The banana tree (Musa spp.), is a plant explored in the majority of the tropical countries. Its fruit is consumed in practically all the countries of the world "in nature" or in form of candies, compotes, tinned, flakes, etc., which had to its energy value, caloric and wealth in vitamins and leaves minerals. The banana tree can be attacked by some pathogen, being distinguished fungus Fusarium oxysporum f. sp. cubense (Foc) that cause Panama disease, one of the main diseases of the culture. The search for alternative methods of control has been searched and in special the use of essential oils and vegetables extracts. This work was developed in laboratory conditions and greenhouse of the CECA/UFAL, with objectives to evaluate the fungitoxici effect "in vitro", "in vivo" and as inductive resistance of essential oils of Eucalyptus spp. (eucalypt citriodora variety), Cymbopogon winteranus (citronella), Caryophyllus aromaticus L. (india crave) and Piper aduncum L. (pepper-of-monkey) and of watery vegetables extracts of Caryophyllus aromaticus L. (india crave), Cinnamomum zeylanicum (cinnamon), Allium sativum L. (garlic), Ocimum basilicum (basil), Zingiber officinale Rosc. (ginger) and Ruta graveolens (arruda) and of the commercial products the ASM and Ecolife®. The oils had been added to the way of BDA in the 1,25 concentrations; 2,5; 3,75 and 5% and the watery vegetables extracts had been added to the way of BDA, in the concentrations of 5; 10; 15 and 20 % for watery vegetables extracts and 0,25; 0,5; 0,75 and 1 % for Ecolife®. For evaluation the index of intensity of the disease (ID), spread plants of banana tree of the variety apple had been sprayed with essential oils (1,25%) and with extracts of garlic and cinnamon (20%), extract of crave (15%) and Ecolife® (0,75%).After, the plants had been eight days inoculated with the suspension of Foc (104 con.mL-1), for the method of immersion of roots, for one hour. The oil of citronella, eucalypt, in all the tested concentrations, and the extracts of crave (15 and 20%) and Ecolife® (0,75 and 1%) had inhibited in 100% the mycelial growth of fungus, thus constituting the best treatments. While that, the oils of crave and pepper-of-monkey and the extracts of garlic and cinnamon had inhibited the mycelial growth partially, in all the tested concentrations, being that the extract of garlic to the measure that increased the concentration showed an increment in the PIC of fungus. In the in vivo evaluation, the oil of citronella presented the best one resulted (ID=25%), differing from the witness, the oils of crave and of eucalypt ID had presented = 95 and 75%, respectively. The extracts of crave and garlic, and Ecolife® ID had presented = 29, 25 and 8,33%. The total or partial inhibition of the mycelial growth of F. oxysporum f. sp. cubense, evaluated "in vivo", for essential oils and watery vegetables extracts, in the sample composites of antifúngica action. The activities of the phenylalanine enzyme ammonia lyase (PAL) were determined in plants submitted to the treatments, collected to the 24, 48, 72, 144 and 288 hours after the inoculation. Significant difference between the inductors and the witness, being distinguished Ecolife®, extract of basil was observed, oil of citronella, oil and extract of crave, and extract of arruda that had provided to the lesser indices of disease in the apple banana changes, respectively with 8,33; 12,5; 25,0; 29,17; 25,0; e 41,67 %.The Ecolife® inductor presented the best performance in relation to the other tested inductors and the witness, providing a control of 83,7% of the disease. All the analyzed treatments had presented the presence of the enzyme in accordance with, varying the level the date of extraction and the extract or used oil as inductive of resistance. The extract of crave presented one high enzymatic activity in extraction 24 and 