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Na natureza, as orquídeas são totalmente dependentes de fungos micorrízicos para germinar. Estes fungos podem penetrar nas células das raízes e formar pélotons, os quais, quando digeridos pela planta, providenciam açúcares simples para o embrião. Durante a fase aclorofilada de desenvolvimento da plântula, orquídeas são obrigatoriamente dependentes dos fungos; algumas continuam assim por toda vida enquanto outras se tornam facultativamente responsivas à colonização. O objetivo deste trabalho foi identificar quantos clados de fungos podem estabelecer associação micorrízica com Coppensia doniana (sin. Oncidium donianum), uma orquídea amplamente distribuída nos arredores de Campos do Jordão e, demonstrar como as características morfológicas dos isolados, quando analisadas com ferramentas de estatística multivariada, podem ser úteis para a taxonomia destes fungos. Dez plantas foram amostradas em um sítio com vegetação típica de campos de altitude, junto ao Parque Estadual de Campos do Jordão. Fungos foram isolados pela transferência asséptica de cortes de raízes contendo pélotons para meios de cultura BDA modificados. Três clados de fungos foram formados, tanto analisando as características qualitativas das culturas quanto as quantitativas. Os clados foram identificados como dois morfotipos do gênero Ceratorhiza (fase anamórfica de Ceratobasidium) e uma Rhizoctonia-uninucleada. O sequenciamento da região ITS produziu resultados idênticos a estes, mostrando os mesmos três clados. Todas as sequências tiveram alta correlação com sequências de Ceratobasidium depositadas no Genbank, o que sugere uma alta afinidade de Coppensia doniana com este gênero. Também ficou demonstrado que os dados morfológicos, quando associados à estatística multivariada são uma ferramenta útil na taxonomia polifásica de Rhizoctonia spp. As sequências dos isolados de Coppensia doniana também foram comparadas com as de isolados de outras orquídeas, dentro da subtribo Oncidiinae, incluindo: Ionopsis utricularioides e Psygmorchis pussila, coletadas na região do Valle del Cauca Colômbia e isolados de 10 Ionopsis utricularioides, Oncidium altissimum e Tolumnia variegata, estudados por Otero (2002, 2004, 2007), em diferentes regiões de Porto Rico, Costa Rica, Cuba e Panamá. Esta última análise veio a comprovar a preferência de orquídeas da subtribo Oncidiinae por fungos do gênero Ceratobasidium, apesar de que os clados obtidos no Brasil e na Colômbia foram distantes filogeneticamente dos clados previamente estudados na América Central. Representantes dos três clados obtidos de C. doniana em Campos do Jordão foram também testados quanto à capacidade de induzir germinação em suas sementes. Todos isolados testados tiveram sucesso na germinação das sementes, levando as plântulas a estádios avançados de desenvolvimento após 30 dias, o que indica um alto potencial para utilização biotecnológica destes isolados para a germinação das sementes destas orquídeas, tanto para a floricultura comercial quanto para programas de reintrodução de espécies de orquídeas ameaçadas de extinção. / In nature, orchids are fully dependent on mycorrhizal fungi for germination. These fungi can penetrate root cells and form pelotons, whose digestion provides simple sugars for the embryo. During the achlorophyllous seedling stage, orchids are obligatory dependent on the fungi, and some species remain so through life, while others become facultatively responsive to fungal infection. The aim of this study was to identify how many fungal clades can establish mycorrhizal associations with Coppensia doniana, a widespread orchid from Campos do JordãoBrazil, and to demonstrate how their morphological features, analyzed with multivariate statistics, can be useful for classification. Ten plants were sampled in an Araucaria forest near Campos do Jordão. Fungi were isolated by transferring surface disinfected root segments containing pelotons to PDA culture medium. Three main fungal clades were formed by qualitative and quantitative morphological data. They were identified as two morphotypes of Ceratorhiza (anamorphic stage of Ceratobasidium) and one uninucleated Rhizoctonia. The ITS sequencing corroborates this identification, since the same three clades were found. All sequences were highly correlated to Ceratobasidium ITS data deposited at the Genebank, suggesting a high affinity between this species of Oncidiinae and Ceratobasidium. It also could be shown that morphological data associated with multivariate statistics can be a useful tool in fungal multi-level taxonomy. C. doniana sequences were also compared to sequences obtained from isolates of other orchids, belonging to the sub-tribe Oncidiinae, including: Ionopsis utricuarioides and Psygmorchis pussila, collected in Valle del Cauca Colombia and isolated from I. utricularioides, Oncidium altissimum and Tolumnia variegata, studied by Otero (2002, 2004, 2007) in different regions of Puerto Rico, Costa Rica and other Caribbean islands. This last analysis confirmed the preference of this Oncidiinae sub-tribe for Ceratobasidium, although isolates obtained in Brazil or Colombia belong to different clades from those previously studied in Puerto Rico, Costa Rica, Panama and Cuba. 12 Fungi representing the three clades obtained from C. doniana in Campos do Jordão were also tested for their ability to induce germination of C. doniana seeds, with a positive response for all of them, being able to bring the seedlings to advanced development stages in 30 days. These results suggest a high biotechnological potential of these isolates, to be used in orchid symbiotic germination for commercial flower production or for the reintroduction of endangered Brazilian orchid species.

