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The establishment of economic enterprises and the demand for coastal areas for leisure purposes exert great pressure on the dunes, stripping them of vegetation and causing the aggravation of coastal erosion processes. The use of arbuscular mycorrhizal fungi (AMF) is a viable alternative to restore dunes, giving their ability to improve soil conditions and plant growth under unfavorable conditions. The aim of this study was to evaluate the effect of AMF on the growth of Canavalia rosea (Sw.) DC. For the experiment, two treatment groups were set up (16 pots with arbuscular mycorrhizal fungi inoculum and 16 pots without) over a period of 10 weeks. It was found that C. rosea (Sw.) DC. responded positively to the inoculation with the AMF and the percentages of colonization were 6.4 and 10.2% in the eighth and tenth week of plant growth, respectively. Also, the growth of Canavalia rosea (Sw.) DC. increased significantly with AMF throughout the experiment. This proved the efficacy of arbuscular mycorrhizal fungi as promoters of dune plants´ growth, and as potential strategy for the rehabilitation of dunes in the coastal areas. Key words: Canavalia rosea, inoculation, mycorrhizal colonization, dune restoration, re-plant.

Conventional agricultural practices based on the application of synthetic fertilizers are increasingly considered as unsustainable. Under a forecasted scenario of drought for the next decades, there is a global demand for innovative and sustainable approaches to ameliorate plant performance. Here, encapsulating beneficial microbes (BMs) to promote plant growth is gaining attention. This study evaluates bacterial encapsulation using polymeric beads of alginate, testing the survival of Pseudomonas libanensis TR1 stored up to 90 days. Produced beads were subjected to different treatments (fresh, air-dried and pulverized), which resulted in a variable size range (1200–860 µm). After storage, bacterial viability was maintained, and air-dried beads displayed a higher number of colony-forming units (2 × 107). Then, a glasshouse experiment investigated the drought resistance (plant growth, biomass, and photosynthetic responses) of Vigna unguiculata plants inoculated with these alginate beads. After 10 days of complete water restriction, turgidity and relative water content of V. unguiculata were still high under drought stress (> 80%). Leaf and root growth and biomass did not evidence significant changes after water restriction even after P. libanensis inoculation. Plant photosynthetic parameters (stomatal conductance, net photosynthetic rate, leaf CO2 concentration, or Fv'/Fm') were slightly affected due to inoculation but the level of stress-induced minimal plant responses. In our experiment, water restriction might have been insufficient to downregulate photosynthetic efficiency and reduce plant growth, limiting our understanding of the role of P. libanensis inoculation in alleviating drought stress in V. unguiculata, but highlighting the important relationship between the stress level and agricultural benefits of using encapsulated BMs.

Although arbuscular mycorrhizal (AM) fungi are known to promote growth and yield of agricultural crops, inoculation methods for effective scaling up from greenhouse to the field are still underexplored. The application of single or mixed beneficial AM fungal isolates is hindered by the lack of experimental reproducibility of findings at different scales and the cost-effectivity of inoculation methods. Seed coating has been considered a feasible delivery system of AM fungal inocula for agricultural crops. In this study, the impact of single and multiple AM fungal isolates applied via seed coating on chickpea productivity was evaluated under greenhouse and field conditions. Overall, plants inoculated with multiple AM fungal isolates had better performance than those inoculated with single AM isolate under greenhouse and field conditions. While plants in greenhouse displayed higher shoot dry weight (14%) and seed individual weight (21%), in field, inoculation with multiple AM isolates increased pod (160%), and seed (148%) numbers, and grain yield (140%). Under field conditions, mycorrhizal root colonization was significantly higher in chickpea plants inoculated with multiple AM fungal isolates compared to other treatments. These findings highlight the potential of field-inoculation with multiple AM fungal isolates via seed coating as a sustainable agricultural practice for chickpea production.

