No description available.

Arbuscular mycorrhizal fungi (AMF) play an important role in nutrient cycling and growth of flax (Linum usitatissimum L.). However, limited information is available regarding the symbiotic association between flax and AMF in field environments. A study was conducted to survey AMF communities colonizing flax grown in Saskatchewan. Additionally, field and growth chamber studies investigated the impact of AMF inoculation on nutrient uptake and growth of flax. Eighteen commercial flax fields were surveyed to assess mycorrhizal colonization of flax and to assess the impact of agricultural practices and soil abiotic factors on AMF activity. The flax root-associated AMF communities were explored using a 454 sequencing method, together with microscopic-based measurements of root AMF colonization and soil spore density. High levels of root colonization were detected in most flax fields. Of the 222 AMF operational taxonomic units (OTUs) identified in flax roots, 181 OTUs clustered as Funneliformis-Rhizophagus, 19 as Claroideoglomus, 14 as Paraglomus, six as Diversisporales and two as Archaeospora. Results suggest that tillage influenced the composition of AMF communities colonizing flax, and reduced relative AMF abundance and species richness. Additionally, AMF community characteristics were related to soil abiotic factors such as pH, EC, available phosphorus and nitrogen. Field experiments were conducted over two years (two sites per year) using a commercial AMF inoculant applied at three rates (0, 1X, and 2X the recommended rate) with or without P fertilizer (16.8 kg ha-1). The response of flax cultivars to AMF inoculation was examined in a growth chamber experiment. In addition, 454 sequencing was employed to examine the impact of AMF inoculation on root-associated AMF communities. Under field conditions, only one site showed increased root colonization with AMF inoculation. Flax responded to AMF inoculation differently under different field conditions. At the two sites with intermediate initial soil P level, evidence of increased above-ground biomass and plant nutrient uptake with AMF inoculation was observed. However, such an effect was not detected when P fertilizer was combined with the inoculation. At a low P site and an irrigated site, P application accounted for all of the increases in plant nutrient uptake and biomass of flax, whereas no responses to AMF inoculation were detected. The 454 sequencing revealed different inoculation-induced changes in the diversity and composition of root-associated AMF communities between sites, which was possibly related to different field environments and native AMF communities. In the growth chamber, AMF inoculation resulted in general increases of plant nutrient uptake among cultivars, but only one cultivar showed enhanced biomass with inoculation. The diversity of AMF communities colonizing different flax cultivars was generally reduced by AMF inoculation. Community composition shifted under AMF inoculation, and the shifts appeared to be cultivar specific. These results suggested that benefits of AMF inoculation in flax production are limited and currently not predictable, and the degree of response is likely dependent on a myriad of soil and environmental conditions.

Thesis (MSc)--University of Stellenbosch, 2005. / ENGLISH ABSTRACT: The Mountain Fynbos biome, a division of the Cape Floristic Region (CFR), is home to round-leafed Buchu [Agathosma betulina (Berg.) Pillans], one of South Africa’s best-known endangered herbal medicinal plants. Agathosma betulina is renowned as a traditional additive to brandy or tea, which is used for the treatment of a myriad of ailments. In its natural habitat, A. betulina thrives on mountain slopes in acid and highly leached gravelly soils, with a low base saturation and low concentrations of organic matter. To adapt to such adverse conditions, these plants have formed mutualistic symbioses with arbuscular mycorrhizal (AM) fungi. In this study, the effect of indigenous AM taxa on the physiology of A. betulina is investigated. In addition, the AM taxa responsible for these physiological responses in the plant were identified using morphological and molecular techniques. Agathosma