A doença cancro cítrico é considerada uma das mais importantes doenças da citricultura devido ao seu poder de proliferação nas fazendas, e aos danos causados às plantas e frutos. Os prejuízos causados pela presença da doença são consideravelmente preocupantes, pois as principais medidas de controle pelos órgãos responsáveis envolvem a erradicação de plantas infectadas e demais plantas vizinhas, inviabilizando economicamente grandes áreas produtivas. A legislação brasileira exige um extenso protocolo de atividades que necessita ser realizado antes da confirmação do diagnóstico. Atrasos na confirmação do diagnóstico favorecem a proliferação da doença. Assim, qualquer esforço em acelerar esta detecção deve com certeza ter um grande impacto nesta área. Esta é a motivação de nosso trabalho, onde aplicamos a técnica de espectroscopia por imagens de fluorescência em folhas de culturas cítricas com a intenção de avaliar a capacidade de diagnóstico desta técnica em plantas assintomáticas contaminadas no laboratório com cancro cítrico. O objetivo é determinar o instante de tempo mínimo necessário entre a infecção e o diagnóstico preciso da doença. Este estudo foi aplicado para experimentos envolvendo amostras destrutivas e não-destrutivas. Os resultados mostram a possibilidade de aplicar tal técnica na detecção de cancro cítrico. / The citrus canker disease is considered one of the most important citrus diseases due to its ability to spread on farms, and to damage plants and fruits. The damage, caused by the citrus canker, can be devastating, because the main control actions involve the eradication of infected plants and other plants nearby, causing large economic losses. Brazilian law requires an extensive testing protocol to confirm the diagnosis. Delays in diagnosis tests allows the spread of the disease. Therefore, any effort to accelerate this procedure will have a major impact in this area. This is the motivation of our work, where we apply the fluorescence imaging spectroscopy technique on citrus leaves with the goal to evaluate the diagnostic capability of this technique in asymptomatic plants infected with citrus canker in the laboratory. The goal is to determine the minimum time delay between infection and accurate diagnosis of the disease. This study was applied to experiments involving non-destructive and destructive samples. The results show the possibility of applying this technique in the detection of citrus canker.

Leptosphaeria maculans and L. biglobosa co-infect winter oilseed rape plants to cause phoma stem canker disease. The sexual spores of both species are produced in pseudothecia on infected winter oilseed rape stem debris after harvest and this is the most important source of inoculum for infection of newly-emerged plants in autumn. Field experiments investigated the effects of host resistance on proportions of pathogens Leptosphaeria maculans and L. biglobosa in most leaf and stem tissues during 2011/2012, 2012/2013 and 2013/2014 growing seasons and on the pseudothecial development on crop debris on nine winter oilseed rape cultivars; cvs Adriana (Rlm4 + quantitative resistance (QR)), Bilbao (Rlm4), Capitol (Rlm1), Drakkar (no R gene against L. maculans), DK Cabernet (Rlm1 + (QR), Es-Astrid (QR), Excel (Rlm7), Roxet (Rlm7) and NK Grandia (QR). Cultivars with a combination of R-gene resistance and QR [Adriana (Rlm4 + QR), DK Cabernet (Rlm1 + QR)] or cultivars with only QR [(Es-Astrid and NK Grandia)] had more numbers of L. maculans leaf spots than other cultivars in autumn but less stem canker damage. There was greater number of L. biglobosa leaf spots on leaves of cvs Roxet and Excel with resistance gene Rlm7 than those of other cultivars and later more L. biglobosa DNA was detected in their stems than in those of other cultivars. In all cultivars in the three growing seasons, there was a greater amount of L. biglobosa DNA than L. maculans DNA in basal stem canker and upper stem lesions. The cv. Drakkar (no R gene against L. maculans) was susceptible in all three growing seasons, with a great number of L. maculans and L. biglobosa leaf spots and severe stem cankers. There were four cultivars (Adriana, Bilbao, Drakkar and NK Grandia) selected for the study of pseudothecial development under natural conditions with different times of exposure and in controlled environment conditions (20oC, continuous wetness). The fastest development was on the susceptible cv. Drakkar (no R gene against L. maculans), followed by Bilbao (Rlm4), Adriana (Rlm4 + QR) and NK Grandia (QR) for stem base cankers and upper stem lesions in controlled conditions. Results for pseudothecial development on stems of the nine winter oilseed rape cultivars that were exposed in natural conditions at Bayfordbury support the controlled environment results, with pseudothecia on stems of cultivars with a combination R-gene and QR consistently maturing later than those on