Submitted by Marlene Santos (marlene.bc.ufg@gmail.com) on 2017-09-29T19:13:24Z No. of bitstreams: 2 Dissertação - William Andrés López Arboleda - 2015.pdf: 1579315 bytes, checksum: 378fd0e65f3b2919d56630143950ed75 (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) / Approved for entry into archive by Luciana Ferreira (lucgeral@gmail.com) on 2017-10-02T13:35:40Z (GMT) No. of bitstreams: 2 Dissertação - William Andrés López Arboleda - 2015.pdf: 1579315 bytes, checksum: 378fd0e65f3b2919d56630143950ed75 (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) / Made available in DSpace on 2017-10-02T13:35:40Z (GMT). No. of bitstreams: 2 Dissertação - William Andrés López Arboleda - 2015.pdf: 1579315 bytes, checksum: 378fd0e65f3b2919d56630143950ed75 (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) Previous issue date: 2015-03-27 / Sclerotinia sclerotiorum is a phytopathogenic fungus that infects more than 400 plant species, including common bean. Genetic variability studies in connection with phenotypic traits of agronomic interest are important to drive the control strategies against this pathogen. The aims of this study were: to evaluate the genetic variability, fungicide sensitivity, aggressiveness and to determine the proportion of MAT (Mating Type) alleles of 79 isolates of S. sclerotiorum distributed in four populations from common bean. Two populations represented a single sampling location in two different times (2000 and 2013 growing seasons). To evaluate the fungicide sensitivity a cell viability test based on the alamarBlue dye using mycelial growth was standardized. Dose-response curves for fluazinam, procymidone and benomyl were estimated using this test and were compared with dose-response curves estimated by the mycelial growth inhibition on PDA plate and the FRAC (Fungicide Resistance Action Committee) protocol. Despite the differences to assess the fungicide sensitivity between the three methods, the dose-response curves showed similar trends for the three fungicides. The fungicide sensitivity assessment at the four populations showed low sensitivity to benomyl in the Planaltina population. Furthermore, this population presented a principally clonal population structure, with a haplotype represented by 18 out of 20 isolates. Significant population differentiation in all pairwise comparisons of phi, except the comparison between EV_2013-NH, was detected. Five genetically homogeneous groups were inferred by the DAPC analysis. No group was conformed by isolates from the four populations. Only two haplotypes between the two populations from the same sampling location were shared. The hypothesis of random mating was rejected at the four populations; however this hypothesis was not rejected at the two major populations inferred by the DAPC analysis. The screening of mating type locus showed a dominance of Inv+ isolates and a high proportion of Inv+/Inv- isolates (presumable heterokaryons). / Sclerotinia sclerotiorum é um fungo fitopatogênico capaz de colonizar mais de 400 hospedeiras, sendo o agente causal do mofo branco no feijoeiro. Estudos de variabilidade genética associados a características fenotípicas de interesse agronômico, como a sensibilidade a fungicidas, oferecem informações importantes para direcionar estratégias de controle sobre este patógeno. Os objetivos deste trabalho foram avaliar a variabilidade genética, sensibilidade a fungicidas e agressividade de 79 isolados de S. sclerotiorum distribuídos em quatro populações procedentes de culturas de feijoeiro em pivô central. Duas destas populações representaram um único local de coleta em duas épocas diferentes (2000 e 2013). Para avaliar a sensibilidade a fungicidas foi padronizado um teste de viabilidade celular baseado no corante alamarBlue® sobre o crescimento micelial em microplaca de 96 poços. Curvas de dose-resposta para os fungicidas fluazinam, procimidona e benomyl, usando um isolado de S. sclerotiorum, foram estimadas com este método, e comparadas com curvas de dose-resposta obtidas com os métodos de inibição do crescimento em placa e o proposto pelo Fungicide Resistance Action Committee (FRAC). Apesar das diferentes abordagens as curvas dose-resposta mostraram tendências semelhantes para os três fungicidas. A avaliação da sensibilidade a fungicidas nas quatro populações indicou uma alta insensibilidade ao benomyl na população de Planaltina. Por outro lado, a estrutura populacional foi principalmente clonal com um haplótipo representado por 18 dos 20 isolados desta população. Diferenciação populacional significativa foi detectada em todas as comparações par a par do phi, com a exceção da comparação EV-2013-NH. A analise DAPC identificou cinco grupos geneticamente homogêneos. Nenhum dos grupos esteve constituído por isolados das quatro populações. Só dois haplótipos foram compartilhados pelas populações EV_2000 e EV_2013. A hipótese de acasalamento aleatório foi rejeitada nas quatro populações, no entanto não foi rejeitada nas duas maiores populações sugeridas pelo DAPC. O screening do Mating type locus (MAT) mostrou uma prevalência de isolados Inv+ e uma alta proporção de isolados Inv+/Inv-