Phytoremediation has been considered as a promising technique to decontaminate polluted soils. However, climatic stress particularly salinity, is a potential threat to soil properties and plant growth, thus restricting the employment of this technology. The aim of this study was to access the impact of microbial inoculation on phytoremediation of nickel (Ni) contaminated saline soils using Helianthus annuus. Salt resistant plant beneficial bacterium (PBB) Pseudomonas libanensis TR1 and arbuscular mycorrhizal fungus (AMF) Claroideoglomus claroideum BEG210 were used. Inoculation of P. libanensis alone or in combination with C. claroideum significantly enhanced plant growth, changed physiological status (e.g. electrolyte leakage, chlorophyll, proline and malondialdehyde contents) as well as Ni and sodium (Na+) accumulation potential (e.g. uptake and translocation factor of Ni and Na+) of H. annuus under Ni and salinity stress either alone or in combination. These results revealed that bioaugmentation of microbial strains may serve as a preferred strategy for improving phytoremediation of metal-polluted saline soils.

Chickpea (Cicer arietinum L.) is a widely cropped pulse and an important source of proteins for humans. In Mediterranean regions it is predicted that drought will reduce soil moisture and become a major issue in agricultural practice. Nitrogen (N)-fixing bacteria and arbuscular mycorrhizal (AM) fungi have the potential to improve plant growth and drought tolerance. The aim of the study was to assess the effects of N-fixing bacteria and AM fungi on the growth, grain yield and protein content of chickpea under water deficit.; Results: Plants inoculated with Mesorhizobium mediterraneum or Rhizophagus irregularis without water deficit and inoculated with M. mediterraneum under moderate water deficit had significant increases in biomass. Inoculation with microbial symbionts brought no benefits to chickpea under severe water deficit. However, under moderate water deficit grain crude protein was increased by 13%, 17% and 22% in plants inoculated with M. mediterraneum, R. irregularis and M. mediterraneum + R. irregularis, respectively.; Conclusion: Inoculation with N-fixing bacteria and AM fungi has the potential to benefit agricultural production of chickpea under water deficit conditions and to contribute to increased grain protein content. © 2017 Society of Chemical Industry.; © 2017 Society of Chemical Industry.

Background Plant-growth-promoting rhizobacteria (PGPR) and arbuscular mycorrhizal (AM) fungi have the ability to enhance the growth, fitness, and quality of various agricultural crops, including cowpea. However, field trials confirming the benefits of microbes in large-scale applications using economically viable and efficient inoculation methods are still scarce. Microbial seed coating has a great potential for large-scale agriculture through the application of reduced amounts of PGPR and AM fungi inocula. Thus, in this study, the impact of seed coating with PGPR, Pseudomonas libanensis TR1 and AM fungus, Rhizophagus irregularis (single or multiple isolates) on grain yield and nutrient content of cowpea under low-input field conditions was evaluated. Results Seed coating with P. libanensis + multiple isolates of R. irregularis (coatPMR) resulted in significant increases in shoot dry weight (76%), and in the number of pods and seeds per plant (52% and 56%, respectively) and grain yield (56%), when compared with non-inoculated control plants. However, seed coating with P. libanensis + R. irregularis single-isolate (coatPR) did not influence cowpea grain yield. Grain lipid content was significantly higher (25%) in coatPMR plants in comparison with control. Higher soil organic matter and lower pH were observed in the coatPMR treatment. Conclusions Our findings indicate that cowpea field productivity can be improved by seed coating with PGPR and multiple AM fungal isolates under low-input agricultural systems. © 2019 Society of Chemical Industry.

