The specificity of fungal isolates from endemic Western Australian orchid species and hybrids in the tribe Diurideae was investigated using symbiotic seed germination and analysis of the fungal DNA by amplified fragment length polymorphism (AFLP). The distribution of the fungal isolates in the field was also assessed using two different seed baiting techniques. The information from these investigations is essential for developing protocols for reintroduction and translocation of orchid species. Two groups of orchids in the tribe Diurideae were studied. Firstly, a number of Caladenia species, their natural hybrids and close relatives from the southwest of Western Australia were selected because orchid species from the genus Caladenia are considered to have among the most specific mycorrhizal relationships known in the orchid family – an ideal situation for the investigation of mycorrhizal specificity. Secondly, species of Drakaea and close relatives, from the southwest of Western Australia and elsewhere in Australia, which are never common in nature and occur in highly specialised habitats, were selected to investigate the influence of habitat on specificity. Seed from the common species Caladenia arenicola germinated on fungal isolates from adult plants of both C. arenicola and its rare and endangered relative C. huegelii, while seed from C. huegelii only germinated on its own fungal isolates. The AFLP analysis grouped the fungal isolates into three categories: nonefficaceous fungi, C. huegelii type fungi, and C. arenicola type fungi. The group of C. huegelii type fungi included some fungal isolates from C. arenicola. An analysis of the AFLP fingerprints of C. arenicola fungal isolates from different collection locations showed that some, but not all, populations were genetically distinct, and that one population in particular was very variable. Despite being thought to have very specific mycorrhizal relationships, Caladenia species hybridise frequently and prolifically in nature, often forming self-perpetuating hybrid lineages. Five natural hybrids within Caladenia and its closest relatives were investigated. Symbiotic cross-germination studies of parental and hybrid seed on fungi from the species and the naturally occurring hybrids were compared with AFLP analyses of the fungal isolates to answer the question of which fungi the hybrids use. The germination study found that, while hybrid seeds can utilise the fungi from either parental species under laboratory conditions, it is likely that the natural hybrids in situ utilise the fungus of only one parental species. Supporting these observations, the AFLP analyses indicated that while the parental species always possessed genetically distinct fungal strains, the hybrids may share the mycorrhizal fungus of one parental species or possess a genetically distinct fungal strain which is more closely related to the fungus of one parental species than the other. The work on Caladenia hybrids revealed that C. falcata has a broadly compatible fungus that germinated seeds of C. falcata, the hybrid C. falcata x longicauda, and species with different degrees of taxonomic affinity to C. falcata. In general, germination was greater from species that were more closely related to C. falcata: seeds from Caladenia species generally germinated well on most C. falcata isolates; species from same subtribe (Caladeniinae) germinated well to the stage of trichome development on only some of the fungal isolates and rarely developed further; and seeds from species from different subtribes (Diuridinae, Prasophyllinae, Thelymitrinae) or tribes (Orchideae, Cranichideae) either germinated well to the stage of trichome development but did not develop further, or did not germinate at all. The AFLP analysis of the fungal isolates revealed that the fungi from each location were genetically distinct. In situ seed baiting was used to study the introduction, growth and persistence of orchid mycorrhizal fungi. A mycorrhizal fungus from Caladenia arenicola was introduced to sites within an area from which the orchid and fungus were absent, adjacent to a natural population of C. arenicola. In the first growing season, the fungus grew up to 50 cm from its introduction point, usually persisted over the summer drought into the second season and even into the third season, stimulating germination and growth to tuber formation of the seeds in the baits. Watering the inoculated areas significantly increased seed germination. Mycorrhizal relationships in Drakaeinae were less specific than in Caladeniinae. A