This study addressed the possibility of rice blast fungus (Magnaporthe grisea) as a potential bioherbicide for the control of crabgrass (Digitaria sanguinalis), in turf/lawn plantings. Experiments were: (i) evaluation of the effect of bioherbicide inoculum dosage (titer) on disease severity in crabgrass; (ii) competition between crabgrass and tall fescue (Festuca arundinacea Schreb. 'Fawn') and the additive effect of rice blast bioherbicide on the competitiveness of crabgrass; (iii) survival of rice blast spores under non-conducive infection conditions on crabgrass; and (iv) effect of multiple inoculations of rice blast fungus and subsequent disease on crabgrass.Spore concentrations of 5 x 105 to 1 x 106 conidia/mI had no significant difference in effect on mean biomass reduction of treated crabgrass. All produced tissue death and loss of vigor in treated crabgrass, but no plant death. In one trial, a spore concentration of 2 x 106 conidia/ml produced 100% biomass loss in treated crabgrass.Competition studies indicated crabgrass will out-compete fescue 'Fawn' in plots of equal numbers of each plant, i.e., 3 fescue and 3 crabgrass. In 10 x 10 cm mini-lawns of fescue interspersed with 5 crabgrass plants, fescue will out-compete the crabgrass. In mini-lawns of fescue and crabgrass, infection of crabgrass with non-lethal doses of rice blast fungus improved fescue's ability to out-compete crabgrass. Three methods of investigating competition were tested. Mini-lawns sown with 0.5 g fescue seed (approx. 200 seeds) with crabgrass seed sown into centers of the four quadrants and in center of the mini-lawn, worked best.Survival of Magnaporthe grisea conidia under non-conducive conditions was tested by drying inoculated crabgrass seedlings for 0-48 hours prior to dew-deposition. Treatments verified spores remain viable at least 24 hours on crabgrass leaves under greenhouse conditions (25-40° C and 25-75% relative humidity).Two successive inoculations (at 3 days and 7 days after primary inoculation) of crabgrass with M. grisea conidia (5 x 105 conidia/ml) resulted in significant reduction in growth compared to a single spore application.Results suggested Magnaporthe grisea exhibits potential for future development as a commercial bioherbicide to control crabgrass (Digitaria sanquinalis). Current studies investigated integration in lawn grass, but should be applicable to situations anywhere crabgrass is considered a weed e.g., in corn (Zea mans) or soybeans (Glycine max). / Department of Biology

Thesis (Ph. D.)--Ohio State University, 2005. / Available online via OhioLINK's ETD Center; full text release delayed at author's request until 2006 June 1.

München, Techn. Univ., Diss., 2004.

