Extent of intra-isolate genetic polymorphism in glomus etunicatum using a molecular genetic approach

Mémoire numérisé par la Division de la gestion de documents et des archives de l'Université de Montréal

Étude comparative des propagules extraracinaires et intraracinaires du champignon mycorhizien Glomus irregulare

La germination des spores est une étape essentielle dans le cycle de vie de la majorité des champignons filamenteux. Les champignons mycorhiziens à arbuscules (CMA) forment un certain nombre de propagules infectieuses différentes qui augmentent leur potentiel à coloniser les racines. Parmi elles se trouvent les spores extraracinaires et intraracinaires. La paroi cellulaire des spores joue un rôle majeur dans la survie de ces propagules en étant une barrière physique et osmotique. Puisque une cellule peut faire des ajustements considérables dans la composition et la structure de sa paroi, en réponse aux conditions environnementales, il est possible que les parois des spores intraracinaires et extraracinaires montrent des propriétés mécaniques et osmotiques différentes affectant leur germination et leur survie. Pourtant, contrairement à la connaissance de la génétique moléculaire et de la formation de la paroi cellulaire des CMA, peu d’information est disponible au sujet de ces propriétés mécaniques. Les informations sur la germination des CMA dans des conditions hypertoniques sont aussi rares, et les modèles expérimentaux ne séparent généralement pas les effets directs de la forte pression osmotique externe sur la germination des champignons et les effets attribuables aux plantes. Cette étude avait pour but de répondre à deux importantes séries de questions concernant le comportement des spores mycorhiziennes. Nous avons d'abord déterminé la relation entre la composition de la paroi cellulaire, la structure et les propriétés mécaniques du champignon modèle Glomus irregulare (isolat DAOM 197198). La micro-indentation a été utilisée pour mesurer quantitativement les propriétés mécaniques de la paroi cellulaire. La composition (contenu de chitine et de glomaline) de la paroi cellulaire a été quantifiée par immunofluorescence tandis que la microscopie optique a été utilisée pour mesurer l'épaisseur de la paroi cellulaire. La densité locale en glomaline et l’épaisseur de la paroi étaient significativement plus élevées pour les parois des spores extraracinaires alors que la densité locale en chitine et la rigidité n’ont pas montré de variations entre les spores extraracinaires et intraracinaires. La grande variabilité dans les paramètres étudiés nous a empêchés de cibler un facteur principal responsable de la force totale de la paroi lors de la compression. La diminution des concentrations de chitine et de glomaline a été corrélée à l'évolution de la paroi du champignon au cours de son cycle de vie. On a aussi observé une composition différentielle des couches de la paroi: les polymères de chitine et de glomaline furent localisés principalement dans les couches externes et internes de la paroi, respectivement. Dans la deuxième partie de notre travail, nous avons exploré les effets directs d'engrais, par rapport à leur activité de l'eau (aw), sur la germination des spores et la pression de turgescence cellulaire. Les spores ont été soumises à trois engrais avec des valeurs de aw différentes et la germination ainsi que la cytorrhyse (effondrement de la paroi cellulaire) des spores ont été évaluées après différents temps d'incubation. Les valeurs de aw des engrais ont été utilisées comme indicateurs de leurs pressions osmotiques. L'exposition des spores de Glomus irregulare au choc osmotique causé par les engrais dont les valeurs de aw se situent entre 0,982 et 0,882 a provoqué des changements graduels au niveau de leur cytorrhyse et de leur germination. Avec l'augmentation de la pression de turgescence externe, la cytorrhyse a augmenté, tandis que le taux de germination a diminué. Ces effets ont été plus prononcés à des concentrations élevées en éléments nutritifs. La présente étude, bien qu’elle constitue une étape importante dans la compréhension des propriétés mécaniques et osmotiques des spores de CMA, confirme également que ces propriétés dépendent probablement de plusieurs facteurs, dont certains qui ne sont pas encore identifiés.

