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 2016-12-23T14:37:30Z (GMT). No. of bitstreams: 1 Tatiane Paulino da Cruz.pdf: 1055660 bytes, checksum: 58a56273fc2f252688911c2700a070e5 (MD5) Previous issue date: 2013-02-20 / The decline of guava has been responsible for the eradication of many orchards nationwide, resulting in high economic loss to producers. Due to increasing pressure from society for healthier products, free from chemicals and to increase the importance of essential oils in the management of plant diseases, the aim of this study was to determine the chemical composition and fungitoxic evaluate the effects of essential oils of Eucalyptus citriodora (eucalyptus), E. grandis (eucalyptus), Cymbopogon citratus (lemon grass), Cymbopogon winterianus (lemongrass), Cinnamomum zeylanicum (Cinnamon), Eucalyptus commercial Rosmarinos officinalis (rosemary), Ocimum gratissimum (basil-clove) and Syzygium aromaticum (clove india ) on three isolates of Fusarium solani and M. enterolobii. The essential oil was obtained by hydrodistillation using a Clevenger type apparatus, and its yield was determined relative to the dry weight of the plant. The analysis by gas chromatography and gas chromatography with mass detector of essential oils led to the identification of compounds. To evaluate the effect of essential oils on mycelial growth, sporulation and germination of spores of the fungus were used aliquots of 5, 10, 15, 20, 25 and 30 μl of essential oils that were distributed on the surface of PDA culture medium contained in Petri dishes before subculturing the fungus. To evaluate the effect of oil on hatching and mortality of second stage juveniles (J2) of the nematode were used aqueous suspensions containing 200 eggs or 200 J2 which were added in the same rates of essential oils from botanicals mencinadas the previously. The experimental design was completely randomized with a 9 x 6 + 1, (nine oils, six aliquots and an additional control) with 5 repetitions. The lowest rate of essential oils of clove, cinnamon and clove basil-inhibited by more than 90% spore germination, mycelial growth and sporulation of the fungus isolates as well as the emergence of J2, as for oils eucalyptus, lemongrass, citronella inhibition was greater than 90% from the rate of 15 μl, rosemary oil showed the worst result in 50% inhibiting the development of the fungus isolates / O declínio da goiabeira tem sido responsável pela erradicação de muitos pomares em âmbito nacional, o que resulta em elevada perda econômica aos produtores. Devido a crescente pressão da sociedade por produtos mais saudáveis, livres de produtos químicos e com aumento da importância dos óleos essenciais no manejo de doenças de plantas, objetivou-se com este trabalho determinar a composição química e avaliar os efeitos fungitóxicos dos óleos essenciais de Eucalyptus citriodora (eucalipto), E. grandis (eucalipto), Cymbopogon citratus (capim-limão), Cymbopogon winterianus (citronela), Cinnamomum zeylanicum (Canela), Eucalipto comercial, Rosmarinos officinalis (alecrim), Ocimum gratissimum (alfavaca-cravo) e Syzygium aromaticum (cravo-da-india) sobre três isolados de Fusarium solani e M. enterolobii. O óleo essencial foi obtido por hidrodestilação, utilizando um aparelho tipo Clevenger, e o seu rendimento foi determinado em relação à massa seca da planta. As análises de cromatografia gasosa e cromatografia gasosa com detector de massas de óleos essenciais levaram à identificação dos compostos. Para avaliar o efeito dos óleos essenciais no crescimento micelial, esporulação e germinação de esporos do fungo, foram utilizadas alíquotas de 5; 10; 15; 20; 25 e 30 μL dos óleos essenciais que foram distribuídas na superfície do meio de cultura BDA contido em placas de Petri antes da repicagem do fungo. Para a avaliação do efeito dos óleos sobre a eclosão e mortalidade de juvenis de segundo estádio (J2) do nematóide foram utilizadas suspensões aquosas contendo 200 ovos ou 200 J2 nas quais foram adicionadas as mesmas alíquotas dos óleos essenciais das espécies botânicas mencinadas anteirormente. O delineamento experimental foi inteiramente casualizado num esquema fatorial 9 x 6 + 1, (nove óleos, seis aliquotas e uma testemunha adicional) com 5 repetições. A menor alíquota dos óleos essenciais de cravo-da-índia, canela e alfavaca-cravo inibiram em mais de 90% a germinação de esporos, o crescimento micelial e a esporulação dos isolados do fungo assim como a eclosão de J2, já para os óleos de eucalipto, capim limão, citronela a inibição foi superior a 90% a partir da alíquota de 15 μl, o óleo de alecrim foi o que apresentou o pior resultado inibindo em 50% o desenvolvimento dos isolados do fungo