48 hours after the inoculation of inoculate it. / Fundação de Amparo a Pesquisa do Estado de Alagoas / A bananeira (Musa spp.), é uma planta explorada na maioria dos países tropicais. Seu fruto é consumido em praticamente todos os países do mundo in natura ou em forma de doces, compotas, enlatados, flocos, etc., devido ao seu valor energético, calórico e riqueza em vitaminas e sais minerais. A bananeira pode ser atacada por vários patógenos, destacando-se o fungo Fusarium oxysporum f. sp. cubense (Foc) que causa o mal-do-Panamá, uma das principais doenças da cultura. A busca por métodos alternativos de controle tem sido pesquisada e em especial o uso de óleos essenciais e extratos vegetais. Este trabalho foi desenvolvido em condições de laboratório e casa-de-vegetação do CECA/UFAL, com os objetivos de avaliar o efeito fungitóxico in vitro , in vivo e como indutores de resistência de óleos essenciais de Eucalyptus spp. (eucalipto variedade citriodora), Cymbopogon winteranus (citronela), Caryophyllus aromaticus L. (cravo-da-índia) e Piper aduncum L. (pimenta-de-macaco) e dos extratos vegetais aquosos de Caryophyllus aromaticus L. cravo-da-índia, Cinnamomum zeylanicum (canela), Allium sativum L. (alho), Ocimum basilicum (manjericão), Zingiber officinale Rosc. (gengibre) e Ruta graveolens (arruda) e dos produtos comerciais o ASM e Ecolife®. Os óleos foram adicionados ao meio de BDA nas concentrações de 1,25; 2,5; 3,75 e 5% e os extratos vegetais aquosos foram adicionados ao meio de BDA, nas concentrações de 5; 10; 15 e 20 % para os extratos vegetais aquosos e 0,25; 0,5; 0,75 e 1 % para Ecolife®. Para avaliação o índice de intensidade da doença (ID), plantas micropropagadas de bananeira da variedade maçã foram pulverizadas com os óleos essenciais (1,25%) e com os extratos de alho e canela (20%), extrato de cravo (15%) e Ecolife® (0,75%). Após oito dias, as plantas foram inoculadas com a suspensão de Foc (104 con.mL-1), pelo método de imersão de raízes, por uma hora. O óleo de citronela, eucalipto, em todas as concentrações testadas, e os extratos de cravo (15 e 20%) e Ecolife® (0,75 e 1%) inibiram em 100% o crescimento micelial do fungo, constituindo assim os melhores tratamentos. Enquanto que, os óleos de cravo e pimenta-de-macaco e os extratos de alho e de canela inibiram parcialmente o crescimento micelial, em todas as concentrações testadas, sendo que o extrato de alho à medida que aumentava a concentração mostrava um incremento na PIC do fungo. Na avaliação in vivo , o óleo de citronela apresentou o melhor resultado (ID=25%), diferindo da testemunha, os óleos de cravo e de eucalipto apresentaram um ID= 95 e 75% , respectivamente. Os extratos de cravo e alho, e Ecolife® apresentaram um ID= 29, 25 e 8,33%. A inibição total ou parcial do crescimento micelial de F. oxysporum f. sp. cubense, avaliado in vivo , pelos óleos essenciais e extratos vegetais aquosos, nos mostra compostos de ação antifúngica. As atividades da enzima fenilalanina amônia liase (FAL) foi determinado em plantas submetidas aos tratamentos, coletadas às 24, 48, 72, 144 e 288 horas após a inoculação. Foi observada diferença significativa entre os indutores e a testemunha, destacando-se Ecolife®, extrato de manjericão, óleo de citronela, óleo e extrato de cravo, e extrato de arruda que proporcionaram os menores índices de doença nas mudas de banana maça, respectivamente com 8,33; 12,5; 25,0; 29,17; 25,0; e 41,67 %. O indutor Ecolife® apresentou o melhor desempenho em relação aos outros indutores testados e a testemunha, proporcionando um controle de 83,7% da doença. Todos os tratamentos analisados apresentaram a presença da enzima, variando o nível de acordo com a data de extração e o extrato ou óleo utilizado como indutor de resistência. O extrato de cravo apresentou uma alta atividade enzimática na extração 24 e 48 horas após a inoculação do inoculo.

