UNIVERSITE SAINT ESPRIT DE KASLIK FACULTE DES SCIENCES AGRONOMIQUES

UNIVERSITE SAINT ESPRIT DE KASLIK

FACULTE DES SCIENCES AGRONOMIQUES

CONTRIBUTION A L'ASSAINISSEMENT DU FIGUIER LIBANAIS VIS-A-VIS DE «FIG LEAF MOTTLE-ASSOCIATED VIRUS ^t» ET «FIG LEAF

MOTTLE-ASSOCIATED VIRUS 2» PAR LES TECHNIQUES DE CULTURE IN VITRO

Presente par Tala FATT AL

Pour

obtention du Master en Biodiversite vegetale et Biotechnologie

Kaslik - Liban

Join 2009

Resume

Ce travail s'inscrit dans le cadre d'une strategie d'assainissernent de trois varietes locales de figuier infectees par deux virus de la marbrure du figuier, FLMa V- 1 et FLMaV-2. Le present travail a consiste a experimenter trois procedures pratiquees dans les conditions in vitro : la culture d'apex associee ou non a la thermotherapie et la culture de meristemes.

L'initiation des cultures a ete partiellement reussie a partir des apex soumis ou non ala thermotherapie (35°C pendant 30 jours), ainsi qu'a partir des meristemes; les taux de reprise ont ete modestes sans depasser 38% dans le meilleur des cas et ce en raison des contaminations bacteriennes et myceliennes et d'un phenomene

•

d' oxydation conduisant a Ia perte to tale des explants atteints.

Les plantules regenerees a partir des trois traiternents d'assainissernent ont subi le controle sanitaire par un test RT-PCR et ce sur la base du sequenc;age du gene HSP70 qui permet de reveler une bande de 352 pb et 360 pb pour FLMaV-1 et FLMaV-2 respectivement chez le materiel infecte. Les taux d'assainissement ont varie entre zero et 100% et ce selon les traitements appliques et le virus en question. ll est important de noter que FLMaV-2 serait plus facile a eliminer que FLMaV-1 quelle que soit la technique utilisee. FLMaV-2 serait meme plus sensible ala thermotherapie que FLMaV-1.

Les resultats obtenus sont relativernent originaux et demontrent, sous reserve d 'une confirmation ulterieure, 1 'efficacite de Ia culture d'apex associee a la thermotherapie d 'une part et de la culture de meristemes d' autre part, pour l'assainissement des figuiers libanais vis-a-vis des virus de la marbrure du figuier FLMaV-1 et FLMaV-2.

Mots cles: figuier, FLMaV-1, FLMaV-2, culture d'apex, culture de menstemes, thermotherapie

Abstract

This work joins within the framework of a strategy of purification of three local varieties of fig tree infected by two viruses (Closteroviridea) of the marbling of the fig tree FLMa V -1 and FLMa V -2. The present work consisted in experimenting three procedures practiced in the in vitro conditions: the culture of associated stem cutting or not in the thermotherapy and the shoot tip culture.

The initiation of the cultures was partially successful from stem cuttings ' subjected or not to the thermotherapy (35°C during 30 days), as well as from shoot tips; the rates of resumption were modest without exceeding 38 % in the best case because of the bacterial and fungi contaminations and of a phenomenon of oxidation leading to the total loss of the affected plantlets.

Plantlets regenerated from three treatments of purification underwent the sanitary control by RT-PCR, on the basis of the sequencing ofthe gene HSP70 which allows revealing a band of 352 pb and 360 pb for FLMaV-1 and FLMaV-2 respectively in the infected material. The rates of purification varied between zero and 1 00 % and it is according to the applied treatments and the virus in question. It is important to note that FLMaV-2 would be easier to eliminate that FLMaV-1 whatever is the used technique. FLMaV-2 would be even more susceptible to the thermotherapy than FLMaV-1.

The obtained results are relatively original and demonstrate, subject to a later confirmation, the efficiency of both the culture of stem cuttings associated with thermotherapy and the culture of shoot tips, for the purification of the Lebanese fig trees towards the viruses of the marbling of the fig tree FLMaV-1 and FLMaV-2.

Keywords: fig, FLMaV-1, FLMaV-2, stem cutting culture, shoot tip culture, thermotherapy.

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REFERENCES BIBLIOGRAPHIQUES

40

REFERENCES BIBLIOGRAPHIQUES

Anonyme, sans date, Ficus carica linnaeus, Flora of North America vol. 3. 6 fevrier 2009.

(http:ijwww .efloras.org/florataxon.aspx?flora id= 1&taxon id=200006351) Anonyme, 2005. Principaux produits agricoles et alirnentaires et producteurs.