other cultivars, regardless of the weather conditions in three growing seasons. Ascospores produced in pseudothecia are the primary inoculum that initiate phoma stem canker epidemics in autumn. Ascospore release was later in autumn 2011 than in autumn/winter 2012/2013 or 2013/2014 because of dry weather. The pattern of ascospore release had a peak, or maximum in autumn/winter 2011/2012 (4958 spores/m-3 on 22 Jan 2012) and several maxima in autumn/winter 2012/2013 (1307 spores/m-3 on 5 Nov 2012, 1291 spores/m-3 on 15 Nov 2012, 1306 spores/m-3 on 25 Dec 2012) and 2013/2014 (4575 spores/m-3 on 27 Oct 2013, 4619 spores/m-3 on 3 Nov 2013, 3674 spores/m-3 on 9 Nov 2013, 3521 spores/m-3 on 12 Dec 2013). Results from the qPCR showed that ascospores of L. maculans were released earlier than ascospores of L. biglobosa at Bayfordbury in the 2013/2014 growing season. There were differences in phenotype of isolates amongst ninety-five isolates of L. maculans and forty-eight isolates of L. biglobosa obtained from different sources (phoma leaf spots, upper stem lesions or basal stem cankers) on different cultivars. Cotyledon tests showed that the resistance genes Rlm4, Rlm5, Rlm6 and Rlm7 are still effective in England. Most isolates from phoma leaf spots carried avirulent AvrLm4 (39 isolates; 97.5%), AvrLm5 (39 isolates; 97.5%) and AvrLm6 alleles (36 isolates; 90%) and all 40 isolates carried the avirulent allele AvrLm7 (100%). Fewer isolates from basal stem cankers carried avirulent AvrLm4 (4 isolates, 16.7%) or AvrLm6 alleles (16.7%) but all 24 isolates carried the avirulent AvrLm7 (100%). Fewer isolates from upper stem lesions carried the avirulent AvrLm4 allele (5 isolates; 16.1%), but 15 isolates carried avirulent AvrLm5 (48.4%), 21 isolates carried AvrLm6 (67.7%) and all 31 isolates carried AvrLm7 (100%). By contrast, all isolates were virulent against Rlm1, Rlm2, Rlm3 and Rlm9. This knowledge, together with knowledge about R genes present in current winter oilseed rape cultivars, should be useful to provide recommendations on cultivar selection to growers based on regional frequencies of avirulent alleles of Avr allele genes in the L. maculans populations (races) and improved understanding of the race structure of L. maculans. There is a need to further investigate any R genes that operate against L. biglobosa (possibly from wild brassicas) and to study if any R genes or QR can provide resistance against both L. maculans and L. biglobosa.

A doença cancro cítrico é considerada uma das mais importantes doenças da citricultura devido ao seu poder de proliferação nas fazendas, e aos danos causados às plantas e frutos. Os prejuízos causados pela presença da doença são consideravelmente preocupantes, pois as principais medidas de controle pelos órgãos responsáveis envolvem a erradicação de plantas infectadas e demais plantas vizinhas, inviabilizando economicamente grandes áreas produtivas. A legislação brasileira exige um extenso protocolo de atividades que necessita ser realizado antes da confirmação do diagnóstico. Atrasos na confirmação do diagnóstico favorecem a proliferação da doença. Assim, qualquer esforço em acelerar esta detecção deve com certeza ter um grande impacto nesta área. Esta é a motivação de nosso trabalho, onde aplicamos a técnica de espectroscopia por imagens de fluorescência em folhas de culturas cítricas com a intenção de avaliar a capacidade de diagnóstico desta técnica em plantas assintomáticas contaminadas no laboratório com cancro cítrico. O objetivo é determinar o instante de tempo mínimo necessário entre a infecção e o diagnóstico preciso da doença. Este estudo foi aplicado para experimentos envolvendo amostras destrutivas e não-destrutivas. Os resultados mostram a possibilidade de aplicar tal técnica na detecção de cancro cítrico. / The citrus canker disease is considered one of the most important citrus diseases due to its ability to spread on farms, and to damage plants and fruits. The damage, caused by the citrus canker, can be devastating, because the main control actions involve the eradication of infected plants and other plants nearby, causing large economic losses. Brazilian law requires an extensive testing protocol to confirm the diagnosis. Delays in diagnosis tests allows the spread of the disease. Therefore, any effort to accelerate this procedure will have a major impact in this area. This is the motivation of our work, where we apply the fluorescence imaging spectroscopy technique on citrus leaves with the goal to evaluate the diagnostic capability of this technique in asymptomatic plants infected with citrus canker in the laboratory. The goal is to determine the minimum time delay between infection and accurate diagnosis of the disease. This study was applied to experiments involving non-destructive and destructive samples. The results show the possibility of applying this technique in the detection of citrus canker.

