Reproductive isolation is an essential stage in speciation. In Ascomycetes, the ubiquitous distribution of many species suggests that sympatric speciation through assertive mating should be an important factor. The MAT locus and the pheromone/receptor system could both potentially contribute to the development of such sexual isolation. Alterations at the MAT loci could lead to distinct reproductive habits or a change in mating system, both of which can reduce gene-flow between species. However, if deliberate pre-mating sexual preferences exist, they are more likely to be determined by the pheromone/receptor system. This study of Fusarium reproductive genes, and comparisons with other Ascomycetes, has yielded numerous interesting findings regarding the evolution of these mate-recognition mechanisms and the implications thereof. The G. fujikuroi and F. graminearum species complexes have offered an interesting comparison between heterothallic and homothallic MAT locus evolution. The value of comparative sequence analysis has been demonstrated in the discovery of a previously unknown gene, MAT1-2-3, which may be specific to members of the Order Hypocreales. While all MAT genes share similar regulatory elements, this is the first report of evidence that a transition to homothallism can be accompanied by the recruitment of distinct elements that could facilitate alternate expression of MAT genes. The MAT genes are also highly divergent between Fusarium spp., largely due to relaxed selective constraint, particularly in homothallic species. However, inter-specific gene-flow could curb MAT gene divergence among homothallic species. Despite strong reproductive barriers in the G. fujikuroi complex, the F. sacchari MAT1-1 sequence appears to have been acquired through lateral transfer from a distant relative. Analytical analysis of the MAT locus novelties reported here, including the new MAT gene, will be necessary to determine their biological significance. To investigate the extent of pheromone diversity in the Ascomycetes, and to gain clues as to its biological importance, pheromone peptides from seventy ascomycete species were compared. A number of reproductively incompatible species, such as those in the G. fujikuroi complex, share identical pheromones; which implies that another mechanism must be responsible for the observed reproductive barriers. However, on the whole, pheromones are highly divergent among species. Both adaptive and non-adaptive evolution could have contributed to this pattern. In fact the structure of the á-class pheromone precursor gene, which consists of multiple repeats of the pheromone module, could facilitate rapid diversification through “birth-and-death” evolution. Within species, selection maintains pheromone peptides, implying that much of the inter-specific variation is functionally relevant. This further suggests that pheromone evolution could contribute to the generation of reproductive isolation between species. The most general trend in the findings of this study is that ascomycete reproductive genes are highly divergent. This is in agreement with findings in other Kingdoms. A number of evolutionary forces are probably involved but weaker selective constraint, resulting from the fact that reproduction is not essential in these fungi, appears to be a common factor. This reproductive gene variability could be directly linked to speciation and, therefore, the great diversity in Ascomycetes. Additional information on the appendices is available on a CD, stored at the Merensky Library on Level 3 / Dissertation (MSc)--University of Pretoria, 2011. / Genetics / Unrestricted

Nitrilases are enzymes which catalyze the hydrolysis of nitriles to corresponding carboxylic acids. These enzymes have a great potential in biocatalysis, for example in the synthesis of mandelic acid and mandelamide, because of their chemo- and enantioselectivity. As bioremediation agents they are also applicable to sites contaminated with organic nitriles. In this work, activities of recombinant strains of E. coli expressing hypothetical nitrilases from fungi Giberella moniliformis and Nectria haematococca mpVI 77-13-4 were studied, as well as the biodegradation potential of bacteria from Rhodococcus and Nocardia genera towards benzonitrile herbicides dichlobenil (2,6-dichlorobenzonitrile), ioxynil (3,5-diiodo-4- hydroxybenzonitrile) and bromoxynil (3,5-dibromo-4-hydroxybenzonitrile). The hypothetical fungal nitrilases were expressed as functional enzymes. Nitrilase from G. moniliformis showed highest activity towards benzonitrile (30.9 U/mg protein), total activity yield was 2,560 U/l cell culture. The preferred substrate of the nitrilase from N. haematococca was phenylacetonitrile (12.3 U/mg prot.), total activity yield was 28,050 U/l cell culture. Nitrilase from N. haematococca was also able to hydrolyze mandelonitrile (5.9 U/mg prot.). Soil bacteria Rhodococcus rhodochrous PA-34, Nocardia globerula...

