Grasslands in the western United States face severe environmental threats including those brought about by climate change, such as changes in precipitation regimes and altered fire cycles; land-use conversion and development; and the introduction, establishment, and spread of non-native species. Lehmann’s lovegrass (Eragrostis lehmanniana) was introduced to the southwestern United States in the early 1900s. Since its introduction, it has become the dominant grass in the mid-elevation grasslands of southern Arizona, including the Santa Rita Experimental Range (SRER), where it has displaced native grasses including Arizona cottontop, three awns, and gramas. Like all plants in terrestrial ecosystems, this grass harbors fungal symbionts that can be important for its establishment and persistence. This thesis focuses on fungal symbionts of Lehmann’s lovegrass and has two components. First, the diversity and distributions of endophytes in Lehmann’s lovegrass are evaluated in the context of biotic and abiotic factors in the SRER. Culturing from roots and shoots of Lehmann’s lovegrass at points beneath and outside the canopy of native mesquites, which are encroaching on grasslands over time, provides insight into how a single plant species can exhibit local variation in the composition of its symbionts. Second, the thesis is used as the basis for engagement of students in science, technology, engineering, and mathematics (STEM) through the development and implementation of classroom- and field activities centered on endophytes, which help high school students address core learning aims while also gaining real research experience. Engaging students in important questions relevant to their local environment can catalyze interest in science and help students cross the threshold into research. The contributions of such approaches with respect to learning not only fulfills key next-generation science standards and common core objectives, but provides students with a meaningful introduction to the excitement, importance, and accessibility of science.

Fungi are rich sources of structurally diverse secondary metabolites, some of which possess biological activities of importance to medicine and agriculture. Research in our group focuses on application of ecological considerations to the selection of underexplored fungi for chemical analysis in search of new natural products with potentially important bioactivities. The work described in this thesis involved studies of members of two different fungal ecological groups, fungicolous and endophytic fungi. Fungicolous fungi colonize other fungi and are viewed as potential sources of antifungal agents due to the negative effects often exerted on the host organisms. Our prior studies of such fungi have proven them to be productive sources of new bioactive natural products. Fractionation of extracts produced from eleven fungicolous fungal isolates resulted in the isolation and identification of 24 different, structurally-diverse natural products, many of which were bioactive. Most of these were previously known, but six were new compounds, with one being a distinctive new peptaibol-type metabolite. Endophytic fungi colonize the inner tissues of host plants, often asymptomatically, and occur widely in most plant species. Some endophytes may benefit the host, possibly through production of protective secondary metabolites, but in most cases, their roles are not well understood. Stenocarpella maydis is a widely occurring fungal endophyte and pathogen of corn and is associated with diplodiosis (a neuromycotoxicosis) of cattle. Investigations of extracts of S. maydis and S. macrospora cultures led to the identification of seven compounds, including diplodiatoxin, diplosporin, chaetoglobosins K, L, M, and O, and (all-E) trideca-4,6,10,12-tetraene-2,8-diol, none of which were previously known from U.S. Stenocarpella isolates. Diplodiatoxin was detected as a major component of S. maydis-rotted maize grain, stalks, and stalk residues, and chaetoglobosin M was detected in extracts of naturally-diseased S. maydis-rotted maize seeds collected in the field. Chaetoglobosin K displayed potent antifungal and antiinsectan activity, while diplodiatoxin displayed phytotoxicity. Because of their well-known cytotoxic effects, we proposed that mixtures of chaetoglobosins are responsible, at least in part, for inducing diplodiosis in livestock. Our prior investigations of another common corn endophyte, Acremonium zeae, led to isolation of the pyrrocidines, which display potent antifungal and antibacterial activities against a range of corn pathogens. Further studies of these extracts led to the isolation and identification of three resorcylic acid lactone (RAL)-type compounds, one of which was new. These RAL's, did not exhibit antifungal activity, but demonstrated mild phytotoxicity to maize leaves in assays. They are also members of a compound class we have encountered among other endophytes that inhibit the heat shock protein Hsp90, and have been proposed to play a role in the colonization process. These chemical investigations provide further knowledge that may be useful in determining the roles that endophytes, in particular S. maydis and A. zeae, play in maize. The metabolites encountered during these investigations represented different biosynthetic pathways, and included polyketides, terpenoids, and compounds of mixed biogenetic origin. Structures were characterized by analysis of 1D and 2D NMR data, mass spectrometry, chemical degradation or derivatization reactions, and/or X-ray diffraction analysis. Absolute configuration assignments were determined using Mosher's method, Marfey's method, or chiral amino acid analysis methods.

