FundaÃÃo Cearense de Apoio ao Desenvolvimento Cientifico e TecnolÃgico / Lasiodiplodia theobromae à um fungo fitopatogÃnico responsÃvel por inÃmeras doenÃas em variadas plantas, sendo um fungo tipicamente de regiÃes tropicais e subtropicais. O fungo atacadiversas plantas tropicais, dentre elas destacam-se a mangueira, as Spondiassp., o coqueiro, o cajueiro, entre tantas outras. Seu controle à basicamente genÃtico realizado com o plantio de clones resistentes, entretanto, para sua obtenÃÃo torna-se necessÃrio o conhecimento das caracterÃsticas do patÃgeno. As informaÃÃes disponÃveissobre a variabilidade genÃtica de L. theobromaesÃo insuficientes para assegurar o sucesso em qualquer programa de melhoramento genÃtico visando à resistÃncia a este patÃgeno. Levando-se em conta que as proteÃnas sÃo produtos funcionais dos genes, torna-se importante conhecÃ-las, visando um melhor entendimento do modo de aÃÃo dos patÃgenos, sendo este entendimento, Ãtil como estratÃgia a ser utilizada no melhoramento vegetal buscando a resistÃncia genÃtica. Assim, o objetivo desse estudo foi realizar um estudo genÃtico molecular em uma populaÃÃo de L. theobromae e uma anÃlise proteÃmica diferencial do fungo entre isolados, mais e menos agressivos, visando identificar proteÃnas responsÃveis por essa agressividade.Uma populaÃÃo composta de 105 isolados foi usada na caracterizaÃÃo molecular, extraindo-se o DNA a partir do micÃlio do fungo crescido em meio lÃquido. Cada amostra foi submetida à reaÃÃo em cadeia da polimerase (PCR) com 15 pares de primers especÃficos para essa espÃcie, alÃm de um par de primer da regiÃo ITS e outro da regiÃo EF-1α. Os produtos amplificados foram visualisados em gel de agarose, corados com brometo de etÃdio e os dados tabulados em planilha binÃria e foram analisados pelo mÃtodo de agrupamento nÃo balanceado baseado na mÃdia aritmÃtica (UPGMA) utilizando o programa MVSP. As similaridades genÃticas foram estimadas pelo coeficiente de Jaccard. Os resultados indicaram uma grande variabilidade genÃtica da populaÃÃo avaliada. Jà o estudo proteÃmico foi realizado visando avaliar diferenÃas qualitativas, ou seja, a presenÃa/ausÃncia de uma determinada proteÃna, em relaÃÃo ao grupo antagÃnico. Para tal, utilizaram-se dois isolados do mesmo fungo, diferenciando-se quanto a sua agressividade, em que um era altamente agressivo, enquanto o outro apresentava uma baixa agressividade quando inoculados em mudas de cajueiro. Quando o perfil eletroforÃtico foi analisado, foram evidenciados 96 spots diferencialmente expressos. AtravÃs da LC-ESI-Q-TOF MS/MS, foram identificadas 84 proteÃnasapresentando diversas funÃÃes celulares.Com essa abordagem foi possÃvel a caracterizaÃÃo preliminar do perfil proteico deste fungo, obtendo-se alguns indÃcios dos mecanismos envolvidos na sua agressividade. Este à o primeiro estudo buscando conhecer as proteÃnas responsÃveis pela agressividade de L. theobromae. / Lasiodiplodia theobromae is a plant pathogenic fungus responsible for many diseases in various plants, is a fungus typically tropical and subtropical regions. The fungus attacks many tropical plants, among them, including mango, Spondias sp., coconut, cashew, and many others. His control is basically genetic performed by planting resistant clones, however to obtain it becomes necessary to know the pathogen characteristics. The information available on the genetic variability of L. theobromae is restricted to ensure success in any breeding program for resistance to this pathogen. Taking into account that proteins are functional products of genes, it is important to know them, to improve the understanding of the mode of action of pathogens, with this understanding, useful as a strategy to be used in plant breeding seeking genetic resistance. The objective of this study were to conduct a genetic study molecular in a population of L. theobromae and a differential proteomic analysis of the fungus among isolates, more and less aggressive, to identify proteins responsible for this aggressivity. A population consisting of 105 strains was used for molecular characterization, extracting the DNA from mycelia grown in liquid medium. Each sample was subjected to polymerase chain reaction (PCR) with 15 pairs of primers specific for the species, and a primer pair of the ITS region and other EF-1α region. The amplified products were visualisados agarose gel, stained with ethidium bromide and spreadsheet data in binary tabulated and analyzed by unbalanced grouping method based on the arithmetic mean (UPGMA) using the MVSP program. Genetic similarities were estimated by Jaccardâs coefficient. The results indicated a high genetic variability of the studied population. Since the proteomic study was conducted to assess qualitative differences, that is, the presence/absence of a specific protein in relation to the opposite group. To this end, we used two isolates of the same fungus, differing as their aggressiveness, in which one was highly aggressive, whereas the other had a low aggressiveness when inoculated seedlings of cashew. When the electrophoretic profile was analyzed, 96 were detected differentially expressed spots. By LC-ESI-Q-TOF MS / MS, 84 proteins were identified having the most diverse cellular functions. With this approach it was possible preliminary characterization of the protein profile of this fungus to give some evidence of the mechanisms involved in their aggressiveness. This is the first study seeking to know the proteins responsible for the aggressiveness L. theobromae.