O mofo-branco causado pelo fungo Sclerotinia sclerotiorum se encontra amplamente disperso pelas áreas de produção em todo pais e afeta mais de 400 espécies de planta, causando perdas na produção e redução na qualidade. O controle da doença é considerado difícil, pois o patógeno forma escleródios (estruturas de sobrevivência), que podem permanecer viáveis por vários anos. Normalmente exige-se um manejo integrado, que envolve o controle químico, biológico e diversas práticas culturais, uma vez que cultivares resistentes não estão disponíveis. As adoções de estratégias sustentáveis de manejo do mofo branco dependem da compreensão do patógeno, sua etiologia, morfologia, agressividade e dinâmica populacional. Foram avaliados 150 isolados, coletados em cultivos de cenoura, couve, girassol, nabo, soja e feijão. O índice de velocidade de crescimento micelial (IVCM), a coloração da colônia e densidade do micélio, o tempo necessário para a formação do primeiro escleródio, o número de escleródios por placa, o peso em mg e a forma dos escleródios foram utilizados na caracterização dos isolados. O critério adotado nas avaliações da agressividade dos isolados foi o diâmetro das lesões após diversos intervalos de tempo, usando o método da folha destacada, em feijão e soja. Os valores foram usados para calcular a área abaixo da curva de progresso de doença. A população avaliada mostrou ampla variabilidade para as características morfológicas, fisiológicas e na agressividade. O controle químico é a estratégia mais utilizada no controle do mofo branco. No Brasil, onze ingredientes ativos estão registrados, no entanto, três são mais utilizados: fluazinam, procimidona e tiofanato metílico. O uso intensivo de fungicidas, especialmente os que agem em um unico sitio de ação, podem selecionar isolados resistentes e, consequentemente, levar a falhas de controle. A avaliação da sensibilidade de S. sclerotiorum para os fungicidas mais utilizados é fundamental para monitorar o comportamento da população. Foram avaliadas a sensibilidade de isolados brasileiros de Sclerotinia sclerotiorum a fluazinam, procimidona e tiofanato metílico, atraves de concentrações discriminatórias e valores de CI50 (concentração do fungicida que inibe em 50% o crescimento micelial). Um total de 150 isolados foi testado e nenhum se mostrou resistente a fluazinam ou procimidona. O valor da CI50 para fluazinam variou de 0,00173 ppm a 0,01284 ppm, com média de 0,007 ppm. Para procimidona, a CI50 desses isolados variou de 0,12223 ppm a 0,35916 ppm, com média de 0,21834 ppm. Foram encontrados sete isolados resistentes ao tiofanato metílico, com CI50 variando de 990-2667 ppm. Na população sensível o valor variou de 0,49 - 3,73 ppm. Foi verificada a adaptabilidade dos isolados resistentes, e esta se mostrou comparável a dos isolados sensíveis, sugerindo que os isolados possuem aptidão parasitária suficiente para competir com os isolados sensíveis em condições de campo. / The white mold caused by the fungus Sclerotinia sclerotiorum is widely dispersed in production areas in the whole country and affects more than 400 species, causing production losses and reduced quality. Disease control is considered difficult because the pathogen form sclerotia (survival structure) which can remain viable for several years. Typically this requires a integrated pest management, which involves chemical, biological control and various cultural practices, since resistant cultivars are not available. The adoption of sustainable management strategies for white mold depends on understanding the pathogen, its etiology, morphology, aggressiveness and population dynamics. We evaluated 150 isolates collected from carrots, cabbage, sunflower, turnip, soybeans and beans. Mycelial growth rate index (MIG), colony color and mycelium density, time required to form the first sclerotia, the number of sclerotia per plate, the weight in mg and sclerotia shape were used for the isolates characterization. The criteria used in the isolates aggressiveness evaluations were lesions diameter after various time intervals, using the detached leaf method in bean and soybean. The values were used to calculate the area under the disease progress curve (AUDPC). The population evaluates showed wide variability for morphological, physiological and aggressive characteristics. Chemical control is the most commonly strategy used in the control of white mold. In Brazil, eleven ingredients active are registered, however, three are most commonly used, and they are: fluazinam, procymidone and thiophanate-methyl. The intensive use of fungicides, especially those which act on at a single site of action, can select resistant isolates, and consequently lead to failure control. The evaluation of the sensitivity of S. sclerotiorum to the most commonly used fungicides is critical to monitor the behavior of the population. We assessed the sensitivity of Brazilian Sclerotinia sclerotiorum isolated to fluazinam, procymidone and thiophanate-methyl, through discriminatory concentrations and IC50 values (the fungicide concentration which inhibits 50% of the mycelial growth). A total of 150 strains have been tested, and none were resistant to fluazinam or procymidone. The IC50 value for fluazinam ranged from 0.00173 to 0.01284 ppm ppm, with an average of 0.007 ppm. For procymidone, the IC50 of these isolates ranged from 0.12223 to 0.35916 ppm ppm, averaging 0.21834 ppm. Seven isolates were found resistant to thiophanate-methyl with IC50 ranging from 990-2667 ppm. In sensitive people the value ranged from 0.49 to 3.73 ppm. Adaptability of resistant isolates was observed, and this proved to be comparable to the susceptible isolates, suggesting that the isolates have parasitic fitness enough to compete with susceptible isolates in field conditions.