published_or_final_version / Biological Sciences / Doctoral / Doctor of Philosophy

As one of the most diverse and ecologically important clades of life, fungi are best known as pathogens, saprotrophs, mycorrhizae, and lichens. Yet an enormous amount of previously unknown diversity occurs among endophytic and endolichenic fungi--species-rich, horizontally transmitted fungi that live within asymptomatic photosynthetic structures such as leaves and lichens. Here, I explore the biodiversity of these understudied symbiotrophs and the ecological and biogeographic factors influencing their communities.To evaluate methods currently used in ecological studies of environmental samples of fungi, I assessed inter- and intraspecific divergence of a fast-evolving locus for four genera commonly found as endophytes, and compared analytical methods for identifying and delimiting OTUs. Then I used the most robust methods to show that after soil contact, seeds of a focal tree species contain diverse fungi that are closely related to endophytes and pathogens.To explore the ecological specificity of symbiotrophic fungi, I examined endophytic, endolichenic, and saprotrophic communities inhabiting physically proximate hosts in a biotically rich area of southeastern Arizona. I found that endolichenic fungi are largely distinct from plant-associated fungi, with the exception of a group of ecologically flexible symbionts that occur in lichens and mosses. Although numerous endophytes were found in non-living leaves, fungi that were highly abundant in leaf litter were seldom found as endophytes.To assess symbiotroph biodiversity and ecological specificity at a broad geographic and phylogenetic scale, I isolated>4100 endophytic and endolichenic fungi from diverse communities of plants and lichens across five climatic regions in North America. I found that the abundance, diversity, and composition of these nearly ubiquitous fungi differ as a function of climate, locality, and host. Differences among communities reflect environmental characteristics more strongly than geographic distance.Last, I addressed a series of hypotheses regarding the ecological specificity of fungi inhabiting living and non-living leaves. I show that like endophytes, saprotrophic communities are structured by environmental characteristics, and at small spatial scales by host and leaf status. Yet, differences in communities between living leaves and leaf litter suggest that most endophytes either rapidly complete their life-cycle or are out-competed by robust saprotrophs once leaves senesce.

Orientador: Dulce Helena Siqueira Silva / Banca: Ian Castro-Gamboa / Banca: Geraldo Humberto Silva / Resumo: O presente trabalho apresenta os resultados obtidos sobre o estudo químico e biológico dos fungos endolíticos N odulisporium sp . e M icroascus sp . associados à alga vermelha Asparagopsis taxiformis . Para tanto, fez - se uso das técnicas cromat ográficas de sepa ração CLAE (cromatografia líquida de alta eficiência), CG - EM (cromatografia gasosa acoplada a espectrometria de massas) e cromatografia em coluna (CC), além da avaliação dos potenciais antifúngico, anticolinesterásico e citotóxico dos extratos brutos, fraç ões e subfrações de N odulisporium sp . e M icroascus sp. O estudo químico levou ao isolamento de quatro substâncias pertencentes à classe das diidroisocumarinas (substâncias S 1, S 2, S 10 e S 11 ), duas dicetopiperazinas (substâncias S 3 e S 4 ), um composto fenólico ( S 12 ), uma isoflavona ( S 8 ), um derivado hidroxiimino ( S 9 ), uma mistura contendo uma amida ( S 5 ) e um diol vicinal ( S 6 ), além do isolamento de um composto aromático (substância S 7 ), ainda em fase de identificação. As estruturas das substâncias isoladas foram identificadas com base nos dados espectroscópicos de ressonância magnética nuclear uni e bidimensional (RMN de 1 H, RMN de 13 C, TOCSY - 1D, HSQC, HMBC, COSY e HSQC - TOCSY), UV - Vis e espectrometria de massas (EM). C omporta