Made available in DSpace on 2014-07-29T15:16:29Z (GMT). No. of bitstreams: 1 Eriston Vieira Gomes.pdf: 1068459 bytes, checksum: 42c6dd1935f5ad9ce49cfd644bc60d66 (MD5) Previous issue date: 2009-09-30 / 79 Sclerotinia sclerotiorum isolates were collected under central pivot irrigations sistems, from Brazilian fields, divided in 4 populations (S2, S3, A and M) and analyzed to determine the genetic variability among and between populations using molecular markers based on microsatelittes. 10 primers were used and the amplification products were separate in poliacrilamida gels and the bands were silver stained. A total of 102 different haplotypes were identified, and the amount of haplotypes varied from 6 to 18 for locus. The genotypic diversity ranged from 65% to 91%. Analyses based on genetic diversity and fixation indices which indicate the variability between populations was 28.79% (FST = 28793) and the variability among populations was 71.21%. The Jaccard similarity index indicated that the populations S2 and A is genetically closer. The population S3 presented a similarity index of 0.44 compared with the populations S2 and A. The population M, originated from several collection sites, was considered genetically more distant showing a index of 0.46 compared with the others populations . The high variability between and among populations can indicate that, besides the possible introduction of new genotypes in the analyzed fields, could be having clonal and sexual reproduction in isolated of S. sclerotiorum from Brazilian cerrado. / Foram coletados 79 isolados de S. sclerotiorum, sob sistemas de irrigação por pivô central, provenientes da região do entorno de Brasília DF, divididos em 4 populações (S2, S3, A e M) e analisados, quanto a sua variabilidade intra e interpopulações utilizando marcadores moleculares baseados em microssatélites. Foram utilizados 10 conjuntos de oligonucleotídeos e o produto de amplificação desses marcadores foram separados em géis de poliacrilamida e corados com nitrato de prata. Um total de 102 alelos foram identificados, sendo que a quantidade de alelos variou de 6 a 18 por lócus e a diversidade alélica variou de 65% a 91%. A variabilidade entre as populações foi de 28.79% (Fst = 28793) e a variabilidade dentro das populações foi de 71.21%. O índice de similaridade de Jaccard indicou que as populações S2 e A são as populações geneticamente mais próximas, a população S3 apresentou um índice de similaridade de 0.44 em relação as populações S2 e A e a população M proveniente de vários sítios de coleta foi considerada a população geneticamente mais distante. A alta variabilidade dentro e entre populações pode indicar que esteja havendo reprodução tanto sexuada como assexuada em isolados de S. sclerotiorum da região do entorno de Brasília DF.

Pseudomonas chlororaphis strain PA23 has demonstrated biocontrol of Sclerotinia sclerotiorum (Lib.) de Bary, a fungal pathogen of canola (Brassica napus L.). The objectives of this research were two-fold: to optimize PA23 phyllosphere biocontrol and to investigate PA23’s influence in the rhizosphere. PA23 demonstrated longevity when inoculated on B. napus under greenhouse conditions. Carbon source differentially effected growth rate and antifungal metabolite production of PA23 in culture. Carbon source did not have a significant effect on in vivo biocontrol. PA23 demonstrated biocontrol ability of the fungal root pathogens Rhizoctonia solani J.G. Kühn and Pythium ultimum Trow in radial diffusion assays. PA23’s ability to promote seedling root growth was demonstrated in sterile growth pouches, but in a soil system these results were reversed. This research is essential for developing PA23 into an effective biocontrol agent in the phyllosphere and it opens the door for use of PA23 as a rhizosphere seed treatment. / October 2016

