Astrocytes contain a vesicular compartment that is competent for regulated exocytosis of glutamate.

Astrocytes establish rapid cell-to-cell communication through the release of chemical transmitters. The underlying mechanisms and functional significance of this release are, however, not well understood. Here we identify an astrocytic vesicular compartment that is competent for glutamate exocytosis. Using postembedding immunogold labeling of the rat hippocampus, we show that vesicular glutamate transporters (VGLUT1/2) and the vesicular SNARE protein, cellubrevin, are both expressed in small vesicular organelles that resemble synaptic vesicles of glutamatergic terminals. Astrocytic vesicles, which are not as densely packed as their neuronal counterparts, can be observed in small groups at sites adjacent to neuronal structures bearing glutamate receptors. Fluorescently tagged VGLUT-containing vesicles were studied dynamically in living astrocytes by total internal reflection fluorescence (TIRF) microscopy. After activation of metabotropic glutamate receptors, astrocytic vesicles underwent rapid (milliseconds) Ca(2+)- and SNARE-dependent exocytic fusion that was accompanied by glutamate release. These data document the existence of a Ca(2+)-dependent quantal glutamate release activity in glia that was previously considered to be specific to synapses.

Crescimento de Milhos Transgênico (Bt) e Não Transgênico Inoculados com Fungos Micorrízicos Arbusculares em Solo Contaminado por Cádmio

RESUMO Com o crescimento populacional, aumenta-se a necessidade da produção de alimentos; paralelamente, observa-se o uso indiscriminado de fertilizantes e incorporação de plantas transgênicas no sistema produtivo. O objetivo deste trabalho foi avaliar o efeito do cádmio no crescimento de milho transgênico Bt e não transgênico, inoculados com fungos micorrízicos arbusculares em condições controladas. Para isso, instalou-se um experimento em esquema fatorial 5 × 4 × 2, sendo cinco doses de Cd (0; 28,5; 71,50; 142,50; e 285 mg kg-1Cd), inoculação individual de espécies de fungos micorrízicos arbusculares - FMAs (Glomus clarum, Acaulospora scrobiculatae Scutellospora heterogama) e um controle não inoculado; duas cultivares de milho Monsanto, DKB-390 (Bt) e DKB-S, e três repetições por tratamento, totalizando 120 unidades experimentais compostas de recipientes plásticos de 500 mL cada. O experimento foi conduzido por 45 dias, avaliando-se, em seguida, atributos relacionados ao crescimento vegetativo e de colonização micorrízica. As doses de Cd não influenciaram o crescimento de milho transgênico Bt e não transgênico, independentemente da inoculação de FMAs. O milho transgênico Bt não respondeu à inoculação, com colonização micorrízica entre 14 e 33 %, quando comparada a do milho não transgênico, entre 32 e 74 %.

Occurrence and Structure of Arbuscular Mycorrhizal Fungal Communities in Cassava after Cultivation of Cover Crops as Observed by the “PCR-DGGE” Technique

ABSTRACT Cassava (Manihot esculenta Crantz) is a highly mycotrophic crop, and prior soil cover may affect the density of arbuscular mycorrhizal fungi (AMFs), as well as the composition of the AMFs community in the soil. The aim of this study was to evaluate the occurrence and the structure of AMFs communities in cassava grown after different cover crops, and the effect of the cover crop on mineral nutrition and cassava yield under an organic farming system. The occurrence and structure of the AMFs community was evaluated through polymerase chain reaction (PCR) and denaturing gradient gel electrophoresis (DGGE). A randomized block experimental design was used with four replications. Six different cover crop management systems before cassava were evaluated: black oats, vetch, oilseed radish, intercropped oats + vetch, intercropped oats + vetch + oilseed radish, plus a control (fallow) treatment mowed every 15 days. Oats as a single crop or oats intercropped with vetch or with oilseed radish increased AMFs inoculum potential in soil with a low number of propagules, thus benefiting mycorrhizal colonization of cassava root. The treatments did not affect the structure of AMFs communities in the soil since the AMFs communities were similar in cassava roots in succession to different cover crops. AMFs colonization was high despite high P availability in the soil. The cassava crop yield was above the regional average, and P levels in the leaves were adequate, regardless of which cover crop treatments were used. One cover crop cycle prior to the cassava crop was not enough to observe a significant response in variables, P in plant tissue, crop yield, and occurrence and structure of AMFs communities in the soil. In the cassava roots in succession, the plant developmental stage affected the groupings of the structure of the AMF community.

