Inoculation with free-living diazotrophic bacteria is well documented to enhance vegetative growth and yield increases of various crops coupled with suppression of sublethal pathogens. The use of microbial inoculants has been identified as an alternative or supplement to use of nitrogenous fertilizers and agrochemicals for sustainable agriculture. The search for effective free-living diazotrophic bacterial strains for formulation as biofertilizers has been on going since the 1970’s and a number of inoculant biofertilizers have been developed and are commercially available. In the current study, 250 free-living diazotrophic bacteria were isolated from soils collected from the rhizosphere and leaves of different crops in different areas within KwaZulu-Natal, province, Republic of South Africa. These were evaluated for plant growth-promotion and biological control of damping-off fungi initially by in vitro screening. The growth promotion traits tested included, phosphate-solubilization, production of indole-3-acetic acid, production of ammonia and acetylene reduction. Biocontrol traits evaluated included siderophore-production, antibiosis, and production of hydrogen cyanide (HCN). Biochemical and molecular bioassay tests were conducted to identify the twenty most promising isolates selected in the in vitro study. The twenty isolates were further tested in combination with various levels of nitrogenous fertilizer for growth-promotion of maize (Zea mays L.) and wheat (Triticum aestivum L.) under greenhouse conditions. The five most promising isolates identified for growth promotion under greenhouse conditions for each crop were assessed for their effects on the germination of maize in vitro and growth and yields of two maize and two wheat cultivars, when combined with a low dose of nitrogenous fertilizer in field trials. The five Bacillus subtilis (Ehrenberg) Cohn isolates that suppressed the growth of a wide range of pathogenic fungi in vitro were tested for their efficacy against damping-off of wheat caused by Rhizoctonia solani Kühn in the greenhouse. These isolates were further studied for their modes of action against R. solani in vitro. The modes of action tested included antibiosis, production of siderophores, extracellular enzymes, production of hydrogen cyanide (HCN) and antibiotic resistance. The twenty most promising bacterial isolates identified from the in vitro screening reduced acetylene to ethylene, produced indole-3-acetic acid and siderophores, one isolate solubilized phosphate, and 9 inhibited the growth of R. solani. These twenty isolates enhanced growth of maize and wheat above the Uninoculated Control under greenhouse conditions. The growth enhancements varied with bacterial isolate x crop species interactions, which identified five different isolates for each of the two crops. Relative to the Uninoculated Control, the best five isolates significantly (P = 0.001) enhanced the growth of maize and wheat at all fertilizer levels for a number of growth parameters: increased chlorophyll levels and heights of maize, shoot dry biomass of maize and wheat, and enhanced root development of maize in the greenhouse. Inoculation of maize and wheat with the two most promising isolates identified from the field trial for each crop, in combination with 65% and 50% of the recommended amount of nitrogenous fertilizer for maize and wheat, respectively, caused the same increases in shoot biomass as the Fully Fertilized Control. Application of a combination of the best bacterial isolates and 35% nitrogenous fertilizer resulted in the same or greater shoot dry biomass and yields of both maize and wheat under field conditions. Shoot dry biomass of wheat increased by 75% above the Uninoculated Control and 30% above the Fully Fertilized Control. The wheat yield increased by 95% above the Uninoculated Control and 43% above the Fully Fertilized Control. Seed inoculation with the best isolates combined with 35% N increased yields of maize by 41% above the Uninoculated Control and 15% above the Fully Fertilized Control. The best isolates significantly (P < 0.001) increased plant height, chlorophyll levels and shoot biomass of maize relative to the Uninoculated Control. There was a positive correlation between chlorophyll level and yield, chlorophyll level and shoot dry biomass, height and shoot dry biomass and height and yield of maize at P = 0.01 with r values of 0.87, 0.77, 0.92 and 0.81, respectively. The isolates that exhibited multiple plant-growth promoting traits in vitro, increased shoot biomass of both maize and wheat in the greenhouse and field, and caused yield increases in the two crops under field conditions. Five B. subtilis isolates inhibited the growth of some of the pathogenic fungi tested in vitro up to 95%. Seed inoculation with the same isolates significantly (P = 0.001) suppressed R. solani damping-off of wheat under greenhouse conditions and exhibited multiple mechanisms of disease control in vitro. The use of microbial inoculants in combination with low doses of nitrogenous fertilizers can enhance crop production without compromising the yields. The B. subtilis isolates obtained in this study can effectively control R. solani damping-off of wheat, fix nitrogen and enhance plant growth. The use of microbial inoculants can contribute to the integrated production of cereal crops with reduced nitrogenous fertilizer inputs, as a key component of sustainable agriculture. Key words: Free-living bacteria; plant growth-promotion; diazotrophs; biological nitrogen fixation; phosphate-solubilization; siderophores; indole-3-acetic acid; biocontrol; damping-off / Thesis (Ph.D.)-University of KwaZulu-Natal, Pietermaritzburg, 2013.

