Some plant-beneficial bacteria with biopesticide potential produce antimicrobial compounds that are tightly involved in multitrophic interactions occurring in the phytosphere among which antagonism toward a diverse range of phytopathogens. However, there is a crucial need for an accurate assessment of antibiotic production rate in this environment colonized by these bacteria. In this context, we conducted the present thesis work with the scope to better understand how two different Bacillus genus strains produce lipopeptides in the rhizosphere of plants. In the first part, one of the strains (B. amyloliquefaciens S499) was selected out of other isolates for its technological traits and in vitro inhibition growth activity of plant pathogenic fungi. We further demonstrated its biocontrol potential in tomato open-field experiments where plantings have been devastated by a local fungus preliminary identified as Fusarium semitectum. In a second part, we combined two mass spectrometry-based approaches (electrospray ionization and imaging) to analyze the pattern of surfactin, iturin and fengycin lipopeptide families produced in planta by strain S499. Our results show that rhizosphere conditions are conducive for surfactin synthesis but not for other types of lipopeptides and that the lipopeptide pattern can be markedly influenced by nutritional factors, biofilm formation and oxygen availability. In a last part, surfactin gene expression (srfA) level was evaluated in situ on tomato root using the reporter gene (LacZ) inserted in B. subtilis strain BGS3. Results showed effective expression of srfA and production of surfactin in biologically important level quantities upon establishment of bacterial population on roots. Our results also demonstrate that BGS3 developing in colonies, efficiently utilizes the main substrates from plant exudates to produce surfactins. The production may also be favored in bacteria growing slowly in the rhizosphere. Globally, this work contributes to better appreciate the impact of some environmental factors on the in situ biosynthesis of lipopeptides by strains of Bacillus which is probably an essential step for improving the level and reliability of their efficacy as biological agents for the control of plant diseases.

El fuego bacteriano, causado por Erwinia amylovora, es una enfermedad muy importante a nivel comercial y económico porque afecta a plantas de la familia de las rosáceas y es especialmente agresiva en manzano (Pyrus malus) y peral (Pyrus communis), así como en plantas ornamentales (Crataegus, Cotoneaster o Pyracantha). Esta enfermedad está distribuida por todo el mundo en zonas climáticas templadas de Amércia del Norte, Nueva Zelanda, Japón, Israel, Turquí y Europa. En España, el fuego bacteriano fue detectado por primera vez en 1995 en el norte del País (Euskadi) y más tarde en nuevos focos aparecidos en otras áreas. La enfermedad puede ser controlada comercialmente mediante la aplicación de pesticidas quimicos (derivados de cobre, antibioticos). Sin embargo, muchos de los productos químicos presentan baja actividad o causan fitotoxicidad, y la estreptomicina, el producto más eficaz, esta prohibido en muchos países, incluyendo España. Por tanto, en ausencia de apropiados agentes químicos, el control biológico se contempla como una buena alternativa. En el presente trabajo, un agente de control biológico, Pseudomonas fluorescens EPS62e, ha sido seleccionada de entre 600 aislados de las especies P. fluorescens y Pantoea agglomerans obtenidos de flores, frutos y hojas de plantas de la familia de las rosáceas durante una prospección llevada a cabo en varias áreas geográficas de España. La cepa ha sido seleccionada por su capacidad de suprimir la infecciones producidas por E. amylovora frutos inmaduros, flores y brotes de peral en condiciones de ambiente controlado, presentando unos niveles de control similares a los obtenidos mediante el control químico usando derivados de cobre o antibióticos. La cepa además ha mostrado la capacidad de colonizar y sobrevivir en flores y heridas producidas en frutos inmaduros en condiciones de ambiento