The use of matrix assisted laser desorption ionization time-oflight mass spectrometry, Fourier transform infrared spectroscopy, and other analytical means of identifying and differentiating microorganisms hold much promise. These analytical tools have been extensively assessed for their ability to differentiate bacteria and fungi. Most of this research has been coordinated in medically relevant microorganisms, but the technology can work just as well with agriculturally important microorganisms. In this thesis, these technologies were reviewed and then subsequently studied for their ability to differentiate Aspergillus species (that devastate corn and other crops yearly with aflatoxin contamination), as well as Macrophomina phaseolina and Thielaviopsis basicola which limit yields on soybean and other crops yearly. With the use of these technologies, harmful plant pathogens could be identified and subsequently treated to improve crop yields and also help to protect our nation and state’s food supply.

Thielaviopsis basicola (Berk. and Br.) is a widespread, soil-borne, pathogenic fungus which causes a root rot on tobacco (Nicotiana tabacum L.). Presently, control is achieved primarily through the use of resistant cultivars of tobacco or chemicals. In order to develop a disease prediction program, disease-inoculum density relationships must be determined. It was the purpose of this study to develop an improved procedure for estimating the populations of T. basicola in naturally infested soil. A second objective was to determine the horizontal inoculum pattern of T. basicola in tobacco field soil, and the third objective was to determine the relationship between black root rot of tobacco and inoculum density of T. basicola in naturally infested soil. Two fields in southwestern Virginia were randomly and systematically sampled to assay the population density of T. basicola in the soil. Populations of T. basicola were isolated using a modified carrot-disc baiting technique and calculated using a colonization correction factor. Frequencies of calculated population densities of T. basicola in soil samples were compared, using a Chi-square test, to the expected frequencies indicated by the Poisson, Neyman Type A, and negative binomial distributions. Temperature-tank studies were conducted to investigate inoculum density-disease relationships. Two cultivars of tobacco were grown in soil naturally infested with T. basicola. The soil was thoroughly mixed before use to preclude inoculum clumping. The results obtained with either systematic or random sampling indicated that propagules of T. basicola are clumped or aggregated in tobacco field soil, as there was a good fit of the data to the negative binomial distribution. Also, the variance to mean ratios were significantly (P = 0.05) greater than 1.0 which is indicative of a clumped pattern. Other indexes of aggregation indicated a low to moderate degree of inoculum clumping in soil. For soil temperature-tank tests, root rot incidence was 96-100% at all natural population densities for both slightly resistant cv. Burley 21 and susceptible cv. Judy's Pride plants, and T. basicola was consistently associated with rotted roots. Low R² values were obtained in regression analyses of T. basicola inoculum density versus mean percent root rot per Burley 21 plant and per Judy's Pride plant. / Master of Science

Submitted by (lucia.rodrigues@ufrpe.br) on 2017-03-13T14:20:21Z No. of bitstreams: 1 Iwanne Lima Coelho.pdf: 1395783 bytes, checksum: bfb69bfcc0a83cb3f0e7ff59aade864a (MD5) / Made available in DSpace on 2017-03-13T14:20:21Z (GMT). No. of bitstreams: 1 Iwanne Lima Coelho.pdf: 1395783 bytes, checksum: bfb69bfcc0a83cb3f0e7ff59aade864a (MD5) Previous issue date: 2013-07-30 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - CAPES / The occurrence of stem bleeding, caused by Ceratocystis paradoxa (Anamorphic: Thielaviopsis paradoxa), compromises the productivity of green dwarf coconut and poses a threat to the entire production chain of green coconut. There are no fungicides registered in the Ministério da Agricultura Pecuária e Abastecimento (MAPA) for the management of this disease, the indiscriminate use of the fungicide thiophanate methyl may result in the emergence of resistant pathogenic strains, environmental contamination and to human health. Biological control using yeast, promising biocontrol agents, may represent an efficient and applicable solution when aggregated to crop management. Work on yeasts not recovered in control of the causal agent of this disease, as well as studies related to its field control. The study aimed to select yeasts from fruits of the green dwarf coconut, with potential for biocontrol of stem bleeding coconut on the seedlings of green dwarf coconuts. Thirty-seven yeasts, isolated from immature coconuts, were evaluated in vivo for their potential to reduce the symptoms of stem bleeding in