Monterey pine (Pinus radiata), a native tree to California and two Mexican islands, is important both ecologically and economically. Outside native stands, Monterey pines are grown for landscaping in California and on plantations around the world. Pitch canker, a disease caused by the fungus Gibberella circinata Nirenberg & O’Donnell (Fusarium circinatum Nirenberg and O'Donnell) is threatening the survival of Monterey pines. The disease currently affects Monterey pines in many parts of the world including the native stands. No effective chemical or biological control is available but some Monterey pines show resistance to the disease. The purpose of this project was to develop a working protocol for producing genetic clones of the resistant pines through micropropagation. These genetic clones will be used for outplanting in places outside the native stands for ornamental and plantation purposes. This project analyzes the results of ten trials with varied parameters and bases the final protocol on the parameters used in the trial that induces the growth of new shoots. The final protocol developed in this project describes, step-by-step, the media preparation for the initiation, plant material collection, surface sterilization of plant material, plating in media and initiation of shoots on explants. The protocol calls for collecting shoot tips with hardened buds that have not yet elongated, then washing the shoot tips in sterile water with Tween 20 for 15 minutes. The shoots tips are then surface sterilized in a 50% bleach solution for 20 minutes. The explants are broken into disks (to minimize damage to the cells) by inserting the tip of a scalpel and tilting it slightly. The initiation media shown to induce growth consists of ½ strength LePoivre basal salt mixture, 5mg/L benzylaminopurine, 3% sucrose and 0.8% agar and is adjusted to a pH of 5.7, then autoclaved for 20 minutes. The explants are inserted into solidified media and incubated in a growth chamber programmed for 16 hours of light and 8 hours of dark with temperatures of 27ºC and 22ºC and light irradiance of 80µEm-2s-1. After 1 month the protocol calls for transferring the growing shoots to elongation media with full LP basal salts and transferring every month. When the number of desired shoots has been reached the forthcoming protocol for rooting can be followed.

The future resilience of Pinus radiata D. Don (Monterey pine) is dependent upon the development of a silviculture program inclusive of either preventative or management techniques for the potentially fatal pitch canker disease (Fusarium circinatum Nirenberg and O’Donnell [=F.subglutinans (Wollenw. & Reinking) Nelson et al. f. sp. Pini). As an ecologically and commercially valued species, a myriad of factors threaten the genetic resources of the geographically limited natural range. This study evaluated the effectiveness of uneven-aged forest management for regeneration success in the native, pitch canker infected Año Nuevo stand at Swanton Pacific Ranch in Davenport, California. Seedling survival and growth averages were used to evaluate the impact of the variables of gap size (0.20-hectare, 0.10-hectare, and 0.05-hectare), site-preparation treatment (pile and burn, lop and scatter), and parent tree (13 local seed sources). Pitch canker symptoms were quantified to conclude if there is indeed a range of expressed resistance according to parent tree. Statistical models (i.e. ‘mixed effect’) yielded moderately significant differences in odds of survival between site treatments, with pile and burn plots producing higher survival rates (p=0.066). No significant difference in survival was detected between gap-sizes (p=0.936 and 0.803, 0.05- and 0.10-hectare gaps respectively to 0.20-hectare reference). Significant variation was expressed (estimate=0.1219, SE=0.057) between the 13 parent trees, implicating usefulness in predicting seedling survival. ‘Mixed effect’ models only detected a significant effect from gap-size on growth measures: 0.20-hectare gaps yielded height and diameter measures significantly larger than 0.05-hectare gaps (p=0.027 and 0.0081 respectively). Contingency tables detected significant association between gap-size and number of pitch canker branch symptoms. The range of infection rates produced by the 13 parent trees did not prove significant by analysis. Pile and burn site preparation treatment increases the probability of survival for artificially regenerated seedlings and 0.20-hectare gaps yield significantly higher growth averages over 0.05-hectare gaps.