controlado pero también en flores en condiciones de campo. La exclusión de E. amylovora medinate la colonización de la superficie, el consumo de nutrientes, y la interacción entre las células del patógeno y del agente de biocontrol es la principal causa de la inhibición del fuego bacteriano por la cepa EPS62e. Estas características constituyen aspectos interesantes para un desarrollo efectivo de la cepa EPS62e como un agente de biocontrol del fuego bacteriano en condiciones comerciales. / Fire blight, caused by Erwinia amylovora is a serious disease of rosaceous plants with great commercial and economic interest that is distributed over the world. Disease may be controlled commercially by the application of chemicals (copper compounds, antibiotics). Many chemical agents have low activity or cause phytotoxicity, and streptomycin, the most effective antibiotic, is not approved for use in many countries, including Spain. Therefore, in the absence of suitable chemical control agents, biological control could provide a useful alternative. In the present work, a biological control agent, Pseudomonas fluorescens EPS62e, has been selected among 600 isolates of P. fluorescens and Pantoea agglomerans obtained from flowers, fruits and leaves of rosaceous plants in a survey performed through several geographic areas of Spain. This strain has been selected for its capacity to suppress immature fruit, blossom and shoot infections caused by E. amylovora, under controlled environment conditions, providing control levels similar to chemical control with copper or antibiotic compounds. The strain has also shown the capacity to colonize and survive well in flowers and wounds on immature fruit under controlled environment conditions but also in flowers under natural conditions. Pre-emptive exclusion of the pathogen E. amylovora by surface colonization and nutrients depletion, and cell-to-cell interaction appear to be the main mechanisms of biocontrol. These characteristics constitute interesting traits for an effective development as a fire blight biological control agent under commercial conditions.

Pseudomonas fluorescens EPS62e es va seleccionar com a agent de biocontrol del foc bacterià per la seva eficàcia en el control de Erwinia amylovora. En aquest treball es van desenvolupar mètodes de traçabilitat que van permetre la seva detecció específica i quantificació. Mitjançant les tècniques RAPD i U-PCR es van obtenir fragments d'amplificació diferencial per EPS62e que es van seqüenciar i caracteritzar com marcadors SCAR per dissenyar una PCR en temps real. La PCR a temps real es va utilitzar simultàniament amb mètodes microbiològics per estudiar l'adaptabilitat epifítica de EPS62e en pomera i perera. L'ús combinat de mètodes microbiològics i moleculars va permetre la identificació de tres estats fisiològics de EPS62e: la colonització activa, l'entrada en un estat de viable però no cultivable, i la mort cel·lular. Aquest treball mostra que EPS62e està ben adaptada a la colonització de flors a camp, encoratjant la seva utilització dins d'una estratègia de control biològic contra el foc bacterià. / Pseudomonas fluorescens EPS62e was selected as a reliable biological control agent of fire blight for its high efficacy controlling Erwinia amylovora infections. In the present work, monitoring methods which allowed EPS62e specific detection and quantification were developed. RAPD and U-PCR fingerprints were used to obtain differential amplified fragments from EPS62e that were sequence characterized as SCAR markers. A real-time PCR was developed on the basis of the strain-specific SCAR markers, and was used simultaneously with microbiological methods to study the environmental fate of EPS62e in apple and pear orchards. The combined use of both microbiological and molecular methods permitted the identification of three physiological states for EPS62e, which consisted of active colonization, survival and entry into a viable but nonculturable state, and cell death. The present work shows that EPS62e is well adapted for blossom colonisation in the field, and encourages its utilisation in a fire blight.

