<p>Polyunsaturated fatty acids can be bioactivated by two families of dioxygenases, which either contain non-heme iron (lipoxygenases) or heme (cyclooxygenases, linoleate diol synthases and α-dioxygenases).</p><p>Lipoxygenases and their products play important roles in the pathophysiology of plants and fungi. The only known lipoxygenase with catalytic manganese (Mn-lipoxygenase) is secreted by a devastating root pathogen of wheat, the Take-all fungus <i>Gaeumannomyces graminis</i>. Its mycelia also contains linoleate diol synthase (LDS), which can oxidize linoleic acid to sporulation hormones.</p><p>Mn-lipoxygenase belongs to the lipoxygenase gene family. Recombinant Mn-lipoxygenase was successfully expressed in the yeast <i>Pichia pastoris</i> with an expression level of 30 mg/L in fermentor culture. The tentative metal ligands of Mn-lipoxygenase were studied by site-directed mutagenesis. The results show that four residues His-274, His-278, His-462 and the C-terminal Val-602 likely coordinate manganese, as predicted by sequence alignments with Fe lipoxygenases.</p><p>Mn-lipoxygenase (~100 kDa) contains an Asp-Pro peptide bond in the N-terminal region, which appears to hydrolyze during storage and in the acidic media during Pichia expression to an active enzyme of smaller size, mini-Mn-lipoxygenase (~70 kDa). The active form of Mn-lipoxygenase can oxygenate fatty acids of variable chain length, suggesting that the fatty acids enter the catalytic site with the ω-end (“tail first”).</p><p>Mn-lipoxygenase is an <i>R</i>-lipoxygenase with a conserved Gly316 residue known as a determinant of stereospecificity in other <i>R/S</i> lipoxygenases. The Gly316Ala mutant showed an increased hydroperoxide isomerase activity and transformed 18:3n-3 and 17:3n-3 to epoxyalcohols.</p><p>The genome of the rice blast fungus, <i>Magnaporthe grisea</i>, contains putative genes of lipoxygenases and LDS. Mycelia of <i>M. grisea</i> were found to express LDS activity. This enzyme was cloned and sequenced and showed 65% amino acid identity with LDS from <i>G.graminis</i>. </p><p>Take-all and the rice blast fungi represent a constant threat to staple foods worldwide. Mn-lipoxygenase and LDS might provide new means to combat these pathogens.</p>

Polyunsaturated fatty acids can be bioactivated by two families of dioxygenases, which either contain non-heme iron (lipoxygenases) or heme (cyclooxygenases, linoleate diol synthases and α-dioxygenases). Lipoxygenases and their products play important roles in the pathophysiology of plants and fungi. The only known lipoxygenase with catalytic manganese (Mn-lipoxygenase) is secreted by a devastating root pathogen of wheat, the Take-all fungus Gaeumannomyces graminis. Its mycelia also contains linoleate diol synthase (LDS), which can oxidize linoleic acid to sporulation hormones. Mn-lipoxygenase belongs to the lipoxygenase gene family. Recombinant Mn-lipoxygenase was successfully expressed in the yeast Pichia pastoris with an expression level of 30 mg/L in fermentor culture. The tentative metal ligands of Mn-lipoxygenase were studied by site-directed mutagenesis. The results show that four residues His-274, His-278, His-462 and the C-terminal Val-602 likely coordinate manganese, as predicted by sequence alignments with Fe lipoxygenases. Mn-lipoxygenase (~100 kDa) contains an Asp-Pro peptide bond in the N-terminal region, which appears to hydrolyze during storage and in the acidic media during Pichia expression to an active enzyme of smaller size, mini-Mn-lipoxygenase (~70 kDa). The active form of Mn-lipoxygenase can oxygenate fatty acids of variable chain length, suggesting that the fatty acids enter the catalytic site with the ω-end (“tail first”). Mn-lipoxygenase is an R-lipoxygenase with a conserved Gly316 residue known as a determinant of stereospecificity in other R/S lipoxygenases. The Gly316Ala mutant showed an increased hydroperoxide isomerase activity and transformed 18:3n-3 and 17:3n-3 to epoxyalcohols. The genome of the rice blast fungus, Magnaporthe grisea, contains putative genes of lipoxygenases and LDS. Mycelia of M. grisea were found to express LDS activity. This enzyme was cloned and sequenced and showed 65% amino acid identity with LDS from G.graminis. Take-all and the rice blast fungi represent a constant threat to staple foods worldwide. Mn-lipoxygenase and LDS might provide new means to combat these pathogens.

