Phoma stem canker is a disease of oilseed rape (Brassica napus) caused by closely related plant pathogens Leptosphaeria maculans and L. biglobosa. It is an economically important disease, causing annual yield losses of approximately £770 million worldwide. When colonising oilseed rape, L. maculans and L. biglobosa exist in close proximity on the leaf, competing for resources as they move through the main leaf vein and into the stem. Fungicides are commonly used to decrease severity of phoma stem canker on oilseed rape. However, the efficacy and longevity of active chemicals is under threat from evolution of resistance in pathogen populations and government legalisation. Moreover, it has been suggested that both L. maculans and L. biglobosa differ in their sensitivity to azoles, and important class of fungicides that are used to control the disease through the inhibition of lanosterol 14-α demethylase (erg11, CYP51). This project aims to further understand the role that fungicides have in controlling phoma stem canker by investigating their efficacy against L. maculans and L. biglobosa in crops, in vitro and in planta. In field experiments, established in Cambridgeshire across four cropping seasons, the fungicide mixture penthiopyrad (SDHI) plus picoxystrobin (QoI) was as effective at controlling phoma leaf spotting and phoma stem canker in winter oilseed rape as prothioconazole (DMI), suggesting that both fungicides could be used to reduce phoma stem canker symptoms. The two pathogens differed in their growth rates in vitro, with L. biglobosa growing faster than L. maculans when untreated or treated with lower fungicide concentrations. Fungicide sensitivity assays suggest that L. maculans and L. biglobosa are both sensitive to DMI, SDHI and QoI fungicides and that differences between the species are minor. Prothioconazole and penthiopyrad + picoxystrobin had a similar efficacy on oilseed rape cotyledons colonised with either L. maculans or L. biglobosa. There was no difference between species on prothioconazole treated plants, although there was a difference between L. maculans and L. biglobosa when treated with 20 μg/ml penthiopyrad + picoxystrobin. Heterologous yeast expression of LmCYP51B and LbCYP51B with fungicide sensitivity testing of the yeast transformants suggests that LmCYP51B and LbCYP51B are similarly sensitive to azole fungicides flusilazole, prothioconazole-desthio and tebuconazole. These findings are supported by homology protein modelling, which predicts that LmCYP51B and LbCYP51B are structurally very similar, specifically at the azole-binding site. In conclusion, fungicides are still an effective control method for reducing phoma stem canker symptoms caused by Leptosphaeria species in the UK, and a useful tool to in the sustainable production of oilseed rape.

As the Eucalyptus globulus plantation industry expands and matures in southwestern Australia (WA), the impact of disease within the plantation environment is predicted to increase. This thesis investigated the most abundant canker-causing pathogen associated with branch and stem cankers, Endothiella eucalypti the anamorph of Cryphonectria eucalypti. Endothiella eucalypti was widespread, although at low incidence, throughout the WA plantation estate and was frequently observed sporulating on the bark of healthy hosts in the absence of disease. Regions with a long (approximately 20 years) plantation history, such as Bunbury, had the highest incidence of this pathogen. A high degree of variability in pathogenicity, growth rate and colony morphology was observed between WA isolates of En. eucalypti. In the glasshouse, a significant variation in susceptibility of seven E. globulus provenances to En. eucalypti was observed. Although an interaction between the E. globulus provenance and En. eucalypti isolate was recorded, some provenances were generally more susceptible than others. In two 18-month-old plantations, the susceptibility of three provenances to En. eucalypti was significantly influenced by environmental conditions. Visual assessment of general tree health indicated that less healthy trees had smaller lesions than healthy trees. It is hypothesised that the selection of E. globulus provenances to suit site conditions in the future should decrease the risk of serious disease, especially on marginal sites. Endothiella eucalypti caused disease in intact stems of two-year-old E. globulus under glasshouse conditions. This suggests that En. eucalypti may not require a wound to infect in the field. Vegetative compatibility groupings between WA En. eucalypti isolates indicated a relatively high degree of genotypic diversity within the WA asexual