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Submitted by (ana.araujo@ufrpe.br) on 2017-02-07T18:23:56Z No. of bitstreams: 1 Fabiana Aparecida Cavalcante Silva.pdf: 934712 bytes, checksum: d4f03c8904e031e8ea5d8cd19ffcfece (MD5) / Made available in DSpace on 2017-02-07T18:23:56Z (GMT). No. of bitstreams: 1 Fabiana Aparecida Cavalcante Silva.pdf: 934712 bytes, checksum: d4f03c8904e031e8ea5d8cd19ffcfece (MD5) Previous issue date: 2008-02-26 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - CAPES / Cotton is a crop of considerable importance worldwide, the production of which is affected by diverse biotic and abiotic factors. Stress caused by insect attacks and phytopathogens stand out among the biotic factors, the control of which is significantly hindered due the variability of these organisms and their consequent capacity for adaptation. Fungi are the phytopathogens that most cause damage to cotton plants, especially Colletotrichum gossypii South var. cephalosporioides A.S. Costa, a ramulosis-causing disease characterized by dwarfism and an over-budding of the branches, affecting the development of the buds and number of bolls. The main form of dissemination is through contaminated seeds and the most effect method of control is by means of chemical fungicides or resistant varieties which are obtained after exhaustive tests conducted in fieldwork and greenhouses. The use of biochemical and molecular tools that assist in the identification of resistant genotypes in improvement programs is of considerable importance, as they offer reliability and reduce selection costs in the development process of new varieties. In the present study, a biochemical/molecular study was carried out on cotton varieties submitted toinfection with the ramulosis fungus with the aim of identifying markers associated to the resistance process. In the first assay, biochemical descriptors were assessed in four infected cotton varieties. The plants were inoculated 20 days after cultivation, receiving a fungal concentration of 1 x 106 conidias/mL. Evaluations occurred at 3, 15 and 30 days following inoculation. Proline, peroxidase and catalase levels were quite expressive in the resistant BRS Antares cultivar, exhibiting a rapid response at 3 days after inoculation. Proline and catalase levels are indicated as useful tools for the identification of ramulosis-resistant varieties in cotton crop selection studies. The second assay sought to identify differentially expressed transcripts in cotton plants submitted to infection with the ramulosis fungus. Two varieties with opposing traits regarding resistance to this disease were used: BRS Antares and CNPA Precoce I, respectively resistant and susceptible. Ten RAPD oligonucleotides were used in the RT-PCR reactions at an annealing temperature of 35 ºC. Following an analysis of the band patterns in the control and inoculated plants, 12 down-regulated transcripts, 13 overregulated transcripts and ten activated transcripts were identified. A band ofapproximately 200 bp, obtained with the V10 oligonucleotide, was sequenced and exhibited 86% homology with the GmChl 3 gene, which codifies for thechlorophyllase III enzyme in soybean. This enzyme has considerable importance for vegetal cells submitted to oxidative damage, as it reduces this damage and modulates different defense pathways. The data obtained in the present study are applicable to studies on cotton plant responses to pathogen attacks and offers relevant information for crop improvement programs. / O algodão é uma cultura de grande importância mundial cuja produção é afetada por diversos fatores bióticos e abióticos. Dentre os bióticos destacam-se os estresses provocados pelo ataque de insetos e fitopatógenos, cujo controle é bastante difícil devido à variabilidade destes organismos e,conseqüente, a capacidade adaptativa. Entre os fitopatógenos, os fungos são os que causam mais danos ao algodoeiro,destacando-se o Colletotrichum gossypii South var. cephalosporioides A.S. Costa, causador da ramulose, doença caracterizada pelo nanismo e super-brotamento dos ramos, que afeta o desenvolvimento das maçãs e o número de capulhos. A principal forma de disseminação é via sementes contaminadas e o modo de controle mais efetivo é por meio de fungicidas químicos ou de cultivares resistentes que são obtidas por meio de exaustivos testes conduzidos em campo e casa de vegetação. A utilização de ferramentas bioquímicas e moleculares que auxiliem na identificação de genótipos resistentes nos programas de melhoramento são de grande relevância uma vez que, além da confiabilidade, reduz os custos de seleção nos processos de desenvolvimento de novas cultivares. No presente trabalho procedeu-se a um estudo bioquímico e molecular em cultivares de algodão submetido à infecção com o fungo da ramulose, visando identificar marcadores associados ao processo de resistência. No primeiro