Finger millet is the second most important cereal in Uganda after maize. The yields however, have remained low due to several constraints, such as finger millet blast disease and limited technology options. Therefore breeding investigations were conducted to determine farmer preferred traits, genetic variation, combining ability and genetic effects for head blast disease and head shapes, and other quantitative traits in finger millet. Among other traits, farmers preferred high grain yield potential, brown seed colour, compact head shape, tolerance to blast disease, high tillering ability, medium plant height, early maturity, tolerance to shattering and ease of threshing in new finger millet varieties. Path coefficient analysis indicated that the most important traits were grain mass head-1, tillering ability and reaction to head blast disease. Overall, the high heritabilities and genetic advance (GA) as a percentage of mean revealed the existence of variability which can be utilised through selection and/or hybridisation. The genotype x environment interaction (GEI) and stability analysis showed significant differences due to genotypes (58%), environments (10%) and GEI (32%). Twelve genotypes that combined high yield potential and stability were identified for advancement in the program. Both general (GCA) and specific combining ability (SCA) were significant for most traits, but GCA effects were more important for all the traits except for number of fingers head-1, finger width and panicle width. The Hayman genetic analysis confirmed importance of additive gene action for most of the traits and that additive-dominance model was adequate for explaining genetic variation in finger millet. The results also indicated that yield was controlled by recessive genes whereas blast resistance was controlled by dominant genes. At least two genes, probably three gene pairs and their interactions seemed to control head shape in finger millet. The interactions observed suggest recessive and dominant epistasis, and probably an inhibitor were involved. Seemingly, the gene for curving of fingers, when present in a dominant form prohibits opening of the heads; whereas the recessive form leads to open head shape irrespective of the gene conditions in the other loci. This study forms the baseline for future investigations and the basis for devising breeding strategy on finger millet head shapes. / Thesis (Ph.D.)-University of KwaZulu-Natal, Pietermaritzburg, 2013.

Finger millet (Eleusine coracana) productivity in East Africa has remained low in all production agro-ecologies for decades owing to the low yielding potential of existing that are susceptible to the blast disease caused by the fungus Magnaporthe grisea (Hebert) Barr. and the limited research on the crop. The region holds large finger millet germpasm collections whose value is not yet been fully exploited. However, with the ongoing breeding efforts through hybridization, there is a need to comprehensively characterize the germplasm to identify valuable traits to address biotic and abiotic stresses that affect finger millet productivity. Studies on gene action and inheritance of key traits that contribute to yield improvement are also required to help formulate an effective breeding strategy for finger millet improvement. The objectives of this study were to (i) determine the genetic diversity in a set of germplasm from East Africa (ii) determine association between grain yield and its component traits (iii) identify genotypes for target production agro-ecologies (iv) identify blast resistant finger millet genotypes for use in breeding and production and (v) generate information on the inheritance of blast, grain yield and yield components for the development of an effective breeding strategy. A total of 340 finger millet accessions were collected from three countries in East Africa: Kenya, Tanzania and Uganda and 80 global minicore accessions sourced from ICRISAT-India. High phenotypic variability in the germplasm was recorded for 23 quantitative traits, blast reaction and five qualitative traits. Both morphological and molecular characterization (using SSR markers) of the 340 accessions revealed higher diversity within than among the countries Kenya, Tanzania and Uganda. Seven morphological clusters and three major genetic clusters were detected. Morphological diversity delineation was largely influenced by leaf sheath length, plant height, peduncle length, panicle exertion and grain yield. The mean polymorphic information content (PIC) of 19 polymorphic markers was 0.606 with mean alleles of 195 with sizes that ranged from 148-474 base pairs. The Kenyan and Tanzanian accessions had higher diversity than the Ugandan with the Kenyan and Ugandan, and the Kenyan and Tanzanian accessions being closely related than the Tanzanian and Ugandan. The low diversity in the Ugandan accessions could be attributed to higher research intervention in the country leading to the promotion and use of improved cultivars. Efforts have to be directed towards collection and conservation of valuable diversity before it is lost. The