population of En. eucalypti, whereas inter-simple sequence repeats PCR (ISSR-PCR) analysis indicated a lower level of genotypic diversity. Discrepancies between traditional and molecular techniques, such as ISSR-PCR, was attributed to the more specific gene-togene analysis afforded by molecular techniques. ISSR-PCR successfully distinguished variability within the En. eucalypti population and with the teleomorph, isolated in South Africa. It also separated Cryphonectria cubensis isolates from the C. eucalypti isolates. As copper is the micronutrient most limiting growth of E. globulus in WA, its role in the resistance of two E. globulus provenances was examined in a glasshouse trial. Lesion extension or defence responses of E. globulus to En. eucalypti did not differ between Cu-adequate and Cu-deficient plants. It is suggested that constitutive levels of host defence enzymes played a more important role in providing protection for the host against En. eucalypti than the external supply of copper. A reduction in the canopy volume of E. globulus within plantations due to insect herbivory or foliar pathogens, such as Mycosphaerella spp., has been reported to predispose the host to disease caused by non-aggressive canker-causing fungi. Under two separate glasshouse trials, conditions of 100% defoliation and 80% defoliation maintained over six weeks prior to inoculation, were required to significantly increase lesion extension caused by En. eucalypti in E. globulus stems. The ability of defoliated E. globulus to retain a degree of resistance to En. eucalypti was attributed to the rapid replacement of foliage and up-regulation of photosynthesis in remaining leaves. The carbohydrate reserves of the plant were depleted following defoliation and remained depressed regardless of the length of time the trees remained defoliated. In conclusion, the endophytic habit of En. eucalypti poses a threat to highly stressed trees, however it does not appear to be an immediate threat to WA plantation health. Although En. eucalypti has not yet been responsible for a major disease outbreak in WA, the impact of this disease on plantation-grown eucalypts elsewhere in Australia and worldwide serves as an indication of its potential to affect WA plantations.

Thesis (Ph.D.)(Microbiology))--University of Pretoria, 2004. / Summary in English. Includes bibliographical references.

Thesis (M.Sc.). / Written for the Dept. of Natural Resource Sciences. Title from title page of PDF (viewed 2009/06/29). Includes bibliographical references.

Made available in DSpace on 2014-06-11T19:27:24Z (GMT). No. of bitstreams: 0 Previous issue date: 2011-05-06Bitstream added on 2014-06-13T19:56:03Z : No. of bitstreams: 1 soaresjunior_j_me_rcla.pdf: 752217 bytes, checksum: cb7bfc540971b4b49bd12620984c3d88 (MD5) / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) / O cancro cítrico é uma doença de importância mundial para o cultivo de laranjas e seu agente causador é a bactéria Xanthomonas axonopodis pv. citri. Embora todos os genes presentes nessa bactéria já tenham sido sequenciados, grande parte destas sequências não apresenta qualquer informação experimental sobre a contribuição para o sucesso da instalação do patógeno na planta hospedeira. Por outro lado, torna-se cada vez mais evidente que várias bactérias fitopatogênicas utilizam mecanismos de adaptação de origem genética que podem induzir efeitos fisiológicos na planta e adequar o ambiente de tal forma que permita a ocorrência da doença. Dentre estes mecanismos, está a possibilidade da bactéria produzir substâncias que se assemelhem a hormônios vegetais, como a auxina (AIA). O objetivo deste trabalho foi desenvolver metodologia genômica de busca para investigar se há genes que codificam para a produção de compostos iguais ou semelhantes a este hormônio em X. axonopodis pv. citri e se a sua participação no processo de patogênese é fundamental para o sucesso da infecção. Para tal, foi produzida coleção de mutantes genéticos funcionais aleatoriamente em X. axonopodis pv. citri, com posterior submissão desta mesma coleção a teste de produção de auxina utilizando o reagente Salkowski. Após produção e análise de grupo de mutantes dirigidos e coleção de aproximadamente 14200 mutantes aleatórios não foi detectada linhagem defectiva para a produção de AIA ou composto semelhante. Os resultados obtidos neste estudo, no entanto, mostram que a biossíntese do composto em X. axonopodis pv. citri é dependente do triptofano e que existe uma concentração ótima deste aminoácido a ser adicionado no meio de cultura para que a bactéria realize a biossíntese. Este é o primeiro estudo... / The citrus canker is a disease of global significance