seedlings of green dwarf coconuts, packed in a greenhouse under 40 ± 2 ° C and 60 % RH, for two methods of application: spray (10 mL de 1x107 cells mL-1) and injection (1 mL de 1x107 cells mL-1); and in vitro (28 ± 2 °C, 12 h photoperiod), as for inhibition of mycelial growth, volatile metabolic action, parasitism, antibiosis and "killer" activity. Fifteen yeast strains in vivo reduced the percentage of the area damaged by C. paradoxa isolated Moju - PA. In vitro, the isolates identified as Pichia guilliermondii (LEV 25) and Candida dosseyi (LEV47), stood out as potentially promising antagonists, because of the diameter of the inhibited mycelial growth , competing for space and nutrients, and reduced production of conidia by the action of volatile metabolites on pathogenic isolates of Moju - PA, Neópolis- SE and Sousa –PB. This is the first report of reduction of the symptoms of stem bleeding in seedlings of green dwarf coconuts by the antagonistic action of yeast on C. paradoxa. The isolates of P. guilliermondii and C. dosseyi have potential as a component of integrated management of coconut stem bleeding / A ocorrência da resinose, causada por Ceratocystis paradoxa (Anamorfo: Thielaviopsis paradoxa), compromete a produtividade do coqueiro-anão-verde e representa ameaça para toda a cadeia produtiva do coco-verde. Não há registros no Ministério da Agricultura Pecuária e Abastecimento (MAPA) de fungicidas para o manejo dessa doença, o uso indiscriminado do fungicida tiofanato metílico pode resultar no surgimento de linhagens patogênicas resistentes, contaminação ambiental e à saúde humana. O controle biológico utilizando-se leveduras, promissores agentes de biocontrole, pode representar uma solução eficiente e aplicável quando agregada ao manejo da cultura. Não foram recuperados trabalhos sobre leveduras no controle do agente causal dessa enfermidade, assim como estudos referentes ao seu controle em campo. O trabalho teve por objetivo selecionar leveduras, obtidas de frutos do coqueiro-anão-verde, com potencial para o biocontrole da resinose do coqueiro, em mudas de coqueiro-anão-verde. Trinta e sete leveduras, isoladas de cocos imaturos, foram avaliadas in vivo quanto ao potencial de redução dos sintomas de resinose em mudas de coqueiro-anão-verde, acondicionadas em telado sob 40±2 °C e 60% UR, por dois métodos de aplicação: aspersão (10 mL de 1x107 células mL-1) e injeção (1 mL de 1x107 células mL-1); e in vitro (28±2 °C, 12 h fotoperíodo), quanto inibição de crescimento micelial, ação de metabólitos voláteis, parasitismo, antibiose e atividade “killer”. Quinze cepas de leveduras, reduziram in vivo o percentual de área lesionada por C. paradoxa isolado de Moju-PA. In vitro, os isolados identificados como Pichia guilliermondii (LEV 25) e Candida dosseyi (LEV 47), destacaram-se como os antagonistas potencialmente promissores, pois inibiram o diâmetro de crescimento micelial, competindo por espaço e nutrientes, e reduziram a produção de conídios, pela ação de metabólitos voláteis sobre os isolados patogênicos de Moju-PA, Neópolis-SE e da Sousa-PB. Este é o primeiro relato da redução dos sintomas de resinose em mudas de coqueiro-anão-verde pela ação antagônica de leveduras sobre C. paradoxa. Os isolados de P. guilliermondii e C. dosseyi apresentam potencialidade como componente do manejo integrado da resinose do coqueiro.

Conselho Nacional de Desenvolvimento Científico e Tecnológico / Soil is a structurally complex environment and an important reservoir of microbial diversity. Only a small portion of this diversity has been recovered and cultured by conventional techniques, which limits the access to genetic and metabolic information in such microorganisms. Soil bacteria synthesize a wide variety of secondary metabolites, particularly of antibiotics. Polyketide synthase (PKS) and non-ribosomal peptide synthetase have been highlighted as important enzymatic systems for the biosynthesis of these compounds. The detection of genes associated with these two biosynthetic systems in bacterial isolates has become a strategy to screening for antagonists with high biological control activity. Therefore, the objective of this study was to evaluate bacterial isolates for their in vitro antagonistic activity against plant pathogens of agricultural interest and to analyze their genomes for the presence of genes associated with the synthesis of bioactive compounds activity. Methods of soil sample pre-treatment with chloramine-T and phenol were used for selective isolation and followed by preliminary qualitative antibiosis tests of isolates against the fungal pathogens Thielaviopsis paradoxa and Plenodomus destruens. Extracellular extracts from isolates selected in this previous step were further evaluated for the in vitro antagonist potential against Thielaviopsis paradoxa, Plenodomus destruens and Xanthomonas campestris and compared with the active ingredient of a commercial