Organisation des Nations Unies pour !'Alimentation et !'Agriculture, Departement economique et social, la division de la statistique. 12 fevrier 2009.

(http://www.fao.org/es/ess/top/commodity.html?lang=fr&item=569&year=2005) Anonyme, 2006. Document du travail sur 1 'elaboration d 'une concentration maximale pour les aflatoxines presentes dans les figues sechees. In: comite du codex sur "Les additifs alirnentaires et les contaminants", trente-huitieme session, LaHaye, Pays-Bas, 24-28 Avril, 2006, p 1-9.

(ftp://ftp.fao.org/codex/ccfac38/fa38 40f.pdf)

Anonyme, 2008. Le figuier et sa culture. Blog dedie

a

cultivees et ornementales. 16 fevrier 2009.

(http://ennemisdesvegetaux.sport24.com/r13393/Figuier)

Anonyme, 2008. figues et figuiers. Huiles d'olive et vins/ gite ecologique. 16 fevrier 2009.

(b.ttp://cbql. unblog.fr/tag/en-savoir -plus-sur -les-figues)

Appiano, A., Conti, M. et Lovisolo, 0., 1990. Mosaico del fico: stato attuale delle conoscenze e nuove osservazioni.Agricoltura Ricerca 112-113: 109-112.

Astier, S., Albouy, J., Maury, Y., Robaglia, C., et Lecoq. H., 2007. principles of plant virology. Science publishers, USA. 472pp.

Auge, R., Beauchenese, G., Boccon-Gibbod, J., Decopurtye, L., Digat, B., Jalouzet, R., Minier, R., Morand, J., Reynoird, J.-P., Strllu, D.-G. et Vidalie, H., 1989. La culture in vitro et ses applications horticoles, Edition Tee & Doc, 3eme edition, 224 pp.

Balasingam, G., Ellison, N., Milne, K. S., Forester, R. L. S., 1988. Sensitive and specific detection of two filamentous viruses from nerins using cloned eDNA probes. Acta Hort., 234: 267-174.

Ben Abdallah, F., Fnayou, A., Grenan, S. et Gborbel, A., 1996. Contribution

a

41

Bertaccini, A., Botti, S., Tabanelli, D., Dradi, G., Fogher, C., Previati, A. et Da Re, F., 2004. Micropropagation and establishement of Mite-Borne virus-free garlic (Allium Sativum). Acta Horticulturae, 631: XXVI International Horticultural Congress: Issues and Advances in Transplant Production and Stand Establishment Research.

Biniam, T. et Tadesse M. 2008. A survey of viral status on potatoes grown in Eritrea and in vitro virus elimination of a local variety 'Tsaeda embaba'. African Journal of Biotechnology, 4 (7) pp. 397-403.

Blodgett, E.C and GOme~, 1967. Fig mosaic. Plant Disease, 51: 893-896.

Boxus, P., 1998. Plant biotechnology applied to horticultural crops, Agricultural Research Center, Biotechnology Department, Gembloux- Belgium World conference on horticultural research, 1998, Rome-Italy.

Carraro, L., Osler, R., Loi, N., Ermacora, P. et Refatti, E., 1998. Fruit tree phytoplasma diseases diffused in nature by psyllids, Acta Horticulturae, 550: 345- 350.

Castellano, M. A., Gattoni, G., Minafra, A., Conti, M. et Martelli, G. P., 2007. Fig mosaic in Mexico and South Mrica. Journal of Plant Pathology, 89 (3) : 441-444.

Chalak L., 2002. Rapport annuel, unite de culture des tissus, 40-54 pp.

Chalak, L., Chehade, A., Mattar, E. et Khadri, B., 2008. Morphological characterization of fig accessions cultivated in Lebanon. Acta Horticulturae, 798 : 49- 52.

Chalak, L., Elbitar, A., Masaad, W. et Choueiri, E., 2004. Assainissement de la pomme de terre infectee par le virus pyyNTN par culture de meristemes. Journal scientifique libanais, 1(5): 37- 44.

Chalak, L., Elbitar, A., Rizk, R., Choueiri, E., Salar, P. et Bove, J.M., 2005.

Attempts to eliminate Candidatus phytoplasma phoenicium from infected Lebanese almond varieties by tissue culture techniques combined or not with thermotherapy.

European Journal of Plant Pathology,! (112) : pp. 85-89.

Cheng, S., Fockler, C., Barnes, W.M. et Higuchi, R., 1994. Effective amplification of long targets from cloned inserts and human genomic DNA. Proc Natl Acad Sci, 91: 5695-5699.