This study was a part of the international collaborative IMPACT project, which aims to address the potential threat that the pitch canker disease poses to the use of Pinus radiata D. Don in plantations in New Zealand, Australia, and Chile. A field trial of 264 seedstocks was planted adjacent to a native stand of pitch canker infected P. radiata on the central coast of California, and disease symptom development was recorded over a period of 3 years. The results did not correlate with a greenhouse study of the same seedstocks inoculated with Fusarium circinatum Nirenberg & O'Donnell, the causal agent of pitch canker. Three main types of symptoms were identified (branch flagging, pitchy buds, and chlorotic tips), and preliminary isolation analyses suggest that the disease observed is actually caused by Diplodia pinea (Desm.) Kickx. Survival analysis showed that the effect of tree genetic origin was significant to its time to disease, and that spatial location in the plantation was also significant. Average nearest neighbor analysis showed disease distribution to be significantly clustered, which also suggests that the disease is not pitch canker, but diplodia blight. This experiment illustrates the difficulty in performing naturally infected field trials when another similar-looking fungal disease is also present. It also provides data on seedstock resistance to diplodia blight, another fungal disease important to P. radiata forest managers.

Thesis (MScAgric )--Stellenbosch University, 2011. / ENGLISH ABSTRACT: Pine pitch canker, caused by Fusarium circinatum, is a major constraint to the production of high quality timber in the Western Cape Province of South Africa. Under nursery conditions the pathogen causes a serious root and collar rot disease, and in plantations it causes stem cankers and branch die-back. In this thesis, sources of inoculum within a forestry nursery were investigated and novel approaches developed to eradicate the pathogen. Plant material was evaluated for plantation establishment in the Western Cape Province, and epidemiological studies conducted to better understand the infection, disease development, spore release and dissemination of F. circinatum in pine plantations. Fusarium circinatum was first reported in a forestry nursery in the Mpumalanga Province of South Africa in 1990, and now causes significant economic losses to most forestry nurseries in the country. The irrigation system, planting tray inserts, seeds and air at Karatara nursery were screened as potential sources of F. circinatum. Both the irrigation water and planting tray inserts were found to serve as sources of inoculum. An oxidation reduction potential (ORP)-based system, using hydrogen peroxide, was tested to sanitize irrigation water and planting tray inserts. An ORP level of 400 mV for 6 hours was effective in killing F. circinatum spores in irrigation water without being phytotoxic to pine seedlings. An ORP value of >500 mV cleaned planting tray inserts of all inoculum. Pinus radiata is the principal pine species grown in the Western Cape Province, but this species is also highly susceptible to F. circinatum. There are numerous different varieties or families of P. radiata available that can be planted, and it is known that tolerance levels between these varieties and families vary. Pinus radiata families, as well as other Pinus spp. that could potentially be planted in the Western Cape Province in future, were evaluated for tolerance to the pitch canker fungus. The P. radiata families were moderately to highly susceptible to F. circinatum. Pinus taeda was the Pinus spp. most tolerant, while P. elliottii also had good tolerance to the pathogen. None of the P. radiata families screened showed tolerance comparable to that of either P. elliottii or P. taeda. Pine pitch canker was first reported