Intracellular pH in Arbuscular Mycorrhizal Fungi1 : A Symbiotic Physiological Marker

A method was developed to perform real-time analysis of cytosolic pH of arbuscular mycorrhizal fungi in culture using dye and ratiometric measurements (490/450 nm excitations). The study was mainly performed using photometric analysis, although some data were confirmed using image analysis. The use of nigericin allowed an in vivo calibration. Experimental parameters such as loading time and concentration of the dye were determined so that pH measurements could be made for a steady-state period on viable cells. A characteristic pH profile was observed along hyphae. For Gigaspora margarita, the pH of the tip (0–2 μm) was typically 6.7, increased sharply to 7.0 behind this region (9.5 μm), and decreased over the next 250 μm to a constant value of 6.6. A similar pattern was obtained for Glomus intraradices. The pH profile of G. margarita germ tubes was higher when cultured in the presence of carrot (Daucus carota) hairy roots (nonmycorrhizal). Similarly, extraradical hyphae of G. intraradices had a higher apical pH than the germ tubes. The use of a paper layer to prevent the mycorrhizal roots from being in direct contact with the medium selected hyphae with an even higher cytosolic pH. Results suggest that this method could be useful as a bioassay for studying signal perception and/or H+ cotransport of nutrients by arbuscular mycorrhizal hyphae.

Congruence of fatty acid methyl ester profiles and morphological characters of arbuscular mycorrhizal fungi in Gigasporaceae.

Arbuscular mycorrhizal (AM) fungi (Order Glomales, Class Zygomycetes) are a diverse group of soil fungi that form mutualistic associations with the roots of most species of higher plants. Despite intensive study over the past 25 years, the phylogenetic relationships among AM fungi, and thus many details of evolution of the symbiosis, remain unclear. Cladistic analysis was performed on fatty acid methyl ester (FAME) profiles of 15 species in Gigaspora and Scutellospora (family Gigasporaceae) by using a restricted maximum likelihood approach of continuous character data. Results were compared to a parsimony analysis of spore morphological characters of the same species. Only one tree was generated from each character set. Morphological and developmental data suggest that species with the simplest spore types are ancestral whereas those with complicated inner wall structures are derived. Spores of those species having a complex wall structure pass through stages of development identical to the mature stages of simpler spores, suggesting a pattern of classical Haeckelian recapitulation in evolution of spore characters. Analysis of FAME profiles supported this hypothesis when Glomus leptotichum was used as the outgroup. However, when Glomus etunicatum was chosen as the outgroup, the polarity of the entire tree was reversed. Our results suggest that FAME profiles contain useful information and provide independent criteria for generating phylogenetic hypotheses in AM fungi. The maximum likelihood approach to analyzing FAME profiles also may prove useful for many other groups of organisms in which profiles are empirically shown to be stable and heritable.

The Effects of Arbuscular Mycorrhizal Fungi on four Legume Hosts in South Florida Pine Rockland Soils

This study addressed the effects of salinity and pot size on the interaction between leguminous plant hosts and arbuscular mycorrhizal fungi in four pine rockland soils using a shade house trap-plant experiment. Little is known about the belowground diversity of pine rocklands and the interactions between aboveground and belowground biota – an increased understanding of these interactions could lead to improved land management decisions, conservation and restoration efforts. Following twelve weeks of growth, plants were measured for root and shoot dry biomass and percent colonization by arbuscular mycorrhizal fungi. Overall, arbuscular mycorrhizal fungi had positive fitness effects on the four legume species (Cajanus cajan, Chamaecrista fasciculata, Tephrosia angustissima and Abrus precatorius), improving their growth rate, shoot and root biomass; pot size influenced plant-fungal interactions; and percent colonization by arbuscular mycorrhizal fungi was influenced by soil type as well as salinity.

Resposta de sabiá Mimosa caesalpiniaefolia Benth. a inoculações com rizóbio e micorriza em diferentes níveis de fósforo em solo de restinga degradado.

Para avaliar os efeitos da inoculação com Bradyrhizobium, fungos micorrízicos arbusculares (FMA) e níveis de fósforo em mudas de sabiá Mimosa caesalpiniaefolia Benth, plantadas em um solo espodossolo humilúvico de restinga degradada, adubado com cinco níveis de fósforo e duas fontes fosfatadas, no crescimento, nodulação, colonização micorrízica e teores e acúmulos de macro e micronutrientes, foi realizado um experimento em vasos plásticos, utilizando um delineamento experimental inteiramente casualizado, em esquema fatorial completo 2 X 4 X 5 com 4 repetições.

Mineral Nutrition Of Campos Rupestres Plant Species On Contrasting Nutrient-impoverished Soil Types.

