Resumo Fungos do complexo Rhizoctonia são micorrízicos de orquídeas e podem germinar suas sementes e desenvolver as plântulas. Neste contexto, nosso objetivo foi selecionar um isolado fúngico para promover a germinação e o desenvolvimento de plântulas de Cyrtopodium saintlegerianum. Foram encontrados pelotons nas raízes desta orquídea e foram identificados três fungos micorrízicos. Nós testamos os isolados obtidos das raízes de C. saintlegerianum e seis outros isolados micorrízicos assim como três isolados patogênicos (de arroz e feijão) para germinar as sementes em meio de aveia-agar. Sementes não inoculadas foram usadas como controle. Os isolados En07 (Waitea circinata), Cs10 (Tulasnella sp.) e Ro88 (Rhizoctonia oryzae) foram eficientes para promover a germinação, mas somente o En07diferiu estatisticamente do controle. O isolado não específico En07 promoveu 81% de germinação e o isolado específico (Cs10) promoveu 60%, evidenciando a associação micorrízica não específica durante a germinação. Plântulas axênicas foram inoculadas com quatro fungos micorrízicos (plântulas não inoculadas - controle). Após seis meses, os isolados En07 e Cs10 foram eficientes na interação com as plântulas, mas não diferiram do controle. Portanto, nossos resultados sugerem que fungos do complexo Rhizoctonia podem ser usados na germinação e no desenvolvimento de plântulas de C. saintlegerianum.

Abstract Fungi of Rhizoctonia complex are mycorrhizal of orchids and may to germinate yours seeds and development the seedlings. In this context, our objective was to select a fungal isolate to promote seed germination and seedling development of Cyrtopodium saintlegerianum. Pelotons were found in the roots and three mycorrhizal fungi were isolated. We tested mycorrhizal isolates obtained from C. saintlegerianum roots and six mycorrhizal fungi from other orchids as well three pathogenic isolates (of rice and bean) to germinate the seeds in oatmeal-agar medium. Seeds not inoculated were used as control. The isolates En07 (Waitea circinata), Cs10 (Tulasnella sp.) and Ro88 (Rhizoctonia oryzae) were efficient to promote seed germination, but only En07 differing statistically of the control. The non-specific isolate En07 promoted germination in 81% of seeds and the specific isolate (Cs10) promoted 60%, evidencing the non-specificity mycorrhizal association in this orchid during germination. Axenic seedlings were inoculated with four mycorrhizal fungi (non-inoculated seedlings - control). After six months, the isolates En07 and Cs10 were efficient in the interaction with the seedlings, but did not differ to the control. Therefore, our results suggested that fungi of the Rhizoctonia complex can be used in the germination and seedling development of C. saintlegerianum.

Cyrtopodium glutiniferum is an endemic orchid of Brazil with potential medicinal and ornamental applications. As mycorrhizal fungi are essential for the initiation of the orchid life cycle, the aim of this study was to determine the strains of mycorrhizal fungi suitable for seed germination and protocorm development of C. glutiniferum and to characterize the symbiotic development of protocorms. Seeds of C. glutiniferum were inoculated with nine mycorrhizal fungi, Epulorhiza spp., Ceratorhiza spp., Rhizoctonia sp., originally isolated from Brazilian neotropical orchids. Only Epulorhiza isolates promoted seed germination and protocorm development. Three Epulorhiza isolates (M1, M6 = E. epiphytica, M20 = Epulorhiza sp.) promoted protocorm development until leaf production at 63 days. The protocorms are comprised of parenchyma cells delimited by a unistratified epidermis; the parenchyma cells of the upper part of the protocorms are smaller than those located more towards the base. Intact and digested pelotons were observed inside of protocorms implying that the seedlings were capable of mycotrophy. Additionally, the development of a bud primordium only occurred after colonization by fungus. This study suggests that C. glutiniferum has a preference for strains of Epulorhiza and that fungus digestion is essential to protocorm development.

In Orchidaceae, association with symbiotic fungi is required for seed germination and seedling development, thereby being the main energy source during the first steps of germination. Colombia is one of the countries with the greatest biodiversity of orchids, with an estimated 3,200 species, but few studies on orchid mycorrhiza have been conducted. In our study, we isolated and sequenced the internal transcribed spacer rDNA region of fungi from two co-occurring Colombian epiphytic orchids, I. utricularioides and P. pusilla, both belonging to the subtribe Oncidiinae. All sequences were recognized as belonging to the genus Ceratobasidium, known to be a common orchid mycorrhizal fungus in both tropical and temperate orchids. One sequence was 100% similar to fungi isolated from I. utricularioides in Costa Rica in a previous study. I. utricularioides was confirmed to be a specialist, associating with only one clade of mycorrhizal fungi. However, P. pusilla was shown to be a generalist, associating with three clades. This finding indicates that the variation in mycorrhizal specificity could be an important factor in the co-existence of orchids. The high affinity between the subtribe Oncidiinae and Ceratobasidium was also reinforced.