betulina was grown under glasshouse conditions in its native rhizosphere soil containing a mixed population of AM fungi. Control plants, grown in the absence of AM fungi, were included in the experimentation. In a time-course study, relative growth rate (RGR), phosphorus (P)-uptake, P utilization cost, and carbon (C)-economy of the AM symbiosis were calculated. The data showed that the initial stages of growth were characterized by a progressive increase in AM colonization. This resulted in an enhanced P-uptake in relation to non-AM plants once the symbiosis was established. Consequently, the lower P utilization cost in AM plants indicated that these plants were more efficient in acquiring P than non-AM plants. When colonization levels peaked, AM plants had consistently higher growth respiration. This indicated that the symbiosis was resulting in a C-cost to the host plant, characterized by a lower RGR in AM plants compared to non-AM plants. Arbuscular mycorrhizal colonization decreased with increasing plant age that coincided with a decline in P-uptake and growth respiration, along with increases in RGR to a level equal to non-AM plants. Consequently, the AM benefit was only observed during the initial stages of growth. In order to identify the AM fungi in planta, morphological and molecular techniques were employed, which indicated colonization by AM fungi belonging to the genera Acaulospora and Glomus. Phylogenetic analyses of a dataset containing aligned 5.8S ribosomal RNA gene sequences from all families within the Glomeromycota, including sequences obtained during the study, supported the above mentioned identification. / AFRIKAANSE OPSOMMING: Die Fynbos bergbioom, ‘n onderafdeling van die Kaapse Floristiese Streek, huisves rondeblaar Boegoe [Agathosma betulina (Berg.) Pillans], een van Suid Afrika se bekendste bedreigde medisinale plante. Agathosma betulina is bekend vir sy gebruik as tinktuur vir die behandeling van verskeie kwale. Die plant kom voor in bergagtige streke, in suur en mineraal-arm grond, met ‘n lae organiese inhoud. Gevolglik, om aan te pas by hierdie ongunstige kondisies, vorm die plante simbiotiese assosiasies met blaasagtige, struikvormige mikorrisa (BSM). In die huidige studie is die effek van hierdie BSM op die fisiologie van A. betulina ondersoek. Die identiteit van die BSM is ook gevolglik met morfologiese en molekulêre identifikasie tegnieke bepaal. Agathosma betulina plante is onder glashuis kondisies in hul natuurlike grond gekweek, wat ‘n natuurlike populasie van BSM bevat het. Kontroles is ook in die eksperiment ingesluit en hierdie stel plante is met geen BSM geïnokuleer nie. Gevolglik is die relatiewe groeitempo, fosfor opname, fosfor verbuikerskoste asook die koolstof ekonomie van die plante bereken. Die data het getoon dat die eerste groeifase gekarakteriseer is deur toenames in BSM kolonisasie vlakke. Dit het tot ‘n hoër fosfor opname in BSM geïnokuleerde plante gelei. Die laer fosfor verbuikerskoste gedurende hierdie fase het aangedui dat die plante wat geïnokuleer is met BSM oor beter meganismes beskik het om fosfor uit die grond te bekom. Toe BSM kolonisasie vlakke gepiek het, was groei respirasie hoër in BSM geïnokuleerde plante as in die kontroles. Dit het aangedui dat die BSM kolonisasie van plante tot hoër koolstof kostes vir hierdie plante gelei het, wat weerspieël is in die laer groeitempo van die BSM geïnokuleerde plante. Die BSM kolonisasie vlakke het gedaal met toenemende ouderdom van hul gasheer plante, wat gekarakteriseer is deur ‘n laer opname van fosfor en laer groei respirasie, tesame met ‘n toename in relatiewe groeitempo tot vlakke soortgelyk aan die van die kontrole plante. Die BSM voordele vir die plant is dus net gedurende die eerste groeifase waargeneem. Die BSM wat verantwoordelik is vir hierdie fisiologiese veranderinge is gevolglik geïdentifiseer met behulp van morfologiese en molekulêre tegnieke en dit is gevind dat BSM wat behoort tot die genera Acaulospora en Glomus binne hierdie plante voorkom. Filogenetiese analise gegrond op opgelynde 5.8S ribosomale RNA geen volgordes afkomstig van al die families binne Glomeromycota asook volgordes gevind in die studie, het die bogenoemde identifikasie gestaaf.