Glomus intraradices and Gigaspora margarita arbuscular mycorrhizal associations differentially affect nitrogen and potassium nutrition of Plantago lanceolata in a low fertility dune soil

Two controlled microcosm experiments aimed at a critical re-assessment of the contributions of divergent arbuscular mycorrhizal (AM) fungi to plant mineral nutrition were established that specifically targeted Plantago lanceolata–Glomus intraradices (B.B/E) and –Gigaspora margarita (BEG 34) symbioses developed in a native, nutrient limited, coastal dune soil. Plant tissue nitrogen (N), phosphorus (P) and potassium (K) status as well as plant growth parameters and levels of mycorrhizal colonization were assessed at harvest. In addition to the general well-established mycorrhizal facilitation of P uptake, the study was able to demonstrate a G. intraradices-specific contribution to improved plant nitrogen and potassium nutrition. In the two respective experiments, G. intraradices-inoculated plants had 27.8% and 40.8% more total N and 55.8% and 23.3% more total K when compared to Gi. margarita inoculated counterparts. Dissimilar overall contribution of the two isolates to plant nutrition was identified in AM-genus specific differences in plant tissue N:P:K ratios. G. intraradices inoculated and non-mycorrhizal plants generally exhibited N:P:K ratios indicative of P limitation whereas for Gi.margarita mycorrhizal plants, corresponding ratios strongly implied either N or K limitation. The study provides further evidence highlighting AM functional biodiversity in respect to plant nutrient limitation experienced by mycorrhizal P. lanceolata in an ecologically relevant soil system.

Glomus clarum e G. etunicatum: cultivo em solo e aeroponia

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

Aspergillus niger e Glomus clarum incrementam a disponibilidade de fósforo em Latossolos sob Urochloa brizantha

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Transcriptome and Functional Analysis of Carotid Body Glomus Cells

The carotid body (CB) is a major arterial chemoreceptor containing glomus cells that are activated by changes in arterial blood contents including oxygen. Despite significant advancement in the characterization of their physiological properties, our understanding on the underlying molecular machinery and signaling pathway in CB glomus cells is still limited.

To overcome these limitations, in chapter 1, I demonstrated the first transcriptome profile of CB glomus cells using single cell sequencing technology, which allowed us to uncover a set of abundantly expressed genes, including novel glomus cell-specific transcripts. These results revealed involvement of G protein-coupled receptor (GPCR) signaling pathway, various types of ion channels, as well as atypical mitochondrial subunits in CB function. I also identified ligands for the mostly highly expressed GPCR (Olfr78) in CB glomus cells and examined this receptor’s role in CB mediated hypoxic ventilatory response.

Current knowledge of CB suggest glomus cells rely on unusual mitochondria for their sensitivity to hypoxia. I previously identified the atypical mitochondrial subunit Ndufa4l2 as a highly over-represented gene in CB glomus cells. In chapter 2, to investigate the functional significance of Ndufa4l2 in CB function, I phenotyped both Ndufa4l2 knockout mice and mice with conditional Ndufa4l2 deletion in CB glomus cells. I found that Ndufa4l2 is essential to the establishment of regular breathing after birth. Ablating Ndufa4l2 in postnatal CB glomus cells resulted in defective CB sensitivity to hypoxia as well as CB mediated hypoxic ventilatory response. Together, our data showed that Ndufa4l2 is critical to respiratory control and the oxygen sensitivity of CB glomus cells.

