftcr initial symptom express ion. A section of the stem of ringspot-affected oil palms showed a purplish colour in the vascular system. The roots and fruil bunches of diseased young palms often showed systemic necrosis. Five year-old and older oí! palms were not affected. The lethal ringspot disease re-emerged around 1973 in the north-western oil palm-producing region of Ecuador, where it was referred to as '(elhal yellowing' and 'bud mottle' (Dzido et al .. 1978, Chávez, 1988). Disease incidence varied between 3 and 40%, bUl sorne lots suffered the 1055 of up to 95% of the palms 18-24 months old. In 1985, a similar lethal disease reached the neighbouring oí! palm prodllction region of Tllmaco in south-weSlern Colombia (Jiménez, 1988). In this region, however, the characteristic symptoms consist of elongated yeJlow patches, which eventuaJly appear as streaks on the basal leaflets and raquis of affected oil palm leaves. By 1988, the disease was affecting young plantations up lo two and a half years old in an area of approximately 4.500 heclares. Disease incidence ranged from 2-45% and affected palms were usually found in fod (Jiménez, 1988). Previous investigations conducted in the United States, France and Latin America on the etiology of Ihis disease failed to demonstrate the presence of any identifiable 3 pathogen (Dzido el al., 1978: Dolle! et aL, 1980; Renard and Quillee, 1984; Arévalo, 1988; Corley and Wood, 1990). This investigation was conducted in response to the increasing incidence of the lethal 'ringspot' disease in south-western Colombia, and the threat it poses to the remaining 130.000 hectares of African oí! palm grown in this country, the major praducer of tbis erap in Latin America. This disease is also a major concern for other eountries in Latin America, sucb as Brazil, Venezuela, Suríname, Costa Rica, Guatemala, Honduras, Mexico and Nicaragua, which cultívate approximately J 60.000 additional hectares of Afriean oil palm. Ecuador and Peru already bave over 100.000 bectares of African oil palm al risk. Tbis researeh may al so benefil small-scaJe African oil palm pradueers in West Africa, the centre of origin of E. guineensis (Sauer, 1994), where similar symptoms have been ohserved lo affeet this important oil erap (Renard and Quillec, 1984). Materials and methods Virus isolates Al! virus isolates used in this investigation were obtained from symplomatic spear lea ves of 1-2 year old Afriean oi! palms showing Ihe characteristic foliar yellowing (Figure l) and streaking of the younger leaves and rachis in Ihe munieipality of Tumaco, Naríño, Colombia. Tbe Slems of all diseased palms selected for this investigation were díssected to observe the characteristic purplisb rings associated witb tbe lelbal ringspot disease (Figure 2). Stem samples were also tesled in Ihis invesligation. Selecled lissue was processed fresb or conserved at -80 co until needed. A lcaf sample obtaíned from a wild palm belonging lo the genus Bactris (tentatively identified as B. setldosa), was also • assayed here because the palm was found in ¡he municipality of Tumaeo, showing folíar yellowíng symptoms similar to those deseribed for 'ríngspot' in Afrícan oí! palm. Pathogenicity tests The followíng plant species were mechaníeally inoculated with extraets obtained from ringspot-affected leaf tissue diluted 1:4 or 1: lO (w/v) in sterile distilled water or buffers prepared as 0.5 or O. l M potassium phosphate buffer, pH 7.5, eontaining 0.5 g of sodium sulphite and l mM sodium diethyl-dithioearbamale (DIECA). Chenopodium a/bum, C. amaran/ie%r, C. quinoa, C. mura/e. Elaeis guineensis, Nicotiann ben/hamiana, N. tabacum 'Samsun', Sorghum bicolor 'Rio·. and Zea mays. Infected African oí! palm extraels were inoculated with sterile gauze, sterile píns and syringes to healthy seedlíngs of Ihis spedes, using 10 seedlings per test. Eleclron microscopy Leaf extraets from symplomatic oi! palms were negatively staíned in 2% uranyl acetate, pH 3.7, and examined for the presenee of virus partíc!es using a JEOL JEM-IOIO eleclron microscope. Leaf tissue of diseased oil palms, was prepared for cytology as described earlier (Morales el al., 1990). Thin sections were cut with a diarnond knife using a MT 6000 Sorval ultramicrotome. , I 5 Electrophoresis Double-stranded RNAs were extracted from healthy and ringspot-affected African oi! palm plants according to the