The Koala bears housed and maintained by the Pretoria Zoo are relying on 20 different Eucalyptus species in the Eucalyptus arboretum in Pretoria for food. Species of the Botryosphaeriaceae are well known endophytes and pathogens of Eucalyptus in South Africa and elsewhere. This study was aimed at characterizing the Botryosphaeriaceae infecting these trees planted for the Koala bears. The experimental set-up also provided the opportunity to broaden our knowledge regarding the Eucalyptus species preferences of the Botryosphaeriaceae, as well as to reveal their potential movement to and from surrounding trees. In Chapter 1, the literature is reviewed regarding the biology of fungal endophytes, with a specific focus on Botryosphaeriaceae on Eucalyptus. Such a review clearly shows that, despite much previous work, we still only have a limited understanding of the biology and ecology of most fungal endophytes. This is particularly true for tree endophytes. Botryosphaeriaceae are clearly important and widespread canker and dieback pathogens of Eucalyptus. The taxonomic confusion that plagued the group, however, hindered a clear understanding of their diversity and biology. The use of molecular tools together with morphological characteristics has improved the ability to separate these fungi at species level. These tools are important for future work to better understand the true diversity. This would be a prerequisite if we are to gain a better understanding of the biology of these fungi and design with management strategies to control them. In Chapter 2, five species of Botryosphaeriaceae were identified from Eucalyptus species in the Pretoria arboretum, South Africa. Two species were described here as new species of Botryosphaeriaceae, within Neofusicoccum, namely N. ursorum prov. nom. and N. crypto-australe prov. nom. This is the first report of the latter species on Eucalyptus in South Africa. The other species included N. parvum, N. eucalypti comb. nov. (previously known as Dichomera eucalypti) and B. dothidea. Neofusicoccum parvum was the most common species isolated, followed by N. ursorum and N. eucalypti. The identifications were all based on the morphological characteristics, including cultural and conidial morphology, and DNA sequence data of the internal transcribed spacer region (ITS 1 and 4), and the translation elongation factor 1-α. The closely related species N. parvum and N. ribis were distinguished using a previously designed PCR-RFLP technique. As for the distinction between the cryptic species N. crypto-australe and N. australe, it was necessary to analyze a third gene region (β- tubulin) to confirm their separation using the phylogenetic species concept. All isolates obtained from this study produced lesions on stems of Eucalyptus camaldulensis (clone ZG-14) in the pathogenicity trial conducted under green house conditions. Of all species, N. eucalypti and N. crypto-australe were found to be the most pathogenic and B. dothidea the least pathogenic. There was, however, also significant variation in virulence between isolates of the same species. The results clearly show the potential threat of species of Botryosphaeriaceae to Eucalyptus. Field trials should be conducted in future studies to validate the findings in the greenhouse trials. Isolates representing different species identified in Chapter 2, were used for designing molecular tools for the in vitro and in vivo identification of Botryosphaeriaceae (Chapter 3). Sequences of the translation elongation factor 1-α were compared, and unique polymorphisms identified. Species specific primers were designed around these polymorphisms. All the primers designed were proven to be specific enough to distinguish the five different species from each other. The sensitivity of all primers were shown to detect fungal DNA concentration between 50 ng/μL and 0.01ng/μL. Preliminary tests of these primers on Eucalyptus leaves were done, and latent infections of N. parvum (the most common species) could be identified. More samples are likely to reveal the latent infections of other species using these tools. Future studies could now use these tools for the rapid identification of the fungi on Eucalyptus. It could be expanded to other hosts and more species of the Botryosphaeriaceae as well. The results presented in this study provided detailed information on species of the Botryosphaeriaceae from the 20 different species of Eucalyptus in a Pretoria arboretum. It yielded unique species, as well as well known pathogens of this host. All species isolated in this study were found to be pathogenic on Eucalyptus. The knowledge foundation, data and tools provided by this study can now be applied to characterize the infection biology, fine scale distribution and population diversity of these fungi. It is likely to bring new insights into the ecology of these organisms, their potential origin and movement between hosts. The presence of these fungi in other parts of South Africa should also be considered. / Dissertation (MSc)--University of Pretoria, 2009. / Microbiology and Plant Pathology / Unrestricted

The Botryosphaeriaceae (Ascomycetes), with more than 2000 species (http://www.indexfungorum.com), represents one of most widely distributed groups of fungal plant pathogens. These species are known to infect both economically important crops and native plants. In this study species of the Botryosphaeriaceae associated with native woody hosts in South Africa and Western Australia were investigated. Based on ITS rDNA sequence comparisons, combined with phenotypic characters and PCR-RFLP analyses, eight species were identified on native Syzygium cordatum in South Africa. These included Neofusicoccum parvum, N. ribis, N. luteum, N. australe, N. mangiferae, Botryosphaeria dothidea, Lasiodiplodia gonubiensis and L. theobromae. Three additional cryptic species were identified in the N. parvum / N. ribis complex from S. cordatum using five gene genealogies and the genealogical concordance phylogenetic species recognition (GCPSR). These are the first species of the Botryosphaeriaceae described using fixed single nucleotide polymorphisms (SNPs) as a defining character, and are described as N. cordaticola, N. kwambonambiense and N. umdonicola. The analysis of microsatellite marker data supported the distinction of these species. These data were also used to characterise the distribution of the latter three species and N. parvum on S. cordatum. Finding the same haplotypes of N. parvum on S. cordatum and closely related, planted Eucalyptus indicates movement of this pathogen between these hosts. Since all of the species recognised from S. cordatum were pathogenic to Eucalyptus, and the newly described species were more virulent than N. parvum and N. ribis on S. cordatum, their movement between hosts can pose a serious treat to both native and non-native plants. From Western Australia, molecular sequence data and morphological analyses revealed seven new species of the Botryosphaeriaceae from baobab and other native trees. These included Dothiorella longicollis, Fusicoccum ramosum, Lasiodiplodia margaritacea, Neoscytalidium novaehollandiae, Pseudofusicoccum adansoniae, P. ardesiacum and P. kimberleyense. In the literature review, which also considers work done in this thesis, the influence of molecular tools on the taxonomy of the Botryosphaeriaceae during the last decade, with a particular focus on cryptic species recognition, is considered. This study clearly showed that a polyphasic approach in species identification, as well as investigation of less well studied native flora, will reveal numerous new and cryptic species in the Botrysphaeriaceae and improve our knowledge of this group of important plant pathogens in the future. / Thesis (PhD)--University of Pretoria, 2011. / Microbiology and Plant Pathology / Unrestricted

