Identificação e relações filogenéticas, potencial de uso de isolados de Trichoderma no controle do mofo branco e como promotores de crescimento do feijoeiro

Tese (doutorado)—Universidade de Brasília, Instituto de Ciências Biológicas, Departamento de Fitopatologia, Programa de Pós-Graduação em Fitopatologia, 2013.Trichoderma constitui um gênero de microfungos mundialmente distribuído na natureza. Nos solos, estão presentes como microorganismos de vida livre ou colonizando raízes de plantas. Podem também colonizar endofiticamente os tecidos vegetais, como simbiontes. Por apresentarem ação antagonista contra fitopatógenos, linhagens de diversas espécies desse gênero vem sendo estudadas como agentes de biocontrole de doenças de plantas, dentre as quais, o mofo branco, causado por Sclerotinia sclerotiorum, em diversas espécies vegetais. A caracterização molecular desse grupo de antagonistas tem grande importância, tanto em estudos de biodiversidade e distribuição das espécies, como na identificação de potenciais agentes de biocontrole, e de possíveis patógenos oportunistas a humanos, a exemplo de Trichoderma longibrachiatum. O objetivo desse trabalho foi identificar 148 isolados em nível de espécie, por sequenciamento das regiões ITS do DNA ribossomal e avaliar a biodiversidade desses isolados com referência à origem geográfica. Foi realizada seleção in vitro de isolados com potencial de biocontrole, os quais foram avaliados, em casa de vegetação, quanto à capacidade de supressão do mofo branco e como promotores de crescimento do feijoeiro. Os 29 isolados selecionados foram também submetidos à técnica de MALDI-TOF MS (Matrix-assisted laser desorption/ionization-time of flight mass spectrometry), para confirmação das espécies, correlacionando esta técnica de identificação com a técnica molecular empregada. Filtrados de culturas do isolado CEN201, com potencial de inibição do crescimento micelial do patógeno supracitado, foram submetidos a técnicas de extração e purificação de metabólitos secundários. A capacidade de inibição do crescimento micelial de S. Sclerotiorum, por esses compostos, foi confirmada. No capítulo 1 são relatados resultados desses estudos, mostrando que 17,4% dos isolados apresentaram alto nível de antagonismos em pareamento de culturas e dois grupos apresentaram 83,3 a 88,9% de inibição micelial do patógeno por metabolitos não voláteis. Outro ponto ressaltado nesta pesquisa foi o índice de biodiversidade relativa aos 144 isolados de Trichoderma procedentes de sete estados brasileiros, que alcançou 0,076 com 11 espécies. Já o capítulo 2 relata a supressão do mofo branco com 19 dos 29 isolados selecionados in vitro no capítulo 1. Observou-se, também, promoção de crescimento de plantas de feijão com 7 isolados estudados. Foram ainda mostrados os resultados obtidos na identificação dos isolados por espectrometria de massa (MALDI-TOF MS). Esses resultados, confrontados com os dados obtidos na identificação molecular verificada no capítulo anterior, indicaram a convergência das duas técnicas, confirmando o potencial de MALDI-TOF MS como método rápido para identificação de espécies de Trichoderma. O capítulo 3 está focado nos estudos com metabólitos não voláteis produzidos pelo isolado CEN201 (T.asperellum). As técnicas utilizadas, Cromatografia líquida e MALDI-TOF MS possibilitaram, por homologia de massa/carga, inferir a presença de dois compostos com ação antibiótica, Asperelina B e Trichocompactina, no filtrado de culturas estudado. _______________________________________________________________________________________ ABSTRACTTrichoderma composes a genus of microfungi distributed worldwide. In soil, these fungi may be present as free life or colonizing plant roots. Can also to colonize plant tissues endophytically like symbionts. Some species have been studied as biocontrol agents of plant diseases, among which, white mold caused by Sclerotinia sclerotiorum in several plant species. Their mechanisms of action include the production of secondary metabolites with antibiotic effect, mycoparasitism and competition for space and nutrients. The molecular characterization of this group of antagonists is very important, both in studies of biodiversity and species distribution and for identification of potential biocontrol agents, and possible opportunistic pathogens to humans like Trichoderma longibrachiatum. The aim of this study was to identify 148 the isolates of Trichoderma on species level by sequencing the ITS region of Ribossomal and to evaluate their biodiversity with reference to geographical originan. Was performed, in vitro, selection of isolates with potencial biocontrol, which were evaluated in greenhouse as the ability of white mold supression and as beans growth promoters. The 29 selected isolates were also subjected to the technique of MALDI-TOF MS (Matrix-assisted laser desorption / ionization-time of flight mass spectrometry) to confirm the species, correlating this technique in identification with molecular technique employed. Culture filtrates of isolate CEN 201, with potential to inhibit the mycelial growth of the pathogen above, were subjected to techniques of extraction and purification of secondary metabolites. The capacity of mycelial growth inhibition of S. Sclerotiorum by these compounds was confirmed. In chapter 1, are reported results of these studies showing that 17,4% of the isolates showed a high level of antagonism in pairing cultures and two groups showed 83,3 to 88,9% of inhibition of the pathogen by volatile metabolites. Another point emphasized in this study was the index of biodiversity on 144 Trichoderma isolates from seven Brazilian states, which reached 0,076 with 11 species. Already Chapter 2 reports the suppression of white mold with 19 of the 29 isolates selected in vitro in Chapter 1. It was observed, also, promote growth of bean plants with 7 isolates tested. Also shown are the results obtained by identification of the isolated mass spectrometry (MALDI-TOF MS). These results, compared with the data obtained in the molecular identification verified in the previous chapter showed the convergence of the two techniques, confirming the potential of MALDI-TOF MS as a rapid method for identification of Trichoderma species. The Chapter 3 is focused on studies of non-volatile metabolites produced by isolated CEN201 (T.asperellum). The techniques used, liquid chromatography and MALDI-TOF MS was enable, by homology mass/charge, to infer the presence of two compounds with antibiotic activity, Trichocompactin and Asperelin B in culture filtrates studied