Podocarpus cunninghamii is an endemic New Zealand conifer that, in pre-human times, formed extensive forest communities across the eastern South Island high country. Anthropogenic disturbances have reduced the distribution of Podocarpus cunninghamii communities such that they now exist mainly as small and isolated remnants within a highly modified, predominantly pastoral landscape. Very little is known of the ecology of high country Podocarpus cunninghamii communities, and without this information it is not possible to develop an ecological basis for their restoration. This thesis explores the ecology of Podocarpus cunninghamii in the eastern South Island high country, investigating factors that potentially affect the restoration of Podocarpus cunninghamii within this environment, with special attention paid to the role of arbuscular mycorrhizal fungi (AMF). Field investigations of Podocarpus cunninghamii communities showed that they contain a high degree of floristic and structural variation determined by soil and climatic variables. Analysis of age and size class distributions suggest that Podocarpus cunninghamii has more than one regeneration strategy, and can regenerate within intact forest following the opening of small canopy gaps or can undergo large-scale recruitment following catastrophic disturbance. Field and glasshouse experiments investigating growth and nutrient responses of Podocarpus cunninghamii to different AMF inoculants found that Podocarpus cunninghamii responses are dependent on both AMF type and grass competition. Finally, investigation of Podocarpus cunninghamii carbon stocks showed that they are less than that of other New Zealand forest types, but are greater than that of grazed pastures. Successful restoration of high country Podocarpus cunninghamii communities will require the incorporation of associated species based on local environmental conditions, and will also need to allow for disturbance processes. AMF may have an important role to play in restoration by reducing seedling production times and by increasing the competitiveness of Podocarpus cunninghamii when in competition with exotic grasses.

Soil phosphorus (P) is often unavailable in SK soils due to immobilization by microbial biomass and complexation with cations. The prohibition of synthetic fertilizer use in organic systems means farmers rely on crop rotation or approved inputs to supply P for crops. Legumes in crop rotation add P to soil through decomposition and deposition, and approved fertilizers such as bone meal (BM), rock phosphate (RP), and composted manure add P to soil through dissolution. Arbuscular mycorrhizal fungi (AMF) improve crop access to soil P. The fungi colonize roots of host crops, allowing roots to reach immobile pockets of soil P. Colonization by AMF is usually decreased or delayed following partial fallow periods, non-host plants, and the addition of soluble P fertilizers. This thesis consists of two studies. For the first, the effects of crop rotation were tested on AMF colonization and soil P dynamics. Colonization by AMF of mycorrhizal crops was examined following a non-mycorrhizal crop, a partial fallow period, and mycorrhizal crops. All crops were colonized evenly (63-70%) at flowering despite non-mycorrhizal and partial fallow periods, and the sequence most depleted in soil N (wheat-barley) had the lowest colonization in August (36%). The second study evaluated soil P and plant N and P after applications of BM, hydroxyapatite (HAP), and sheep manure compost. Compost application increased plant P uptake compared to the control (1.26 vs. 0.71 mg pot-1), while applications of BM and HAP alone did not. Compost application did not affect AMF colonization of wheat (Triticum aestivum L.). Overall this research highlights the importance of legumes and composted manure use in organic systems. Legume use in crop rotation simultaneously increased soil P deposition and may have preserved AMF communities despite fallow periods and non-host crops in rotation. Conditions normally affecting AMF colonization in conventional systems did not apply. The use of composted manure in the greenhouse study resulted in the greatest P uptake and concentrations in wheat. Thus the use of legumes and composted manure may increase P availability to crops directly and indirectly: directly through soil P deposition and perhaps indirectly through the preservation of AMF communities.