A produção de culturas de feijão, soja, algodão, batata, girassol, vêm sofrendo perda de até 70% ocasionada pelo fungo de solo denominado mofo-branco, da espécie Sclerotinia sclerotiorum, filamentoso e necrotrófico. Esse fungo na forma de escleródio apresenta-se como grânulo que se confunde com os grãos nas etapas de colheita e limpeza, podendo chegar ao consumidor final. Entretanto, ainda não são conhecidas as consequências do consumo desse fungo na forma de escleródios nos alimentos que são base da alimentação humana. Assim, a presente proposta visa o maior conhecimento da composição e aspectos toxicológicos desse fungo tão comum nos principais produtos agrícolas disponibilizados para o consumidor. Para identificação de solventes adequados à extração de compostos dos escleródios foram realizados testes cromatográficos que apontaram a escolha do acetato de etila para produção do extrato. Foram preparadas rações fúngicas contendo 6, 60 e 600 mg do extrato em 100g de ração, resultando num consumo de 25, 240 e 2600 mg de extrato por kg de peso corpóreo. No estudo in vivo, o consumo de ração fúngica não causou alterações nutricionais nos animais. O teste de cometa apresentou aumento no comprimento da cauda de cometas em 106,34; 174,77 e 131,90% para sangue; aumento de Tail Moment de 166%, 380% e 271% (linfócitos) e 660%, 639% e 429% (fígado) e aumento de 129%, 212% e 160% (linfócitos) e 284%, 296% e 260% (fígado) no % de DNA na cauda. Os testes de micronúcleos de medula óssea e cólon apresentaram aumento da frequência de micronúcleos em 186,95%; 147,82%; 239,13% para eritrócitos e 202,63; 173,68 e 223,68% em células do cólon. Este resultado também foi significativo para o teste de apoptose demonstrando aumento de 568,88, 457,77e 513,33% no numero de células apoptóticas. Estes resultados demonstram a ação mutagênica de escleródios do fungo Sclerotinia sclerotiorum, que podem ser desde a quebra do DNA, quanto a alterações nas fibras do fuso ou rearranjos cromossômicos associados ou não a fragmentação do DNA. Esses aspectos tornam os escleródios compostos que devem ser alvo de mais estudo sobre esses efeitos lesivos, uma vez que os mesmos podem chegar ao consumo alimentar de homens e animais por meio de alimentos e rações contaminadas, respectivamente. / The crop of soybean, beans, canola, cotton, peas, lettuce, potatoes, currently represent one of the most important agricultural activities in Brazil. These crops have been threatened, leading to losses of up to 70% of its total volume by the presence of soil fungus called popularly white mold or rot soil, the species Sclerotinia sclerotiorum. This fungal species is characteristic to be filamentous and cause necrosis in their hosts, and often find themselves in the granules form (sclerotia) which mix themselves with the seeds in the stages of harvesting and cleaning, thereby they can reach the final consumer in batches containing up to half of its grains contaminated by dormant mycelium of the fungus For identification of suitable solvents for extraction of compounds of sclerotia chromatographic tests were performed which indicated the choice of ethyl acetate to extract production. fungal diets containing 6, 60, and 600 mg of extract in 100 g of feed were prepared, resulting in a consumption of 25, 240 and 2600 mg of extract per kg body weight. In the in vivo study, the consumption of fungal feed caused no nutritional changes in animals. The comet test showed an increase in the length of comet tail 106,34; 174.77 and 131.90%. in blood; increase of Tail Moment 166%, 380% and 271% (lymphocytes) and 660%, 639% e 429% (liver) and increase of 129%, 212% and 160% (lymphocytes) and 284%, 296% and 260% (liver) in % of DNA in tail. Micronucleus tests in bone marrow and colon showed increased micronuclei frequency 186.95%; 147.82%; 239.13% to 202.63 and erythrocytes; 173.68 and 223.68% in colon cells. This result was also significant for apoptosis test showing increased 568.88, 513.33 457,77e% in the number of apoptotic cells. These results demonstrate the mutagenic action of sclerotia of Sclerotinia sclerotiorum, which may be from the DNA breakage, as changes in spindle fiber or chromosomal rearrangements associated or not with DNA fragmentation. These aspects render the compounds sclerotia that should be subject to further study on these damaging effects, as they can reach the food intake of humans and animals through contaminated food and feed, respectively.and the other half mixed with the sclerotia. / Fundação de Amparo à Pesquisa do Estado de Minas Gerais - FAPEMIG