Orientador: Paulo Cezar Ceresini / Resumo: Rhizoctonia solani AG-1 IA é um patógeno habitante do solo que causa doenças em pastagens e plantas cultivadas, em vários estados brasileiros. Para penetrar e colonizar os tecidos do hospedeiro, o microrganismo utiliza enzimas que podem ser de interesse biotecnológico e industrial. O objetivo do presente trabalho foi produzir e caracterizar a α-amilase secretada por R. solani AG-1 IA utilizando produtos e resíduos agroindustriais como substratos para fermentação. Para todos os ensaios foi adotado o processo de cultivo em estado sólio (CES) à temperatura de 25 °C e umidade inicial de 60%. Inicialmente, nove isolados de R. solani provenientes de diferentes hospedeiros (arroz, braquiária e soja) foram comparados quanto à produção amilolítica. As maiores atividades enzimáticas foram proporcionadas pelos isolados TO_022 e MT_SO85, procedentes da cultura da soja. Na etapa seguinte, foi avaliado o efeito do substrato sobre a produção enzimática. Farelo de trigo (12,98 U mL-1; TO_022), farelo de soja (3,97 U mL-1; MT_SO85) e braquiária lavada (4,95 U mL-1; TO_022) induziram eficientemente a produção amilolítica. O perfil de produção enzimática no farelo de trigo, melhor substrato avaliado no cultivo, indicou o tempo de 216 h como o mais apropriado para a obtenção de α-amilases pelo isolado TO_022 (22,14 U mL-1), e 240 h para o isolado MT_SO85 (22,84 U mL-1). Para caracterização físico-química da enzima foi utilizado o extrato enzimático bruto do isolado TO_022. A α-amilase de R. sola... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: Rhizoctonia solani AG-1 IA is a soil-borne pathogen that causes diseases in pastures and cultivated plants in several Brazilian states. To penetrate and colonize host tissues, the microorganism uses enzymes that may be of biotechnological and industrial interest. The aim of the present work was to produce and characterize the α-amylase secreted by R. solani AG-1 IA using agroindustrial products and residues as substrates for fermentation. For all the tests, the solid state cultivation process (SSC) at 25 °C and initial humidity of 60% was adopted. Initially, nine isolates of R. solani from different hosts (rice, Brachiaria grass and soybean) were compared for amylolytic production. The major enzymatic activities were provided by the isolates TO_022 and MT_SO85, from the soybean crop. In the next step, the effect of the substrate on the enzymatic production was evaluated. Wheat bran (12.98 U mL-1; TO_022), soybean meal (3.97 U mL-1, MT_SO85) and washed ruzigrass (4.95 U mL-1; TO_022) efficiently induced amylolytic production. The enzymatic production profile in wheat bran, the best substrate evaluated in the culture, indicated the time of 216 h as the most appropriate to obtain α-amylases by the TO_022 isolate (22.14 U mL-1), and 240 h for the isolated MT_SO85 (22.84 U mL-1). For the physicochemical characterization of the enzyme, the crude enzymatic extract of the TO_022 isolate was used. The α-amylase of R. solani TO_022 exhibited maximum activity at 55 °C and pH 5.5, and ma... (Complete abstract click electronic access below) / Mestre

Bibliography: leaves 172-197.

Bibliography: leaves 172-197. / xvii, 197 leaves : ill. (chiefly col.) ; 30 cm. / Title page, contents and abstract only. The complete thesis in print form is available from the University Library. / Thesis (Ph.D.)--University of Adelaide, Dept. of Soil and Water, 2002?