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

Les espèces du groupe Piptadenia sont des légumineuses endémiques du Brésil, dont la plupart sont des arbres capables de se développer sur des sols peu fertiles faisant d'eux de bons candidats pour le reboisement des terres dégradées. Les Piptadenia établissent une symbiose à la fois avec des champignons mycorhiziens à arbuscules (CMA) et des rhizobiums. Ces espèces sont proches du genre Mimosa, connu pour son affinité pour les symbiotes du genre Burkholderia. Dans ce travail de thèse nous décrivons la biodiversité des symbiotes rhizobiums associés au groupe Piptadenia, et élargissons l'affinité des Burkholderia à ce groupe de légumineuses. Les études phylogénétiques sur des marqueurs neutres et symbiotiques montrent une origine stable et ancienne de la symbiose Burkholderia/Mimoseae. Les études de spécificité d'association entre espèces de Burkholderia et espèces de Piptadenia montrent que cette dernière est lâche, les patterns d'association étant davantage liées aux sites prospectés au Brésil plutôt qu'à une sélection par l'hôte. Dans un second temps, nous avons étudié l'association tripartite entre plusieurs génotypes de Burkholderia, un CMA (Glomus clarum), et l'espèce Piptadenia gonoacantha, décrite dans la littérature comme formant une nodulation mycorhize-dépendante. Nos travaux montrent que la nodulation n'est pas CMA-dépendante, mais par contre l'efficience symbiotique des nodules dépend de la mycorhization pour certains génotypes de Burkholderia. Nous décrivons également des interactions entre symbiose rhizobienne et mycorhizienne au sein des nodules (présence du CMA dans les nodules avec sporulation dans certaines combinaisons de symbiotes). Ces travaux soulèvent la nécessité de prendre en compte les interactions génotype-génotype entre symbiotes rhizobiens et mycorhiziens lors de la sélection des inoculums dans le cadre des programmes de revégétalisation au Brésil par des arbres du groupe Piptadenia. / The Piptadenia group comprise endemic species from Brazil of which many are trees able to develop on poorly fertile soils and are good candidates for revegetation programs. Piptadenia species establish symbioses with both arbuscular mycorrhizal fungi (AMF) and rhizobia. These species are phylogenetically close to the Mimosa genus, known for its affinity for Burkholderia rhizobial symbionts. In this thesis we describe the biodiversity of rhizobial symbionts associated to the Piptadenia group, and enlarge the affinity towards Burkholderia to this group of legumes. Phylogenetic studies on neutral and symbiotic markers show a stable and ancient symbiosis Burkholderia/Mimoseae. Specificity studies between Burkholderia and Piptadenia group species show that specificity is not strong, and that patterns of associations between partners are isolation site dependent rather than linked to the host legume. In the second part of this thesis we have studied the tripartite association between several Burkholderia genotypes, an AMF (Glomus clarum), and Piptadenia gonoacantha (Pg), a legume species described as making an AMF-dependent nodulation (Jesus et al., 2005). Our experiments show that nodulation in Pg is not AMF-dependent, but that symbiotic efficiency of nodules rely on AMF presence for specific Burkholderia genotypes. We also describe interactions between rhizobial and mycorrhizal symbiosis (AMF presence in nodules, with sporulation in several symbionts combinations). Our work underlines the necessity to consider genotype-genotype interactions between rhizobial and AMF symbionts for the selection of synergistic inoculums in revegetation programs using Piptadenia group species in Brazil.

A Araucaria angustifolia possui alto valor madeireiro e resinífero, é fonte de alimento e espécie característica da Floresta Ombrófila Mista, um ecossistema rico em espécies endêmicas e detentor de alta diversidade. Devido à intensa exploração durante décadas, atualmente a araucária é considerada espécie em perigo crítico de extinção, sendo fundamental a compreensão deste ecossistema para sua preservação e regeneração. As minhocas, os fungos micorrízicos arbusculares (FMA) e as bactérias diazotróficas são organismos edáficos que desempenham funções essenciais no processo de ciclagem de nutrientes e podem favorecer o desenvolvimento vegetal. Muitos trabalhos avaliam o efeito de cada um destes organismos separadamente, mas o estudo das interações