Made available in DSpace on 2017-07-10T17:37:37Z (GMT). No. of bitstreams: 1 Josiel_Pereira_Nascimento.pdf: 909698 bytes, checksum: 3cb3ef0ef8bdf27d1c63a9fb56af8240 (MD5) Previous issue date: 2009-08-29 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / The non tillage planting and the cultivation of safrinha (short season) corn are factors that may provide alterations in the microclimate and in the biology of agro ecosystem, increasing the populations of fungus like Gibberella zeae, which is the etiologic agent of one of the maize ear rots. This work had the objective to identify inoculation methods that can be useful in the election and in the characterization of genotypes tolerant to G. zeae. The experiment was conducted in Toledo/PR during the 2008 safrinha season in a randomized complete block design with five replications, on a factorial scheme where hybrids were the first factor (2B710; 30F45; DKB 214; 30F98; DKB 390; GAC 9020; 30F80; 30R50 and 30K73) and the inoculation methods were the second factor. The hybrids had been grouped as susceptible (2B710; 30F45; DKB 214; 30K73), tolerant (DKB 390; GAC 9020; 30F80; 30R50) and moderately susceptible to giberela (30F98). The 12 evaluated methods consisted of simulating the fungus natural infection on the silk or on the damage caused by insects in the lateral of the ears, and also varying by the absence of covering on the ear (humid chamber), by covering the ear for 24 hours and by covering the ear for 48. The methods with inoculation of 2 mL in the lateral of the ear without covering (3), with covering for 24 hours (7) and with covering for 48 hours (11) had shown efficiency in the characterization of tolerant and susceptible hybrids and could be used on a large scale in the identification and selection of maize varieties with tolerance to ear rot caused by G. zeae. As a result, the seed industry can provide superior materials and the farmers will reduce their losses due to kernel quality. Moreover, the whole society will consume better maize products, with less mycotoxins / O plantio direto e o cultivo de milho safrinha são fatores que podem proporcionar alterações no microclima e na biologia do agroecossistema, aumentando as populações de fungos como Gibberella zeae, agente etiológico da doença denominada giberela ou podridão-vermelha-da-espiga. Esse trabalho teve como objetivo identificar métodos de inoculação que auxiliassem na seleção e na caracterização de genótipos tolerantes à G. zeae. O experimento foi conduzido no município de Toledo/PR durante a safrinha de 2008. O delineamento experimental foi em blocos ao acaso com cinco repetições, em esquema fatorial tendo como primeiro fator os híbridos (2B710; 30F45; DKB 214; 30F98; DKB 390; AG 9020; 30F80; 30R50 e 30K73) e como segundo fator os métodos de inoculação. Os híbridos foram agrupados como suscetíveis (2B710; 30F45; DKB 214; 30K73), tolerantes (DKB 390; AG 9020; 30F80; 30R50) e moderadamente suscetível a giberela (30F98). Os 12 métodos avaliados consistiam em simular a infecção natural do fungo pelo estigma ou por danos causados por insetos nas laterais das espigas, variando ainda em ausência de cobertura (câmara úmida), cobertura por 24 horas e por 48 horas. Os métodos com inoculação de 2 mL na lateral da espiga sem cobertura (3), com cobertura por 24 horas (7) e com cobertura por 48 horas (11) se mostraram eficientes na caracterização de híbridos tolerantes e suscetíveis e poderão ser empregados em larga escala na identificação e seleção de variedades de milho tolerantes à doença. Mais do que isso, a sociedade poderá consumir produtos de milho com qualidade superior, com menos micotoxinas

Made available in DSpace on 2016-12-08T16:44:51Z (GMT). No. of bitstreams: 1 PGPV14MA180.pdf: 350816 bytes, checksum: 21cfa9750f5bc20f9040d6522a471d04 (MD5) Previous issue date: 2014-12-11 / In southern Brazil, Fusarium Head Blight (FHB) caused by the fungus Fusarium graminearum is severe disease because the fungus is abundant in winter and summer crop residues, which helps inoculation in susceptible maize hybrids when have excessive periods of rain after pollination phase. This research aimed to determine the best time to spray fungicide during maize silking to control FHB. The experiment was conducted in field at the experimental area of Agroveterinarias Sciences Center at the Santa Catarina State University (CAV/UDESC) in Lages, Santa Catarina. Were seeded simple hybrids P32R48 H and Maximus TL TG Viptera during two periods in the growing seasons of 2012/13 and 2013/14. After five days of stigma emergence, the ears were artificially inoculated with 1 x 105 macroconídios.mL-1 concentration of F. graminearum injecting 2 ml of inoculum solution in stigma-style channel. Inoculations happen in 03/08/2013, 03/22/2013, 12/26/2013 and 01/19/2014. The fungicide was a mixture of azoxystrobin + cyproconazole (300 mL c.p/ha) + carbendazim (1 L c.p/ha) + nimbus mineral oil (0.5% v.v/ha). The treats were six fungicide spray times: 144, 96 and 48 hours before inoculation; 48, 96 and 144 hours after inoculation and a control, only inoculated without fungicide application. The experiment design was randomized block with four replications. The fungicide was applied with a manual CO2 gas sprayer. The variables evaluated were severity of FHB in the ears (SGE), percentage of damaged kernels (PGA), incidence of F. graminearum in the grains (IFgG) and grain yield (RG). Data were subjected to variance analysis, Tukey´s test and simple correlation analysis. The hybrid P32R48H was more susceptible than Maximus TL TG Viptera to FHB. Sprays 48 hours before and after inoculation were effective to reduce SGE in approximately 50% and 41%; to reduce PGA by 33% and 50%; to reduce IFgG by 27%; and to increase RG by 9% and 7%, respectively when compared to control. There is a negative linear correlation between SGE, PGA, IFgG and RG variables. The yield and grain quality were improved by fungicide spray between the 3rd and 7th day period after stigma issue / Na região Sul do Brasil, a podridão de espiga de giberela, causada pelo fungo Fusarium graminearum, é uma doença preocupante, pois o fungo é abundante nos resíduos culturais das gramíneas de inverno e de verão, o que facilita a sua inoculação nas espigas de híbridos suscetíveis em safras com excesso de chuva após a fase da polinização. Este trabalho