Currently, 74% of cotton acres in the United States are planted with glyphosatetolerant varieties. The average glyphosate-tolerant cotton crop is treated with glyphosate 2.1 times each year in addition to other herbicides, insecticides, and fungicides. The primary objectives of this research were to: 1) describe the influence of glyphosate and pesticides commonly applied at or near the time of cotton planting on soil microbial activity and biomass; 2) study the effect of glyphosate on fluometuron degradation; 3) evaluate the response of Rhizoctonia solani to glyphosate and fluometuron; 4) study changes in glyphosate metabolism that occur as a result of repeated glyphosate applications; and 5) define shifts in the soil microbial community. Additionally, methods for accelerated solvent extraction (ASE) of fluometuron from soils were developed. In one experiment, the addition of glyphosate reduced C-mineralization in soils treated with fluometuron, aldicarb, or mefenoxam + PCNB formulations. However, in a second experiment, C-mineralization increased when glyphosate was applied with fluometuron relative to fluometuron applied alone. Accelerated solvent extraction was used in experiments which demonstrated that application of glyphosate with fluometuron increased the rate of fluometuron degradation in soil relative to fluometuron alone. When glyphosate was added to minimal medium, degradation of fluometuron by R. solani was reduced and less fungal biomass was produced. The total amount of 14C-glyphosate mineralized was reduced when glyphosate was applied 5 times relative to 1, 2, 3, or 4 times. Incorporation of 14Cglyphosate residues into soil microbial biomass was greater following five glyphosate applications than one application 3 and 7 days after application (DAA). Soil fatty acid methyl ester (FAME) profiles were altered by five glyphosate applications relative to one application. Additionally, FAMEs common to gram-negative bacteria were present in higher concentrations following five applications relative to 1, 2, 3, or 4 applications both 7 and 14 DAA. These studies indicated that: 1) glyphosate altered the soil microbial response to other pesticides; 2) fluometuron-degrading microorganisms in soil responded differently to glyphosate; 3) changes in the dissipation or distribution of glyphosate following repeated glyphosate applications were associated with changes in the structural diversity of the soil microbial community.

The increasing concerns about chemical pesticides that are environmentally hazardous and the continuous development of resistance by palhogens to chemical pesticides have led to this study. Many studies have shown that some Gram-negative bacteria, such as Pseudomonas flouresens, control plant diseases and promote plant growth. In this study Gram positive bacteria, Bacillus sp., were chosen because of their ability to produce endospores. Endospores can be used in stable, dry formulations. The advantage of using endospores is their ability to survive harsh conditions such as droughts and high temperatures, which give a long shelf life to the biological control agent. Bacillus isolates were recovered from the rhizosphere of 12 different crops, and were subsequently screened in vitro for their antimicrobial activity. Of 130 isolates, 87 exhibited antimicrobial activity against the test organisms: Rhizoctonia solani, Pythium sp., Phytophthora cinnamoni, Fusarium sp., and single representatives of Gram negative and Gram positive bacteria, namely, Erwinia carotovora and Staphylococcus aureus respectively. The Bacillus isolates B77, B81 and B69 inhibited all the test organisms investigated, which suggests that they produced broad spectrum antimicrobial compounds or more than one antimicrobial compound. Of the isolates that showed antimicrobial activity, 78 of them did not inhibit Trichoderma harzianum K D, which is a registered biological control agent; indicating their potential for combined application. Selected Bacillus isolates were tested for the biological control of R. solani under greenhouse conditions in wheat, cabbage, tomato, maize, and cucumber seedlings. Bacillus isolates were applied as seed treatments, and the inoculated seeds were planted in R. solani infested speedling trays. Shoot dry weight measurement of seedlings indicated that 12 out of 19 Bacillus isolates showed significantly different shoot dry weight in wheat whereas all the isolates tested in tomato and cucumber gave significantly different shoot dry weight. No significantly different shoot dry weight was obtained for maize or cabbage. Seed emergence findings indicated that none of the Bacillus isolates gave significantly different emergence percentage on wheat, cabbage, tomato, and maize but all of them showed significantly different emergence percentage on cucumber. The results indicate that both the pathogen and the biological control agents exhibited varying levels of specificity on each crop tested. The biological control potential of the best Bacillus isolates was tested on bean and maize crops in the field. Green bean and maize seeds were coated with the selected Bacillus isolates and then sown under field conditions. For each isolate, four replicate treatment plots were established, with and without a R. solani inoculum. Percentage emergence, plant survival levels to harvesting and yield of maize cobs and green beans pods were measured. For all parameters measured the positive and negative controls were not significantly different thereby rendering the results for the entire field study inconclusive. However, Bacillus isolates B77, BII, R5 and R7 improved green bean pod yield and Bacillus Isolate B8I increased maize yield, indicating their potentials as plant growth promoting rhizobacteria (PGPR). / Thesis (M.Sc.)-University of Natal, Pietermaritzburg, 2003.

