Atividade ATPásica e pirofosfatásica em microssomos de raízes de milho colonizadas com fungos micorrízicos arbusculares

No presente trabalho, foi avaliada a influência de duas espécies de fungos micorrízicos arbusculares (FMAs) Glomus clarum e Gigaspora margarita sobre as atividades ATPásica e pirofosfatásica de microssomos obtidos por meio de fracionamento celular de raízes de milho colonizadas aos 20, 30, 40 e 60 dias do plantio. Ambos os fungos proporcionaram aumentos significativos nas atividades das ATPases e pirofosfatases; entretanto, as maiores atividades foram observadas nas raízes colonizadas pelo fungo G. clarum. Os dados cinéticos indicaram a presença de uma ativação diferencial das H+-ATPases e H+-pirofosfatases presentes nas membranas das células radiculares, dependendo da espécie fúngica e do estádio da colonização das raízes pelos FMAs. Como indicadores da eficiência da micorrização, foram avaliados a altura e o conteúdo de nutrientes na parte aérea das plantas. Os dados obtidos mostraram a primeira evidência cinética de estimulação de atividade pirofosfatásica em membranas microssomais de raízes colonizadas e descreveram um padrão inédito de ativação diferencial para a hidrólise de ATP, dependendo da espécie (G. clarum ou G. margarita) e do estádio de colonização.

Does wheat genetically modified for disease resistance affect root-colonizing pseudomonads and arbuscular mycorrhizal fungi?

This study aimed to evaluate the impact of genetically modified (GM) wheat with introduced pm3b mildew resistance transgene, on two types of root-colonizing microorganisms, namely pseudomonads and arbuscular mycorrhizal fungi (AMF). Our investigations were carried out in field trials over three field seasons and at two locations. Serial dilution in selective King's B medium and microscopy were used to assess the abundance of cultivable pseudomonads and AMF, respectively. We developed a denaturing gradient gel electrophoresis (DGGE) method to characterize the diversity of the pqqC gene, which is involved in Pseudomonas phosphate solubilization. A major result was that in the first field season Pseudomonas abundances and diversity on roots of GM pm3b lines, but also on non-GM sister lines were different from those of the parental lines and conventional wheat cultivars. This indicates a strong effect of the procedures by which these plants were created, as GM and sister lines were generated via tissue cultures and propagated in the greenhouse. Moreover, Pseudomonas population sizes and DGGE profiles varied considerably between individual GM lines with different genomic locations of the pm3b transgene. At individual time points, differences in Pseudomonas and AMF accumulation between GM and control lines were detected, but they were not consistent and much less pronounced than differences detected between young and old plants, different conventional wheat cultivars or at different locations and field seasons. Thus, we conclude that impacts of GM wheat on plant-beneficial root-colonizing microorganisms are minor and not of ecological importance. The cultivation-independent pqqC-DGGE approach proved to be a useful tool for monitoring the dynamics of Pseudomonas populations in a wheat field and even sensitive enough for detecting population responses to altered plant physiology.

Arbuscular mycorrhizal fungi: genetics of multigenomic, clonal networks and its ecological consequences

Arbuscular mycorrhizal fungi are thought to have remained asexual for 400 million years although recent studies have suggested that considerable genetic and phenotypic variation could potentially exist in populations. A brief discussion of these multigenomic organisms is presented. (C) 2003 The Linnean Society of London.

Identification and isolation of two ascomycete fungi from spores of the arbuscular mycorrhizal fungus Scutellospora castanea.

Two filamentous fungi with different phenotypes were isolated from crushed healthy spores or perforated dead spores of the arbuscular mycorrhizal fungus (AMF) Scutellospora castanea. Based on comparative sequence analysis of 5.8S ribosomal DNA and internal transcribed spacer fragments, one isolate, obtained from perforated dead spores only, was assigned to the genus Nectria, and the second, obtained from both healthy and dead spores, was assigned to Leptosphaeria, a genus that also contains pathogens of plants in the Brassicaceae. PCR and randomly amplified polymorphic DNA-PCR analyses, however, did not indicate similarities between pathogens and the isolate. The presence of the two isolates in both healthy spores and perforated dead spores of S. castanea was finally confirmed by transmission electron microscopy by using distinctive characteristics of the isolates and S. castanea. The role of this fungus in S. castanea spores remains unclear, but the results serve as a strong warning that sequences obtained from apparently healthy AMF spores cannot be presumed to be of glomalean origin and that this could present problems for studies on AMF genes.