de pesquisa teve como objetivo definir o melhor momento para pulverização de fungicida durante o estádio do espigamento do milho para controlar a podridão de espiga de giberela. O experimento foi conduzido à campo, na área experimental do Centro de Ciências Agroveterinárias da Universidade do Estado de Santa Catarina (CAV/UDESC) no município de Lages, Santa Catarina. Foram semeados os híbridos simples P32R48 H e Maximus TL TG Viptera, em duas épocas nas safras agrícolas de 2012/13 e 2013/14. Após cinco dias da emissão dos estigmas, as espigas foram inoculadas artificialmente com uma concentração de 1 x 105 macroconídios.mL-1 de F. graminearum injetando 2 mL da solução de inóculo no canal do estilo-estigma. As inoculações foram realizadas nos dias 08/03/2013, 22/03/2013, 26/12/2013 e 19/01/2014. O fungicida utilizado foi a mistura de azoxistrobina + ciproconazole (300 mL p.c/ha) + carbendazim (1 L p.c/ha) + oléo mineral Nimbus (0,5% v.v./ha). Os tratamentos constituiram-se em seis momentos de pulverização do fungicida: 144, 96 e 48 horas antes da inoculação; 48, 96 e 144 horas depois da inoculação e uma testemunha, somente inoculado sem aplicação de fungicida. A distribuição dos tratamentos seguiu o modelo do delineamento em blocos casualizados, com quatro repetições. O fungicida foi aplicado com o auxílio de pulverizador costal com pressão produzida por gás CO2. As variáveis avaliadas foram severidade da giberela nas espigas (SGE), porcentagem de grãos ardidos (PGA), incidência de F. graminearum nos grãos (IFgG) e rendimento de grãos (RG). Os dados obtidos foram submetidos à análise de variância, ao teste de Tukey e a análise de correlação simples. O híbrido P32R48H apresentou-se mais suscetível a podridão de giberela. As pulverizações 48 horas antes e depois da inoculação foram eficientes em reduzir a SGE em aproximadamente 50% e 41%; em reduzir a PGA em 33% e 50%; em reduzir a IFgG em 27%; e em aumentar o RG em 9% e 7%, respectivamente quando comparados ao tratamento sem fungicida. Existe associação entre as variáveis SGE, PGA, IFgG e RG. Pode-se aumentar o rendimento e melhorar a qualidade de grãos com a aplicação de fungicida no período do 3º ao 7º dia após a emissão dos estigmas

Made available in DSpace on 2016-12-06T17:42:07Z (GMT). No. of bitstreams: 1 PGPV14DA016.pdf: 1791236 bytes, checksum: 435d76b4d49498dd4bdc194423dac320 (MD5) Previous issue date: 2014-10-17 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / The gibberella ear rot caused by fungus Fusarium graminearum, causes damage in productivity and quality of maize kernel being disease of difficult to control due to the unavailability of resistant hybrids and low efficacy of cultural practices. Three studies were conducted, the aim of the first was to compare four methods of inoculation (injection silkchannel, sprinkling on silk, kernel- wounding and deposition in the ear leaf sheath) and a control treatment in four hybrids (P30R50H, P30B30H, P3989, P32R48H) based on disease severity and kernel yield; the second study determined the reaction of 20 hybrids to gibberella ear rot by quantifying the incidence of the disease, rot kernels and F. graminearum in the kernels and their correlations; and the third study characterized the genetic resistance of hybrids to gibberella ear rot through preparation of a reaction scale involving classes and disease intensity and their correlation with kernel yield and inoculation methods. The experiments of first study were conducted in 2011/12 harvest in Cruz Alta, RS and Lages. The experiments of the second and third studies were conducted in two environments in the season 2012/13 in Cruz Alta. The design of the experiments was a randomized complete blocks design with treatments arranged in a factorial with four replications. In the first study there was no difference in disease severity between hybrids and inoculation methods. The hybrid P32R48H presented higher disease severity, distinguishing from the others. Inoculations in silk-channel and wounding the kernels provided higher disease severity and lower kernel yield. In the second study, the method of the wounding the kernels provided increases above 40% in the incidence of gibberella ear rot, rot kernels and occurrence of F. graminearum in the grain when compared to the injection silk-channel method. There was an effect of hybrids and the values were 37.2% and 95.6% for the incident of gibberella ear rot, 2.1% to 17.0% for rot kernels and 4.0% to 29.3% for incidence of F. graminearum in the kernels. Significant and positive correlations (r = 0.70) were found between incidence of F. graminearum in the kernels and rots kernels. In the third study it was watched that the kernel yield was affected by hybrids, regardless of the inoculation method. The P30F35YH and BG7049H hybrids obtained better yields, regardless of the environment and inoculation method, being classified as moderately susceptible to gibberella ear rot. The AG8025PRO, DKB250PRO2, AS1555PRO, Status, 30K64HNSR and P4285 were classified as moderately resistant considering the intensity of gibberella ear rot and the involvement of resistance to colonization of the stigma (tip of the ear) and kernel resistance. There were significant and positive relationship between productivity with injection in the silk-channel (r = 0.93) and wounding the kernels with productivity (r = 0.94) in environment 1 and 2. It was found that although there are no resistant hybrids there is genetic variability and the involvement resistance of stigma mechanisms and the kernels, and the giberella ear rot affected more kernel quality than quantity / A podridão de giberela causada pelo fungo Fusarium graminearum, provoca danos na produtividade e qualidade de grãos de milho sendo doença de difícil controle devido à indisponibilidade de híbridos resistentes e baixa eficácia de práticas culturais. Foram realizados três estudos, o objetivo do primeiro foi comparar quatro métodos de inoculação (injeção no canal do estilo-estigma, aspersão nos estigmas, ferimento dos grãos no centro da espiga e deposição na bainha da folha da espiga) e um tratamento controle em quatro híbridos (P30R50H, P30B30H, P3989, P32R48H) com base na severidade da doença e produtividade de grãos; o segundo estudo determinou à reação de 20 híbridos a podridão de giberela pela quantificação da incidência da doença, grãos