biofungicide. The taxonomic affiliation of the antagonistic isolates was assessed by sequencing of 16S rDNA gene. For the detection of PKS and NRPS genes, degenerate primers described in the literature were used. Two hundred and fifty-seven bacterial isolates, in their majority belonging to the genus Bacillus or similar, were obtained. Out of those, 84 (33%) were simultaneously bioactive for both fungi in qualitative tests. Fourty-eight bacterial isolates had extracellular extracts with inhibitory effects against Thielaviopsis paradoxa, with eight of these with inhibition ≥80%, when compared to the control. Plenodomus destruens was inhibited by the extracellular extracts of 63 isolates, of which 10 inhibited ≥80% of this pathogen growth. The extract of a Bacillus isolate from a commercial biofungicide strongly inhibited P. destruens (95%), but had no effect on T. paradoxa. Thirty antagonistic isolates from this study did not differed from this commercial isolate regarding in vitro growth inhibition of P. destruens by their extracts of 27 isolates inhibited X. campestris (inhibition rates varying from 45 to 100%), of which six completely suppressed the growth of this bacteria. There was no relationship between either the presence of PKS genes or the joined occurrence of NRPS and PKS and the inhibitory action of the strains on the relative growth of the pathogens. Only the presence of NRPS genes was associated with the effect on the relative growth of Thielaviopsis paradoxa. The isolation of bacteria with promising bioactive potential associated with experiments at greenhouse and field attesting to the feasibility of its use, will enable the achievement of new biocontrol agents. / O solo é um ambiente estruturalmente complexo e um importante reservatório da diversidade microbiana. Somente uma pequena proporção desta diversidade tem sido recuperada e cultivada por técnicas convencionais, o que limita o acesso às informações genéticas e metabólicas desses micro-organismos. Bactérias do solo sintetizam uma ampla variedade de metabólitos secundários, em particular de antimicrobianos. Sintases de policetídeo (PKS) e da sintetases de peptídeo não-ribossomal (NRPS) têm se destacado como importantes sistemas enzimáticos para a biossíntese destes compostos. A detecção de genes associados com estes dois sistemas biossintéticos em isolados de bactérias é uma estratégia para a seleção de antagonistas com alta atividade de controle biológico. Diante disso, o objetivo deste estudo foi isolar e avaliar o potencial dos isolados quanto à atividade antagônica in vitro frente a fitopatógenos de interesse agrícola, e analisar em seus respectivos genomas a presença de genes associados à síntese de compostos bioativos. Métodos de pré-tratamentos das amostras de solo com cloramina T e fenol foram utilizados para o isolamento e seguidos por testes de antibiose qualitativos frente aos fitopatógenos fúngicos Thielaviopsis paradoxa e Plenodomus destruens. Filtrados extracelulares dos isolados selecionados nesta etapa foram quantitativamente avaliados a seguir quanto à capacidade de inibição de T. paradoxa, P. destruens e Xanthomonas campestris, e comparados com o ingrediente ativo de um biofungicida comercial. A afiliação taxonômica dos isolados antagonistas foi realizada com base nas sequências parciais do gene DNAr 16S. Para a detecção dos genes PKS e NRPS foram utilizados primers degenerados descritos na literatura. Dos 257 isoladas bacterianos obtidos, a grande maioria foi pertencente ao gênero Bacillus ou outros proximamente afiliados. Destes, 84 (33%) foram inibitórios de ambos os fungos nos testes qualitativos. Quarenta e oito isolados bacterianos apresentaram filtrados extracelulares com efeitos inibitórios contra T. paradoxa, com oito destes com inibições de mais de 80% comparativamente ao controle sem filtrado. P destruens foi inibido pelo filtrado extracelular de 63 isolados, dos quais 10 inibiram mais de 80% do crescimento deste patógeno. O extrato do isolado de Bacillus do biofungicida comercial apresentou forte inibição sobre P. destruens (95%), mas não afetou T. paradoxa. Trinta isolados antagonistas deste estudo não diferiram deste isolado comercial com relação à inibição do crescimento in vitro de P. destruens pelos extratos. Extratos de 27 isolados inibiram X. campestris (inibições entre 45 e 100% do crescimento), dos quais seis suprimiram totalmente o crescimento desta bactéria. A presença dos genes PKS ou a presença conjunta de genes de NRPS e PKS não foram associadas à ação inibitória dos isolados em relação ao crescimento relativo dos três fitopatógenos. Somente a presença do NRPS associou-se ao efeito sobre o crescimento relativo do Thielaviopsis paradoxa. O isolamento de bactérias com potencial bioativo promissor associado a experimentos em casa-de-vegetação e em campo que atestem a viabilidade do seu uso possibilitará a obtenção de novos agentes de biocontrole.