for the cultivation of oranges and its causative agent is the bacterium Xanthomonas axonopodis pv. citri. Although all the genes present in this bacterium have been sequenced, most of these sequences have no experimental information regarding the contribution to the successful installation of the pathogen on the host plant. On the other hand, it has been clearly demonstrated that several pathogenic bacteria use adaptative mechanisms that can induce physiological effects in plants and adjust the environment in a way that allows the occurrence of the disease. Among these mechanisms there is the possibility the bacteria produce substances that are similar to plant hormones such as auxin (IAA). The objective of this study was to develop a screening methodology to investigate the presence of genes that code for a compound identical or similar to auxins in X. axonopodis pv. citri as well as if it plays a role during the pathogenesis process. The main goal of this study was to produce a collection of random and directed genetic mutant strains of X. axonopodis pv. citri for the screening of auxin production by using the Salkowski reagent. After production and analysis of the group of mutants, including a collection of approximately 14,200 random mutant strains, defective production of IAA or similar compound was not detected. On the other hand, the results of this study show that such compound mimics effects of hormonal properties in X. axonopodis pv. citri and is dependent of tryptophan. In addition, it is shown that there is an optimum concentration of this amino acid to be added in the culture medium for the bacteria to carry out biosynthesis. To our knowledge, this is the first study to investigate systematically the production of auxin-like compounds in Xanthomonas axonopodis pv. citri.

Resumo: A caracterização da estrutura genética de populações clonais de patógenos bacterianos tem sido feita, entre outros métodos, por "Southern blot" empregando-se como sondas seqüências de inserção ou genes avr. O presente trabalho objetivou o desenvolvimento de oligonucleotídeos iniciadores e sondas de DNA baseadas em genes de patogenicidade para estudo da diversidade genética de isolados de Xanthomonas citri subsp. citri, Xanthomonas fuscans subsp. aurantifolii e Xanthomonas alfalfae subsp. citrumelonis. Observou-se a presença dos genes avrXacE1, avrXacE2 e lrp em todos isolados das subsp. citri e aurantifolii e a ausência do gene avrXacE2 no isolado da subsp. citrumelonis. Os perfis de restrição gerados por PCR-RFLP não revelaram polimorfismo nos genes amplificados e o uso de genes avr como sondas para "Southern blot" mostrou-se efetiva na diferenciação das espécies de Xanthomonas patogênicas a citros e na identificação de relativo polimorfismo entre isolados da mesma espécie. A diversidade haplotípica foi maior com o gene avrXacE1. Com exceção à sonda correspondente ao gene lrp, o número de haplótipos identificados (17) variou de acordo com a endonuclease utilizada em "Southern blot". Os isolados da subsp. citri apresentaram um maior número de cópias dos genes avr em comparação com isolados das subsp. aurantifolii e citrumelonis. O estado de São Paulo, que adota uma campanha de erradicação do cancro cítrico, apresentou a menor diversidade haplotípica, provavelmente em decorrência do curto período em que o patógeno interage com o hospedeiro. / Abstract: The genetic structure of clonal populations of bacterial pathogens has been characterized, among other methods, by Southern blot using insertion sequences or avr genes as probes. The present work aimed the development of DNA primers and probes based on pathogenicity genes to study the genetic diversity of Xanthomonas citri subsp. citri, Xanthomonas fuscans subsp. aurantifolii, and Xanthomonas alfalfae subsp. citrumelonis strains. The presence of avrXacE1, avrXacE2 and leucine rich protein (lrp) genes was observed in all citri and aurantifolii strains, as well as the absence of the avrXacE2 gene in citrumelonis isolate. The restriction profiles generated by PCR-RFLP did not show polymorphism in the amplified genes and the use of avr genes as Southern blot probes showed to be effective to differentiate Xanthomonas species pathogenic to citrus and to identify a relative polymorphism between strains belonging to the same species. The haplotype diversity was higher with the avrXacE1 gene. Excepting the probe corresponding to lrp gene, the number of haplotypes identified (17) varied according to the endonuclease used on Southern blot. The strains of the citri species presented a higher number of copies of the avr genes compared to aurantifolii and citrumelonis. The state of São Paulo, that adopts a campaign of eradication of the citrus canker, presented the smaller haplotype diversity, probably as result of the short period where the pathogen interacts with the host. / Orientadora: Maria Inês Tiraboschi Ferro / Coorientador: José Belasque Júnior / Banca: João Martins Pizauro Junior / Banca:Julio Cezar Franco de Oliveira / Mestre