ardidos e F. graminearum nos grãos e suas correlações; e o terceiro estudo caracterizou a resistência genética de híbridos à podridão de giberela pela elaboração de uma escala de reação envolvendo classes e intensidade de doença e suas correlações com produtividade de grãos e métodos de inoculação. Os experimentos do primeiro estudo foram conduzidos na safra 2011/12 em Cruz Alta, RS e Lages, SC. Os experimentos do segundo e terceiro estudos foram realizados em dois ambientes na safra agrícola 2012/13, em Cruz Alta. O delineamento dos experimentos foi em blocos casualizados com tratamentos arranjados num fatorial com quatro repetições. No primeiro estudo houve diferença na severidade da doença entre híbridos e métodos de inoculação. O híbrido P32R48H apresentou maior severidade da doença, diferindo dos demais. Inoculações no canal do estilo-estigma e ferimento dos grãos proporcionaram maior severidade da doença e menores produtividades de grãos. No segundo estudo o método de ferimento dos grãos proporcionou acréscimos superiores a 40% na incidência de espigas giberelas, incidência de grãos ardidos e incidência de F. graminearum nos grãos quando comparado ao método de injeção no canal do estilo estigma. Houve efeito de híbridos e os valores foram de 37,2% a 95,6% para incidência de espigas giberelas, 2,1% a 17,0% para grãos ardidos e 4,0% a 29,3% para incidência de F. graminearum nos grãos. Correlações significativas e positivas (r = 0,70) foram obtidas entre incidência de F. graminearum nos grãos e grãos ardidos. No terceiro estudo foi observado que a produtividade de grãos foi afetada pelos híbridos, independentemente do método de inoculação. Os híbridos P30F35YH e BG7049H obtiveram melhores produtividades, independente do ambiente e método de inoculação, sendo classificados como moderadamente suscetível à podridão de giberela. Os híbridos AG8025PRO, DKB250PRO2, AS1555PRO, Status, 30K64HNSR e P4285 foram classificados como moderadamente resistente, considerando a intensidade de giberela e o envolvimento da resistência à colonização do estigma (ponta da espiga) e resistência do grão. Houve relações significativas e positivas entre produtividade com injeção no canal do estilo-estigma (r = 0,93) e produtividade com ferimento dos grãos (r = 0,94) nos ambientes 1 e 2. Foi constatado que apesar de não haver hibridos resistentes existe variabilidade genética e o envolvimento de mecanismos de resistência do estigma e dos grãos, e que as podridões de giberela afetaram mais a qualidade dos grãos do que a quantidade

Gibberella zeae (anamorph Fusarium graminearum) is a homothallic ascomycete pathogen that is responsible for causing Fusarium head blight (FHB) of wheat and small grains. In addition to causing a reduction in yield, harvested grain is frequently contaminated with trichothecene mycotoxins that are harmful for human and animal health. Use of wheat varieties with resistance to FHB is an important strategy to lower its impact. In order to produce varieties with durable resistance, we must understand the origin and degree of genetic diversity present in the pathogen population. In my research, I focused my efforts on an investigation of the role of mating and sexual development in the generation of genotypic and phenotypic variability in G. zeae. The goal of one part of my work was to develop new genetic markers that can be used to monitor out-crossing and genetic diversity in the population. I also optimized gene deletion protocols for G. zeae so that I could produce mutant and control strains to address my research hypothesis that MAT genes play a direct role in pathogenicity. Application of novel repetitive RFLP probes to a group of G. zeae isolates originating from and near Kentucky revealed a surprisingly high degree of diversity in these local populations. Diversity between locations was greater than that within locations, suggesting the relative importance of local inoculum sources. The probes were also useful as genetic markers for segregation analysis. I crossed two genetically closely related, and commonly used, laboratory strains of G. zeae and found that this resulted in transgressive segregation for both aggressiveness and toxigenicity. I showed that the very high and very low levels of aggressiveness and toxigenicity in transgressive segregants are heritable. I also showed that selfing produced a higher degree of diversity in these traits among the progeny than was observed among conidial progeny. This suggests the presence of epigenetic factors that impact pathogenicity. Sexual behavior in G. zeae is under the control of MATing type genes. I deleted the complete MAT1 locus, and the MAT1-1-1, and MAT1-2-1 genes separately. Deletion of each of the targeted sequences produced the expected shifts in fertility phenotype. The mat1KO strains became asexual, while mat1-1-1KO and mat1-2-1KO strains shifted to obligate heterothallism. Deletion of the MAT1-1-1 and MAT1-2-1 genes had a negative effect on aggressiveness and mycotoxin production in planta, but deletion of the complete MAT1 locus had no effect. The set of mutant and ectopic control strains that I generated will be a useful asset that will be made available to the research community.

Submitted by (lucia.rodrigues@ufrpe.br) on 2017-02-17T12:29:16Z No. of bitstreams: 1 Andre Angelo Medeiros Gomes.pdf: 644128 bytes, checksum: cd67aaf23e82d52d2fc876105888dc75 (MD5) / Made available in DSpace on 2017-02-17T12:29:16Z (GMT). No. of bitstreams: 1 Andre Angelo Medeiros Gomes.pdf: 644128 bytes, checksum: cd67aaf23e82d52d2fc876105888dc75 (MD5) Previous issue date: 2013-02-18 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - CAPES / Brazil is one of the biggest producers and consumers of maize (Zea mays L.) in the world and the fusariosis caused by F. verticillioides, on ears and or on stalk, is a recurrent problem when environmental conditions favor epidemics in the field or in storage. The present study aimed to characterize through crosses the F. verticillioides isolates from the three main climatic zones where maize is grown in Brazil. The fertility and effective population size were calculated from data generated by crossing field isolates with female fertile testers of F. verticillioides. For the entire population, 231 out of 300 isolates were cross-fertile with tester isolates. MAT-1 and MAT-2 idiomorphs