42

Choueiri, E., Jreijiri, F., Verdin, E., Salar P., Danet J.L., Bove J. et Garnier M., 2002. First report of grapevine "Bois Noir'' Disease and of a New phytoplasma infecting Solanaceous Plants in Lebanon. Plant Disease, 86: 697.

Choueiri, E., Verdin, E., Danet, J.L., Jreijiri, F., El Zammar, S., Salard, P., hove, J.M. et Garnier, M., 2003. Phytoplasma disease of almond in Lebanon. In:

Options Mediterraneennes, 45: Virus and virus-like diseases of stone fruits, with particular reference to the Mediterranean region, Myrta A, Di Terlizzi B., and Savino V. ed., pp.123-125.

Clark, M. F., Adams, A. N., Thresh, J. N., Casper, R., 1976. detection of plum pox and other viruses by ELISA. Acta Horticulturae, 67: 51-57.

Comlekcioglu, S., Kuden, A. B., Kacar Y. A., Kamberoglu, M.A., 2007.

Meristem culture of two fig cultivars in Turkey. Fruits, 62 : 125-131.

Condit, I. J. et Home, W. T., 1933. A mosaic of the fig in California.

Phytopathology, 23: 887-897.

Cousin, M. T., 1995. Phytoplasmes et phytoplasmoses, Agronomie, 5 (15):

245-264.

Davies, D. R. et Allen, Y., 1973. In vitro propagation of freesia, ann. Rep. John Innes lnst., pp. 67-68.

Deogratias, J. M., Lutz, A. et Dosba, F., 1985. In vitro shoot-tips micrografting from juvenile and adult Prunus avium L. and Prunus persica Batsch to produce virus-free plants. Acta Horticulture, 13^1h International Symposium on Fruit Tree Virus Disease.

Doctrinal, M., 2000, La culture de meristemes, un outil pour assainir les varietes, INRA, centre de recherche de Bordeaux.

Doctrinal, M., 2001, Les techniques de culture in vitro. INRA, centre de recherche de Bordeaux.

English, H., 1960. Experiment on the control of Phomopus canker in kodata fig trees. In: Proc. 14th Ann. Res. Conf. Calif.. Fig Jnst.:13-15.

English, H., 1962. Canker and dieback disorders of fig trees. In: Proc. 16th Ann. Res. Conf. Calif. Fig Inst.: 13-15.

Elbeaino T., Digiaro M., Alabdullah A. K., De Stradis, A., Minafra, A., Mielke, N., Castellano M. A. et Martelli G. P., 2009. A multipartite negative-sense

43

single-stranded RNA virus is the putative agent of fig mosaic disease. Journal of General Virology, 90 (5): 1281-1288.

Elbeaino T., Digiaro, M., Nahdi S., Alabdullah A. et Martelli G. P., 2009.

Fig leaf mottle-associated virus 1 and Fig leaf mottle-associated virus 2 in the Mediterranean region and study of sequence variation of the hsp70 gene. Journal of Plant Pathology, (Manuscript N°: JPP485RP, In Press).

Elbeaino, T., Digiaro, M., De Stradis, A. and Martelli, G. P., 2007.

Identification of a second closterovirus in mosaic-diseased figs. Journal of Plant Pathology. 89 (1): 119-124.

Elbeaino, T., Digiaro, M., De Stradis, A. et Martelli, G. P., 2006. Partial characterization of a closterovirus associated with a chlorotic mottling of fig. Journal of Plant Pathology, 88: 187-192.

Elbeaino T., Choueiri, E., Hobeika C. et Digiaro, M., 2007. Presence of FLMaV 1 and 2 in Lebanese fig orchards (short communication). Journal of Plant Pathology, 89 (3), 409-411.

Faccioli, G., 2001. Control of potato viruses using meristem and stem-cutting cultures, thermotherapy and chemotherapy in virus and virus-like diseases of potatoes and seed-potatoes. Eds. Loebenstein, G, Berger, P.-H., A.-A., Brunet, R.G., Lawsan, Kluwer Academic publishers, Dodrescht: 365-390.

Garrett, R. H., Grisham, C. M., Lubochinsky, B., 2000. Biochimie, DeBoeck universite, p 419.

Gattoni G., Minafra A., Castellano M. A., De Stradis A., Boscia D., Elbeaino T., Digiaro M. et Martelli G.-P., 2009. Fig latent virus 1, a new member of the family Flexiviridae. Journal of Plant Pathology (In Press).

George, E.F., 1993. Plant propagation by tissue culture. The Technology, Part I, 147 p.