ressaltar que o diol vicinal ( S 6 ) está sendo descrito pela primeira vez na literatura e que as demais substâncias isoladas não haviam sido relatadas para os gêneros Nodulisporium ou Microascus . A avaliação das atividades antifúngica e anticolineste rásica evidenciaram o potencial de bioatividade de extratos e frações de N odulisporium sp . e M icroascus sp, sendo reveladas atividades forte e moderada frente à s enzimas acetilcolinesterase humana e de enguia elétrica. Este é o primeiro estudo químico e biológico relatado na literatura para os endófitos Nodulisporium sp . e Microascus... / Abstract: This work present s results from the chemical and biological study of endophytic fungal strains of Nodulisporium sp and Microascus sp associated to the red alga Asparagopsis taxiformis . Chromatographic separation techniques as HPLC, GC - MS and CC have been used in addition to bioassays for evaluation of the antifungal, anticholinesterasic and cytotoxic potential of crude extracts, fractions and subfractions of Nodulisporium sp and Microascus sp fungal strains. The c hemical study led to the isolation of four dihydroisocoumarins (compounds S1, S2, S10 and S11 ), two diketopiperazines (compounds S3 and S4 ), one phenolic compound ( S1 2 ), one isoflavone ( S8 ), o ne hydroxyl - imino derivative ( S9 ), a mixture containing one amide ( S5 ) and one novel vicinal diol ( S6 ), in addition to one aromatic compound (compounds S7 ) still under structural investigation. Their structures were identified based on 1D and 2D NMR spectroscopic data ( 1 H and 13 C NMR, TOCSY - 1D, HSQC, HM BC, COSY and HSQC - TOCSY), UV - Vis and mass spectrometry (MS) . Compound S 6 is an aliphatic vicinal diol and is herein described as a novel compound whereas the remaining compounds had not been described before neither for Nodulisporium nor Microascus genera. Antifungal and anticholinesterase activities evaluation of extracts and fractions of Nodulisporium sp e Microascus sp evidenced their strong and moderate potential against human and electric eel acetylcholinesterase enzymes. This is the first report on th e chemical and biological studies of endophytic fungi Nodulisporium sp and Microascus sp, associated to the red algae Asparagopsis taxiformis. Their promising results highlight the outstanding chemodiversity of extracts and fractions from such fungal strai ns from marine environment as well as their bioactivity potential. They also evidence the importance of further investigating such fungal strains aiming at rele... / Mestre

Árvores evolutivas de espécies de Peperomia baseadas nas regiões de ITS e matK foram confrontadas com perfis metabólicos de extratos brutos obtidos por RMN 1H, HPLC-DAD e LC-ESI-MS. A análise de clusters (HCA) e a análise discriminante de componentes principais (DAPC) suportaram em parte os agrupamentos observados por ITS e matK. As principais classes de metabólicos secundários observadas foram policetídeos, meroterpenos, cromenos, fenilpropanoides e amidas, sendo que foram ainda isolados um policetídeo (malabaricona D) de P. megapotamica; o fenilpropanoide elemicina e a lignana tetraidrofurânica diyangambina de P. rotundifolia; e as flavonas 5,6,7-trimetoxiflavona e 4\',5,6,7- tetrametoxiflavona de P. sincorana. As PKS tipo chalcona sintase (CHS) de peperomias, diferiram daquelas de famílias filogeneticamente próximas como Myristicaceae (Virola) e Lauraceae (Aniba). Como muitos meroterpenos de espécies de Peperomia possuem como núcleo básico o ácido orselínico, produto típico de líquens e bactérias, o gene ácido orselínico sintase (OSAS) foi investigado nas plantas e nos 27 fungos endofíticos isolados de espécies de Peperomia (identificados pelas sequências de ITS1-5.8S-ITS2). Foram amplificadas pelo menos uma PKS (NR, HR e PR) em cada uma das linhagens e AOS em várias delas (UR1, UC3, UF1, UF2, UF3, UF4, GC3, NR1, NC1, NC2, NF1, AR1, AC1, AC4, OC1 e OF1). A homologia de 60% da OSAS amplificada de UR1 com