study of the species Spiculaea ciliata revealed that this species, when germinated symbiotically, develops very rapidly and has photosynthetic protocorms, unlike all other members of the Drakaeinae. An AFLP analysis of the fungal isolates of this species grouped the isolates according to whether they had been isolated from adult plants or reisolated from protocorms produced in vitro. Isolates were genetically distinct when compared before germination and after reisolation. A cross-species symbiotic germination study of seeds of three Drakaea species and one Paracaleana species against fungal isolates from the same species and several other Drakaeinae species revealed lower specificity in this group than previously thought. A number of fungal isolates from Drakaea and Paracaleana species germinated two or more seed types, while all seed types germinated on fungal isolates from other species and the seed of Drakaea thynniphila germinated to some extent on every fungal isolate tested. An AFLP analysis of the Drakaeinae fungal isolates supported this information, revealing little genetic differentiation between the fungi of different orchid species. An ex situ seed baiting technique was used to examine the role of mycorrhizal fungi in microniche specialisation in the narrow endemic Drakaea. Soil samples from within and outside two Drakaea populations were tested for germination of the relevant seed types. In both cases, germination was significantly higher on soil samples from within than outside the populations, suggesting that the relevant mycorrhizal fungi may be restricted to the same microniches as the Drakaea species. The presence of similar fungi at distant, disjunct locations may be related to the extreme age and geological stabilityof the Western Australian landscape. The information from these investigations is essential for developing protocols for reintroduction and translocation of orchid species. It appears that the mycorrhizal relationships in these groups of orchids are not as specific as was previously thought. For reintroduction work, a broad sampling strategy is necessary, as it cannot be assumed that the same orchid species has the same fungus at different locations. A broadly compatible fungus may be of considerable utility in conservation work, such as in situations where a specific fungus appears to have poor saprophytic competence or where soil conditions have been altered. Seed baiting studies provide additional data on fungal distribution in situ. In general, molecular data do not provide information about efficacy or fungal distribution, so research programs that combine symbiotic germination studies with seed baiting investigations and genetic analyses of the fungi will provide the maximum benefit for designing more effective conservation programs.

Submitted by Luciana Ferreira (lucgeral@gmail.com) on 2018-11-12T14:09:32Z No. of bitstreams: 2 Dissertação - Kellen Cristhina Inácio Sousa - 2012.pdf: 5012340 bytes, checksum: 5d359d0996e1d7af5f7a16ae553d8b85 (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) / Approved for entry into archive by Luciana Ferreira (lucgeral@gmail.com) on 2018-11-12T14:26:47Z (GMT) No. of bitstreams: 2 Dissertação - Kellen Cristhina Inácio Sousa - 2012.pdf: 5012340 bytes, checksum: 5d359d0996e1d7af5f7a16ae553d8b85 (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) / Made available in DSpace on 2018-11-12T14:26:47Z (GMT). No. of bitstreams: 2 Dissertação - Kellen Cristhina Inácio Sousa - 2012.pdf: 5012340 bytes, checksum: 5d359d0996e1d7af5f7a16ae553d8b85 (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) Previous issue date: 2012-06-28 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - CAPES / In Brazil, 2500 to 3000 species of orchids have been reported to occur, of which 300 are found in the “cerrado” region. The orchids Cyrtopodium saintlegerianum (epiphyte) and Epidendrum nocturnum (rupicola) occur in areas anthropized of cerrado and cerrado rupestre, respectively. The orchids posses mycorrhizal association with rhizoctonia-like and endophytic fungi. The identification of these fungi can be done by microscopic and morphological characters. Root infecting fungi also are utilized for in vitro symbiotic germination of orchid seeds, aiming the conservation of plant species and fungi. The objective of the present investigation was isolation and morphological characterization of mycorrhizal and endophytic fungi originating from roots of C. saintlegerianum and E. nocturnum, as well as symbiotic in vitro seed germination. Three isolates of Epulorhiza