Grbelja,

J.

Acta Bot. Croat., 42 : 11-14.

Griffiths, A. J. F., Miller, J.H., Suzuki, D. T., lewontin, R. C. et Gelbart, W.

M., 1996. An introduction to genetic analysis. W.H. Freeman and Co. (ed.), New- York.

44

Guesdon, J. L., Chevrier, D., Tbam, T. N. Thierry, D., 1991. Nonradioactive molecular hybridization. Bulletin de la Societe de pathologie exotique, 84: 697-703.

Higuchi, R., Dollinger, G., Walsh, P. S. et Griffith, R., 1992. Simultaneous amplification and detection of specific DNA sequences. Biotechnology, 10: 413--417.

irget, M.E., Aksoy, U., Okur, B., Ongun, A.R. et Tepecik, M., 2008. Effect of calcium based fertilization on dried fig (Ficus carica L. cv. Sarzlop) yield and quality. Scientia horticulturae, 4 (118): 308-313.

Jonard, R., 2002. Le micro-greffage et ses applications en arboriculture fruitiere, Laboratoire des cultures de tissus vegetaux, Universite des Sciences et Techniques du Languedoc, Montpellier, pp. 14-19.

Kartesz, J., 1999. Ficus L., ITIS Report. 5 fevrier 2009.

(http:Uwww.itis.gov/servlet/SingleRpt/SingleRpt?search topic=TSN&search_value=

19081)

Kester, D. E., 1970. Growth in vitro of tissue of almond, almond hybrids and some other Prunus. Hortscience, 5(4):349.

Lemoine, J., 2000. Maladies de degenerescence des arbres fruitiers

a

a

Li, L. Y., Procter, C. H., 1944. A virus disease of fig in New Zealand. N. Z. J.

Sci. Techno[., 26: 88-90.

Limasset, P. et Comuet, P., 1949. Recherche du virus de la mosalque du tabac dans les meristemes des plantes infectees, C.R.Acad. Sci., 235: 1324-1325.

Maragara, J., 1982. Bases de la multiplication vegetative, les meristemes et l'organogenese. INRA, Paris, 261 pp.

Martelli, G.P., 1994, Production and distribution of certified propagative material with special reference to fruit crop. In: "the European and Mediterranean experience", ^yth Arab Congress on plant protection, Maroc.

Martelli, G. P., Castellano, M.A. et Lafortezza R., 1993. An ultrastructural study of fig mosaic. Phytopath. Medit., 32 : 33-43.

Ministere de l' Agriculture Libanaise, 2008. Statistiques agricoles. Liban.

Morel, G. et Martin, C., 1952, Guerison de dahlias d'une maladie

a

Acad. Sci., 235 : 1324-1325.

45

Mosella, C., Signoret, P. A. et Jonard, R., 1980. Sur la mise au point de technique d'apex en vue de !'elimination de deux types de particules virales chez le pecher (Prunus persica Batsch), C.R. Acad. Sci., 290 : 287-290.

Mullis, K., Faloona, F., Scharf, S., Saiki, R., Hom, G. Et Erlich, H., 1986.

Specific enzymatic amplification of DNA in vitro: the polymerase chain reaction.

Cold Spring Harb Symp Quant Bioi., 1: 263-73.

Murashige, T. et Skoog, F., 1962. A revised medium for rapid growth and bioassay with tobacco tissue cultures. Physiol Plant, (15): 473-479.

Myrta A., Di Terlizzi B., Boscia D., Choueiri E., Gatt M., Gavriel L., c;;aglayan K., Varveri C., Zeramdini H., Aparicio F., Pallas V. et Savino V., 2001.

Serological characterisation of Mediterranean Prunus Necrotic Ring Spot Virus Isolates. Journal of Plant Pathology, 83: 45-49.

Nahdi S., Elbeaino T., Digiaro M. and Martelli G. P., 2006. First record of fig leaf mottle-associated virus 1 in Tunisia. Journal of Plant Pathology, 88 (3, supplement): 70.

Navarro, L. et Juarez, J., 1977. Tissue culture techniques used in Spain to recover virus-free Citrus plants, Acta Hort., 78: 425-435.

Nicoli, M., 1985. La regeneration des agrumes en Corse par la technique du micro-greffage des meristemes «in vitro,, Fruits, 40 (2):113-136.

Obenauf, G. L., 1978. Review of fig spoilage research. Proc. Calif Fig Res.

Inst., pp. 48-51.