of the fertile isolates segregated in a 105:126 rate. Female isolates (hermaphrodites) were 96 out of 231 fertile isolates, while 135 were male only isolates. The Ne(mt) was 99% of the count for the Brazilian population when the mating type idiomorphs were used as predictors in the estimative of the population size. But when this calculation was made on the basis of the female fertile isolates the Ne(f) was 83%. When the total population is divided into sub-populations representing climatic zones, the frequencies of female fertile isolates were 33 out of 80 fertile isolates from the Equatorial Tropical Zonal Climate (ETZC); 14 out of 78 fertile isolates from the Temperate Zonal Climate (TZC); and 49 out of 73 fertile isolates from the Tropical Central Brazil Zonal Climate (TCBZ). The effective population size expressed in percentage of the total number of individual in each population for the three populations represented by isolates from the ETZC, TZC and TCBZ were Ne(mt) = 99, 100, 97 and Ne(f) = 83, 52, 96 respectively. The Brazilian population of F. verticillioides shows high fertility and high effective population size, suggesting the possibility of frequent sexual reproduction in the field. Apparently there is a tendency of higher fertility in populations established at low latitudes, represented by the tropical climate. / O Brasil é um dos maiores produtores e consumidores de milho (Zea mays L.) do mundo e a fusariose, causada por Fusarium verticillioides, seja em espigas ou no colmo, é um problema recorrente quando condições ambientes favorecem o surgimento de epidemias no campo ou em armazenamento. O presente trabalho teve como objetivos caracterizar por meio de cruzamentos sexuais, isolados de F. verticillioides dos três principais climas zonais que se cultiva milho no Brasil. Através de cruzamentos de isolados do campo de F. verticillioides, com testadores de G. moniliformis; e determinar a taxa de fertilidade, frequência de hermafroditismo, e tamanho efetivo da população. Dos 300 isolados estudados, 231 tiveram cruzamento fértil. Os tipos de acasalamento MAT-1 e MAT-2 segregaram numa proporção 105:126. Entre os 231 isolados férteis, 96 se comportaram como hermafroditas e 135 como fêmeas estéreis. Na população total, do Brasil, levando em consideração o tipo de acasalamento, o tamanho efetivo da população Ne(mt), foi de 99% da população total, e tomando por base a frequência de hermafrodita , o tamanho efetivo Ne(f) foi de 83% da população total. Quando o número total de isolados é subdividido em três populações distintas, baseado nas condições de clima de local de coleta dos isolados, as frequências de hermafroditas foram; 33 hermafroditas dentre 80 isolados férteis do Clima Zonal Tropical Equatorial (ZTE), com 47 se comportando como fêmea estéril; 14 hermafroditas dentre 78 isolados férteis do Clima Zonal Temperado (ZT), com 64 se comportando como fêmea estéril; e 49 hermafroditas dentre 73 isolados do Clima Zonal Tropical Brasil Central (ZTBC), com apenas 24 se comportando como fêmea estéril. O tamanho efetivo das populações representadas por isolados dos climas ZTE, ZT e ZTBC, foram Ne(mt) = 99, 100, 97 e Ne(f) = 83, 52, 96 respectivamente, expresso em porcentagem do número total de indivíduos de cada população. A população brasileira de F. verticillioides analisada apresenta alto índice de fertilidade e tamanho efetivo, sugerindo a possibilidade de frequente reprodução sexuada no campo. Aparentemente, existe uma tendência de maior fertilidade da população em latitudes menores, representadas pelo clima tropical.

Os fungos endofíticos tornam-se valiosa fonte para descoberta de novas moléculas de interesse terapêutico e biotecnológico. O objetivo deste trabalho consistiu em avaliar o potencial bioativo do produto da fermentação do fungo endofítico Gibberella moniliformis 99(3) e identificar as moléculas responsáveis por essa bioatividade. Inicialmente, o endófito isolado das folhas de Laguncularia racemosa foi submetido ao processo de fermentação em caldo Czapeck por 20 dias/ 28ºC em condições estáticas. Após, o caldo fermentado foi particionado com diferentes solventes, gerando os extratos acetato de etila (JAc: 238 mg) e n-butanólico (JBu: 27,2 mg). Estes foram biomonitorados pelo ensaio de difusão em ágar frente diferentes patógenos (S. aureus ATCC 6538, E. coli ATCC 25922 e C. albicans ATCC 10231) e, diante do potencial antimicrobiano observado, foi determinada também a concentração inibitória mínima (CIM) e microbicida mínima (CMM). É pertinente destacar os resultados para JAc (CIM: 125-250 µg/mL) e para JBu (CIM: 25-50 µg/mL; CMM: 100-200 µg/mL) contra C. albicans ATCC 10231. Procedeu-se estudo de fracionamento bioguiado com o extrato JAc, obtendo 27 frações. Estas foram reagrupadas em quatro (J1, J2, J3 e J4), às quais foram biomonitoradas quanto ao potencial antimicrobiano, sendo o maior destaque evidenciado pela fração J3. No estudo cromatográfico, J3 revelou-se positivo com reagente de Dragendorff e luz UV 254 nm. Os estudos espectroscópicos com esta fração (H¹ RMN, C¹³ RMN, COSY, HSQC, HMBC, EM e IV), permitiram a caracterização de um alcaloide piridínico denominado Ácido 5-butilpiridina-2-carboxílico, também conhecido como Ácido fusárico. Investigou-se o potencial biológico deste composto por meio de seis ensaios biológicos in vitro. No estudo de viabilidade celular, pelo ensaio do MTS frente quatro linhagens humanas tumorais (A549, Hep G2, HT 144 e MCF 7), observou-se seletividade moderada para duas linhagens (MCF 7 e HT 144) e baixa seletividade para a linhagem normal (CCD-1059Sk). A redução da viabilidade celular de 60 % (MCF 7) e 50 % (HT 144), ocorreu por efeito antiproliferativo (IC50: 60 µg/mL), conforme constatado no ensaio por exclusão com o azul de tripano. Nos estudos antimicrobianos, o alcaloide piridínico evidenciou forte potencial antifúngico frente C. albicans ATCC 10231 (CIM: 25-50 µg/mL; CFM: 200-400 µg/mL). Neste sentido, o conjunto dos dados permitiu sugerir tratamento tópico seguro para o paciente com candidíase, uma vez que a CIM esteve bem abaixo de uma possível concentração tóxica para os fibroblastos de pele (CCD-1059Sk). Ainda, diante do perfil antifúngico observado contra linhagens provenientes de ambiente hospitalar, este composto poderia ser indicado como higienizador em fontes secundárias de contaminação (CIM contra