Laboratory bioassay was used to determine the pathogenicity of nine isolates of fungi of the genera Metarhizium, Beauveria and Clonostachys to the parasitic mite, Varroa destructor. All nine isolates were pathogenic to V. destructor with Metarhizuim anisopliae UAMH 9198, Clonostachys rosea UAMH 9161 and Beauveria bassiana GHA being the most pathogenic within their respective species. Metarhizium anisopliae UAMH 9198 was more lethal to V. destructor than B. bassiana GHA and C. rosea UAMH 9161 with LC50 values of 1.6 x 10(5), 9.6 x 10(6) and 5.4 x 10(6) conidia/mL, respectively. Metarhizium anisopliae and B. bassiana significantly affected brood and adult honey bee survivorship and their immune responses. They were lethal to the bees with LC50s of 3.70 x 10(6) and 2.62 x 10()5 conidia/mL, respectively. The effect of temperature and thymol on conidia germination, production and colony growth of the fungal isolates was determined. Temperature significantly affected conidia germination, production and colony growth (P < 0.05) but thymol did not. Efficacy of the two most promising isolates (M. anisopliae UAMH 9198 and B. bassiana GHA) as potential bio-control agents against V. destructor in hives was evaluated. Fungal inocula were applied as dry formulation, with corn flour as carrier, using dispenser tray and dusting applications. Treatments were either applied alone or in combination with thymol to determine any synergistic effects. All treatments significantly increased mite mortality (P < 0.05), however, the mite control efficacy varied between fungal treatments and application methods. Combined treatments of fungi and thymol caused significantly higher mite mortality than single fungal treatments, which showed control levels of ≤61%. Significant differences in mite mortality were found between the two delivery methods with the differences depending on the fungal isolate. The results suggest that M. anisopliae UAMH 9198 would be a more effective bio-control agent for the management of V. destructor in honey bee colonies than the other isolates tested when dispensed continuously in hives using delivery methods such as a dispenser tray. However, future research is needed to improve delivery methods and investigate the effect of carriers used in the formulation on the efficacy of such entomopathogenic fungi. / Ontario Ministry of Agriculture and Food (OMAF), University of Guelph

Nitrogen (N) fertilization is known to influence the susceptibility of many plants to a variety of diseases. In the case of diseases caused by Botrytis cinerea, the role of N fertilization appears to be variable, with high levels either fostering or reducing severity depending on the studies. To test whether this variability could be due to possible differences in the host plants, inoculum pressure or in the behavior of different strains of the pathogen, studies were carried out to investigate the effect of different N fertilization regimes on the susceptibility of tomato and lettuce to six isolates of B. cinerea. Possible epidemiological effects of N fertilization through the sporulation of the pathogen and on the pathogenicity of resulting secondary inoculum were also investigated on tomato. Plants were grown in a soil-less drip-irrigation system. Differential N nutrition ranging from 0.5 to 30 mM NO3- was applied for the last four weeks prior to inoculation on the leaves (lettuce) or on leaf pruning wounds (tomato) and incubation of the plants in conditions conducive to disease development. On the tomato stems, disease onset was delayed and overall severity was lower for all isolates on plants with higher N inputs, regardless of inoculum concentration. However, the rate of stem lesion expansion was differentially affected depending on the strains, decreasing with increasing N fertilization levels for the more aggressive isolates, while increasing for the less aggressive isolates.In contrast with tomato, high N fertilization increased disease severity on lettuce for all isolates tested. On tomato plant tissue, sporulation of B. cinerea decreased significantly with increasing N fertilization up to 15-30 mM NO3- and the pathogenicity of the spores was significantly influenced by the nutritional status of their production substrate. It was highest for spores produced