Micro PIXE mapping proves a differential distribution and concentration of trace elements in fungal structures of Rhizophagus intraradices.

Arbuscular mycorrhizal (AM) fungi can sequester different potentially toxic elements, such as trace elements (TEs), within their structures to alleviate the toxicity for its host plant and themselves. To elucidate the role of AM fungi in TEs immobilization in the rhizosphere of host plants, it is important to know the TEs distribution in AM fungal structures. In the present study, we investigated the distribution and concentration of TEs within extraradical spores and mycelium of the AM fungus Rhizophagus intraradices, collected from the rhizosphere of Senecio bonariensis plants grown in a soil polluted with multiple TEs, by using Particle-Induced X-ray Emission with a micro-focused beam (micro PIXE). This technique enabled the simultaneous micrometric mapping of elements in a sample. The calculated values were compared with those in the polluted substrate, measured by the Wavelength Dispersive X-ray Fluorescence technique. The highest concentrations of Fe, P, Ti, Mn, Cr, Cu and Zn were found in AM fungal spores, where they were accumulated, while extraradical mycelium was enriched in Cu. Finally, we demonstrated that AM fungi can simultaneously accumulate high amounts of different TEs in their structures, thus reducing the toxicity of these elements to its host plant.

Arbuscular mycorrhizal fungi activity in the rhizosphere of tree seedlings subjected to residual herbicides / Atividade de fungos micorrízicos arbusculares na rizosfera de mudas de árvores submetidas a herbicidas residuais

Abstract Trees occurring on the margins of agricultural areas can mitigate damage from residual herbicides. Rhizospheric microbial activity associated with trees is one of the main remedial capacity indicators. The objective of this study was to evaluate the rhizospheric microbiological activity in tree species subjected to the herbicides atrazine and sulfentrazone via the rhizosphere. The experiment was designed in four blocks and a 6 × 3 factorial scheme. The first factor consisted of six tree species from Brazil and the second of atrazine, sulfentrazone, and water solutions. Four herbicide applications were performed via irrigation. The total dry mass of the plants, mycorrhizal colonization, number of spores, basal respiration of the rhizospheric soil, and survival rate of bioindicator plants after phytoremediation were determined. Trichilia hirta had higher biomass when treated with atrazine and sulfentrazone. Herbicides decreased the microbial activity in Triplaris americana and did not affect the microbiological indicators of Myrsine gardneriana, Schizolobium parahyba, and Toona ciliata. Fewer bioindicator plants survived in soil with Triplaris americana and sulfentrazone. Microbiological indicators were influenced in different ways between species by the presence of herbicides in the rhizosphere.

Resumo As árvores que ocorrem nas margens das áreas agrícolas podem mitigar os danos dos herbicidas residuais. A atividade microbiana rizosférica associada às árvores é um dos principais indicadores de capacidade corretiva. O objetivo deste trabalho foi avaliar a atividade microbiológica rizosférica em espécies arbóreas submetidas aos herbicidas atrazina e sulfentrazone via rizosfera. O experimento foi estruturado em quatro blocos e esquema fatorial 6 × 3. O primeiro fator consistiu em seis espécies de árvores do Brasil e o segundo em soluções de atrazine, sulfentrazone e água. Quatro aplicações de herbicidas foram realizadas via irrigação. Foram determinados a massa seca total das plantas, colonização micorrízica, número de esporos, respiração basal do solo rizosférico e taxa de sobrevivência de plantas bioindicadoras após fitorremediação. Trichilia hirta apresentou maior biomassa quando tratada com atrazina e sulfentrazone. Os herbicidas diminuíram a atividade microbiana em Triplaris americana e não afetaram os indicadores microbiológicos de Myrsine gardneriana, Schizolobium parahyba e Toona ciliata. Menos plantas bioindicadoras sobreviveram no solo com Triplaris americana e sulfentrazone. Os indicadores microbiológicos foram influenciados de formas distintas entre as espécies pela presença dos herbicidas na rizosfera.

Arbuscular mycorrhizal fungi activity in the rhizosphere of tree seedlings subjected to residual herbicides.

