Na cultura da soja, a ocorrência de doenças fúngicas como o mofo branco e a ferrugem asiática, tem reduzido a produtividade de grãos significativamente nos últimos anos. Esta pesquisa objetivou: a) avaliar o potencial de cruzamentos de soja para a obtenção de progênies com boas características agronômicas, com ênfase em produtividade, resistência ao mofo branco e tolerância à ferrugem; b) avaliar a reação ao fungo causador do mofo branco nos genótipos estudados e c) avaliar a tolerância à ferrugem em gerações precoces por meio da estimação do efeito ferrugem. O material genético foi oriundo de um dialelo parcial, com 50 cruzamentos, entre o grupo I (dez genitores representados por linhagens experimentais (USP) com alta produtividade de grãos) e o grupo II (cinco genitores - com indicações na literatura de possível resistência ao mofo branco). Em 2012/13, dois experimentos foram conduzidos, sendo o primeiro com os 50 cruzamentos (geração F2) e três testemunhas comuns e o segundo envolvendo os 15 genitores e as mesmas três testemunhas. Em 2013/14 e 2014/15, a fim de se estimar o efeito ferrugem (nível de tolerância) por meio da diferença (perda) entre as médias de produtividade de grãos (PG) e tamanho das sementes (peso de cem sementes ou PCS), os experimentos com os cruzamentos (gerações F3 e F4) e os genitores foram esquematizados em quatro experimentos (dois manejos para cruzamentos e dois manejos para genitores): manejo O&P, com aplicações de fungicidas para o controle da ferrugem e outras doenças fúngicas; manejo D, com aplicações de fungicida para controle de doenças fúngicas, exceto a ferrugem. Separadamente, dez plantas F4 de cada cruzamento e os genitores foram avaliados quanto à reação ao mofo branco utilizando o método da inoculação de folhas destacadas, a fim de se estimar a área abaixo da curva de progresso da doença. De modo geral, para todas as gerações avaliadas foi observada superioridade de 2,2% para PG dos cruzamentos em relação aos genitores. As perdas médias dos genótipos na presença da ferrugem foram 2,1% e 5,5% para PG e PCS, respectivamente. Considerando a PG, a resistência ao mofo branco e a tolerância à ferrugem, os cinco melhores cruzamentos foram: 111 (USP70.006 x MSOY6101), 131 (USP70.080 x MSOY6101), 133 (USP70.080 x A4725RG), 113 (USP70.006 x A4725RG) e 126 (USP 70.057 x MSOY 6101). Os melhores genitores foram A4725RG e USP70.080. Como padrões de resistência ao mofo branco foram identificados os cruzamentos 142 (USP70.109 x PI153.282) e 111 (USP70.006 x MSOY6101) e os genitores MSOY6101 e Caiapônia; e de suscetibilidade, os cruzamentos 114 (USP70.006 x EMGOPA313) e 135 (USP70.080 x Caiapônia) e os genitores USP14-01-20 e USP70.004. Além disso, as seguintes conclusões foram obtidas: a) as análises dialélicas foram eficazes em identificar populações F2 de maior potencial de ganho genético na seleção; b) a estratégia de se usar diferentes tipos de fungicidas mostrou-se eficiente para estimar a reação das plantas à ferrugem, em termos de PG e PCS; c) a metodologia da folha destacada foi útil em diferenciar os genótipos quanto à resistência das plantas ao mofo branco. / In soybean, the occurrence of fungal diseases such as white mold and Asian rust has reduced seed yield significantly over the past years. This work aimed to: a) assess the potential of soybean crosses to obtain progenies with good agronomic traits, with emphasis on seed yield, white mold resistance and tolerance to rust; b) assess the reaction of genotypes to Sclerotinia sclerotiorum, fungus that cause white mold; c) assess tolerance to rust in early generations through methods of estimation of the rust effect. The genetic material was derived from a partial diallel with 50 crosses between the group I (ten parents - experimental lines, USP, with high seed yield), and group II (five parents - with evidences from the literature of certain resistance to white mold). In 2012/13, two experiments were conducted, the first with 50 crosses (F2) and three common checks; and, the second one involving the 15 parents and the same three checks. In the 2013/14 and 2014/15, in order to estimate the rust effect (tolerance level) by the difference (loss) between the seed yield (PG) and one hundred-seed weight (PCS), experiments with crosses (F3 and F4 generations) and parents were designed in four experiments (two disease managements to crosses and two disease managements to parents); management O&P, with applications of fungicides to control rust and other fungal diseases; management D, with applications of one fungicide for controlling the late season leaf diseases, except rust. Separately, ten F4 plants of each cross and the parents were evaluated for resistance to white mold using the method of inoculation in detached leaves in order to estimate the area under the disease progress curve. In general, for all evaluated generations it was observed superiority of 2,2% in the PG of the crosses, related to the parents. The average loss of PG was 2,1% on the genotypes under study in the presence of rust and the average loss in seed size was 5,5%. Considering the resistance to white mold, tolerance rust and high means of PG, the five best crosses were: 111 (USP 70.006 x MSOY6101), 131 (USP 70.080 x MSOY6101), 133 (USP 70 .080 x A4725RG), 113 (USP 70.006 x A4725RG) and 126 (USP 70.057 x MSOY 6101). The best parents were A4725RG and USP 70.080. As resistance standards to white mold were identified the crosses 142 (USP 70.109 x PI 153282) and 111 (USP 70.006 x MSOY6101) and the parents MSOY6101 and Caiapônia; Susceptibility standards were the crosses 114 (USP 70.006 x EMGOPA 313) and 135 (USP 70.080 x Caiapônia) and the parents USP14-01-20 and USP 70.004. In addition, the following conclusions were obtained a) the diallel analyzes were effective in identifying F2 populations with higher potential for selection gain; b) the strategy of using different managements of fungicides was efficient to estimate the response of plants to rust in terms of PG and PCS; c) the methodology of detached leaf was useful in differentiating the genotypes for resistance to white mold.