diversity in plant height, maturity, yield and blast reaction and the cluster groups detected in the germplasm should provide a basis for finger millet improvement through hybridization and selection. Higher genotypic than phenotypic correlations were recorded for most of the traits studied with grain yield having high positive correlations with finger width, grains per spikelet, threshing percent, peduncle length and panicle exertion. Both grain yield and days to flowering had negative correlations with all three blast types (leaf, neck and finger). Path coefficient analysis revealed that productive tillers per plant, 1000 grain mass, grains per spikelet and threshing percent had positive direct genetic effects on grain yield with strong indirect effects from several of the other traits which necessitates simultaneous selection for those traits with strong direct effects and those with strong indirect effects for grain yield improvement. High broad sense heritability estimates and high genetic advance as percent of mean were recorded in fingers per panicle, flag leaf sheath length, 1000 grain mass, finger length, peduncle length, panicle exertion, number of leaves per plant and leaf sheath length probably indicating the predominance of additive gene effects in controlling these traits hence the potential for improvement through selection. Adaptation and stability analysis using the GGE biplot model identified Lanet 2012 long rains, Serere 2012 long rains and Miwaleni 2012 long rains as the most discriminating environments for the low temperature, sub-humid mid altitude and dry lowland areas, respectively. Alupe 2012 long rains was the ideal environment for genotype discrimination for blast while Lanet 2012 long rains was best for grain yield. Genotypes G3, G5, G17, G25, G28, G36 and G71 were identified as being stable across environments and G1, G18, G19, G37, G54, G61, G74, G75, and G77 were found ideal for specific adaptation. Disease severity scores were highly negatively (P<0.01) correlated with days to flowering and grain yield suggesting that early lines suffered more disease damage leading to reduced yield. Resistant genotypes were slow blasting (probably associated with horizontal resistance) which may enable them to withstand blast pathogen variability for longer periods. Nine genotypes were identified with high resistance to blast and will be useful for breeding as blast resistance sources. Resistant genotypes had low AUDPC values and disease severity rating for the three blast types and vice-versa for susceptible genotypes. Further investigations need to be carried out to determine the possibility of the three blast types being controlled by the same genes. Early maturing blast susceptible genotypes with good yield potential could be utilized in areas with low blast prevalence. To understand the gene action for inheritance of the various traits 16 F2 families plus their four female and four male parents were evaluated at Alupe and Kakamega western Kenya under artificial blast inoculation. Significant additive genetic effects were recorded for all traits (except for finger width and grains per spikelet) meaning that improvement for these traits would be possible through the common selection methods for self pollinating crops. Parent lines KNE 392, and KNE 744 and IE 11 were found to be suitable for blast resistance breeding while Okhale 1 was found to be suitable for high grain yield and blast resistance improvement due to their high desirable GCA effects. Most of the F2 families showed transgressive segregation for the three blast types in either direction which gives hope for the development new pure lines with better blast resistance than the parents. Crosses IE 3104 x KNE 796, KAT FM 1 x Okhale 1, IE 11 x Okhale, IE 11 x P 224 and KNE 744 x KNE 392 have potential to generate lines with blast resistance due to their high desirable SCA effects. The F2 segregation distributions for blast indicated quantitative inheritance. However the one to four minimum number of genes (effective factors) detected for resistance control in all the three blast types was not in sync with the segregation patterns in the F2 families and further investigations are required. There were differences in segregation patterns between crosses which may suggest the presence of different resistance genes in the different parents used. This would call for gene pyramiding for durable resistance. These results confirm the potential of sourcing valuable parental stocks in the local germplasm for the development of genotypes to improve finger millet productivity in East Africa. Already some of the high yielding and blast resistant genotypes identified here have been incorporated in the regional cultivar trials. The diversity information generated will facilitate effective conservation and utilization of this germplasm. Results of gene action for inheritance of the various traits from this study will enable breeders to develop sound breeding strategies for finger millet improvement in the region. / Thesis (Ph.D.)-University of KwaZulu-Natal, Pietermaritzburg, 2013.