procedure of Dodds and Bar-Joseph (1993). Approx-imate ds-RNA weíghts were estimated using a I Kb ladder (Bethesda Research Laboratory). The Bactrís sp. sample was processed following ¡he same procedure. Nucleic aeid extrae/ion and cDNA synthesis Double stranded RNA extractions were performed as described aboye for the electrophoresis methodology. The ds-RNA species were separated in Iow melting point agarose gels (Gíbco BRL) and then re-extracted using a RNeasy Kit (Qiagcn) according to instructions. Approximately 3 ¡.tg of ds-RNA in a 13 ¡.tI volume, were denatured with an equal volume of 40 mM methylmercuric hydroxide, in ¡he presence of 500 ng of random prímers and 500 ng of an 18mer-oligo(dT) (Gihco), according lo the method of Jelkman and co-workers (1989). cDNA synlhcsis was calculaled for a final volume of 40 ,.11 of 50 mM Tris-HCI, pH 8.3; 75 mM Mg Ch. 10 mM DTT, 0.5 mM each dATP, dCTP, dGTP, d1lP; 40 U RNasin (promega), and 400 U of SuperScript Il RT (Gíbco, BRL). The mixture was incubated for 60 min at 37° C prior lo adding 200 U of enzyme with 30 addítional minutes of incubation. The reaction proceeded at 70" C for I min and finally stopped by immersion in ice-water. For the second strand reaetíon, the 40 ¡.tI of the first reaction, were taken up lo 1501-11 in a final mixture of 25 mM Tris-HCl, pH 7.5, 100 mM 6 Kel, 5 mM Mg eh, 10 mM (NH4hS04, 0.15 mM S-NAD+, 0.25 mM dATP, deTP, dGTP, dITP, 1.2 mM DIT, 25 U Eschericia coli Ligase, 40 U E, coli Polymerase, and 4 U E. coli RNase H. The mixture was incubated for 3 h al 16° e, adding 30 U T4 DNA Polymerase prior to further incubatíon al 16° e for 10 min. The reaetion was stopped by addíng 10 /JI 0.5 M EDTA, ph 8.0, and then the nucJeic acíd was extracted with phenol:chloroform:ísoamyl alcohol (25:24: 1). The cDNA was precípitated with a 1110 volume of 7 M Ammonium acetate and 2.5 volumes of elhano/. Nucleic acid hybridisation Random-primed c-D;-'¡A was labelled with [a31 PldATP. First strand cDNA was purified through Sephacryl S-400 (Pharmacia). Dot blot hybridizations were performed using 100 mg of young oil palm tissue were homogenized in Iiquid nitro gen. Total Rt"lA extraction was performed according to the instructions provided for the RNeasy kit (Qiagen). One ¡Jg of total RNA per sample was applíed onto Hybond-N+ nylon membranes (Amersham, Pharmacia Biotech). The membranes with the samples were treated with 0.05 N NaOH-0.15 M NaC! for 5 min and, then, another 5 min with 0.1 M Tris. pH 7.5-0.15 M NaC!. prior to being fixed in a UV-Stratalinker (Stratagene). The hybridization was carried out for 16 h al 50" e in 40% formamide, 5X SSPE. 6X Denhardt's and lOO fl/ml salmon DNA. Membranes were washed twice at 52° e with 0.5% SDS and O.5X SSc. Besides the ringspot-affected and disease-free African oíl palm controls, ¡WO potyviruses, Sugareane masaie virus and Bean common mosaie virus, maintained in African oH palm and common bean. respectively, were also used as control,. I j I • 7 Molecular cloníng The cDNA was Iigated iuto the SmaI sitc of pBJueScrípt JI (+) pJasmids (Stratagene), and then used to heat shock transform E. coli cells DH5a (Stratagene). Transformed E. coli cells were seJected using agar-ampícillin, X-GaJ, and IPTG (Stratagene). To identify baeteriaJ coJonies containing transformed pJasmids, a sample of every white co10ny observed was re-suspended in 50 flJ of a PCR mix containing J flJ JO mM dNTPs, 5 I.¡] 25 mM Mg Ch, 0.5 fll T3 primer (10 pmol, Stratagene), 1 U Taq DNA polymerase (Promega),5 fl1 PCR 10X buffer (Promega) and distílled water to reach the final volume. Samples were amplified in a PTC 100 thermocycler (M! Research) followíng a 30 cycle program (94 CO/3 min, 94 Cn/30 s, 72 CO/2 min, and a final extensíon al 72 CO/5 min. The producls obtained were analysed by e1ectrophoresis in a 1.3% agarose geL Sequencing ScquencÍng was performed with an ABI PRISM 377 DNA automatcd sequencer (Perkin E1mer). Sequences obtained were unaJysed using the BLAST program of the NationaI Center for Biotechnology Informalion (Natíonal Instítutc of HeaJth, Bethesda, MD), • < j 8 Results Pathogenidty tests None of the test plants mechanically inoculated in this study developed symptoms or was shown by electron mieroscopy to contain virus-like particles. The ínoculated African oil palm seedlings were maintained under observatíon for over ayear, bul none of the 10 seedlings