The ecological significance of arbuscular mycorrhizal fungal effects on clonal reproduction in plants

The population ecology of clonal plants depends on the number and distribution of ramets formed during growth. Variation in clonal reproduction has previously been explained by variation in effects of abiotic resource heterogeneity and by plant genotypic variation. Different co-occurring species of the mutualistic arbuscular mycorrhizal fungi (AMF) have been shown to differentially alter growth traits of Prunella vulgaris which we hypothesize would lead to changes in clonal reproduction. Two experiments were carried out to test whether different co-occurring mycorrhizal fungi significantly influence clonal reproduction of P. vulgaris whether this effect also occurs when P. vulgaris is growing in an artificial plant community and how the effects compare with plant genotype effects on clonal growth of P. vulgaris. In the first experiment the number of ramets of P. vulgaris growing in a plant community of simulated calcareous grassland was significantly affected by inoculation with different mycorrhizal fungi. The number of ramets produced by P. vulgaris differed by a factor of up to 1.8 with different mycorrhizal fungi. The fungal effects on the number of new ramets were independent of their effects on the biomass of P. vulgaris. In a second experiment 17 different genotypes of P. vulgaris were inoculated with different mycorrhizal fungi. There were significant main effects of genotypes and mycorrhizal fungi on clonal reproduction of P. vulgaris. The effect of different mycorrhizal fungi contributed more than the effect of plant genotype to variation in size and ramet production. However mean stolon length and spacer length which determine the spatial arrangement of ramets were only significantly affected by plant genotype. There were no mycorrhizal fungal X plant genotype interactions on clonal growth of P. vulgaris indicating that there is no obvious evidence that selection pressures would favor further coevolution between P. vulgaris and mycorrhizal fungal species. In natural communities plants can be colonized by several different AMF at the same time. The effect of the mixed AMF treatment on the growth and clonal reproduction of P. vulgaris could not be predicted from the responses of the plants to the single AMF To what extent however the patterns of colonization by different AMF differ among plants in a natural community is unknown. Since the effects of AMF on growth and clonal reproduction occur on a population of P. vulgaris in a microcosm plant community and because the effects are also as great as those caused by plant genotypic variation we conclude that the effects are strong enough to potentially affect population size and variation of clonal plants in communities.

The effects of arbuscular mycorrhizal fungi inoculation on Euterpe oleracea mart. (açaí) seedlings

With the objective of verifying the response of Euterpe oleracea seedlings to seven arbuscular mycorrhizal fungi species, an experimental trial was carried out under greenhouse conditions. Seeds of E. oleracea were sown in carbonized rice husk. Germinating seeds were initially transferred to plastic cups, containing fumigated Reddish Yellow Quartz Sand and inoculated with arbuscular mycorrhizal fungi. Two months later, seedlings were transferred to 2 kg black plastic bags, containing the same soil without fumigation. Plant growth and mineral nutrients were evaluated nine months after mycorrhizal inoculation. Differential effects were observed among the species tested, with Scutellispora gilmorei being the most effective ones in promoting growth and nutrient content of E. oleracea seedlings. The increment resulted from inoculation with S. gilmorei were 92% in total plant height, 116% in stem diameter, 361% in dry matter production, 191% in N, 664% in P, 46% in K, 562% in Ca, 363% in Mg and 350% in Zn contents, comparing to uninoculated controls. Infected root length was positively correlated to nutrient content and plant growth. It was concluded that growth and nutrient uptake of E. oleracea seedlings could be significantly improved by inoculation of effective arbuscular mycorrhizal fungi.

Changes in arbuscular mycorrhizal fungal phenotypes and genotypes in response to plant species identity and phosphorus concentration.

* Arbuscular mycorrhizal fungi (AMF) are plant symbionts that improve floristic diversity and ecosystem productivity. Many AMF species are generalists with wide host ranges. Arbuscular mycorrhizal fungi individuals are heterokaryotic, and AMF populations are genetically diverse. Populations of AMF harbor two levels of genetic diversity on which selection can act, namely among individuals and within individuals. Whether environmental factors alter genetic diversity within populations is still unknown. * Here, we measured genetic changes and changes in fitness-related traits of genetically distinct AMF individuals from one field, grown with different concentrations of available phosphate or different host species. * We found significant genotype-by-environment interactions for AMF fitness traits in response to these treatments. Host identity had a strong effect on the fitness of different AMF, unearthing a specificity of response within Glomus intraradices. Arbuscular mycorrhizal fungi individuals grown in novel environments consistently showed a reduced presence of polymorphic genetic markers, providing some evidence for host or phosphate-induced genetic change in AMF. * Given that AMF individuals can form extensive hyphal networks colonizing different hosts simultaneously, contrasting habitats or soil properties may lead to evolution in the population. Local selection may alter the structure of AMF populations and maintain genetic diversity, potentially even within the hyphal network of one fungus.