Submitted by Marco Antônio de Ramos Chagas (mchagas@ufv.br) on 2017-04-27T11:34:04Z No. of bitstreams: 1 texto completo.pdf: 589394 bytes, checksum: a51b7716ef15e4adbbdcd4469c379e41 (MD5) / Made available in DSpace on 2017-04-27T11:34:04Z (GMT). No. of bitstreams: 1 texto completo.pdf: 589394 bytes, checksum: a51b7716ef15e4adbbdcd4469c379e41 (MD5) Previous issue date: 2004-02-16 / Conselho Nacional de Desenvolvimento Científico e Tecnológico / Isolados pré-selecionados de rizobactérias foram empregados para microbiolização de diferentes substratos. A compatibilidade entre isolados foi determinada pelo método do antibiograma, enquanto o possível efeito sinérgico foi investigado pelo tratamento do substrato com os isolados de rizobactérias aplicados individualmente ou em misturas, empregando-se a mesma proporção de inóculo dos co- inoculantes. Considerando-se os clones de eucalipto responsivos ao tratamento do substrato com rizobactérias, o ganho médio para enraizamento e biomassa radicular foi de 20,4 e 73,0%, respectivamente. No entanto, levando-se em consideração a interação entre o(s) melhor(es) isolado(s) de rizobactéria e clone(s) de eucalipto, observou-se incremento médio de 21,4% e 78,0%, respectivamente para índice de enraizamento e biomassa de raízes. Os isolados mais promissores foram o VC2 e Ca para enraizamento e o VC2 e 3918 para biomassa radicular. Nos estudos envolvendo mistura de isolados não foram observados efeitos significativos de incremento de misturas sabidamente compatíveis e vice-versa. Além disso, constatou-se especificidade da interação entre isolados de rizobactérias e clones de eucalipto. Quanto à forma de veiculação, os resultados variaram de acordo com o clone, forma de aplicação e isolado empregado. Em uma última etapa, avaliou se in vitro e em casa de enraizamento o efeito de rizobactérias sobre o controle biológico de Cylindrocladium candelabrum e Rhizoctonia solani. Considerando o efeito dos meios de cultivo e para os dois patógenos, os isolados FL2, 3918, S1 e S2 foram os mais efetivos. Ao final de 25 dias, constatou-se ao observar as miniestacas mantidas em casa de enraizamento maior incidência de podridão (71%) causada por C. candelabrum em relação à mela de miniestacas causada por R. solani. Para o primeiro patógeno, o tratamento do substrato com a rizobactéria Ca foi responsável pela redução de 33 e 26,7% em termos médios, quando comparado aos tratamentos testemunha e imersão de miniestacas em calda fungicida (epoxyconazole + pyraclostrobin 0,6 mL/L do i.a.), respectivamente. Em relação à mela de miniestacas, não foram constatadas reduções significativas, embora para o isolado Ca observou-se tendência de menor intensidade da doença. O monitoramento periódico da população dos patógenos em substrato artificialmente infestado evidenciou para C. candelabrum diferenças significativas no tempo necessário para estabilização da população e no nível populacional atingido ao longo do tempo de incubação. A partir de 10 dias, notou-se maior população do patógeno no tratamento testemunha. Entre os tratamentos com rizobactérias, a partir de 15 dias de incubação, o isolado Ca destacou-se dos demais por suprimir a população do patógeno. Ao final de 30 dias, a diferença entre a população do patógeno no substrato rizobacterizado com este isolado em relação ao tratamento testemunha foi de 26,7%. No ensaio realizado in vitro, quando se avaliou a sensibilidade de isolados de rizobactérias a fungicidas, tebuconazole inibiu 80% dos isolados e a mistura dos fungicidas epoxyconazole e pyraclostrobin inibiu apenas o crescimento do isolado S1. O isolado mais sensível aos princípios ativos foi o S1, e os menos afetados, o 3918 e MF2. Verifica-se, diante desses resultados, que é necessário realizar estudos que viabilizem a produção e formulação do produto biológico à base de rizobactérias para seu emprego em escala comercial para o eucalipto. Os depósitos de patente foram feitos junto ao INPI (PI 0101400-5 e 001409). / Pre-selected rhizobacteria isolates were used for microbiolization of different substrates. The compatibility among isolates was determined by the antibiogram method, and the synergic effect was investigated by treating the substrate with isolates individually or mixed. The average increase in rooting and root biomass production were 20.4% and 73.0%, respectively, considering the clones responsive to the treatment with rhizobacteria. However, taking into consideration the interaction among the best isolates and eucalyptus clones, it was observed an increase of 21.4% and 78.0% on rooting and dry root biomass production, respectively. The most promising isolates were VC2 and Ca for rooting and VC2 and 3918 for root biomass production. In studies involving mixing