Microbiologia
URI permanente desta comunidadehttps://locus.ufv.br/handle/123456789/11840
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Item Production and regeneration of protoplasts from orchid Mycorrhizal Fungi Epulorhiza repens and Ceratorhiza sp.(Brazilian Archives of Biology and Technology, 2010-01) Coelho, Irene da Silva; Queiroz, Marisa Vieira de; Costa, Maurício Dutra; Kasuya, Maria Catarina Megumi; Araújo, Elza Fernandes deThe aim of this work was to study the standardization of conditions to obtain and regenerate Epulorhiza repens and Ceratorhiza sp. protoplasts. For E. repens, the largest number of protoplasts (8.0 × 106 protoplasts/mL) was obtained in 0.6 M KCl, using 15 mg/mL of Lysing Enzymes, and 2-day-old fungal mycelium. When 0.5 M sucrose was used as osmotic stabilizer, the highest frequency of regeneration was achieved (8.5 %); 80.0 % of protoplasts were nucleated, and 20.0 % anucleated. For Ceratorhiza sp., the largest number of protoplasts (4.0 × 107 protoplasts/mL) was achieved in 0.6 M NaCl, when 15 mg/mL of Lysing Enzymes and 15mg/mL of Glucanex, with 2-day-old fungal mycelium were used. The highest frequency of regeneration was 6.7 % using 0.5 M sucrose as osmotic stabilizer; 88.8 % of protoplasts were nucleated, and 11.2 % anucleated.Item In vitro culture of Gigaspora decipiens and Glomus clarum in transformed roots of carrot: the influence of temperature and pH(Acta Scientiarum. Agronomy, 2013-07) Kasuya, Catarina Megumi; Costa, Francilina Araújo; Haddad, Lydice Sant'Anna Meira; Oton, Wagner Campos; Costa, Maurício Dutra; Borges, Arnaldo ChaerMonoxenic cultures of arbuscular mycorrhizal fungi (AMF) in transformed roots have been used to better understand the symbiosis with these fungi, but few species have been successfully established in vitro. The object was to establish monoxenic cultures of Gigaspora decipiens and Glomus clarum and to verify the effects of temperature and pH on spore formation. Cultures were established from superficially disinfested spores that were germinated on an agar and water. After germination, the spores were transferred to Petri dishes containing transformed carrot roots. After 4-6 days formed newly formed spores and structures typical. The effects of temperature (22, 25, 28 or 32ºC) and pH (4.0, 4.5, 5.5 or 6.5) on the production of spores were assessed over three months, resulting in sigmoidal growth curves. The spore increased from 22ºC, reaching higher values 28oC and 32oC there was a reduction in the production. The highest spore production of G. decipiens occurred in a pH of 6.5, whereas for G. clarum was pH of 4.0. The cultivation of other species is still necessary to elucidate particular aspects of the symbiosis that so far are unclear, including the effects of environmental factors on the production of spores of different AMF species.Item Mechanisms of phosphate solubilization by fungal isolates when exposed to different P sources(Annals of Microbiology, 2014-03) Mendes, Gilberto de Oliveira; Freitas, André Luiz Moreira de; Pereira, Olinto Liparini; Silva, Ivo Ribeiro da; Vassilev, Nikolay Bojkov; Costa, Maurício DutraThe use of phosphate-solubilizing fungi is a promising biotechnological strategy in the management of phosphorus (P) fertilization, as it enables the utilization of rock phosphates (RP) or the recovery of P fixed in soil particles. The objective of our study was to evaluate fungal isolates for mechanisms of solubilization of P-bearing compounds, such as AlPO4, FePO4, Ca3(PO4)2, Araxá RP, and Catalão RP. Four fungal isolates obtained from Brazilian soils were characterized in liquid media: Aspergillus niger FS1, Penicillium canescens FS23, Eupenicillium ludwigii FS27, and Penicillium islandicum FS30. A. niger FS1 was the only isolate able to solubilize all of the P sources, solubilizing 71, 36, 100, and 14 % of the P from AlPO4, FePO4, Ca3(PO4)2, and RPs, respectively. Medium acidification was an effective solubilization mechanism, particularly for Ca3(PO4)2. The other P sources were mainly solubilized through organic acids produced by the fungi. Oxalic acid, produced exclusively by A. niger FS1, and citric acid were decisive