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.

Du fait de la réduction de l'usage des produits phytosanitaires, les bioagresseurs du sol, notamment les champignons Sclerotinia et les nématodes à galles Meloidogyne, sont de plus en plus préoccupants en maraîchage sous abri. Des techniques alternatives existent, mais leurs efficacités partielles et variables nécessitent la recherche de nouvelles stratégies pour leur gestion.Cette étude vise à déterminer l'intérêt de combiner deux techniques, la solarisation et l'engrais vert (Sorgho sudanense) et les influences des successions culturales et des fréquences d'utilisation de ces techniques sur leurs efficacités.Pour cela, un dispositif en parcelles de producteurs a permis de suivre l'évolution des incidences et sévérités de Sclerotinia et Meloidogyne dans différents systèmes de culture.Les effets des cultures de melon sont prédominants sur ceux des techniques alternatives, de façon opposée pour ces deux bioagresseurs. Les successions incluant du melon favorisent les plus fortes attaques de Meloidogyne alors qu'elles réduisent celles de Sclerotinia. Pour ce pathogène, l'utilisation de solarisation tous les ans ou un an sur deux en combinaison avec l'engrais vert en assure le contrôle le plus efficace. Par contre, la culture de melon étant très sensible à Meloidogyne, elle masque les effets des techniques alternatives.Des propositions d'amélioration de systèmes de culture, basées sur la modification des fréquences de culture du melon et sur l'optimisation de l'usage des techniques alternatives, ont été faites pour la gestion commune de Sclerotinia et Meloidogyne.Ces résultats montrent les interactions entre les différentes pratiques culturales utilisées sur une parcelle et l'intérêt des études agronomiques systémiques. De plus, les effets opposés observés d'un même système de culture soulignent l'intérêt d'études multi-bioagresseurs, pour ne pas faire de propositions qui résolvent un problème mais en aggravent d'autres. / In the current context of the reduction of pesticide use, soilborne pests and diseases, and especially the fungi Sclerotinia and the root-knot nematodes Meloidogyne, become more and more worrying in vegetable production under shelter in Southern France. Alternative techniques exist, but their partial and varying efficiencies require the search for new strategies of management of these pests and diseases fit for producers.Therefore, the aim of this study is to determine the benefit of combinations of two techniques, soil solarisation and green manure (Sorghum sudanense). It is also to determine the influences of crop successions and frequences of these techniques on their individual efficiencies.To this effect, a setup of producers' plots has been designed. The spatiotemporal evolution of incidences and severities of Sclerotinia and Meloidogyne in several cropping systems has been observed on this setup.The presence of a melon crop has a major influence on the efficiency of the alternative techniques. Opposite effects for the two considered pests and diseases appeared. Crops successions with melon are most favourable to highest infestations of Meloidogyne whereas, Sclerotinia infections are reduced. For this pathogen, a solarisation every year or every two years ensures the most effective control. On the contrary, in the case of Meloidogyne, the melon crop is very sensitive to this pest, a characteristic that masks the positive effects of alternative techniques.

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.

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.