sp. from each one of C. saintlegerianum, and E. nocturnum and two of Rhizoctonia sp. from E. nocturnum were obtained. Also, two isolates of Xylaria sp. were obtained from roots of C. saintlegerianum. There were, however, differences among isolates Epulorhiza sp. of C. saintlegerianum and of the E. nocturnum, in relation to morphological and enzymatic characters. The fungus was localized in root tissues of both species by optical and scanning electronic microscopes. The seed viability was tested by tetrazolium chloride and found 80.3 and 32.33% viable embryos of C. saintlegerianum and E. nocturnum, respectively. There was no in vitro symbiotic germination of E. nocturnum due to low seed viability. Two experiments of symbiotic germination of C. saintlegerianum seeds were conducted, both under photoperiods of 16/8 h (light/dark) at 26°C ± 2°C. The treatments were two isolates of Xylaria sp., three plant pathogens (Rhizoctonia solani of common beans and R. oryzae of rice ), and three mycorrhiza of C. saintlegerianum, two of Cyrtopodium vernum, and one ofE. nocturnum. Of three culture media tested, germination was obtained only in oat meal agar culture. The isolate En07 of Rhizoctonia sp. from E. nocturnum was found better for seed germination with 81.64 and 90.73% of germination of experiments 1 and 2, respectively. One non-specific isolate, two plant pathogenic isolates and one specific isolate were efficient for seed germination of C. saintlegerianum. On the other hand, for symbiotic in vitro seed germination of C. saintlegerianum, there was no specificity between this orchid and only one rhizoctonia-like fungus. These results showed that C. saintlegerianum can be propagated utilizing different root infecting fungi, which facilitates future programs of reintroduction and commercialization of species. / O Brasil possui entre 2500 e 3000 espécies de orquídeas, das quais cerca de 300 são encontradas em áreas de Cerrado. Cyrtopodium saintlegerianum (epífita) e Epidendrum nocturnum (rupícola) ocorrem em áreas antropizadas de Cerrado e Cerrado Rupestre, respectivamente. As orquídeas possuem associação micorrízica com fungos rizoctonioides, e fungos endofíticos também podem ser encontrados nessas plantas. Os fungos rizoctonioides são utilizados para a germinação simbiótica in vitro de sementes de orquídeas, tendo em vista a conservação das espécies vegetais e fúngicas. O presente trabalho objetivou o isolamento e a caracterização morfológica de fungos oriundos do sistema radicular de C. saintlegerianum e E. nocturnum, visando a germinação simbiótica in vitro de suas sementes. Foram obtidos três isolados de Epulorhiza sp. para C. saintlegerianum, e para E. nocturnum obteve-se três de Epulorhiza sp. e dois de Rhizoctonia sp. Das raízes de C. saintlegerianum também foram obtidos dois isolados de Xylaria sp. Houve diferença entre os isolados de Epulorhiza sp. oriundos de C. saintlegerianum e os originários de E. nocturnum quanto as características morfológicas e enzimáticas. Os fungos micorrízicos foram histolocalizados, por microscopia óptica e eletrônica de varredura, em raízes de ambas as espécies. A viabilidade de sementes foi realizada pelo teste do cloreto de tetrazólio e verificou-se 80,3 e 32,33% de embriões viáveis para C. saintlegerianum e para E. nocturnum, respectivamente. Não houve germinação simbiótica in vitro de E. nocturnum devido a baixa viabilidade das sementes. Para as sementes de C. saintlegerianum foram realizados dois experimentos de germinação simbiótica, ambos em fotoperíodo de 16/8 h (Luz/Escuro) a 26°C ± 2°C. Os tratamentos foram dois isolados de Xylaria sp., três fitopatogênicos (Rhizoctonia solani de feijão e R. oryzae de arroz), e três micorrízicos de C. saintlegerianum, dois de Cyrtopodium vernum Rchb.f. & Warm., e um de E. nocturnum. Foram testados três meios de cultivo, mas somente o meio agar aveia padrão proporcionou a germinação. Verificou-se que o isolado En07 de Rhizoctonia sp. oriundo de E. nocturnum foi o melhor para germinar as sementes com 81,64 e 90,73% de germinação nos experimentos 1 e 2, respectivamente. Observou-se que um isolado não específico, dois isolados fitopatogênicos e um específico foram eficientes em germinar as sementes de C. saintlegerianum. Portanto, para a germinação simbiótica in vitro de C. saintlegerianum não houve especificidade entre esta orquídea e um único fungo rizoctonioide. Estes resultados indicam que C. saintlegerianum pode ser propagada utilizando fungos rizoctonioides, o que facilita futuros programas de reintrodução e comercialização da espécie.