Les maladies du bois de la vigne, par exemple le syndrome de l’esca et l’eutypiose, sont dues à des champignons qui se développent dans le xylème et dégradent les tissus ligneux. Elles sont en recrudescence depuis une vingtaine d’année dans tous les vignobles du monde. A ce jour, aucun traitement curatif n’est disponible alors que ces maladies engendrent généralement la mort de la plante. L’étude des relations hôte-parasite et notamment celle des réponses de défense de plante, est nécessaire pour la compréhension et le développement de nouvelles stratégies de lutte. Parmi les molécules de défense de la vigne, les stilbènes sont connus pour leur forte activité antifongique.Notre travail, basé sur des approches biochimiques et moléculaires apporte de nouvelles données sur le rôle potentiel des stilbènes en tant que molécules de défense contre les maladies du bois. Nous avons identifié et quantifié les stilbènes majoritaires des tissus ligneux de V. vinifera, évalué la sensibilité des agents de maladie du bois à l’effet antifongique de ces stilbènes et suivi leur éventuelle induction suite à l’inoculation d’agents de maladie du bois. Nous avons souligné l’importance des oligomères de stilbènes dont l’activité antifongique est élevée et montré qu’ils sont fortement induits dans le bois inoculé par un champignon pathogène de la famille des Botryosphaeriaceae. La comparaison des réponses de défenses entre des cépages tolérants et sensible, a montré que les cépages tolérants répondent globalement plus fortement et plus rapidement que le cépage sensible à une élicitation par un filtrat de milieu de culture d’un agent de l’esca. / Grapevine wood diseases, as esca syndrome and eutypiosis, are worldwide afflictions. They are due to xylem inhabiting fungi that cause the decay of woody tissues and finally the death of the plant. No curative treatment has been successfully developed until now. Studying grapevine-wood disease pathogen interaction is a prerequisite to the development of protection strategies. Phenolics, particularly stilbenoids, are antimicrobial molecules involved in the resistance of grapevine to fungal pathogens. Our work, based on biochemical and molecular approaches, reveals new data on the possible role of stilbenoids in grapevine defence against trunk diseases. We identified and quantified major stilbenoids of woody canes from V. vinifera. These compounds, especially stilbenoid oligomers, displayed in vitro antifungal activity against trunk disease agents and were induced in cuttings inoculated with an agent of the Botryosphaeriaceae family. We also compared the defence response of susceptible or tolerant cultivars to wood decay based on an elicitation assay with a culture filtrate of an esca pathogen. Defence responses of tolerant cultivars were more effective than those of the susceptible cultivar.

Los hongos de la familia Botryosphaeriaceae causan daños en la madera de la vid en regiones vitícolas de todo el mundo. Para caracterizar las distintas enfermedades de madera de la vid en planta adulta y para conocer el impacto de estos patógenos en Cataluña, se llevó a cabo un muestreo en viñedos afectados de diferentes denominaciones de origen catalanas. Los resultados confirmaron la existencia en Cataluña de las tres enfermedades de la vid más comunes en planta adulta (yesca, eutipiosis y BDA [black dead arm]), y evidenciaron asimismo la dificultad para diagnosticarlas, debido a la múltiple y frecuente concurrencia de éstas sobre una misma planta. Las especies fúngicas aisladas con mayor frecuencia fueron Phaeomoniella chlamydospora, Fomitiporia mediterranea, Eutypa lata y distintas especies de Botryosphaeriaceae. Dentro de esta familia se identificaron seis especies, siendo por orden de abundancia Diplodia seriata, Neofusicoccum parvum, Dothiorella viticola, Botryosphaeria dothidea, N. luteum y N. vitifusiforme. La controversia taxonómica que presenta la familia Botryosphaeriaceae, debido a las similitudes morfológicas entre taxones, se resolvió con el estudio combinado de la morfología de los conidios de los anamorfos y la información molecular de distintas regiones del ADN. Mediante la técnica de la PCR-cooperativa y el empleo posterior de una sonda de hibridación específica, se desarrolló una herramienta molecular de gran sensibilidad para la detección rápida y rutinaria de las especies de Botryosphaeriaceae. La sonda se diseñó sobre la región nuclear del ITS (espaciadores internos de transcripción) del ARNr, e identificó de forma específica a un grupo de siete especies de Botryosphaeriaceae (las seis aisladas de vides catalanas más Diplodia mutila). Profundizando en la caracterización de la familia, se estudió la capacidad de cinco especies (B. dothidea, Di. seriata, Do. viticola, N. luteum y N. parvum) para producir micotoxinas. Todos los aislados estudiados produjeron compuestos hidrofílicos fitotóxicos, aunque N. luteum y N. parvum destacaron por la fitotoxicidad que también mostraron sus compuestos lipofílicos. Esto corroboró los resultados de las pruebas de patogenicidad, en las que N. luteum y N. parvum mostraron una gran virulencia. Dothiorella viticola, N. vitifusiforme y Di. seriata mostraron una virulencia débil o nula, mientras que B. dothidea mostró un carácter patogénico intermedio. Sobre la susceptibilidad del hospedador a los distintos patógenos, se observó que el portainjerto Ru140 fue el menos susceptible y el 41B el más susceptible, mientras que las seis variedades de uva ensayadas (Cabernet-Sauvignon, Garnacha, Macabeo, Parellada, Tempranillo y Xarel·lo) no mostraron diferencias claras de susceptibilidad. Se constató que algunas especies vegetales que comparten su hábitat con la vid, como almendros, encinas, jaras, melocotoneros y nogales, son susceptibles a algunas especies de Botryosphaeriaceae, y que por tanto podrían actuar como hospedadores alternativos de los patógenos. De los factores abióticos que podrían afectar al desarrollo de las enfermedades se estudió el estrés hídrico. Los resultados obtenidos mostraron que tanto el estrés hídrico como el patógeno N. parvum alteraron parámetros fisiológicos y de crecimiento de la vid, aunque no se observó un efecto combinado de ambos