ensaio, avaliaram-se descritores bioquímicos em quatro cultivares de algodão infectado com o fungo causador da ramulose. As plantas foram inoculadas aos 20 dias após o cultivo, recebendo uma concentração fúngica de 1 x 106 conídios/mL. As avaliações ocorreram aos 3, 15 e 30 dias após a inoculação. Observou-se que os teores de prolina, peroxidase e catalase foram bastante expressivos na cultivar resistente BRS Antares apresentando resposta rápida logo aos 3 dias após a inoculação. Os teores de prolina e catalase foram indicados como ferramentas úteis para identificação de cultivares resistentes a ramulose nos trabalhos de seleção na cultura do algodão. No segundo ensaio buscou-se identificar transcritos diferencialmente expressos em plantas de algodão submetidas a infecção com o fungo da ramulose. Duas cultivares antagônicas em relação à resistência a esta doença foram utilizadas, BRS Antares e CNPA Precoce I, respectivamente, resistente e susceptível. Dez oligonucleotídeos RAPD foram utilizados nas reações de RT-PCR a uma temperatura de anelamento de 35 ºC. Após análise dos padrões de banda nas plantas controle e inoculada foram identificados doze transcritos sub-regulados, treze super-regulados e dez ativados. Uma banda de aproximadamente 200 pb obtida com o oligonucleotídeo V10 na cultivar resistente foi sequenciada e a apresentou homologia de 86 % com o gene GmChl 3 que codifica para enzima clorofilase III em soja. Esta enzima apresenta grande importância para as células vegetais submetidas a danos oxidativos uma vez que reduz estes estragos além de modular diferentes vias de defesa. Os dados obtidos no presente trabalho são de grande aplicabilidade em estudos de resposta do algodão ao ataque de patógenos além de oferecer informações relevantes para os programas de melhoramento da cultura.

Fusarium oxysporum f. sp. vasinfectum (Fov) was first identified in Australia in 1993, and has since become one of the most significant threats to the country’s thriving cotton industry. The interaction between a unique Australian biotype of Fov and cotton hosts with varying susceptibilities to Fusarium wilt was studied. This research described the infection process and associated host defence mechanisms of two commercial cotton varieties after inoculation with Fov, and quantified their subsequent accumulation of antimicrobial terpenoids. / A rapid, reliable glasshouse bioassay that correlated with field resistance was developed for the study of Fusarium wilt of cotton. Detailed observations of the infection process obtained through light microscopy were used to formulate the disease cycle of Australian Fusarium wilt cotton. Using pathogen growth assays, varietal differences in root exudates and vascular tissues in the cotton hosts were documented. Root diffusate from the most susceptible cotton variety to Fusarium wilt, Siokra 1-4, contained a lipophilic compound that promoted the germination of Fov microconidia. On the other hand, a lipophilic compound present in diffusate from the least susceptible variety, Sicot 189, inhibited the growth of Fov germ tubes. / A bioassay using inoculated whole plants showed that Fov colonisation of the vascular tissues of Sicot 189 was restricted after 3 days. The basis for this inhibition was investigated further using light and transmission electron microscopy. Infection induced the reorganisation of contact cells in host vascular tissue, including an increase in cytoplasmic content and the partitioning of vacuoles, which was concurrent with the accumulation of materials in adjacent vessel lumens, via pits. Histochemical analysis indicated these globular materials secreted into the vessels were terpenoids. These structural and terpenoid responses in Siokra 1-4 and Sicot 189 were similar, however, they were more intense and rapid in the latter, less susceptible variety. The responses in Sicot 189 also corresponded to the time period that pathogen inhibition was observed. Thus, a correlation was demonstrated between the rapid and intense induction of both structural and biochemical responses with decreased susceptibility to Fusarium wilt. Detailed HPLC analysis of vascular tissues confirmed that terpenoids accumulated more rapidly and at higher concentrations in the less susceptible cotton variety. These findings provided strong evidence for the involvement of antimicrobial terpenoids in the determination of Fusarium wilt susceptibility of Australian cotton varieties. / This work represents the most complete survey to date of the interaction of Australian biotypes of Fov with cotton. These insights can contribute to future cotton breeding efforts and cultural management of Fusarium wilt in the field. Thus, each part of this study has advanced complementary facets of our understanding of Fov, and has provided a framework from which future studies on phytoalexins and other putative cotton defences can be studied.