Made available in DSpace on 2014-07-29T15:16:30Z (GMT). No. of bitstreams: 1 DISSERTACAO DE MESTRADO FINAL.pdf: 1117246 bytes, checksum: 91bcbcfc247218ffb970c121596f7b0c (MD5) Previous issue date: 2010-08-30 / The vegetal biomass consists mainly of cellulose, hemicellulose and lignin. Cellulose is the most abundant polymer and xylan is the main component of hemicellulose. The conversion of cellulose and xylan to glucose and xylose can be realized by an enzymatic complex found in secretions of microorganisms such as fungi and bacteria. Enzymatic hydrolysis is an important step to the bioconversion of cellulosic and hemicellulosic fraction from lignocellulosic wastes. The thermophilic fungus Humicola grisea var thermoidea produces an efficient complex of cellulolytic enzymes (endoglucanases, cellobiohydrolases and &#946;-glucosidase) and xylanolytic (endoxylanase and &#946;-xylosidase) with high thermostability when grown on different lignocellulosic substrates. The aim of this study was to analyze the kinetics of production of cellulases and xylanase by the fungus H. grisea cultivated on medium containing rice straw (RS), corncob (CC), crushed cane sugar bagasse (CSB) and wheat bran (WB) as carbon source and subsequently analyze the profile of proteins with cellulolytic and xylanolytic activity secreted by the fungus when grown in minimal medium, by liquid fermentation, containing the substrates at concentrations of 1, 2 and 3% and maintained at 42 ° C, 120 rpm for different times. The best results were obtained when the fungus was grown in 3% BCA and FT, the peaks of FPase (0.17 U / mL) and CMCase (3.54 U / mL) were observed after 192 h of growth with 3% BCA , peak avicelase (0,195 U / mL) after 48 h with 3% FT and peak xylanase (23.75 U / mL) after 216 h with 3% FT. The results showed that the best inducer of enzyme production with FPase and CMCase activity was the CSB and the best inducer of enzymes production with xylanase and avicelase activity was the WB. In profile analysis of proteins secreted by H. grisea by SDS-PAGE (216 h) and zymogram (144 h), no band was seen when the fungus was grown in the presence of glucose, suggesting catabolite repression. However, two very strong protein bands corresponding to HXYN2 (23 kDa) and CBH1.2 (47 kDa) were visualized in the gels containing CSB (2 to 3%) and WB (2 and 3%). These enzymes are the main xylanolytic and cellulolytic systems of the fungus, respectively. Were monitored by recombinant enzymes from H. grisea (in gels), an endoxylanase HXYN2r (23 kDa), an cellobiohydrolase CBH1.2r (47 kDa). The masses full profile of H. grisea can be seen in Figures 13, 14, 15, 16, 18 and 19. / A biomassa vegetal é constituída principalmente de celulose, hemicelulose e lignina. A celulose é o polímero mais abundante e a xilana o principal componente hemicelulósico. A conversão da celulose e da xilana à glicose e xilose pode ser realizada por um complexo enzimático encontrado nas secreções de microrganismos tais como fungos e bactérias. A hidrólise enzimática é um importante passo para a bioconversão da fração celulósica e hemicelulósica de resíduos lignocelulósicos. O fungo termofílico Humicola grisea var thermoidea produz um eficiente complexo de enzimas celulolíticas (endoglicanases, celobiohidrolases e &#946;-glicosidases) e xilanolíticas (endoxilanases e &#946;-xilosidase) com alta termoestabilidade quando cultivado em diferentes substratos lignocelulósicos. O objetivo desse trabalho foi analisar a cinética de produção de celulases e xilanases pelo fungo H. grisea cultivado em meio contendo palha de arroz (PA), sabugo de milho (SM), bagaço de cana-de-açúcar (BCA) e farelo de trigo (FT) como fonte de carbono e posteriormente analisar o perfil de proteínas com atividade celulolítica e xilanolítica secretadas pelo fungo quando cultivado em meio mínimo, por fermentação líquida, contendo os substratos nas concentrações de 1, 2 e 3%, e mantidos a 42 °C, 120 rpm por diferentes tempos. Os melhores resultados foram obtidos quando o fungo foi cultivado em 3% de BCA e FT, sendo que os picos de FPase (0.17 U/mL) e CMCase (3.54 U/mL) foram observados após 192 e 240 h respectivamente de crescimento com 3% de BCA, o pico de Avicelase (0.195 U/mL) após 48 h com 3% de FT e o pico de xilanase (23.75 U/mL) após 216 h com 3% de FT. Os resultados demonstraram que o melhor indutor da produção de enzimas com atividade de FPAse e CMCase foi o BCA e o melhor indutor da produção de enzimas com atividade de Avicelase e xilanase foi o FT. Na análise do perfil de proteínas secretadas pelo H. grisea por SDS-PAGE (216 h) e zimograma (144 h), nenhuma banda foi visualizada quando o fungo foi cultivado na presença de glicose, sugerindo repressão catabólica. Entretanto, duas bandas protéicas muito fortes, correspondentes à HXYN2 (23 kDa) e CBH1.2 (47 kDa) foram visualizadas nos géis contendo BCA (2 e 3%) e FT (2 e 3%); e representam as principais enzimas dos sistemas xilanolítico e celulolítico do fungo, respectivamente. Estas foram monitoradas pelas enzimas recombinantes do H. grisea (nos geis): uma endoxilanase HXYN2r (23 kDa) e uma celobiohidrolase CBH1.2r (47 kDa). As massas do perfil completo do H. grisea podem ser vistas nas Figuras 13-19.