Effects of arbuscular mycorrhizal fungi and phosphorus fertilization on post vitro growth of micropropagated Zingiber officinale roscoe

The rhizomes of Zingiber officinale Roscoe (ginger) are widely used for their medicinal and flavoring properties, whereas the influence of root symbionts on their growth is poorly understood. In this study, the effects of phosphate fertilization and inoculation with a mixture of arbuscular mycorrhizal fungi (AMF) (isolates Glomus clarum RGS101A, Entrophospora colombiana SCT115A and Acaulospora koskei SPL102A) on survival, growth and development of micropropagated ginger were investigated. After transplanting to post vitro conditions, the ginger microplants were subjected to the following treatments: a) AMF mixture, b) P addition (25 mg kg-1), c) AMF + P, and d) non-mycorrhizal control without P addition. After eight months of growth, survival ranged from 86 to 100 % in the AMF and AMF+P treatments versus 71 % survival in control and P treatments. In the AMF, P and AMF+P treatments, the shoot, root and rhizome biomass production were significantly larger than in the control plants. In the non-mycorrhizal control plants the leaf number, leaf area, number of shoots/plants, and shoot length were significantly lower than in the AMF, P and AMF+P treatments. Root colonization ranged from 81 to 93 % and was not affected by P application. The data confirmed the response of several growth variables of micropropagated ginger to mycorrhizal colonization and P addition.

Intraspecific genetic variation in arbuscular mycorrhizal fungi and its consequences for molecular biology, ecology, and development of inoculum

It has been known for some time that different arbuscular mycorrhizal fungal (AMF) taxa confer differences in plant growth. Although genetic variation within AMF species has been given less attention, it could potentially be an ecologically important source of variation. Ongoing studies on variability in AMF genes within Glomus intraradices indicate that at least for some genes, such as the BiP gene, sequence variability can be high, even in coding regions. This suggests that genetic variation within an AMF may not be selectively neutral. This clearly needs to be investigated in more detail for other coding regions of AMF genomes. Similarly, studies on AMF population genetics indicate high genetic variation in AMF populations, and a considerable amount of variation seen in phenotypes in the population can be attributed to genetic differences among the fungi. The existence of high within-species genetic variation could have important consequences for how investigations on AMF gene expression and function are conducted. Furthermore, studies of within-species genetic variability and how it affects variation in plant growth will help to identify at what level of precision ecological studies should be conducted to identify AMF in plant roots in the field. A population genetic approach to studying AMF genetic variability can also be useful for inoculum development. By knowing the amount of genetic variability in an AMF population, the maximum and minimum numbers of spores that will contain a given amount of genetic diversity can be estimated. This could be particularly useful for developing inoculum with high adaptability to different environments.

Genetic exchange in an arbuscular mycorrhizal fungus results in increased rice growth and altered mycorrhiza-specific gene transcription.

Arbuscular mycorrhizal fungi (AMF) are obligate symbionts with most terrestrial plants. They improve plant nutrition, particularly phosphate acquisition, and thus are able to improve plant growth. In exchange, the fungi obtain photosynthetically fixed carbon. AMF are coenocytic, meaning that many nuclei coexist in a common cytoplasm. Genetic exchange recently has been demonstrated in the AMF Glomus intraradices, allowing nuclei of different Glomus intraradices strains to mix. Such genetic exchange was shown previously to have negative effects on plant growth and to alter fungal colonization. However, no attempt was made to detect whether genetic exchange in AMF can alter plant gene expression and if this effect was time dependent. Here, we show that genetic exchange in AMF also can be beneficial for rice growth, and that symbiosis-specific gene transcription is altered by genetic exchange. Moreover, our results show that genetic exchange can change the dynamics of the colonization of the fungus in the plant. Our results demonstrate that the simple manipulation of the genetics of AMF can have important consequences for their symbiotic effects on plants such as rice, which is considered the most important crop in the world. Exploiting natural AMF genetic variation by generating novel AMF genotypes through genetic exchange is a potentially useful tool in the development of AMF inocula that are more beneficial for crop growth.

Arbuscular mycorrhizal inoculation increases biomass of Euterpe edulis and Archontophoenix alexandrae after two years under field conditions

Inoculation with arbuscular mycorrhizal fungi (AMF) of tree seedlings in the nursery is a biotechnological strategy to improve growth, survival after transplanting, biomass production and to reduce the use of fertilizers. Archontophoenix alexandrae and Euterpe edulis are palm species used in southern Brazil to produce the palm heart, the latter being included in the list of threatened species due to the overexploitation of its native population. The purpose of this paper was to evaluate the effect of mycorrhizal inoculation on growth and physiological parameters of A. alexandrae and E. edulis. After germination, the seedlings were inoculated (AMF) or not (CTL) with AMF in the treatments. Values of chlorophyll content, biomass and shoot phosphorus were not statistically different between the AMF and CTL treatments, after five months in the greenhouse. Inoculation with AMF significantly increased the levels of starch and soluble carbohydrates in shoots and roots of both species. Under field conditions, AMF had no effect on stem diameter and height after 12 and 24 months, but total plant biomass and leaf, stem and root biomass were greater in AMF than in CTL plants. The data indicated that AMF inoculation in the nursery has a strong effect on biomass accumulation after growing for 24 months under field conditions. Therefore, AMF inoculation should be considered an important strategy to increase growth and production of these economically important tropical palm species.

