Pisolithus is an ectomycorrhizal (ECM) fungal genus that exists in native and plantation forests worldwide. Despite many aspects of the genus having been studied over the years, there is limited knowledge of the genetic population structure of Pisolithus spp. The major aim of this study was to begin to elucidate the genetic structure of populations of Pisolithus native to the eastern states of Australia. Initially, the number of species represented in the University of Western Sydney Pisolithus culture collection was investigated using ITS-RFLP and previously developed simple sequence repeat (SSR) markers. From these analyses, two clear groupings emerged with the RFLP groups corresponding to the SSR groups identified. To study genetic population structure, appropriate markers were required and SSR markers were considered to be most suited to this end. The developed SSR markers were successfully used to amplify P. microcarpus and P. albus DNA from ECM root tips and soil samples. Therefore, these markers will be a useful tool for future investigations into the population structure of above and below-ground structures of P. microcarpus and P. albus populations. / Doctor of Philosophy (PhD)

Submitted by Nathália Faria da Silva (nathaliafsilva.ufv@gmail.com) on 2017-06-14T14:33:21Z No. of bitstreams: 1 resumo.pdf: 21140 bytes, checksum: f257be804d9a30cfb29d742fcc325158 (MD5) / Made available in DSpace on 2017-06-14T14:33:21Z (GMT). No. of bitstreams: 1 resumo.pdf: 21140 bytes, checksum: f257be804d9a30cfb29d742fcc325158 (MD5) Previous issue date: 2005-01-14 / O objetivo deste trabalho foi caracterizar a formação de basidiósporos de Pisolithus sp., bem como os eventos de meiose, mitose pós-meiótica e o acúmulo de reservas lipídicas durante o desenvolvimento dos basidiocarpos fúngicos. Os estudos foram conduzidos em corpos de frutificação frescos, coletados sob florestas de Eucalyptus spp., na Universidade Federal de Viçosa, Viçosa, MG. Os eventos envolvidos na formação dos basidiósporos foram analisados utilizando-se técnicas de microscopia ótica, microscopia de fluorescência, microscopia eletrônica de varredura e cromatografia gasosa. Pisolithus sp. produz oito esporos por basídio, envolvidos em matriz gelatinosa, no interior dos peridíolos constituintes do basidioma. Os esporos apresentam de 6 a 8 μm de diâmetro, são globosos, equinatos, com espículas embotadas. Previamente à formação dos basidiósporos, as células das hifas constituintes do peridíolo diferenciam-se em basídios clavados com 12 a 18 μm de comprimento e 6 a 8 μm de diâmetro. Os basidiocarpos apresentam abundante quantidade de lipídeos totais distribuídos nos basídios e nos esporos. A análise da composição de ácidos graxos revelou a presença de 17 compostos com cadeias de 14 a 20 carbonos, na maioria insaturados, contendo uma ou duas insaturações. A composição e o conteúdo em ácidos graxos variou de acordo com o estádio de desenvolvimento dos peridíolos. Nos basidiósporos livres, os ácidos graxos predominantes foram 16:0, 16:1w5c, 18:1w9c e a mistura 18:2w6,9c/18:0ante, sugerindo que o ácido oléico seja o principal ácido graxo constituinte das reservas lipídicas dos basidiósporos. Na formação do diplóide, a cariogamia pode ocorrer tanto no basídio quanto nas células das hifas sub- basidiais ou sub-apicais, seguindo-se os eventos característicos da divisão meiótica. Após a formação da tétrade meiótica nos basídios, há a ocorrência de uma rodada de divisão nuclear mitótica, resultando na produção de oito núcleos na célula basidial. Os núcleos formados migram para dentro dos basidiósporos de forma assincrônica. Durante os eventos de migração, os núcleos haplóides adquirem a forma de fuso, sugerindo a participação de microtúbulos no transporte dessas organelas. Este estudo constitui o primeiro relato dos processos de divisão e migração nuclear em Pisolithus durante a formação dos basidiósporos e da presença e distribuição de ácidos graxos no basidiocarpo desse fungo. O conhecimento das etapas de formação dos basidiósporos e das características das reservas de carbono presentes nesses propágulos permitirá o desenvolvimento de estratégias que visem elucidar os fatores que influenciam o processo de germinação dos basidiósporos de Pisolithus sp., viabilizando a utilização desses esporos como inoculantes, em viveiros florestais, e na pesquisa genética focalizada na associação ectomicorrízica. / The objective of this work was to characterize the formation of Pisolithus sp. basidiospores as well as the events of meiosis, post-meiotic mitosis, and the accumulation of lipid reserves during the development of the fungal basidiocarps. This study was conducted in fresh fruit bodies collected under Eucalyptus spp. forests at Universidade Federal de Viçosa, Viçosa, MG. The events involved in the development of the basidiospores were analyzed using light microscopy, fluorescent microscopy, scanning electron microscopy, and gas chromatography. Pisolithus sp. produces eight spores per basidium imbibed in a gelatinous matrix inside the peridioles constituting the basidiome. The spores are, approximately, 6 to 8 μm in diameter, globose, echinate, with blunt spines. Previously to the formation