Expanding global populations, unequal food distribution and disease pressure suggest food poverty is increasing. Consequently, much attention is focussed on alternative natural methods in which to increase agricultural yield. Previously, it was observed that Trichoderma hamatum strain GD12 and its respective N-acetyl-β-D-Glucosamine mutant ∆Thnag:hph promoted plant biomass and fitness that, as a result, may provide a credible natural alternative to synthetic fertilisers. However, on a molecular level, the manner in which this is achieved has not been fully elucidated. In this thesis, I report the biofertiliser effect of GD12 and mutant ∆Thnag::hph once applied to autoclaved peat microcosms as sole applications. Furthermore, I demonstrate the biocontrol ability of GD12 when co-inoculated with Sclerotinia sclerotiorum or Rhizoctonia solani and reveal, that once mycelium co-inoculation has occurred, GD12 increase plant biomass and provide protection; whilst ∆Thnag::hph does not. Consequently, I challenged the biocontrol effects of Trichoderma metabolite extract where I validate that both Trichoderma wild type GD12 and mutant ∆Thnag::hph are incapable of suppressing pathogen growth. Subsequently, I characterised the up-regulated signatures associated with GD12 and ∆Thnag::hph using LC-MS techniques where unique compounds were discovered from each strain of Trichoderma. In conclusion, I provide evidence that N-acetyl-β-D-Glucosamine mutation bring about metabolomic changes that affect the fungal secretome which, in turn, alters plant phenotype, fitness and germination. Furthermore, I have shown that these effects are species specific and depend upon pathogen, plant and fungal properties. However, further investigations are needed to fully elucidate the compound(s) responsible for biocontrol and biofertilisation; especially plant-specific effects that take place as a consequence of fungal activity.