Na natureza, as orquídeas são totalmente dependentes de fungos micorrízicos para germinar. Estes fungos podem penetrar nas células das raízes e formar pélotons, os quais, quando digeridos pela planta, providenciam açúcares simples para o embrião. Durante a fase aclorofilada de desenvolvimento da plântula, orquídeas são obrigatoriamente dependentes dos fungos; algumas continuam assim por toda vida enquanto outras se tornam facultativamente responsivas à colonização. O objetivo deste trabalho foi identificar quantos clados de fungos podem estabelecer associação micorrízica com Coppensia doniana (sin. Oncidium donianum), uma orquídea amplamente distribuída nos arredores de Campos do Jordão e, demonstrar como as características morfológicas dos isolados, quando analisadas com ferramentas de estatística multivariada, podem ser úteis para a taxonomia destes fungos. Dez plantas foram amostradas em um sítio com vegetação típica de campos de altitude, junto ao Parque Estadual de Campos do Jordão. Fungos foram isolados pela transferência asséptica de cortes de raízes contendo pélotons para meios de cultura BDA modificados. Três clados de fungos foram formados, tanto analisando as características qualitativas das culturas quanto as quantitativas. Os clados foram identificados como dois morfotipos do gênero Ceratorhiza (fase anamórfica de Ceratobasidium) e uma Rhizoctonia-uninucleada. O sequenciamento da região ITS produziu resultados idênticos a estes, mostrando os mesmos três clados. Todas as sequências tiveram alta correlação com sequências de Ceratobasidium depositadas no Genbank, o que sugere uma alta afinidade de Coppensia doniana com este gênero. Também ficou demonstrado que os dados morfológicos, quando associados à estatística multivariada são uma ferramenta útil na taxonomia polifásica de Rhizoctonia spp. As sequências dos isolados de Coppensia doniana também foram comparadas com as de isolados de outras orquídeas, dentro da subtribo Oncidiinae, incluindo: Ionopsis utricularioides e Psygmorchis pussila, coletadas na região do Valle del Cauca Colômbia e isolados de 10 Ionopsis utricularioides, Oncidium altissimum e Tolumnia variegata, estudados por Otero (2002, 2004, 2007), em diferentes regiões de Porto Rico, Costa Rica, Cuba e Panamá. Esta última análise veio a comprovar a preferência de orquídeas da subtribo Oncidiinae por fungos do gênero Ceratobasidium, apesar de que os clados obtidos no Brasil e na Colômbia foram distantes filogeneticamente dos clados previamente estudados na América Central. Representantes dos três clados obtidos de C. doniana em Campos do Jordão foram também testados quanto à capacidade de induzir germinação em suas sementes. Todos isolados testados tiveram sucesso na germinação das sementes, levando as plântulas a estádios avançados de desenvolvimento após 30 dias, o que indica um alto potencial para utilização biotecnológica destes isolados para a germinação das sementes destas orquídeas, tanto para a floricultura comercial quanto para programas de reintrodução de espécies de orquídeas ameaçadas de extinção. / In nature, orchids are fully dependent on mycorrhizal fungi for germination. These fungi can penetrate root cells and form pelotons, whose digestion provides simple sugars for the embryo. During the achlorophyllous seedling stage, orchids are obligatory dependent on the fungi, and some species remain so through life, while others become facultatively responsive to fungal infection. The aim of this study was to identify how many fungal clades can establish mycorrhizal associations with Coppensia doniana, a widespread orchid from Campos do JordãoBrazil, and to demonstrate how their morphological features, analyzed with multivariate statistics, can be useful for classification. Ten plants were sampled in an Araucaria forest near Campos do Jordão. Fungi were isolated by transferring surface disinfected root segments containing pelotons to PDA culture medium. Three main fungal clades were formed by qualitative and quantitative morphological data. They were identified as two morphotypes of Ceratorhiza (anamorphic stage of Ceratobasidium) and one uninucleated Rhizoctonia. The ITS sequencing corroborates this identification, since the same three clades were found. All sequences were highly correlated to Ceratobasidium ITS data deposited at the Genebank, suggesting a high affinity between this species of Oncidiinae and Ceratobasidium. It also could be shown that morphological data associated with multivariate statistics can be a useful tool in fungal multi-level taxonomy. C. doniana sequences were also compared to sequences obtained from isolates of other orchids, belonging to the sub-tribe Oncidiinae, including: Ionopsis utricuarioides and Psygmorchis pussila, collected in Valle del Cauca Colombia and isolated from I. utricularioides, Oncidium altissimum and Tolumnia variegata, studied by Otero (2002, 2004, 2007) in different regions of Puerto Rico, Costa Rica and other Caribbean islands. This last analysis confirmed the preference of this Oncidiinae sub-tribe for Ceratobasidium, although isolates obtained in Brazil or Colombia belong to different clades from those previously studied in Puerto Rico, Costa Rica, Panama and Cuba. 12 Fungi representing the three clades obtained from C. doniana in Campos do Jordão were also tested for their ability to induce germination of C. doniana seeds, with a positive response for all of them, being able to bring the seedlings to advanced development stages in 30 days. These results suggest a high biotechnological potential of these isolates, to be used in orchid symbiotic germination for commercial flower production or for the reintroduction of endangered Brazilian orchid species.