Orientador: Antonio Nivaldo Hespanhol / Banca: Edgar Aparecido da Costa / Banca: Sonia Maria N. Marangoni Montes / Resumo: Esta pesquisa tem como objetivo analisar a citricultura paulista a partir do aparecimento da doença de citros denomin ada cancro cítrico no município de Presidente Prudente - SP, no ano de 1957. O combate ao cancro cítrico mobilizou os poderes públicos e privados no Estado de São Paulo, desencadeando o maior embargo fitossanitário da história da agricultura brasileira. As conseqüências desse embargo refletem até os dias atuais um quadro de exclusão frente aos poucos produtores da Região de Presidente Prudente - SP. Além disso, provocou um intenso debate na região a respeito do papel desempenhado pelo FUNDECITRUS, em suas a ções de erradicação do cancro cítrico na região, promovendo assim de acordo com os produtores um tratamento desigual para os produtores da região em questão e os produtores da região nobre da citricultura ao Norte do Estado. / Abstract: The objective of this research is to analyze the citriculture State São Paulo starting from the emergence of the disease of citric denominated citrus canker in the municipal district of Presidente Prudente - SP, in the year of 1957. The combat to the citrus canker mobilized the public and private powers in the State of São Paulo, unchaining the largest seizure phytosanitary of the history of the Brazilian agriculture. The consequences of that seizure contemplate little by little until the current days a picture and exclusion front producing Region of Presidente Prudente - SP. Besides, it provoked an intense debate in the area regarding the paper carried out by FUNDECITRUS, in their action s of erradication of the citrus canker in the area, promoting like this in agreement with the producers an unequal treatment for the producing of the area in subject and the producing of the noble area of the citriculture to the North of the State. / Mestre

Orientadores: Marcos Antonio Machado, Alexandre Morais do Amaral / Dissertação (mestrado) - Universidade Estadual de Campinas, Instituto de Biologia / Made available in DSpace on 2018-08-12T03:30:01Z (GMT). No. of bitstreams: 1 Homem_RafaelAugusto_M.pdf: 2248507 bytes, checksum: 707bfda4edde12e0c0ac988130077aa3 (MD5) Previous issue date: 2008 / Resumo: O cancro cítrico é uma das mais sérias doenças de citros no mundo, sobretudo nos países onde a citricultura exerce papel influente na geração de empregos e divisas, como o Brasil, o maior produtor mundial de laranjas. Por esse motivo, o agente causal dessa doença, a bactéria Xanthomonas axonopodis pv. citri (Xac), teve seu genoma completamente sequenciado. No genoma de Xac foram identificados dois agrupamentos gênicos (operons), xcsCDEFGHIJKLMN e xpsEFGHIJKLMND, que codificam para o sistema de secreção do tipo II (SSTII), mecanismo altamente envolvido no processo de patogenicidade em algumas bactérias causadoras de doenças em plantas. Até o momento, não há evidência da função desempenhada por cada conjunto gênico do SSTII de Xac e da relação desses com o processo de interação com a planta de citros. Neste estudo foram analisadas as funcionalidades dos dois agrupamentos gênicos do SSTII de Xac e sua atividade durante a interação com a planta hospedeira. As análises revelaram que a bactéria utiliza os dois sistemas de forma distinta e com relevância diferenciada durante a interação com a planta, com repercussão no crescimento da bactéria no tecido foliar, sobretudo o operon xps. Adicionalmente, foram identificadas contribuições distintas na atividade de degradação dos compostos amido, carboximetilcelulose e proteína. Exames de microscopia revelaram que a bactéria tem sua organização estrutural do biofilme influenciada pelos dois SSTII e análises de expressão gênica revelaram que o operon xps apresenta atividade extremamente maior em relação ao operon xcs. Estes resultados mostram pela primeira vez a influência independente de ambos SSTII na capacidade patogênica de Xac. / Abstract: The citrus canker is a major threat to the citrus industry worldwide, especially where the citriculture plays an important role in employments and revenues, like United States and Brazil, the largest orange producing country. Therefore, the causal agent of citrus canker, the Gram negative bacterium Xanthomonas axonopodis pv. citri (Xac), had