factors in the solubilization of AlPO4 and FePO4. Penicillium isolates produced more gluconic acid than A. niger FS1 in all treatments. However, this higher production did not result in higher solubilization for any of the P sources, showing that gluconic acid contributes little to the solubilization of the P sources evaluated. The higher capacity of medium acidification and the production of organic acids with stronger metal-complexation activity are characteristics that confer to A. niger FS1 a wider action on insoluble P sources. Consequently, this isolate qualifies as a promising candidate for application in the management of P fertilization.Item Calcium oxalate crystals in eucalypt ectomycorrhizae: morphochemical characterization(Plos One, 2013-07) Pylro, Victor Satler; Freitas, André Luiz Moreira de; Otoni, Wagner Campos; Silva, Ivo Ribeiro da; Borges, Arnaldo Chaer; Costa, Maurício DutraEctomycorrhizal fungi are ubiquitous in forest ecosystems, benefitting plants principally by increasing the uptake of water and nutrients such as calcium from the soil. Previous work has demonstrated accumulation of crystallites in eucalypt ectomycorrhizas, but detailed morphological and chemical characterization of these crystals has not been performed. In this work, cross sections of acetic acid-treated and cleared ectomycorrhizal fragments were visualized by polarized light microscopy to evaluate the location of crystals within cortical root cells. Ectomycorrhizal sections were also observed by scanning electron microscopy (SEM) coupled with energy dispersive x-ray (EDS) microprobe analysis. The predominant forms of crystals were crystal sand (granules) and concretions. Calcium, carbon and oxygen were detected by EDS as constituent elements and similar elemental profiles were observed between both crystal morphologies. All analyzed crystalline structures were characterized as calcium oxalate crystals. This is the first report of the stoichiometry and morphology of crystals occurring in eucalypt ectomycorrhizas in tropical soils. The data corroborates the role of ectomycorrhizae in the uptake and accumulation of calcium in the form of calcium oxalate crystals in hybrid eucalypt plants.Item Relative importance of soil physico-chemical characteristics and plant species identity to the determination of soil microbial community structure(Applied Soil Ecology, 2015-07) Bonduki, Victor Hugo A.; Melo, Christiane Augusta D.; Tótola, Marcos R.; Ferreira, Francisco A.; Costa, Maurício Dutra; Massenssini, André M.The structure of soil microbial communities is affected by biotic and abiotic environmental factors, such as plant community composition and soil chemical characteristics, among others. However, little is known about the relative importance of these factors on soil microbial community structure. The objective of this study was to verify which factor, soil chemical characteristics or plant species identity, is more important to the determination of soil microbial community structure. For this, a factorial experiment with four soil chemical conditions and five plant species were set in a greenhouse. After 80 days of cultivation, the rhizospheric soil microbial community structure was accessed by a multiplex T-RFLP, and the mycorrhizal colonization of roots and plant shoot dry mass were estimated. Plant species showed similar growth responses to different soil chemical conditions, but exhibited different patterns in the control of root mycorrhizal colonization. A principal component analysis (PCA) was performed using the T-RFLP data set and showed that soil chemical condition is the main factor defining the structure of soil microbial community. Archaeal and bacterial communities showed to be more sensitive to changes in the soil chemical environment, suggesting a greater importance of these microbial groups in plant adaptation.Item Basidiosporogenesis, meiosis, and post-meiotic mitosis in the ectomycorrhizal fungus Pisolithusmicrocarpus(Fungal Genetics and Biology, 2010-03-03) Campos, André Narvaes da Rocha; Costa, Maurício DutraPisolithus microcarpus (Cooke and Massee) G. Cunn. is a model organism for the studies on