A cultura de trigo (Triticum aestivum L.) é a segunda maior do mundo e o Brasil ocupa o segundo lugar de produção na América do sul. Entretanto, a produtividade desta cultura pode ser limitada devido à ocorrência de doenças como a brusone, causada pelo fungo Pyricularia grisea e a doença denominada giberela, causada pelo fungo Fusarium graminearum Populações bacterianas associadas à rizosfera de trigo podem apresentar potencial como agentes de controle biológico de diferentes fitopatógenos. Neste contexto, esta pesquisa foi direcionada ao estudo da composição da comunidade bacteriana rizosférica do trigo e a busca por micro-organismos com potencial para o controle biológico da brusone e da giberela. Assim, para melhor compreensão das comunidades associadas ao trigo, foram realizadas coletas em duas regiões diferentes no Brasil, sendo possível a obtenção de 606 estirpes entre bactérias e actinobactérias da rizosfera do trigo e de solo de cultivo da mesma cultura. Destas, 16 apresentaram, em testes in vitro, potencial antagonista diante dos fungos fitopatogênicos Pyricularia grisea e Fusarium graminearum com diferentes porcentagens de inibição. Dez dos isolados selecionados apresentaram similaridade com a família Streptomycetaceae, porém, quatro linhagens necessitam de estudos mais detalhados, pois a similaridade foi baixa, podendo indicar uma espécie ainda não descrita; quatro linhagens demonstraram similaridade com a família Bacillaceae e dois com a família Paenibacillaceae. Na avaliação de produção de metabólitos secundários com efeito inibitório, apenas dez apresentam potencial, porém estudos mais detalhados se fazem necessários para a confirmação deste mecanismo. A análise de diversidade bacteriana demonstrou uma maior abundância do filo Actinobacteria, seguido pelo filo Proteobacteria e Acidobacteria em ambas as áreas amostradas, entretanto, o filo Acidobacteria foi o que demonstrou a maior variação entre as classes presentes nas diferentes regiões estudadas, indicando uma seleção da comunidade de acordo com a variedade do cultivar e o estádio de desenvolvimento do vegetal. A comunidade bacteriana de trigo apresenta micro-organismos com potencial para a inibição dos fungos causadores da brusone e da giberela, porém o efeito destas linhagens deve ser melhor investigado em condições de campo. A compreensão das comunidades bacterianas associadas ao trigo pode se apresentar como uma importante ferramenta para direcionar a busca por antagonistas. / Wheat (Triticum aestivum) is the second largest crop in the world and Brazil is in the second position in the ranking of production in South America. However, its productivity can be limited due to the occurrence of diseases like wheat blast, caused by the fungus Pyricularia grisea and the disease called Fusarium head blight (FHB), caused by the fungus Fusarium graminearum. Bacterial populations associated to wheat rhizosphere may have potential to act as biological control agents of different plant pathogens. In this context, this research aimed to look at wheat rhizosphere bacterial community and the pursuit of microorganisms with potential for the biological control of wheat blast and FHB. Given this, in order to study wheat bacterial communities, data collection was carried out in two different regions in Brazil, returning 606 bacterial and actinomycetes isolates from wheat rhizosphere and bulk soil. Among these,, 16 strains revealed antagonistic potential against both plant pathogens Pyricularia grisea and Fusarium graminearum, with different percentages of inhibition. Ten strains were selected out of the 16 and showed similarity with the family Streptomycetaceae, whereas four of them displayed a low similarity, requiring a deeper analysis and might indicate new species. Four isolates showed similarity with the family Bacillaceae and two with the family Paenibacillaceae. On the assessment of production of secondary metabolites with inhibitory effects, only ten strains were positive, but more detailed studies are necessary to confirm this mechanism. The analysis of bacterial diversity revealed a larger abundance of the phylum Actinobacteria, followed by the phylum Proteobacteria and Acidobacteria in both areas, however, the phylum Acidobacteria revealed more variation among its classes when both araes were compared, indicating a selection of the community according to the cultivar and the developmental stage. Wheat bacterial community presents microorganism with inhibition potential against fungi responsible for wheat blast and FHB, yet the effect of such strains should be investigated closely under field conditions. The understanding of bacterial communities associated to wheat may be seen as an important tool to help in the search for antagonists.

A cultura de trigo (Triticum aestivum L.) é a segunda maior do mundo e o Brasil ocupa o segundo lugar de produção na América do sul. Entretanto, a produtividade desta cultura pode ser limitada devido à ocorrência de doenças como a brusone, causada pelo fungo Pyricularia grisea e a doença denominada giberela, causada pelo fungo Fusarium graminearum Populações bacterianas associadas à rizosfera de trigo podem apresentar potencial como agentes de controle biológico de diferentes fitopatógenos. Neste contexto, esta pesquisa foi direcionada ao estudo da composição da comunidade bacteriana rizosférica do trigo e a busca por micro-organismos com potencial para o controle biológico da brusone e da giberela. Assim, para melhor compreensão das comunidades associadas ao trigo, foram realizadas coletas em duas regiões diferentes no Brasil, sendo possível a obtenção de 606 estirpes entre bactérias e actinobactérias da rizosfera do trigo e de solo de cultivo da mesma cultura. Destas, 16 apresentaram, em testes in vitro, potencial antagonista diante dos fungos fitopatogênicos Pyricularia grisea e Fusarium graminearum com diferentes porcentagens de inibição. Dez dos isolados selecionados apresentaram similaridade com a família Streptomycetaceae, porém, quatro linhagens necessitam de estudos mais detalhados, pois a similaridade foi baixa, podendo indicar uma espécie ainda não descrita; quatro linhagens demonstraram similaridade com a família Bacillaceae e dois com a família Paenibacillaceae. Na avaliação de produção de metabólitos secundários com efeito inibitório, apenas dez apresentam potencial, porém estudos mais detalhados se fazem necessários para a confirmação deste mecanismo. A análise de diversidade bacteriana demonstrou uma maior abundância do filo Actinobacteria, seguido pelo filo Proteobacteria e Acidobacteria em ambas as áreas amostradas, entretanto, o filo Acidobacteria foi o que demonstrou a maior variação entre as classes presentes nas diferentes regiões estudadas, indicando uma seleção da comunidade de acordo com a variedade do cultivar e o estádio de desenvolvimento do vegetal. A comunidade bacteriana de trigo apresenta micro-organismos com potencial para a inibição dos fungos causadores da brusone e da giberela, porém o efeito destas linhagens deve ser melhor investigado em condições de campo. A compreensão das comunidades bacterianas associadas ao trigo pode se apresentar como uma importante ferramenta para direcionar a busca por antagonistas. / Wheat (Triticum aestivum) is the second largest crop in the world and Brazil is in the second position in the ranking of production in South America. However, its productivity can be limited due to the occurrence of diseases like wheat blast, caused by the fungus Pyricularia grisea and the disease called Fusarium head blight (FHB), caused by the fungus Fusarium graminearum. Bacterial populations associated to wheat rhizosphere may have potential to act as biological control agents of different plant pathogens. In this context, this research aimed to look at wheat rhizosphere bacterial community and the pursuit of microorganisms with potential for the biological control of wheat blast and FHB. Given this, in order to study wheat bacterial communities, data collection was carried out in two different regions in Brazil, returning 606 bacterial and actinomycetes isolates from wheat rhizosphere and bulk soil. Among these,, 16 strains revealed antagonistic potential against both plant pathogens Pyricularia grisea and Fusarium graminearum, with different percentages of inhibition. Ten strains were selected out of the 16 and showed similarity with the family Streptomycetaceae, whereas four of them displayed a low similarity, requiring a deeper analysis and might indicate new species. Four isolates showed similarity with the family Bacillaceae and two with the family Paenibacillaceae. On the assessment of production of secondary metabolites with inhibitory effects, only ten strains were positive, but more detailed studies are necessary to confirm this mechanism. The analysis of bacterial diversity revealed a larger abundance of the phylum Actinobacteria, followed by the phylum Proteobacteria and Acidobacteria in both areas, however, the phylum Acidobacteria revealed more variation among its classes when both araes were compared, indicating a selection of the community according to the cultivar and the developmental stage. Wheat bacterial community presents microorganism with inhibition potential against fungi responsible for wheat blast and FHB, yet the effect of such strains should be investigated closely under field conditions. The understanding of bacterial communities associated to wheat may be seen as an important tool to help in the search for antagonists.