Canola/rapeseed (Brassica napus L.) is a major oilseed crop worldwide. However, its production is largely affected by the fungal disease Sclerotinia stem rot as well as seed glucosinolates. So far the genetic mechanisms controlling these two traits have been poorly understood. In the present study, three bi-parental doubled haploid B. napus populations M730, M692 and ZT were grown in either natural or artificial environments and genotyped using the Brassica 60K Infinium® SNPs and/or sequence related amplified polymorphisms. Three genetic linkage maps covered 2,597.7 cM, 2,474.1 cM and 1,731.6 cM in 19 chromosomes for M730, M692 and ZT, respectively. Plants were inoculated with Sclerotinia sclerotiorum mycelia on stems at the reproductive stage to evaluate their resistivity. Four aliphatic glucosinolates and one indolic glucosinolate were detected in the seeds using high-performance liquid chromatography. 4-hydroxy-3-indolylmethyl predominated over aliphatic glucosinolates in canola, but inversely constituted a small portion of total glucosinolate content in semi-winter rapeseed. In rapeseed, 2-hydroxy-3-butenyl predominated in 4C aliphatic glucosinolates, which in turn predominated in total aliphatic glucosinolates, which likewise predominated in total glucosinolate content. QTLs regulating major glucosinolates were located on chromosome A9 for high glucosinolate content populations M730 and ZT, and on chromosome C7 for low glucosinolate content population M692. Major QTLs for Sclerotinia stem rot resistance were located on chromosomes A7 and C6 in M730, on chromosomes A3 and A7 in ZT, while no major QTLs were found in M692. Additive genetic effect was the major factor explaining phenotypic variations of the two traits. No direct genetic relationship was observed between Sclerotinia stem rot resistance in adult plants and seed glucosinolates in B. napus. The findings in the studies could be used to formulate breeding and research strategies in B. napus and the major QTLs controlling the two traits and their closely linked SNP markers could be validated over wide germplasm and used in marker assisted selection. / October 2016

This thesis investigates two aspects of phytoalexin metabolism by the phytopathogenic fungus <i>Sclerotinia sclerotiorum</i> (Lib) de Bary: (i) determination of detoxification pathways of structurally different molecules; (ii) design and synthesis of potential inhibitors of enzyme(s) involved in detoxification steps.<p>First, the transformations of important cruciferous phytoalexins by the economically important stem rot fungus, <i>S. sclerotiorum</i>, were investigated. During these studies a number of new metabolic products were isolated, their chemical structures were determined using spectroscopic techniques, and further confirmed by synthesis. The metabolic products did not show detectable antifungal activity against <i>S. sclerotiorum </i> which indicated that these metabolic transformations were detoxification processes. Overall, the results of these transformations suggested that <i>S. sclerotiorum</i> produces various enzymes that can detoxify cruciferous phytoalexins via different pathways. While the detoxifications of strongly and moderately antifungal phytoalexins such as brassilexin, sinalexin, and 1-methoxybrassinin were fast and led to glucosylated products, the transformations of the weakly antifungal phytoalexins brassicanal A, spirobrassinin and 1-methoxyspirobrassinin were very slow and yielded non-glucosylated compounds.<p>Next, the design of potentially selective inhibitors of the brassinin detoxification enzyme, BGT, was sought. Two sets of potential inhibitors of BGT were designed: (i) a group was based on the structure of brassinin, where the indole ring of brassinin was replaced with benzofuran, thianaphthene, 7-azaindole and pyrazolo[1,5-a]pyridine and/or the position of side chain was changed from C-3 to C-2; and (ii) another group based on the structure of camalexin where the thiazole ring of camalexin was replaced with a phenyl group. The syntheses and chemical characterization of these potential detoxification inhibitors, along with their antifungal activity, as well as screening using fungal cultures and cell-free extracts of <i>S. sclerotiorum</i>, were examined. The results of these screening indicated that 3-phenylindoles, 3-phenylbenzofuran, 5-fluorocamalexin, methyl (indol-2-yl)methyl-dithiocarbamate, methyl (benzofuran-3-yl)methyldithiocarbamate and methyl (benzo-furan-2-yl)methyldithiocarbamate could slow down the rate of detoxification of brassinin in fungal cultures and also in cell-free extracts of <i>S. sclerotiorum</i>. Among the designed compounds, 3-phenylindole appeared to be the best inhibitor both in fungal cultures and in cell-free extracts. Metabolism studies of all the designed compounds using fungal cultures of <i>S. sclerotiorum</i> indicated that they were metabolized by <i>S. sclerotiorum</i> to glucosyl derivatives, although at much slower rates.<p>It is concluded that some inhibitors that can slow down the rate of metabolism of brassinin could be good leading structures to design more active inhibitors of BGT.