In nature, orchid seeds obtain the nutrients necessary for germination by degrading intracellular fungal structures formed after colonization of the embryo by mycorrhizal fungi. Protocols for asymbiotic germination of orchid seeds typically use media with high concentrations of soluble carbohydrate and minerals. However, when reintroduced into the field, seedlings obtained via asymbiotic germination have lower survival rates than do seedlings obtained via symbiotic germination. Tree fern fiber, the ideal substrate for orchid seedling acclimatization, is increasingly scarce. Here, we evaluated seed germination and protocorm development of Cyrtopodium glutiniferum Raddi cultivated in asymbiotic media (Knudson C and Murashige & Skoog) and in oatmeal agar (OA) medium inoculated with the mycorrhizal fungus Epulorhiza sp., using non-inoculated OA medium as a control. We also evaluated the performance of tree fern fiber, pine bark, eucalyptus bark, corncob and sawdust as substrates for the acclimatization of symbiotically propagated plants. We determined germination percentages, protocorm development and growth indices at 35 and 70 days of cultivation. Relative growth rates and the effects of substrates on mycorrhizal formation were calculated after 165 days of cultivation. Germination efficiency and growth indices were best when inoculated OA medium was used. Corncob and pine bark showed the highest percentages of colonized system roots. The OA medium inoculated with Epulorhiza sp. shows potential for C. glutiniferum seedling production. Corncob and pine bark are promising substitutes for tree fern fiber as substrates for the acclimatization of orchid seedlings.

Na natureza, as espécies de Orchidaceae estão associadas a fungos micorrízicos. A compreensão da especificidade fungo-planta nessa associação pode auxiliar no desenvolvimento de programas para propagação simbiótica das orquídeas. Fungos micorrízicos Epulorhiza spp. têm sido isolados de Epidendrum secundum Jacq. com maior freqüência. Assim, o objetivo deste trabalho foi avaliar a germinação de sementes e o desenvolvimento de protocormos de E. secundum associados a diferentes isolados de fungos do gênero Epulorhiza. Utilizou-se 16 fungos Epulorhiza spp. isolados de diferentes populações de E. secundum. Após 44 dias, todos os fungos testados induziram a germinação das sementes. Entretanto, observou-se diferença na eficiência desses fungos em promover o desenvolvimento dos protocormos, mesmo entre os fungos que apresentam semelhanças morfológicas. O estágio de desenvolvimento mais avançado dos protocormos e o maior índice de crescimento foram observados quando as sementes foram inoculadas com o isolado M65. Conclui-se que a germinação das sementes e o desenvolvimento dos protocormos de E. secundum dependem do fungo micorrízico e que, apesar da grande freqüência de associação entre essa espécie de orquídea e fungos do gênero Epulorhiza, é importante a seleção de isolados que apresentem maior eficiência simbiótica. Isolados eficientes são úteis na produção de mudas de orquídeas de importância econômica e ornamental e de espécies ameaçadas de extinção.

In nature, species of Orchidaceae are always associated with mycorrhizal fungi. The comprehension of fungal-plant specificity in this association can assist with the establishment of programs that focus on symbiotic propagation of orchids. Epulorhiza mycorrhizal fungi have frequently been isolated from Epidendrum secundum Jacq. Thus, the objective of this study was to evaluate the germination of E. secundum seeds and protocorm development associated with different isolates of Epulorhiza spp. Sixteen Epulorhiza fungi obtained from different E. secundum populations were used. After 44 days, all isolates promoted seed germination of this species. However, differences were observed in the efficiency in promoting seed germination and protocorm development, even among the isolates that had morphological similarities. The highest stage of protocorm development and the highest growth indexes were obtained in the presence of isolate M65. The results suggest that seed germination and protocorm development of E. secundum depend on the mycorrhizal fungi and, despite the high frequency of association between this plant and Epulorhiza, it is important to select isolates with high efficiency to establish symbiosis. Efficient isolates are useful for the production of seedlings that have economic and ornamental importance, as well as for endangered orchids.

Os fungos rizoctonioides são os principais fungos micorrízicos de orquídeas. A caracterização morfológica e a análise de sequências conservadas do DNA total são as principais estratégias empregadas na identificação e no estudo da diversidade desses fungos. Contudo, fungos rizoctonioides fitopatogênicos têm sido caracterizados e identificados, de forma confiável e precisa, pela determinação da composição de seus ácidos graxos. Com o objetivo de avaliar a utilização da composição de ácidos graxos na caracterização e identificação de fungos rizoctonioides micorrízicos de orquídeas, três fungos Epulorhiza spp., micorrízicos de Epidendrum secundum, dois fungos não identificados, isolados de Epidendrum denticulatum, e um fungo fitopatogênico Ceratorhiza sp. AGC foram agrupados com base no perfil de seus ácidos graxos, utilizando as distâncias euclidiana e de Mahalanobis e o método UPGMA. Os dendrogramas gerados possibilitaram a distinção entre o isolado fitopatogênico de Ceratorhizasp. AGC e os fungos micorrízicos estudados. Os simbiontes de E. secundum foram distribuídos em dois clados: o primeiro contendo os isolados de Epulorhiza sp.1, e o segundo, o isolado de Epulorhiza sp.2. A similaridade entre os simbiontes de E. denticulatum e os fungos Epulorhizaspp. sugere a identificação desses simbiontes como Epulorhiza sp. Esses resultados foram semelhantes aos obtidos pela análise da região ITS (Internal Transcribed Spacer) do rDNA. O dendrograma construído a partir da distância de Mahalanobis apresentou melhor resolução, distinguindo claramente os clados descritos. A análise da composição de ácidos graxos mostrou ser uma ferramenta útil na caracterização e identificação dos fungos micorrízicos rizoctonioides estudados.