Composite Medicago truncatula plants harbouring Agrobacterium rhizogenes-transformed roots reveal normal mycorrhization by Glomus intraradices

Composite plants consisting of a wild-type shoot and a transgenic root are frequently used for functional genomics in legume research. Although transformation of roots using Agrobacterium rhizogenes leads to morphologically normal roots, the question arises as to whether such roots interact with arbuscular mycorrhizal (AM) fungi in the same way as wild-type roots. To address this question, roots transformed with a vector containing the fluorescence marker DsRed were used to analyse AM in terms of mycorrhization rate, morphology of fungal and plant subcellular structures, as well as transcript and secondary metabolite accumulations. Mycorrhization rate, appearance, and developmental stages of arbuscules were identical in both types of roots. Using Mt16kOLI1Plus microarrays, transcript profiling of mycorrhizal roots showed that 222 and 73 genes exhibited at least a 2-fold induction and less than half of the expression, respectively, most of them described as AM regulated in the same direction in wild-type roots. To verify this, typical AM marker genes were analysed by quantitative reverse transcription-PCR and revealed equal transcript accumulation in transgenic and wild-type roots. Regarding secondary metabolites, several isoflavonoids and apocarotenoids, all known to accumulate in mycorrhizal wild-type roots, have been found to be up-regulated in mycorrhizal in comparison with non-mycorrhizal transgenic roots. This set of data revealed a substantial similarity in mycorrhization of transgenic and wild-type roots of Medicago truncatula, validating the use of composite plants for studying AM-related effects.

Bacteriosomes in axenic plants: endophytes as stable endosymbionts

Observations of cells of axenic peach palm (Bactris gasipaes) microplants by light microscopy revealed movements of small particles within the cells. The phenomenon was characterized initially as Brownian movement, but electron microscopy revealed the presence of an intracellular bacterial community in these plants. Microscopy observations revealed the particular shapes of bacterial cells colonizing inner tissues of analyzed plants. Applying a molecular characterization by polymerase chain reaction and denaturing gradient gel electrophoresis, it was revealed the existence of bacterial rRNA within the plants. Sequencing of the rRNA identified three different phylogenetic groups; two bands had a high degree of similarity to sequences from Moraxella sp. and Brevibacillus sp., and a third sequence was similar to a non-cultivated cyanobacterium. The presence of those endosymbionts, called bacteriosomes, in axenic peach palm microplants raises the question of whether these stable endosymbionts were acquired in the process of evolution and how could they benefit the process of plants micropropagation.

Zn uptake, physiological response and stress attenuation in mycorrhizal jack bean growing in soil with increasing Zn concentrations

The influence of arbuscular mycorrhizal fungi (AMF) inoculation on Canavalia ensiformis growth. nutrient and Zn uptake, and on some physiological parameters in response to increasing soil Zn concentrations was studied. Treatments were applied in seven replicates in a 2 x 4 factorial design, consisting of the inoculation or not with the AMF Glomus etunicatum, and the addition of Zn to soil at the concentrations of 0, 100, 300 and 900 mg kg(-1). AMF inoculation enhanced the accumulation of Zn in tissues and promoted biomass yields and root nodulation. Mycorrhizal plants exhibited relative tolerance to Zn up to 300 mg kg(-1) without exhibiting visual symptoms of toxicity, in contrast to non-mycorrhizal plants which exhibited a significant growth reduction at the same soil Zn concentration. The highest concentration of Zn added to soil was highly toxic to the plants. Leaves of plants grown in high Zn concentration exhibited a Zn-induced proline accumulation and also an increase in soluble amino acid contents; however proline contents were lower in mycorrhizal jack beans. Plants in association or not with the AMF exhibited marked differences in the foliar soluble amino acid profile and composition in response to Zn addition to soil. In general, Zn induced oxidative stress which could be verified by increased lipid peroxidation rates and changes in catalase, ascorbate peroxidase, glutathione reductase and superoxide dismutase activities. In summary, G. etunicatum was able to maintain an efficient symbiosis with jack bean plants in moderately contaminated Zn-soils, improving plant performance under those conditions, which is likely to be due to a combination of physiological and nutritional changes caused by the intimate relation between fungus and plant. The enhanced Zn uptake by AMF inoculated jack bean plants might be of interest for phytoremediation purposes. (C) 2009 Elsevier Ltd. All rights reserved.