Made available in DSpace on 2014-07-29T15:26:20Z (GMT). No. of bitstreams: 1 Tese Wagner Rodrigues de Carvalho.pdf: 2607795 bytes, checksum: 19e8481b5eb319af71b9e1f22666f1d9 (MD5) Previous issue date: 2008-06-30 / Xylanases have been used in the biobleaching of paper pulps, in bioconversion of plant biomass, for food and feed industries, among others. For successful selection of xylanases suitable for specific industrial applications it is important to characterize enzymes isolated from different sources. The thermophilic fungus Humicola grisea var. thermoidea is described as a good producer of extracellular endoxylanases and the Hxyn2 gene from this fungus was isolated and expressed in yeast Pichia pastoris. The aim of this project was focused on the production, purification and biochemical characterization of the recombinant HXYN2 (HXYN2r) enzyme and application on enzymatic hydrolysis of lignocellulosics substrates. The culture conditions of P. pastoris in flasks, using the 12.3 transformant and BMMY-U medium, were optimized and the best xylanolytic activity was 478.2 U/mL after 96 h, with a protein concentration of 100 mg/L, using 2.34% (w/v) of nitrogen sources (yeast extract plus urea 1.34:1.0% - w/v), 1% (v/v) of methanol, and OD600 of 10. The HXYN2r enzyme was purified by gel filtration chromatography with a yield of 6.4% and showed optimum pH and temperature values of 6.5 and 60ºC, respectively, maintaining 100% of initial activity after 4 h of incubation at pH 5.5, 6.5 and 7.5. The half-life time was 18 min at 60ºC and the enzyme was 100% stable after 3 h of incubation at 50ºC. Km and Vmax values were 7.9 mg/mL e 235.4 μmol/(mL.min), respectively. The HXYN2r enzyme were used on the enzymatic hydrolysis of sugarcane bagasse (SCB), corn cob (CC) and foliar sample of sugarcane (FSC) alone or with the enzymes of xylanolytic and cellulolytic system produced by H. grisea fungus. For enzymatic hydrolysis experiments, the substrates were milled and pretreated with 0.25% (w/v) of H2SO4 by 30 min. On the enzymatic hydrolysis the best conversion yield of hemicellulosic fractions were obtained using PpHXYN2r supplemented with EHg: 42.8% to CC, 9.6% to SCB and, a mean of 20% to foliar samples. / As endoxilanases têm sido utilizadas no biobranqueamento da polpa de papel, na bioconversão da biomassa de plantas, nas indústrias alimentícias e de ração animal, entre outras. Para a seleção de endoxilanases que possam ser empregadas em processos industriais específicos é importante caracterizar enzimas isoladas de diferentes fontes. O fungo termofílico Humicola grisea var. thermoidea é um bom produtor de endoxilanases extracelulares e o gene que codifica uma das endoxilanases (HXYN2) deste fungo foi isolado e expresso na levedura Pichia pastoris. O presente trabalho se concentrou na produção, purificação e caracterização bioquímica da endoxilanase recombinante HXYN2 e na sua aplicação na hidrólise enzimática de substratos lignocelulósicos. As condições de cultivo da levedura P. pastoris em frascos, utilizando o transformante 12.3 e o meio BMMY-U foram otimizadas e obteve-se uma atividade xilanolítica de 478,2 U/mL após 96 h de cultivo, com uma concentração de 100 mg/L de proteínas, utilizando 2,34% (p/v) de fontes de nitrogênio (extrato de levedura e urea 1,34:1,0% - p/v), 1% (v/v) de metanol e DO600 de 10. A enzima HXYN2r foi purificada por cromatografia de filtração em gel com um rendimento de 6,4% e apresentou pH e temperatura ótimos de 6,5 e 60ºC, respectivamente, mantendo 100% da atividade inicial após 4 h de incubação nos pH s 5,5, 6,5 e 7,5. O tempo de meia-vida foi de 18 min a 60ºC e a enzima manteve 100% da atividade após 3 h de incubação a 50ºC. Os valores de Km e Vmáx foram 7,9 mg/mL e 235,4 μmol/(mL.min), respectivamente. A enzima HXYN2r foi aplicada na hidrólise enzimática de sabugo de milho (SM), bagaço de cana-de-açúcar (BCA) e palha de cana-de-açúcar (PCA) sozinha ou em conjunto com as enzimas do sistema xilanolítico e celulolítico produzidas pelo fungo H. grisea. Para os experimentos de hidrólise enzimática os substratos testados foram moídos e submetidos a uma etapa de pré-tratamento com 0,25% (p/v) de H2SO4 por 30 min. Nos ensaios de hidrólise enzimática as melhores taxas de conversão da fração hemicelulósica dos substratos foram obtidas utilizando-se a PpHXYN2r suplementado com o EHg: 42,8% para o SM; 9,6% para o BCA e, em média 20% para as amostras foliares.