its genome completely sequenced. Throughout the genome of Xac, two operons (xps EFGHIJKLMND and xcsCDEFGHIJKLMN) that encompass 11 and 12 different proteins, respectively, for the type two secretion systems (TIISS) were identified. These mechanisms are highly involved in pathogenicity and virulence in some bacteria that cause disease in plants. However, so far, there are no studies on the function of these operons in Xac and their relationship for the infection process in the citrus plant. In this study, the functions of the two operons that code for the TIISS in Xac and their activity during the interaction with the plant host were analyzed. The analyses revealed that the bacterium uses both TIISS, however in a different fashion during the contact with the leaf tissue and the xps operon is highly more active. In addition, the operons were found to be differentially effective on the degradation of starch, carboxymethilcellulose and proteins. Confocal microscopy investigations found that the bacterial organization (biofilm) is influenced by both TIISS and gene expression analyses showed a much higher activity of the xps operon as compared to the xcs group. These results provide the first clear evidence of the independent influence of both TIISS in the pathogenic process of Xac. / Mestrado / Genetica de Microorganismos / Mestre em Genética e Biologia Molecular

Cryphonectria cubensis is an ascomycetous fungus that causes a serious canker disease on Eucalyptus trees in many parts of the world. The importance of the disease has led to numerous studies involving the taxonomy, genetic diversity and the control of Cryphonectria canker. However, there remain many questions pertaining to the disease that have not been considered. The objectives of the studies presented in this thesis were, therefore, to investigate the possibility of biological control of Cryphonectria canker, to evaluate the currently used disease screening strategy in South Africa and to establish a transfection system with dsRNA elements in Diaporthe, which is closely related to Cryphonectria. The introductory chapter of this thesis provides a review of the literature pertaining to Cryphonectria cubensis. In addition literature on hypovirulence in fungi is also extensively reviewed, with a special emphasis on the genus Cryphonectria. The aim of study in the second chapter of the thesis was to screen the South African C. cubensis population for the presence of dsRNA viruses. Two viruses were identified and the full sequence of these elements showed a strong homology to the mitochondrial viruses (mitoviruses) within the family Narnaviridae. We, therefore, named the viruses Cryphonectria cubensis mitovirus 1 (CcMV1) and Cryphonectria cubensis mitovirus 2 (CcMV2). The two viral genomes are 2601 nucleotides and 2639 nucleotides in size respectively and encode for a protein that probably functions as an RNA-dependant RNA polymerase (RdRp). Pathogenicity studies indicated that the viruses do not result in a significant reduction in pathogenicity of C. cubensis. In the third chapter, results of a study to consider whether different Eucalyptus clones responded similarly to various South African C. cubensis isolates, are presented. The aim was, therefore, to evaluate the current C. cubensis resistant screening method used on Eucalyptus spp. in South Africa. The statistical analysis of the inoculation data showed a significant isolate x clone interaction. This data also suggest the possibility of vertical resistance, which is different to previous assumptions. Transfection studies (Chapter 4) involving a positive stranded RNA virus, Diaporthe RNA virus (DaRV) from a South African D. perjuncta isolate are presented here. In this study, a virus free D. perjuncta isolate, a virulent C. cubensis isolate and a hypovirulent C. cubensis isolate containing the hypovirus CHV1-EP713 were chosen to be transfected with DaRV. By using electroporation, it was possible to infect a virus free D. perjuncta isolate with the Diaporthe RNA virus, thus extending the transfection range of this virus. The resulting transfection led to altered colony morphology but did not lead to a reduction in pathogenicity. We were also not successful in attempts to transfect isolates of C. cubensis with DaRV, indicating that the virus does not replicate in this host. In a previous study a virulent South African C. cubensis isolate was transfected with the Cryphonectria parasitica hypovirus CHV1-EP713. This resulted in the fungus becoming hypovirulent. Chapter five of this thesis presents the results of a study to evaluate the potential use of this virus in the biological control of Cryphonectria canker in