Micorriza arbuscular e a tolerância das plantas ao estresse

Fungos micorrízicos arbusculares (FMAs) são fungos de solo, biotróficos obrigatórios e formadores da simbiose mutualista mais comum na natureza: a micorriza arbuscular (MA). Essa associação ocorre nas raízes da maioria das plantas terrestres, promovendo melhorias no crescimento, desenvolvimento e aumento na tolerância e, ou, resistência das plantas a vários agentes ambientais adversos. Além disso, os FMAs podem ser utilizados como potenciais agentes de controle biológico de doenças de plantas. Esses fungos produzem ainda glomalina, uma proteína que desempenha papel fundamental na estabilidade do solo e bioestabilização de solos contaminados. As diferentes respostas das plantas a essa simbiose podem ser atribuídas à diversidade funcional das MAs, em função da interação FMA-planta-condições ambientais. O estabelecimento e funcionamento da MA durante as condições de estresse envolvem um complexo processo de reconhecimento e desenvolvimento, concomitantemente às alterações bioquímicas, fisiológicas e moleculares em ambos os simbiontes. Além disso, a colonização micorrízica das raízes tem impacto significativo na expressão de genes de diversas plantas que codificam proteínas presumivelmente envolvidas na tolerância ao estresse. Nesse contexto, considerando que os FMAs são essenciais no estabelecimento e adaptação das plantas em locais perturbados, nesta revisão são abordados os mecanismos fisiológicos e moleculares da associação MA responsáveis por essa adaptação e pela maior tolerância das plantas ao estresse.

Molecular players shaping the arbuscular mycorrhizal symbiosis of rice

SUMMARY : The arbuscular mycorrhizal (AM) symbiosis is an evolutionarily ancient association between most land plants and Glomeromycotan fungi that is based on the mutual exchange of nutrients between the two partners. Its structural and physiological establishment is a multi-step process involving a tightly regulated signal exchange leading to intracellular colonization of roots by the fungi. Most research on the molecular biology and genetics of symbiosis development has been performed in dicotyledonous model legumes. In these, a plant signaling pathway, the common SYM pathway, has been found to be required for accommodation of both root symbionts rhizobia and AM fungi. Rice, a monocotyledon model and the world's most important staple crop also forms AM symbioses, has been largely ignored for studies of the AM symbiosis. Therefore in this PhD work functional conservation of the common SYM pathway in rice was addressed and demonstrated. Mycorrhiza-specific marker genes were established that are expressed at different stages of AM development and therefore represent readouts for various AM-specific signaling events. These tools were successfully used to obtain evidence for a yet unknown signaling network comprising common SYM-dependent and -independent events. In legumes AM colonization induces common SYM signaling dependent changes in root system architecture. It was demonstrated that also in rice, root system architecture changes in response to AM colonization but these alterations occur independently of common SYM signaling. The rice root system is complex and contains three different root types. It was shown that root type identity influences the quantity of AM colonization, indicating root type specific symbiotic properties. Interestingly, the root types differed in their transcriptional responses to AM colonization and the less colonized root type responded more dramatically than the more strongly colonized root type. Finally, in an independent project a novel mutant, inhospitable (iho), was discovered. It is perturbed at the most early step of AM colonization, namely differentiation of the AM fungal hyphae into a hyphopodium at the root surface. As plant factors required for this early step are not known, identification of the IHO gene will greatly contribute to the advance of mycorrhiza RÉSUMÉ : La symbiose mycorhizienne arbusculaire (AM) est une association évolutionnairement ancienne entre la majorité des plantes terrestres et les champignons du type Glomeromycota, basée sur l'échange mutuel d'éléments nutritifs entre les deux partenaires. Son établissement structural et physiologique est un processus en plusieurs étapes, impliquant des échanges de signaux étroitement contrôlés, aboutissant à la colonisation intracellulaire des racines par le champignon. La plupart des recherches sur la biologie moléculaire et la génétique du développement de la symbiose ont été effectuées sur des légumineuses dicotylédones modèles. Dans ces dernières, une voie de signalisation, la voie SYM, s'est avérée nécessaire pour permettre la mise en place de la symbiose mycorhizienne. Chez les plantes monocotylédones, comme le riz, une des céréales les plus importantes, nourrissant la moitié de la population mondiale, peu de recherches ont été effectuées sur les bases de la cette symbiose. Dans ce travail de thèse, la conservation fonctionnelle de la voie commune SYM chez le riz a été étudiée et démontrée. De plus, des gènes marqueurs spécifiques des différentes étapes du développement de l'AM ont été identifiés, permettant ainsi d'avoir des traceurs de la colonisation. Ces outils ont été utilisés avec succès pour démontrer l'existence d'un nouveau réseau de signalisation, comprenant des éléments SYM dépendant et indépendant. Chez les légumineuses, la colonisation par les AM induit des changements dans l'architecture du système racinaire, via la signalisation SYM dépendantes. Cependant chez le riz, il a été démontré que l'architecture de système racinaire changeait suite à la colonisation de l'AM, mais ceux, de façon SYM indépendante. Le système racinaire du riz est complexe et contient trois types différents de racines. Il a été démontré que le type de racine pouvait influencer l'efficacité de la colonisation par l'AM, indiquant que les racines ont des propriétés symbiotiques spécifiques différentes. De façon surprenante, les divers types de racines répondent de différemment suite à colonisation par l'AM avec des changements de la expression des gènes. Le type de racine le moins colonisé, répondant le plus fortement a la colonisation, et inversement. En parallèle, dans un projet indépendant, un nouveau mutant, inhospitable (iho), a été identifié. Ce mutant est perturbé lors de l'étape la plus précoce de la colonisation par l'AM, à savoir la différentiation des hyphes fongiques de l'AM en hyphopodium, à la surface des racines. Les facteurs d'origine végétale requis pour cette étape étant encore inconnus, l'identification du gène IHO contribuera considérablement a accroître nos connaissance sur les bases de la mise en place de cette symbiose.