Uso de esterco no desenvolvimento de mudas de mamoeiro colonizadas com fungos micorrízicos

O trabalho objetivou a adequação de esterco na composição do substrato para produção de mudas de mamoeiro (Carica papaya L.), que permita o melhor estabelecimento da simbiose micorrízica e sua expressão no desenvolvimento da muda. Foram testadas cinco doses de esterco no substrato (0, 5, 10, 20 e 30%) e a inoculação ou não de Glomus etunicatum. Após 50 dias de cultivo concluiu-se que: o esterco promove máximo desenvolvimento das mudas nas doses de 20% e 30%; a adição de P e K não resulta em benefícios adicionais no crescimento das mudas; a inoculação de G. etunicatum é eficiente para o desenvolvimento da muda até a dose de 10% de esterco; o fungo coloniza as mudas em doses de até 30% de esterco, mas com tendência decrescente; a combinação de 10% de esterco e inoculação do fungo micorrízico arbuscular promove a formação de plantas sadias, sem sintomas de deficiência nutricional e apropriadas para o transplantio ao campo.

Arbuscular mycorrhiza: the challenge to understand the genetics of the fungal partner.

Arbuscular mycorrhizal symbioses occur between fungi and the majority of plant species. They are important for plant nutrition, plant growth, protection from pathogens, plant diversity, nutrient cycling, and ecosystem processes. A key goal in research is to understand the molecular basis of the establishment, regulation, and functioning of the symbiosis. However, lack of knowledge on the genetics of the fungal side of this association has hindered progress. Here, we show how several key, recently discovered processes concerning the genetics of arbuscular mycorrhizal fungi could be essential for ultimately understanding the molecular genetics of this important symbiosis with plants.

Recombination in Glomus intraradices, a supposed ancient asexual arbuscular mycorrhizal fungus

Background: Arbuscular mycorrhizal fungi (AMF) are important symbionts of most plant species, promoting plant diversity and productivity. This symbiosis is thought to have contributed to the early colonisation of land by plants. Morphological stasis over 400 million years and the lack of an observed sexual stage in any member of the phylum Glomeromycota led to the controversial suggestion of AMF being ancients asexuals. Evidence for recombination in AMF is contradictory. Results: We addressed the question of recombination in the AMF Glomus intraradices by sequencing 11 polymorphic nuclear loci in 40 morphologically identical isolates from one field. Phylogenetic relationships among genotypes showed a reticulate network pattern providing a rationale to test for recombination. Five statistical tests predicted multiple recombinant regions in the genome of a core set of isolates. In contrast, five clonal lineages had fixed a large number of differences. Conclusion: Our data show that AMF from one field have undergone recombination but that clonal lineages coexist. This finding has important consequences for understanding AMF evolution, co-evolution of AMF and plants and highlights the potential for commercially introduced AMF inoculum recombining with existing local populations. Finally, our results reconcile seemingly contradictory studies on whether AMF are clonal or form recombining populations.

Fosfato e micorriza na estabilidade de agregados em amostras de latossolos cultivados e não-cultivados

Nos trópicos, existe escassez de informação quanto à contribuição de espécies fúngicas do solo na formação e estabilização de agregados. Este estudo teve como objetivo avaliar o efeito do histórico de uso, níveis de P, de inoculação micorrízica e cultivo com braquiária e soja em casa de vegetação, sobre o diâmetro médio geométrico dos agregados (DMG), o índice de floculação das partículas, a matéria seca das raízes, a colonização micorrízica e o comprimento total de hifas, em amostras de Latossolo Vermelho distrófico e Latossolo Vermelho distroférrico. Amostras dos dois solos, previamente cultivados por longos períodos, e de solos não-cultivados, foram trazidas para casa de vegetação, submetidas a inoculação, e a dois níveis de P, e então cultivadas com braquiária e soja, em dois cultivos sucessivos. Os resultados mostraram que o solo previamente cultivado apresentou menor comprimento total de hifas, menor estabilidade de agregados (menor diâmetro médio de agregados) e menor índice de floculação. A inoculação propiciou maior estabilidade dos agregados, e este efeito é condicionado ao nível de P do solo e ao histórico de uso. A presença de P promoveu, indiretamente, maior agregação, por propiciar maior comprimento total das hifas e matéria seca de raízes.

Interações microbianas na disponibilidade e absorção de manganês por soja

O objetivo deste trabalho foi avaliar o efeito da interação entre fungos micorrízicos arbusculares e a comunidade microbiana do solo, sobre a disponibilidade, a absorção e a atenuação da toxidez de Mn em soja. As plantas foram micorrizadas com Glomus etunicatum ou G. macrocarpum, com e sem o restabelecimento da comunidade microbiana do solo autoclavado. Um tratamento não micorrizado teve a comunidade microbiana restabelecida, enquanto outro foi mantido como controle absoluto. As plantas micorrizadas apresentaram maior crescimento e atenuação da toxidez de Mn, principalmente quando se restabeleceu a comunidade microbiana. Nesse caso, houve aumento da disponibilidade de Fe e diminuição da disponibilidade de Mn no substrato. O teor de P foi maior nas plantas micorrizadas. Houve menor concentração de Fe e Mn na parte aérea das plantas micorrizadas, mais evidente quando a comunidade microbiana foi restabelecida. Nas raízes, esse comportamento foi o mesmo quanto ao Mn e inverso quanto ao ferro. A diminuição da concentração de Mn na parte aérea das plantas micorrizadas foi atribuída à diminuição de sua disponibilidade no substrato, enquanto em relação ao Fe foi atribuída à sua retenção nas raízes.