C. albicans isoladas de lençol e jaleco: 50-100 µg/mL). Também foi constatado forte potencial bacteriostático contra E. coli (CIM: 50-100 µg/mL) e moderado contra S. aureus (CIM: 200-400 µg/mL). Por meio das imagens de MEV foi possível observar que a bactéria S. aureus sofreu evidentes alterações morfológicas quando tratada com 200 e 400 µg/mL de Ácido fusárico. Ao investigar o efeito sinérgico da associação do composto com antimicrobianos de uso comercial, verificou-se frente a E. coli efeito parcialmente sinérgico (ICIF = 0,625), com potencialização em oito vezes da atividade antimicrobiana da estreptomicina. Frente S. aureus foi verificado efeito aditivo na associação do alcaloide e amoxicilina (ICIF = 1). Em estudo da ação antituberculose, observou-se CIM90 > 25 µg/mL. Conclui-se que o fungo endofítico G. moniliformis é promissor na produção de compostos de interesse e os estudos realizados no presente trabalho abrem novas perspectivas de conhecimento para o Ácido fusárico que até então não haviam sido explorados na literatura. / The endophytic fungi become valuable sources for discovering new molecules with therapeutic and biotechnological interest. The present study aimed to evaluate the bioactive potential of the products obtained in the fermentation of Gibberella moniliformis 99(3) endophytic fungus and to identify the molecules responsible for this bioactivity. Initially, the endophyte isolated from leaves of Laguncularia racemosa (Brazilian mangrove) was submitted to the fermentation in Czapek broth for 20 days / 28°C, under static conditions. Then, the fermented broth was partitioned with different solvents resulting in the ethyl acetate (JAc: 238 mg) and n-BuOH (JBu: 27.2 mg) extracts. These extracts were biologically monitored by agar diffusion assay against different pathogens (S. aureus ATCC® 6538™, E. coli ATCC® 25922™ and C. albicans ATCC® 10231™). Since an antimicrobial potential was observed, the minimum inhibitory concentration (MIC) and minimal microbicidal concentration (MMC) were also determined. It is pertinent to highlight the results of JAc (CIM: 125-250 µg/mL) and to JBu (CIM: 50-100 µg/mL; CMM: 100-200 µg/mL) against C. albicans ATCC 10231. A bioguided fractionation study was realized with JAc extract obtaining 27 fractions, which were grouped into four fractions (J1, J2, J3 e J4). The antimicrobial potential was biomonitored, with more emphasis to J3. In chromatographic study, the J3 fraction was positive to Dragendorff reagent and UV 254 nm. In the spectroscopic studies with J3 (H¹ NMR, C¹³ NMR, COSY, HSQC, HMBC, MS and IR) was possible the characterization of a pyridine alkaloid called 5-butylpyridine-2-carboxylic acid also known as fusaric acid. The bioactive potential of this compound was investigated applying six biological assays in vitro. The cell viability study was realized by colorimetric assay MTS, using four human tumor lines (A549, Hep G2, HT 144 and MCF 7), was observed a moderate selectivity for the lines MCF 7 and HT 144 and low selectivity was assessed on normal fibroblast strains (CCD-1059Sk). The reduction of cellular viability of 60 % (MCF 7) and 50 % (HT 144) occurs by antiproliferative effect (IC50: 60 µg/mL), according to test by exclusion with trypan blue. In antimicrobials studies, the pyridine alkaloid showed strong antifungic potential against C. albicans ATCC 10231 (MIC: 25-50 µg/mL; MFC: 200-400 µg/mL). In this sense, a safe and topical treatment for patients with candidiasis could be suggested based on data set, once the MIC was well below toxic concentration for a possible skin fibroblasts (CCD-1059Sk ). Besides that, this compound could be suitable for use as a sanitizer on secondary sources of hospital infection due to the fungistatic profile observed in C. albicans isolated from lab coat and sheet (MIC : 50-100 µg/mL). It was also observed strong bacteriostatic potential against E. coli (MIC: 50-100 µg/mL) and moderate against S. aureus (MIC: 200-400 µg/mL). With the SEM images was noted that S. aureus suffered obvious morphological changes when treated with 200 and 400 µg/mL of fusaric acid. Investigating the synergistic potential of the combination between fusaric acid and commercially used antimicrobials, it was observed partially synergistic effect (ICIF = 0,625) for E. coli, with eight times potentiation of the streptomycin antimicrobial activity. It was checked for S. aureus an additive effect in the combination of alkaloid and amoxicillin (ICIF = 1). Studying antimycobacterial activity, the compound was tested against Mycobacterium tuberculosis, with MIC90 > 25 µg/mL. The present work consistently demonstrated that the fungus endophyte G. moniliformis is promising in the production of compounds of interest and the data presented in this study open up new avenues of knowledge for fusaric acid which until then had not been explored in the literature.

Submitted by Marco Antônio de Ramos Chagas (mchagas@ufv.br) on 2016-08-22T11:29:51Z No. of bitstreams: 1 texto completo.pdf: 956629 bytes, checksum: 1096a842439a74fe8b122975f58e42bd (MD5) / Made available in DSpace on 2016-08-22T11:29:51Z (GMT). No. of bitstreams: 1 texto completo.pdf: 956629 bytes, checksum: 1096a842439a74fe8b122975f58e42bd (MD5) Previous issue date: 2016-02-23 / Conselho Nacional de Desenvolvimento Científico e Tecnológico / Os fungicidas são normalmente aplicados para o manejo da giberela com o objetivo de garantir a produtividade e reduzir níveis de micotoxinas associadas à doença. Vários ingredientes ativos estão disponíveis no Brasil, sendo que os mais comumente recomendados pertencem ao grupo dos inibidores da demetilação (DMI) e benzimidazóis (MBC), ou usados em mistura com fungicidas do grupo das estrobilurinas (QoI). Dada a importância crescente de epidemias de giberela no Brasil, o uso de fungicidas vêm aumentando consideravelmente e, geralmente, duas aplicações têm sido recomendadas. Parcelas de campo em 16 municípios na região de Guarapuava, Sul do Paraná foram monitoradas durante quatro anos (2011 a 2014) e um total de 227 isolados semelhantes a espécies do complexo de espécies de Fusarium graminearum foram obtidos a partir de espigas de trigo sintomáticas. Uma amostra de 173 isolados foi identificada quanto ao genótipo tricoteceno com base em PCR. Entre eles, 67% foram identificados como 15- (A)cetil-desoxinivalenol(DON), 29% do