Banana is the most important fruit crop in the world but ironically one of the crops least studied. This fruit constitutes a major staple food for millions of people in developing countries and also it is considered the highest selling fruit in the world market making this crop a very important export commodity for the producing countries. At the present time, one of the most significant constraints of banana production that causes significant economical losses are fungal diseases. Among these, Panama disease, also known as Fusarium wilt has been the most catastrophic. Panama disease is caused by the soil-borne fungus Fusarium oxysporum formae specialis (f.sp) cubense (FOC), which infects susceptible bananas through the roots causing a lethal vascular wilt. To date, the race 4 of this pathogen represents the most serious threat to banana production worldwide since most of the commercial cultivars are highly susceptible to this pathogen. Introduction of FOC resistance into commercial cultivars by conventional breeding has been difficult because edible bananas are sterile polyploids without seeds. Genetic transformation of banana, which has already been established in various laboratories around the world has the potential to solve this problem by transferring a FOC race 4 resistance gene into susceptible banana cultivars (eg. Cavendish cultivars). However, a FOC resistant (R) gene has not been isolated. Genes that confer resistance to Fusarium oxysporum have been isolated from tomato and melon using a map-based positional cloning approach. The tomato I2 and melon Fom-2 genes belong to the non-Toll/interleukin like receptors (TIR) subclass of nucleotide-binding site and leucine-rich repeat (NBS-LRR) R genes. These genes confer resistance only to certain races of F. oxysporum in their corresponding plant families limiting their use in other plant families. The fact that these two Fusarium resistance genes share the same basic non-TIR-NBS-LRR structure suggests a similar Fusarium resistance mechanism is shared between the families Solanaceae and Cucurbitaceae. This observation opens the possibility to find similar Fusarium resistance genes in other plant families including the Musaceae. A remarkable discovery of a population of the wild banana Musa acuminata subspecies (ssp.) malaccensis segregating for FOC race 4 resistance was made by Dr. Ivan Buddenhagen (University of California, Davis) in Southeast Asia. Research carried out at Queensland Department of Primary Industries (Australia) using this plant material has demonstrated that a single dominant gene is involved in FOC race 4 resistance (Dr. Mike Smith, unpublished results). Tissue-culture plantlets of this FOC race 4 segregating population were kindly provided to the Plant Biotechnology Program (Queensland University of Technology) by Dr. Mike Smith to be used in our research. This population holds the potential to assist in the isolation of a FOC race 4 resistance gene and other potential Fusarium resistance genes. The overall aims of this research were to isolate and characterise resistance gene candidates of the NBS-type from M. acuminata ssp. malaccensis and to identify and characterise potential Fusarium resistance genes using a combination of bioinformatics and gene expression analysis. Chapter 4 describes the isolation by degenerate PCR of five different classes of NBS sequences from banana (Musa acuminata ssp malaccensis) designated as resistance gene candidates (RGCs). Deduced amino acid sequences of the RGCs revealed the typical motifs present in the majority of known plant NBS-LRR resistance genes. Structural and phylogenetic analyses showed that the banana RGCs are related to non-TIR subclass of NBS sequences. The copy number of each class was estimated by Southern hybridisation and each RGC was found to be in low copy number. The expression of the RGCs was assessed by RT-PCR in leaf and root tissues of plants resistant or susceptible to Fusarium oxysporum f. sp. cubense (FOC) race 4. Four classes showed a constitutive expression profile whereas no expression was detected for one class in either tissue. Interestingly, a transcriptional polymorphism was found for RGC2 whose expression correlated with resistance to FOC race 4 suggesting a possible role of this gene in resistance to this devastating FOC race. Moreover, RGC2 along with RGC5 showed significant sequence similarity to the Fusarium resistance gene I2 from tomato and were chosen for further characterisation. The NBS sequences isolated in this study represent a valuable source of information that could be used to assist the cloning of functional R genes in banana. Chapter 5 describes the isolation and characterisation of the full open reading frame (ORF) of RGC2 and RGC5 cDNAs. The ORFs of these two banana RGCs were predicted to encode proteins that showed the typical structure of non-TIR-NBS-LRR resistance proteins. Homology searches using the entire ORF of RGC2 and RGC5 revealed significant sequence similarity to the Fusarium resistance gene I2 from tomato. Interestingly, the phylogenetic analysis showed that RGC2 and RGC5 were grouped within the same phylogenetic clade, along with the Fusarium resistance genes l2 and Fom-2. These findings suggest that the banana RGC2 and RGC5 are potential resistance gene candidates that could be associated with Fusarium resistance. The case of RGC2 is more remarkable because its expression was correlated to FOC race 4 resistance (Chapter 4). As a first step to test whether RGC2 has a role in FOC race 4 resistance, different expression constructs were made with the ORF of this sequence. One of the constructs contains a RGC2 putative promoter region that was successfully cloned in this work. These constructs will be used to transform susceptible banana plants that can then be challenged with FOC race 4 to assess whether resistance has been acquired by genetic complementation. The results of this thesis provide interesting insights about the structure, expression and phylogeny of two potential Fusarium resistance genes in banana, and provide a rational starting point for their functional characterisation. The information generated in this thesis may lead to the identification of a Fusarium resistance gene in banana in further studies and may also assist the cloning of Fusarium resistance genes in other plant species.