Rhizoctonia-like fungi are the main mycorrhizal fungi in orchid roots. Morphological characterization and analysis of conserved sequences of genomic DNA are frequently employed in the identification and study of fungi diversity. However, phytopathogenic Rhizoctonia-like fungi have been reliably and accurately characterized and identified through the examination of the fatty acid composition. To evaluate the efficacy of fatty acid composition in characterizing and identifying Rhizoctonia-like mycorrhizal fungi in orchids, three Epulorhiza spp. mycorrhizal fungi from Epidendrum secundum, two unidentified fungi isolated from Epidendrum denticulatum, and a phytopathogenic fungus, Ceratorhiza sp. AGC, were grouped based on the profile of their fatty acids, which was assessed by the Euclidian and Mahalanobis distances and the UPGMA method. Dendrograms distinguished the phytopathogenical isolate of Ceratorhiza sp. AGC from the mycorrhizal fungi studied. The symbionts of E. secundum were grouped into two clades, one containing Epulorhiza sp.1 isolates and the other the Epulorhiza sp.2 isolate. The similarity between the symbionts of E. denticulatum and Epulorhiza spp. fungi suggests that symbionts found in E. denticulatum may be identified as Epulorhiza. These results were corroborated by the analysis of the rDNA ITS region. The dendrogram constructed based on the Mahalanobis distance differentiated the clades most clearly. Fatty acid composition analysis proved to be a useful tool for characterizing and identifying Rhizoctonia-like mycorrhizal fungi.

Fungos micorrízicos rizoctonioides Epulorhiza spp. têm sido isolados de orquídeas do gênero Epidendrum e vêm sendo utilizados na germinação simbiótica das sementes de orquídeas. Epidendrum secundum é uma orquídea largamente distribuída em campos de altitude do Parque Estadual da Serra do Brigadeiro (PESB), Minas Gerais, e pouco se sabe sobre a associação micorrízica dessa espécie nesse parque. O objetivo deste trabalho foi avaliar a diversidade morfológica dos fungos micorrízicos rizoctonioides isolados de quatro populações de E. secundum em três regiões de um campo de altitude localizado na subserra Totem Deitado, PESB. Vinte e seis isolados fúngicos foram obtidos, todos pertencentes ao gênero Epulorhiza. As características morfológicas qualitativas e quantitativas avaliadas revelaram, de modo geral, baixa variabilidade entre os isolados obtidos de uma mesma população e de populações localizadas na mesma região, porém grande variabilidade foi observada entre os isolados obtidos das populações de diferentes regiões. Com base nessas características morfológicas, os isolados foram divididos em quatro grupos: o primeiro constituído pelos fungos obtidos das populações I e II da região A, o segundo pelos fungos da população III da região B, o terceiro pelo isolado M61 da população II da região A, e o quarto pelo único isolado obtido na população IV da região C. A variabilidade morfológica observada é um indicativo da diversidade dos fungos Epulorhiza spp. associados a E. secundum no PESB.

Rhizoctonia-like mycorrhizal fungi Epulorhiza spp. have been isolated from orchids of the genus Epidendrum and have been used to promote the symbiotic germination of orchid seeds. Epidendrum secundum is a widely distributed orchid in campo de altitude (high elevation grassy vegetation) regions of the State Park of Serra do Brigadeiro (PESB), Minas Gerais, Brazil, and little is known about the mycorrhizal relationships of this species in the park. The objective of this study was to evaluate the morphological diversity of Rhizoctonialike mycorrhizal fungi isolated from four E. secundum populations growing in three regions of a campo de altitude, in the region of Totem Deitado Mountain, PESB. Twenty-six fungal isolates were obtained, all belonging to Epulorhiza. The quantitative and qualitative morphological characteristics revealed, in general, low variability among the fungi isolated from the same orchid population and among isolates from the same region, but high variability among the fungi isolated from the populations from different regions. Based on these morphological characteristics, the isolates were separated into four groups. The first consisted of the fungi from populations I and II from region A, the second of isolates from population III from region B, the third of isolate M61 from population II from region C, and the fourth of a single isolate obtained from population IV from region C. The morphological variability observed indicates the existence of diversity of Epulorhiza spp. associated with E. secundum in PESB.