In Brazil, the campos rupestres occur over the Brazilian shield, and are characterized by acidic nutrient-impoverished soils, which are particularly low in phosphorus (P). Despite recognition of the campos rupestres as a global biodiversity hotspot, little is known about the diversity of P-acquisition strategies and other aspects of plant mineral nutrition in this region. To explore nutrient-acquisition strategies and assess aspects of plant P nutrition, we measured leaf P and nitrogen (N) concentrations, characterized root morphology and determined the percentage arbuscular mycorrhizal (AM) colonization of 50 dominant species in six communities, representing a gradient of soil P availability. Leaf manganese (Mn) concentration was measured as a proxy for carboxylate-releasing strategies. Communities on the most P-impoverished soils had the highest proportion of nonmycorrhizal (NM) species, the lowest percentage of mycorrhizal colonization, and the greatest diversity of root specializations. The large spectrum of leaf P concentration and variation in root morphologies show high functional diversity for nutritional strategies. Higher leaf Mn concentrations were observed in NM compared with AM species, indicating that carboxylate-releasing P-mobilizing strategies are likely to be present in NM species. The soils of the campos rupestres are similar to the most P-impoverished soils in the world. The prevalence of NM strategies indicates a strong global functional convergence in plant mineral nutrition strategies among severely P-impoverished ecosystems.

Notes on Acaulospora bireticulata Rothwell & Trappe and first record of Acaulospora koskei Blask. from Brazil)

(Notes on Acaulospora bireticulata Rothwell & Trappe and first record of Acaulospora koskei Blask. from Brazil). Our knowledge of species distribution in arbuscular mycorrhizal fungi (AMF) is still limited and morphological variations found in isolates of one species may provide additional insight for understanding the characters used in the taxonomy of this group. The aims of this work were to expand biogeographical data regarding Acaulospora koskei Blaszk. and Acaulospora bireticulata Rothwell & Trappe, both found in an Araucaria Forest in Sao Paulo state, Brazil, as well as to compare the descriptions of these species with those of other similar AMF.

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.

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.

delta N-15 values of tropical savanna and monsoon forest species reflect root specialisations and soil nitrogen status

A large number of herbaceous and woody plants from tropical woodland, savanna, and monsoon forest were analysed to determine the impact of environmental factors (nutrient and water availability, fire) and biological factors (microbial associations, systematics) on plant delta(15)N values. Foliar delta(15)N values of herbaceous and woody species were not related to growth form or phenology, but a strong relationship existed between mycorrhizal status and plant delta(15)N. In woodland and savanna, woody species with ectomycorrhizal (ECM) associations and putative N-2-fixing species with ECM/arbuscular (AM) associations had lowest foliar delta(15)N values (1.0-0.6parts per thousand), AM species had mostly intermediate delta(15)N values (average +0.6parts per thousand), while non-mycorrhizal Proteaceae had highest delta(15)N values (+2.9 to +4.1parts per thousand). Similar differences in foliar delta(15)N were observed between AM (average 0.1 and 0.2parts per thousand) and non-mycorrhizal (average +0.8 and +0.3parts per thousand) herbaceous species in woodland and savanna. Leguminous savanna species had significantly higher leaf N contents (1.8-2.5% N) than non-fixing species (0.9-1.2% N) indicating substantial N acquisition via N-2 fixation. Monsoon forest species had similar leaf N contents (average 2.4% N) and positive delta(15)N values (+0.9 to +2.4parts per thousand). Soil nitrification and plant NO3- use was substantially higher in monsoon forest than in woodland or savanna. In the studied communities, higher soil N content and nitrification rates were associated with more positive soil delta(15)N and plant delta(15)N. In support of this notion, Ficus, a high NO3- using taxa associated with NO3- rich sites in the savanna, had the highest delta(15)N values of all AM species in the savanna. delta(15)N of xylem sap was examined as a tool for studying plant delta(15)N relations. delta(15)N of xylem sap varied seasonally and between differently aged Acacia and other savanna species. Plants from annually burnt savanna had significantly higher delta(15)N values compared to plants from less frequently burnt savanna, suggesting that foliar N-15 natural abundance could be used as marker for assessing historic fire regimes. Australian woodland and savanna species had low leaf delta(15)N and N content compared to species from equivalent African communities indicating that Australian biota are the more N depauperate. The largest differences in leaf delta(15)N occurred between the dominant ECM Australian and African savanna (miombo) species, which were depleted and enriched in N-15, respectively. While the depleted delta(15)N of Australian ECM species are similar to those of previous reports on ECM species in natural plant communities, the N-15-enriched delta(15)N of African ECM species represent an anomaly.

Growth and nutrient accumulation in mycorrhized papaya seedlings cultivated in a phosphorus-fertilized substrate

ABSTRACT The indiscriminate use of mineral fertilizers in papaya orchards has increased production costs, and the use of arbuscular mycorrhizal fungi is a promising alternative to reduce such expenses. Therefore, the present research aimed at studying the efficiency of arbuscular mycorrhizal fungi (AMF) on dry matter and nutrient accumulation in Sunrise Solo papaya seedlings, by applying doses of P2O5 (triple superphosphate) that are harmful to the symbiosis. The experiment was carried out in a protected environment and was set up in a randomized block design with four replications, and consisted of four P2O5 doses (0, 672, 1386 and 2100 mg dm-3), three mycorrhizal fungi species (Gigaspora margarita, Entrophospora colombiana and Scutellospora heterogama) and the control treatment (mycorrhiza-free). Shoot and root dry matter as well as nitrogen, phosphorus and potassium contents in leaf and root tissues were assessed. Mycorrhizal inoculation promoted a 30% increase in shoot dry matter in relation to the control treatment. Mycorrhizal fungi promoted increases in leaf and root nitrogen content up to 672 mg dm-3 P2O5. Inoculation of E. colombiana favored the highest gains in root and shoot dry matter. P2O5 fertilization increased foliar and root phosphorus content.