Bioprospecção de Trichoderma spp. sobre o crescimento micelial de Colletotrichum cliviae e C. truncatum

The use of antagonists in the control of plant pathogens has been extensively studied in recent decades, especially fungi from genus Trichoderma spp., due to their ability to act as biocontrolling agents and inducers of plant resistance against diseases. Thus, we aimed to select Trichoderma spp. with potential to control growth of pathogenic fungi. We used 56 Trichoderma strains from the isolate bank of the microbiology laboratory of Federal University of Tocantins. The strains were isolated from soil samples collected in southern Tocantins state, Brazil. The studied pathogens of foliar diseases were Colletotrichum cliviae and Colletotrichum truncatum, both of which were isolated from soybean plants showing disease symptoms. To evaluate the antagonistic potential of  Trichoderma spp., we used the dual culture assay and expressed the results as percentage of colonization and grade scale. Grade evaluation revealed that 46 Trichoderma strains were effective antagonists of the two pathogens. Regarding the percentage of pathogen growth inhibition, effective antagonists were represented by 22 strains showing more than 75% inhibition of  Colletotrichum cliviae and three strains showing more than 90% inhibition of Colletotrichum truncatum.Patógenos de plantas podem provocar prejuízos severos com perdas significativas na produtividade. O uso de antagonista no controle de fitopatógenos tem sido bastante estudado nas últimas décadas, destacando-se os fungos do gênero Trichoderma spp. por serem capazes de atuarem como agentes de biocontrole e indutores de resistência de plantas a doenças. Diante disso, este trabalho teve como objetivo selecionar isolados de Trichoderma spp. com potencial para o controle do crescimento de fungos fitopatogênicos. Foram utilizados 56 isolados de Trichoderma spp. que fazem parte do banco de isolados do laboratório de microbiologia da Universidade Federal do Tocantins, isolados de amostras de solo coletados em áreas da região sul do Estado do Tocantins. Os patógenos causadores de doenças foliares foram Colletotrichum cliviae e Colletotrichum truncatum, isolados de plantas de soja com os sintomas da doença. Para avaliação do potencial antagônico dos isolados de Trichoderma spp. foi utilizada a técnica de pareamento de colônias do patógeno, com resultados de percentagem de colonização e escala de notas de acordo com os critérios propostos por Bell com adaptações. Pela avaliação das notas, 46 isolados foram eficientes antagonistas para os dois fitopatógenos. Quanto a percentagem de inibição do crescimento dos patógenos, 22 isolados foram eficientes antagonistas, com mais de 75% de inibição do Colletotrichum cliviae e três isolados com mais de 90% do Colletotrichum truncatum

Identification and phylogeny of Trichoderma species by MALDI-TOF analysis and characterization of antagonistic activities against Sclerotinia scleroti