We identified a synthetic hexapeptide that blocks Magnaporthe grisea appressorium formation, in artificial hydrophobic surface. The results suggest that peptides interfere with surface recognition. M. grisea non pathogenic pth1 mutants were complemented by N. crassa orthologous gene suggesting that the biochemical function of pth1 has not evolved specifically to play a role in appressorium development.

Tobacco phylloplanins (T-phylloplanin) are a group of closely-related glycoproteins that are formed and disposed at the interface between the plant aerial surface (the phylloplane) and the atmosphere. They are synthesized in short procumbent trichomes and are secreted to aerial surfaces where they are thought to serve the plant as a first line of defense against fungal pathogens. Here it is shown using in vitro and in planta assays that tobacco and sunflower phylloplanins have broad-spectrum antifungal activities against spores - and also hyphae for two species - of several true fungi. Field tests show that T-phylloplanin reduces diseases caused by three important fungal pathogens of turf grasses. Tobacco phylloplanins are distinct proteins but they have properties in common with small, membrane-pore-forming, antimicrobial peptides formed by other organisms. To directly determine if T-phylloplanin has pore-forming activity we monitored conductivity change and specific ion leakage from spores and hyphae in suspension. Results indicate that phylloplanin causes fungal membrane disruption that leads to ion depletion and cell death. Having observed broad efficacy of T-phylloplanin against spores and/or hyphae of several true fungi, but no activity towards hyphae of the oomycetes, Pythium and Peronospora parasitica, we tested for possible effects on zoospores of the latter two pathogens. T-phylloplanin was shown to be effective against their zoospores, extending the efficacy of T-phylloplanin to include water molds. In the course of these experiments we also tested the effects of the diterpene cis-abienol that is secreted from tall trichomes of tobaccos and found this compound impacted zoospores and could prevent black shank disease caused by P. parasitica when applied to soil-grown tobaccos as a root drench. Thus, results of these studies with phylloplanins and cis-abienol, two different tobacco surface accumulated compounds are consistent with their serving the plant as first line of defense systems against a wide array of invading fungal pathogens. Phylloplanins and cis-abienol may be useful for controlling fungal diseases in tobacco float beds. The efficacy shown here for T-phylloplanin control of fungal pathogens of turf grasses in the field suggests that this natural product may find use in IPM of turf and other crops.

Thesis--Auburn University, 2009. / Abstract. Vita. Includes bibliographical references (p. 62-71).

Thesis--Auburn University, 2009. / Abstract. Vita. Includes bibliographical references (p. 78-88).