of compatibles and non-compatibles isolates it was not observed any significant effect on increment, but it was observed a specificity of interaction among rhizobacteria isolates and eucalyptus clones. The response to the inoculation method varied according to the clone, the method of application and the isolate used. Finally, the effect of the rhizobacteria on the biological control of Cylindrocladium candelabrum and Rhizoctonia solani was evaluated in vitro and in greenhouse. Considering the effect of culture media for these pathogens, the isolates FL2, 3918, S1 and S2 were the most effective. After 25 days, the rotting caused by C. candelabrum (71%) on minicuttings grown in nurseries was higher than the web blight caused by R. solani. The treatment of minicuttings with the isolate Ca promoted an average reduction of 33% and 26.7% when compared to the non-inoculated treatment and to the treatment with immersion of the minicutting in fungicide broth (epoxyconazole + pyraclostrobin 0.6 mL/L of a.i.), respectively. It was not observed significant reduction on minicuttings web blight, although the isolate Ca have showed a tendency to reduce the disease intensity. The periodic monitoring of pathogen populations in artificially infested substrate showed significant differences in the time needed for stabilization of the population and in the population level of C. candelabrum along the incubation time. It was observed a greater pathogen population on the control treatment from 10 days after inoculation. After 15 days of incubation, the isolate Ca was the most effective in suppressing the pathogen population. After 30 days, the difference between the pathogen population in the substrate infested with C. candelabrum and the control treatment was 26.7%. In the in vitro assay, tebuconazole (Folicur) inhibited 80% of the isolates and the mixture of the fungicides epoxyconazole and pyraclostrobin (Opera) only inhibited the isolate S1. This isolate (S1) was the most sensitive to the active ingredients tested and 3918 and MF2 the least affected ones. These results indicate the need of studies to produce and formulate the bioproduct based on rhizobacteria at a commercial scale. Patent deposits were made at INPI (PI 0101400-5 e 001409). / Dissertação importada do Alexandria

Orientador: Paulo Cezar Ceresini / Resumo: O controle biológico é um dos métodos do manejo integrado de doenças de plantas, sendo uma das únicas alternativas viáveis para minimizar o impacto da queima-das-folhas e morte súbita das pastagens do gênero Urochloa (anteriormente Brachiaria). A queima-das-folhas e morte de pastagens é causada pelo fungo basidiomiceto Rhizoctonia solani grupo de anastomose AG-1 IA. Em nosso estudo determinamos se isolados de espécies de Pseudomonas fluorescentes obtidos de solos supressivos do bioma Amazônico possuem potencial como agentes de biocontrole de R. solani AG-1 IA. Especificamente objetivamos: i. Efetuar o screening de isolados de Pseudomonas quanto ao potencial de biocontrole in vitro; ii. Realizar a identificação molecular dos isolados de Pseudomonas fluorescentes selecionados como potenciais agentes de biocontrole e iii. Determinar se as bactérias selecionadas in vitro mantém, in vivo, o potencial de biocontrole da queima-das-folhas e morte da braquiária. Três isolados foram selecionados como potenciais agentes biocontroladores nos testes de antagonismo in vitro (Amana, Poti e Yara), pois diminuíram significativamente tanto o crescimento micelial quanto a germinação de escleródios do patógeno. Com base em análises filogenéticas, os isolados Amana e Yara foram agrupados ao clado de Pseudomonas putida enquanto o isolado Poti foi agrupado em clado distinto de todos os demais, e possivelmente se constitui numa nova espécie. Nos testes in vivo, a inoculação via sementes não resultou... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: Biological control is one of the methods of the integrated management of plant diseases, which is one of the only viable alternative to minimizes foliar blight impact and sudden death of pastures from genus Urochloa (previously known as Brachiaria). Foliar blight and death of pastures are caused by the fungus Rhizoctonia solani AG-1 IA. In our study, it was determined whether isolates from florescent Pseudomonas obtained from suppressive soils on the Amazon biome had potential as biocontrol agents of Rhizoctonia solani AG-1 IA. Specifically, we aim to: i. Carry out a screening of isolates from Pseudomonas to evaluate their potential as in vitro biocontroler; ii. Perform molecular identification of selected fluorescent Pseudomonas isolates as potential biocontrol agents; and iii. Determine whether in vitro selected bacterial strains maintain the biocontrol ability in vivo of foliar blight and sudden death of braquiária. Three isolates were selected as potential biocontoller agents in vitro antagonism tests (Amana, Poti e Yara), which reduced significantly mycelial growth and sclerotia germination of pathogen. Based on phylogenetic analyzes, Amana and Yara isolates were grouped to the clade belonging to Pseudomonas putida and Poti isolated was grouped in a distinct clade, and should to constitute a new species. For in vivo tests, seed inoculation did not resulted in R. solani AG-1 IA control. On the other hand, in foliar application of biocontrol agent, Amana isolated was the m... (Complete abstract click electronic access below) / Mestre