on plants with very low or very high N fertilization (0.5 or 30 mM NO3-) and lowest for those from plants with moderate levels of N fertilization. Plant fertilization also strongly affected the efficacy of two biocontrol agents (Trichoderma atroviride and Microdochium dimerum) to protect pruning wounds of tomato against B. cinerea. The highest levels of protection were obtained with high N fertilization and related to a delay in symptom development on the stems, sometimes associated with a slowdown in lesion expansion. Histological studies showed that the decrease in disease severity at high N fertilization was associated to structural alteration of Botrytis mycelial cells. In the presence of a biocontrol agent, the effect on the pathogen was further associated to vacuolisation, glycogen deposition and mycelial cell death. Hypotheses to explain these results are discussed in light of the possible physiological effects of nitrogen fertilization on nutrient availability for the pathogen in the host tissue and of possible production of defense metabolites by the plant. These results also open new possibilities for including the manipulation of N fertilization as a tool for the integrated protection of vegetable crops

The study examined interactions between morogo plants, arbuscular mycorrhizal fungi (AMF) and Fusarium species. Morogo refers to traditional leafy vegetables that, together with maize porridge, are dominant staple foods in rural areas of the Limpopo Province such as the Dikgale Demographic Surveillance Site (DDSS). Morogo plants grow either as weeds (often among maize), occur naturally in the field or are cultivated as subsistence crops by rural communities. Botanical species of morogo plants consumed in the DDSS were determined. Colonisation of morogo plant roots by AMF and Fusarium species composition in the immediate soil environment were investigated in four of eight DDSS subsistence communities, Isolated AMF were shown to belong to the genera Acaulospora and Glomus. Twelve Fusarium species were isolated from soil among which Fusariurn verticilliodes and Fusarium proliferaturn occurred predominantly. Greenhouse pot trials were conducted to examine the effect of AMF on morogo plant growth (cowpea; Mgna unguiculata) and Fusarium proliferatum levels in soil, Interaction between plants and AMF, as well as tripartite interactions of cowpea plants, AMF and Fusarium proliferatum were investigated. Non-inoculated cowpea plants served as controls for the following inoculations of cowpea in pots: (i) Fusarium proliferatum; (ii) commercial AMF from Mycoroot (PTY) Ltd. (a mixture of selected indigenous Glomus spp referred to commercial AMF for the purpose of this study); (iii) indigenous AMF obtained from DDSS soil (referred to iocal AMF for the purpose of this study); (iv) commercial AMF plus Fusarium proliferatum; (v) local AMF plus Fusariurn proliferatum. Results showed reduced root colonization by local as well as commercial AMF when Fusarium proliferatum were present. Local AMF significantly enhanced cowpea growth while commercial AMF apparently reduced the level of Fusarium proliferatum in the rhizosphere and surrounding soil. Results suggest that AMF may have potential as biological growth enhancers and bioprotective agents against Fusarium proliferatum. / Thesis (M. Environmental Science (Water Science))--North-West University, Potchefstroom Campus, 2007.

ABSTRACT Assessing the impact of herbivory on plant growth and reproduction is important to predict the success of biocontrol of invasive plants. Leaf area production is most important, as photosynthesis provides the foundation for all plant growth and fitness. High levels of defoliation generally reduce the productivity of plants. However, leaf area production fluctuates during the season and compensational growth may occur, which both complicate accurate estimations of defoliation impacts. Under field conditions the interaction with neighbouring species and the availability of soil nutrients need to be assessed in order to gauge long term effects of weed invasions on natural environments. In this thesis I have investigated seasonal leaf area dynamics in Buddleia davidii following repeated artificial defoliation, to quantify compensational leaf production and to understand