entre eles é escasso. Portanto, este trabalho teve como objetivo avaliar o efeito da inoculação de minhocas invasoras Amynthas corticis, do FMA Gigaspora rosea e de um isolado de bactéria diazotrófica sobre o desenvolvimento de mudas de araucária, considerando suas interações. O experimento foi realizado em vasos, em casa de vegetação, sob delineamento em blocos casualizados, em esquema fatorial 2x2x2, sendo: presença e ausência de minhoca (A. corticis), presença e ausência de FMA (G. rosea), e presença e ausência de bactéria diazotrófica (isolado S32274), com oito repetições cada, sendo que a metade dos vasos foi avaliada 150 dias após o transplantio (Época 1) e o restante aos 250 dias após o transplantio (Época 2) das mudas de araucária. Os parâmetros de avaliação foram: altura e diâmetro das plantas, massa seca das raízes e da parte aérea, concentração e conteúdo total de nitrogênio e fósforo da parte aérea, densidade de esporos de G. rosea no substrato, taxa de colonização radicular pelo FMA e sobrevivência, reprodução e massa fresca das minhocas. A biomassa e o conteúdo de N e P das plantas foram afetados pela interação dos organismos. A inoculação de G. rosea propiciou melhor desenvolvimento das mudas de araucária, entretanto, não houve efeito significativo da inoculação de somente diazotróficos. As plantas inoculadas com minhocas apresentaram tendência de menor biomassa e a sobrevivência das minhocas foi baixa. O estudo das interações de microrganismos e macrofauna deve ser considerado na pesquisa e desenvolvimento de práticas de manejo mais sustentáveis para reflorestamento e manutenção de florestas de araucária. / Araucaria angustifolia is a tree of high lumber and resin values, a source of food, and the most important tree in the Mixed Subtropical Ombrophilous forest, a rich ecosystem with many endemic species. However, due to intense exploration for decades, the Araucaria is presently considered a critically endangered species, and the understanding of this ecosystem is fundamental for conservation and regeneration purposes. Soil organisms such as earthworms, arbuscular mycorrhizal fungi (AMF) and diazotrophic bacteria play an essential role in nutrient cycling and can increase plant performance. Many studies have evaluated the impact of each of these organisms independently, but few have studied their interactions. Therefore, the aim of this study was to evaluate the inoculation effects of invasive earthworms (Amynthas corticis), AMF (Gigaspora rosea) and an isolated of diazotrophic bacteria, and the interactions between each of these, on the performance of A. angustifolia seedlings. The plants were grown in greenhouse conditions in a block-type randomized design with a factorial layout of 2x2x2, being: presence and absence of earthworms (A. corticis), presence and absence of AMF (G. rosea) and presence and absence of diazotrophic bacteria (isolated S32274), in eight replicates, evaluated at 150 and 250 d after transplanting. The following parameters were evaluated: plant height and diameter, shoot and root biomass, shoot nutrient content (P, N), number of AMF spores, root mycorrhizal colonization rate and earthworm survival, reproduction and fresh biomass. Plant biomass and shoot nutrient content were affected by the three-way interaction of earthworms, AMF and bacteria. AMF inoculation enhanced A. angustifolia performance, while bacterial inoculation alone had no significant effect on plants and earthworms had low survival rates and tended to reduce plant biomass. Microbial-faunal interactions can lead to important changes in plant growth and should be considered in future research and development of sustainable Araucaria forest management practices.

As plantas apresentam diversas adaptações fisiológicas à baixa disponibilidade de fósforo (Pi) do solo. Este trabalho discute os custos fisiológicos e energéticos associados com essas estratégias, focado nas respostas da cana-de-açúcar (Saccharum spp.) à disponibilidade de Pi durante a simbiose com micorrizas arbusculares (Glomus clarum). Esses custos são importantes componentes para a adaptação a solos com baixo Pi, afetando a aquisição e conteúdo de fósforo; o crescimento e concentração de açúcares em tecidos vegetais. Plantas de cana-de-açúcar foram cultivadas em vasos com ou sem micorrizas (Glomus clarum), e sob a disponibilidade de baixo (20 mg kg-1) ou alto (202 mg kg-1) fósforo. Raízes e parte-aérea foram