nivalenol (NIV) e 4% como genótipo 3-ADON. Uma amostra de 35 isolados foi selecionada para determinar a concentração dos fungicidas tebuconazol e carbendazim que reduz em 50% do crescimento de micelial (EC 50 ), usando as seguintes doses (0; 0,5; 1,0; 2,0 e 4,0 μg / ml). As medias estimadas de EC 50 , independente do genótipo, foram 0,39 μg/ml (0,0004 a 3,0) e 1,25 μg/ml (0,91 a 2,65) para tebuconazol e carbendazim, respectivamente. As medias de EC 50 foram 1,32 μg/ml e 1,21 μg/ml para carbendazim e 0,58 μg/ml e 0,05 μg/ml para tebuconazol, para os isolados 15-ADON e NIV ou 3-ADON, respectivamente. O teste não paramétrico Kolmogorov-Smirnov mostrou diferença entre os isolados 15-ADON e os NIV+3-ADON quanto ao tebuconazol (P = 0,002), mas não para o carbendazim (P = 0,514). As doses discriminatórias de 1,4 μg/ml e 2,0 μg/ml foram usadas para identificar isolados menos sensíveis à carbendazim e tebuconazol, respectivamente, em toda coleção. Baseado nessas doses, dois isolados menos sensíveis ao tebuconazol e seis isolados menos sensíveis ao carbendazim foram encontrados, sendo todos originários do munícipio de Guarapuava ao longo de três safras e possuem o genótipo 15-ADON. Houve correlação significativa entre os valores de EC 50 dos dois fungicidas (r = 0,45; P = 0,007). Um único isolado apresentou os maiores valores de EC 50 para ambos fungicidas e apresentou uma mutação (R31K) no gene cyp51A. A presença da reduzida sensibilidade e um isolado resistente, reportado pela primeira vez no Brasil, sugerem que a seleção pode conduzir mudanças nas populações com consequências no manejo da doença. Estudos futuros devem se concentrar em atributos de fitness, competitividade e experimentos in vivo para verificar se a eficácia do fungicida é prejudicada na presença de isolados menos sensíveis. Em estudos de controle químico da giberela, resultados sobre a eficácia têm sido inconsistentes e variáveis para um mesmo fungicida. Uma revisão sistemática de estudos de eficácia dos fungicidas pode ser útil para apontar para os produtos mais eficazes e cenários onde a eficácia pode ser melhorada. Os objetivos deste trabalho foram a) revisar sistematicamente estudos de controle químico para o manejo da giberela realizados no Brasil durante os últimos 15 anos (2000-2015), publicados em revistas com e sem corpo editorial e b) conduzir uma metanálise da eficácia de controle e retorno em produtividade de fungicidas selecionados. Uma busca na literatura identificou 18 estudos que relataram a severidade da giberela (o mesmo que índice giberela) para pelo menos um tratamento com fungicida em comparação com um tratamento controle sem o fungicida, e também uma medida da variância amostral do ensaio. Apenas três e dois estudos relataram valores médios de grãos giberelados e deoxinivalenol (DON), respectivamente. Os estudos foram analisados e oito tratamentos com fungicidas, com pelo menos dez entradas foram selecionados para o estudo de metanálise. Foram incluídos os tratamentos com os fungicidas dos grupos: DMI (tebuconazol, metconazol e propiconazol) e MBC (carbendazim) formulados individualmente e uma pré-mistura de DMI + QoI (azoxistrobina + tebuconazol, piraclostrobina + metconazol, trifloxistrobina + prothioconazol e trifloxistrobina + tebuconazol). Foram testadas uma aplicação (×1) e duas aplicações (×2), sendo a primeira na floração plena e a segunda dez dias após. Um modelo metanalítico multivariado foi ajustado para o log das médias para cada fungicida e o tratamento controle de cada ensaio. A diferença nas estimativas médias do log da severidade entre um tratamento com fungicida e o controle foram usadas para calcular a razão de resposta média (R) e então transformadas para porcentagem de controle (C) e porcentagem de retorno em produtividade (P). A média de C e P variou de 48,94 a 70,39% e de 7,41 a 21,29%, respectivamente. Metconazol (×2) teve o melhor desempenho para reduzir a severidade da doença (C = 70,39%) e tebuconazol (×1) em retorno em produtividade (P = 21,29%). Propiconazol foi o menos eficaz entre os DMIs (C = 48,94%), seguido de carbendazim (C = 53,90%). Em geral, os fungicidas mais eficazes resultaram em maior retorno de rendimento. Quando os grupos de fungicidas foram comparados, em geral, os DMIs e as misturas tiveram o melhor desempenho, especialmente em ensaios conduzidos sob elevada pressão da doença (> 7% no tratamento controle). Os resultados mostraram que a eficácia de controle média em estudos brasileiros quando se utiliza duas aplicações são mais elevadas do que quando se utiliza apenas uma aplicação, como relatado em outro estudo de metanálise na América do Norte. Estudos futuros deverão centrar-se no efeito sobre os níveis de DON, especialmente para as misturas, que estão estrategicamente utilizadas para o controle de doenças foliares. / Fungicides are commonly applied for managing Fusarium head blight (FHB) with the aim of protecting yield and improving grain quality, but especially targeting mycotoxin reduction. Several active ingredients are available in Brazil and the most common belong to the demethylation-inhibitor (DMI) and benzimidazoles (MBC) groups, with the former commonly used in mixture with fungicides of the quinone-outside inhibitor (QoI) group. Given the increasing importance of FHB epidemics in Brazil, the use of fungicides is increasing considerably and usually two sprays are recommended. Experimental trials where fungicides have been applied for FHB control were monitored in 16 municipalities at the Guarapuava region, Southern Paraná, during four years (2011 to 2014). A total of 227 isolates resembling those of the Fusarium graminearum species complex were obtained from wheat heads with FHB symptoms. Of those, 173 isolates were assigned to trichothecene genotypes based on PCR assays. A random sample of 35 isolates from 2011 to 2013 was selected to determine the concentration of tebuconazole and carbendazim fungicides that reduces 50% of the mycelial growth (EC 50 ). The following doses were used: 0, 0.5, 1.0, 2.0 and 4.0 μg/ml. In the PCR assays, most isolates (67%) were of the 15-(A)cetyl-deoxynivalenol (DON), 29% of the nivalenol (NIV) and 4% of the 3-ADON genotype. The estimated means of the EC 50 , irrespectively of the genotype, were 0.39 μg/ml (0.0004 to 3.0) and 1.25 μg/ml (0.91 to 2.65) for tebuconazole and carbendazim, respectively. The mean