Trees occurring on the margins of agricultural areas can mitigate damage from residual herbicides. Rhizospheric microbial activity associated with trees is one of the main remedial capacity indicators. The objective of this study was to evaluate the rhizospheric microbiological activity in tree species subjected to the herbicides atrazine and sulfentrazone via the rhizosphere. The experiment was designed in four blocks and a 6 × 3 factorial scheme. The first factor consisted of six tree species from Brazil and the second of atrazine, sulfentrazone, and water solutions. Four herbicide applications were performed via irrigation. The total dry mass of the plants, mycorrhizal colonization, number of spores, basal respiration of the rhizospheric soil, and survival rate of bioindicator plants after phytoremediation were determined. Trichilia hirta had higher biomass when treated with atrazine and sulfentrazone. Herbicides decreased the microbial activity in Triplaris americana and did not affect the microbiological indicators of Myrsine gardneriana, Schizolobium parahyba, and Toona ciliata. Fewer bioindicator plants survived in soil with Triplaris americana and sulfentrazone. Microbiological indicators were influenced in different ways between species by the presence of herbicides in the rhizosphere.

Effector proteins of Rhizophagus proliferus: conserved protein domains may play a role in host-specific interaction with different plant species.

Arbuscular mycorrhizal (AM) fungi show high promiscuity in terms of host. Effector proteins expressed by AM fungi are found important in establishing interaction with host. However, the mechanistic underlying host-specific interactions of the fungi remain unknown. The present study aimed (i) to identify effectors encoded by Rhizophagus proliferus and (ii) to understand molecular specificity encoded in effectors for interaction with specific plant species. The effectors predicted from the whole genome sequence were annotated by homology search in NCBI non-redundant protein, Interproscan, and pathogen-host interaction (PHI) databases. In total, 416 small secreted peptides (SSPs) were predicted, which were effector peptides with presence of nuclear localization signal, small cysteine-rich, and repeat-containing proteins domains. Similar to the functionally validated SP7 effectors in Rhizophagus irregularis, two proteins (RP8598 and RP23081) were identified in R. proliferus. To understand whether interaction between SP7 and the plant target protein, ERF19, is specific in nature, we examined protein-peptide interaction using in silico molecular docking. Pairwise interaction of RP8598 and RP23081 with the ethylene-responsive factors (ERF19) coded by five different plant species (Lotus japonicus, Solanum lycopersicum, Ocimum tenuiflorum, Medicago truncatula, Diospyros kaki) was investigated. Prediction of high-quality interaction of SP7 effector with ERF19 protein expressed only by specific plant species was observed in in silico molecular docking, which may reiterate the role of effectors in host specificity. The outcomes from our study indicated that sequence precision encoded in the effector peptides of AM fungi and immunomodulatory proteins of host may regulate host specificity in these fungi.

Lime and phosphate application as mycorrhizae stimulation to enhance growth and yield of Marandu grass / Calagem e fosfatagem associadas a estimulante de micorrização no crescimento e produção do capim-Marandu

Pastures are important food sources for Brazilian cattle herds. However, inadequate management of soil fertility has emerged as a major cause of low yield rates and of progressive degradation of these areas. The objective of the present study was to evaluate growth, by means of morphogenetic and structural characteristics, and yield of Brachiaria brizantha Stapf. cv. Marandu as functions of the application of the mycorrhizae stimulant formononetin, associated with lime and phosphate application. The experimental design was completely randomised with four replications, and the treatments were arranged in a 2 x 2 x 5 factorial scheme, consisting of two liming treatments (with and without limestone), two formononetin treatments (with and without application) and five P2O5 doses (0, 25, 50, 100 and 200 mg dm-3). Three shoot cuttings were carried out after a first standardisation cutting to evaluate the morphogenetic and structural characteristics as well as the dry matter yield of different morphological fractions. Liming and phosphate application at the dose of 141 mg dm-3 P2O5 increased growth and yield of Marandu grass, and these practices are essential for the cultivation of this pasture grass in Yellow Latosol of the Cerrado region of Piauí, Brazil. The application of formononetin increased stem elongation rate, total number of tillers and green stem dry matter, and decreased dead dry matter of Marandu grass, which are effects that contribute to the improvement of pasture quality.

