FundaÃÃo Cearense de Apoio ao Desenvolvimento Cientifico e TecnolÃgico / O mamoeiro (Carica papaya) à uma importante fruteira tropical e sua produÃÃo està aumentando no Nordeste Brasileiro. O Papaya lethal yellowing virus (PLYV) à encontrado infetando o mamoeiro somente no Nordeste brasileiro, aonde vem constituindo sÃrio problema para os produtores. O PLYV à bastante estÃvel e pode ser transmitido por inoculaÃÃo mecÃnica, solo, Ãgua de irrigaÃÃo, mÃos e instrumentos de corte contaminados. A presente atividade de pesquisa teve por objetivo avaliar os efeitos de produtos quÃmicos e de extratos de plantas de uso medicinal sobre a infectividade do PLYV; acompanhar a distribuiÃÃo do vÃrus em plantas inoculadas e avaliar sua interaÃÃo com o Papaya ringspot virus (PRSV). Extratos de plantas infetadas com PLYV foram misturados com igual volume de um dos seguintes produtos quÃmicos: Ãlcool, n-butanol, sabÃo lÃquido comercial, Triton X-100, sÃdio dodecilsulfato (SDS), hipoclorito de sÃdio e carbonato de sÃdio e com extratos de 23 espÃcies vegetais de uso medicinal. As misturas foram incubadas por 30 min em condiÃÃes de laboratÃrio e mecanicamente inoculadas em plantas de mamoeiro. Amostras iguais de tecidos de plantas inoculadas com PLYV foram avaliadas por ELISA indireto para determinar o tempo necessÃrio para sua infecÃÃo sistÃmica. De acordo com os resultados, somente SDS, hipoclorito de sÃdio e carbonato de sÃdio inativaram o vÃrus, mas o SDS ocasionou danos nas plantas e o carbonato ocasionou pequenas pontuaÃÃes esbranquiÃadas. De outra parte, nenhum dos extratos de plantas inativou completamente o PLYV, mas o extrato de Schinus terebinthifolius retardou os sintomas do vÃrus. Tais resultados demonstram a importÃncia do uso de soluÃÃes de hipoclorito de sÃdio para a inativaÃÃo do PLYV na superfÃcie de ferramentas agrÃcolas. Estudos da movimentaÃÃo do vÃrus em plantas inoculadas indicaram que a presenÃa do PLYV pode ser detectada por sorologia nas folhas inoculadas trÃs a quatro dias apÃs a inoculaÃÃo, seis dias no caule, 10 dias nas raÃzes, 15 dias nas folhas mais jovens e a planta inteira foi infetada sistemicamente somente apÃs 25 a 30 dias. Estudos de gama de hospedeiros confirmaram que o PLYV nÃo infeta espÃcies das famÃlias: Amaranthaceae, Brassicaceae, Fabaceae, Pedaliaceae e Solanaceae, nem ocasiona lesÃes locais nas indicadoras Chenopodium amaranticolor; C. murale e C. quinoa. Os estudos sobre a interaÃÃo entre PLYV e PRSV demonstraram a existÃncia de sinergismo entre os dois vÃrus em mamoeiro. A tÃcnica de imunoprecipitaÃÃo de RT-PCR (IP-RT-PCR) demonstrou ser um mÃtodo prÃtico e especÃfico para amplificaÃÃo do RNA do vÃrus, reduzindo problemas de contaminaÃÃo com o RNA de plantas. / Papaya (Carica papaya) is an important tropical fruit crop and its production is increasing every year in Northeastern Brazil. Papaya lethal yellowing virus (PLYV) is found infecting papaya only in Northeastern Brazil where it has become a serious problem for the papaya producers. PLYV is very stable and it can be readily transmitted by human actions including contaminated hands, agricultural tools, soil and irrigation water. The present study had the objective to evaluate the effects of chemical products and the extracts from 23 medicinal plants on the infectivity of PLYV in greenhouse experiments; to determine the distribution and movement of PLYV in mechanically inoculated plants and evaluate its interaction with Papaya ringspot virus (PRSV). Extracts from PLYV infected plants were mixed with equal amount of either one of the following chemical products: alcohol, n-butanol, commercial liquid soap, Triton X-100, sodium dodecilsulfate (SDS), sodium hypochlorite and sodium carbonate, and with equal amount of extracts from 23 medicinal plant species. The mixtures were incubated for 30 min at room temperature and mechanically inoculated in healthy papaya. Equal amounts of tissue from each part of papaya plants inoculated with PLYV were serologically evaluated to demonstrate how long the virus takes to infect systemically inoculated plants. According to the results only SDS, sodium hypochlorite and sodium carbonate inactivated the virus, but SDS caused damage in the plants and carbonate caused small whitish points on the treated leaves. On the other hand, neither one of the used medicinal plant extracts inactivated completely the PLYV infectivity, but extracts from Schinus terebinthifolius inhibited the symptoms induced by the virus. Those results demonstrated the importance of sodium hypochlorite to inactivate PLYV in contaminated agriculture tools. The presence of PLYV in inoculated plants was serologically detected three to four days after inoculation in the inoculated leaves, only after six days in the stem, ten days in the roots, 15 days in the younger leaves and the plants were systemically infected only 25 to 30 days after inoculation. Host range studies confirmed that PLYV does not infect plant species from the families: Amaranthaceae, Brassicaceae, Fabaceae, Pedaliaceae and Solanaceae, neither cause local lesions in Chenopodium amaranticolor; C. murale and C. quinoa. Interaction studies indicated a synergistic effect between PLYV and PRSV in papaya. The technique of RT-PCR immunoprecipitation (IP-RT-PCR) has proven to be practical and specific for amplification of PLYV RNA, reducing problems of contamination with plant RNAs.