the regulatory mechanisms involved. The impact of defoliation on photosynthesis, seed production, germination and nitrogen translocation patterns were analysed. Finally, possible facilitation between B. davidii and a native nitrogen fixer, Coriaria arborea, and the impact of B. davidii on soil nutrient availability were investigated. In defoliated B. davidii, increased node production (34%), leaf size (35%) and leaf longevity (12%) resulted in 52% greater total emergent leaf area in the short term. However, with time and diminishing tissue resources the compensation declined. No upregulation of photosynthesis was observed in pre-existing leaves. Compensational leaf area production occurred at the expense of reproduction but the germination capacity of individual seeds was unaffected. In B. davidii, nitrogen reserves are stored in old leaves. Thus, the defoliation-induced decline in tissue reserves led to changes in the remobilisation pattern and increased the importance of soil uptake but biomass production especially that of roots had declined significantly (39%). Slight facilitation effects from the neighbouring nitrogen fixer and VA-mycorrhizae were observed on B. davidii in the field, while its impact on soil chemistry during spring was negligible. Defoliation of B. davidii resulted in priority allocation of resources to compensational leaf growth and a concomitant reduction in flower and seed production. The compensational leaf production greatly increased the demand for nitrogen, while continued leaf removal decreased the pool of stored nitrogen. This led to changes in nitrogen remobilisation and an increased importance of root uptake. However, the significant decline in root growth will likely impair adequate nutrient uptake from the soil, which is especially important where B. davidii invades nutrient poor habitats and will increase the success of biocontrol of the species. While mycorrhizae increase nutrient accessibility for B. davidii, it is likely that the additional stress of defoliation will negate the small facilitative effects from nitrogen-fixing species like C. arborea. This research provides new insights into the mechanisms regulating leaf area dynamics at the shoot level and systemic physiological responses to defoliation in plants, such as nitrogen translocation. The compensation in leaf area production was considerable but only transitory and thus, the opportunity to alleviate effects of leaf loss though adjustment of light capture limited. However, to ascertain that photosynthesis at whole plant level does not increase after defoliation, more detailed measurements especially on new grown leaves are necessary. In general, defoliation had greatly reduced plant growth and performance so that an optimistic outlook for controlling this species can be given. Conclusions about the wider impacts of B. davidii on soil chemistry and community function will require further research.

O uso de bactérias promotoras de crescimento vegetal ou agentes de biocontrole como inoculantes agrícolas é uma alternativa importante e ecologicamente correta, com grandes benefícios na agricultura para substituir, ou ao menos suplementar, a excessiva utilização de fertilizantes e pesticidas. Neste trabalho avaliamos a capacidade de biocontrole e de competência rizosférica de três bactérias com características de promoção de crescimento vegetal (Plant growth promoting - PGP): Bacillus mycoides B38V, Paenibacillus riograndensis SBR5 e Burkholderia sp. 89. As três bactérias avaliadas apresentaram grande versatilidade na utilização de substratos, o que poderia lhes garantir uma vantagem competitiva no ambiente rizosférico. Porém, inconsistências foram observadas nos ensaios em câmara de crescimento, ou seja, as características de PGP e de biocontrole observadas in vitro não se refletiram em benefícios para a planta. A linhagem 89 destacou-se pela produção de um metabólito estável com ampla atividade contra fungos fitopatogênicos. Através de abordagens genômicas e de análises multilocus, descrevemos Burkholderia sp. 89 como uma nova espécie membro do complexo Burkholderia cepacia, denominada de B. catarinensis 89T. O sequenciamento de seu genoma, seguido de uma análise pela ferramenta AntiSMASH, revelou a presença de um agrupamento gênico de peptídeo