Dosage-dependent shift in the spore community of arbuscular mycorrhizal fungi following application of tannery sludge

The controlled disposal of tannery sludge in agricultural soils is a viable alternative for recycling such waste; however, the impact of this practice on the arbuscular mycorrhizal fungi (AMF) communities is not well understood. We studied the effects of low-chromium tannery sludge amendment in soils on AMF spore density, species richness and diversity, and root colonization levels. Sludge was applied at four doses to an agricultural field in Rolandia, Parana state, Brazil. The sludge was left undisturbed on the soil surface and then the area was harrowed and planted with corn. The soil was sampled at four intervals and corn roots once within a year (2007/2008). AMF spore density was low (1 to 49 spores per 50 cm(3) of soil) and decreased as doses of tannery sludge increased. AMF root colonization was high (64%) and unaffected by tannery sludge. Eighteen AMF species belonging to six genera (Acaulospora, Glomus, Gigaspora, Scutellospora, Paraglomus, and Ambispora) were recorded. At the sludge doses of 9.0 and 22.6 Mg ha(-1), we observed a decrease in AMF species richness and diversity, and changes in their relative frequencies. Hierarchical grouping analysis showed that adding tannery waste to the soil altered AMF spore community in relation to the control, modifying the mycorrhizal status of soil and selectively favoring the sporulation of certain species.

Can a N-2-fixing Gluconacetobacter diazotrophicus association with sugarcane be achieved?

Several published studies claim that high rates of N-2 fixation occur in sugarcane and sorghum, and have ascribed this result to infection by the bacterium Gluconacetobacter diazotrophicus, abetted by arbuscular mycorrhizal infection ( Glomus clarum). These results have not been confirmed within Australia. In this study, G. diazotrophicus was detected in stalks of field-grown sugarcane in Australia ( based on phenotypic tests, and a PCR test using species-specific primers developed to amplify a fragment of the G. diazotrophicus 16S rRNA gene). Isolates were nitrogenase positive ( acetylene reduction assay) in vitro. However, in glasshouse trials involving inoculation of sugarcane setts with G. diazotrophicus, co-inoculation with mycorrhizae, and plant growth under low N status, recovery of bacteria from maturing plants was variable. At 165 days from planting, no appreciable N-2-fixation, as assessed by dry weight increment, N budget, or N-15 ratio, of either an Australian or a Brazilian cultivar of sugarcane, or a sorghum cultivar, was achieved. We conclude that a N-2-fixing sugarcane - G. diazotrophicus association is not easily achievable, being primarily limited by a lack of infection.

Responses of sugarcane, maize, and soybean to phosphorus and vesicular-arbuscular mycorrhizal fungi

The presence of vesicular-arbuscular mycorrhizal (VAM) fungi in long-term cane-growing fields associated with yield decline led to the supposition that VAM fungi may be responsible for the poor yields. A glasshouse trial was established to test the effectiveness of a species of VAM fungi, Glomus clarum, extracted from one of these North Queensland fields on the growth of sugarcane (Saccharum interspecific hybrid), maize (Zea mays), and soybean (Glycine max) for 6 phosphorus (P) rates (0, 2.7, 8.2, 25, 74, 222 mg/kg). For maize and soybean plants that received VAM (+ VAM), root colonisation was associated with enhanced P uptake, improved dry weight (DW) production, and higher index tissue-P concentrations than those without VAM (-VAM). By comparing DW responses of maize and soybean for different P rates, savings in fertiliser P of up to 160 and 213 kg/ha, respectively, were realised. Sugarcane plants were generally less responsive. Apart from a 30% DW increase with VAM when 2.7 mg P/kg was added, DW of +VAM plants was equivalent to, or worse than in the case of 222 mg P/kg, DW of -VAM plants. For all 3 host species, colonisation was least at the highest P application, presumably from excessive P within the plant tissue. Critical P concentrations for the 3 host species were below those reported elsewhere, and for soybean and sugarcane, the critical concentration for +VAM plants was lower than that of -VAM plants. There are 3 implications that arise from this study. First, VAM fungi present in cane-growing soils can promote the growth of maize and soybean, which are potential rotation crops, over a range of P levels. Second, the mycorrhizal strain taken from this site did not generally contribute to a yield decline in sugarcane plants. Third, application of P fertiliser is not necessary for sugarcane when acid-extractable P is