of the basidiospores, hyphal cells forming the peridioles differentiate into clavate basidia with 12 to 18 μm in length, and 6 to 8 μm in width. The basidiocarps present abundant quantities of total lipids distributed in hyphae, basidia, and basidiospores. Composition analyses of fatty acids revealed the presence of 17 compounds with 14 to 20 carbon atoms, mostly insaturated, containing one or two insaturations. The composition and content of fatty acids varied according to the developmental stage of the peridioles. Inside free basidiospores, the predominant fatty acids were 16:0, 16:1w5c,18:1w9c, and the mixture 18:2w6,9c/18:0ante, suggesting that oleic acid is the main fatty acid making up the lipid reserves of the spores. ixDuring diploid formation, karyogamy can take place either in the basidia as well as in subbasidial or subapical hyphal cells, followed by the typical events of meiotic division. After the formation of the meiotic tetrad in the basidia, one round of mitotic nuclear division occurs, resulting in the production of eight nuclei per basidial cell. The newly-formed nuclei migrate into basidiospores asynchronously. During the migration events, the haploid nuclei acquire a fusiforme shape, suggesting the participation of microtubules in the nuclear transport into spores. This work is the first report on the nuclear division and migration in Pisolithus during the formation of basidiospores and on the presence and distribution of fatty acids in the basidiomata of this fungus. A thorough understanding of the main stages of basidiospore formation and of the main characteristics of the carbon reserves present within these spores will allow the development of strategies aiming at the elucidation of the factors that influence the germination of Pisolithus sp. basidiospores. This will ultimately make possible the use of these spores as inoculants in forest nurseries and in the genetic research focused on the ectomycorrhizal association.

Made available in DSpace on 2015-03-26T13:51:51Z (GMT). No. of bitstreams: 1 texto completo.pdf: 528396 bytes, checksum: 2b516aa2ea3ea48bfb173971c8b65f4c (MD5) Previous issue date: 2010-08-18 / Conselho Nacional de Desenvolvimento Científico e Tecnológico / The low germination percentages of P. microcarpus basidiospores represents a major drawback for obtaining monokaryotic strains for quantitative genetic studies on the mycorrhizal associations, the use of spores in mutagenesis experiments aiming at the identification of genes important to the symbiosis, and the use of these propagules as inoculants in forest nurseries. The characterization of the mRNA present within the fungal basidiospores may provide information on the level of preparedness of the spores to germinate and sustain initial hyphal growth. The aim of this work was to characterize the mRNA present in the basidiospores of the ectomycorrhizal fungus P. microcarpus after basidiosporogenesis. Total RNA was extracted from the mature basidiospores and mycelium and mRNA was used for cDNA synthesis. An analysis of the presence of 14 gene transcripts involved in lipid metabolism, glycogen mobilization, trehalose mobilization, nitrogen assimilation, glycolysis, pentose phosphate pathway and glucan degradation using cDNA from the fungal basidiospores and dikaryotic mycelium was done. qPCR analysis was performed to compare the amount of transcripts of the genes d15fa, ntrh, and ag13 which code respectively, for Δ15 fatty acid desaturase, neutral α-trehalase, and α-1,3-glucosidase in the dikaryotic mycelium and basidiospores. The 14 transcripts studied were present both in the dikaryotic mycelium and in the basidiospores, indicating a preparedness of these propagules to initiate and sustain germination. qPCR analysis indicated a higher amount of transcripts of genes the d15fa, ntrh, and ag13 inside the basidiospores when compared to the dikaryotic mycelium. These results suggest a higher need for enzymes involved in the synthesis of unsaturated fatty acids, trehalose mobilization, and glucan degradation during basidiospore germination. A cDNA library was constructed from basidiospore mRNAs and 288 clones were sequenced. One hundred and nineteen sequences were obtained, resulting in a total of 12 ESTs (expressed sequence tags). The amino acid sequence deduced from the EST corresponding to clone 277 showed significant similarity to a protein involved in respiratory metabolism and may be important during the germination process. Other ESTs showed significant identity to unknown ESTs deposited in the NCBI database. These ESTs may code for proteins that are specific of the basidiospores of P. microcarpus. However, to confirm this hypothesis, other clones must be sequenced to better characterize the cDNA library. / As baixas percentagens de germinação dos basidiósporos do fungo ectomicorrízico Pisolithus microcarpus dificultam a obtenção de estirpes monocarióticas, material para estudos de genética quantitativa da associação micorrízica, inviabilizam a utilização de esporos em experimentos de mutagênese visando à identificação de genes importantes para a simbiose, além de dificultar a utilização desses propágulos como inoculantes em viveiros