Plant beneficial microbes (PBMs), such as plant growth-promoting bacteria, rhizobia, arbuscular mycorrhizal fungi, and Trichoderma, can reduce the use of agrochemicals and increase plant yield, nutrition, and tolerance to biotic–abiotic stresses. Yet, large-scale applications of PBM have been hampered by the high amounts of inoculum per plant or per cultivation area needed for successful colonization and consequently the economic feasibility. Seed coating, a process that consists in covering seeds with low amounts of exogenous materials, is gaining attention as an efficient delivery system for PBM. Microbial seed coating comprises the use of a binder, in some cases a filler, mixed with inocula, and can be done using simple mixing equipment (e.g., cement mixer) or more specialized/sophisticated apparatus (e.g., fluidized bed). Binders/fillers can be used to extend microbial survival. The most reported types of seed coating are seed dressing, film coating, and pelleting. Tested in more than 50 plant species with seeds of different dimensions, forms, textures, and germination types (e.g., cereals, vegetables, fruits, pulses, and other legumes), seed coating has been studied using various species of plant growth-promoting bacteria, rhizobia, Trichoderma, and to a lesser extent mycorrhizal fungi. Most of the studies regarding PBM applied via seed coating are aimed at promoting crop growth, yield, and crop protection against pathogens. Studies have shown that coating seeds with PBM can assist crops in improving seedling establishment and germination or achieving high yields and food quality, under reduced chemical fertilization. The right combination of biological control agents applied via seed coating can be a powerful tool against a wide number of diseases and pathogens. Less frequently, studies report seed coating being used for adaptation and protection of crops under abiotic stresses. Notwithstanding the promising results, there are still challenges mainly related with the scaling up from the laboratory to the field and proper formulation, including efficient microbial combinations and coating materials that can result in extended shelf-life of both seeds and coated PBM. These limitations need to be addressed and overcome in order to allow a wider use of seed coating as a cost-effective delivery method for PBM in sustainable agricultural systems.

Cowpea (Vigna unguiculata L. Walp) is an important legume grown primarily in semi-arid area. Its production is generally inhibited by various abiotic and biotic stresses. The use of beneficial microorganisms (e.g., plant growth promoting bacteria (PGPB) and arbuscular mycorrhizal fungi (AMF)) can enhance agricultural production, as these microorganisms can improve soil fertility and plant tolerance to environmental stresses, thus enhancing crop yield in an eco-friendly manner. Application of PGPB and AMF in large scale agriculture needs to be improved. Thus, the use of seed coating could be an efficient mechanism for placement of inocula into soils. The aim of this study was to evaluate the effects of the AMF Rhizophagus irregularis BEG140 and the PGPB Pseudomonas libanensis TR1 alone or in combination on the biomass and physiological traits of cowpea. Four treatments were set: (i) non-inoculated control, (ii) PGPB, (iii) AMF applied via seed coating, and (iv) PGPB + AMF applied via seed coating. Cowpea plants inoculated via seed coating with R. irregularis and those inoculated with R. irregularis + P. libanensis showed root mycorrhizal colonization of 21.7% and 24.2%, respectively. PGPB P. libanensis was efficient in enhancing plant biomass and seed yield. There was no benefit of single (AMF) or dual (PGPB + AMF) inoculation on plant growth or seed yield. The application of beneficial soil microorganisms can be a viable approach for sustainable cowpea production in precision agriculture scenarios.

A plant growth-promoting bacterial (PGPB) strain SC2b was isolated from the rhizosphere of Sedum plumbizincicola grown in lead (Pb)/zinc (Zn) mine soils and characterized as Bacillus sp. based on (1) morphological and biochemical characteristics and (2) partial 16S ribosomal DNA sequencing analysis. Strain SC2b exhibited high levels of resistance to cadmium (Cd) (300 mg/L), Zn (730 mg/L), and Pb (1400 mg/L). This strain also showed various plant growth-promoting (PGP) features such as utilization of 1-aminocyclopropane-1-carboxylate, solubilization of phosphate, and production of indole-3-acetic acid and siderophore. The strain mobilized high concentration of heavy metals from soils and exhibited different biosorption capacity toward the tested metal ions. Strain SC2b was further assessed for PGP activity by phytagar assay with a model plant Brassica napus. Inoculation of SC2b increased the biomass and vigor index of B. napus. Considering such potential, a pot experiment was conducted to assess the effects of inoculating the metal-resistant PGPB SC2b on growth and uptake of Cd, Zn and Pb by S. plumbizincicola in metal-contaminated agricultural soils. Inoculation with SC2b elevated the shoot and root biomass and leaf chlorophyll content of S. plumbizincicola. Similarly, plants inoculated with SC2b demonstrated markedly higher Cd and Zn accumulation in the root and shoot system, indicating that SC2b enhanced Cd and Zn uptake by S. plumbizincicola through metal mobilization or plant-microbial mediated changes in chemical or biological soil properties. Data demonstrated that the PGPB Bacillus sp. SC2b might serve as a future biofertilizer and an effective metal mobilizing bioinoculant for rhizoremediation of metal polluted soils.