Oukabli, A., 2003. Le figuier un patrimoine genetique diversifie

a

Peaucelle, A., Laufs, P., 2006. Des Casques Bleus chez les plantes : un microARN, miR164, stabilise les frontieres dans le meristeme I The UN peace- keeping force in the plants: A microRNA, miR164, stabilizes the boundary domain in the meristem. MIS : medecine sciences, 10 (22) : 796-798. 23 mars 2009.

(http:ijid.erudit.org/iderudit/013801ar)

Plavsic, B. et Milicic, D., 1980. Intracellular changes in trees infected with fig mosaic. Acta Hortic.,110: 281-286.

Putz, C., 1971, Obtention de framboisiers (Bois blanc) sans virus par la technique des cultures de meristemes. Ann. Phytopathology, 3: 493-501.

46

Quacquarelli, A., 1971. ll mosaico del fico e il virus latente del Chenopodium.

Phytopath. Medit, 10: 283-286.

Quak, F., 1970, Review of heat treatment and meristem-tip culture as a method to obtain virus free plants, In: Proc. XVIll int Congr. Tel Aviv, 3, 12-25.

Salasse, J. P., sans date. Glossaire du figuier, les Ecologistes de 1 'Euziere. 5 mars 2009.

(http://www.educ-

envir .org/ -euziere/science/article.php3 ?id article=296#haut_page)

Skiredj, A., Walali, D.M.L. et El Attir, H., 2007. Le figuier (Ficus carica L.), L'art de produire des legumes et des fruits au Maroc. 20 janvier 2009.

(http:ljwww .legume-fruit -maroc.com/figuier .php)

Stone, 0. M., 1963. Factors effecting the growth of carnation plants from shoot apices. Ann. appl. Bioi., 52: 199-209.

Thomas C., Meyer D., WoltT M., llimber C., Alioua M. et Steinmetz A., 2003. Molecular characterization and spatial expression of the sunflower ABP1 gene.

Plant Mol. Bioi., 52(5):1025-1036.

VanDerMeer, F. A., 1980. Witches broom in Populars, Populier. 17: 42-49.

Vidaut, J., Baccaunaud, M., Caraglio, Y., Hutin, C., et Roger, J.-P., 1997.

Le figuier. CTIFL Publishers, Paris, France, p. 263.

Viennot-Bourgin, M., 1981. Thermotherapie et multiplication in vitro d'apex de vigne - application a la separation ou !'elimination de diverses maladies de type viral eta I' evaluation des degats. Seance de 1' Academie d' Agriculture de France.

Vlugt, C.I.M. V.D., Linthorst, H.J.M., Schadewuk, A.R. V. et Bol, J.F.,1988. Detection of tobacco rattle virus in different parts of tulip by ELISA and eDNA hybridisation assays. Neth J. Plant Pathol, 94 (3): 149-160.

Walia, J. J., Salem, N. M., et Falk, B. W., 2009. Partial sequence and survey analysis identify a multipartite, negative-sense RNA virus associated with fig mosaic.

Plant disease, 1 (93): 4-10.

Walkey, D. G. A, 1980, production of virus-free plants by tissue culture, pp.

109-117 in Ingram and Helgeson ( eds ).

Wallali, L. D., Skiredj, A., El Attir, H., 2003. Fiches techniques l'amandier, l'olivier, le figuier, le grenadier. Transfert de technologie en agriculture. 105 p 3.

47

Walter, B., 1998. Viruses and virus-diseases of the grapevine: diagnosis and control methods. Virologie, 6 (2): 435-44, Novembre - Decembre 1998, Revues.

Wang, W., Tronchet, M., Dorion, N., Albouy, J., 1988. Production of virus- free Dahlia: meristeme-culture and virus detection through eDNA probes and ELISA . . Acta Hort., 234: 421-428.

Xu, H., Nie, J., et De BoerS. H., 2005. Differentiation and molecular detection of Canadian necroticstrains of Potato Virus Y. The Canadian J oumal of Plant Pathology. Volume 27, numero 1, janvier- mars 2005, ISSN 0706-0661. p125-131.

Zohary, D. et Spiegel-Roy P., 1975. Beginnings of fruit growing in the Old World. Science, 187: 319-327.

Zcyd J.P., Brettell, R., Cerreudre, J., Duhoux, E., Gaspar, T., Gazeau, C.

M., Holsters, M., Jacobs, M., Monnier, M., Negrutiu, I., Paszkowski, J., Pelletier, G., Potrykus, I., Saul, M. W., Shillito, R. D., Van Den Eede, Get Vemaded, D., 1988, Cultures de cellules, tissus et organes vegetaux, fondements theoriques et utilisations pratiques, Edition Tee & Doc, 1 ere edition, 305 pp.

48 ~.