a sequência de Streptomyces viridochromogenes é sugestivo de transferência horizontal de bactéria para fungos. / Phylogenetic tree of Peperomia species based on ITS and matK were compared to the metabolic profiling of crude extracts using 1H NMR, HPLC-DAD and LC-ESI-MS. Cluster analysis (HCA) and discriminant analysis of principal componentes (DAPC) supported significantly the clustering observed by ITS and matK. The major classes of secondary metabolites were polyketides, meroterpenes, chromenes, phenylpropanoids and amides and besides, one polyketide (malabaricone D) of P. megapotamica; the phenylpropanoid elemicin and the tetrahydrofuran lignan diyangambin from P. rotundifolia; and the flavones 5,6,7- trimethoxyflavone and 4\',5,6,7-tetramethoxyflavone were isolated from P. sincorana. The PKS chalcone synthase type (CHS) from peperomia, differed from phylogenetically related families Myristicaceae (Virola) and Lauraceae (Aniba). Since several meroterpenes from Peperomia species have the orsellinic acid as an aromatic core, typical of liquens and bacteria, the orsellinic acid (OSAS) was investigate in the plants and 27 endophytes isolated from Peperomia (identified based on ITS1-5.8S-ITS2 sequences). At least one PKS (NR, HR and PR) were isolated from each strains and AOS were isolated from several strains (UR1, UC3, UF1, UF2, UF3, UF4, GC3, NR1, NC1, NC2, NF1, AR1, AC1, AC4, OC1 and OF1). The 60% of homology observed for OSAS amplified from UR1 with that of Streptomyces viridochromogenes is suggestive of a horizontal transferring from bacteria to fungi.

Fungos endofíticos constituem uma fonte promissora e ainda pouco explorada de novas substâncias potencialmente bioativas, que podem ter aplicações na medicina, agricultura e biologia química. Neste trabalho foram isolados cinco fungos endofíticos das folhas de Tithonia diversifolia, os quais foram cultivados em pequena escala e avaliados em diversos bioensaios. O fungo endofítico Phoma sorghina foi cultivado em escala ampliada, produzindo nove substâncias, sendo três derivados antraquinônicos inéditos na literatura. Três fungos endofíticos associados à Viguiera robusta foram estudados. De Colletotrichum gloeosporioides foram isoladas oito substâncias, sendo um derivado dicetopiperazínico e um derivado aminoaçúcar inéditos na literatura. Diaporthe phaesolorum produziu três derivados dicetopiperazinicos, um inédito na literatura. Chaetomium globosum produziu treze substâncias, sendo oito derivados azaphilônicos inéditos. Algumas linhagens endofíticas foram avaliadas em experimentos de biotransformação de três naftoquinonas e da 1-tetralona, catalisando reações de oxidação e redução. O fungo Diaphorte phaseolorum catalisou uma reação de metilação inédita na literatura de biotransformação. / Endophytic fungi are a promising and untapped source of potential biologically active compounds that might be useful as therapeutic and agrochemical agents, or chemical biology tools. In this thesis five endophytic fungi were isolated from the leaves Tithonia diversifolia. All the strains have been cultivated in small scale and the extracts submitted to bioassays. The culture of the endophyte Phoma sorghina was scaled up, affording six known compounds and three novel anthraquinone derivatives. Three endophytes from Viguiera robusta have also been studied as sources of natural products. Colletotrichum gloeosporioides produced six known compounds, one novel aminosugar and one new diketopiperazine derivative. Diaporthe phaesolorum biosynthesized three diketopiperazine derivatives, one novel in the literature. Five known compounds and eight novel azaphilone derivatives were isolated from Chaetomium globosum. Some endophytic strains have been evaluated in biotransformation experiments of three naphthoquinones and 1-tetralone, catalyzing oxidation and reduction reactions. The endophyte Diaphorte phaseolorum catalyzed a novel methylation reaction in the biotransformation literature.