Fungi have been considered a potential source of natural anticancer drugs. However, studies on these organisms have mainly focused on compounds present in the sporocarp and mycelium. The aim of this study was to assess the anticancer potential of fungal spores using a bioassay-guided fractionation with cancer and normal cell lines. Crude extracts from spores of the basidiomycetous fungus Pisolithus tinctorius were prepared using five solvents/solvent mixtures in order to select the most effective crude extraction procedure. A dichloromethane/methanol (DCM/MeOH) mixture was found to produce the highest extraction yield, and this extract was fractionated into 11 fractions. Crude extracts and fractions were assayed for cytotoxicity in the human osteocarcinoma cell line MG63, the human breast carcinoma cell line T47D, the human colon adenocarcinoma cell line RKO, and the normal human brain capillary endothelial cell line hCMEC/D3. Cytotoxicity was assessed by the 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) reduction assay. The results showed a reduction in cancer cell viability of approximately 95% with 4 of 11 fractions without a significant reduction in viability of hCMEC/D3 cells. Data demonstrated that spores of P. tinctorius might serve as an interesting source of compounds with potential anticancer properties.

Arbuscular mycorrhizal (AM) fungi and plant growth-promoting rhizobacteria, responsible for enhancing plant nutrition, vigour and growth, may be used to reduce dosages of chemical fertilisers. Technologies that allow an economically viable and efficient application of these beneficial microbes in large scale agriculture must be studied. Seed coating is a potential delivery system for efficiently introducing minor amounts of bioinoculants. Despite the dramatic reduction on inoculum dose per plant, inoculation of AM fungi via seed coating was as effective as conventional soil inoculation. Fertilisation and inoculation had a significant impact on maize shoots nutrient concentrations. Different fertilisation regimes did not influence mycorrhizal colonisation. Plants without fertilisation and singly inoculated with R. irregularis showed shoot nutrient concentration increments of 110, 93, 88 and 175% for nitrogen, phosphorus, potassium and zinc, respectively, comparing with non-inoculated controls. Plants singly inoculated with P. fluorescens via seed coating under full fertilisation, presented enhancements of 100, 75 and 141% for magnesium, zinc and manganese, respectively, comparing with non-inoculated controls. Seed coating is a promising tool for delivering microbial inoculants into the soil, while promoting sustainable production of maize. This technology is particularly pertinent in low input agriculture, with potential environmental profits and food quality improvements.