Na natureza, as orquídeas são totalmente dependentes de fungos micorrízicos para germinar. Estes fungos podem penetrar nas células das raízes e formar pélotons, os quais, quando digeridos pela planta, providenciam açúcares simples para o embrião. Durante a fase aclorofilada de desenvolvimento da plântula, orquídeas são obrigatoriamente dependentes dos fungos; algumas continuam assim por toda vida enquanto outras se tornam facultativamente responsivas à colonização. O objetivo deste trabalho foi identificar quantos clados de fungos podem estabelecer associação micorrízica com Coppensia doniana (sin. Oncidium donianum), uma orquídea amplamente distribuída nos arredores de Campos do Jordão e, demonstrar como as características morfológicas dos isolados, quando analisadas com ferramentas de estatística multivariada, podem ser úteis para a taxonomia destes fungos. Dez plantas foram amostradas em um sítio com vegetação típica de campos de altitude, junto ao Parque Estadual de Campos do Jordão. Fungos foram isolados pela transferência asséptica de cortes de raízes contendo pélotons para meios de cultura BDA modificados. Três clados de fungos foram formados, tanto analisando as características qualitativas das culturas quanto as quantitativas. Os clados foram identificados como dois morfotipos do gênero Ceratorhiza (fase anamórfica de Ceratobasidium) e uma Rhizoctonia-uninucleada. O sequenciamento da região ITS produziu resultados idênticos a estes, mostrando os mesmos três clados. Todas as sequências tiveram alta correlação com sequências de Ceratobasidium depositadas no Genbank, o que sugere uma alta afinidade de Coppensia doniana com este gênero. Também ficou demonstrado que os dados morfológicos, quando associados à estatística multivariada são uma ferramenta útil na taxonomia polifásica de Rhizoctonia spp. As sequências dos isolados de Coppensia doniana também foram comparadas com as de isolados de outras orquídeas, dentro da subtribo Oncidiinae, incluindo: Ionopsis utricularioides e Psygmorchis pussila, coletadas na região do Valle del Cauca Colômbia e isolados de 10 Ionopsis utricularioides, Oncidium altissimum e Tolumnia variegata, estudados por Otero (2002, 2004, 2007), em diferentes regiões de Porto Rico, Costa Rica, Cuba e Panamá. Esta última análise veio a comprovar a preferência de orquídeas da subtribo Oncidiinae por fungos do gênero Ceratobasidium, apesar de que os clados obtidos no Brasil e na Colômbia foram distantes filogeneticamente dos clados previamente estudados na América Central. Representantes dos três clados obtidos de C. doniana em Campos do Jordão foram também testados quanto à capacidade de induzir germinação em suas sementes. Todos isolados testados tiveram sucesso na germinação das sementes, levando as plântulas a estádios avançados de desenvolvimento após 30 dias, o que indica um alto potencial para utilização biotecnológica destes isolados para a germinação das sementes destas orquídeas, tanto para a floricultura comercial quanto para programas de reintrodução de espécies de orquídeas ameaçadas de extinção. / In nature, orchids are fully dependent on mycorrhizal fungi for germination. These fungi can penetrate root cells and form pelotons, whose digestion provides simple sugars for the embryo. During the achlorophyllous seedling stage, orchids are obligatory dependent on the fungi, and some species remain so through life, while others become facultatively responsive to fungal infection. The aim of this study was to identify how many fungal clades can establish mycorrhizal associations with Coppensia doniana, a widespread orchid from Campos do JordãoBrazil, and to demonstrate how their morphological features, analyzed with multivariate statistics, can be useful for classification. Ten plants were sampled in an Araucaria forest near Campos do Jordão. Fungi were isolated by transferring surface disinfected root segments containing pelotons to PDA culture medium. Three main fungal clades were formed by qualitative and quantitative morphological data. They were identified as two morphotypes of Ceratorhiza (anamorphic stage of Ceratobasidium) and one uninucleated Rhizoctonia. The ITS sequencing corroborates this identification, since the same three clades were found. All sequences were highly correlated to Ceratobasidium ITS data deposited at the Genebank, suggesting a high affinity between this species of Oncidiinae and Ceratobasidium. It also could be shown that morphological data associated with multivariate statistics can be a useful tool in fungal multi-level taxonomy. C. doniana sequences were also compared to sequences obtained from isolates of other orchids, belonging to the sub-tribe Oncidiinae, including: Ionopsis utricuarioides and Psygmorchis pussila, collected in Valle del Cauca Colombia and isolated from I. utricularioides, Oncidium altissimum and Tolumnia variegata, studied by Otero (2002, 2004, 2007) in different regions of Puerto Rico, Costa Rica and other Caribbean islands. This last analysis confirmed the preference of this Oncidiinae sub-tribe for Ceratobasidium, although isolates obtained in Brazil or Colombia belong to different clades from those previously studied in Puerto Rico, Costa Rica, Panama and Cuba. 12 Fungi representing the three clades obtained from C. doniana in Campos do Jordão were also tested for their ability to induce germination of C. doniana seeds, with a positive response for all of them, being able to bring the seedlings to advanced development stages in 30 days. These results suggest a high biotechnological potential of these isolates, to be used in orchid symbiotic germination for commercial flower production or for the reintroduction of endangered Brazilian orchid species.