florestais. A caracterização do mRNA presente no interior desses basidiósporos poderá fornecer informações sobre o nível de preparação que têm para iniciar o processo de germinação e sustentar o crescimento inicial das hifas. O objetivo deste trabalho foi o de caracterizar os mRNA presentes nos basidiósporos do fungo ectomicorrízico P. microcarpus após a basidiosporogênese. O RNA total foi extraído de basidiósporos maduros e micélio, procedendo posteriormente a obtenção mRNA para a síntese de cDNA. Determinou-se a presença de transcritos de 14 genes envolvidos no metabolismo de lipídeos, na mobilização de glicogênio, na mobilização de trealose, na assimilação de nitrogênio, na via glicolítica, na via das pentoses fosfato e na degradação de glicanas. A análise por qPCR foi realizada visando comparar a quantidade de transcritos dos genes d15fa, ntrh e ag13 que codificam respectivamente as enzimas ácido graxo-Δ15 desaturase, α- trealase neutra e 1,3-α-glicosidase, nos basidiósporos e no micélio dicariótico. Os 14 x transcritos avaliados foram encontrados tanto no micélio dicariótico quanto nos basidiósporos, sugerindo a preparação desses propágulos para iniciar e sustentar o processo de germinação. A análise por qPCR indicou maior quantidade de transcritos dos genes d15fa, ntrh e ag13 no interior dos basidiósporos quando comparado ao micélio dicariótico. Esse resultado sugere a participação de enzimas responsáveis pela síntese de ácidos graxos insaturados, mobilização de trealose e degradação de glicanas durante o processo de germinação. Uma biblioteca de cDNA foi construída a partir dos fragmentos de cDNA dos basidiósporos, sendo caracterizada por meio do sequenciamento de 288 clones. Foram obtidas 119 sequências que, ao serem agrupadas, resultaram em 12 ESTs (expressed sequence tags). A sequência de aminoácidos deduzida da EST referente ao clone 277 apresentou similaridade significativa com proteína envolvida no metabolismo respiratório, podendo ser importante durante o processo de germinação. Outras ESTs apresentaram identidade significativa com ESTs depositadas no banco de dados do NCBI ainda não identificadas. Alternativamente, sugere-se que essas ESTs possam ser codificadoras de proteínas específicas dos basidiósporos de P. microcarpus, importantes para a etapa de germinação desses propágulos.

Made available in DSpace on 2015-03-26T13:52:01Z (GMT). No. of bitstreams: 1 texto completo.pdf: 1165886 bytes, checksum: 9908ee426c5cfd8f17fea52b7f132085 (MD5) Previous issue date: 2011-08-23 / Conselho Nacional de Desenvolvimento Científico e Tecnológico / The basidiospores of Pisolithus show low germination percentages despite possessing conspicuous reserve compounds for the provision of carbon and energy during the initial growth of the fungal hyphae. The highly hydrophobic and resistant cell wall of the basidiospore can contribute to basidiospores recalcitrance to germination. Thus, this study aimed at demonstrating the presence of glycogen, polysaccharides, lipids, RNA and proteins in mature basidiospores of Pisolithus microcarpus, determining the fatty acid profile of the basidiospores of ectomycorrhizal fungi, and evaluating methods of permeabilization of the basidiospore cell wall. Hystochemical techniques used in the detection of different reserve compounds in the basidiospores revealed the presence of glycogen, polysaccharides, proteins, RNA, lipids, lipoproteins, neutral lipids, and acidic lipids. The presence of 11 different fatty acids in the basidiospores of P. microcarpus, nine in those of Scleroderma sp. and 16 in the basidiospores of Suillus sp. was observed in our study. The most abundant fatty acids in the fungal basidiospores were 18:1w9c and 16:0. Besides oleic and palmitic acids, the following fatty acids showed significant contents: 16:1w5c 16:0, 18:1w5c for P. microcarpus; 12:1 AT11-12, 18:00 for Scleroderma sp.; 15:00 and 18:00 for Suillus sp. The fatty acid profile of the basidiospores of P. microcarpus showed 36.8 % similarity to that of Scleroderma sp. The fatty acid profile of these two species showed a similarity of only 5.2 % compared to that of Suillus sp. The use of chitinase, cellulase, and lyticase for 1, 2, 3, 4, 6, 8, 12, and 24 h, at 30 °C, under stirring at 80 rpm, and of sulfuric acid at concentrations of 5, 10, 25, 50 % (v / v) for 5, 10, 25, 50 or 80s did not promote cell wall permeability. The treatment with sodium hypochlorite at concentrations of 5, 10, 20, 30, 40, 50, 60, 70, 90 and 100 % of original concentration of active chlorine (20 g L-1) and contact times of 5, 10 , 20, 30, 40, 50, 60, 70, and 80s promoted the permeabilization of the basidiospore cell wall as evaluated by the staining of internal fatty acids with Sudan Black B. The highest percentage of permeabilized basidiospores, 97 %, was obtained with the treatment with hypochlorite at 100 % for 80s. However, the viability tests and the observation of basidiospores by scanning electron microscopy revealed the loss of viability and cell wall integrity. The exposure of the basidiospores to sodium hypochlorite at 5 % for 40s promoted the highest percentages of permeabilized basidiospores, 83 %, without loss of viability and