Made available in DSpace on 2015-04-20T12:31:11Z (GMT). No. of bitstreams: 1 Rogerio Eiji Hanada.pdf: 659208 bytes, checksum: 3edd1deee7d95cb812891d0f6918caa6 (MD5) Previous issue date: 2006-03-30 / Black pod rot of cacao (Theobroma cacao) is one of the most important diseases affecting the crop worldwide. It is responsible for reducing over than 30% of the cocoa production every year. The disease is caused by Phytophthora spp. being P. palmivora (Butler) Butler one of the most common species in the cocoa growing area of Bahia, Brazil. Fungal endophytes were collected from healthy plants of cacao and cupuaçu (Theobroma grandiflorum (willd. ex Spreng.) Shum. from the states of Amazonas and Bahia, Brazil. The main objectives of this project were to isolate and to select endophytic fungi with biocontrol potential against P. palmivora. Three hundred fungal endophytes were obtained, being 104 screened against P. palmivora based on artificial inoculations of attached pods. Field experiments were performed at Almirante Cacau (Bahia-Brazil). Evaluations of disease severity were possible by using a disease rating scale specifically developed. Eight fungal isolates were selected and tested in the axenic cocoa seedlings for proving the endophytic behavior. From 103 fungi tested in the field, a Trichoderma viride Pers. ex S.F. Gray isolate was selected as the most promising candidate. This fungus was applied on cocoa plants in the field for evaluating its life span in several plant organs. The optimum spore concentration was established and formulation studies for a high performance application were evaluated. The compatibility for a tank mix application with chemical fungicides was tested and also several methods for mass production by solid state fermentation. Studies on the mode of action was carried out by evaluating the production of chitinase, b1,3 glucanase and celulase. With the exception of Tolypocladium sp., all the isolates tested: Three Fusarium, 2 Pestalotiopsis, one Curvularia and T. viride were recovered from the axenic cocoa plants. T. viride on adult cocoa plants showed an efficient epiphytic colonization on the pod surface and inside the trunk as an endophyte, been recovered after 35 and 50 days after application. The disease severity reduced by increasing the inoculum concentration up to 107 conídios.ml-1. The addition of an emulsified vegetable oil was responsible to increase the antagonist efficacy. The T. viride population over the pods decreased after 20 days. After 40 days the fungus was more dificult yor recoveretion. Disease severity increased by decreasing the antagonist population. T. viride germination was not affected by the Cooper hydroxide and Fosetil. A spore concentration of over 108 was achieved using rice as a substrate. The antagonist could grow and secrete b1,3 glucanase and chytinase in all the liquid media tested, however only on media supplemented with carboxymetilcelulose and P. palmivora mycelium were able to induce the production of celulase. / A podridão-parda do fruto de cacaueiro (Theobroma cacau) é considerada mundialmente a doença mais importante da cultura. Várias espécies de Phytophthora estão associadas à doença, sendo P. palmivora a de maior ocorrência na Bahia. O trabalho teve como objetivo isolar e selecionar fungos endofíticos com potencial de controlar a podridãoparda. A viabilização da aplicação no campo e o provável modo de ação também foram estudados. Os fungos endofíticos foram isolados de cacaueiros e cupuaçuzeiros (T. grandiflorum) dos estados do Amazonas e Bahia. A seleção foi conduzida em plantio comercial da Empresa Almirante Cacau, em Itajuípe, BA. Posteriormente, oito isolados selecionados, em campo, como os mais eficientes foram submetidos ao estudo de comprovação endofítica utilizando cacaueiros axênicos. O fungo endofítico mais eficiente no controle da doença, foi aplicado em plantas adultas e a sua sobrevivência foi avaliada nos órgãos da planta. Concentrações de 104, 105, 106, 107 e 5,0 x 107 conídios.ml-1, formulações em óleo agrícola vegetal emulsionado (Nortox) 2 % e sacarose 2%, comportamento da severidade da doença com o declínio da população do antagonista, compatibilidade com os fungicidas hidróxido de cobre e fosetil Al, produção massal de inóculo utilizando substrato de arroz e a produção extracelular de quitinase, b1,3 glucanase e celulase, em meio de cultura líquido, foram também avaliados. Foram testados 103 isolados de fungos endofíticos, sendo que Trichoderma viride foi o mais eficiente no controle da doença. Exceto Tolypocladium sp., os demais fungos: três isolados de Fusarium, dois de Pestalotiopsis, um isolado de Curvularia sp. e um de T. viride foram recuperados após a inoculação em cacaueiros axênicos. Trichoderma viride, em planta adulta, apresentou boa colonização epifítica, na parte externa