South Africa. A field trial was established and existing cankers were treated with the transfected isolate. The treatment of the cankers did not lead to a significant reduction in canker size, but did alter the morphology of the cankers. The virus was also shown to be transmitted via hyphal anastomosis to the virulent canker causing isolates. In addition the co-inoculation on single trees with both the virulent and virus-containing isolate, resulted in a significant reduction in the size of the lesions. This study also showed that the transfected C. cubensis isolate are characterised by significantly smaller lesions than those associated with the virulent, virus-free isolate. Cryphonectria cubensis and the associated canker disease of Eucalyptus threaten the forestry industry in South Africa. The overall aims of the studies presented in this thesis were to gain a more complete understanding of this fungus and to evaluate potential control strategies. Each of these chapters should contribute towards a better understanding of the viruses associated with C. cubensis and other important aspects of Cryphonectria canker, which will hopefully lead to enhanced control strategies of the disease in South Africa. / Thesis (PhD)--University of Pretoria, 2008. / Microbiology and Plant Pathology / PhD / Unrestricted

O cancro cítrico (Xanthomonas axonopodis pv. citri) é uma das mais importantes doenças dos citros e seu controle é feito com medidas de exclusão e erradicação do patógeno. As chances de detecção de plantas doentes diminuem em pomares com baixa incidência da doença. Considerando-se as dificuldades da diagnose visual do cancro cítrico e as vantagens de uma tecnologia que possa melhorar o processo de detecção desta doença no campo, procurou-se identificar características espectroscópicas únicas de uma planta com esta doença. Os objetivos do presente trabalho foram verificar as características espectroscópicas de folhas com cancro cítrico e correlacioná-las às alterações na eficiência fotossintética do tecido foliar doente e relacionar severidade da doença à eficiência fotossintética. Os perfis espectroscópicos e a eficiência fotossintética de plantas das espécies Citrus sinensis (?Hamlin?), Citrus reticulata (?Ponkan?) e Citrus limonia (?Cravo?) foram avaliados antes e após a inoculação com Xanthomonas axonopodis pv. citri. O modelo y = (1 - x) ?, onde y representa a assimilação líquida relativa de CO2 e x, a severidade da doença, foi ajustado aos dados por meio de regressão não-linear. O modelo monomolecular foi ajustado à relação entre assimilação líquida relativa de CO2 e razões espectroscópicas da região do vermelho, também por regressão não-linear. Concluiu-se que a doença afeta a eficiência fotossintética de ?Ponkan? e ?Hamlin?. Em ?Hamlin?, a doença não apenas reduz a área foliar, mas também reduz a fotossíntese do tecido verde remanescente. Há relação positiva entre as razões espectroscópicas da região do vermelho e a fotossíntese nas variedades testadas. / Citrus canker (Xanthomonas axonopodis pv. citri) is one of the most important diseases of citrus. Regulatory control is applied by means of exclusion and eradication of the pathogen. Possibilities of detection of infected trees decrease in orchards with low disease incidence. Considering the difficulties in visual diagnosis of citrus canker and the advantages of a technology that can improve the process of its detection in the field, single spectroscopic characteristics of a plant with this disease was searched. The objectives of the present work were to verify the spectroscopic characteristics of leaves with citrus canker and to correlate them to photosynthetic efficiency of these diseased leaves, and to relate disease severity to photosynthetic efficiency. Spectroscopic profiles and photosynthetic efficiency of Citrus sinensis (?Hamlin?), Citrus reticulata (?Ponkan?) and Citrus limonia (?Cravo?) were assessed before and after the inoculation with Xanthomonas axonopodis pv. citri. The model y = (1 - x) ?, in which y represents relative net assimilation of CO2 and x, disease severity, was fitted to the data by nonlinear regression. The monomolecular model was fitted to the relationships between relative net assimilation of CO2 and spectroscopic reasons of the red region, also by nonlinear regression. It was concluded that the disease affects the photosynthetic efficiency of ?Ponkan? and ?Hamlin?. In ?Hamlin?, the disease not only reduces the amount of leaf area, but also reduces the photosynthesis of the remaining green leaf tissue. There is a positive relationship between spectroscopic reasons of the red region and photosynthesis in ?Hamlin?, ?Ponkan?, and ?Cravo?.