Fungos micorrízicos arbusculares em sistema de aléias no Estado do Maranhão, Brasil

Sistemas em aléias podem consistir numa solução para o uso da terra em regiões do Trópico Úmido. A relação dessa forma de manejo com a dinâmica dos fungos micorrízicos arbusculares (FMA) ainda é pouco compreendida. O objetivo desse estudo foi verificar a influência de leguminosas arbóreas em um sistema em aléias na capacidade infectiva e diversidade de FMA nativos em São Luís, Maranhão. Amostras de solo coletadas do sistema em aléias no campus experimental da Universidade Estadual do Maranhão (UEMA) - São Luís, em duas épocas do ano (Julho/2006 e Abril/2007), a duas distâncias (0 m e 0,5 m) do tronco de três leguminosas (Leucaena leucocephala, Clitoria fairchildiana e Acacia mangium) e em área testemunha (sem leguminosas) na profundidade de 0 - 20 cm. O solo coletado foi utilizado para avaliar a capacidade infectiva dos FMA nativos, densidade e identificação de glomerosporos. O sistema em aléias aumenta o potencial de infectividade dos FMA nativos dependendo da espécie de leguminosa arbórea associada, estação de coleta e proximidades das árvores. Dezesseis espécies de FMA foram encontradas na área distribuídas em cinco gêneros, sendo Scutellospora o mais representativo.

Efeito do alumínio e de Glomus leptotichum Shenck e Smith no desenvolvimento de cultivares de Leucaena leucocephala (Lam.) de Wit tolerante e intolerante ao alumínio

Com o objetivo de avaliar a interação micorrízica X Al tóxico no substrato efetuou-se um experimento fatorial 2x2x2, em casa de vegetação, no Departamento de Genética da ESALQ/USP, em Piracicaba. Foram empregadas duas cultivares de Leucaena leucocephala, uma Al - tolerante (Planta 49 - Estrada do Bongue) e outra Al-intolerante (NO749), cultivadas em areia lavada e esterilizada, com adição de solução nutritiva. A micorriza foi estabelecida com a inoculação de Glomus teptotichum Shenck e Smith no substrato, mantendo-se como testemunha a ausência do fungo. Dois níveis de Al foram avaliados, 0 e 9 ppm, adicionados à solução nutritiva. Aos 65 dias após o transplante das plântulas procedeu-se à colheita e foram determinados o peso da metária seca da parte aérea, altura de planta, porcentagem de colonização radicular, teor e quantidade acumulada de N, P, K, Ca, Mg e Al na parte aérea. Observou-se um acentuado efeito promotor da micorriza em Leucaena, o que geroulhe incrementos tanto no desenvolvimento da planta como na absorção de nutrientes. A cultivar intolerante ao Al tóxico mostrou-se mais dependente da micorriza e inclusive mais beneficiada,apresentando os maiores acréscimos na produção de matéria seca e acúmulo de nutrientes na parte aérea. Nas condições do experimento não houve acentuada diferença entre as doses de Al empregadas e, apesar do maior acúmulo do elemento em plantas micorrizadas, não foi observado interferência no desenvolvimento das cultivares.

Glomus intraradices induces changes in root system architecture of rice independently of common symbiosis signaling

? Arbuscular mycorrhizal fungi colonize the roots of most monocotyledons and dicotyledons despite their different root architecture and cell patterning. Among the cereal hosts of arbuscular mycorrhizal fungi, Oryza sativa (rice) possesses a peculiar root system composed of three different types of roots: crown roots; large lateral roots; and fine lateral roots. Characteristic is the constitutive formation of aerenchyma in crown roots and large lateral roots and the absence of cortex from fine lateral roots. Here, we assessed the distribution of colonization by Glomus intraradices within this root system and determined its effect on root system architecture. ? Large lateral roots are preferentially colonized, and fine lateral roots are immune to arbuscular mycorrhizal colonization. Fungal preference for large lateral roots also occurred in sym mutants that block colonization of the root beyond rhizodermal penetration. ? Initiation of large lateral roots is significantly induced by G. intraradices colonization and does not require a functional common symbiosis signaling pathway from which some components are known to be needed for symbiosis-mediated lateral root induction in Medicago truncatula. ? Our results suggest variation of symbiotic properties among the different rice root-types and induction of the preferred tissue by arbuscular mycorrhizal fungi. Furthermore, signaling for arbuscular mycorrhizal-elicited alterations of the root system differs between rice and M. truncatula.