do fruto, e como endofítico no interior do caule, sendo recuperado, com maior freqüência, em até 35 a 50 dias após a aplicação. A severidade da doença foi inversamente proporcional às concentrações de inóculo do antagonista, até 107 conídios.ml-1 e a adição do óleo vegetal na suspensão aumentou a eficiência do bioagente. A população de T. viride aplicado no fruto diminuiu rapidamente, em 20 dias de aplicação e, aos 40 dias, a recuperação tornou-se difícil. Por outro lado, a severidade da doença aumentou, à medida que a população de T. viride diminuiu. Trichoderma viride foi mais sensível ao hidróxido de cobre, porém a germinação dos conídios se manteve acima de 90% em solução fungicida na concentração recomenda pelo fabricante. Concentração acima de 108 conídios.g-1 do antagonista foi produzida em grãos de arroz. O bioagente cresceu e secretou b1,3 glucanase e quitinase em todos os meios de cultura, no entanto, somente os meios suplementados com carboximetilcelulose e micélio de P. palmivora foram capazes de induzir o fungo a secretar celulase.

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / The sudden death syndrome (SDS), caused by species of Fusarium, is a disease difficult to control and there are no management techniques and resistant cultivars that give long-term positive results. This study aimed to assess populations of Fusarium spp. and Trichoderma spp. associated to eight soybean genotypes, cultivated under no tillage; to determine if the control of Fusarium varies among isolates of Trichoderma; to identify isolates of Fusarium spp. obtained from different genotypes, and to determine their toxicity and pathogenicity to two genotypes of soybean. Soil and roots were collected from experimental plots located at the Fundação Centro de Experimentação e Pesquisa (FUNDACEP), obtaining isolates of Fusarium spp. and Trichoderma spp. There were no differences in the population of Fusarium spp. in plants with symptoms of SDS among the different genotypes. For Trichoderma spp. there was a significant difference among genotypes from plants with and without symptoms of SDS and the genotype CEPS 06006 RR had the biggest population of the antagonist. In the direct confrontation test, eight isolates of Trichoderma spp. obtained the highest score (1) in relation to the control of Fusarium spp. and isolates of Trichoderma spp. from soil of areas with the SDS symptom were more aggressive. Nine species of Fusarium: F. avenaceum, F. solani, F. equiseti, F. Acuminatum, F. kyushuense, F. graminum, F. subglutinans, F. verticillioides and F. lateritium were identified. All isolates promoted internerval chlorosis, necrosis and death of seedlings in the toxicity test. In the pathogenicity test in plants inoculated with the pathogen there was an increase in leaves and roots dry weight and in the number of pods, indicating that there may be species specificity of the species studied in relation to genotype. / A Podridão Vermelha da Raiz da soja (PVR), causada por espécies de Fusarium, é uma doença de difícil controle, não existindo técnicas de manejo e cultivares resistentes que tenham resultados positivos a longo prazo. Assim, este trabalho objetivou quantificar populações de Fusarium spp. e Trichoderma spp. associadas à oito genótipos de soja, cultivados em sistema plantio direto, observar se o biocontrole de Fusarium spp. difere entre isolados de Trichoderma spp., identificar isolados de Fusarium spp. obtidos de diferentes genótipos e verificar sua toxicidade e patogenicidade a dois genótipos de soja. Amostras de solo e raízes foram coletadas em parcelas experimentais na Fundação Centro de Experimentação e Pesquisa (FUNDACEP), obtendo-se isolados de Fusarium spp. e Trichoderma spp. Não houve diferença na população de Fusarium spp. em plantas com sintomas de PVR entre os diferentes genótipos. Para Trichoderma spp., houve diferença entre as populações nos genótipos de plantas com e sem sintomas de PVR, tendo o genótipo CEPS 06006 RR apresentado maior população do antagonista. No teste de confronto direto, oito isolados de Trichoderma spp. obtiveram a melhor nota (um), em relação a Fusarium spp. e isolados de Trichoderma spp. oriundos de solo em áreas com o sintoma de PVR foram mais eficientes. Foram identificadas nove espécies de Fusarium: F. avenaceum, F. solani, F. equiseti, F. acuminatum, F. kyushuense, F. graminum, F. subglutinans, F. verticillioides e F. lateritium. Todos os isolados de Fusarium spp. provocaram clorose internerval, necrose e morte de plântulas no teste de toxicidade. No teste de patogenicidade, em plantas inoculadas com o patógeno houve aumento na massa seca de parte aérea e de raízes, no número de vagens, indicando que pode haver especificidade das espécies estudadas em relação ao genótipo.