sobre la planta. Además, la longitud de las necrosis vasculares causadas por N. parvum no aumentó significativamente con el estrés hídrico, confirmando que N. parvum es un patógeno primario y no un patógeno de debilidad. / Species in the fungal family Botryosphaeriaceae are known for causing grapevine trunk diseases in grape-producer regions worldwide. Declining vineyards were surveyed in Catalonia (NE Spain) to characterize those diseases on adult plants and to identify their causing agents. Most comon grapevine trunk diseases (esca, Eutypa dieback and black dead arm disease) were confirmed to occur in Catalonia. However, concurrence of symptoms in the same plant, belonging to at least two different diseases, often difficults disease diagnosis. Most frequently isolated fungal pathogens were Phaeomoniella chlamydospora, Fomitiporia mediterranea, Eutypa lata and several species of Botryosphaeriaceae. Six botryosphaeriaceous species were identified, in decreasing abundance order: Diplodia seriata, Neofusicoccum parvum, Dothiorella viticola, Botryosphaeria dothidea, N. luteum and N. vitifusiforme. The controversy in the taxonomy of Botryosphaeriaceae due to confusing morphological similarities among the species was resolved by combining the study of conidial morphology and multigene nucleotide data. A sensitive tool for a rapid detection of Botryosphaeriaceae species was developed based on the co-operational PCR technique and the use of a hybridization probe. The probe was designed to target the ITS region (internal transcribed spacers) of the rRNA repeat. It identified specifically the above Botryosphaeriaceae species isolated from grapevine plus Diplodia mutila. Five botryosphaeriaceuos species were tested for mycotoxin production, namely B. dothidea, Di. seriata, Do. viticola, N. luteum and N. parvum. All species produced hydrofilic phytotoxic compounds, and N. luteum and N. parvum were also shown to produce low-weight lipophilic toxins. These results support the high virulence showed by N. luteum and N. parvum in the patogenicity test. Virulence of Dothiorella viticola, N. vitifusiforme and Di. seriata was weak to low, while B. dothidea showed an intermediate virulence. In terms of host susceptibility, rootstock 140Ru was the less susceptible to the botryosphaeriaceous fungi tested while 41B was the most susceptible one. However, no great differences in susceptibility were observed among the six grapevine varieties tested (Cabernet Sauvignon, Grenache, Macabeo, Parellada, Tempranillo and Xarel·lo). Some plant species from the surroundings of vineyards, namely almond, peach and walnut trees, holm oak and rockroses, were shown to be susceptible to some Botryosphaeriaceae species. It is hypothesized that these species could act as alternative hosts for those pathogens. Hydric stress in combination with N. parvum infection was studied as a potential factor that could affect disease development. Physiological and growth parameters of grapevine were modified by both hydric stress and N. parvum main effects, but no combined effects were detected. Vascular necroses were similar in both stressed and non-stressed grapevines, therefore N. parvum is not considered as a weakness pathogen.

MELO, José Glauber Moreira. Análise molecular e proteômica de Lasiodiplodia theobromae associado a fruteiras tropicais. 2014. 88 f. Tese (Doutorado em agronomia)- Universidade Federal do Ceará, Fortaleza-CE, 2014. / Submitted by Elineudson Ribeiro (elineudsonr@gmail.com) on 2016-08-29T20:06:07Z No. of bitstreams: 1 2014_tese_jgmmelo.pdf: 2467689 bytes, checksum: e9a0fc6142e5ef3db09ac0252d32f4e7 (MD5) / Approved for entry into archive by Jairo Viana (jairo@ufc.br) on 2016-08-31T23:39:17Z (GMT) No. of bitstreams: 1 2014_tese_jgmmelo.pdf: 2467689 bytes, checksum: e9a0fc6142e5ef3db09ac0252d32f4e7 (MD5) / Made available in DSpace on 2016-08-31T23:39:17Z (GMT). No. of bitstreams: 1 2014_tese_jgmmelo.pdf: 2467689 bytes, checksum: e9a0fc6142e5ef3db09ac0252d32f4e7 (MD5) Previous issue date: 2014 / Lasiodiplodia theobromae is a plant pathogenic fungus responsible for many diseases in various plants, is a fungus typically tropical and subtropical regions. The fungus attacks many tropical plants, among them, including mango, Spondias sp., coconut, cashew, and many others. His control is basically genetic performed by planting resistant clones, however to obtain it becomes necessary to know the pathogen characteristics. The information available on the genetic variability of L. theobromae is restricted to ensure success in any breeding program for resistance to this pathogen. Taking into account that proteins are functional products of genes, it is important to know them, to improve the understanding of the mode of action of pathogens, with this understanding, useful as a strategy to be used in plant breeding seeking genetic resistance. The objective of this study were to conduct a genetic study molecular in a population of L. theobromae and a differential proteomic analysis of the fungus among isolates, more and less aggressive, to identify proteins responsible for this aggressivity. A population consisting of 105 strains was used for molecular characterization, extracting the DNA from mycelia grown in liquid medium. Each sample was subjected to polymerase chain reaction (PCR) with 15 pairs of primers specific for the species, and a primer pair of the ITS region and other EF-1α region. The amplified products were visualisados agarose gel, stained with ethidium bromide and spreadsheet data in binary tabulated and analyzed by unbalanced grouping method based on the arithmetic mean (UPGMA) using the MVSP program. Genetic similarities were estimated by Jaccard’s coefficient. The results indicated a high genetic variability of the studied population. Since the proteomic study was conducted to assess qualitative differences, that is, the presence/absence of a specific protein in relation to the opposite group. To this end, we used two isolates of the same fungus, differing as their aggressiveness, in which one was highly aggressive, whereas the other had a low aggressiveness when inoculated seedlings of cashew. When the electrophoretic profile was analyzed, 96 were detected differentially expressed spots. By LC-ESI-Q-TOF MS / MS, 84 proteins were identified having the most diverse cellular functions. With this approach it was possible preliminary characterization of the protein profile of this fungus to give some evidence of the mechanisms involved in their aggressiveness. This is the first study seeking to know the proteins responsible for the aggressiveness L. theobromae. / Lasiodiplodia theobromae é um fungo fitopatogênico responsável por inúmeras doenças em variadas plantas, sendo um fungo tipicamente de regiões tropicais e subtropicais. O fungo atacadiversas plantas tropicais, dentre elas destacam-se a mangueira, as Spondiassp., o coqueiro, o cajueiro, entre tantas outras. Seu controle é basicamente genético realizado com o plantio de clones resistentes, entretanto, para sua