sintetases não ribossomais (NRPS) relacionadas com a biossíntese do sideróforo ornibactina e um agrupamento híbrido NRPS-policetídeo sintetase responsável pela biossíntese do glicolipopeptideo cíclico com atividade antifúngica burkholdina. Como estratégia de purificação de metabólitos secundários foi utilizada a metodologia da mineração de genoma combinada com fracionamento guiado por bioensaios seguida de análises em espectrômetro de massas. Desta forma, purificamos com sucesso duas variantes de ornibactina, D e F (761 e 789 Da, respectivamente), e detectamos a variante ornibactina B (m/z= 733) e as moléculas sinalizadoras homoserina lactonas C6-HSL, 3OH-C8-HSL e C8-HSL. Análises de espectrometria de massas demonstraram a presença de um grupo de metabólitos com massas de 1240, 1254, 1268, 1216, 1244 e 1272 Da, que, provavelmente, são novas variantes do antifúngico burkoldina. Sendo assim, B. catarinensis 89T possui potencial biotecnológico com possíveis aplicações farmacêuticas e agronômicas para o biocontrole de fungos fitopatogênicos. / The use of plant growth promotion bacteria or biocontrol agents as agricultural inoculants is an important eco-friendly alternative to substitute, or at least supplement, the excessive use of fertilizers and pesticides. In this work, we evaluated the biocontrol potential and rhizosphere competence of three bacteria that had shown plant growth promotion (PGP) abilities: Bacillus mycoides B38V, Paenibacillus riograndensis SBR5 and Burkholderia sp. 89. All three bacteria presented great versatility in their substrate utilization, which could enable them to survive in a competitive rhizosphere environment. However, inconsistencies were observed in the greenhouse experiments, whereas their interesting abilities observed in vitro did not result in benefits to the plants. Strain 89 produces a stable metabolite with a wide range of antifungal activity. Genomic comparisons and multilocus sequence analysis revealed Burkholderia sp. 89 as a new species of the Burkholderia cepacia complex and we described it as B. catarinensis 89T. We sequenced its genome and analyzed it with the AntiSMASH tool. This in silico prediction revealed the presence of a nonribosomal peptide synthetase (NRPS) cluster, which is related to the production of the siderophore ornibactin. Moreover, a hybrid NRPS- polyketide synthetase cluster for the production of the antifungal cyclic glicolipopeptide burkholdin was also found. A genome mining combined with a bioassay-guided fractionation with further mass spectrometry analysis was applied for the purification of these compounds. This approach enabled us to purify and characterize two variants of the siderophore ornibactin, D and F (761 and 789 Da, respectively). Also, we could detect the variant ornibactin B (m/z= 733) and the quorum sensing molecules homoserine lactones C6-HSL, 3OH-C8-HSL and C8-HSL in the supernatant of B. catarinensis 89T. Mass spectrometry analysis showed the presence of a group of metabolites with the masses 1240, 1254, 1268, 1216, 1244 and 1272 Da, which are probably new variants of the antifungal metabolite burkoldin. Therefore, B. catarinensis 89T has a great biotechnological potential for the production of metabolites with pharmaceutical and agricultural applications for the biocontrol of phytopathogenic fungi.

Made available in DSpace on 2015-03-26T13:37:51Z (GMT). No. of bitstreams: 1 texto completo.pdf: 1284492 bytes, checksum: 553a1e66f159f200babfafbb0bac0ccd (MD5) Previous issue date: 2013-02-28 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / In vitro and greenhouse tests were carried out in order to: (i) assess the compatibility of the nematophagous fungi Pochonia chlamydosporia and Duddingtonia flagrans; (ii) evaluate the control of Meloidogyne javanica by the nematophagous fungi applied together and separately; (iii) evaluate the development of tomato plants inoculated with the nematophagous fungi and study the action of the fungi on plant nutritional status. All the compatibility tests, with the exception of an antibiosis test, indicated that P. chlamydosporia and D. flagrans are compatible to be applied toghether. An experiment in microcosm soil, in which predation of M. javanica by D. flagrans was assessed, the number of juveniles reduced in