coletadas para as análises após 14, 30, 44 e 58 dias pós-inoculação (dpi) com Glomus clarum . A condição de BP causou a deficiência de Pi nas plantas, micorrízicas ou não. As plantas sob AP continham um teor foliar de Pi adequado, e partir dos 44 dpi acumularam pelo menos 6 vezes mais Pi parte-aérea, do que as cultivadas sob BP, efeito mais evidente nas micorrízicas. A eficiência de absorção, indicada pelo acúmulo de Pi na parte-aérea a uma dada biomassa da raiz, foi igual para todos os tratamentos, sugerindo que as eficiências radicular e micorrízica da absorção de Pi foram similares, independentemente da doses de Pi. A disponibilidade de fósforo não afetou a biomassa total das plantas, sendo as cultivadas sob BP mais eficientes na utilização deste nutriente. Por outro lado, as plantas micorrízicas suplementadas com BP apresentaram maior crescimento da raiz e redução na parte-aérea, resultando no aumento da proporção raiz:parte-aérea. Aos 58 dpi, a glicose, frutose e sacarose presente nas folhas de plantas micorrízicas foi 3,8, 2,3 e 2,4 vezes respectivamente mais concentrada do que nas não micorrízicas. Esses resultados sugerem que, nestas condições experimentais, o estabelecimento da simbiose não foi uma associação mutualística típica, afetando o perfil de crescimento e a alometria da cana cultivada com BP. As concentrações de fotoassimilados na folhas de planta micorrízicas indicam que houve aumentos nas taxas fotossintéticas, mas isso não resultou no maior crescimento do macrosimbionte. A tecnologia de amplificação quantitativa de transcritos reversos (RT-PCR) se tornou uma opção para a validação funcional de genes, com alta sensibilidade, acurada quantificação e eficácia. A quantificação relativa da expressão gênica é conseqüentemente fácil e determina a expressão de um gene em relação a outro expresso e relativamente constante. Neste trabalho foi analisada a variabilidade de expressão dos genes de cana-de-açúcar codificando a actina (Actina), gliceraldeido fosfato desidrogenase (GAPDH), tubulina (Tubulina), e ubiquitinas (UbiQ1 e UbiQ2) em diversos tecidos, e comparou-se a variabilidade obtida utilizando os programas Genorm e NormFinder. Em seguida, foram realizadas análises de expressão gênica utilizando o programa REST para a validação estatística da expressão de genes de cana-de-açúcar. O gene UbiQ1 foi mais estável nos tecidos ou órgãos testados: meristema, inflorescência, folha, colmo e raízes tratadas com alto e baixo fósforo. Tendo o gene UbiQ1 como referência, a expressão relativa dos genes transportadores de fosfato de alta afinidade de cana-de-açúcar PT7 e PT8 foi avaliada em amostras de raiz fertilizadas com alto ou baixo Pi, inoculadas ou não com fungo micorrízico e coletadas aos 58 dpi. Esses dois genes pertencem à família Pht1 de transportadores de fosfato e são similares aos ortólogos de arroz ORYsa;tPht1;7 e ORYsa;Pht1;8. Sob a deficiência de Pi, o transportador de fosfato PT7 foi induzido em raízes não colonizadas; já nas micorrízicas foi pouco expresso, sendo que altas taxas de colonização radicular suprimiram a expressão do PT7. O gene PT8 pouco variou sua expressão, sendo sutilmente mais expresso em plantas micorrízicas do que nas não micorrízicas sob o suprimento de BP. Estes resultados indicam que o PT7 é induzido em raízes sob estresse por fósforo e provavelmente associado à absorção radicular de Pi. Enquanto o gene PT8 possui uma modulação pouco variável provavelmente envolvido na manutenção do fluxo ou homeostase de Pi, possivelmente associado com a absorção radicular e micorrízica de fosfato. O PT7 e o PT8 foram expressos em tratamentos de médio/longo prazo, apresentando expressão ou indução em resposta a privação por Pi, o que é consistente com a função proposta de aquisição e mobilização de Pi para esta família de transportadores. A cana-de-açúcar micorrízica mostrou alta plasticidade de resposta ao BP / Plants display a wide array of physiological adaptations to low soil phosphorus (Pi) availability. This work discussed physiological and energetic costs associated with these strategies, focusing on sugarcane responses to Pi availability during the development of arbuscular mycorrhizal (AM) symbiosis. Such costs are important components of adaptation to low phosphorus soils affecting phosphorus acquisition and leve, growth and soluble sugars concentration in plant tissues. Sugarcane plants were grown in pots, with or without AM (Glomus clarum), and