obtenção torna-se necessário o conhecimento das características do patógeno. As informações disponíveissobre a variabilidade genética de L. theobromaesão insuficientes para assegurar o sucesso em qualquer programa de melhoramento genético visando à resistência a este patógeno. Levando-se em conta que as proteínas são produtos funcionais dos genes, torna-se importante conhecê-las, visando um melhor entendimento do modo de ação dos patógenos, sendo este entendimento, útil como estratégia a ser utilizada no melhoramento vegetal buscando a resistência genética. Assim, o objetivo desse estudo foi realizar um estudo genético molecular em uma população de L. theobromae e uma análise proteômica diferencial do fungo entre isolados, mais e menos agressivos, visando identificar proteínas responsáveis por essa agressividade.Uma população composta de 105 isolados foi usada na caracterização molecular, extraindo-se o DNA a partir do micélio do fungo crescido em meio líquido. Cada amostra foi submetida à reação em cadeia da polimerase (PCR) com 15 pares de primers específicos para essa espécie, além de um par de primer da região ITS e outro da região EF-1α. Os produtos amplificados foram visualisados em gel de agarose, corados com brometo de etídio e os dados tabulados em planilha binária e foram analisados pelo método de agrupamento não balanceado baseado na média aritmética (UPGMA) utilizando o programa MVSP. As similaridades genéticas foram estimadas pelo coeficiente de Jaccard. Os resultados indicaram uma grande variabilidade genética da população avaliada. Já o estudo proteômico foi realizado visando avaliar diferenças qualitativas, ou seja, a presença/ausência de uma determinada proteína, em relação ao grupo antagônico. Para tal, utilizaram-se dois isolados do mesmo fungo, diferenciando-se quanto a sua agressividade, em que um era altamente agressivo, enquanto o outro apresentava uma baixa agressividade quando inoculados em mudas de cajueiro. Quando o perfil eletroforético foi analisado, foram evidenciados 96 spots diferencialmente expressos. Através da LC-ESI-Q-TOF MS/MS, foram identificadas 84 proteínasapresentando diversas funções celulares.Com essa abordagem foi possível a caracterização preliminar do perfil proteico deste fungo, obtendo-se alguns indícios dos mecanismos envolvidos na sua agressividade. Este é o primeiro estudo buscando conhecer as proteínas responsáveis pela agressividade de L. theobromae.

Submitted by Mario BC (mario@bc.ufrpe.br) on 2016-11-24T13:20:38Z No. of bitstreams: 1 Mariote dos Santos Brito Netto.pdf: 3540374 bytes, checksum: ee89d890558eed9701de9441d70f0cc0 (MD5) / Made available in DSpace on 2016-11-24T13:20:38Z (GMT). No. of bitstreams: 1 Mariote dos Santos Brito Netto.pdf: 3540374 bytes, checksum: ee89d890558eed9701de9441d70f0cc0 (MD5) Previous issue date: 2016-07-29 / The gummosis out as a major disease reported in all producing regions of cashew in Brazil. In this study we identify, characterize and evaluate the comparative epidemiology of species Botryosphaeriaceae associated with gummosis in Anacardium in Brazil. A total of 138 isolates were sampled and identified using a combination of morphological analysis and phylogenetic based on the partial sequence of the translation elongation factor 1-α sequence (EF-1α), internal transcribed spacers (ITS) and sequence of the β-tubulin. ten species were identified Botryosphaeriaceae: Lasiodiplodia brasiliense, L. euphorbicola, L. gonubiensis, L. iraniensis, L. jatrophicola, L. gravistriata sp. nov., L. pseudotheobromae, L. theobromae, Neofusicoccum batangarum and Pseudofusicoccum stromaticum. Only L. theobromae previously described in cashew tree, while all other species are reported for the first time in association with cashew trees in Brazil and worldwide. Lasiodiplodia theobromae was the prevalent species. All species of pathogenic Botryosphaeriaceae were highlighted in branches of cashew. There were significant differences between species with N. batangarum, L. iraniensis, L. jatrophicola and L. gravistriata being the most aggressive species, while L. euphorbicola, L. pseudotheobromae were less aggressive. All species of Botryosphaeriaceae caused symptoms in alternative hosts tested except P. stromaticum, L. brasiliense and L. iraniensis had the highest injury in avocado, banana, guava, papaya, mango and passion fruit. L. jatrophicola showed lower aggressive in the hosts, as N. batangarum was not pathogenic in passion fruit and only L. gravistriata caused symptoms in melon. Our results suggest that these alternate hosts serve as a potential source of inoculum. Species Botryosphaeriaceae demonstrated reduction in mycelial growth in the presence of Thiophanate-methyl fungicides, difenoconazole and azoxystrobin. The response sensitivity present variation according to the species of the fungicides and Botryosphaeriaceae. / A gomose destaca-se como uma das principais doenças relatadas em todas as regiões produtoras de caju no Brasil. No presente estudo identificamos, caracterizamos e avaliamos a epidemiologia comparativa das espécies de Botryosphaeriaceae associadas a gomose em Anacardium no Brasil. Um total de 138 isolados foram amostrados e identificados usando uma combinação de analise morfológica e filogenética baseados na sequencia parcial do translation elongation factor 1-α sequence (EF-1α), internal transcribed spacers (ITS) e sequencia do β-tubulin. dez espécies de Botryosphaeriaceae foram identificadas: Lasiodiplodia brasiliense, L. euphorbicola, L. gonubiensis, L. iraniensis, L. jatrophicola, L. gravistriata sp. nov., L. pseudotheobromae, L. theobromae, Neofusicoccum batangarum e Pseudofusicoccum stromaticum. Somente L. theobromae foi previamente descrito em cajueiro, enquanto que todas outras espécies são reportadas pela primeira vez em associação com cajueiro no Brasil e no mundo. Lasiodiplodia theobromae foi a espécie prevalente. Todas espécies de Botryosphaeriaceae foram patogênicas em ramos destacados de cajueiro. Houve diferença significativa entre as espécies, com N. batangarum, L. iraniensis, L. jatrophicola e L. gravistriata sendo as espécies mais agressivas, enquanto L. euphorbicola, L. pseudotheobromae foram as menos agressivas. Todas as espécies de Botryosphaeriaceae causaram sintomas nos hospedeiros alternativos testados exceto, P. stromaticum. L. brasiliensee L. iraniensis apresentaram as maiores lesões em abacate, banana, goiaba, mamão, manga e maracujá. L. jatrophicola mostrou os menores valores de agressividade nos hospedeiros, já N. batangarum não foi patogênico em maracujá e somente L. gravistriata causou sintomas em melão. Nosso resultado sugere que esses hospedeiros alternativos servem com uma fonte de inóculo potencial. L. gravistriata apresentou crescimento nas temperaturas de 5°C e 10°C. As espécies de Botryosphaeriaceae demostraram redução no crescimento micelial na presença dos fungicidas Tiofanato-metilico, difenoconazole e azoxistrobin, apresentando diferentes níveis de sensibilidade a cada um dos princípios ativos utilizados.