Plants have evolved mechanisms to defend themselves against pathogen attack. These defense mechanisms consist of a series of inducible responses (including specific recognition of pathogen invasion, signal transduction and defense gene activation) that result in resistance. Plants responses to pathogen invasion also result in the suppression of various housekeeping activities of the cells, thus diverting the cellular resources to defense responses. Systemic acquired resistance (SAR), an inducible defense response enhanced as a result of initial infection with a necrotising pathogen, lead to long-term resistance in a plant. Differential gene expression of genes related to defense in cultured cotton cells and leaf disks that have been challenged with a purified elicitor from Verticillium dahliae, as well as a chemical inducer of defense responses, DL-b-amino-n-butyric acid, were investigated. The mRNA differential display reverse transcriptase polymerase chain reaction (DDRT-PCR) was used to identify differentially expressed genes 5 h after application of either 50 mg mL-1 Verticillium dahliae elicitor or 1 mM DL-b-amino-n-butyric acid to cotton cell suspension cultures and leaf disks. Identified cDNAs up- or down-regulated for this study were classified into seven groups: ‘Transcription factor’, ‘Ubiquitin and Proteasome’, ‘Mitochondria’, ‘Protein kinase/Receptor-like kinase’, ‘Defense/Resistance’, ‘Carbohydrate metabolism/Cell wall’ and ‘Other’. The identified cDNAs up-regulated after Verticillium dahliae elicitor treatment, classified in the ‘Transcription factor’ group, coded for a MYB family transcription factor, zinc finger protein and a RMA1 RING zinc finger protein. The identified cDNA classified in the ‘Mitochondria’ group coded for a cytochrome C oxidase subunit I and II and the cDNA classified in the ‘Protein kinase/Receptor-like kinase’ group coded for a serine/threonine protein kinase. The identified cDNA classified in the ‘Defense/Resistance’ group coded for a disease resistance protein family and the cDNAs classified in the ‘Carbohydrate metabolism/Cell wall’ group coded for a beta-1,4-Nacetylglucosaminyltransferase, a cellulose synthase-like protein, a 3-deoxy-D-manno-octulosonic acid transferase-like protein and a hydroxyproline-rich glycoprotein homolog. In addition, a cDNA classified in the ‘Other’ group, coded for a urea active transporter-like protein. The cDNA identified that was down-regulated after Verticillium dahliae elicitor treatment, classified in the ‘Carbohydrate metabolism/Cell wall’ group, coded for a proline-rich protein family and cDNAs classified in the ‘Other’ group coded for a thioredoxin reductase1 and ‘hookless1’ homologue. Among the identified cDNAs up-regulated after DL-b-amino-n-butyric acid treatment, classified in the ‘Ubiquitin and Proteasome’ group, were a 20S proteasome subunit alpha type 5 and an ubiquitin. The identified cDNA classified in the ‘Mitochondria’ group coded for a NADH dehydrogenase subunit 6, a mitochondrial DNA product. The identified cDNAs classified in the ‘Other’ group coded for an armadillo repeat containing protein and a phosphoinositide-specific phospholipase C. The cDNA identified that was down-regulated after DL-b-amino-n-butyric acid treatment, classified in the ‘Protein kinase/Receptor-like kinase’ group, coded for a casein kinase I like protein. The identified cDNA classified in the ‘Carbohydrate metabolism/Cell wall’ group, coded for a putative glycine rich protein. Also, the identified cDNA classified in the ‘Other’ group, coded for a NADH dehydrogenase subunit F that is coded for by chloroplast DNA. The differential expression of the cDNAs up-regulated after the Verticillium dahliae elicitor treatment was confirmed for seven of the nine cDNA clones with a Reverse Northern dot blot. Also, the differential expression of two cDNAs up-regulated after DL-b-amino-n-butyric acid treatment was confirmed and the induction kinetics was followed with a Reverse Northern dot blot. The mRNAs corresponding to C8B5, the gene encoding an ubiquitin, were detectable after 2.5 h and showed a significant increase in expression up to 7.5 h, after which the expression levels decreased to levels similar to those detected at 2.5 h. The mRNAs corresponding to L4B4, a homologue of an a-type subunit of 20S proteasome, were detectable after 2.5 h with an gradual increase in expression levels up to 7.5 h after which the expression levels decreased to levels similar to those detected at 2.5 h. This study facilitated a better understanding of differential gene regulation during triggering of defense responses in cotton following elicitation with the Verticillium dahliae elicitor and DL-b-aminon- butyric acid. / Prof. I.A. Dubery

