Submitted by Cássia Santos (cassia.bcufg@gmail.com) on 2017-01-10T09:15:32Z No. of bitstreams: 2 Dissertação - Nayane Oliveira Almeida - 2016.pdf: 4246837 bytes, checksum: 2ee0e2f0ece0d114ea4032c91be4556f (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) / Approved for entry into archive by Luciana Ferreira (lucgeral@gmail.com) on 2017-01-10T10:58:54Z (GMT) No. of bitstreams: 2 Dissertação - Nayane Oliveira Almeida - 2016.pdf: 4246837 bytes, checksum: 2ee0e2f0ece0d114ea4032c91be4556f (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) / Made available in DSpace on 2017-01-10T10:58:54Z (GMT). No. of bitstreams: 2 Dissertação - Nayane Oliveira Almeida - 2016.pdf: 4246837 bytes, checksum: 2ee0e2f0ece0d114ea4032c91be4556f (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) Previous issue date: 2016-03-22 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - CAPES / The problems caused by nematodes and by the Panama disease on banana plantations are responsible for production losses and limiting to its cultivation. In the state of Goiás there is few information about the nematode genus that affect this crop, and its relationship with the incidence of the fungus Fusarium oxysporum f. sp. cubense (Foc). This research aimed to survey the occurrence of plant parasitic nematodes, the incidence of Foc and soil attributes, and determine if there is a correlation among these factors. In January 2015, twelve banana producing regions in the state of Goiás were sampled: Anápolis, Caiapônia, Goiatuba, Itaguaru, Itumbiara (two areas), Jataí, Morrinhos, Ouro Verde, Palestina, Taquaral and Uruana. All sampled areas, except Morrinhos, revealed contamination with Foc, and all had different genus of nematodes. Meloidogyne sp., Helicotylenchus sp. and Rotylenchus sp. were the main genus of plant parasitic nematodes present in the banana plantations, with Meloidogyne sp. and Rotylenchus sp. the most dominant and abundant genus. We found that Pratylenchus sp. increases the population levels of F. oxysporum and that Helicotylenchus sp. has been affected by the concentration of P, Ca, Mn and the soil pH. / Os problemas fitossanitários causados por nematoides e pela doença mal-do-Panamá, na cultura da banana, são responsáveis por grandes perdas de produção ou são fatores limitantes de seu cultivo. Em Goiás, são escassas as informações sobre os gêneros de nematoides que afetam a bananicultura, bem como sua relação com a incidência do fungo Fusarium oxysporum f. sp. cubense (Foc), agente causal do mal-do-Panamá. Assim, esse trabalho teve por objetivo fazer levantamento da ocorrência de fitonematoides, da incidência de Foc e dos atributos dos solos, e determinar se há correlação entre estes fatores. Em janeiro de 2015, foram amostradas doze regiões produtoras de banana no estado de Goiás, distribuídas em onze municípios: Anápolis, Caiapônia, Goiatuba, Itaguaru, Itumbiara, Jataí, Morrinhos, Ouro Verde, Palestina, Taquaral e Uruana. Todas as áreas amostradas, exceto a do município de Morrinhos, apresentaram-se contaminadas com Foc, e todas apresentaram diversos gêneros de fitonematoides. Meloidogyne sp., Helicotylenchus sp. e Rotylenchus sp. foram os principais gêneros de fitonematoides presentes nos bananais no estado de Goiás, sendo Meloidogyne sp. e Rotylenchus sp. os gêneros mais dominantes e abundantes. Foi constatado que a presença de Pratylenchus sp. aumenta o nível populacional de F. oxysporum e que Helicotylenchus sp. é afetado pelos teores de P, Ca, Mn e pelo pH do solo.

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.

El objetivo de esta Tesis Doctoral es estudiar diferentes herramientas para el manejo de plagas y enfermedades del cultivo de la platanera. Entre las herramientas que vamos a desarrollar, se van a analizar los compuestos orgánicos volátiles (COVs) fúngicos derivados de hongos entomopatógenos (HE) y nematófagos, como fuente de metabolitos con actividad antagónica contra el picudo negro (PN) de la platanera, Cosmopolites sordidus, para su control y manejo en el campo. Así mismo, vamos a estudiar las respuestas de cultivares de plataneras a quitosano, un polisacárido biodegradable, para evaluar su posible uso en el campo como estimulante y protector de las plantas frente a plagas y patógenos, como Fusarium oxysporum f. sp. cubense. El conjunto de capítulos de esta tesis pretende sentar las bases de una estrategia de manejo sostenible de plagas y enfermedades del cultivo de la platanera, basada en el uso de COVs derivados de hongos presentes de forma natural en los cultivos, en combinación con la suplementación de quitosano en el riego, para un efecto de protección y activación de las defensas de las plataneras antes de cualquier infección de plagas y/o enfermedades. El objetivo principal de esta Tesis Doctoral es encontrar nuevas fórmulas para la gestión integrada de plagas como Cosmopolites sordidus y enfermedades de la platanera en condiciones de campo. En esta Tesis Doctoral hemos ideado enfoques sostenibles para la gestión de las plagas y enfermedades de las plataneras. Nuestros objetivos son: a) Cosmopolites sordidus (picudo negro de la platanera, PN), la principal plaga de los cultivos de plátano y, b) el hongo del marchitamiento Fusarium oxysporum f. sp. cubense Raza Tropical 4 (FocTR4), agente causante de una nueva variante extremadamente virulenta de la enfermedad del “Mal de Panamá”, que se está extendiendo rápidamente por todo el mundo. Nuestras herramientas de gestión sostenible son: a) los hongos entomopatógenos (HE, conocidos por su uso como agentes de control biológico, ACBs) aislados de campos comerciales de plátanos, b) sus compuestos orgánicos volátiles (COVs) y, c) el quitosano, un compuesto biodegradable y elicitor de la inmunidad de las plantas con actividad antimicrobiana. Damos evidencia de que los COVs de los hongos agentes de control biológico son repelentes del PN. Pueden utilizarse en los cultivos de platanera mediante estrategias de push and pull para gestionar la plaga de forma sostenible. El quitosano puede utilizarse en el riego para prevenir las defensas de la platanera local y sistémicamente. Por lo tanto, este polímero, con probada actividad antimicrobiana frente a otros patógenos de marchitamiento de Fusarium spp., podría utilizarse contra la actual pandemia en las plataneras causada por FocTR4. La capacidad de inducir reguladores del crecimiento de las plantas sostiene también el papel fertilizante del quitosano. La inducción de compuestos relacionados con la respuesta sistémica adquirida (RSA) hace que el riego con quitosano sea una herramienta para manejar también las plagas de las plataneras sobre el suelo (PN) y las enfermedades (Sigatoka). De esta manera, los COVs y el quitosano podrían ayudar a reducir el uso de agroquímicos tóxicos en los cultivos de platanera en todo el mundo.