This work focused on the biodegradation of three structurally related fluoroacetates (FAs), mono- (MFA), di- (DFA) and trifluoroacetate (TFA), using as microbial inocula samples collected from a site with a long history of industrial contamination and activated sludge obtained from a municipal wastewater treatment plant. Biodegradation experiments were carried out under different modes of substrate supplementation, which included (i) FAs fed as sole carbon sources; (ii) FAs (only for DFA and TFA) fed in co-metabolism with sodium acetate; and (iii) mixtures of MFA with DFA or TFA. Biodegradation of the target compounds was assessed through fluoride ion release. Defluorination was obtained in the cultures fed with MFA, while DFA and TFA were recalcitrant in all tested conditions. When present in mixture, DFA was shown to inhibit biodegradation of MFA, while TFA had no effect. A total of 13 bacterial isolates obtained from MFA degrading cultures were found to degrade 20 mgL −1 of this compound, as single strains, when supplemented as a sole carbon source. Sequencing of the 16S rRNA gene indicated that among these degrading bacteria only Delftia acidovorans had been previously reported to be able to degrade MFA. This work shows that, despite their similar chemical structures, biodegradation of the three tested FAs is very distinct and draws attention to the unknown impacts that the accumulation of DFA and TFA may have in the environment as a result of their high recalcitrance. © 2017 Elsevier R.S. Oliveira wishes to acknowledge the support of Fundação para a Ciência e a Tecnologia (FCT) through the research grant SFRH/BPD/85008/2012 and Fundo Social Europeu (FSE) . M.F. Carvalho and F. Pereira acknowledge Investigator FCT program supported by FCT , FSE and Programa Operacional Potencial Humano . This work was financed by national funds through FCT within the scope of the project IF/00791/2013/CP1197/CT0002 .

Ectomycorrhizal fungi (ECMF) play an important role in forest ecosystems, often mitigating stress factors and increasing seedling performance. The aim of this study was to investigate the effects of a nursery inoculation on Pinus pinaster growth and on the fungal communities established when reforesting burned areas. Inoculated P. pinaster saplings showed 1.5-fold higher stem height than the non-inoculated controls after a 5 year growth period, suggesting that fungal inoculation could potentiate tree growth in the field. Ordination analysis revealed the presence of different ECMF communities on both plots. Among the nursery-inoculated fungi, Laccaria sp., Rhizopogon sp., Suillus bovinus and Pisolithus sp. were detected on inoculated Pinus saplings on both sampling periods, indicating that they persisted after field establishment. Other fungi were also detected in the inoculated plants. Phialocephala sp. was found on the first assessment, while Terfezia sp. was detected on both sampling periods. Laccaria sp. and Rhizopogon sp. were identified in the control saplings, belonging however to different species than those found in the inoculated plot. Inocybe sp., Thelephora sp. and Paxillus involutus were present on both sampling periods in the non-inoculated plots. The results suggest that ECMF inoculation at nursery stage can benefit plant growth after transplantation to a post-fire site and that the inoculated fungi can persist in the field. This approach has great potential as a biotechnological tool to aid in the reforestation of burned areas.

It is recognized that inoculation with ectomycorrhizal (ECM) fungi can have a positive effect on the establishment of nursery grown seedlings and influence their survival during the first years. However, an increased effort is needed to understand their role and behavior in abandoned burned areas. We investigated tree performance of pre-inoculated Quercus rubra seedlings on a site with fire history and monitored fungal persistence and succession dynamics of the ectomycorrhizal community established. Nursery grown Q. rubra seedlings inoculated with a mixture of 6 ECM fungi ( Cenococcum geophilum , Hebeloma crustuliniforme , H. mesophaeum , H. velutipes , Paxillus involutus and Scleroderma citrinum ) were transplanted to Serra da Cabreira, Northern Portugal. Non-inoculated control oak seedlings were also transplanted. Two years after planting, biometric and mycorrhizal parameters were determined. The presence of the inoculated ECM fungi and the dominant ECM fungal community established was assessed by DGGE-cloning. Results showed that the bioinoculants improved the growth performance of outplanted oak seedlings, promoting a c.a. 1.6-fold increase in height and a 2.9-fold increase in the number of ECM root tips. After two years, H. mesophaeum , H. velutipes , P. involutus and S. citrinum persisted in the root system of inoculated plants. Ordination analysis demonstrated that ECM fungal communities of inoculated and non-inoculated samples were significantly different. The present work reinforces the benefits of using nursery inoculated plants in forest reclamation processes.