A cana-de-açúcar é uma das mais importantes culturas para o Brasil e vem recebendo significativa atenção devido à crescente substituição de combustíveis fósseis por fontes renováveis e menos poluentes de energia como o etanol. Consequentemente, os aspectos biotecnológicos oriundos da interação de bactérias e cana-de-açúcar devem ser mais bem avaliados, além disso, estudos de interação planta-microrganismo desenvolvidos em países tropicais são ainda incipientes. As bactérias interagindo com as plantas de interesse agronômico podem acarretar os mais diversos benefícios, com ênfase na promoção do crescimento vegetal e da fitossanidade. Dentre essas bactérias, diversas linhagens de Pantoea agglomerans têm sido isoladas intimamente associadas a várias espécies vegetais. As bactérias pertencentes ao gênero Pseudomonas também têm sido exploradas biotecnologicamente, pois produzem uma vasta gama de antimicrobianos que podem ser utilizados no controle de pragas e fitopatógenos. A partir desses fatos, uma linhagem de P. agglomerans, 33.1, previamente isolada endofiticamente de Eucalyptus grandis e promotora de crescimento dessa cultura, foi avaliada quanto aos aspectos biotecnológicos durante associação com cana-de-açúcar. A linhagem 33.1 foi capaz de promover o crescimento de cana-de-açúcar e também induziu a produção de proteínas de resistência pelas plantas inoculadas. Dentre os mecanismos avaliados na promoção de crescimento vegetal, a linhagem apresentou alta produção de fitormônio e fosfatases. Foi também confirmado que a interação planta-bactéria afetou o metabolismo da linhagem 33.1. Um plasmidio integrativo, pNKGFP, foi desenvolvido e inserido na linhagem 33.1. A linhagem bacteriana marcada foi monitorada durante interação com cana-de-açúcar. Por microscopia de fluorescência, re-isolamento e PCR quantitativo (qPCR) foi confirmada a capacidade de colonização cruzada de cana-deaçúcar pela linhagem 33.1:pNKGFP, abrindo assim precedentes para utilização dessa linhagem em outras culturas. Posteriormente, a linhagem 33.1 foi geneticamente modificada para produção da proteína Cry. Por meio de bioensaios em dieta artificial e colmos de cana-de-açucar foi provado o parcial controle de Diatraea saccharalis, uma importante praga de cana-de-açúcar, pela linhagem endofítica expressando heterologamente o gene cry1a7c. Vários antibióticos e sideróforos produzidos pela bactéria P. fluorescens, linhagem Pf-5, foram avaliados quanto ao controle de Fusarium spp., incluindo isolados de F. verticillioides causadores de Pokkah boeng em cana-deaçúcar. Por mutações sitio-dirigido em genes envolvidos na biossíntese de antimicrobianos foi provado que os compostos 2,4-diacetil-floroglucinol e rizoxina são os principais responsáveis no controle Fusarium spp. Por HPLC e RT-qPCR foi também provado que ácido fusárico afeta a produção de antibióticos pela Pf-5. / Sugarcane is one of the most important Brazilian crops. Its importance is increasing due to the gradual substitution of fossil fuel for renewable and cleaner energy sources, such as ethanol Consequently, the biotechnological aspects that mediate sugarcane-bacterium interactions must be evaluated; studies from tropical countries are incipiente Bacterium-plant interactions can often improve the growth and fitness of associated plants. One of these beneficial bacteria, Pantoea agglomerans, has been isolated from many vegetal species. The Pseudomonas genus also has a record of biotechnological exploration due to its wide range of antibiotic production. These compounds can be used on pest and plant-pathogen control. For these reasons, P. agglomerans, strain 33.1, previously isolated from Eucalyptus grandis and a known eucalyptus growth-promoter, was evaluated about regarding the biotechnological aspects of sugarcane-bacterial association. 33.1 strain was able to promote sugarcane growth and also to induce the production of resistance proteins for inoculated plants. The plant-hormone and phosphatase production by 33.1 were associated with sugarcane growth-promotion bacterial mechanisms. An integrative plasmid, pNKGFP, was constructed and used to transform 33.1. The marked strain was monitored during sugarcane association. The sugarcane cross-colonization by 33.1:pNKGFP was confirmed by fluorescence microscopy, re-isolation and quantitative PCR (qPCR), suggesting that 33.1 should be used in association with other crops. 33.1 was genetically modified to produce the Cry protein. The partial biocontrol of Diatraea saccharalis, the sugarcane borer, by the endophytic strain expressing heterologously the cry1a7c gene, was confirmed in bioassays using artificial diet and sugarcane stalks. Several antibiotics and siderophores produced by P. fluorescens Pf-5 were tested against Fusarium spp.; including isolates of F. verticillioides responsible for the sugarcane Pokkah boeng disease. By deriving site-directed mutants of Pf-5, with single and multiple mutations, in the bio-synthetic gene clusters of the antifungal compounds, it was proved that 2,4-diacetyl-phoroglucinol and rhizoxin are the major compounds responsible for the Fusarium spp. suppression. The fusaric acid effects on Pf-5 antibiotic production was proved by HPLC and RT-qPCR.