up to 72.7%. In a second assay, in microcosm soil, with different doses of the fungi applied together and separately, the application of P. chlamydosporia and D. flagrans reduced in up to 81.35% the number of juveniles when 10.000 chlamydospores of each fungus were applied per g of soil. Two experiments were conducted in a greenhouse to evaluate the effect of different concentration of P. chlamydosporia and D. flagrans in the control M. javanica and growth of tomato. The number of galls, number of eggs, height and dry weight of plants were evaluated. In both experiments the antagonists reduced the number of galls and egg production of root-knot nematode and did not affect plant height. In the first experiment, the addition of P. chlamydosporia increased plant dry matter, what was not observed in second one. An assay, in greenhouse conditions, was also carried out to evaluate growth promotion and mineral nutrients content in the tomato plants depending on the application of P. chlamydosporia and D. flagrans in combination and separated. The content of N, P, K, Ca, Mg and S and dry matter of the plants were evaluated. The two fungi increased total dry matter and mineral nutrients content in tomato plants. The combination of the two antagonists provides greater control of root-knot nematode and proved to be a promising strategy in the biological control of plant nematodes. Growth promotion and the positive effect in the nutritional status of the plants inoculated with the studied fungi indicated the great potential of these organisms for agricultural use, not only for the control of nematodes but also by its direct effect on plant growth and for providing better use of fertilizers. / Testes in vitro e em casa de vegetação foram realizados com os objetivos de: (i) avaliar a compatibilidade dos fungos nematófagos Pochonia chlamydosporia (Pc) e Duddingtonia flagrans (Df); (ii) avaliar a influência do nematoide Meloidogyne javanica e de ambos os fungos nematófagos no desenvolvimento de plantas de tomate; (iii) estudar a ação dos fungos sobre o estado nutricional das plantas. Nos ensaios de compatibilidade de Pc e Df in vitro, com exceção do teste de antibiose, todos os testes de compatibilidade dos isolados indicaram que os organismos são compatíveis. No primeiro experimento no solo em microcosmo em que foi avaliado a predação de M. javanica por Df, houve redução no número de juvenis em até 72,7%. No segundo experimento em microcosmo, em que foram avaliadas doses dos fungos em conjunto e em separado, a aplicação em conjunto dos dois organismos aumentou o controle, reduzindo em até 81,35% o número de juvenis quando 10.000 clamidósporos de cada fungo foram aplicados por g de solo. Dois experimentos foram realizados em casa de vegetação para avaliar o efeito de diferentes concentrações dos fungos nematófagos Pc e Df, isoladamente ou em mistura, no controle de M. javanica e no crescimento do tomateiro. Foram avaliadas as variáveis: número de galhas g-¹ de raiz, número de ovos g-¹ de raiz, altura e massa seca das plantas. Em ambos os experimentos observou-se o efeito dos dois antagonistas na redução do número de galhas e de ovos do nematoide das galhas. Os tratamentos não influenciaram a altura das plantas nos dois experimentos. No experimento 1, a adição de Pc aumentou a massa seca das plantas, o que não ocorreu no experimento 2. Um ensaio em casa de vegetação também foi realizado para avaliar a promoção de crescimento e aquisição de nutrientes minerais no tomateiro em função da aplicação de ambos os fungos. Após 60 dias avaliou-se a massa seca das plantas e o conteúdo de N, P, K, Ca, Mg e S. Houve efeito positivo dos dois fungos aplicados sobre a massa seca total do tomateiro e conteúdo de nutrientes minerais nas plantas. Dos resultados obtidos, conclui-se que a ação conjunta dos dois antagonistas proporciona maior controle do nematoide das galhas, mostrando-se uma estratégia promissora no controle biológico de fitonematoides. Adicionalmente, a promoção de crescimento vegetal pelos dois fungos estudados, associada à melhoria do estado nutricional das plantas, indica o potencial de uso agrícola desses organismos, não apenas para o controle de nematoides, mas também por sua ação direta no desenvolvimento do tomateiro e na economia de fertilizantes.