This work describes the study chemical and pharmacological of the extracts and compounds isolated from two species of Rubiaceae (Sabicea grisea Cham. & Schltdl. var. grisea and Psychotria barbiflora DC.). Extracts and fractions of S. grisea, and fractions which showed positive reactions record to alkaloids of P. barbiflora, exhibited a significant reduction in the nociceptive response, with inhibition upper or comparable to the reference drug, indomethacin. The chemical study of fractions from leaves and stems of S. grisea, guided by pharmacological tests, resulted to the isolation of two saturated alcohols, octacosanol and hexacosanol, two pentacyclic triterpenes oleanane serie, siaresinolic and 3-O-α-L-rhamnopyranosyl-(1→2)-α-Larabinopyranosylsiaresinolic acids, vanillic and salicylic acids, scopoletin, ethyl caffeate and two phytosteroids, β-sitosterol and 3-O-β-D-glucopyranosylsitosterol. It is noteworthy that no previous reports about the presence of these compounds in the genus Sabicea. Moreover, no record was found on the 3-O-α-L-rhamnopyranosyl-(1→2)-α-L-arabinopyranosyl-siaresinolic acid, named Gottliebside, an simple tribute to the greatest Natural Products Chemistry of the Brazil, Prof. Otto Richard Gottlieb, therefore, a new natural product. The chemical study of alkaloidal fractions from leaves and stems of P. barbiflora led to the isolation of two β-carboline alkaloids, harman and stricnosidinic acid, whose occurrences corroborate the inclusion of this species in the subgenus Heteropsychotria. In the pharmacological tests, octacosanol (10 and 1 mg/kg, i.p) and siaresinolic acid (1 and 0.1 mg/kg, i.p) showed peripheral antinociceptive effects mediated by α2-adrenergic receptors and channel K+ ATPdependent, respectively. Furthermore, these compounds inhibited the accumulation of inflammatory cells, as well as TNF-α in the model of carrageenan-induced inflammation, indicating an anti-inflammatory action. In addition, these compounds (≤200 μg/ml and ≤ 50 μg/ml, respectively) were unable to induce cell death in when evaluated in the reduction of 3-(4,5-dimethythiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) test, demonstrating the absence of cytotoxicity of these compounds. / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / Este trabalho descreve o estudo químico e farmacológico dos extratos e compostos isolados de duas espécies de Rubiaceae (Sabicea grisea Cham. & Schltdl. var. grisea e Psychotria barbiflora DC.). Extratos e frações de S. grisea, bem como frações com reações positivas para alcaloides obtidas de P. barbiflora, exibiram redução significativa da resposta nociceptiva, com inibição superior ou comparável ao fármaco de referência, indometacina. O estudo químico das frações das folhas e caule de S. grisea, guiados pelos ensaios farmacológicos, resultou no isolamento de dois alcoóis saturados, octacosanol e hexacosanol, dois triterpenos pentacíclicos da série oleanano, ácidos siaresinólico e 3-O-α-L-rhamnopiranosil-(1→2)-α-Larabinopiranosilsiaresinólico, os ácidos salicílico e vanílico, a escopoletina, o cafeato de etila, além dos fitoesteroides, o β-sitosterol e 3-O-β-D-glicopiranosilsitosterol. Não há relatos sobre a presença destes compostos no gênero Sabicea e não foi encontrado registro sobre o ácido 3-O-α-L-rhamnopiranosil-(1→2)-α-Larabinopiranosilsiaresinólico, nomeado de Gottliebsídeo, uma singela homenagem ao maior Químico em Produtos Naturais do Brasil, Prof. Otto Richard Gottlieb, sendo, portanto, um novo produto natural. O estudo químico das frações alcaloídicas das folhas e caule de P. barbiflora conduziu, pela primeira vez, ao isolamento de dois alcaloides β-carbolínicos, harmana e ácido estrictosidínico, cujas ocorrências corroboram com a inclusão desta espécie no subgênero Heteropsychotria. Nos ensaios farmacológicos, o octacosanol (10 e 1 mg/kg, i.p) e o ácido siaresinólico (1 e 0,1 mg/kg, i.p) apresentaram efeitos antinociceptivos periféricos mediado por receptores α2-adrenérgicos e canais de K+ATP-dependente, respectivamente. Além disso, estes compostos inibiram o acúmulo de células inflamatórias, bem como os níveis de TNF-α no modelo de inflamação induzida por carragenina, indicando uma ação anti-inflamatória. Em adição, o octacosanol (≤ 200 μg/ml) e o ácido siaresinólico (≤ 50 μg/ml) não foram capazes de induzir morte nas célula quando avaliados no teste de redução do brometo de 3-(4,5-dimetiltiazol-2-il)-2,5-difeniltetrazólio (MTT), revelando ausência de citotoxicidade destes compostos.