Jasmonate sind wichtige zelluläre Mediatoren, die eine essentielle Rolle in der Pflanzen-entwicklung und Abwehr von biotischem und abiotischem Stress spielen. Jasmonsäure (JA) als zentrales Intermediat kann dabei auf unterschiedlichste Art und Weise metaboli-siert werden. Eine Möglichkeit der Metabolisierung ist die Veresterung der JA zu Methyljasmonat (MeJA) durch die Jasmonsäure-Carboxyl-Methyltransferase (JMT). Interessanterweise ist diese Reaktion reversibel, da die Methyljasmonatesterase (MJE) die Hydrolyse zu JA katalysiert. Obwohl die Funktion diverser Metabolite, wie die des biologisch aktivsten Metaboliten JA-Isoleucin, aufgeklärt wurde, ist die Rolle der reversiblen Methylierung noch weitestgehend unklar. Aufgrund der differenten physikochemischen Eigenschaften von JA und MeJA wird diskutiert ob MeJA von JA unterschiedliche biologische Eigenschaften aufweist und/oder als transzelluläres oder systemisches Signal fungiert. Anhand der Generierung transgener Pflanzen, in denen die Umwandlung zwischen JA und MeJA gestört ist, sollten diese Hypothesen überprüft werden. Dazu wurden Tomatenlinien hergestellt, die die Enzyme der reversiblen Methylierung (JMT, MJE) überexprimieren (JMT-OE, MJE-OE) sowie Linien, in denen durch ,,Post transcriptional gene silencing“ die MJE verringert exprimiert wird. Basierend auf MJE-RNAi-Linien, die eine reduzierte in vitro MeJA-Hydrolyseaktivität auf-weisen, sollte durch exogene Applikation von MeJA analysiert werden, ob MeJA selbst biologische Aktivitäten aufweist oder zunächst mittels MJE in JA umgewandelt werden muss. Es war kein reproduzierbarer Unterschied der Reaktion auf die MeJA-Applikation über die wässrige Phase und über die Gasphase zwischen Kontroll- und MJE-RNAi-Linien festzustellen. Um zu eruieren, ob MeJA als transzelluläres, parakrines Signal agiert, wurden die Gen-expression und das Oxylipinprofil in verwundeten Blättern der transgenen Linien unter-sucht. Ob MeJA als systemisches Signal dient, sollte anhand der Verwundung von Pfropfkombinationen aus Kontrolllinie als Unterlage und MJE-RNAi als Spross anhand der Analyse der Genexpression in distalen Bereichen festgestellt werden. Die Ergebnisse der Studien deuten daraufhin, dass MeJA weder als transzelluläres Signal noch als sys-temisches Signal bei der Verwundungsantwort agiert. Zudem kann ausgeschlossen wer-den, dass die MJE in distalen Bereichen der Pflanze in der Perzeption des systemischen Signals involviert ist. Da Jasmonate an der Abwehr von Pathogenen beteiligt sind, wurde die Empfindlichkeit gegenüber dem nekrotrophen Pilz S. sclerotiorum getestet. Alle transgenen Linien mit Veränderung der reversiblen Methylierung wiesen erhöhte Läsionsgrößen, stärkeres Pilzwachstum und erhöhte JA-Isoleucin und 12-OH-JA-Isoleucin Spiegel auf. Allerdings war keine Korrelation zwischen den erhöhten JA-Isoleucin Spiegeln und der Expression von Abwehrgenen wie PINII und JA-Biosyntheseenzymen wie AOC zu verzeichnen. Somit scheint die reversible Methylierung eine Rolle in der Pathogenabwehr gegenüber S. sclerotiorum zu spielen jedoch sind die Mechanismen, die zur erhöhten Suszeptibilität der Linien führen, noch unklar. / Jasmonates are essential cellular mediators that play an important role in development and defense of biotic and abiotic stress stimuli. Jasmonic acid (JA) as central intermedi-ate can be metabolised in different ways. One possibility is the esterification of JA via jasmonic acid carboxyl methyltransferase (JMT) to methyl jasmonate (MeJA). Interest-ingly, this reaction is reversible as methyl jasmonate esterase (MJE) is able to hydrolyse MeJA into JA. Although the function of several JA metabolites such as JA-isoleucine, the biological most active one was elucidated, the in vivo function of reversible methylation is still unknown. Because JA and MeJA harbour different physicochemical properties, MeJA is assumed to function as transcellular and/or even systemic signal. This hypothe-sis was addressed by the analysis of transgenic plants disturbed in JA and MeJA conver-sion. Tomato plants overexpressing the enzymes of the reversible methylation as well as transgenic plants with reduced MJE expression induced via post transcriptional gene silencing of MJE were generated. Based upon MJE-RNAi-lines with reduced MeJA in vitro cleaving activity, it was analysed if MeJA is biologically active per se or if it only exerts its effects after hydrolysis to JA. There was no reproducible difference between control line and MJE-RNAi-line observed in response to MeJA application by vapor and liquid phase. To evaluate the role of MeJA as transcellular or paracrine signal, wounded leaves of all transgenic lines were analysed with respect to changes in gene expression and oxylipin profile. To analyse if MeJA may act as a systemic signal, wounding experiments with grafting combinations of wildtype stock and MJE-RNAi scion were performed and distal gene expression was observed. The experimental results suggest that MeJA acts neither as transcellular nor as systemic signal. Furthermore, it can be excluded that MJE is involved in the perception of the systemic signal in distal tissues of the plant. Because jasmonates are involved in pathogen defense, the susceptibility against the necrotrophic pathogen S. sclerotiorum was also tested. All transgenic lines with alterations in reversi-ble methylation exhibit a higher lesion diameter, enhanced growth of fungi and higher JA-isoleucine and 12-OH-JA-isoleucine levels. However, there was no correlation be-tween high JA-isoleucine levels and expression of defense genes such as PINII and JA-biosynthesis genes, e.g. AOC. Hence, reversible methylation seems to be involved in the pathogen response towards S. sclerotiorum but the mechanisms responsible for the higher susceptibility are still unknown.