This diploma thesis is concerned on protease inhibitors isolated from potato (Solanum tuberosum L.) tubers and evaluation of their antifungal properties. Theoretical part of the thesis deals with protease inhibitors which have an antifungal effect. Tubers of potato cultivars Adéla, Ornella, Eurostarch - were used for protease inhibitors isolation. Antifungal activity of isolated protein fractions were evaluated versus fungi from genus Rhizoctonia and Fusarium that are important pathogens in agriculture. Their activity was also evaluated statistically.

Resumo: O fungo Thanatephorus cucumeris é responsável por várias doenças foliares em culturas de importância agrícola ou em espécies nativas na região Amazônica do país. Entre as doenças relatadas, a mancha areolada de Thanatephorus é considerada uma das mais importantes para a região. Neste estudo, baseando-se na ausência de informações sobre quais os grupamentos de anastomose (AG) de Rhizoctonia solani estão associados a plantas hospedeiras na Amazônia, foi testada a hipótese de que os isolados de T. cucumeris oriundos de seringueira e citros e outras espécies cultivadas ou nativas pertencem a grupamentos de anastomose distintos. Assim, os isolados de T. cucumeris foram caracterizados citomorfologicamente, por meio da caracterização cultural, grupamento de anastomose e molecular. Esta última baseou-se na observação da variação associada a seqüências da região ITS-5,8S do rDNA. Também não há informação sobre a patogenicidade cruzada, à seringueira, de isolados provenientes de outras espécies de plantas da Amazônia. Então, por meio do teste de patogenicidade cruzada foi testada uma segunda hipótese, a de que isolados de T. cucumeris de hospedeiros distintos são patogênicos também à seringueira. O estudo teve como objetivo elucidar aspectos importantes sobre a etiologia do patógeno, que podem ser relevantes para o manejo da doença. De forma importante, concluiu-se que a seringueira hospeda não apenas um, mas sim vários grupos de anastomose de R. solani como agente causal da mancha areolada. / Abstract: The fungus Thanatephorus cucumeris is responsible for causing several foliar diseases on important agricultural crops or on native species in the Amazonian area of the country. Among the diseases, Thanatephorus aerial blight is considered one of the most important in that region. In this study, based on the lack of information on which anastomosis groups (AG) of R. solani are associated with distinct hosts in the Amazon, we tested the hypothesis that T. cucumeris isolates from rubber tree, citrus and other cultivated or native species from the area belong to distinct AGs. So, T. cucumeris isolates were characterized based on cytomorphology, cultural characteristics, by anastomosis grouping, and molecularly. This last one was based on the observation of the variation associated with sequences of the ITS-5.8S region of the rDNA. There is no information about the cross pathogenicity (to rubber tree) of isolates from other Amazonian plant species. Based on cross-pathogenicity tests, a second hypothesis was tested stating that T. cucumeris isolates from distinct hosts are also pathogenic to rubber tree. This study had as objective to elucidate important aspects of the aetiology of the pathogen that can be relevant for managing the disease. We have found that the rubber tree hosts not only one, but several anastomosis groups of R. solani as causal agent of the aerial blight. / Orientador: Paulo Cezar Ceresini / Coorientador: Alcebíades Ribeiro Campos / Banca: Edson Luiz Furtado / Banca: Cesar Junior Bueno / Mestre