L'azote est connu pour influencer la sensibilité de certaines plantes à diverses maladies. Dans le cas des maladies causées par Botrytis cinerea, le rôle de la fertilisation azotée semble être variable, avec des niveaux élevés favorisant ou réduisant la gravité en fonction des études. Pour vérifier si cette variabilité pourrait être due à des différences possibles entre plantes hôtes, à la pression d'inoculum ou à un comportement différent de différentes souches de l'agent pathogène, des études ont été menées pour évaluer l'effet de différents régimes de fertilisation azotée sur la sensibilité de la tomate et de la laitue à six isolats de B. cinerea. Des effets épidémiologiques éventuels de la fertilisation azotée à travers la sporulation du pathogène et la pathogénicité de l'inoculum secondaire ont également été étudiés sur la tomate. Les plantes ont été cultivées dans un système hors-sol fertirrigué au goutte à goutte. Une nutrition azotée différentielle allant de 0,5 à 30 mM de nitrate a été appliquée pendant les quatre dernières semaines avant l'inoculation des plantes sur des feuilles (laitue) ou sur des plaies d'effeuillage (tomates). Après inoculation, les plantes ont été incubées dans des conditions propices au développement de la maladie. Sur la tomate, l'apparition de la maladie a été retardée et la sévérité globale des symptômes était plus faible pour tous les isolats aux doses de fertilisation azotée les plus élevées, indépendamment de la concentration d'inoculum. Toutefois, le taux d'expansion des lésions sur tige a été affecté différemment selon les souches, diminuant avec des niveaux croissants de fertilisation azotée pour les isolats les plus agressifs, mais augmentant pour les isolats moins agressifs. En contraste avec la tomate, la fertilisation azotée a augmenté la sévérité de la maladie sur la laitue pour tous les isolats testés. La sporulation de B. cinerea sur tomate a diminué significativement avec l'augmentation de la fertilisation azotée des plantes jusqu'à 15-30 mM de nitrate et la pathogénicité des spores a été fortement influencée par l'état nutritionnel de leur substrat de production. Elle était la plus élevée pour les spores produites sur des plantes ayant reçu des niveaux de fertilisation azotée très faibles ou très élevés (0,5 ou 30 mM nitrate) et la plus faible pour celles produites sur des plantes ayant reçu une fertilisation azotée modérée. La fertilisation des plantes a aussi fortement affectée l'efficacité de deux agents de lutte biologique (Trichoderma atroviride et Microdochium dimerum) à protéger les plaies d'effeuillage de la tomate contre B. cinerea. Les plus hauts niveaux de protection ont été obtenus avec la fertilisation azotée élevée et ceci a pu être lié à un retard dans le développement des symptômes sur les tiges, parfois associé à un ralentissement de l'expansion des lésions. Des études histologiques ont montré que la diminution de la gravité de la maladie sous fertilisation azotée élevée a été associée à une altération structurelle des cellules du mycélium de Botrytis. En présence d'un agent de lutte biologique, l'effet de l'agent pathogène a été en outre associé à une vacuolisation, dépôt de glycogène et mort des cellules mycéliennes. Les hypothèses pour expliquer ces résultats sont discutées à la lumière des effets physiologiques possibles de la fertilisation azotée sur la disponibilité des nutriments pour l'agent pathogène dans les tissus de l'hôte et de la production possible de métabolites de défense de la plante. Ces résultats ouvrent de nouvelles perspectives pour manipuler la fertilisation azotée comme un outil pour la protection intégrée des cultures maraîchères / 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 damping-off and stem rot disease syndrome is harmful to many cultivated crops. Damping-off and stem rot caused by Sclerotium rolfsii Sacc. on cowpea results in yield losses with serious socio-economic implications. The objectives of the present study were to investigate the occurrence of the diseases in Benin, study etiology and factors influencing the diseases, and develop strategies for the control of the diseases in the field. Results showed that the diseases are distributed countrywide. Sclerotium rolfsii was the main causal agent but minor pathogens, namely Pythium sp., Rhizoctonia solani Kühn and Phoma pomorum Thüm were also recorded. In the Ouémé valley, the diseases were favoured by soil moisture and S. rolfsii initial inoculum that