A cultura de cana-de-açúcar ocupa posição de destaque nos cenários nacional e internacional devido principalmente a produção de etanol como fonte de energia renovável e menos nociva ao ambiente. Entretanto um dos obstáculos à produtividade é a ocorrência de várias doenças dentre elas a escaldadura das folhas causada por Xanthomonas albilineans. Bactérias endofíticas e rizosféricas pertencentes ao gênero Burkholderia tem sido isoladas com alta frequência em diferentes culturas como, por exemplo, a cana-de-açúcar. Nas últimas décadas, estas bactérias têm recebido especial atenção devido ao seu potencial como promotoras de crescimento vegetal, como agentes de biorremediação, mas muito pouco é explorado quanto ao potencial biotecnológico dessas bactérias como agentes de biocontrole de doenças. Visto que essas bactérias vivem em um ambiente altamente competitivo e sujeito a flutuações ambientais, representam uma fonte altamente significativa de metabólitos secundários bioativos, como as bacteriocinas sintetizadas ribossomicamente e outros peptídeos não ribossomais. Portanto, os objetivos principais deste trabalho foram determinar a frequência de linhagens de Burkholderia spp. endofíticas e da rizosfera de cana-deaçúcar capazes de produzir bacteriocinas e outros metabólitos secundários e por meio de mutagênese aleatória por transposon, identificar genes associados a produção desses metabólitos. Os resultados de caracterização permitiram concluir que as bactérias endofíticas e rizosféricas pertencentes ao gênero Burkholderia avaliadas apresentaram grande potencial em produzir metabólitos com atividade antibacteriana in vitro; sendo capazes de controlar X. albilineans importante patógeno da cultura de cana-de-açúcar. Para uma das linhagens foi obtida uma biblioteca de mutantes, a qual foi parcialmente caracterizada quanto à alteração da atividade antibacteriana. Foram identificados doze mutantes que apresentaram perda da atividade antibacteriana. A análise das sequências flanqueadoras do transposon para os doze mutantes permitiu a identificação de genes associados a produção de bacteriocinas, a regulação da expressão gênica, a enzimas possivelmente associadas ao metabolismo secundário, ao metabolismo geral da célula e a proteínas hipotéticas. A identificação e clonagem de tais genes permitirão uma maior compreensão da produção desses compostos e futuras aplicações biotecnológicas. / The sugarcane crop has an important role in international and national scenery mainly because the ethanol production as a sustainable energy source and less harmful to the environment. However one of the obstacles to the productivity is the occurrence of several diseases among them leaf scald caused by Xanthomonas albilineans. Endophytic and rhizospheric bacteria that belong to the Burkholderia genus have been isolated in high frequency in different cultures, such as sugarcane. In the last decades, these bacteria have been receiving attention due their potential as plant growth promoters, bioremediation agents. However, the biotechnological potential of these bacteria as agents of diseases biocontrol is very poorly evaluated. Since these bacteria live in a highly competitive environment and subject to environmental fluctuations, they may represent a highly significant source of bioactive secondary metabolites, as ribosomal synthesized bacteriocins and other nonribosomal peptides. Therefore, the main aim of this work was to determine the frequency of bacteriocin and secondary metabolites production by endophytic and rhizospheric isolates of Burkholderia spp. from sugarcane. Also, the genes associated to synthesis of these metabolites were identified by random mutagenesis based on Tn5 transposon. The results showed that endophytic and rhizospheric Burkholderia spp. present in vitro potential to production of metabolites with antibacterial activity; being inhibited X. albilineans, an important pathogen of sugarcane crops. For one of the Burkholderia strain it was obtained a mutant library, which was partially characterized according to antibacterial activity. Twelve mutants that showed the loss of antibacterial activity were identified and further evaluated. Also, the analysis of the transposon flanking sequences for these mutants indicated that genes associated to the bacteriocin production, regulation of gene expression, enzymes possibly associated to the secondary metabolism, general metabolism of the cell and hypothetical proteins are related to loss of inhibition ability. The identification and cloning of such genes will allow a better understanding of the production of theses compounds and further biotechnological applications.