inoculated by the standard, pricking or injection methods expressed any symptoms. Electron microscopy Direct observation of negatively staíned spear leaf tissue extraets prepared from ringspot-affected African oi! palms consistently revealed ¡he presence of flexuous, filamentous vims-Iike particles approxímately 800 nm in length and 15 nm in diameter (Figure 2), albeit al a very low concentratíon. Longer partieles of similar morphology were occasionally observed in these tissue extraet., suggesting the occurrence of end-to-end aggregation. Similar filamentous virus-like particles were observed in total disarray in the cytoplasm of leaf cells obtained frorn ringspot-affected African oíl palrns (Figure 3). These particles were localised in particular areas of the mesophyll, but did not form any particular typc of inclusion or viroplasrn. .. 9 Electrophoresis The exlractíon and electrophoresis of ds-RNA from ringspot-affected African oil palms, revealed the presencc of up lo Ihree bands approximately 8034, 7171 and 5854 bp in size (average of nine eleclrophoresis tests for an equal number of ringspot-affected palms, over a !hree year period). The 8034 band predominated in mOSl tests (Figure 4), whereas the concentratíon of the two lower bands varied appreciably, suggesting ¡he existence of only one ribonucleic acid species and two degraded sub-products. The leaf sample of !he symptomatic Bactris sp. palm tested here, yielded a similar band pattern, with a predominan! ds-RNA species of approximately 8135 bp. Nucleic acid hybridisation Positive hybridisation reactions were observed with leaf and stem tissue extracts prepared from ringspot-affected African oil palms, but not with similar extracts prepared from disease-free palms. Very mi Id background reactions were observed wilh a potyvirus !hal infects African oil palm (Figure 5). Molecular cloning and sequencing A total of 10 clones were sequenced and seJected for further analysis. Eight overlapping clones with a continuous sequence of 1667 bases, showed nucleotide seguence identities of 59.8% and 59.7% with corresponding terminal regions in ORF I (RNA-dependent 10 RNA polymerase) of Cherry green ring motile virus (CGRMV) and Cherry necrotic rusty maule virus (CNRMV), respectively. This clone shows a 49% nucleotíde sequence identity in Ihis genomic region with Apple stem pitting virus (ASPV). An 816 nucleotide long section of this clone, shows a 67% amino acid sequence identity with the corresponding sequences of CGRMV and CNRMV. An additional clone of 916 bp located in the central portion of ORF 1, had nuc1eotide sequence identities of 50.7% alld 47.5%, and amino acid sequence identilies of 52% and 39%, with the corresponding region, of CGRMV and ASPV, respective!y. FinaI1y. a 671 bp clone was showll lo contain a 469 bp fragment with a 43.7% and 48% nucleotide and amino acid sequence identity, respective!y, 10 the corresponding region in ORF 4 (putative movement protein 3 of lhe triple-gene block) ol' CGRMV. The remaining 202 bp fragment had a 43% and 35% amino acid sequence identities with corresponding regions of the coal protein gene (ORF 5) of CNRMV and CGRMV. Discussion Results obtained in Ihis investigation showed thal the 'ringspot' disease of African oi! palm in Colombia is consistently associated wíth a filamentous virus related to species in the genus Foveavirus (Regenmonel el al., 2000; Rott and Jelkmann, 2001; Zhang et al., 1998). The African oíl palm virus, hereafter referred to as African oil palrn ringspot virus (AOPRVl, would be ¡he firsl foveavirus found in the tropies. Similar tropical, filamentous viruses possessing genomic affinities to allexi-, carla-, fovea- and potexviruses, namely, Indian citrus ringspot virus (Rustici et al., 2000) and Banana rnild · . 