Paragangliomas timpânicos: relato de casos

Os tumores glômicos, também chamados de paragangliomas, são formados por células não cromafins. O tumor é tipicamente vascular formado por vasos capilares e pré-capilares, interposto por células epiteliais. Para a sua abordagem inicial, deve-se ressaltar que os sintomas mais comumentes encontrados são o zumbido pulsátil e hipoacusia. A investigação através de imagem (tomografia computadorizada e ressonância magnética) se faz necessária. Apresentaremos neste trabalho 5 pacientes portadores de paragangliomas timpânicos atendidos no Hospital (de 1995 a 2001). O sexo predominante foi o feminino, a idade variou de 48 a 60 anos, com média de 50 anos. A queixa predominante foi o zumbido pulsátil e a hipoacusia. A conduta foi cirúrgica em todos os casos.

Incremento no desenvolvimento do porta-enxerto de pessegueiro 'Okinawa', promovido por fungos micorrízicos arbusculares autóctones

Fungos micorrízicos arbusculares (FMA) são organismos comuns da rizosfera, que se associam às raízes das plantas, incrementando a absorção nutricional e estimulando o seu crescimento. Objetivou-se avaliar a influência de três espécies de FMA (Gigaspora margarita, Glomus clarum e Glomus etunicatum), isolados de pomares de pessegueiros, no crescimento vegetativo, absorção de nutrientes e acúmulo de substâncias de reserva, de plantas do porta-enxerto de pessegueiro cv. Okinawa (Prunus persica (L.) Batsch). O delineamento experimental utilizado foi o de blocos casualizados, com quatro repetições por tratamento e 15 plantas por parcela. As plantas que foram inoculadas com G. etunicatum apresentaram maior altura, diâmetro de caule, área foliar, biomassa fresca e seca da parte aérea, total de nutrientes absorvidos e substâncias de reserva acumuladas, enquanto as inoculadas com G. clarum apresentaram crescimento intermediário, superior às inoculadas com G. margarita, que apresentaram resultados semelhantes às plantas não inoculadas. O desempenho foi relacionado com as taxas de colonização das raízes que, nas plantas inoculadas com G. etunicatum e G. clarum, foram de 93 e 91%, respectivamente, enquanto, nas com G. margarita foi somente de 37%.

Fungos micorrízicos arbusculares afetando a produção de mudas de pinhão-manso na região sul do Estado de Tocantins, Brasil

O presente trabalho teve como objetivo avaliar os efeitos da inoculação de quatro espécies de fungos micorrízicos arbusculares no crescimento e nutrição de mudas de pinhão-manso. Foram utilizados 12 tratamentos e 10 repetições, sendo: a inoculação individual (Scutellospora calospora, Glomus clarum, Gigaspora margarita, Acaulospora morrowiae), em dupla das espécies (S. calospora + G. clarum, S. calospora + G. margarita, S. calospora + A. morrowiae, G. clarum + G. margarita, G. clarum + A. morrowiae e G. margarita + A. morrowiae), com a mistura das quatro espécies de micorrizas (MIX) e a testemunha sem inoculação com micorrizas. Para os teores de macro e micronutrientes, 75 dias após a emergência das plantas, diferenças significativas (p < 0,05) foram observadas para os teores de nitrogênio (N), fósforo (P), potássio (K), magnésio (Mg), enxofre (S), manganês (Mn) e boro (B), com destaque para a inoculação com G. clarum e A. morrowiae isoladamente e em dupla inoculação. Para a biomassa da parte aérea e raiz, colonização micorrízica e eficiência micorrízica, as maiores médias (p > 0,005) foram observadas para os tratamentos com G. clarum e A. morrowiae inoculados isoladamente e para a dupla inoculação de S. calospora + G. clarum e G. clarum + A. morrowiae.