Made available in DSpace on 2015-03-26T13:51:48Z (GMT). No. of bitstreams: 1 texto completo.pdf: 3770731 bytes, checksum: c7215eb027111ce798d9f027c87afb98 (MD5) Previous issue date: 2009-12-11 / Conselho Nacional de Desenvolvimento Científico e Tecnológico / One of the new strategies developed to preserve food is bioconservation and within this concept, the use of bacteriocins stands out for these ingredients are considered natural food. Gram-negative bacteria are generally resistant to the action of bacteriocins of gram-positive for these are less permeable due to the presence of the outer membrane, which acts as an effective barrier. The aim of this study was to evaluate the effectiveness of bovicina HC5 associated with chelating agents or surfactants on the growth of Salmonella Typhimurium. In this work, the growth of Salmonella was evaluated in tests on microplates and by count of viable number (CFU) on agar plate. The morphology of the cells was observed in scanning electron microscopy and atomic force and leakage of potassium was measured by flame spectrophotometry. Bovicin HC5 did not significantly alter the growth of Salmonella Typhimurium, but when combined with chelating agents or surfactants, it promoted inhibition or reduction in growth rate. The association of bovicin HC5 and EDTA exerted greater antimicrobial effect by suppressing the growth of Salmonella, even with the lower concentration of bovicin HC5 that was used, 50 AU.mL-1. The association of bovicin HC5 with chelating agents, diethanolamine, gluconic acid, nitrilotriacetic acid, lactic acid and sodium citrate and the surfactant, Tween 80 and ramnolipid, reduced the growth rate. Potassium phosphate and calcium acetate did not have antimicrobial effect, even associated with bovicin HC5. Treatment with bovicin HC5, regardless of the presence of EDTA, causes leakage of potassium. Concentrations of 50 AU.mL-1 of bovicin HC5 associated with low concentrations of EDTA (1,6 mmol.L-1) exerted a bactericidal effect on Salmonella Typhimurium with a reduction of three logarithmic cycles on cell viable number. Cells treated with EDTA and bovicin HC5 and were visualized by atomic force microscopy and showed changes in their cell envelope, with a reduction in height and increase in roughness. These data demonstrated the possibility of using bovicin HC5 in control of Salmonella Typhimurium from that associated with substances that dismantled the outer membrane. / Uma das novas estratégias desenvolvidas para conservação de alimentos é a bioconservação e dentro deste conceito, o uso de bacteriocinas se destaca por estas serem consideradas ingredientes naturais dos alimentos. Bactérias gram-negativas, geralmente, são resistentes à ação de bacteriocinas de bactérias gram-positivas por essas serem menos permeáveis pela presença da membrana externa, que atua como uma efetiva barreira. O objetivo deste estudo foi avaliar a eficácia de bovicina HC5 associada a agentes quelantes ou tensoativos sobre o crescimento de Salmonella Typhimurium. O crescimento de Salmonella foi avaliado em ensaios em microplacas e por contagem de unidades formadoras de colônias (UFC) em placas. A morfologia das células foi observada em microscopia de varredura e de força atômica e o extravasamento de potássio foi mensurado por espectofotometria de chama. Bovicina HC5 não alterou significativamente o crescimento de Salmonella Typhimurium, mas quando associada a agentes quelantes ou tensoativos inibiu ou reduziu a velocidade de crescimento. A associação de bovicina HC5 com EDTA exerceu maior efeito antimicrobiano, por suprimir o crescimento de Salmonella na concentração de 50 AU.mL-1 da bacteriocina. A associação de bovicina HC5 com os quelantes, dietanolamina, ácido glucônico, ácido nitrilotriacético, ácido lático e citrato de sódio e com os agentes tensoativos, Tween 80 e ramnolípideo, reduziu a velocidade do crescimento bacteriano. Fosfato de potássio e acetato de cálcio foram os únicos agentes avaliados que não apresentaram efeito antimicrobiano, mesmo associados à bovicina HC5. O tratamento com bovicina HC5, independente da presença de EDTA, ocasionou extravasamento de potássio. Concentrações de 50 AU.mL-1 de bovicina HC5 associada a baixas concentrações de EDTA (1,6 mmol.L-1) exerceram efeito bactericida sobre Salmonella Typhimurium com redução de três ciclos logarítmicos no número de células viáveis. Células tratadas com bovicina HC5 e EDTA foram visualizadas por microscopia de força atômica e apresentaram alterações no envelope celular, com redução na altura e aumento nas rugosidades. Estes dados demonstraram a possibilidade de utilização de bovicina HC5 no controle de Salmonella Typhimurium desde que associada a substâncias que desestruturem a membrana externa.