Common bean (Phaseolus vulgaris L.) is an economically important food commodity in Brazil. However, it can suffer serious damage by white mould disease caused by the pathogen Sclerotinia sclerotiorum. Species of the genus Trichoderma can act as biocontrol agents against of this pathogen. This study describes the identification based on morphological, MALDI-TOF MS and molecular analysis of these 29 Trichoderma sp. isolates and their in vitro antagonistic behaviour against S. sclerotiorum. In order to evaluate the disease incidence greenhouse experiments were also performed using bean seedling. According to the results obtained, MALDI-TOF MS technique was appropriate for all Trichoderma species identification confirming the morphological and molecular-based identifications through analysis of rDNA ITS sequence data. In vitro inhibition experiments showed that 38% of Trichoderma isolates colonised the entire surface of the medium (grade 1 evaluation score) in dual cultures against S. sclerotiorum. Pathogen exposition to non-volatile metabolites produced by Trichoderma resulted in the inhibition of their mycelia growth between c.a. 83 and 100%. Moreover, 19 Trichoderma isolates enabled a total suppression of disease in bean seedlings when evaluated in greenhouse experiments. Two Trichoderma isolates identified as Trichoderma asperellum (CEN201 and CEN162) were responsible to the highest rates of growth promotion in bean plants, which ranged from 26 to 34%. The MALDI-TOF technique was appropriate for species designation for the majority of Trichoderma species, confirming most molecular-based identifications through analysis of rDNA ITS sequence data

Evaluation of inhibitory effects of Trichoderma asperellum metabolites on mycelial growth of Sclerotinia sclerotiorum

Common bean (Phaseolus vulgaris L.) is an economically important food commodity in Brazil. However, it can suffer serious damage by white mould disease caused by the pathogen Sclerotinia sclerotiorum. Species of the genus Trichoderma can act as biocontrol agents against of this pathogen. This study describes the identification based on morphological, MALDI-TOF MS and molecular analysis of these 29 Trichoderma sp. isolates and their in vitro antagonistic behaviour against S. sclerotiorum. In order to evaluate the disease incidence greenhouse experiments were also performed using bean seedling. According to the results obtained, MALDI-TOF MS technique was appropriate for all Trichoderma species identification confirming the morphological and molecular-based identifications through analysis of rDNA ITS sequence data. In vitro inhibition experiments showed that 38% of Trichoderma isolates colonised the entire surface of the medium (grade 1 evaluation score) in dual cultures against S. sclerotiorum. Pathogen exposition to non-volatile metabolites produced by Trichoderma resulted in the inhibition of their mycelia growth between c.a. 83 and 100%. Moreover, 19 Trichoderma isolates enabled a total suppression of disease in bean seedlings when evaluated in greenhouse experiments. Two Trichoderma isolates identified as Trichoderma asperellum (CEN201 and CEN162) were responsible to the highest rates of growth promotion in bean plants, which ranged from 26 to 34%. The MALDI-TOF technique was appropriate for species designation for the majority of Trichoderma species, confirming most molecular-based identifications through analysis of rDNA ITS sequence data

BIOLOGICAL CONTROL OF PLANT PATHOGENS BY Bacillus subtilis in vitro

Considerando as perdas causadas por doenças em diversas culturas, as rizobactérias se apresentam como uma alternativa para manejar esses patógenos. O trabalho teve por finalidade avaliar a potencialidade antagonista de isolados de Bacillus subitilis aos fungos fitopatogênicos Fusarium subglutinans, Curvularia lunata e Bipolaris spp.. Foram estudados sete isolados de B. subtilis e seus efeitos antagônicos em fungos fitopatogênicos utilizando quatro métodos: técnica de cultura fúngica sobre cultura antagonista, pareamento direto, pareamento com risco no centro da placa e técnica de círculo. Também foram avaliados os efeitos de metabólitos voláteis e termoestáveis desses isolados. O delineamento experimental utilizado foi inteiramente casualizado através de três repetições. As avaliações foram feitas aos três, seis, nove e doze dias para os quatro métodos e aos seis e doze dias para avaliação de metabólitos voláteis e termoestáveis, onde foram medidos o diâmetro da colônia e calculado a porcentagem de inibição de crescimento (PIC) do patógeno. Os isolados de B. subilis UFTBs 03, UFTBs 05, UFTBs 06 e UFTBs 07 foram eficazes na inibição de crescimento micelial dos fungos patogênicos F subglutinans, C lunata e Bipolaris spp. pelos quatro métodos. Os isolados UFTBs 02, UFTBs 03, UFTBs 06 e UFTBs 07 inibiram por metabólitos voláteis o crescimento micelial de F. subglutinans. Os isolados UFTBs 01, UFTBs 04, UFTBs 05, UFTBs 06 e UFTBs 07 foram capazes de inibir o crescimento micelial de F. subglutinans, C. lunata e Bipolaris spp. por metabólitos termoestáveis.Palavras-chave: Biocontrole, Rizobactérias, Antagonismo, Bacillus subitilis.Considering the losses caused by diseases in many cultures, rhizobacteria present themselves as an alternative for managing these pathogens. The work aimed to evaluate the potential antagonist of Bacillus subtilis isolates to pathogenic fungi Fusarium subglutinans, Curvularia lunata and Bipolaris spp. Seven were studied isolates of B. subtilis and its antagonistic effect on pathogenic fungi using four methods: fungal culture technique on culture antagonist, direct pairing, pairing with risk in the center of the plate and circle technique. Was also evaluated effect of volatile metabolites and thermostable these isolates. The experimental design was completely randomized with three repetitions. The assessments took the tree, six, nine and twelve day for the four methods and the six and twelve day to evaluate volatile metabolites and thermostable, which were measured the diameter of the colony and calculated the percentage growth inhibition (PGI) of the pathogen. B. subtilis isolates UFTBs 03, UFTBs 05, UFTBs 06 and UFTBs 07 were effective in inhibiting mycelial growth of pathogenic fungi F. subglutinans, C. lunata and Bipolaris spp. by four methods. Isolated UFTBs 02, UFTBs 03, UFTBs 06 and UFTBs 07 inhibited by volatile metabolites mycelial crecimento of F. subglutinans. Isolated UFTBs 01, UFTBs 04, UFTBs 05, UFTBs 06 and UFTBs 07 they were able to inhibit the mycelial growth of F. subglutinans, C. lunata and Bipolaris spp. by thermostable metabolites, so considering the antibiose the mechanism of action.Keywords: Biocontrol; Rhizobacteria; Antagonism; Bacillus subitilis