Endophytic actinobacteria have the capacity to establish intimate associations with plants and colonize their inner tissues without causing apparent disease symptoms. They can protect plants by producing bioactive compounds that act as plant growth promoters or biological control agents and, in return, obtain nutrition and protection from the host plant. The application of endophytic actinobacteria in agriculture has attracted increasing attention. We address isolation and identification methods and the occurrence and diversity of endophytic actinobacteria in agricultural crops. Attention is given to the roles of endophytic actinobacteria in plant growth and development and health promotion for sustainable agriculture is discussed.

Diflubenzuron (DFB) is an insecticide commonly used to control forest pests. The objectives of this study were to assess the effect of diflubenzuron on the development of Pinus pinaster seedlings and Pisolithus tinctorius under laboratory conditions and to study the possible protective role of this ectomycorrhizal fungus against the effects of diflubenzuron. In vitro experiments revealed that diflubenzuron inhibited fungal growth at all tested concentrations (0.01, 0.1, 1, 10 and 100 mg L(-1)). Root growth was inhibited at the two highest diflubenzuron concentrations. The activity of the antioxidant defence system of non-inoculated P. pinaster increased at 1 and 10 mg DFB kg(-1) substrate, and inoculation increased the threshold to the highest concentration. The protective role of the ectomycorrhizal fungus was seen in the increase of CAT activity. This study revealed that despite causing no mortality, diflubenzuron has the ability to cause sub-lethal damage to P. pinaster. The disproportionate use of this insecticide may lead to higher amounts of its residues in soil and the biosphere, endangering trees, fungi and their symbiosis.

Plants and microbes coexist or compete for survival and their cohesive interactions play a vital role in adapting to metalliferous environments, and can thus be explored to improve microbe-assisted phytoremediation. Plant root exudates are useful nutrient and energy sources for soil microorganisms, with whom they establish intricate communication systems. Some beneficial bacteria and fungi, acting as plant growth promoting microorganisms (PGPMs), may alleviate metal phytotoxicity and stimulate plant growth indirectly via the induction of defense mechanisms against phytopathogens, and/or directly through the solubilization of mineral nutrients (nitrogen, phosphate, potassium, iron, etc.), production of plant growth promoting substances (e.g., phytohormones), and secretion of specific enzymes (e.g., 1-aminocyclopropane- 1-carboxylate deaminase). PGPM can also change metal bioavailability in soil through various mechanisms such as acidification, precipitation, chelation, complexation, and redox reactions. This review presents the recent advances and applications made hitherto in understanding the biochemical and molecular mechanisms of plant–microbe interactions and their role in the major processes involved in phytoremediation, such as heavy metal detoxification, mobilization, immobilization, transformation, transport, and distribution.

Application of hyperaccumulator-endophyte symbiotic systems is a potential approach to improve phytoremediation efficiency, since some beneficial endophytic bacteria are able to detoxify heavy metals, alter metal solubility in soil, and facilitate plant growth. The objective of this study was to isolate multi-metal resistant and plant beneficial endophytic bacteria and to evaluate their role in enhancing plant growth and metal accumulation/translocation. The metal resistant endophytic bacterial strain E6S was isolated from stems of the Zn/Cd hyperaccumulator plant Sedum plumbizincicola growing in metalliferous mine soils using Dworkin and Foster salts minimal agar medium with 1-aminocyclopropane-1-carboxylate (ACC) as the sole nitrogen source, and identified as homologous to Achromobacter piechaudii based on morphological and biochemical characteristics, partial 16S rDNA sequence and phylogenetic analysis. Strain E6S showed high level of resistance to various metals (Cd, Zn, and Pb). Besides utilizing ACC, strain E6S exhibited plant beneficial traits, such as solubilization of phosphate and production of indole-3-acetic acid. Inoculation with E6S significantly increased the bioavailability of Cd, Zn, and Pb in soil. In addition, bacterial cells bound considerable amounts of metal ions in the following order: Zn > Cd > Pb. Inoculation of E6S significantly stimulated plant biomass, uptake and bioaccumulation of Cd, Zn, and Pb. However, E6S greatly reduced the root to shoot translocation of Cd and Zn, indicating that bacterial inoculation assisted the host plant to uptake and store heavy metals in its root system. Inoculation with the endophytic bacterium E6S homologous to A. piechaudii can improve phytostabilization of metalliferous soils due to its effective ability to enhance in situ metal rhizoaccumulation in plants.