Orientador: Nilton Luiz de Souza / Banca: Edson Luiz Furtado / Banca: Marli Teixeira de Almeida Minhoni / Banca: Luiz Eduardo Aranha de Camargo / Banca: Eiko Kuramae / Resumo: Rhizoctonia solani Kuhn (teleomorfico: Thanatephorus cucumeris (Frank) Donk) e um fungo basidiomycota, fitopatogeno anamorfico com uma ampla gama de hospedeiros, incluindo mais de 500 generos de plantas. Causam doencas em varias culturas importantes no mundo, infectando sementes, raizes, folhas, hastes e frutos. A diversidade genetica de 274 isolados de Rhizoctonia spp. coletados, em diversas partes do mundo, foram caracterizados utilizando-se a analise de sequencia da regiao ITS1-5.8S rDNA-ITS2, para avaliar o grau de variabilidade genetica dentro e entre grupos de anastomose (AGs), incluindo os isolados padroes dos respectivos AGs. A arvore filogenetica gerada pela analise das sequencias da regiao ITS foi suportada por 66% gbootstraph para a separacao em nove maiores grupos. De maneira geral, o agrupamento dos isolados de Rhizoctonia spp. ocorreu de acordo com os grupos de anastomose ja previamente determinados. A similaridade de sequencias entre isolados de mesmo hospedeiro, mas de diferente origem geografica foi elevada. Por exemplo, isolados de melao do Brasil e Espanha mostraram 97,2% de similaridade na regiao ITS1 e de 98,6 a 99,3 % na regiao ITS2. Isolados de batata do Brasil e da Espanha mostram similaridade de sequencia de 94%. Para melhorar a qualidade das sequencias da regiao ITS1-5.8S rDNA-ITS2 dos isolados CBS316.84 (Waitea circinata) e CBS569.83 (Ceratobasidium globisporum) foram gerados clones, e a analise das sequencias dos clones apresentaram variacao de 94,9 a 100 % de identidade 2 entre os clones do isolado CBS316.84 e de 91,9 ate 100 % para os clones do isolado CBS569.83. Os 44 isolados selecionados previamente, a partir do cladograma gerado da regiao ITS1-5.8S-ITS2 foram submetidos ao sequenciamento dos genes ATP sintase subunidade 6 mitocondrial (ATP6), fator de elongacao 1-alpha (EF-1ƒ¿), RNA polimerase 2 (RPB2) e a regiao ITS. As arvores filogeneticas baseadas na analise gneighbor-joiningh geradas a partir das... / Abstract: Rhizoctonia solani Kuhn (teleomorph: Thanatephorus cucumeris (Frank) Donk) is an anamorphic plant pathogenic basidiomycota fungus with a wide host range, including more than 500 genera of higher plants. Species cause disease in several important crops worldwide by infecting the seeds, roots, leaves, stems and fruits. Two hundred seventy four isolates from plant pathogenic fungus Rhizoctonia spp. collected worldwide was characterized using ITS1-5.8S rDNA-ITS2 sequence analysis to assess the degree of genetic variability within and among anastomosis groups (AGs), including tester isolates of the respective AGs. Within the ITS phylogenetic tree, there was support (66% bootstrap value) for the separation into nine major groups. In general the clustering isolates of Rhizoctonia species agreed with previously determined anastomosis groups. Nucleotide sequence similarity between isolates from the same host but from different geographic origin was high e.g. isolates from melon from Brazil and Spain showed from 97.2 % similarity in ITS1 region and 98.6 to 99.3 % in ITS2 region. Potato isolates from Brazil and Spain showed a sequence similarity about 94%. Several isolates from the same geographic origin were found closely related to different anastomosis groups such as isolates from potato, tomato, soybeans, beans, sugar beet and have been reported different symptoms around the world. Were generated cloning of ITS region from CBS 316.84 (Waitea circinata) and CBS 569.83 (Ceratobasidium globisporum) isolates to improve the sequence quality. The clones showed 94.9 to 100% and 91.9 to 100% identity respectively. Fourty four isolates were selected from the ITS1-5.8S rDNA-ITS2 phylogenetic tree for sequencing using the genes of ssembling the Fungal tree of Life (AFTOL) such as ATP 4 synthase subunit 6 mitocondrial (ATP6), elongation factor 1-alpha (EF-1ƒ¿) and RNA polymerase 2 (RPB2). The phylogenetic tree generated by sequences of EF-1ƒ¿ and RPB2 in general. / Doutor