Made available in DSpace on 2014-07-29T12:03:35Z (GMT). No. of bitstreams: 1 Tese_Cristine_Cysneiros.pdf: 1284054 bytes, checksum: c3e0f947790961725c6b67bf30fac547 (MD5) Previous issue date: 2009-04-03 / The objective of this work was to produce and characterize four multi enzyme complexes from the fungus Humicola grisea var. thermoidea, maintained for 96 hours at 42oC in growth media containing different carbon sources: Marandu grass; soybean seedcoats; Tifton 85 hay; and corn forage. Different amounts of cellulase, xylanase and &#61538;-glucosidase enzymes were obtained depending on the different carbon sources. Cellulase presented increased activity in temperatures between 40ºC and 50ºC. The observed optimum temperature range for xylanase and &#946;-glycosidase was from 50ºC and 60ºC. Optimum pH for cellulase activity was 6.0 when fungus growth occurred in Tifton hay, corn forage, and soybean seedcoats. When the enzyme was obtained from medium containing Marandu grass, optimum enzyme activity occurred at pH 5.5. Regardless of the carbon source, xylanase activity was higher at pH 6.0. As for &#946;-glucosidase, optimum activity was observed at pH 5.5 for Tifton media as compared to pH 6.5 for corn and soybean containing media. For grass Marandu, the activity of the enzyme was maximum in the range 5.5 to 6.5. Cellulase produced from all growth media were maintained stable after incubation for 60 minutes at 39°C. Xylanase presented thermal stability during 240 minute incubation period at 50°C. Activity stability of &#946;-glycosidase varied according to carbon source and presented 66.7 to 125.75% activity at 50°C for 240 minutes. / Enzimas fibrolíticas exógenas são produzidas por cultura específica de bactérias ou fungos. São essenciais aos animais por estarem envolvidas na hidrólise dos componentes complexos das dietas em moléculas orgânicas mais simples como glicose, celobiose, xilose, aminoácidos, ácidos graxos, que são então usadas pelos microrganismos do rúmen e/ou pelo animal. Melhoras no desempenho dos ruminantes devido ao uso de enzimas fibrolíticas são atribuídas principalmente à maior degradação da fibra no rúmen, o que resulta em aumento da ingestão de energia disponível pelos animais. Os objetivos deste trabalho foram os de produzir e caracterizar quatro soluções enzimáticas, utilizando o fungo Humicola grisea var. thermoidea e avaliar seus efeitos por meio da digestibilidade verdadeira in vitro da matéria seca de quatro substratos: capim Marandu, casquinha de soja, feno de Tifton-85 e forragem de milho. As soluções enzimáticas foram produzidas a partir de quatro meios de culturas diferentes, contendo a fonte de carbono específica, durante 96 horas de cultivo, a 42°C. Foi observado que o fungo produziu as enzimas celulases, xilanase e &#61538;-glicosidase em diferentes concentrações, o que foi dependente da fonte de carbono. A caracterização bioquímica mostrou que a celulase produzida apresentou maior atividade em temperatura entre 40ºC e 50°C. A temperatura ótima de xilanase e &#946;-glicosidase foi entre 50 e 60°C. O pH ótimo da enzima celulase foi 6,0, quando o fungo cresceu em feno de Tifton, forragem de milho e casquinha de soja. Para o capim Marandu, a enzima apresentou atividade ótima em pH 5,5. Para as quatro fontes de carbono, a xilanase produzida apresentou pH ótimo de 6,0. Em relação