Fluorinated compounds are finding increasing uses in several applications. They are employed in almost all areas of modern society. These compounds are all produced by chemical synthesis and their abundance highly contrasts with fluorinated molecules of natural origin. To date, only some plants and a handful of actinomycetes species are known to produce a small number of fluorinated compounds that include fluoroacetate (FA), some ω-fluorinated fatty acids, nucleocidin, 4-fluorothreonine (4-FT), and the more recently identified (2R3S4S)-5-fluoro-2,3,4-trihydroxypentanoic acid. This largely differs from other naturally produced halogenated compounds, which totals more than 5000. The mechanisms underlying biological fluorination have been uncovered after discovering the first actinomycete species, Streptomyces cattleya, that is capable of producing FA and 4-FT, and a fluorinase has been identified as the enzyme responsible for the formation of the C–F bond. The discovery of this enzyme has opened new perspectives for the biotechnological production of fluorinated compounds and many advancements have been achieved in its application mainly as a biocatalyst for the synthesis of [18F]-labeled radiotracers for medical imaging. Natural fluorinated compounds may also be derived from abiogenic sources, such as volcanoes and rocks, though their concentrations and production mechanisms are not well known. This review provides an outlook of what is currently known about fluorinated compounds with natural origin. The paucity of these compounds and the biological mechanisms responsible for their production are addressed. Due to its relevance, special emphasis is given to the discovery, characterization and biotechnological potential of the unique fluorinase enzyme. © 2016 Informa UK Limited, trading as Taylor & Francis Group M. F. Carvalho acknowledges Investigator FCT program supported by Fundação para a Ciência e a Tecnologia (FCT), Fundo Social Europeu (FSE), and Programa Operacional Potencial Humano. R. S. Oliveira wishes to acknowledge the support of FCT through the research grant SFRH/BPD/85008/2012, FSE and Programa Operacional Capital Humano.

Zn tissue accumulation in Solanum nigrum grown in a non-contaminated and a naturally contaminated Zn matrix and the effect of inoculation with different arbuscular mycorrhizal fungi (AMF) on metal uptake were assessed. S. nigrum grown in the contaminated soil always presented higher Zn accumulation in the tissues, accumulating up to 1622 mg Zn kg −1 . The presence of both Glomus claroideum and Glomus intraradices enhanced the uptake and accumulation of Zn by S. nigrum (up to 83 and 49% higher Zn accumulation, respectively). The main deposits of the metal were found in the intercellular spaces and in the cell walls of the root tissues, as revealed by autometallography, with the inoculation with different AMF species causing no differences in the location of Zn accumulation. These findings indicate that S. nigrum inoculated with selected heavy metal tolerant AMF presents extracting and accumulating capacities, constituting a potentially suitable remediation method for Zn polluted soils.

A field survey of the arbuscular mycorrhizal status of herbaceous plant species was conducted in a highly alkaline anthropogenic sediment resulting from the disposal of waste from an acetylene and polyvinyl chloride factory. Most plant species found at the site were mycorrhizal and the dominant mycotrophic plant species was Conyza bilbaoana. Fungal species richness was assessed by identification of spores extracted from the sediment and from continuously propagated trap pot cultures. All of the six species of arbuscular mycorrhizal fungi (AMF) found were from the genus Glomus. Glomus intraradices and G. mosseae were found in field-collected sediment samples and also occurred most frequently in trap cultures. To test the symbiotic effectiveness of these two fungi, seedlings of C. bilbaoana were inoculated with either native G. intraradices BEG163 or G. mosseae BEG198 and non-native G. intraradices BEG75 or G. mosseae BEG25 isolates in sterile and non-sterile sediment collected from the study site. All four isolates were able to colonise C. bilbaoana. However, AMF native to the target sediments were generally more effective than the non-native fungi in promoting plant establishment and growth under highly alkaline conditions. The non-native G. intraradices was, however, more effective than the non-native G. mosseae. The results of this study suggest the use of adapted AMF as inoculants for phytorestoration of alkaline anthropogenic-stressed sediments.