This thesis investigates two aspects of phytoalexin metabolism by the phytopathogenic fungus <i>Sclerotinia sclerotiorum</i> (Lib) de Bary: (i) determination of detoxification pathways of structurally different molecules; (ii) design and synthesis of potential inhibitors of enzyme(s) involved in detoxification steps.<p>First, the transformations of important cruciferous phytoalexins by the economically important stem rot fungus, <i>S. sclerotiorum</i>, were investigated. During these studies a number of new metabolic products were isolated, their chemical structures were determined using spectroscopic techniques, and further confirmed by synthesis. The metabolic products did not show detectable antifungal activity against <i>S. sclerotiorum </i> which indicated that these metabolic transformations were detoxification processes. Overall, the results of these transformations suggested that <i>S. sclerotiorum</i> produces various enzymes that can detoxify cruciferous phytoalexins via different pathways. While the detoxifications of strongly and moderately antifungal phytoalexins such as brassilexin, sinalexin, and 1-methoxybrassinin were fast and led to glucosylated products, the transformations of the weakly antifungal phytoalexins brassicanal A, spirobrassinin and 1-methoxyspirobrassinin were very slow and yielded non-glucosylated compounds.<p>Next, the design of potentially selective inhibitors of the brassinin detoxification enzyme, BGT, was sought. Two sets of potential inhibitors of BGT were designed: (i) a group was based on the structure of brassinin, where the indole ring of brassinin was replaced with benzofuran, thianaphthene, 7-azaindole and pyrazolo[1,5-a]pyridine and/or the position of side chain was changed from C-3 to C-2; and (ii) another group based on the structure of camalexin where the thiazole ring of camalexin was replaced with a phenyl group. The syntheses and chemical characterization of these potential detoxification inhibitors, along with their antifungal activity, as well as screening using fungal cultures and cell-free extracts of <i>S. sclerotiorum</i>, were examined. The results of these screening indicated that 3-phenylindoles, 3-phenylbenzofuran, 5-fluorocamalexin, methyl (indol-2-yl)methyl-dithiocarbamate, methyl (benzofuran-3-yl)methyldithiocarbamate and methyl (benzo-furan-2-yl)methyldithiocarbamate could slow down the rate of detoxification of brassinin in fungal cultures and also in cell-free extracts of <i>S. sclerotiorum</i>. Among the designed compounds, 3-phenylindole appeared to be the best inhibitor both in fungal cultures and in cell-free extracts. Metabolism studies of all the designed compounds using fungal cultures of <i>S. sclerotiorum</i> indicated that they were metabolized by <i>S. sclerotiorum</i> to glucosyl derivatives, although at much slower rates.<p>It is concluded that some inhibitors that can slow down the rate of metabolism of brassinin could be good leading structures to design more active inhibitors of BGT.

No description available.

No description available.

Allium white rot (AWR) is a soilborne disease that seriously damages commercial cultivation of onion (Allium cepa) and garlic (Allium sativum) crops. The disease has been found everywhere onions are cultivated and at present no system of control has been found that fully prevents the occurrence of the disease. The fungus responsible for the disease, Sclerotium cepivorum, uses oxalic acid to kill Allium bulb and root tissue in growing onion and garlic plants. Research suggests recombinant oxalate oxidase and oxalate decarboxylase enzymes may be able to degrade this acid and confer resistance against pathogens that rely on it, such as Sm. cepivorum or Sclerotinia sclerotiorum. To test the efficacy of these enzymes against white rot pathogens, three transgenes for wheat oxalate oxidase, barley oxalate oxidase and Flammulina oxalate decarboxylase were transformed into onions and garlic by Agrobacterium-mediated transformation. Allium species are highly recalcitrant to transformation, so these three transgenes were also transformed into tobacco to provide fast-recovering, easy to test transformants to assess the efficacy of the transgenes. Transformed garlic and tobacco lines were analysed to assess the integration and expression of the transgenes, then challenged with Sm. cepivorum or Sa. sclerotiorum, respectively, to assess the bioactivity of recombinant wheat oxalate oxidase, barley oxalate oxidase, and Flammulina oxalate decarboxylase against oxalic acid-dependent pathogens. Results show that one line of tobacco expressing the Flammulina oxalate decarboxylase enzyme was found to be consistently resistant to Sclerotinia sclerotiorum. Garlic lines transformed with this transgene failed to display stable transgene expression or disease resistance, possibly due to silencing of the transgene in recovered transformant tissue.