There are generally two contrasting alternatives to what limits recruitment in plants, namely the availability of seeds (seed limitation) or the quality or quantity of suitable sites (microsite limitation). Dust seeds, the smallest existing seeds, lack or have minimal nutrient reserves. During germination and initial development they consequently parasitize on mycorrhizal fungi. This is called mycoheterotrophy, and can vary in degree of fungal dependency in adult plants from full, partial or initial mycoheterotrophy. The aim of this thesis was to investigate the recruitment ecology of mycoheterotrophic Ericaceae (tribe Pyroleae) species with dust seeds, and to determine what limits their recruitment. The investigated species were: Chimaphila umbellata, Moneses uniflora, Orthilia secunda, Pyrola chlorantha, P. minor and P. rotundifolia. This aim was achieved by combining field experiments (seed sowing) with isotope analysis and fungal host pyrosequencing. Results provide evidence that the species in Pyroleae are heterogeneous, not only with regard to their degree of mycoheterotrophy, but also concerning germination and early seedling development. A combination of microsite and seed limitation is thus likely to be of importance for all studied species, but the relative importance of these limitations varies among species. Despite having adaptations for wind dispersal the majority of the seeds were deposited in close vicinity of the seed source. But with high seed production at least some seeds should be able to disperse long-distance. Seedlings of all studied species were found to associate with a wide range of ectomycorrhizal fungi, at least during their initial developmental stages. There seems to be a tendency for host narrowing in some Pyroleae species, but not as strict as the host specialization seen in fully mycoheterotrophic Monotropa hypopitys, supporting the hypothesis of geographical and developmental host shifts. / <p>At the time of the doctoral defense, the following papers were unpublished and had a status as follows: Paper 3: In press. Paper 4: Manuscript.</p>

Submitted by Michele Mologni (mologni@unoeste.br) on 2018-07-27T17:21:00Z No. of bitstreams: 1 Joice Yuri Minamiguchi.pdf: 1305334 bytes, checksum: ce408a4b1cb0c85010e11bc39b0761d9 (MD5) / Made available in DSpace on 2018-07-27T17:21:00Z (GMT). No. of bitstreams: 1 Joice Yuri Minamiguchi.pdf: 1305334 bytes, checksum: ce408a4b1cb0c85010e11bc39b0761d9 (MD5) Previous issue date: 2017-12-19 / Orchid seeds do not have practically reserve of energy and are not able to absorb what exists, necessitating a symbiosis with mycorrhizal fungi for germination to occur. During the aclorophyll stage of embryo development, these are the dependent on the mycorrhizal association, because fungi provide simple sugars to the plant from digested pelotons. The objective of this work was to isolate mycorrhizal fungi of orchid in cultivated and wild plants, that could promote the germination and development of seedlings of different species. Roots of nine species of orchids were collected for the isolation by peloton, four of natural environment, three from in vitro culture that developed and grew in greenhouse orchid and two who were born spontaneously in the greenhouse orchid. This methodology promoted the isolation of nine anamorphic rhizoctonioid from two species from a natural environment, Prescotia sp and Oeceoclades maculata and of a spontaneous species in greenhouse orchid, Arundina bambusifolia. In pots of three species of orchids, Vanda sp., Cattleya tigrina and Cattleya walkeriana, were placed baiting containing seeds of the same. This methodology obtainedthe isolation of eight anamorphic rhizoctonioid fung from Cattleya tigrina and Cattleya walkeriana. Were made the quantitative and qualitative morphological characterizations of the 17 isolates obtained by the two methodologies, and these characteristics were used for the formation of groups by the grouping method UPGMA. This method together with the morphological characteristics made it possible to classify four