Produção de mudas de acácia colonizadas com micorrizas e rizóbio em diferentes recipientes

Realizou-se um experimento em casa de vegetação, com o objetivo de avaliar diferentes métodos na produção de mudas de Acacia mangium Willd, colonizadas com fungos micorrízicos arbusculares (FMAs) e rizóbio. O delineamento experimental foi o inteiramente casualizado num esquema fatorial 4x2 (controle, FMAs, rizóbio e FMAs + rizóbio x blocos prensados e tubetes de plástico), com seis repetições. Os blocos prensados foram confeccionados com substratos orgânicos (bagaço de cana + torta de filtro de usina açucareira) e vermiculita, colocados em fôrma metálica de 60x40x20 cm e prensados a 10 kgf cm-2, a fim de proporcionar agregação do material. A inoculação do rizóbio foi realizada com estirpe selecionada para a espécie (Br 3609, Br 6009). A inoculação de FMAs foi feita no momento da confecção dos blocos. Mudas de Acacia mangium que receberam inóculo de FMAs + rizóbio e produzidas em blocos prensados apresentaram maior produção de matéria seca e conteúdo de N na parte aérea. O conteúdo de P na parte aérea é significativamente maior somente nas mudas infectadas com os FMAs, independentemente do tipo de recipiente.

Efeito de Glomus etunicatum e fósforo no crescimento inicial de espécies arbóreas em semeadura direta

O objetivo deste trabalho foi avaliar os efeitos da disponibilidade de P no solo, da micorriza formada por Glomus etunicatum e de Mycoform, um estimulante desta última, no crescimento e competição inicial de seis espécies arbóreas semeadas diretamente. O trabalho foi realizado em casa de vegetação com as espécies Senna macranthera (fedegoso), Guazuma ulmifolia (mutamba), Senna multijuga (cássia-verrugosa), Solanum granuloso-leprosum (gravitinga), Schinus terebenthifolius (aroeira) e Trema micrantha (trema), em solo com níveis de P na solução considerados muito baixo, baixo e alto, com inoculação ou não do fungo micorrízico arbuscular G. etunicatum, além do tratamento G. etunicatum + Mycoform. O crescimento das mudas respondeu à inoculação em P muito baixo e baixo. As mudas apresentaram moderada dependência das micorrizas, não respondendo ao G. etunicatum em P alto. Gravitinga morreu em P muito baixo, mas foi dominante com P baixo e alto. Fedegoso foi dominante com P muito baixo, mostrando-se adaptado à baixa fertilidade. G. etunicatum influenciou a dominância das espécies, auxiliando as menos competitivas e gerando maior equilíbrio. Mycoform influenciou pouco o crescimento, nutrição e competição. O crescimento de espécies pioneiras semeadas diretamente é favorecido pela elevação do P e pelas micorrizas, as quais também favorecem o equilíbrio entre espécies.

Rapid genotypic change and plasticity in arbuscular mycorrhizal fungi is caused by a host shift and enhanced by segregation.

Arbuscular mycorrhizal fungi (AMF) are among the most abundant symbionts of plants, improving plant productivity and diversity. They are thought to mostly grow vegetatively, a trait assumed to limit adaptability. However, AMF can also harbor genetically different nuclei (nucleotypes). It has been shown that one AMF can produce genotypically novel offspring with proportions of different nucleotypes. We hypothesized that (1) AMF respond rapidly to a change of environment (plant host) through changes in the frequency of nucleotypes; (2) genotypically novel offspring exhibit different genetic responses to environmental change than the parent; and (3) genotypically novel offspring exhibit a wide range of phenotypic plasticity to a change of environment. We subjected AMF parents and offspring to a host shift. We observed rapid and large genotypic changes in all AMF lines that were not random. Genotypic and phenotypic responses were different among offspring and their parents. Even though growing vegetatively, AMF offspring display a broad range of genotypic and phenotypic changes in response to host shift. We conclude that AMF have the ability to rapidly produce variable progeny, increasing their probability to produce offspring with different fitness than their parents and, consequently, their potential adaptability to new environmental conditions. Such genotypic and phenotypic flexibility could be a fast alternative to sexual reproduction and is likely to be a key to the ecological success of AMF.