The Botryosphaeriaceae is a family of fungi that includes many species, which are well-known as pathogens, saprophytes and endophytes of plants and especially of trees. As a result of their pathogenic nature and potential threat to plantations and agricultural crops, much research has been devoted to their identification. The main focus of studies that make up this thesis has been on the fungal complex referred to as Diplodia pinea sensu lato. These fungi are members of the Botryosphaeriaceae and studies have specifically concentrated on their taxonomy, host associations and mycovirus infections associated with them. Diplodia pinea sensu lato represents a species complex of highly similar morphological types that mainly infect Pinus spp., world-wide. The species complex includes what have in the past been known as the A, B and C morphological types of D. pinea. Multiple gene genealogies based on sequences of partial protein-coding genes and microsatellite markers were used to resolve the species complex into two genera, D. pinea and D. scrobiculata (= B morphotype). Diplodia-like isolates from Australia, Greece and Cyprus were characterized using both morphological and molecular characteristics. Morphologically, these isolates all have dark, thick-walled conidia (Diplodia-like) but phylogenetically, they could belong to three distinct genera of the Botryosphaeriaceae namely Diplodia, Lasiodiplodia and Dothiorella. Results of this study led to the description of Dothiorella casuarini from Casuarina spp. in Australia and they highlight the fact that similar morphological characteristics and disease etiology does not necessarily provide a true reflection of the evolutionary history of a pathogen. Phylogenetic studies on species of the Botryosphaeriaceae with Diplodia-like anamorphs revealed intriguing host association patterns. The availability of sequence data for many species of the Botryosphaeriaceae made it possible to extend the phylogeny to include six of the ten lineages as previously described for the Botryosphaeriaceae. Angiosperms appeared to be the most common, and possibly ancestral, host group of the Botryosphaeriaceae, with the exception of Macrophomina, Guignardia, Saccharata and “Botryosphaeria” quercuum. Infection of gymnosperms most likely occurred more recently, only in specific groups (Diplodia and Lasiodiplodia) via host shifts. Three distinct viruses have now been characterized from isolates of D. pinea sensu lato. Two of these were previously characterized and are known as Sphaeropsis sapinea RNA virus 1 and 2 (SsRV1 and SsRV2). The third dsRNA element more commonly found in association with D. scrobiculata was characterized in this dissertation and named Diplodia scrobiculata RNA virus 1 (DsRV1). It has a genome of 5018 bp with a unique genome organization characterized by two open reading frames (ORFs). One ORF codes for a putative polypeptide similar to proteins of the vacuolar protein-sorting (VPS) machinery and the other one for a RNA dependent RNA polymerase (RdRp). The hypothetical protein probably has a role in transport or protection of this unencapsulated virus into membranous vesicles. Phylogenetically, DsRV1 groups closest to a dsRNA element from Phlebiopsis gigantea (PgV2) and they both group separately from other families in which fungal viruses have been classified. The frequency and distribution of DsRV1, SsRV1 and SsRV2 were determined in a collection of D. pinea and D. scrobiculata isolates using Real-time PCR. Infections with SsRV1 and SsRV2 occurred in both D. pinea and D. scrobiculata, while DsRV1 was mainly found in D. scrobiculata. DsRV1 was also found to always occur in combination with SsRV1 and/or SsRV2. Therefore, DsRV1 probably selected against a coat protein as the result of a fitness trade-off. Although earlier studies indicated that these viruses have no effect on the phenotype or virulence of D. pinea and D. scrobiculata isolates, the presence of specific viruses in their host populations serve as a useful marker in studying movement of fungal pathogens. The ultimate aim of studies making up this dissertation was to expand the base of knowledge regarding species in the D. pinea species complex. This was justified by the fact that D. pinea is one of the most important tree pathogens in South Africa and that an expanded knowledge might contribute to reducing diseases caused by it. Clearly understanding the identity of the fungus must clearly underpin many elements of a management strategy and this was one of the aims of the suite of studies conducted. Furthermore, I attempted to augment the knowledge base regarding dsRNA elements in D. pinea sensu lato. These studies were of a basic nature and relatively far removed from the practical application level. Nonetheless, it is my hope that they have pushed ahead knowledge barriers and that in some way they will contribute to reducing the impact of Diplodia-associated diseases in the future. / Thesis (PhD)--University of Pretoria, 2011. / Microbiology and Plant Pathology / Unrestricted