Biomass efficiency and productivity of soybean inoculated with Trichoderma / Eficiência de biomassa e produtividade de soja inoculada com Trichoderma

This work aimed to evaluate the efficiency of the product TrichoPlus (Trichoderma asperellum) as a plant growth promoter in soybean and the productive performance at the field in a floodplain in the municipality of Formoso do Araguaia, Tocantins, Brazil. Four treatments with different doses of the TrichoPlus product (2, 4, 6 and 8 g per kg of seeds) were used in the experiment, plus two treatments being a positive control with a commercial product based on Trichoderma asperellum and an absolute control (without inoculation). Biomass, stand and productivity data were evaluated. For the treatment with the TrichoPlus product, a powder formulation was used, with active ingredient based on Trichoderma asperellum 201, formulated with a minimum concentration of 2 x108 UFC g-1, having graphite in the composition as inert, being applied directly to the seeds before planting, aiming better adherence and protection of the Trichoderma in direct contact with the seed and protection right after planting. Significant results were observed for the treatments with different doses of TrichoPlus in the characteristics of biomass, stand maintenance and productivity. The best dose for productivity was achieved in 5.1 g kg-1 of seed, with gains 12.6 and 6.0% higher compared with the absolute control and positive control treatments, proving its effectiveness in promoting plant growth and, consequently, in the productivity, in the cultivation in floodplain soil

HIV Infection and Oral Manifestations: An Update

Human immunodeficiency virus (HIV) causes a complete depletion of the immune system; it has been a major health issue around the world since the 1980s, and due to the reduction of CD4+ T lymphocytes levels, it can trigger various opportunistic infections. Oral lesions are usually accurate indicators of immunosuppression because these oral manifestations may occur as a result of the compromised immune system caused by HIV infection; therefore, oral lesions might be initial and common clinical features in people living with HIV. So, it is necessary to evaluate and understand the mechanism, prevalence, and risk factors of oral lesions to avoid the increase morbidity among those with oral diseases

Coinfection with Different Trypanosoma cruzi Strains Interferes with the Host Immune Response to Infection

A century after the discovery of Trypanosoma cruzi in a child living in Lassance, Minas Gerais, Brazil in 1909, many uncertainties remain with respect to factors determining the pathogenesis of Chagas disease (CD). Herein, we simultaneously investigate the contribution of both host and parasite factors during acute phase of infection in BALB/c mice infected with the JG and/or CL Brener T. cruzi strains. JG single infected mice presented reduced parasitemia and heart parasitism, no mortality, levels of pro-inflammatory mediators (TNF-α, CCL2, IL-6 and IFN-γ) similar to those found among naïve animals and no clinical manifestations of disease. On the other hand, CL Brener single infected mice presented higher parasitemia and heart parasitism, as well as an increased systemic release of pro-inflammatory mediators and higher mortality probably due to a toxic shock-like systemic inflammatory response. Interestingly, coinfection with JG and CL Brener strains resulted in intermediate parasitemia, heart parasitism and mortality. This was accompanied by an increase in the systemic release of IL-10 with a parallel increase in the number of MAC-3+ and CD4+ T spleen cells expressing IL-10. Therefore, the endogenous production of IL-10 elicited by coinfection seems to be crucial to counterregulate the potentially lethal effects triggered by systemic release of pro-inflammatory mediators induced by CL Brener single infection. In conclusion, our results suggest that the composition of the infecting parasite population plays a role in the host response to T. cruzi in determining the severity of the disease in experimentally infected BALB/c mice. The combination of JG and CL Brener was able to trigger both protective inflammatory immunity and regulatory immune mechanisms that attenuate damage caused by inflammation and disease severity in BALB/c mice