EC 50 were 1.32 μg/ml and 1.21 μg/ml for carbendazim and 0.58 μg/ml and 0.05 μg/ml for tebuconazole, for isolates of the 15-ADON and NIV + 3-ADON isolates, respectively. A Kolmogorov-Smirnov non-parametric test suggested that the sensitivity to tebuconazole of the 15-ADON and NIV+3-ADON isolates were sampled from populations with different distributions (P = 0.002), but not for carbendazim (P = 0,514). Discriminatory doses of the 1.4 μg/ml and 2.0 μg/ml were used to screen for less- sensitive isolates in the whole sample. Based on those doses, two tebuconazole-less sensitive and six carbendazim-less sensitive isolates were found, all were originated from Guarapuava municipality across three growing seasons and possessed the 15-ADON genotype. There was as significant correlation between the EC 50 values of tebuconazole and carbendazim (r = 0.45; P = 0,007). One isolate exhibited the highest EC 50 values for both fungicides and showed a mutation (R31K) in the cyp51A gene. The presence of less sensitive and a resistant isolate reported for the first time in Brazil suggest that selection may drive changes in the populations with impact in disease management. Future studies should focus on the attributes of fitness, competitiveness and in vivo assays to check whether fungicide efficacy is hindered in the presence of resistant isolates. Furthermore, results on fungicide efficacy have been inconsistent and variable for a same fungicide. Quantitative summary of fungicide data available in the country is lacking and could be useful to point to the most efficacious products and scenarios where efficacy can be improved. The objectives of this work were to a) systematically review the peer- and non- peer reviewed literature on chemical control studies for FHB management conducted in Brazil during the last 15 years and b) conduct a meta-analysis of the control efficacy and yield return of selected fungicides. A search in the literature identified 18 studies that reported FHB severity (same as FHB index) for least one fungicide treatment compared to a non-treated check, and also a measure of sampling variance in the trial. Only three and two studies reported mean values of Fusarium-damaged kernels (FDK) and deoxyninvalenol (DON), respectively. Studies were scrutinized and eight fungicide treatments with at least 10 entries were selected for the meta-analysis study. They included DMI (tebuconazole, metconazole and propiconazole) and MBC (carbendazim) fungicide treatments formulated solo and a pre-mix of DMI+QoI (azoxystrobin + tebuconazole, pyraclostrobin + metconazole, trifloxystrobin + prothioconazole and trifloxystrobin + tebuconazole). One (×1) and two applications (×2) were tested, the first at full flowering and the second 10 days later. A multivariate meta-analytic model was fitted to the log of the means for each fungicide and check treatment of the trial. The difference in the mean estimates of the log of severity between a fungicide treatment and the check were used to calculate the mean response ratio represented by percentage control efficacy (C) and percentage yield return (P). Mean C and P ranged from 48.94 to 70.39% and from 7.41 to 21.29%, respectively. Metconazole (×2) performed best to reduce disease severity (C = 70.39%) and tebuconazole (×1) to increase yield (P = 21.29%). Propiconazole was the least efficacious among the DMIs (48.94%), followed by carbendazim (53.9%). In general, the most efficacious fungicides resulted in higher yield return. When fungicide groups were compared, the DMIs and the mixtures performed best in general, especially in trials conducted under high disease pressure (>7% in the check treatment). Results showed that mean control efficacy in Brazilian studies when using two sprays are higher one spray, as reported in other meta-analysis study in North America. Future studies should focus on the effect on DON levels, especially for the mixtures, which are strategically used for the control foliar diseases.

Doctor of Philosophy / Department of Plant Pathology / Robert L. Bowden / John F. Leslie / Gibberella zeae is an ascomycete filamentous fungus and the major cause of Fusarium head blight, also called scab, in small grains. This dissertation contains three related studies of G. zeae. In the first, the genetic map was aligned with the first assembly of the genomic sequence released by The Broad Institute (Cambridge, MA). Approximately 99% of the sequence was anchored to the genetic map, indicating the high quality of the sequence assembly and validity of the genetic map. The alignments grouped the linkage groups and supercontigs into four sets, which is consistent with the hypothesis that there are four chromosomes in this fungus. In the second, the sex pheromone precursor genes (ppg1 and ppg2) and the pheromone receptor genes (pre1 and pre2) were identified and characterized. Deletion of ppg1 or pre2 ([Delta]ppg1 or [Delta]pre2 strains) reduced the number of perithecia produced by self-fertilization, but did not completely block perithecial formation. The proportion of crosses resulting from outcrossing increased when the [Delta]ppg1 strains were used as the female in crosses with male strains containing an intact ppg1 gene. [Delta]ppg2 and [Delta]pre1 mutants had no discernable effect on morphological phenotype or self-fertilization. Thus, one of the pheromone/receptor pairs (ppg1/pre2) found in many Ascomycetes has a role in, but is not essential for, selfing or outcrossing in G. zeae, whereas the other pheromone/receptor pair (ppg2/pre1) no longer has a detectable function in sexual reproduction. In the third study, spore germination of G. zeae was tested in the presence of α- factor-like pheromone peptides of G. zeae or N. crassa. The pheromone peptide of N. crassa more efficiently inhibited spore germination than did the peptide from G. zeae. Arginine and lysine residues were the most important determinants in blocking spore germination. In conclusion, this research has validated the genetic map and the genomic assembly of G. zeae, characterized sex pheromone functions and characterized pheromone peptide ability to inhibit spore germination. The pheromone peptides of G. zeae and N. crassa may be useful as control agents for G. zeae and pheromone peptide efficacy might be further enhanced by judicious substitutions for some of the amino acids.