the ecology, physiology, and genetics of the ectomycorrhizal associations. However, little is known about the basidiosporogenesis in this species and, in particular, the nuclear behavior after karyogamy. In this work, the events involved in basidiosporogenesis and meiosis in P. microcarpus were analyzed using fluorescence and scanning electron microscopy. The basidia are formed inside peridioles by the differentiation of the cells along the whole hyphae. Basidial cells measure 12–18 μm in length and 6–7 μm in diameter. P. microcarpus produces eight basidiospores per basidium imbibed in a gelatinous matrix in the basidiocarp. The basidiospores are globose, equinate, with blunt spines, and measure 6–8 μm. Karyogamy can take place inside basidia as well as in undifferentiated hyphal cells followed by nuclear migration to a newly developed basidium where meiosis takes place. After the formation of the meiotic tetrad, one round of post-meiotic mitosis occurs, resulting in the production of eight nuclei per basidium. The newly-formed nuclei migrate into the basidiospores asynchronously, resulting in the production of eight uninucleate spores. This corresponds to pattern A of post-meiotic mitosis. This work is the first report on meiosis and post-meiotic mitosis during basidiosporogenesis in P. microcarpus and contributes to clarify some aspects of the biology and genetics of this ectomycorrhizal species.Item Histochemistry and storage of organic compounds during basidiosporogenesis in the ectomycorrhizal fungus Pisolithus microcarpus(World Journal of Microbiology and Biotechnology, 2010-02-25) Campos, André Narvaes da Rocha; Costa, Maurício DutraKnowledge on the distribution and storage of different organic compounds during basidiosporogenesis in P. microcarpus is paramount to a better understanding of basidiospore recalcitrance to germination. The objective of this work was to detect the presence and distribution of phenolics, reducing sugars, starch, glycogen, total polysaccharides, RNA, and proteins during P. microcarpus basidiosporogenesis. Starch and reducing sugars were not detected in the fungal basidiocarps, while other polysaccharides predominated in the extracellular matrix at the base of the basidiocarp containing unconsolidated peridioles. Phenolics were also detected in this region. Glycogen was present inside the hyphae, basidia, and basidiospores and constitutes an important storage compound in the fungal basidiocarps. In mature basidiospores, RNA accumulation occurred at discrete locations in the cytoplasmatic periphery, while polysaccharides and proteins were shown to predominate in the cell wall. The presence of glycogen, RNA, and proteins inside the basidiospores strongly indicates provision for future germination and suggests that other factors may also influence basidiospore recalcitrance.Item Decreased mineral availability enhances rock phosphate solubilization efficiency in Aspergillus niger(Annals of Microbiology, 2014-05-30) Oliveira, Samantha Caixeta de; Mendes, Gilberto de Oliveira; Silva, Ubiana Cássia da; Silva, Ivo Ribeiro da; Ribeiro Júnior, José Ivo; Costa, Maurício DutraMicrobial solubilization of rock phosphate (RP) is mainly achieved by the production of organic acids and medium acidification through H+ release. During RP solubilization, mineral nutrient availability is likely to be critical for determining how much carbon is channeled either for metabolite synthesis or for microbial growth, influencing organic acid release by microorganisms. Thus, the objective of this work was to study the relationships between the concentration of mineral nutrients in the growth medium and the efficiency of RP solubilization by Aspergillus niger FS1. For this, the fungus was grown in Czapek medium containing 0, 1, 2, 10, 50, and 100 % of its original concentration of mineral nutrients. Decreasing mineral availability in the growth medium led to decreases in fungal biomass and solubilized P, and increases in titratable acidity and solubilization efficiency as expressed by mg solubilized P per g fungal biomass (YP/B), indicating a shift in fungal metabolism from biomass production to organic acid