were higher closer to the river. Sclerotium rolfsii isolates collected in the valley showed genetic diversity in terms of pathogenicity, mycelial compatibility groups and ITS rDNA sequences. A paper-based screening method was found to be a rapid laboratory method for screening for resistance in cowpea cultivars. Furtheremore, Moringa oleifera L. leaf extracts, Trichoderma Kd 63 and Trichoderma IITA 508 significantly reduced the disease incidence. The best disease control was recorded in the field when M. oleifera seed treatment was integrated with a soil sprinkle of Trichoderma. The present work provides information on damping-off and stem rot of cowpea in Benin and control strategies for ecologically sustainable cowpea production. / Thesis (DPhil (Microbiology and Plant Pathology))--University of Pretoria, 2006. / Microbiology and Plant Pathology / unrestricted

Les plantes interagissent en permanence avec une grande diversité de microorganismes qu'elles sélectionnent entre autre au niveau de leurs racines. Certaines bactéries, qualifiées de PGPR (Plant Growth-Promoting Rhizobacteria), sont capables de stimuler la croissance et la santé de la plante, grâce à l'expression d'une large panoplie de propriétés phytobénéfiques. L'hypothèse actuelle serait que les PGPR possédant un nombre maximal de ces fonctions auraient un plus fort impact bénéfique sur le végétal. Toutefois, l'occurrence de ces PGPR multifonctions dans la rhizosphère n'est pas connue. De plus, les possibles interactions entre propriétés co-occurrentes au sein d'une même PGPR et la résultante de ces interactions sur la plante sont relativement peu documentées. Dans ce contexte, ce projet de thèse a eu comme objectif général de mieux comprendre la distribution des propriétés phytobénéfiques chez un groupe bactérien possédant un large éventail de ses propriétés, celui des Pseudomonas fluorescents et d'évaluer chez une PGPR modèle multifonction, Pseudomonas fluorescens F113, si des interactions fonctionnelles existent entre ces propriétés et déterminer leurs contributions respectives à l'effet phytostimulateur. L'étude d'environ 700 isolats de Pseudomonas provenant de la rhizosphère de deux cultivars de maïs et de sol nonrhizosphérique, a mis en évidence que ces cultivars sélectionnent majoritairement des Pseudomonas fluorescents (i) arborant un nombre réduit de propriétés phytobénéfiques (1 à 5 propriétés) et (ii) appartenant à des sous-groupes taxonomiques particuliers. Par ailleurs, l'étude de génomique comparative que nous avons menée au sein du groupe des Pseudomonas fluorescents souligne le lien entre phylogénie et les profils de propriétés phytobénéfiques possédées par les Pseudomonas. Enfin, chez P. fluorescens F113, les propriétés co-occurrentes ne contribuent pas de façon égale à l'effet observé sur la plante. L existence d'interactions entre ces propriétés co-occurrentes a également été mise en évidence. De ce réseau d'interactions, résultera un effet bénéfique observé sur la plante particulier. Ces résultats sont importants pour mieux comprendre la place et le rôle de ces PGPR multifonctions dans la rhizosphère. Ils permettent d'approfondir nos connaissances à propos du fonctionnement écologique des Pseudomonas fluorescents au sein du rhizomicrobiote / Plants are constantly interacting with a huge diversity of microorganisms, especially in the rhizosphere, where plant roots (through rhizodeposition) will select particular bacterial populations. Some bacteria, called PGPR (Plant Growth-Promoting Rhizobacteria), are able, in turn, to improve plant growth and health, through the expression of a wide range of plantbeneficial properties. The current hypothesis is that PGPR harboring a maximum number of these plant-beneficial properties would provide a better effect on plant. However, the occurrence of these multi-trait PGPR in the rhizosphere is unknown. In addition, the interaction between co-occurring plant-beneficial properties within the same PGPR and their resulting effects on plant are poorly documented. In this context, the aims of this thesis were to (i) determine the distribution of plant-beneficial properties in the fluorescent Pseudomonas bacterial group, known to harbor a wide range of these properties, and (ii) to evaluate in the PGPR model strain Pseudomonas fluorescens F113, if a crosstalk between plant-beneficial properties occurs and to determine the relative contribution of each co-occurring plantbeneficial properties. The study of 