a &#946;-glicosidase, a atividade enzimática foi maior em pH 5,5, no meio com feno de Tifton. Para capim Marandu, a atividade da enzima foi máxima na faixa de 5,5 a 6,5. Quanto à forragem de milho e casquinha de soja, a enzima exibiu maior atividade em pH 6,5. A celulase produzida, nas quatro fontes de carbono, permaneceu estável após a incubação por 60 minutos, a 39°C. Xilanase produzida apresentou estabilidade térmica durante 240 minutos de incubação, a 50°C. A &#946;-glicosidase, dependendo da fonte de carbono, manteve de 66,7 a 125,75% de sua atividade, a 50°C, durante 240 minutos. Para avaliar o potencial das soluções enzimáticas sobre a digestibilidade in vitro dos substratos, 2,5; 5,0 e 10 mL de cada solução foram aplicados, por aspersão, em 17 g dos seus respectivos substratos, moídos em peneira com malha de 1 mm de diâmetro. Após aspersão, as enzimas ficaram em contato com os substratos por 2 e 24 h (tempo de reação enzima-substrato), antes de serem incubados no rúmen. A digestibilidade in vitro da MS foi avaliada em líquido ruminal tamponado, durante o período de 12, 24, 48 e 96 h. Para cada substrato, foram incubados 34 sacos (4 níveis de enzimas x 4 períodos de incubação x 2 repetições x 1 branco x 1 testemunha). As soluções enzimáticas, em qualquer nível de enzimas, quando comparados aos tratamentos controle, aumentaram a digestibilidade da MS dos substratos, nos tempos de reação enzima-substrato e período de incubação no rúmen. Este estudo mostrou que enzimas fibrolíticas exógenas produzidas por H. grisea tem potencial para uso como aditivo em dietas de ruminantes

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Diversiteten bland gener som ger resistens (R) mot infektionssjukdomar är mycket stor i växtriket, särskilt i den effektorutlösta klassen av försvaret. Denna artikel beskriver interaktioner mellan växters effektorutlösta fösvar och patogeners effektorproteiner och undersöker evolutionära och genetiska mekanismer för uppkomsten av ny resistens. R-genprodukter har en typisk domänstruktur och interagerar både direkt och indirekt med patogeners effektorproteiner.Vid kontakt med ett effektorprotein utlöses en försvarsrespons som kan förhindra fortsatt patogen tillväxt. Flera genetiska mekanismer verkar samtidigt på R-gener, något som resulterar i en hög hastighet för uppkomst av nya R-varianter. Den intima samevolution som existerar hos många patogen-växtsystem formar det evolutionära utvecklingsmönstret av R-gener. Diversifierande positiv selektion via den biologiska kapprustningsmodellen och bevarande negativ frekvensberoende selektion är båda viktiga samevolutionära processer. Denna artikel indikerar att växters immunförsvar är anpassningsbart och robust, men belyser också den kunskapsbrist som råder inom resistensforskningen. / The diversity among genes conferring resistance (R) against infectious diseases is very high in the plant kingdom, especially in the effector triggered class of defense. This article describes the interactions between the plant effector triggered immunity and pathogen effectors and examines the evolutionary and genetic mechanisms for the emergence of new resistance. R gene products have a typical domain structure and interacts both directly and indirectly with pathogen effectors. Upon contact with an effector a defense response is triggered that may prevent the further pathogen growth. Multiple genetic mechanisms act simultaneously on the R-genes, which results in a rapid diversification of novel R variants. The intimate co-evolution of many existing plant-pathogen systems form the evolutionary pattern of R genes. Diversifying positive selection via the biological arms race model and conservative negative frequency-dependent selection are both important coevolutionary processes. This article indicates that the plant immune system is adaptive and robust, but also highlights the lack of knowledge in plant resistance research.