Thesis (M. App. Sc.)--University of Queensland, 2001. / Includes bibliographical references.

No description available.

Sclerotinia sclerotiorum is a widespread plant pathogen that produces structures known as sclerotia. When sclerotia germinate they give rise to infective hyphae, myceliogenic germination, or they produce ascocarps, carpogenic germination. Biological control has usually targeted sclerotia or ascospores. The main objectives of the research presented herein were to observe the effect of a mycoparasite and fungus gnats (Bradysia coprophila) on the survival of sclerotia in vitro and in field conditions, and to study the enzymatic activity of the mycoparasite when in contact with sclerotia damaged by fungus gnats. / In this research several mycoparasites were evaluated for their efficacy to degrade sclerotia in soil. From these tests, an isolate of Trichoderma hamatum, TMCS 3 proved to be the most effective. Larvae of fungus gnats have also been reported to feed on sclerotia. When both organisms were combined in laboratory tests, fewer sclerotia survived than when the organisms acted alone. Sclerotia recovered from this treatment contained fewer viable cells when compared to sclerotia recovered from treatments with TMCS 3 or fungus gnats alone. The results obtained from field trials showed that TMCS 3 was effective at degrading sclerotia. Unfortunately environmental conditions were not always optimal for the establishment of high populations of fungus gnats. Few larvae were observed feeding on sclerotia and no significant differences were found among treatments. / Growth of TMCS 3 was studied using different carbon sources as substrates, including sclerotia of S. sclerotiorum. Biomass obtained from this latter treatment was significantly larger than on the other carbon sources tested. Enzymatic activity was also induced by the presence of sclerotia. In many cases, sclerotial exudates from mechanically damaged sclerotia or sclerotia damaged by larval feeding showed that the concentration of amino acids, carbohydrates, and electrolytes was increased as damage to the sclerotia has increased. Exudation of protein was not different when damaged and undamaged sclerotia were compared. Exudates from sclerotia with the melanized rind completely removed by fungus gnats feeding accelerated the germination of conidia of TMCS 3. These heavily damaged sclerotia also enhanced the growth of TMCS 3 when both organisms were grown together. However enzymatic (i.e. glucanase and chitinase) activity of TMCS 3 was not increased by the damage to the sclerotia. When damaged sclerotia were buried in soil infested with TMCS 3 they were degraded faster when the medulla of sclerotia was completely uncovered by larval feeding.

The fungal pathogen Sclerotinia sclerotiorum (Lib.) de Bary is capable of infecting over 400 plant species including canola (Brassica napus L.). The fungus secretes oxalic acid (OA), which plays an important role in infection and disease progression. An analysis of proteome-level changes associated with infection of susceptible canola leaves by S. sclerotiorum revealed significant changes in the abundance of 32 proteins, including proteins involved in photosynthesis and metabolism, hormone signaling, and antioxidant defense. A similar subset of 37 proteins was affected when leaves were treated with OA alone; this compound also caused a reduction in the activities of a number of antioxidant enzymes, suggesting an OA-mediated suppression of the oxidative burst. To further understand the mechanisms of pathogenesis, the role of Sssp, a predicted secreted protein from S. sclerotiorum, was targeted for analysis. Mutant strains of S. sclerotiorum were generated by disruption of the Sssp gene and characterized for virulence on canola. Based on the extent of symptom development, the virulence of the Sssp-disrupted mutants was significantly reduced relative to the wild-type, indicating that Sssp may play a role in the infection process. Finally, the development of sclerotia, long-term survival structures that serve as a primary source of inoculum for the fungus, was examined. A total of 88 proteins were found to exhibit temporal changes in abundance during sclerotium formation and maturation, including proteins involved in the regulation of melanogenesis. A total of 56 proteins were also identified in the sclerotial exudates, providing a basis for future studies. Collectively, the studies described in this dissertation represent the most comprehensive proteome-level analysis of the canola/S. sclerotiorum interaction and sclerotial development, and could contribute to the development of novel strategies for the management of S. sclerotiorum. / Plant Science

Universiẗat, Diss., 2003--Bonn.

No description available.