11 mosaic virus (Gambley and Thomas, 200 1), have been recenlly deseribed, but remain unassigned 10 any virus genus. The various clones sequenced in this investigatíon consistently showed a closer affinity between AOPRV and recognized or tentative foveaviruses, Ihan to species belonging lO other filamentous virus genera. Whereas jI is possible Ihat further molecular work on AOPRV and future taxonomic developments might require !he re-c1assification of this virus, we propose jt as a tentative member of (he F oveavirus genus. One important aspect of AOPRV is its epidemiology, considering Ihat foveaviruses, such as ASPV, CGRMV and CNRMV, do nol have known biological vector (Regenmortel el al., 2000). African' oi! palm does nOI requíre any cultural Ireatments, such as pnming. during jls susceptible period (first three years of age) Ihal might explaín the transmission ¡he causal virus. The ringspot disease of African oi! palm disseminales rapidly during seasons of relatively low precipitation. when sorne arthropod vectors are more active. The disease also spreads among adjacent palms either in the nursery or in young plantations. Acknowledgements This research was possible thanks to the financial and logistic support of the Colombian Oil Palm Research Centre (CENIPALMA). We are grateful to Mr. Guillenno Guzmán for technical support with the graphic materíal incJuded in this publication. . . .. References Arévalo E (1988) Principales enfennedades de la palma aceitera en Tocache, Peru. In: Ramakrishna B (ed)VI Seminario Problemas Fitopatológicos de la Palma Africana (pp 117-133) Prociandino, Bucaramanga, Colombia Chávez F (1988) Enfennedades presentes en la palma africana en Ecuador y su incidencia. In: Ramakrishna B (ed) VI Seminario Problemas FitopatoJógicos de la Palma Africana (pp 113-116) Prociandino, Bucaramanga. Colombia Corley RHV and Wood BI (1990) Fatal yellowing aud bud rot conditions of oil palm in South Ameriea. Report on a visit lo Brazil. Ecuador and Colombia. Cenipalma Document No. D 0277 Dollet M. Gargani D and Dzido JL (1980) Recherches sur l' etiologie de la maladie des taches annulaires du palmier a huile (Elaeis guineensis) sevissant en Eguateur et au Perou. Cenipalma Document No. 1559 Dzido JL, Genty P and Ollagnier M (1978) Les principales maladies du palmier iI huile en Eguateur. Oléagineux 33: 55-60 Gambley CF and Thomas lE (2001) Molecularcharacterization ofBanana mild mosaic Virus, a new filamentous virus in Musa spp. Archives of Virology 146: 1369-1379 12 • Jiménez OD (\988) Mancha anular de la palma africana de aceite (Elaeis xuineensis lacq.) en Colombia. Ascolfi Informa 14: 55-57 13 Renard JL and Quillec G (1984) Les maladies graves du palmier a huile en Afrique et en Amérique du Sud. Oleagíneux 39: 57-62 Regenmortel, MHV van, Fauquel CM, Bishop DHL, Carstens EB, Eslens MK, Lemon SM, Maníloff J, Mayo MA, McGeoch DJ, Pringle CR and Wickner RB (2000) Virus Taxonomy: Seventh Reporl of Ihe InlernationaJ Commítlee on Taxonomy of viruses, Academic Press, New York, Rott ME and Jelkmann W (200 1) Complete nucleotide sequence of cherry necrotic rusty mottle virus. Archives of Vírology 146:395-401 Rustid G, Accotto GP, Noris Masenga V, Luisoni E and Mílne RG (2000) ludían citrus ringspot virus: a proposed new species with sorne affiníties lo potex-, carla-, fovea- and allexiviruses, Archives ofVirology 145: 1895-1908 Sauer JD (1994) Historícal geography of crop plants: a select roster. CRC Press, London. Zhang YP, Kirkpatrick Be Smar! CD and Uyemoto JK (1998) cDNA c10ning and molecular characterization of cherry green ríng mottle virus, Journal of General Virology 79: 2275-2281 14 Figure Legends Figure l. Young Afriean oH palm showing initial foliar yellowing associated with the 'ringspot' disease. Figure 2. Filamentous particle observed in African oíl palms affected by the 'ringspot' disease. Figure 3. Filamentous particles observed in the cytoplasm of 'ringspot'-affected African oil palm leaf cells. Figure 4. Electrophoresis of double-stranded RNA. Lane 1: healthy oíl palm; Lane 2: oíl palm infected by a potyvirus; Lane 3: 1 Kb markers; Lane 4: oíl palm affected by 'ringspot'. Figure 5: Dot blot of: column 1: Leaf extracts l'rom 'ringspot' -affected oíl palm; Column 2: Extraets from neerotie oil palm stem tissue al'l'ected by 'ríngspot'.; Column 3: healthy oil palm leaf extraet; Column 4: Leal' extraets from potyvirus-infected oil palmo • \ • • Clones de cONA obtenidos a partir de ds-RNA de tejido de palma de aceite con síntomas de Mancha Anular. Se empleó como agente desnaturalizante Hidróxido de Metil Mercurio 20 mM. 

Detection of a foveavirus associated with the lethal `ringspot` disease of African oil palm (Elaeis guineensis Jacq.) in South America

https://cgspace.cgiar.org/bitstream/10568/72108/1/63794.pdf

Detection of a foveavirus associated with the lethal `ringspot` disease of African oil palm (Elaeis guineensis Jacq.) in South America

Abstract

Similar works

Full text

Available Versions

CGSpace (CGIAR)