Vitis vinifera L. représente l’une des cultures les plus répandues dans les pays producteurs de vin, laquelle est soumise à de nombreuses contraintes environnementales pouvant favoriser l’émergence des maladies de dépérissement du bois (MDB). La famille des Botryosphaeriaceae est responsable du Botryosphaeria dieback, provoquant des chancres et des nécroses qui conduisent à une dépréciation de la qualité du vin, voire à la mort des ceps. Les méthodes de lutte sont peu efficaces. Sept espèces sont retrouvées dans le vignoble français dont B. dothidea, D. intermedia, D. mutila, D. seriata, Do. viticola, N. parvum et L. viticola. Nous avons étudié différents traits d’histoire de vie de ces agents pathogènes : (i) leur agressivité in planta, (ii) leur adaptation à des contraintes environnementales (e.g. température, fongicides), (iii) la présence de mycovirus, pour acquérir des connaissances sur leur pouvoir adaptatif face aux contraintes environnementales et expliquer la variabilité de leur agressivité. En complément, l’évaluation de la sensibilité de cultivars de Vitis face à une infection par N. parvum et D. seriata a été réalisée, et le potentiel de défenses de différents cépages (Ugni Blanc, Cabernet-Sauvignon et Merlot) a été étudié. L’ensemble des travaux menés ont permis de révéler des espèces très agressives telles que N. parvum et L. viticola par rapport à D. seriata, lors d’inoculations en serre sur des boutures. Les températures optimales de croissance déterminées montrent que certaines espèces (ex. Lasidiplodia spp.) sont mieux adaptées à des températures élevées (33°C). Par ailleurs, la sensibilité de 65 souches et génotypes a été testée pour 9 fongicides avec des modes d’action différents (inhibiteurs de la respiration mitochondriale, de la biosynthèse des stérols, du cytosquelette, multi-sites, etc.). De nombreuses espèces sont peu ou pas sensibles à certains de ces fongicides et des souches résistantes ont été trouvées avec un facteur de résistance pouvant atteindre plus de 1000. D’autre part, la détection de mycovirus au sein des 65 isolats a permis d’identifier la présence de 6 mycovirus, dont Neofusicoccum luteum mitovirus 1 et Neofusicoccum luteum fusavirus 1. L’évaluation du potentiel de défense des trois cultivars Vitis face à une infection par N. parvum et D. seriata a montré des réponses différentes entre cépages et en fonction de l’agent pathogène. In fine, des analyses croisant les différents traits d’histoire de vie et les interactions plante-pathogènes ont été faites, et l’ensemble des résultats nous a fourni de nouvelles pistes d’étude pour lutter contre ces agents pathogènes. / Vitis vinifera L. is largely cultivated in countries producing wine but an increase in grapevine trunk diseases (GTDs) have been observed due to the attack of several fungal pathogens including those belonging to the Botryosphaeriaceae family (Botryosphaeria dieback). Seven species were isolated in the French vineyards B. dothidea, D. intermedia, D. mutila, D. seriata, Do. viticola, N. parvum and L. viticola. Nowadays, no efficient products are available to control these diseases. Studying, different life traits of 65 strains of different genotypes of the Botryosphaeriaceae’s family, known to have members displaying different aggressiveness, would lead to a better understanding of these wood pathogens. Their in planta aggressiveness, their adaptation towards environmental pressures (temperature, fungicides), and the detection of mycoviruses were carried out. In order to have a better comprehension of the interaction within the plant, the expression analysis of genes involved in the plant defense were assayed upon 3 cultivars (Cabernet Sauvignon, Merlot, and Ugni-Blanc) but also on two species (N. parvum and D. seriata). L. viticola, and N. parvum, were shown to be more aggressive than D. seriata and. L. viticola is more adapted to higher temperature (33°C). Moreover, the strains tested with 9 different fungicides (mitochondrial respiratory, sterol biosynthesis, cytoskeleton or multisite inhibitors, etc.) showed lower sensitivity within some species, (with a resistance factor reaching a 1000). In addition, at least 6 mycoviruses were characterized. Amongst them, two mycoviruses were isolated from a N. luteum strain and were fully sequenced (Neofusicoccum luteum mitovirus 1 and Neofusicoccum luteum fusavirus 1). The three cultivars infected with either N. parvum or D. seriata showed different gene responses between themselves but also between the different strains inoculated. These different studies are giving us more information upon these Botryospheriaceae fungi, to find out new efficient or complementary methods in order to control GTDs

Thesis (MSc)--University of Stellenbosch, 2007. / ENGLISH ABSTRACT: Grapevine trunk diseases are caused by invasive pathogens that are responsible for the slow decline of vines. In particular, Eutypa dieback of grapevine has had a devastating impact on vineyards worldwide, reducing growth and yield, eventually killing the grapevine. The causal organism of Eutypa dieback was first described as Eutypa armeniacae Hansf. & Carter, the pathogen that causes dieback of apricots, but since 1987 this species has been considered a synonym of Eutypa lata (Pers.:Fr.) Tul & C. Tul (anamorph Libertella blepharis A. L. Smith). Recently, it was proposed that at least two species that are capable of infecting grapevines are responsible for Eutypa dieback. Consequently, the molecular identification and characterisation of Eutypa dieback was used to delineate the species occurring on infected grapevines in South Africa. This involved the molecular analyses of three molecular markers, namely, the internal transcribed spacer (ITS) and large subunit (LSU) regions of the ribosomal DNA operon, and the -tubulin gene. The results obtained revealed the presence of a second species, namely, Eutypa leptoplaca (Mont.) Rappaz, that occurred together with E. lata on infected grapevines. Also co-habiting with these pathogens were related fungi form the Diatrypaceae family, Cryptovalsa ampelina (Nitschke) Fuckel and Eutypella vitis (Schwein.) Ellis & Everhart. Pathogenicity tests conducted on isolates representing C. ampelina, E. lata, E. leptoplaca, and E. vitis revealed that all were pathogenic to grapevine. Several species of Botryosphaeriaceae that commonly invade the woody tissue of grapevines are also pathogenic to grapevine. The symptoms in grapevine commonly associated with Botryosphaeriaceae are easily confused with the symptoms produced by Eutypa dieback which prompted