from the Tokai plantation in the Western Cape Province in 2005. In order to develop a disease management strategy for the disease in pine plantations, we investigated the effect of pruning wounds as potential entry sites for the pathogen, and how fast the disease spread under South African climatic conditions. The amount and time of sporulation was also investigated. Pitch canker increased slowly over a 3-year period, and pruning wounds appeared not to serve as primary entry points for F. circinatum. Spore quantification over a 12-month period using quantitative real-time PCR showed that inoculum was produced throughout the year, but that significantly more spores were released during the cold, rainy winter months. The role of insects in pathogen transmission and spread should be investigated in the Tokai plantation. / AFRIKAANSE OPSOMMING: Fusarium circinatum veroorsaak “pine pitch canker”. Hierdie siekte is een van die grootste beperkinge op die Suid Afrikaanse bosbou industrie. In kwekerye veroorsaak die patogeen wortelvrot en wortelkraagvrot. In plantasies veroorsaak F. circinatum kankers op die hoofstam van dennebome en die terugsterwing van sytakke. In hierdie tesis was bronne van inokulum in ‘n bosbou kwekery ondersoek. Nuwe tegnieke was gebruik om die bronne uit te wis. Plantmateriaal wat in die Wes-Kaap geplant kan word, was geëvalueer vir toleransie teen F. circinatum. Epidemiologiesie studies was uitgevoer om die infeksieproses, siekte-ontwikkeling, spoorvrystelling, en verspreiding van F. circinatum binne denneplantasies beter te verstaan. Die eerste verslag van F. circinatum in ‘n bosboukwekery was in 1990 in die Mpumalanga Provinsie. Verliese word tans veroorsaak deur die patogeen in die meeste bosbou kwekerye in Suid Afrika. Die besproeiingstelsel, planthouers en saad was getoets as potensiële bronne van inokulum. Daar was gevind dat beide die besproeiingstelsel en die planthouers dien as bronne van F. circinatum inokulum. ‘n Oksidasie reduksie potensiaal (ORP)-gebaseerde sisteem waar waterstofperoksied gebruik was, was getoets of dit die besproeingwater en planthouers kan ontsmet. ‘n ORP-vlak van 400 mV vir ‘n blootstellingstydperk van 6 ure is effektief om al die F. circinatum spore dood te maak, sonder dat dit fitotoksies is teenoor die dennesaailinge. ‘n ORP-vlak van >500 mV het die F. circinatum in die planthouers uitgewis. Pinus radiata is die belangrikste dennespesie wat geplant word in die Wes-Kaap Provinsie, maar hierdie spesie is hoogs vatbaar vir F. circinatum. Verskillende variteite van P. radiata met verskillende tolleransie vlakke bestaan. Pinus radiata families en ander spesies wat in die toekoms potensieel geplant kan word in die Wes-Kaap was geevalueer vir toleransie teen F. circinatum. Die P. radiata families was matig to hoogs vatbaar vir F. circinatum. Pinus taeda het die hoogste toleransie vlak gehad en P. elliottii was matig tolererant. Nie een van die P. radiata families wat getoets was het toleransie vlakke gehad wat vergelykbaar was met die toleransie vlakke van P. taeda of P. elliottii nie. In 2005 was F. circinatum vir die eerste keer geraporteer in die Tokai plantasie op volwasse dennebome in die Wes-Kaap Provinsie. Die rol van snoeiwonde in die infeksieproses van F. circinatum was bestudeer asook hoe vinning die siekte ontwikkel en versprei. Die hoeveelheid en tyd van sporulasie was ook bestudeer. “Pitch canker” het stadig toegeneem oor die 3-jaar moniteringsperiode en dit blyk asof snoeiwonde nie die primêre ingangspunt vir die patogeen is nie. Kwantifisering van die sporulasie van F. circinatum deur middel van qPCR het aangedui dat spore reg deur die jaar geproduseer word. Meer spore word geproduseer in die koue nat wintersmaande. Die rol van insekte in die infeksieproses en verspreiding van F. circinatum in die Tokai plantasie moet ondersoek word.