morphological damages to the spore cell wall. These results contribute to the understanding of the variables that may play a role in the process of germination of basidiospores of P. microcarpus and allow the development of strategies to improve the isolation of monokaryons in vitro. / Os basidiósporos Pisolithus apresentam baixas percentagens de germinação, embora disponham de compostos de reserva para a provisão de carbono e energia durante o crescimento inicial das hifas. Neles, a parede celular altamente hidrofóbica e resistente pode contribuir para a recalcitrância à germinação. Dessa forma, o presente trabalho teve como objetivo demonstrar a presença de glicogênio, polissacarídeo, lipídeos, RNA e proteínas em basidiósporos maduros de Pisolithus microcarpus, determinar o perfil de ácidos graxos de basidiósporos de fungos ectomicorrízicos e avaliar métodos de permeabilização da parede celular dos basidiósporos. As técnicas histoquímicas utilizadas na detecção de diferentes compostos de reserva nos basidiósporos evidenciaram a presença de glicogênio, polissacarídeos, proteínas, RNA, lipídeos totais, lipoproteínas, lipídeos neutros e lipídeos ácidos. Detectou-se a presença de onze ácidos graxos distintos nos basidiósporos de P. microcarpus, nove nos de Scleroderma sp. e dezesseis nos de Suillus sp. Os ácidos graxos mais abundantes nas estruturas fúngicas analisadas foram o 18:1w9c e o 16:0. Além do ácido oléico e ácido palmítico, destacaram-se os seguintes ácidos graxos: 16:1w5c, 18:1w5c para P. microcarpus; 12:1at11-12 e 18:00 para Scleroderma sp.; 18:00 e 15:00 para Suillus sp. O perfil de ácidos graxos dos basidiósporos de P. microcarpus apresentou similaridade de 36,8 % com o de Scleroderma sp. O perfil de ácidos graxos dessas duas espécies apresentou similaridade de somente 5,2 % em relação àquele de Suillus sp. O uso de quitinase, liticase e celulase por 1, 2, 3, 4, 6, 8, 12 e 24 h, sob agitação a 80 rpm e a 30 °C, não resultou na permeabilização da parede celular dos basidiósporos. O tratamento dos basidiósporos com ácido sulfúrico nas concentrações de 5, 10, 25, 50 % (v/v), por 5, 10, 25, 50 ou 80 s, também não resultou na permeabilização dos basidiósporos. Os tratamentos com hipoclorito de sódio em concentrações de 5, 10, 20, 30, 40, 50, 60, 70, 90 e 100 % da concentração original de cloro ativo (20 g L- 1) e tempos de contato de 5, 10, 20, 30, 40, 50, 60, 70 e 80 s promoveram a permeabilização da parede celular dos basidiósporos avaliada pela penetração do corante Negro Sudão B. A maior percentagem de basidiósporos permeabilizados, 97 %, foi obtida com o tratamento com hipoclorito a 100 % pelo tempo de 80 s. No entanto, os testes de viabilidade e a observação dos basidiósporos por microscopia eletrônica de varredura evidenciaram a perda da integridade da parede celular e da viabilidade dos esporos quando submetidos a esse tratamento. A exposição dos basidiósporos ao hipoclorito de sódio a 5 % pelo tempo de 40 s foi o tratamento que resultou em maiores percentagens (83 %) de basidiósporos permeabilizados, sem prejuízos na viabilidade e da morfologia desses propágulos. Os resultados obtidos contribuem para a compreensão das variáveis que atuam no processo de germinação dos basidiósporos de P. microcarpus e permitirão o desenvolvimento de estratégias visando melhorar a obtenção de monocários in vitro.

Pisolithus is an important ectomycorrhizal genus world-wide, however to date we remain largely ignorant of the genetic and functional variation that exists within isolates of this genus. Fifty-three isolates of Pisolithus were obtained from various locations in central and eastern Australia and genetic variation within the isolates was assessed using ITS-RFLP and ITS sequencing analyses. RFLP analysis initially grouped the isolates into eight RFLP types. Neighbour-joining analysis of ITS sequences with Pisolithus ITS sequences available in databases clustered the majority of isolates into four groups within two major clades, each comprising isolates of similar basidiospre characteristics. Most Australian isolates correspond with recent provisional descriptions of P. albus or P. marmoratus. One isolate (LJ30) had low sequence identity (61.6-78.0%) to the other isolates and probably represents a separate undescribed Australian species. Significant intraspecific variation was observed in ITS-RFLP profiles for the putative P. albus isolates, suggesting that the sole use of RFLP analysis in diversity assessment may over-estimate Pisolithus species richness. Investigations were also initiated to identify if a relationship exists between genetic and physiological diversity in Australian Pisolithus. It is, however, clear that extensive physiological variation exists in Australian Pisolithus isolates. The size and distribution of genets of Australian Pisolithus species I and II ( putative P. albus and P. marmoratus) was also assessed using microsatellite-primed PCR to gain a better understanding of the likely distribution of underground mycelial networks and possible reproduction strategies in native soils. The data demonstrate that both species have the ability to be long-lived and extend for significant distances in native soils in undisturbed conditions. The field site for Pisolithus species I, however, also contained of a large number of small individuals suggesting that this species may employ a life-history strategy combining r-, C and S characteristics depending on local soil conditions / Doctor of Philosophy (PhD)