As pastagens constituem importante fonte alimentar para os rebanhos brasileiros, entretanto, o manejo inadequado da fertilidade dos solos têm-se destacado como uma das principais causas dos baixos índices produtivos e da progressiva degradação destas áreas. O objetivo do presente estudo foi avaliar o crescimento, através das características morfogênicas e estruturais, e a produção da Brachiaria brizantha Stapf. cv. Marandu em função da aplicação do estimulante de micorrização formononetina associado a calagem e a fosfatagem. O delineamento experimental adotado foi o inteiramente casualizado, com quatro repetições, sendo os tratamentos dispostos num esquema fatorial 2 x 2 x 5, constituídos por dois tratamentos de calagem (com e sem calcário), dois de formononetina (com e sem aplicação) e cinco doses de P2O5 (0, 25, 50, 100 e 200 mg dm-3). Foram realizados três cortes da parte aérea, após um primeiro corte de uniformização, para realização das avaliações das características morfogênicas e estruturais, bem como a produção de massa seca das diferentes frações morfológicas. A calagem e a fosfatagem na dose de 141 mg dm-3 de P2O5 incrementaram o crescimento e a produção do capim-Marandu, devendo ser práticas indispensáveis ao cultivo desta forrageira em Latossolo Amarelo do Cerrado piauiense, Brasil. A aplicação de formononetina promoveu aumento da taxa de alongamento do colmo, número total de perfilho, massa seca de colmo verde e diminuiu a fração da massa seca morta docapim-Marandu, efeitos que contribuem para a melhoria da qualidade das pastagens.

Lime and phosphate application as mycorrhizae stimulation to enhance growth and yield of Marandu grass / Calagem e fosfatagem associadas a estimulante de micorrização no crescimento e produção do capim-Marandu

Pastures are important food sources for Brazilian cattle herds. However, inadequate management of soil fertility has emerged as a major cause of low yield rates and of progressive degradation of these areas. The objective of the present study was to evaluate growth, by means of morphogenetic and structural characteristics, and yield of Brachiaria brizantha Stapf. cv. Marandu as functions of the application of the mycorrhizae stimulant formononetin, associated with lime and phosphate application. The experimental design was completely randomised with four replications, and the treatments were arranged in a 2 x 2 x 5 factorial scheme, consisting of two liming treatments (with and without limestone), two formononetin treatments (with and without application) and five P2O5 doses (0, 25, 50, 100 and 200 mg dm-3). Three shoot cuttings were carried out after a first standardisation cutting to evaluate the morphogenetic and structural characteristics as well as the dry matter yield of different morphological fractions. Liming and phosphate application at the dose of 141 mg dm-3 P2O5 increased growth and yield of Marandu grass, and these practices are essential for the cultivation of this pasture grass in Yellow Latosol of the Cerrado region of Piauí, Brazil. The application of formononetin increased stem elongation rate, total number of tillers and green stem dry matter, and decreased dead dry matter of Marandu grass, which are effects that contribute to the improvement of pasture quality.(AU)

As pastagens constituem importante fonte alimentar para os rebanhos brasileiros, entretanto, o manejo inadequado da fertilidade dos solos têm-se destacado como uma das principais causas dos baixos índices produtivos e da progressiva degradação destas áreas. O objetivo do presente estudo foi avaliar o crescimento, através das características morfogênicas e estruturais, e a produção da Brachiaria brizantha Stapf. cv. Marandu em função da aplicação do estimulante de micorrização formononetina associado a calagem e a fosfatagem. O delineamento experimental adotado foi o inteiramente casualizado, com quatro repetições, sendo os tratamentos dispostos num esquema fatorial 2 x 2 x 5, constituídos por dois tratamentos de calagem (com e sem calcário), dois de formononetina (com e sem aplicação) e cinco doses de P2O5 (0, 25, 50, 100 e 200 mg dm-3). Foram realizados três cortes da parte aérea, após um primeiro corte de uniformização, para realização das avaliações das características morfogênicas e estruturais, bem como a produção de massa seca das diferentes frações morfológicas. A calagem e a fosfatagem na dose de 141 mg dm-3 de P2O5 incrementaram o crescimento e a produção do capim-Marandu, devendo ser práticas indispensáveis ao cultivo desta forrageira em Latossolo Amarelo do Cerrado piauiense, Brasil. A aplicação de formononetina promoveu aumento da taxa de alongamento do colmo, número total de perfilho, massa seca de colmo verde e diminuiu a fração da massa seca morta docapim-Marandu, efeitos que contribuem para a melhoria da qualidade das pastagens.(AU)

Identification of mVOCs from Andean rhizobacteria and field evaluation of bacterial and mycorrhizal inoculants on growth of potato in its center of origin.