700 Pseudomonas isolates from the rhizosphere of two maize cultivars and from non-rhizosphere soils shows that cultivars mostly select fluorescent Pseudomonas (i) displaying few plant-beneficial properties (up to 5 properties) and (ii) belonging to particular taxonomic subgroups. Furthermore, the comparative genomic study we conducted within the fluorescent Pseudomonas group emphasizes the link between phylogeny and plant-beneficial profiles owned by Pseudomonas. Finally, in P. fluorescens F113, co-occurring plant-beneficial properties do not contribute equally to the observed effects on plant. The existence of crosstalks between these co-occurring plant-beneficial properties was also highlighted. This network of functional interactions may lead to specific effects on plant. These results are important for understanding the place and role of multi-trait PGPR in the rhizosphere. They help to deepen our understanding of the ecological functioning of fluorescent Pseudomonas among the rhizomicrobiote

Les interactions entre micro-organismes sous-tendent de nombreux services écosystémiques, y compris la régulation des maladies des plantes cultivées. Un acteur de cette régulation est le pathobiome, défini comme le sous-ensemble des micro-organismes associés à une plante hôte en interaction avec un agent pathogène. L'un des défis actuels consiste à reconstruire les pathobiomes à partir de données de metabarcoding, pour identifier des agents potentiels de biocontrôle et pour surveiller en temps réel leurs réponses aux changements environnementaux. Plusieurs verrous méthodologiques doivent cependant être levés pour atteindre ces objectifs. Tout d’abord, il n’existe pas de consensus concernant l’approche bioinformatique la plus fiable pour déterminer l’identité et l’abondance des micro-organismes présents dans les échantillons végétaux. De plus, les réseaux microbiens construits avec les méthodes actuellement disponibles sont des réseaux d’associations statistiques entre des comptages de séquences, non directement superposables aux réseaux d’interactions (ex : compétition, parasitisme) entre micro-organismes. L’objectif de la thèse était donc de déterminer les approches bioinformatiques et statistiques les plus pertinentes pour reconstruire des réseaux d’interactions microbiennes à partir de données de metabarcoding. Le modèle d’étude était la vigne (Vitis vinifera L. cv. Merlot noir) et l’oïdium de la vigne, Erysiphe necator. Nous avons tout d’abord déterminé l’approche bioinformatique la plus adaptée pour identifier la communauté fongique associée à ce pathogène, en comparant la capacité de 360 pipelines à retrouver la composition d’une communauté artificielle de 189 souches fongiques. DADA2 est apparu comme l’outil le plus performant. Nous avons ensuite évalué l’influence de la pratique culturale (viticulture conventionnelle vs. biologique) sur les communautés fongiques des feuilles et évalué le niveau de réplicabilité des réseaux microbiens construits avec une méthode d’inférence classique, SparCC. La réplicabilité était très faible, jetant ainsi un doute sur l’utilité de ces réseaux pour le biocontrôle et la biosurveillance. Nous avons donc utilisé une nouvelle approche statistique, le modèle PLN, qui permet de prendre en compte la variabilité environnementale, pour explorer finement le pathobiome d’Erysiphe necator. Les interactions microbiennes prédites par le modèle sont en cours de comparaison avec des expériences de confrontations de levures en co-cultures. Une approche alternative, HMSC, a également été testée sur un autre modèle biologique et certaines prédictions ont été confrontées avec succès aux données de la littérature. Les réseaux microbiens, sous réserve d’amélioration des méthodes de reconstruction, pourraient donc être utilisés pour capturer les signaux des interactions biotiques dans le pathobiome. / Interactions between microorganisms underpin many ecosystem services, including the regulation of crop diseases. An actor in this regulation is the pathobiome, defined as the subset of microorganisms associated with a host plant in interaction with a pathogen. One of the current challenges is to reconstruct pathobiomes from metabarcoding data, in order to identify potential biocontrol agents and to monitor in real time their responses to environmental changes. However, several methodological hurdles must be overcomed to achieve these objectives. First, there is no consensus on the most reliable bioinformatics