Abstract Ficus carica L. is considered one of the oldest fruit trees in the Mediterranean basin and is widely grown and harvested for the consumption of its fruits dry and fresh. This species is affected by different virus diseases, especially by Fig mosaic disease (FMD), for which Fig leaf mottle-associated virus 1 (FLMaV-1), Fig leaf mottle-associated virus 2 (FLMaV-2), Fig mild mottling-associated virus (FMMaV), Fig mosaic virus (FMV), Fig latent virus 1 (FLV-1), Fig badnavirus 1 (FBV-1) and Fig fleck-associated virus (FFkaV) are associated. FMD is the most widespread disorder of this species, which represents a threat and a constraint for healthy fig production and germplasm exchange. Thus, the objective of the present doctoral research was the establishment of an efficient and rapid in vitro F. carica propagation, sanitation and conservation of free-FMD plant material for future large-scale commercialization. Initially, FMD-related viruses distribution was screened within the different fig plant organs (buds, leaves, syconia and seeds) of 14 Mediterranean genotypes (Palazzo, Severoni precoce, Bianca, Pilusedda, Dottato bianco, Bifera, Zidi, Baiyadi, Biancu, Brogiotto nero, Catalanisca, Houmairi, Triboiti and Turca 'Serilop') which were utilized afterward as in vitro plant source material. RT-PCR assays revealed that all the aforementioned viruses were present without any exception in seeds, whereas only 4 viruses (FBV, FFkaV, FLMaV-1 and FMV) were detected in buds, leaves and syconia with highly variable infection rates. Moreover, encapsulation technology proved to be a powerful multiplication technique to sustain standard fig tissue culture protocol for three cultivars (Catalanisca, Palazzo and Bifera) and it gave high, almost similar, viability, regrowth and conversion rates. Microcutting rooting in one-step was achieved and conversion rate was comparable for the three cultivars. Furthermore, in order to eliminate FMD associated viruses, with the exception of FBV-1 which resisted to all the sanitation attempts, Caulogenesis and Meristem Tip Culture Protected by the Synthetic Seeds technique (MTC-SS) gave the best sanitation rates. Finally, F. carica (cv. Houmairi) artificial seeds conservation, for final delivery, was achieved. A high viability and moderate regrowth rates were registered with a lesser conversion rate strictly related to the plant growth regulators (PGRs) used. Keywords: Fig, mosaic, RT-PCR, virus distribution, cytokinins, encapsulation, micropropagation, synthetic seed. / Resumen La higuera (Ficus carica L.) es considerada como uno de de los árboles frutales más antiguos de la cuenca mediterránea y es ampliamente cultivado y cosechado para el consumo de sus frutos tanto secos como en fresco. Esta especie se ve afectada por diversas enfermedades virales, especialmente por la denominada "Fig mosaic disease" (FMD) asociada actualemnte a los virus: Fig leaf mottle-associated virus 1 (FLMaV-1), Fig leaf mottle-associated virus 2 (FLMaV-2), Fig mild mottling-associated virus (FMMaV), Fig mosaic virus (FMV), Fig latent virus 1 (FLV-1), Fig badnavirus 1 (FBV-1) y Fig fleck-associated virus (FFkaV). Esta enfermedad representa una amenaza y un obstáculo para la producción de higos y el intercambio de germoplasma. El principal objetivo del presente trabajo fue establecer un método de propagación de higuera in vitro para el saneamiento y la conservación de material vegetal libre de FMD para su posterior comercialización. Inicialmente, se estudió la distribución de los virus implicados en la enfermedad en diversos órganos de 14 genotipos de F. carica (Palazzo, Severoni precoce, Bianca, Pilusedda, Dottato bianco, Bifera, Zidi, Baiyadi, Biancu, Brogiotto nero, Catalanisca, Houmairi, Triboiti y Turca 'Serilop'), los cuales fueron utilizados posteriormente como fuente material vegetal in vitro. Los resultados obtenidos mediante RT-PCR revelaron que todos los virus mencionados estaban presentes