Food security (a pressing issue for all nations) faces a threat due to population growth, land availability for growing crops, a changing climate (leading to increases in both abiotic and biotic stresses), heightened consumer awareness of the risks related to the use of agrichemicals, and also the reliance on depleting fossil fuel reserves for their production. Legislative changes in Europe mean that fewer agrichemicals will be available in the future for the control of crop pests and pathogens. The need for the implementation of a more sustainable agricultural system globally, incorporating an integrated approach to disease management, has never been more urgent. To that end, the Valorizing Andean Microbial Diversity (VALORAM) project (http://valoram.ucc.ie), funded under FP7, examined the role of microbial communities in crop production and protection to improve the sustainability, food security, environmental protection, and productivity for rural Andean farmers. During this work, microbial volatile organic compounds (mVOCs) of 27 rhizobacterial isolates were identified using gas chromatography/mass spectrometry (GC/MS), and their antifungal activity against Rhizoctonia solani was determined in vitro and compared to the activity of a selection of pure volatile compounds. Five of these isolates, Pseudomonas palleroniana R43631, Bacillus sp. R47065, R47131, Paenibacillus sp. B3a R49541, and Bacillus simplex M3-4 R49538 trialled in the field in their respective countries of origin, i.e., Bolivia, Peru, and Ecuador, showed significant increase in the yield of potato. The strategy followed in the VALORAM project may offer a template for the future isolation and determination of putative biocontrol and plant growth-promoting agents, useful as part of a low-input integrated pest management system.

Calcium oxalate crystals in eucalypt ectomycorrhizae: morphochemical characterization.

Ectomycorrhizal fungi are ubiquitous in forest ecosystems, benefitting plants principally by increasing the uptake of water and nutrients such as calcium from the soil. Previous work has demonstrated accumulation of crystallites in eucalypt ectomycorrhizas, but detailed morphological and chemical characterization of these crystals has not been performed. In this work, cross sections of acetic acid-treated and cleared ectomycorrhizal fragments were visualized by polarized light microscopy to evaluate the location of crystals within cortical root cells. Ectomycorrhizal sections were also observed by scanning electron microscopy (SEM) coupled with energy dispersive x-ray (EDS) microprobe analysis. The predominant forms of crystals were crystal sand (granules) and concretions. Calcium, carbon and oxygen were detected by EDS as constituent elements and similar elemental profiles were observed between both crystal morphologies. All analyzed crystalline structures were characterized as calcium oxalate crystals. This is the first report of the stoichiometry and morphology of crystals occurring in eucalypt ectomycorrhizas in tropical soils. The data corroborates the role of ectomycorrhizae in the uptake and accumulation of calcium in the form of calcium oxalate crystals in hybrid eucalypt plants.

First report of the ectomycorrhizal status of boletes on the Northern Yucatan Peninsula, Mexico determined using isotopic methods.

Despite their prominent role for tree growth, few studies have examined the occurrence of ectomycorrhizal fungi in lowland, seasonally dry tropical forests (SDTF). Although fruiting bodies of boletes have been observed in a dry tropical forest on the Northern Yucatan Peninsula, Mexico, their occurrence is rare and their mycorrhizal status is uncertain. To determine the trophic status (mycorrhizal vs. saprotrophic) of these boletes, fruiting bodies were collected and isotopically compared to known saprotrophic fungi, foliage, and soil from the same site. Mean δ(15)N and δ(13)C values differed significantly between boletes and saprotrophic fungi, with boletes 8.0‰ enriched and 2.5‰ depleted in (15)N and (13)C, respectively relative to saprotrophic fungi. Foliage was depleted in (13)C relative to both boletes and saprotrophic fungi. Foliar δ(15)N values, on the other hand, were similar to saprotrophic fungi, yet were considerably lower relative to bolete fruiting bodies. Results from this study provide the first isotopic evidence of ectomycorrhizal fungi in lowland SDTF and emphasize the need for further research to better understand the diversity and ecological importance of ectomycorrhizal fungi in these forested ecosystems.

Natural attenuation in a slag heap contaminated with cadmium: the role of plants and arbuscular mycorrhizal fungi.