Pisolithus is an important ectomycorrhizal genus world-wide, however to date we remain largely ignorant of the genetic and functional variation that exists within isolates of this genus. Fifty-three isolates of Pisolithus were obtained from various locations in central and eastern Australia and genetic variation within the isolates was assessed using ITS-RFLP and ITS sequencing analyses. RFLP analysis initially grouped the isolates into eight RFLP types. Neighbour-joining analysis of ITS sequences with Pisolithus ITS sequences available in databases clustered the majority of isolates into four groups within two major clades, each comprising isolates of similar basidiospre characteristics. Most Australian isolates correspond with recent provisional descriptions of P. albus or P. marmoratus. One isolate (LJ30) had low sequence identity (61.6-78.0%) to the other isolates and probably represents a separate undescribed Australian species. Significant intraspecific variation was observed in ITS-RFLP profiles for the putative P. albus isolates, suggesting that the sole use of RFLP analysis in diversity assessment may over-estimate Pisolithus species richness. Investigations were also initiated to identify if a relationship exists between genetic and physiological diversity in Australian Pisolithus. It is, however, clear that extensive physiological variation exists in Australian Pisolithus isolates. The size and distribution of genets of Australian Pisolithus species I and II ( putative P. albus and P. marmoratus) was also assessed using microsatellite-primed PCR to gain a better understanding of the likely distribution of underground mycelial networks and possible reproduction strategies in native soils. The data demonstrate that both species have the ability to be long-lived and extend for significant distances in native soils in undisturbed conditions. The field site for Pisolithus species I, however, also contained of a large number of small individuals suggesting that this species may employ a life-history strategy combining r-, C and S characteristics depending on local soil conditions / Doctor of Philosophy (PhD)

Submitted by Jos? Henrique Henrique (jose.neves@ufvjm.edu.br) on 2017-10-02T20:42:54Z No. of bitstreams: 2 license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) angela_lais_fernandes_gomes.pdf: 1004005 bytes, checksum: 971c90aeba361b9e02c0d4ce971cfb63 (MD5) / Approved for entry into archive by Rodrigo Martins Cruz (rodrigo.cruz@ufvjm.edu.br) on 2017-10-09T14:09:05Z (GMT) No. of bitstreams: 2 license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) angela_lais_fernandes_gomes.pdf: 1004005 bytes, checksum: 971c90aeba361b9e02c0d4ce971cfb63 (MD5) / Made available in DSpace on 2017-10-09T14:09:05Z (GMT). No. of bitstreams: 2 license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) angela_lais_fernandes_gomes.pdf: 1004005 bytes, checksum: 971c90aeba361b9e02c0d4ce971cfb63 (MD5) Previous issue date: 2016 / Universidade Federal dos Vales do Jequitinhonha e Mucuri (UFVJM) / Coordena??o de Aperfei?oamento de Pessoal de N?vel Superior (CAPES) / Os benef?cios das associa??es ectomicorr?zicas s?o dependentes da planta hospedeira, do isolado f?ngico e do ambiente. O objetivo deste trabalho foi selecionar isolados de Pisolithus sp. que colonizem mudas clonais de eucalipto propagados por miniestaquia e que promovam maior sobreviv?ncia, crescimento e nutri??o das mudas em viveiro comercial. Os clones PT3335 e PT3336 foram inoculados com 18 isolados de Pisolithus sp. e crescidos em substrato com aduba??o fosfatada reduzida, mais os controles n?o inoculados com (Controle) e sem (Comercial) redu??o da aduba??o fosfatada de substrato. A inocula??o dos fungos ectomicorr?zicos aumentou a sobreviv?ncia, enraizamento, coloniza??o, crescimento e teores de Fe em rela??o aos controles, por?m estes efeitos foram dependentes de isolados e clones. Para o PT3335, alguns isolados dobraram a sobreviv?ncia das mini-estacas em rela??o ao Controle e ao Comercial, sendo os melhores o D29, D62, D63, D118 e D216. Para o PT3336 os melhores isolados foram D5, D29 e D88, com aumento da sobreviv?ncia de at? 25 %. Os maiores aumentos de altura foram observados nas mudas do clone PT3335 inoculadas com D15, D16, D95, D184, D198, D206 e D216, sendo de 27 % a 32 % em rela??o ?s mudas do Comercial. Os isolados D63 e D216 aumentaram a massa seca (MS) da parte a?rea dos dois clones em rela??o ao Comercial, sendo os maiores aumentos de 140 % nas mudas inoculadas com o D216 e de 87,5 % naquelas inoculadas com o D63. As mudas do PT3336 inoculadas com o D216 apresentaram maiores MS total (31,6 %) e teores de clorofila (39,9 %) em rela??o ?s mudas do Comercial. Os isolados que mais colonizaram as ra?zes foram D5 (19,5 %), D10 (11,7 %), D216 (10,5 %) e D63 (8,8 %) para o PT3335 e D118 (15,6 %), D206 (11,7 %), D216 (11,1 %) e D63 (10,0 %) para o PT3336. Os teores de Fe nas