approach to determine the identity and abundance of microorganisms present in plant samples. In addition, microbial networks built with currently available methods are networks of statistical associations between sequence counts, not directly related to networks of interactions (e. g. competition, parasitism) between microorganisms. The objective of the thesis was therefore to determine the most relevant bioinformatics and statistical approaches to reconstruct microbial interaction networks from metabarcoding data. The study system was grapevine (Vitis vinifera L. cv. Merlot noir) and the fungal agent of grapevine powdery mildew Erysiphe necator. First, we determined the most appropriate bioinformatics approach to identify the fungal community associated with this pathogen, by comparing the ability of 360 pipelines to recover the composition of an artificial community of 189 fungal strains. DADA2 has emerged as the most powerful tool. We then evaluated the influence of the cropping system (conventional vs. organic viticulture) on foliar fungal communities and assessed the level of replicability of microbial networks built with a standard inference method, SparCC. Replicability was very low, casting doubt on the usefulness of these networks for biocontrol and biomonitoring We therefore used a new statistical approach, the PLN model, which allows us to take into account environmental variability, to finely explore the pathobiome of Erysiphe necator. The microbial interactions predicted by the model are being compared with experiments confronting yeasts in co-cultures. An alternative approach, HMSC, was also tested on another biological model and some predictions were successfully compared with the data in the literature. Microbial networks, provided improved reconstruction methods, could therefore be used to capture signals of biotic interactions in the pathobiome.

<p>  </p> <p><em>Sclerotinia sclerotiorum </em>is a soilborne pathogen of soybean that causes Sclerotinia stem rot, alternatively called white mold. Sclerotinia stem rot can cause significant yield losses under cool and wet environmental conditions. Two biofungicides, <em>Coniothyrium minitans </em>and <em>Bacillus amyloliquefaciens, </em>are currently available and labeled to limit or suppress <em>S. sclerotiorum</em> in soybean. These biofungicides can be applied in place of synthetic foliar fungicides to provide an alternative mode of action for the control of Sclerotinia stem rot. However, limited information is available regarding the efficacy of <em>C. minitans </em>and <em>B. amyloliquefaciens </em>as biocontrol agents of <em>S. sclerotiorum </em>in soybean and the sensitivity of the biofungicides biological activity on <em>S. sclerotiorum </em>to pesticides commonly used in soybean production systems. This research aims to provide management recommendations for <em>S. sclerotiorum </em>in soybean using <em>C. minitans </em>and <em>B. amyloliquefaciens </em>and to develop guidelines for how to incorporate the biofungicides into an established soybean pest management program. To assess the effectiveness of <em>C. minitans </em>and <em>B. amyloliquefaciens </em>as biocontrol agents of <em>S. sclerotiorum </em>dual culture, amended media, and soil plate assays were conducted along with experiments in the growth chamber and field. The presence of a distinct inhibition zone surrounding the <em>B. amyloliquefaciens </em>colony in the dual culture assay and the absence of mycelial growth on the media plates amended with <em>B. amyloliquefaciens </em>confirmed that the bacteria can control the mycelial growth of <em>S. sclerotiorum </em>through antibiosis. The absence of an inhibition zone surrounding the <em>C. minitans </em>isolate in the dual culture assay along with the degradation of sclerotia following treatment with <em>C. minitans </em>in the soil plate assay indicates an inability to limit the mycelial growth of <em>S. sclerotiorum </em>and confirms that the primary mode of action is mycoparasitism. In the growth chamber, <em>B. amyloliquefaciens</em> at 14.03 L/ha applied using the dip method significantly reduced Sclerotinia stem rot lesion length when compared to the non-treated control and resulted in the lowest lesion area under the disease progress curve (lAUDPC). When <em>B. amyloliquefaciens </em>and <em>C. minitans </em>were applied in the field, no differences were observed between treatments for soybean moisture, test weight, or yield. To