the need for the development of a detection method that can correctly identify the presence of multiple pathogens. A reverse dot blot hybridisation (RDBH) method was subsequently applied to provide a rapid, accurate and reliable means of detecting the Eutypa species involved in the Eutypa disease complex, as well as those species of Botryosphaeriaceae known to cause disease in grapevines. The method involved the use of multiplex PCR to simultaneously amplify and label the regions of DNA that are used as pathogen specific probes. Consequently, membrane immobilised species-specific oligonucleotides synthesised from the ITS, - tubulin and LSU molecular data were evaluated during the application of this diagnostic method to detect Eutypa species. It was found that the species-specific oligonucleotides, designed from ITS sequence data, could consistently detect E. lata and E. leptoplaca. The application of the RDBH method for the detection of these Eutypa species, based on -tubulin and LSU sequence data, however, proved to be unsuccessful. Subsequently, a RDBH method, utilising species-specific oligonucleotides designed from elongation factor-1α sequence data, was successfully applied for the detection of Botyrosphaeria dothidea (Moug.:Fr.) Ces. & De Not., Neofusicoccum luteum (Pennycook & Samuels) Crous, Slippers & A.J.L. Phillips, Neofusicoccum parvum (Pennycook & Samuels) Crous, Slippers, A.J.L. Phillips and Neofusicoccum ribis (Slippers, Crous & M.J. Wingf.) Crous, Slippers & A.J.L. Phillips. The method, however, was unsuccessful for the detection of Diplodia seriata De Not. In addition to the above-mentioned shortcomings, the RDBH was not amenable to the detection of pathogens directly from field or environmental samples, but required preparation of DNA from pure cultures. The method, however, allows for the identification of multiple pathogens in a single assay. As DNA extraction methods are amended, improved and honed to obtain DNA from environmental samples, so would it increase the usefulness of RDBH. / AFRIKAANSE OPSOMMING: Wingerd stamsiektes word veroorsaak deur patogene wat die vermoë het om wingerdplante te infekteer en dan stadige agteruitgang van dié wingerde te veroorsaak. Veral Eutypa terugsterwing het ‘n vernietigende effek op wingerde wêreldwyd deurdat dit groeikrag en oesmassa verlaag, maar ook omdat dit uiteindelik wingerdstokke kan dood. Die veroorsakende organisme is aanvanklik as Eutypa armeniacae Hansf. & Carter beskryf, die patogeen wat terugsterf by appelkose veroorsaak, maar sedert 1987 word hierdie spesies beskou as ‘n sinoniem van Eutypa lata (Pers.:Fr.) Tul & C. Tul (anamorph Libertella blepharis A. L. Smith). Dit is egter onlangs voorgestel dat ten minste twee spesies die vermoë het om wingerd te infekteer om Eutypa terugsterwing te veroorsaak. Gevolglik is molekulêre identifikasie- en karakteriseringstudies geloods om te bepaal watter spesies Eutypa terugsterwing in Suid-Afrikaanse wingerde veroorsaak. Dit het die molekulêre analise van drie molekulêre merkers behels, naamlik die interne getranskribeerde spasiëerderarea (“ITS”), die groot ribosomale subeenheid (“LSU rDNA”) en β-tubilien geen. Resultate van die filogenetiese analise dui daarop dat ’n tweede spesies, naamlik Eutypa leptoplaca (Mont.) Rappaz, saam met E. lata in geïnfekteerde plante voorkom. Saam met bogenoemde twee spesies het daar ook verwante spesies van die Diatrypaceae familie voorgekom, naamlik Cryptovalsa ampelina (Nitschke) Fuckel en Eutypella vitis (Schwein.) Ellis & Everhart. Patogenisiteitstudies wat uitgevoer is met verteenwoordigende isolate van C. ampelina, E. lata, E. leptoplaca, en E. vitis dui daarop dat almal patogene van wingerd is. Verskeie Botryosphaeriaceae spesies wat gereeld in houtagtige wingerdweefsel aangetref word, is ook patogene van wingerd. Interne simptome wat algemeen met Botryosphaeriaceae infeksies geassosieer word, kan baie maklik met dié van Eutypa terugsterwing verwar word en dit het die nood laat ontstaan om ‘n opsporingsmetode te ontwikkel wat akkuraat genoeg is om tussen veelvoudige infeksies te onderskei. ’n Omgekeerde-stippelklad-hibridisasie (OSH) metode is gevolglik aangewend om Eutypa spesies betrokke in die Eutypa-siektekompleks op ‘n vinnige, akkurate en betroubare manier op te spoor, sowel as die Botryosphaeriaceae species wat bekend is as patogene van wingerd. Die metode behels ’n saamgestelde PKR vir die vermeerdering en merk van DNS areas wat gebruik word as patogeen spesifieke peilers. Spesies-spesifieke oligonukleotiede ontwikkel vanaf die ITS, -tubilien en LSU molekulêre data is op ‘n membraan vasgeheg en gebruik om ’n diagnostiese toets te ontwikkel vir Eutypa species. Merkers ontwikkel vanaf die ITS kon E. lata and E. leptoplaca konsekwent opspoor. Die opspoor van Eutypa spesies met merkers vanaf die -tubulien en LSU gene met OSH was onsuksesvol. Die OSH metode met merkers vanaf die verlengingsfaktor-1α kon susksesvol gebruik word om Botyrosphaeria dothidea (Moug.:Fr.) Ces. & De Not., Neofusicoccum luteum (Pennycook & Samuels) Crous, Slippers & A.J.L. Phillips, Neofusicoccum parvum (Pennycook & Samuels) Crous, Slippers, A.J.L. Phillips and Neofusicoccum ribis (Slippers, Crous & M.J. Wingf.) Crous, Slippers & A.J.L. Phillips op te spoor. Dié metode kon egter nie Diplodia seriata De Not. opspoor nie. Bykomend tot bogenoemde tekortkominge, kon die omgekeerde-stippelklad-hibridisasie metode ook nie aangepas word om patogene direk vanuit plantmateriaal op te spoor nie en word DNS afkomstig vanaf suiwer kulture benodig. Dié metode laat egter identifikasie van verskeie patogene in ‘n enkele toets toe. Soos DNS ekstraksie metodes aangepas, verbeter en verfyn word om DNS vanuit plantmateriaal te verkry, sal die bruikbaarheid van die omgekeerde stippelklad hibridisasie metode ook verbeter.