genera of mycorrhiza, Epulorhiza, Ceratorhiza, Moniliopsis and Rhizoctonia-like. The isolates were separated into 7 different groups, and one isolate from each group was used for the symbiotic germination of six species of orchids, Cattleya brevicaulis, Cattleya tigrina, Cattleya walkeriana, Cattleya labiata, Dendrobium signatum and Encyclia cordigera. Posteriorly confirmation of the mycorrhizal association was made, of protocorm resulting from symbiotic germination. Ninety days after seeding, seedlings of the six species that germinated were transferred to flasks, and 180 days after seeding were evaluated growth and dry mass variables. The methodology of isolation by peloton promoted the isolation of mycorrhiza from plants of natural environment and from a spontaneous in greenhouse orchid, the baiting methodology was able to promote the isolation of mycorrhiza from plants that were born and developed in a controlled environment. Of the seven inoculum, six were efficient to promote the germination of the six species tested, but only five inoculum presented growth of seedlings. One inoculum is no specific, presenting better results, improving seedling growth in five of the evaluated species. / Sementes de orquídea praticamente não possuem reserva de energia e não são capazes de absorver as que possuem, necessitando de uma simbiose com fungos micorrízicos para que ocorra a germinação. Durante a fase aclorofilada do desenvolvimento do embrião, estes são dependentes da associação micorrízica, pois os fungos fornecem açúcares simples oriundos de pelotons digeridos pela planta. O objetivo deste trabalho foi isolar fungos micorrízicos de orquídeas cultivadas e silvestres, que pudessem promover a germinação e o desenvolvimento de plântulas de diferentes espécies. Raízes de nove espécies de orquídeas foram coletadas para o isolamento por peloton, quatro de ambiente natural, três oriundas de cultivo in vitro aclimatizadas e crescidas em um orquidário e de duas que nasceram espontaneamente no orquidário. Esta metodologia obteve o isolamento de nove fungos rizoctonióides anamorfos de duas espécies oriundas de ambiente natural, Prescotia sp e Oeceoclades maculata e de uma espécie espontânea no orquidário, Arundina bambusifolia. Nos vasos de três espécies Vanda sp., Cattleya tigrina e Cattleya walkeriana, foram colocados pacotes de isca contendo sementes das mesmas. Esta metodologia obteve o isolamento de oito fungos rizoctonióides anamorfos de Cattleya tigrina e Cattleya walkeriana. Foram feitas as caracterizações morfológicas quantitativas e qualitativas dos 17 isolados obtidos pelas duas metodologias, e essas características foram usadas para a formação de grupos pelo método de agrupamento UPGMA. Esse método juntamente com as características morfológicas possibilitou a classificação de quatro gêneros de micorrizas, Epulorhiza, Ceratorhiza, Moniliopsis e Rhizoctonia-like. Os isolados foram separados em sete grupos diferentes, e um isolado de cada grupo foi utilizado para a germinação simbiótica de seis espécies de orquídeas, Cattleya brevicaulis, Cattleya tigrina, Cattleya walkeriana, Cattleya labiata, Dendrobium signatum e Encyclia cordigera. Posteriormente foi feita a confirmação da associação micorrízica, dos protocórmios resultantes da germinação simbiótica. Noventa dias após a semeadura, plântulas das seis espécies que germinaram foram transferidas para frascos, e 180 dias após a semeadura foram avaliadas variáveis de crescimento e de massa seca. A metodologia de isolamento por peloton promoveu o isolamento de micorrizas de plantas de ambiente natural e de uma espontânea em orquidário. A metodologia por isca foi capaz de promover o isolamento de micorrizas de plantas que nasceram e se desenvolveram em ambiente controlado. Dos sete inóculos, seis foram eficientes para promover a germinação de sementes das seis espécies testadas, mas somente cinco inóculos exibiram crescimento de plântulas. Um inóculo demonstrou ser não específico, apresentando melhor resultado melhorando o crescimento de plântulas em cinco das espécies avaliadas.