D.Phil. / The fungus, Verticillium dahliae, is the causative agent of Verticillium wilt, which results in significant cotton (Gossypium hirsutum) crop losses worldwide. This study contributes to the elucidation of cotton defence responses against V. dahliae. The identification, cloning and characterization of three genes that were differentially expressed in response to elicitation with a cell wall-derived (CWD) V. dahliae elicitor are described. It was hypothesized that the molecular architectures of the three characterized genes are supportive of a role in cotton defence against V. dahliae. As one of these genes was present as two homoeologous copies, this study also reports on the molecular characterization of both homoeologs, thus providing further insight into the processes of genomic evolution between homoeologous loci in allotetraploid cotton. The three genes were initially represented as expressed sequence tags (ESTs), obtained from a previous differential display reverse transcription polymerase chain reaction (DDRT-PCR) study by Zwiegelaar (2003), as part of an MSc project. These ESTs, designated C1B10, C4B5 and C4B4, were differentially induced upon elicitation with a CWD V. dahliae elicitor (Zwiegelaar, 2003). In the present study, the genes represented by the three ESTs were identified and characterized by genome walking and 5‘/3‘ rapid amplification of cDNA ends (RACE). Additionally, PCR and reverse-transcription PCR (RT-PCR) were utilized, where necessary, to obtain internal sequences, not covered by the genome walking and RACE reactions. Through the use of these molecular techniques, the full transcript and genomic sequences of each of the three genes was obtained, including their promoters. The promoter of each gene was analyzed for cis-elements driving gene transcription, through bioinformatic analysis. Furthermore, the copy number of each gene was determined through Southern blot analysis. The genes were translated to reveal their encoded protein sequences. The amino acid sequences were submitted to a basic local alignment (BLAST) search of the NCBI database to identify, and align them with, homologous proteins from other plant species (and those from G. hirsutum, if any). An in silico analysis of the encoded protein of each gene was also performed. This examination included domain architecture, post-translational modification, subcellular location and tertiary structure predictions. This study also involved the isolation of the elicitor from the cell walls of V. dahliae fungal cultures. The potency of the freshly-isolated elicitor was investigated with a triphenyltetrazolium chloride (TTC) viability assay on cotton cell suspensions. Its potential to induce PR-proteins was also explored but these results were inconclusive. In addition, expression studies were performed with real-time PCR (q-PCR), to confirm the up- or down-regulation of each gene upon elicitation of cotton cell suspensions with the CWD V. dahliae elicitor, and to investigate the time frame/kinetics of induction. The gene corresponding to the C1B10 EST was designated GhLIPN as this study revealed that it encodes a lipin protein. Lipins are novel proteins with phosphatidate phosphatase 1 (PAP1) activity, exclusive to eukaryotes. They play a fundamental role in the lipid metabolism of organisms ranging in complexity from yeast to animals and plants. In plants, this role includes lipid membrane remodelling during phosphate (Pi) deficiency. During the study of the GhLIPN gene, it was discovered that it occurred as two distinct homoeologous copies from the A- and D-co-resident genomes of allopolyploid G. hirsutum. The GhLIPN homoeologs were named GhLIPN I and N for Insert present and No insert, respectively, based on the presence or absence of a 13 base pair (bp) insertion/deletion (indel) site in intron 6.