mudas do PT3336 inoculadas com D5, D10, D58, D85, D106, D118, D170, D184 e D216 foram de 21 a 79,3 % maiores do que os das mudas do Comercial e de 38,6 % a 110 % maior que os das do Controle. A coloniza??o se correlacionou positivamente com a MS da parte a?rea, das ra?zes e total para o PT3335 e com a sobreviv?ncia e teores de P para o PT3336. A inocula??o com isolados de Pisolithus sp. aumenta a coloniza??o ectomicorr?zica e o crescimento de mudas eucalipto em viveiro comercial, mas isto ? dependente do clone e do isolado. Os isolados D63 e D216 s?o os mais promissores para utiliza??o em programas de inocula??o em viveiro comercial de mudas clonais de eucalipto. / Disserta??o (Mestrado) ? Programa de P?s-Gradua??o em Produ??o Vegetal, Universidade Federal dos Vales do Jequitinhonha e Mucuri, 2016. / The benefits of ectomycorrhizal associations are dependent on the host plant, the fungal isolate and environment. The objective of this work was to select Pisolithus sp. isolates that colonize eucalypt cuttings propagated by minicutting and that promote higher survival, growth and nutrition of cuttings on commercial nursery. The PT3335 and PT3336 clones were inoculated with 18 isolates of Pisolithus sp. and grown in substrate with reduced phosphate fertilization, in addition to non inoculated controls with (Control) and without (Commercial) reduction of the substrate phosphate fertilization. The inoculation of ectomycorrhizal fungi increased the survival, rooting, colonization, growth and Fe contents in relation to controls, but these effects were dependent on isolates and clones. For PT3335, some isolates doubled the survival of minicuttings in relation to Control and Commercial, having D29, D62, D63, D118 and D216 as the best ones. For PT3336 the best isolates were D5, D29 and D88, with survival increased to 25 %. The greatest height increases were observed in the PT3335 cuttings inoculated with D15, D16, D95, D184, D198, D206 and D216, which were from 27 to 32 % in relation to the Commercial cuttings. The D63 and D216 isolates increased the dry mass (DM) of the aerial part of both clones in relation to Commercial, which the largest increases were 140 % times in the cuttings inoculated with D216 and 87.5 % in those inoculated with D63. The PT3336 cuttings inoculated with D216 presented higher total DM (31.6 %) and chlorophyll contents (39.9 %) in relation to the Commercial cuttings. The isolates that most colonized the roots were D5 (19.5 %), D10 (11.7 %), D216 (10.5 %) and D63 (8.8 %) for PT3335 and D118 (15.6 %), D206 (11.7 %), D216 (11.1 %) and D63 (10.0 %) for PT3336. The Fe contents in the PT3336 cuttings inoculated with D5, D10, D58, D85, D106, D118, D170, D184 and D216 were from 21 % to 79.3 % higher than those of Commercial cuttings and from 38.6 % to 110 % higher than those of the Control. The colonization was positively correlated with the DM of the aerial part, of the roots and total for the PT3335 and with the survival and P contents for the PT3336. The inoculation with Pisolithus sp. isolates increases ectomycorrhizal colonization and growth of eucalypt cuttings in commercial nursery, but this is dependent on the clone and isolate. The D63 and D216 isolates are the most promising for usage in inoculation programs in commercial nursery of eucalypt cuttings.

Submitted by Nathália Faria da Silva (nathaliafsilva.ufv@gmail.com) on 2017-06-19T11:16:39Z No. of bitstreams: 1 resumo.pdf: 21429 bytes, checksum: 79ea720a40b5dbef2ed9fcd6826438df (MD5) / Made available in DSpace on 2017-06-19T11:16:39Z (GMT). No. of bitstreams: 1 resumo.pdf: 21429 bytes, checksum: 79ea720a40b5dbef2ed9fcd6826438df (MD5) Previous issue date: 2005-06-19 / Conselho Nacional de Desenvolvimento Científico e Tecnológico / O objetivo deste trabalho foi estudar a formação de ectomicorrizas por isolados monocarióticos e dicarióticos de Pisolithus sp.em Eucalyptus grandis, sob condições de casa-de-vegetação, e investigar as relações entre o estabelecimento da associação e o crescimento e a absorção de P, Ca, Mg, K, Cu e Zn pelas plantas. Caracterizou-se, também, a produção de massa seca micelial e a absorção de nutrientes pelos isolados fúngicos cultivados por 30 dias, a 25°C, em 50 mL de solução Melin-Norkrans modificada. Os isolados fúngicos apresentaram grande variação na produção de massa seca micelial e na capacidade de absorção de macro- e micronutrientes. Em geral, os isolados monocarióticos apresentaram maiores índices de eficiência de utilização de nutrientes do que os dicários. Todos os isolados monocarióticos e dicarióticos de Pisolithus sp. testados foram capazes de formar ectomicorrizas típicas quando associados com E. grandis. As ectomicorrizas formadas pelos isolados monocarióticos apresentaram manto ao redor das raízes laterais e rede de Hartig limitada aos espaços intercelulares da epiderme radicular, evidenciando a compatibilidade entre os monocários e a planta hospedeira. A espessura do manto fúngico variou de 24 a 30 μm para ambos os tipos de isolados. A presença dos isolados fúngicos monocarióticos associados às raízes laterais de E. grandis resultou em aumento do diâmetro radial das células da epiderme radicular, característico das