A field study of the natural attenuation occurring in a slag heap contaminated with high available cadmium was carried out. The aims of this research were: to determine plants colonizing this slag heap; to analyze colonization and morphological biodiversity of spores of arbuscular mycorrhizal fungi (AMF); to determine spore distribution in undisturbed samples; to know mycelium and glomalin abundance in the rhizosphere of these plants, and to investigate glomalin participation in Cd-stabilization. Forming vegetal islands, 22 different pioneering plant species from 11 families were colonizing the slag heap. The most common plants were species of Fabaceae, Asteraceae and Poaceae. Almost all plants were hosting AMF in their roots, and spores belonging to Gigaspora, Glomus, Scutellospora and Acaulospora species were observed. Micromorphological analysis showed that spores were related to decomposing vegetal residues and excrements, which means that mesofauna is contributing to their dispersion in the groundmass. Mycelium mass ranged from 0.11 to 26.3 mg/g, which contained between 13 and 75 mg of glomalin/g. Slag-extracted total glomalin was between 0.36 and 4.74 mg/g. Cadmium sequestered by glomalin extracted from either slag or mycelium was 0.028 mg/g. The ecological implication of these results is that organisms occupying vegetal patches are modifying mine residues, which contribute to soil formation.

Glomalin-related soil protein in a Mediterranean ecosystem affected by a copper smelter and its contribution to Cu and Zn sequestration.

The amount of glomalin-related soil protein (GRSP), a glycoprotein produced by arbuscular mycorrhizal fungi (AMF), its contribution to the sequestering of Cu and Zn in the soil, and the microsite variation of other soil traits (pH, water-stable aggregates--[WSA], soil organic carbon--[SOC]) was studied in a semi-arid Mediterranean ecosystem near a copper smelter and affected by deposit of metal-rich particles since 1964. Rhizospheric (R) and non-rhizospheric (NR) soil of four representative plants (Argemone subfusiformis, Baccharis linearis, Oenothera affinis and Polypogon viridis) was analyzed. The results showed a strong variability in GRSP (6.6-36.8 mg g(-1)), Cu content (62-831 mg kg(-1) for the total Cu and 5.8-326 mg kg(-1) for the available Cu) and pH (4.2-5.5) in the different plant and rhizospheric zones analyzed. A strong relationship between the GRSP with the soil Cu and Zn contents was found (r=0.89 and 0.76 for Cu and Zn respectively, p<0.001). The GRSP-bound Cu ranged from 3.76 to 89.0 mg g(-1) soil and represents 1.44-27.5% of the total Cu content in soil. Moreover, the WSA reached 89% in P. viridis R. For this plant, the C contained in GRSP represented up to 89% of SOC, and this coincided with the most extreme conditions of soil degradation within the ecosystem (the highest content of heavy metals and low pH values). This study provides evidence on the role of the GRSP in Cu and Zn sequestration and suggests a highly efficient mechanism of AMF to mitigate stress leading to stabilization of soils highly polluted by mining activities.

Studies on neutral exopolysaccharides produced by the ectomycorrhiza Thelephora terrestris.

The ectomycorrhizal hymenomycete Thelephora terrestris was grown in synthetic pure culture and the production of extracellular polysaccharide was monitored. The exopolysaccharides were prepared by ethanol precipitation and then fractionated into two components using a DEAE-Sepharose column. A neutral fraction (NeP) was fractionated on Sepharose CL-6B, which resulted in three peaks: NeP1, NeP2 and NeP3. NeP1 was filtered through an exclusion membrane and two polysaccharides were obtained (fractions: NeA, NeB). Fraction NeB was submitted to methylated derivatives and 1H-, 13C- and 2D NMR spectroscopic analyses. These analyses showed a main chain of a (1-->6)-linked alpha-D-Manp units substituted at O-2 by a variety of side chains containing alpha-Fucp, beta-Xylp and beta-Galp residues. The main fraction corresponds to mannan as shown by methylation analysis. Size exclusion chromatography (HPSEC-MALLS) of fraction NeB showed a main component of 15.0 kDa. It contained mannose, galactose, fucose and xylose in a molar ratio of 50:29:11:10. The fractions NeP2 and NeP3 were characterised as a (1-->6)-linked beta-glucan (pustulan) and (1-->3)-linked beta-glucan, respectively.