A presença de bactérias no cultivo in vitro de plantas é normalmente relacionada à contaminação, acarretando o descarte de microplantas de alto valor comercial. No entanto, essa presença pode ser caracterizada como \"manifestações\", decorrentes de possíveis estresses físicos ou nutricionais, da microbiota endofítica, que coloniza ativamente os tecidos vegetais, e que não necessariamente, afeta o desenvolvimento da planta. Sendo assim, o objetivo deste trabalho foi controlar a manifestação bacteriana, preservando as microcepas de Eucalyptus cloeziana em desenvolvimento in vitro, adicionando-se ao meio de cultura, os antibióticos gentamicina, ciprofloxacina, rifampicina, timentin (isolados ou combinados entre si) e o biocida clorometil-isotiazolinona + metil-isotiazolinona. O monitoramento do trabalho foi realizado por meio de avaliações semanais, visando verificar possíveis influências dos agentes antimicrobianos na morfofisiologia do vegetal e a eficiência no controle das manifestações bacterianas. Para tanto, foram avaliados parâmetros referentes ao incremento de massa seca, número de brotações, teor de acúmulo de nutrientes, análises histológicas e histoquímicas das microcepas. Para verificar possíveis flutuações das comunidades bacterianas endofíticas nas microcepas, utilizou-se a técnica de DGGE. Todas as análises foram realizadas aos 15 e 30 dias após a adição dos agentes antimicrobianos no meio de cultura, exceto a avaliação nutricional que foi feita apenas no final do experimento (30 dias). Os resultados obtidos permitiram inferir que ao longo dos 30 dias de cultivo, o antibiótico gentamicina, ocasionou queda no desenvolvimento das microcepas e não foi eficiente no controle das manifestações bacterianas. Os antibióticos rifampicina e ciprofloxacina apesar de não serem efetivos no controle da manifestação bacteriana, não afetaram o desenvolvimento das microcepas. Conclui-se que, o biocida clorometil-isotiazolinona + metil-isotiazolinona efetivamente controlou as manifestações bacterianas, sem alterar a morfofisiologia das microcepas, bem como o timentin, que além de controlar as manifestações, proporcionou maior vigor das microcepas em função da melhor assimilação dos nutrientes. / The in vitro bacterial presence in plant cultivation, usually is related to contamination, resulting in the elimination of the microplants with high commercial value. However, this presence can be characterized as \"manifestations\" arising from possible physical or nutritional stresses, from microbial endophytic that actively colonize plant tissues, and do not necessarily affect plant development. Therefore, the purpose of this study was to control the bacterial manifestation, preserving in vitro developing of Eucalyptus cloeziana microstumps, by adding to the culture medium, the antibiotics gentamicin, ciprofloxacin, rifampicin, timentin (alone or combined) and biocide + chloromethyl isothiazolinone methyl-isothiazolinone. The monitoring work was conducted through weekly evaluations, to verify the possible influence of antimicrobial agents in the plant morphophysiology and the efficient control of bacterial manifestations. Therefore, were evaluated parameters related to the increase in dry weight, number of buds, content of nutrient accumulation, histological and histochemical the microstumps. DGGE technique was used to check possible fluctuations of endophytic bacterial communities in microstumps. All analyzes were performed at 15 and 30 days after the addition of antimicrobial agents in the culture medium, except for the nutritional assessment that was made only at the end of the experiment (30 days). The results allow to infer that over the 30 days of culture, the antibiotic gentamicin, resulted in the microstumps development decrease and was not effective in controlling bacterial manifestations. The antibiotics rifampicin and ciprofloxacin although were not effective in controlling the bacterial manifestation, affected the development of microstumps. It was concluded that the biocide chloromethyl-methyl-Isothiazolinone + Isothiazolinone effectively controlled the bacterial manifestations without modifying the morphophysiology of the microstumps, well as the timentin, which besides controlling the manifestations, provided greater vigor of microstumps due to better assimilation of nutrients.