Arbuscular mycorrhizal fungal communities and soil aggregation as affected by cultivation of various crops during the sugarcane fallow period

Management systems involving crop rotation, ground cover species and reduced soil tillage can improve the soil physical and biological properties and reduce degradation. The primary purpose of this study was to assess the effect of various crops grown during the sugarcane fallow period on the production of glomalin and arbuscular mycorrhizal fungi in two Latosols, as well as their influence on soil aggregation. The experiment was conducted on an eutroferric Red Latosol with high-clay texture (680 g clay kg-1) and an acric Red Latosol with clayey texture (440 g kg-1 clay) in Jaboticabal (São Paulo State, Brazil). A randomized block design involving five blocks and four crops [soybean (S), soybean/fallow/soybean (SFS), soybean/millet/soybean (SMS) and soybean/sunn hemp/soybean (SHS)] was used to this end. Soil samples for analysis were collected in June 2011. No significant differences in total glomalin production were detected between the soils after the different crops. However, total external mycelium length was greater in the soils under SMS and SHS. Also, there were differences in easily extractable glomalin, total glomalin and aggregate stability, which were all greater in the eutroferric Red Latosol than in the acric Red Latosol. None of the cover crops planted in the fallow period of sugarcane improved aggregate stability in either Latosol.

SOIL CHEMICAL PROPERTIES AND GROWTH OF SUNFLOWER (HELIANTHUS ANNUUS L.) AS AFFECTED BY THE APPLICATION OF ORGANIC FERTILIZERS AND INOCULATION WITH ARBUSCULAR MYCORRHIZAL FUNGI

The use of organic fertilizers and the inoculation of mycorrhizal fungi in the cultivation of oil crops is essential to reduce production costs and minimize negative impacts on natural resources. A field experiment was conducted in an Argissolo Amarelo (Ultisol) with the aim of evaluating the effects of fertilizer application and inoculation of arbuscular mycorrhizal fungi on the growth attributes of sunflower (Helianthus annuus L.) and on soil chemical properties. The experiment was conducted at the Federal University of Rio Grande do Norte, Brazil, using a randomized block design with three replicates in a 4 × 2 factorial arrangement consisting of four treatments in regard to application of organic fertilizer (liquid biofertilizer, cow urine, mineral fertilizer, and unfertilized control) and two treatments in regard to arbuscular mycorrhizal fungi (with and without mycorrhizal fungi). The results showed that the physiological attributes of relative growth rate and leaf weight ratio were positively influenced by fertilization, compared to the control treatment, likely brought about by the supply of nutrients from the fertilizers applied. The growth and productivity attributes were positively affected by mycorrhization.

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.

Requisitos nutricionais para o fungo Alternaria alternata

Uma linhagem de Alternaria alternata (ALT A) foi cultivada em meio sintético com diferentes fontes de C, N e vitaminas. O crescimento micelial foi avaliado durante sete dias após a semeadura e a esporulação no décimo dia de incubação. O meio sintético básico suplementado com biotina, NH4Cl e os carboidratos maltose e glicose suportaram um bom crescimento micelial (em média 0,75 cm/dia). Galactose e arabinose induziram significativamente a produção de esporos (mL-1), da ordem de 5,0 x10³ e 4,1 x 10³, respectivamente. Nas diferentes fontes de vitamina e N não houve diferenças significativas quanto ao crescimento micelial e à esporulação.