Soybean (Glycine max (L.) Merr.) is one the major crops produced worldwide. However, soybean is susceptible to many diseases. Sclerotinia stem rot (SSR) disease caused by Sclerotinia sclerotiorum (Lib.) de Bary is considered one of the most important fungal diseases of soybean. It can be controlled by chemicals (e.g. fungicides), by breeding cultivars with disease resistance and by cultural control (e.g. increasing the width between rows, reducing plant populations). A promising and complementary method of controlling SSR disease in the field is the application of biological control agents. Biological control agents introduced in a soil environment will interact with other soil food web organisms, as do the pathogenic organism and infected plants, which may change the genetic and functional diversity in soil microbial communities. Profiling these changes may lead to an improved understanding of the interactions between these players (biological control agents, pathogens, soil biota and plants) in the biological control phenomenom, permiting us to exploit naturally-occurring ecological relationships and develop more sustainable approaches to control soybean diseases. Fatty acid biomarkers analysis was used to profile microbial communities in soils. Two laboratory studies were conducted to evaluate the methods used for extraction and profiling the fatty acid biomarkers from soil samples with a range of soil properties (clay content, organic matter content), The first study investigated the best solvent mixture for recovering fatty acid biomarkers from soil using an automated pressurized solvent extraction (PSE) system. Solvent mixtures containing chloroform and methanol were more efficient at extracting fatty acids from agricultural soils than hexane:2-propanol and acetone. The second study presented an exploratory pyrolysis-mass spectrometry technique to rapidly fingerprint soil lipids extracted from different agroecosystems. Pyrolysis-mass spectrometry discriminated among soils and crop production systems in the same way as the fatty acid profiling. I also report on the efficicacy of biological control agents to control Sclerotinia stem rot disease in soybean. A two-year study was conducted in soybean fields under conventional or no tillage to determine whether Trichoderma virens (SoilGard(TM)) and arbuscular mycorrhizal fungi (a mixture of Glomus intraradices and G. mosseae ), used alone or in combination, could reduce sclerotinia stem rot (SSR) disease incidence. Generally, SSR disease indicators, as well as the soybean yield, were not affected significantly by the biological control treatments. I then studied whether changes in microbial community composition were related to the inoculation of the biological control agents and the disease incidence in soybean fields. Inoculation of biological control agents changes the expression of many soil fatty acids during both years of the trial. Also, in the plots with severely diseased plants, fatty acids biomarkers of gram positive and actinomycetes bacteria were significantly greater than in plots with healthy plants. I conclude that further improvement in laboratory techniques and procedures will permit researchers to efficiently extract and characterize soil lipids, providing new insight into soil organic matter dynamics and soil microbial ecology. Further study will be needed to verify the efficacy and optimize the application method, dose and timing of biocontrol agents to provide protection against SSR disease in soybean fields.

Allium white rot (AWR) is a soilborne disease that seriously damages commercial cultivation of onion (Allium cepa) and garlic (Allium sativum) crops. The disease has been found everywhere onions are cultivated and at present no system of control has been found that fully prevents the occurrence of the disease. The fungus responsible for the disease, Sclerotium cepivorum, uses oxalic acid to kill Allium bulb and root tissue in growing onion and garlic plants. Research suggests recombinant oxalate oxidase and oxalate decarboxylase enzymes may be able to degrade this acid and confer resistance against pathogens that rely on it, such as Sm. cepivorum or Sclerotinia sclerotiorum. To test the efficacy of these enzymes against white rot pathogens, three transgenes for wheat oxalate oxidase, barley oxalate oxidase and Flammulina oxalate decarboxylase were transformed into onions and garlic by Agrobacterium-mediated transformation. Allium species are highly recalcitrant to transformation, so these three transgenes were also transformed into tobacco to provide fast-recovering, easy to test transformants to assess the efficacy of the transgenes. Transformed garlic and tobacco lines were analysed to assess the integration and expression of the transgenes, then challenged with Sm. cepivorum or Sa. sclerotiorum, respectively, to assess the bioactivity of recombinant wheat oxalate oxidase, barley oxalate oxidase, and Flammulina oxalate decarboxylase against oxalic acid-dependent pathogens. Results show that one line of tobacco expressing the Flammulina oxalate decarboxylase enzyme was found to be consistently resistant to Sclerotinia sclerotiorum. Garlic lines transformed with this transgene failed to display stable transgene expression or disease resistance, possibly due to silencing of the transgene in recovered transformant tissue.