with low (20 mg kg-1) or high (200 mg kg-1) phosphorus supply. Roots and shoots were harvest for analysis after 14, 30, 44 and 58 days post-inoculation (dpi) with the fungus Glomus clarum,. The low Pi supply caused Pi deficiency in mycorrhizedl or non-mycorrhizedl plants. The efficiency of Pi absorption, indicated by shoot Pi accumulation in correlation to root biomass, suggested that root and mycorrhizal Pi absorption were similar, regardless of the Pi doses. Phosphorus availability did not affect the whole-plant biomass, and plants under low Pi supply, used more efficiently this nutrient. On the other hand, mycorrhized plants supplemented with low Pi presented the highest root growth and shoot reduction, resulting in high root:shoot ratio. At 58 dpi, glucose, fructose and sucrose concentrations in leaves of mycorrhized plants were 3.8, 2.3 and 2.4-fold higher respectively, than in non-mycorrhizedl plants. These results suggested that, under these experimental conditions, mycorrhizal symbiosis establishment was not a typical mutualistic association affecting sugarcane growth profile and allometry, when cultivated under low Pi. The photosynthate levels of leaves from mycorrhizd plants indicated an increase in photossynthetic rate but withut resulting in higher macrobiont growth. The quantitative amplification of reversed transcripts (RT-PCR) technology has become a method of choice for functional gene validation, with sensitiveness, accurate quantification and high-throughput. The relative quantification is easier determined by relative expression in comparison to a constitutively expressed refernce gene. Here, the expression variability of a sugarcane actin gene (Actin) glyceraldehydo phosphate dehydrogenase (GAPDH), tubulin (Tubulina), and ubiquitins (UbiQ1 and UbiQ2) from various tissues were analysed and compared based on the ranking list from the Genorm and NormFinder softwares. Expression analysis based on the REST software gave the proper statistic validation. UbiQ1 was the most stable gene among the candidate gen-references along the various tissues or organs tested: meristem, inflorescence, leaf, stem and roots treated with high and low phosphorus. Considering UbiQ1 as the reference gene, the relative expression of the sugarcane high-affinity phosphate transporters genes PT7 and PT8 were assessed from roots fertilized with low Pi or high Pi , inoculated or not with the mycorrhizal fungus, harvest 58 dpi. Both genes belong to the Pht1 Pi transporter and share similarity with the rice orthologs ORYsat;Pht1;7 and ORYsat;Pht1;8. Under Pi deficiency, the phosphate transporter PT7 was induced in non-colonized roots, but less expressed in mycorrhizedl ones, with high root colonization rate suppressing PT7 expression. PT8 showed low variability in expression, slightly more expressed in mycorrhized plants than in non-mycorhized plants under low Pi supply. These results indicated that PT7 was induced in Pi stressed roots, and possibly associated with the root Pi uptake, while PT8 had limited modulation in expression and probably involved on Pi fluxes or homeostasis, likely associated with both root and mycorrhizal phosphate uptake pathways. PT7 and PT8 were induced during medium/long-term treatments, showing induction or constant expression in both acquisition and mobilization of Pi in response to Pi deprivation, which is consistent with the proposed role of this tranporter family. Mycorrhizedl sugarcane showed a highly plastic response to low Pi

A formação de micorrizas arbusculares é um processo regulado geneticamente e envolve alterações da expressão gênica da planta e do fungo. Poucos genes essenciais para o desenvolvimento da simbiose foram identificados até o presente. Para identificar genes possivelmente envolvidos no controle da colonização fúngica intrarradicular, o perfil de seqüências expressas em raízes de cana-de-açúcar colonizadas por Glomus clarum, em condições de baixo e alto fósforo (P), foi avaliado através do seqüenciamento em larga escala de ESTs e hibridização em macroarranjos de cDNAs. Plântulas micropropagadas de cana-de-açúcar foram plantadas em vasos contendo substrato composto por areia:vermiculita (3:1, vol/vol) e adubado com 20 ou 200 mg P kg-1 de substrato e inoculadas com Glomus clarum. A biomassa seca da parte aérea e a taxa de colonização micorrízica das raízes foram avaliadas no momento da colheita que foi feita oito ou doze semanas após o plantio. O