ectomicorrizas, indicando que os monocários são capazes de produzir os mesmos reguladores de crescimento que os dicários. O comprimento radial das células da epiderme radicular nas ectomicorrizas variou de 26 a 38 μm e a largura de 11 a 17 μm. As médias de percentagem de colonização radicular para os isolados monocarióticos e dicarióticos foram semelhantes (p<0,05), exceto para o isolado M11. A inoculação de E. grandis com os monocários e dicários de Pisolithus sp. resultou em aumentos na massa seca, altura e absorção de nutrientes das plantas. A absorção de Ca, Mg e K foram estimuladas de forma expressiva pela presença das ectomicorrizas, com aumentos de até 760 vezes, sugerindo que a associação tem papel essencial no suprimento desses macronutrientes, especialmente o Ca, para o eucalipto, no campo. Os isolados dicarióticos foram, em geral, mais eficientes em promover a nutrição das plantas de E. grandis do que os monocários. Alguns isolados monocarióticos destacaram-se entre os demais, sendo tão eficientes quanto os dicários. Observaram-se correlações negativas entre os índices de eficiência de utilização de nutrientes pelo micélio fúngico e os conteúdos de nutrientes na parte aérea de E. grandis, concluindo-se que os isolados a serem selecionados para um programa de micorrização controlada deverão ser aqueles capazes de acumular elevado conteúdo de nutrientes no micélio, mas com baixa imobilização desses elementos na biomassa fúngica. A caracterização dos monocários de Pisolithus sp. permitirá a seleção e o cruzamento dos isolados com características desejáveis visando ao melhoramento genético fúngico e à maior eficiência da associação simbiótica. Este trabalho constitui o primeiro relato da formação de ectomicorrizas por isolados monocarióticos de Pisolithus sp. em Eucalyptus grandis e das interações nutricionais dos monocários com a planta hospedeira. / The objective of this work was to study the formation of ectomycorrhizas by monokaryotic and dikaryotic isolates of Pisolithus sp. in Eucalyptus grandis, under greenhouse conditions, and to investigate the relationships between the establishment of the ectomycorrhizal association and the growth and nutrient uptake of the host plant. Dry mycelial mass production and nutrient uptake by the fungal isolates grown for 30 days, at 25°C, in 50 mL of modified Melin- Norkrans solution were also characterized. The fungal isolates showed wide variation in dry mycelial mass production and in their ability to take up macro- and micronutrients. Generally, monokaryons showed higher indices of nutrient utilization efficiency than the dikaryons. All the monokaryotic and dikaryotic isolates tested were capable of forming typical ectomycorrhizas when associated with E. grandis. The ectomycorrhizas formed by the monokaryotic isolates presented a mantle of fungal hyphae around lateral roots and a Hartig net limited to the intercellular spaces of the root epidermis, evidencing the compatibility between the monokaryons and the host plant. Mantle thickness varied from 24 to 30 μm for both types of isolates. The presence of monokaryotic isolates associated with the lateral roots of E. grandis resulted in the typical increases in the radial diameters of the root epidermal cells, characteristic of ectomycorrhizas, indicating that monokaryons are capable of producing the same array of growth regulator compounds as the dikaryons. The radial length of the root epidermal cells in the ectomycorrhizas varied from 26 to 38 μm and cell width from 11 to 17 μm. The means for root colonization percentages for the monokaryotic and dikaryotic isolates were similar (p<0,05). xiThe inoculation of E. grandis with the monokaryons and dikaryons of Pisolithus sp. resulted in increases in shoot dry weight, height and nutrient content. Ca, K, and Mg uptake was highly stimulated by the presence of ectomycorrhizas, with increases of until 760 time, suggesting that the association must have a significant role in supplying these nutrients, especially Ca, to the host in the field. Generally, the dikaryotic isolates were more efficient at promoting plant nutrition than the monokaryons, leading to higher nutrient contents in the shoots. However, some monokaryotic isolates stood out, being as efficient as the dikaryons. Negative correlations were observed between the indices of nutrient utilization efficiency for the fungal mycelium and the nutrient content of the host plant shoots, indicating that the isolates to be selected for a controlled mycorrhization program should accumulate high nutrient contents in the mycelium and have reduced ability to immobilize these nutrients in the fungal biomass. The characterization of Pisolithus sp. monokaryons will allow the selection and the crossing of isolates with desirable traits aiming at the genetic improvement of fungal strains and a higher efficiency of the ectomycorrhizal symbiotic association. This is the first report on the formation of ectomycorrhizas by monokaryotic isolates of Pisolithus sp. in E. grandis and on the nutritional interactions of monokaryons with the host plant.

Thesis (M.S.)--Ohio University, June, 2007. / Title from PDF t.p. Includes bibliographical references.