O cancro cítrico, considerado uma das mais importantes doenças da cultura dos citros, apresentou um drástico aumento de 607% no índice de talhões contaminados no Estado de São Paulo entre 2009 e 2011, mesmo diante da obrigatoriedade legal da erradicação das plantas sintomáticas. O aumento ocorre logo após mudança na legislação de controle e erradicação da doença, justificando a importância de se avaliar as vantagens econômicas de manter o cancro sob controle no Estado e os impactos potenciais de sua disseminação. O objetivo deste estudo é analisar os benefícios econômicos de manter o cancro cítrico sob controle em São Paulo, e comparar as possíveis alternativas para sua prevenção e controle do ponto de vista da sustentabilidade econômica da produção. A abordagem analítica compreende avaliar os fluxos de custos e de receitas do setor diante de diferentes cenários de prevenção e controle do cancro. Com base em valores presentes líquidos desses fluxos, estimar a relação benefício-custo de prevenir, controlar ou manejar a doença no Estado de São Paulo, em um horizonte de tempo de 20 anos. Foram identificados os custos e benefícios mais expressivos ao produtor rural nos diferentes cenários, comparando-se as diferenças em produção de laranjas e custos de produção em situações de prevenção, controle e expansão da doença. Foram analisados os impactos de redução da produtividade e as variações nos custos de produção em cinco cenários, subdivididos em 12 sub cenários. Consideradas as pressuposições adotadas, os resultados apontam vantagens econômicas em manter o cancro cítrico sob controle em São Paulo, com relações benefício-custo significativamente superiores para cenários de prevenção e de controle comparados aos de expansão da doença. No acumulado de 20 anos, a prevenção ao cancro cítrico evita perdas no valor de R$ 12,82 para cada Real investido. Já no cenário em que 90% dos talhões contaminados não são erradicados, a relação é de apenas R$ 0,35 em perdas evitadas para cada Real investido no manejo, supondo que o preço real da laranja permaneça constante em R$11,00/caixa. Contudo, os resultados demonstram que a prevenção e controle da doença apresentam relação benefíciocusto superior ao manejo da doença no médio e longo prazos. Este resultado é relevante considerando que, atualmente, cerca de 99% dos talhões paulistas estão indenes a doença, embora seriamente ameaçados por crescente taxa de disseminação do cancro. / The citrus canker, one of the most important diseases of citrus, showed a drastic increase of 607% in the index of contaminated plots in São Paulo state, between 2009 and 2011, despite the legal requirement of eradication of symptomatic plants, causing a huge impact for farmers. The boost has occurred just after a change in the regulation on the control and eradication of this disease, which justifies the relevance of assessing the economic advantages of keeping canker under control in the state as well as the potential impacts of its dissemination. The objective of this study is to analyze the economic benefits of keeping the citrus canker under control in São Paulo, and to compare the possible alternatives for its prevention and control in terms of the economic sustainability of citrus production. The analytical approach consists on evaluating the cost and revenue flows of this sector for each different scenario of citrus canker prevention, control or fast dissemination. Based upon the net present values of these flows, the benefit-cost ratios are estimated for scenarios of preventing, controlling or managing the disease in the State of São Paulo, for a 20 years projection. For each scenario, the most important costs and benefits for citrus farmers have been identified and then differences of revenues from orange production and their costs compared for those scenarios. The major impacts analyzed are yield reductions and changes in cost production for five scenarios, divided into 12 sub scenarios. Considering assumptions of this work, results point economic advantages in keeping the citrus canker under control in São Paulo, and benefit-cost ratios significantly higher for scenarios of prevention and control of disease compared to scenarios of disease´s expansion. Considering a 20 years period, the scenario of canker prevention avoids losses amounting to R$ 12.82 for each Real invested. On the other hand, for the scenario where 90% of the contaminated plots are not eradicated, the ratio is only $ 0.35 in avoided losses for each Real invested in management, assuming that real price for orange remains constant at R$ 11.00/box along the whole period. However, results demonstrate that preventing and controlling this disease provide benefit-cost ratios greater than managing it in the medium and long term. This is a very relevant finding considering that about 99% of citrus slots in São Paulo are currently not contaminated yet, although they are seriously threatened by a growing rate of canker dissemination.