RNA poliA foi extraído das raízes e utilizado para a síntese de cDNAs. Neste trabalho, quatro bibliotecas de cDNA e uma biblioteca subtrativa supressiva foram preparadas a partir de cDNAs de raízes de cana-de-açúcar colonizadas por G. clarum. Esta abordagem resultou na identificação de 1,925 ESTs as quais foram agrupadas em 1615 genes supostamente regulados pela micorrização. Estes clones foram analisados por Northern blots eletrônico, e aqueles que foram diferencialmente expressos foram submetidos a hibridização em macroarranjos de cDNAs. Um total de 386 genes foram arranjados em membranas de náilon e sua expressão avaliada por sondas sintetizadas a partir de cDNA extraído de raízes em diferentes condições. Dentre os genes com expressão diferencial significativa estatística em raízes colonizadas por G. clarum, em condições de baixo e alto P, foram detectados genes codificando proteínas putativamente envolvidas na percepção de moléculas sinais (receptor tipo quinase de proteína), transporte de íons (canais de íons), transdução de sinais (quinases de proteína e calmodulina), regulação da transcrição (fatores de transcrição), alterações de parede celular e citoesqueleto (extensina, arabinogalactanas, tubulinas), respostas de defesa e estresse (síntese de fitoalexinas e metalotioneínas), síntese de fitohormônios (nitrilase). Os dados sugerem que a transdução de sinais em MAs se dá através da fosforilação de proteínas e que a indução de respostas anti-oxidantes pode ser importante para o desenvolvimento da simbiose. Da mesma forma, os dados sugerem que a atividade de aspartatoproteases podem ser essenciais para o controle do crescimento fúngico intrarradicular. A caracterização desses genes e seus padrões de expressão em MAs poderá elucidar os mecanismos genéticos envolvidos no controle da simbiose. / The formation of arbuscular mycorrhizae (AM) is a genetically regulated process and involves alteration in both plant and fungus gene expression. Few genes essential for the development of the symbioses have been identified so far. In order to identify genes possible involved in the intraradical fungal colonization control, the profiles of expressed sequences in sugarcane roots colonized roots with Glomus clarum at low or high phosphate (P) conditions were evaluated using large scale EST sequencing and cDNA macroarray hybridization. Sugarcane micropropagated seedlings were planted in pots containing sand:vermiculite (3:1, vol:vol), and fertilized with 20 or 200 mg P kg-1 substrate. Seedlings were inoculated with G. clarum and cultivated under greenhouse conditions for 8 or 12 weeks. After harvesting shoot dry weight and root colonization rates were evaluated. PolyA RNA was extracted from the root tissues and used for cDNA synthesis. In this work, one suppressive subtractive library and four cDNA libraries were synthesized using cDNA from sugarcane roots not-colonized or colonized by G. clarum, at low and high P conditions. This approach resulted in the identification of 1,925 ESTs which were clustered in 1,615 genes. The expression of these genes was evaluated using electronic Northern blots. A total of 386 genes with putative differential expression were spotted into nylon membranes in macroarrays and their expression profiles in roots under different conditions were evaluated using macroarray hybridization. Among the genes with statistically significant differential expression, in roots colonized by G. clarum grown at low or high P conditions, it was detected genes encoding proteins putatively involved in the perception of signal molecules (receptor-like protein kinase), ion transporte (channel-like protein precursor), signal transduction (protein kinases and calmodulin), transcriptional regulation (transcription factor), cell wall and cytoskeleton alterations (extensin, arabinogalactans, and tubulins), defense and stress responses (phythoalexins synthesis and metalothioneins), and phytohormone biosynthesis (nitrilases). Our data suggest that the signal transduction in AM occurs through protein phosphorylation, and that the induction of anti-oxidant responses may be important for the development of the symbioses. Additionally, the data suggest that aspartic-proteases might be essential for the control of intraradical fungal growth. Further characterization of these genes and their expression patterns in AM would contribute to elucidate the genetic mechanisms controlling the symbioses.