release. The transfer of pre-grown biomass to media with or without added minerals confirmed that lower mineral availability increases YP/B and led to the solubilization of 76 % of P present in Patos RP. These observations open new perspectives on improving RP solubilization systems by manipulating mineral nutrient availability in the medium, with consequent gains in cost reduction.Item Diversity and distribution of the endophytic bacterial community at different stages of Eucalyptus growth(Antonie van Leeuwenhoek, 2016-03-24) Miguel, Paulo Sérgio Balbino; Neves, Júlio César Lima; Borges, Arnaldo Chaer; Tótola, Marcos Rogério; Costa, Maurício Dutra; Oliveira, Marcelo Nagem Valério de; Delvaux, Júlio César; Jesus, Guilherme Luiz deThe relationships between plants and endophytic bacteria significantly contribute to plant health and yield. However, the microbial diversity in leaves of Eucalyptus spp. is still poorly characterized. Here, we investigated the endophytic diversity in leaves of hybrid Eucalyptus grandis x E. urophylla (Eucalyptus “urograndis”) by using culture-independent and culture-dependent approaches, to better understand their ecology in leaves at different stages of Eucalyptus development, including bacteria with N2 fixation potential. Firmicutes, Proteobacteria (classes alpha-, beta- and gamma-) and Actinobacteria were identified in the Eucalyptus “urograndis” endophytic bacterial community. Within this community, the species Novosphingobium barchaimii, Rhizobium grahamii, Stenotrophomonas panacihumi, Paenibacillus terrigena, P. darwinianus and Terrabacter lapilli represent the first report these bacteria as endophytes. The diversity of the total endophytic bacteria was higher in the leaves from the ‘field’ (the Shannon–Wiener index, 2.99), followed by the indices obtained in the ‘clonal garden’ (2.78), the ‘recently out from under shade (2.68), ‘under shade’ (2.63) and ‘plants for dispatch’ (2.51). In contrast, for diazotrophic bacteria, the highest means of these indices were obtained from the leaves of plants in the ‘under shade’ (2.56), ‘recently out from under shade (2.52)’ and ‘field’ stages (2.54). The distribution of the endophytic bacterial species in Eucalyptus was distinct and specific to the development stages under study, and many of the species had the potential for nitrogen fixation, raising the question of whether these bacteria could contribute to overall nitrogen metabolism of Eucalyptus.Item Basidiosporogenesis, meiosis, and post-meiotic mitosis in the ectomycorrhizal fungus Pisolithus microcarpus(Fungal Genetics and Biology, 2010-03-03) Campos, André Narvaes da Rocha; Costa, Maurício DutraPisolithus microcarpus (Cooke and Massee) G. Cunn. is a model organism for the studies on the ecology, physiology, and genetics of the ectomycorrhizal associations. However, little is known about the basidiosporogenesis in this species and, in particular, the nuclear behavior after karyogamy. In this work, the events involved in basidiosporogenesis and meiosis in P. microcarpus were analyzed using fluorescence and scanning electron microscopy. The basidia are formed inside peridioles by the differentiation of the cells along the whole hyphae. Basidial cells measure 12–18 μm in length and 6–7 μm in diameter. P. microcarpus produces eight basidiospores per basidium imbibed in a gelatinous matrix in the basidiocarp. The basidiospores are globose, equinate, with blunt spines, and measure 6–8 μm. Karyogamy can take place inside basidia as well as in undifferentiated hyphal cells followed by nuclear migration to a newly developed basidium where meiosis takes place. After the formation of the meiotic tetrad, one round of post-meiotic mitosis occurs, resulting in the production of eight nuclei per basidium. The newly-formed nuclei migrate into the basidiospores asynchronously, resulting in the production of eight uninucleate spores. This corresponds to pattern A of post-meiotic mitosis. This work is the first report on meiosis and post-meiotic mitosis during basidiosporogenesis in P. microcarpus and contributes to clarify some aspects of the biology and genetics of this ectomycorrhizal species.