O cancro cítrico é uma doença que afeta diversas espécies de Citrus, cujo agente causal é Xanthomonas axonopodis pv citri (XAC). O genoma desta fitobactéria consiste de um cromossomo de ~5 Mpb e dois plasmídeos, que juntos codificam 4313 CDS (seqüências codificadoras), das quais 2710 apresentam similaridade com proteínas conhecidas. Neste trabalho realizamos uma análise comparativa detalhada do genoma de XAC com genomas de três fitopatógenos, Xanthomonas campestris campestris, Xylella fastidiosa 9a5c e Xylella fastidiosa temecula. Com esta análise identificamos genes espécie e gênero-específicos, potencialmente relevantes para adaptação aos seus respectivos nichos ou hospedeiros, além de ilhas de inserção e deleção genômica putativas. Também identificamos vias metabólicas relacionadas com osmoproteção/osmorregulação e com degradação de compostos aromáticos em XAC, que possivelmente são determinantes na eficácia de sua interação com o hospedeiro. Analisamos o nível de expressão de 9 CDS após crescimento de XAC em diferentes concentrações de glicose e verificamos que este açúcar modula positivamente a expressão de CDS relacionadas à síntese de goma e ao sistema de osmoproteção. Além disso, descrevemos a construção de microarranjos de DNA representando 2760 CDS de XAC, constituindo-se uma nova ferramenta para estudos de genômica comparativa e expressão gênica deste fitopatógeno. / Xanthomonas axonopodis pv citri (XAC) is the bacterial pathogen that causes citrus canker disease in several species of Citrus plants. XAC genome consists of a main cromosome of ~5 Mpb and two plasmids that together encode 4313 CDS (coding sequences). Approximately 63% of the CDS have assigned biological functions. In this work, we present a detailed genomic comparison between the genomes of XAC and of three other phytopathogens, X. campestris campestris, Xylella fastidiosa 9a5c and X. fastidiosa Temecula. Based on this analysis, we identified species and genus-specific genes that might be relevant for adaptation to their niches and hosts. We mapped putative insertion/deletion regions in the XAC genome possibly related to gene gains and losses during the divergence of the four bacterial lineages. We have identified the metabolic pathways related to osmoprotection/osmoregulation and aromatic compound degradation important for XAC efficient host colonization and interaction. Expression levels of 9 CDS were analyzed after XAC growth under different glucose concentrations revealing that this sugar upregulates the expression of CDS related to gum synthesis and to osmoregulation. In addition, we describe here the construction of a DNA microarray representing 2760 CDS of XAC as a new tool for comparative genomic and gene expression studies in this phytopathogen.

O cancro bacteriano, causado por Clavibacter michiganensis subsp. michiganensis (Cmm), é uma das doenças mais importantes da cultura do tomate (Lycopersicon esculentum Mill.), especialmente para tomateiros estaqueados. O controle está fundamentado na adoção de um conjunto de medidas preventivas, pois poucas são as informações sobre materiais resistentes à doença e não existe um produto químico que a controle eficientemente. Os objetivos desta tese foram determinar um método de inoculação adequado à seleção de genótipos de tomateiro resistentes ao cancro bacteriano; avaliar a reação de genótipos de tomateiro à inoculação com Cmm; e avaliar o efeito de acibenzolar-S-metil e outros produtos fitossanitários no controle da doença. Para o ensaio de metodologia, foram avaliados quatro métodos de inoculação (aspersão de suspensão bacteriana na face inferior das folhas; aspersão, sob pressão, de suspensão bacteriana na face inferior das folhas; corte do sistema radicular, com tesoura, seguido de imersão na suspensão bacteriana; e ferimento na haste com alfinete entomológico transpassando uma gota de suspensão de inóculo depositada na axila foliar) e três concentrações de inóculo (108 ufc/mL, 107 ufc/mL e 106 ufc/mL). Posteriormente, foi avaliado o efeito da inoculação em plantas com diferentes idades, aos 7, 14, 21 e 28 dias após a emergência. Foram utilizados os genótipos Kadá e Rotam-4 e um isolado bacteriano proveniente de Bragança Paulista, SP. A avaliação foi realizada através de uma escala descritiva, atribuindo-se notas que foram convertidas em índice de murcha bacteriana. A inoculação de Cmm, quatorze dias após a emergência das plantas, através da aspersão de suspensão bacteriana (108 ufc/mL) na