Made available in DSpace on 2014-06-11T19:29:44Z (GMT). No. of bitstreams: 0 Previous issue date: 2006-07-17Bitstream added on 2014-06-13T19:39:07Z : No. of bitstreams: 1 campos_aps_me_ilha.pdf: 533716 bytes, checksum: a6a41f1f3c5a72bb618808187efbaa15 (MD5) / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) / O fungo Thanatephorus cucumeris é responsável por várias doenças foliares em culturas de importância agrícola ou em espécies nativas na região Amazônica do país. Entre as doenças relatadas, a mancha areolada de Thanatephorus é considerada uma das mais importantes para a região. Neste estudo, baseando-se na ausência de informações sobre quais os grupamentos de anastomose (AG) de Rhizoctonia solani estão associados a plantas hospedeiras na Amazônia, foi testada a hipótese de que os isolados de T. cucumeris oriundos de seringueira e citros e outras espécies cultivadas ou nativas pertencem a grupamentos de anastomose distintos. Assim, os isolados de T. cucumeris foram caracterizados citomorfologicamente, por meio da caracterização cultural, grupamento de anastomose e molecular. Esta última baseou-se na observação da variação associada a seqüências da região ITS-5,8S do rDNA. Também não há informação sobre a patogenicidade cruzada, à seringueira, de isolados provenientes de outras espécies de plantas da Amazônia. Então, por meio do teste de patogenicidade cruzada foi testada uma segunda hipótese, a de que isolados de T. cucumeris de hospedeiros distintos são patogênicos também à seringueira. O estudo teve como objetivo elucidar aspectos importantes sobre a etiologia do patógeno, que podem ser relevantes para o manejo da doença. De forma importante, concluiu-se que a seringueira hospeda não apenas um, mas sim vários grupos de anastomose de R. solani como agente causal da mancha areolada. / The fungus Thanatephorus cucumeris is responsible for causing several foliar diseases on important agricultural crops or on native species in the Amazonian area of the country. Among the diseases, Thanatephorus aerial blight is considered one of the most important in that region. In this study, based on the lack of information on which anastomosis groups (AG) of R. solani are associated with distinct hosts in the Amazon, we tested the hypothesis that T. cucumeris isolates from rubber tree, citrus and other cultivated or native species from the area belong to distinct AGs. So, T. cucumeris isolates were characterized based on cytomorphology, cultural characteristics, by anastomosis grouping, and molecularly. This last one was based on the observation of the variation associated with sequences of the ITS-5.8S region of the rDNA. There is no information about the cross pathogenicity (to rubber tree) of isolates from other Amazonian plant species. Based on cross-pathogenicity tests, a second hypothesis was tested stating that T. cucumeris isolates from distinct hosts are also pathogenic to rubber tree. This study had as objective to elucidate important aspects of the aetiology of the pathogen that can be relevant for managing the disease. We have found that the rubber tree hosts not only one, but several anastomosis groups of R. solani as causal agent of the aerial blight.

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Les agents phytopathogènes telluriques constituent un problème majeur touchant les productions serricoles de concombre (Cucumis sativus L.) et de tomate (Solanum lycopersicum L.). Les traitements biologiques efficaces sont limités pour lutter contre ces agents pathogènes. Cette étude avait pour objectifs d’évaluer (1) l’effet antagoniste in vitro de Bacillus pumilus Meyer et Gottheil souche PTB180 et Bacillus subtilis (Ehrenberg) Cohn souche PTB185 envers Rhizoctonia solani J.G. Kühn, Pythium ultimum Trow, Sclerotinia sclerotiorum (Lib.) de Bary, Phytophthora capsici Leonian, Fusarium oxysporum Schltdl. et F. oxysporum f.sp. radicis-lycopersici Jarvis & Shoemaker, (2) l’effet répresseur d’une application préventive des bactéries sur la pourriture du collet et la fonte des semis causées par R. solani, sur concombre ou tomate en serre et (3) l’effet des bactéries sur la croissance et la productivité des plants de tomate cultivés en serre. Lors des essais in vitro, PTB180 et PTB185, utilisés seuls et en mélange (1:1), ont fortement inhibé la croissance mycélienne des six agents phytopathogènes. Sur plantules de concombre et de tomate, le mélange (1:1) des deux souches a permis une réduction significative (p<0,05) de la sévérité des symptômes de la pourriture du collet. PTB185 et le mélange (1:1) des deux souches ont augmenté significativement la hauteur et la masse sèche des plants infectés par R. solani. Les essais sur graines de tomate montrent que PTB180 a augmenté significativement le taux de levée des graines et que PTB185 a réduit significativement la mortalité post-levée tout en favorisant la croissance des plantules. Les tests réalisés sur plants de tomate sains n’ont pas montré d’effet significatif de PTB180 et PTB185 sur la croissance et la productivité des plants. Les bactéries à l’étude pourraient éventuellement représenter une alternative aux fongicides de synthèse pour lutter contre les agents phytopathogènes.