evaluate the sensitivity of <em>B. amyloliquefaciens </em>and <em>C. minitans</em> biological activity on <em>S. sclerotiorum </em>to pesticides commonly used in soybean production systems a poison plate assay as well as soil plate, growth chamber, and field experiments were conducted. In the poison plate assay <em>C. minitans </em>was most sensitive to the preemergence herbicide flumioxazin and the synthetic fungicides boscalid and fluazinam, while <em>B. amyloliquefaciens </em>was sensitive only to the synthetic fungicide fluazinam. In the soil plate assay the mycoparasitic activity of <em>C. minitans </em>on sclerotia of <em>S. sclerotiorum </em>was sensitive to flumioxazin, metribuzin, glyphosate, picoxystrobin, and boscalid. In the controlled environment experiments, none of the pesticides tested decreased the efficacy of <em>B. amyloliquefaciens</em>. There were no significant interactions between <em>C. minitans </em>and <em>B. amyloliquefaciens </em>with preemergence herbicides, postemergence herbicides, and synthetic fungicides for soybean moisture, test weight, and yield. This research demonstrates that <em>B. amyloliquefaciens </em>and <em>C. minitans </em>are effective biocontrol agents of <em>S. sclerotiorum </em>in soybean. However, antagonistic relationships exist between the biofungicides and certain preemergence, postemergence, and synthetic fungicides used in soybean production systems.</p>

Benthic filter feeders (BFF) can reduce phytoplankton concentration (abundance) thereby controlling eutrophication in several ecosystems, including rivers. However, experiments suggest warming can alter the relationship between BFF grazing rate and the growth rate of (heterotrophic) planktonic prey. To investigate how eutrophication control by grazers is altered with temperature under the influence of other important abiotic (water depth, and speed, light, and turbidity) and biotic factors (initial phytoplankton concentration [hereafter: Pin value], BFF density and spatial BFF distribution), we developed a spatially-explicit computer simulation model. This model simulates the dynamics of a phytoplankton population traveling through a simplified river channel while being grazed by BFF. Our model includes the thermal responses of BFF grazing and phytoplankton growth. The results show that BFF grazing can qualitatively alter and, in some circumstances, even reverse the response of phytoplankton to warming. Moreover, the response of grazer-controlled phytoplankton to warming, water depth and Pin value is non-linear and phytoplankton can increase steeply with slight changes within some ranges of these variables. In addition, these variables can interact causing their combined effects on eutrophication to differ from what is expected considering their isolated effects. Generally, the effect of most variables, including temperature, Pin value and BFF density and spatial distribution, is larger at shallow waters. Moreover, our study shows that phytoplankton control can be substantially improved by heterogeneous BFF distributions where the BFF are located at the extremes of the river either upstream or downstream instead of homogenously distributed along the whole river. However, warming can cause a switch between these two optimal distributions or even can cause differences among the spatial distributions to disappear. In general, the homogeneous BFF distribution can be used as conservative estimate of eutrophication control. In conclusion, this work shows that trophic control can qualitatively alter the response of eutrophication to warming, supporting previous studies suggesting that the prediction of global warming effects requires considering not only the thermal responses of organisms but also their trophic interactions. In addition to these biotic variables, this thesis reveals that considering the interactions between abiotic and biotic variables and including their spatial distribution are important for eutrophication control. Especially, the detection of thresholds in the response of grazer-controlled phytoplankton to temperature, water depth, Pin value, and spatial BFF distribution indicates that one should be careful with predictions because of potential abrupt changes. Although further studies are needed to make specific recommendations for water quality management, our work provides preliminary suggestions on the conditions where grazers or Pin reductions can be more efficient to control eutrophication.