sin excepción en las semillas, mientras que sólo cuatro de ellos (FBV, FFkaV, FLMaV-1 y FMV) fueron en brotes, hojas y siconios con tasas de infección variables. Además, la tecnología de encapsulación demostró ser una técnica de multiplicación eficaz para poder aplicar el protocolo estándar de cultivo de tejidos de higo para tres cultivares (Catalanisca, Palazzo y Bifera) dando altas tasas de viabilidad, rebrote y conversión. Se logró el enraizamiento de microcortes en un solo paso y el índice de conversión fue comparable para los tres cultivares. La callogénesis y el culñtivo de meristemos con la técnica de la semilla sintética (MTC-SS) fueron las técnicas que proporcionaron mayores tasas de desinfección para los virus estudiados a excepción de con FBV-1, entidad viral que no fue eliminada con ninguna de las técnicas ensayadas. Por último, se logró la conservación de las semillas artificiales de higuera (cv Houmairi), registrándose una alta viabilidad y tasas de rebrote moderadas con un menor grado de conversión estrictamente relacionado con hormonas utilizadas. Palabras clave: Higuera, mosaico, RT-PCR, la distribución de los virus, hormonas, encapsulación, micropropagación, y la semilla sintética. / Resum La figuera (Ficus carica L.) és considerada un dels arbres fruiters més antics de la conca mediterrània i és àmpliament conreat i collit per al seu consum fresc i sec. Les malalties virals, especialment "Fig mosaic disease" (FMD), associada amb els viruses: Fig leaf mottle-associated virus 1 (FLMaV-1), Fig leaf mottle-associated virus 2 (FLMaV-2), Fig mild mottling-associated virus (FMMaV), Fig mosaic virus (FMV), Fig latent virus 1 (FLV-1), Fig badnavirus 1 (FBV-1) i Fig fleck-associated virus (FFkaV). Esta malaltia representa una amenaça per a la producció de figues i l'intercanvi de germoplasma. El principal objectiu d'aquest treball va ser estableixerun mètode de propagació de figuera in vitro per al sanejament i la conservació de material lliure de FMD per a su posterior commercialització. Inicialment, es va estudiar la distribució dels virus associats a FMD en diversos òrgans en 14 genotips de F. carica (Palazzo, Severoni Precoce, Bianca, Pilusedda, Dottato bianco, Bifera, Zidi, Baiyadi, Biancu, Brogiotto diners, Catalanisca, Houmairi, Triboiti i Turca 'Serilop'), els quals van ser utilitzats posteriorment com a font de material vegetal in vitro. Els resultats obtinguts del anàlisis realitzats per RT-PCR van revelar que tots els virus eren presents sense excepció en les llavors, mentre que només quatre virus (FBV, FFkaV, FLMaV-1 i FMV) van ser detectats en brots, fulles i siconis amb taxes d'infecció variables. A més, la tecnologia d'encapsulació va demostrar ser una tècnica de multiplicació eficaç per poder aplicar el protocol estàndard de cultiu de teixits de figa per a tres cultivars (Catalanisca, Palazzo i Bifera) donant taxesadequades de viabilitat, rebrot i conversió. Es va aconseguir l'arrelament de microtalls en un sol pas i l'índex de conversió va ser comparable per als tres cultivars. La calogènesi i el cultiu de meristems protegits per llavors sintètiques (MTC-SS)van ser les tècniques que proporcionarem millores tases de desinfecció per als virus estudiats amb l'excepció de FBV-1 que es va resistir a tots els mètodes de sanejament. Finalment, es va aconseguir la conservació de la llavors artificials de figuera (cv. Houmairi), registrant-ne una alta viabilitat i taxes de rebrot moderades amb un menor grau de conversió estrictament relacionat amb hormones utilitzades. Paraules clau: Figuera, mosaic, RT-PCR, la distribució dels virus, hormones, encapsulació, micropropagació, i la llavor sintètica. / Yahyaoui, E. (2017). Use of standard and setup of non conventional techniques for the elimination of viruses associated with Fig Mosaic Disease (FMD) in fig germplasm (Ficus carica L.) [Tesis doctoral]. Universitat Politècnica de València. https://doi.org/10.4995/Thesis/10251/79876