?rea de concentra??o: Produ??o vegetal. / Submitted by Jos? Henrique Henrique (jose.neves@ufvjm.edu.br) on 2017-01-03T16:10:54Z No. of bitstreams: 2 license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) debora_cintia_santos_avelar.pdf: 1075322 bytes, checksum: 956aee85f03869781bc0c12ae661e4f7 (MD5) / Approved for entry into archive by Rodrigo Martins Cruz (rodrigo.cruz@ufvjm.edu.br) on 2017-01-31T16:25:33Z (GMT) No. of bitstreams: 2 license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) debora_cintia_santos_avelar.pdf: 1075322 bytes, checksum: 956aee85f03869781bc0c12ae661e4f7 (MD5) / Made available in DSpace on 2017-01-31T16:25:33Z (GMT). No. of bitstreams: 2 license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) debora_cintia_santos_avelar.pdf: 1075322 bytes, checksum: 956aee85f03869781bc0c12ae661e4f7 (MD5) Previous issue date: 2016 / Coordena??o de Aperfei?oamento de Pessoal de N?vel Superior (CAPES) / A determina??o da dose de inoculante ? essencial para a obten??o de benef?cios da utiliza??o de fungos ectomicorr?zicos (FEM) em mudas de eucalipto. O objetivo deste estudo foi avaliar a melhor dose de inoculante miceliano de isolados de Pisolithus sp., encapsulados em gel de alginato de c?lcio, visando a promo??o da coloniza??o, nutri??o e do crescimento das mudas de clones de eucalipto em viveiro comercial. Em experimentos independentes e utilizando aduba??o fosfatada reduzida, foram produzidas mudas de dois clones de eucalipto (AEC 2034 e AEC 2233) inoculados com 9, 18 e 36 esferas de inoculante miceliano dos isolados f?ngicos D17, D216, D5 e D95 e um tratamento N?o-inoculado (fatorial 3x5). Al?m disso, foram feitos mais dois tratamentos adicionais com (Controle) e sem (Comercial) redu??o da aduba??o fosfatada do substrato e sem adi??o dos inoculantes. A dose de 36 esferas de inoculante, em geral, promoveu maiores ganhos para as mudas de eucalipto, como maior coloniza??o, massa seca da parte a?rea (MSPA) e de ra?zes (MSR) e teores de K, sendo algumas vezes maior que nas mudas do Controle e Comercial. A maior altura da parte a?rea e di?metro do coleto foram observados com o uso de 18 esferas. A coloniza??o ectomicorr?zica foi maior nas mudas inoculadas e considerada m?dia para os dois clones. A inocula??o pr?via nos dois clones n?o promoveu aumento nos teores de P em rela??o ao Comercial, no entanto para o clone AEC 2233 com a utiliza??o do D216, houve aumento nos teores de P em rela??o ao N?o-inoculado, sendo este isolado o mesmo que proporcionou uma das maiores porcentagens de coloniza??o, contribuindo para maior absor??o desse nutriente. Em rela??o aos isolados, a inocula??o com o D17 em geral aumentou a sobreviv?ncia, altura, MSPA, MSR e teores de K no AEC 2034 e a altura e di?metro no AEC 2233, mas isto foi dependente da dose e do clone de eucalipto. A dose de 18 esferas foi mais apropriada, pois, na maioria dos casos, os maiores benef?cios como sobreviv?ncia, altura, di?metro, MSPA, MSR, coloniza??o, teores de N e K foram obtidos com a adi??o de 18 ou mais esferas por mini-estaca e, em alguns casos, os par?metros altura e coloniza??o foram reduzidos nas maiores doses. O isolado D17 ? promissor para uso em programas de micorriza??o controlada em viveiros comerciais de mudas clonais de eucalipto e a dose de 18 esferas por mini-estaca ? a mais recomendada. / Disserta??o (Mestrado) ? Programa de P?s-Gradua??o em Produ??o Vegetal, Universidade Federal dos Vales do Jequitinhonha e Mucuri, 2016. / The determination of the inoculum dose is essential for obtaining the benefits from the utilization of ectomycorrhizal fungi (EMF) in eucalypt saplings. The aim of this study was to evaluate the best dose of inoculum consists of mycelium from isolates Pisolithus sp., encapsulated in calcium alginate gel, aiming at promoting the colonization, nutrition and growth of the eucalypt rooted cuttings in commercial nursery. In independent experiments and using reduced phosphorus fertilization, were produced rooted cuttings from two eucalypts clones (AEC 2034 e AEC 2233) inoculated with 9, 8 and 36 beads of inoculum from fungal isolates D17, D216, D5 e D95 and one Non-inoculated treatment (factorial 3x5). Furthermore, two more additional treatments were performed with (Control) and without (Commercial) reduction of phosphorus fertilization of the substrate and without the addition of the inoculum.The dose of 36 inoculum beads, in general, has promoted higher benefits for the saplings, such as greater colonization, dry mass of shoot (DMS) and of roots (DMR) and K concentrations, sometimes being higher than in the saplings from Control and Commercial. The greater height of the shoot and collar diameter were observed with the use of 18 beads of inoculum. The ectomycorrhizal colonization was higher in inoculated saplings and considered average for the two clones. The previous inoculation in both clones did not promote an increase in P concentrations in relation to Commercial, however for the AEC 2233 clone with the utilization of D216, there was an increase in the P concentrations in relation to Non-inoculated, this being the same isolate that provided one of the greatest percentages of colonization, contributing to a higher absorption of this nutrient. Regarding the isolates, the inoculation with D17 in general increased the survival, height, DMS, DMR and K concentrations in AEC 2034, and height and diameter in AEC 2233, but this was dependent on the dose and on the eucalypt clone. The dose of 18 beads of inoculum was most appropriate, because in most cases, the greatest benefits as survival, height, diameter, DMS, DMR, colonization, N and K concentrations were obtained with addition of 18 or more beads of inoculum by mini-cutting and, in some cases, the parameters height and colonization were reduced with the highest doses. The isolate D17 is promising for its use in controlled mycorrization programs in commercial nurseries of eucalypt rooted cuttings and the dose of 18 beads by mini-cutting is the most recommended.