face inferior das folhas, com avaliação aos 30 dias após inoculação, foi a metodologia mais apropriada para a avaliar a reação de genótipos de tomateiro. Estabelecida a metodologia, foram avaliados dez genótipos comerciais de tomateiro (Carmen, Débora max, IPA-6, Santa Clara, Alambra, Júpiter, Olimpo, Fanny, Jumbo e Densus) para identificação de materiais resistentes ao cancro bacteriano, tendo-se Kadá e Rotam-4 como controles suscetível e resistente, respectivamente. Alambra e Jumbo apresentaram comportamento semelhante a Rotam-4, sendo materiais promissores para o plantio em áreas com histórico de ocorrência da doença. Para a avaliação de controle do cancro bacteriano foram testados os seguintes produtos: acibenzolar-S-metil (ASM), ASM + mancozeb, ASM + oxicloreto de cobre, mancozeb + oxicloreto de cobre, e oxitetraciclina. Três esquemas de aplicação dos produtos foram adotados: (E1) 4 aplicações (a primeira e a segunda realizadas seis e três dias antes da inoculação; a terceira e a quarta, aos três e seis dias após a inoculação); (E2) apenas as duas aplicações préinoculação; (E3) apenas as duas aplicações pós-inoculação. Esses produtos foram incorporados a meio de cultura para avaliação in vitro de seus efeitos sobre Cmm. Independente da forma de aplicação, o ASM, isolado ou em mistura com fungicidas, proporcionou reduções significativas na severidade da doença e não inibiu o desenvolvimento da bactéria in vitro. / Bacterial canker, caused by Clavibacter michiganensis subsp. michiganensis (Cmm), is one of the most important diseases of tomato (Lycopersicon esculentum Mill.). Its control is based on a set of preventive measures, once there is a few information about resistant materials and the chemical control is not efficient. The objectives of this thesis were to determine a suitable inoculation methodology to bacterial canker resistance studies; to evaluate the reaction of commercial tomato genotypes to the inoculation with Cmm; and to evaluate the acibenzolar-S-methyl and other phytossanitary products effects on the control of that disease. To methodology trial, four inoculation methods were evaluated, including foliar pulverization with bacterial suspension; foliar pulverization under pressure with bacterial suspension; cutting of roots with scissor, followed by their immersion in bacterial suspension; and stem wounding with a pin passing over a drop of inoculum suspension deposited on the insertion point of leaf. Besides the inoculation methods, three inoculum concentrations were investigated: 108 cfu/mL; 107 cfu/mL and 106 cfu/mL. Later, it was evaluated the effect of inoculation on plants with different ages: 7, 14, 21 and 28 days after emergency. To those trials, genotypes Kadá and Rotam-4, and a bacterial isolate from Bragança Paulista, SP, were employed. The evaluation was accomplished through a descriptive scale, being attributed notes that were turned into bacterial wilt index. Inoculation of Cmm, fourteen days after emergency of plants, through aspersion with bacterial suspension (108 cfu/mL) over the inferior face of leaves, and evaluation 30 days after inoculation, was the most appropriated methodology to evaluate resistance to bacterial canker in tomato genotypes. After the establishment of the inoculation methodology, ten commercial tomato genotypes (Carmen, Débora max, IPA-6, Santa Clara, Alambra, Jupiter, Olimpo, Fanny, Jumbo and Densus) were investigated to identify resistant materials to the bacterial canker. Kadá and Rotam-4 were employed as susceptible and resistant controls, respectively. Alambra and Jumbo had a similar behavior to Rotam-4, being the most promising materials for the planting in areas with report of occurrence of bacterial canker. To evaluate the bacterial canker control following products were tested: acibenzolar-S-methyl (ASM), ASM + mancozeb, ASM + copper oxicloride, mancozeb + copper oxicloride, and oxytetracycline. Three schemes of application of the products were adopted: (E1) 4 applications (first and second ones were accomplished six and three days before inoculation; third and fourth ones, three and six days after inoculation); (E2) two applications before inoculation only; (E3) two applications after inoculation only. The effect in vitro of these products on Cmm was also evaluated. ASM, isolated or in mixture with fungicides, provided significant reductions in the disease severity independent of the scheme of application and had no effect on bacterial development in vitro.