The combination of ectomycorrhizal (ECM) fungi and plant growth promoting bacteria (PGPB) has considerable potential in forestry. Here we report on the combined effects of PGPB Mesorhizobium sp. and the ECM fungus Paxillus involutus on the growth of Betula pubescens in two different substrates, a forest soil and an alkaline anthropogenic sediment. Growth, nutrient concentration and mycorrhizal formation of B. pubescens were determined at the end of the experiment and the fungal and bacterial communities were assessed by denaturing gradient gel electrophoresis. The combined effects of ECM and PGPB enhanced root and shoot growth most in the forest soil with Mesorhizobium appearing to stimulate mycorrhizal formation. However, in the alkaline sediment, an antagonistic interaction was detected between P. involutus and Mesorhizobium sp., with Mesorhizobium sp. alone stimulating growth. The significant interaction between the ECM and the PGBP across the two substrates is discussed.

The aim of this study was to assess the effects of inoculation of rhizosphere or endophytic bacteria (Psychrobacter sp. SRS8 and Pseudomonas sp. A3R3, respectively) isolated from a serpentine environment on the plant growth and the translocation and accumulation of Ni, Zn, and Fe by Brassica juncea and Ricinus communis on a multi-metal polluted serpentine soil (SS). Field collected SS was diluted to 0, 25, 50, and 75% with pristine soil in order to obtain a range of heavy metal concentrations and used in microcosm experiments. Regardless of inoculation with bacteria, the biomass of both plant species decreased with increase of the proportion of SS. Inoculation of plants with bacteria significantly increased the plant biomass and the heavy metal accumulation compared with non-inoculated control in the presence of different proportion of SS, which was attributed to the production of plant growth promoting and/or metal mobilizing metabolites by bacteria. However, SRS8 showed a maximum increase in the biomass of the test plants grown even in the treatment of 75% SS. In turn, A3R3 showed maximum effects on the accumulation of heavy metals in both plants. Regardless of inoculation of bacteria and proportion of SS, both plant species exhibited low values of bioconcentration factor (

Roots of Phragmites australis from three polluted soils and sediments (a periodically flooded stream bank containing organic pollutants, a high-pH drying sedimentation pond and an acidic, periodically flooded sand polluted by industrial effluents) were sampled over a 1-year cycle of plant growth to assess the degree of colonisation by arbuscular mycorrhizal fungi (AMF). At the dry sedimentation pond, root samples of Juncus effusus and Salix atrocinerea were also taken to assess the presence of AMF throughout the year. Root colonisation was low (

Purpose: Polycyclic aromatic hydrocarbons (PAHs) are a class of organic compounds commonly found as soil contaminants. Fungal degradation is considered as an environmentally friendly and cost-effective approach to remove PAHs from soil. Acenaphthylene (Ace) and Benzo[a]anthracene (BaA) are two PAHs that can coexist in soils; however, the influence of the presence of each other on their biodegradation has not been studied. The biodegradation of Ace and BaA, alone and in mixtures, by the white rot fungus Pleurotus ostreatus was studied in a sandy soil. Materials and methods: Experimental microcosms containing soil spiked with different concentrations of Ace and BaA were inoculated with P. ostreatus. Initial (t 0) and final (after 15 days of incubation) soil concentrations of Ace and BaA were determined after extraction of the PAHs. Results and discussion: P. ostreatus was able to degrade 57.7% of the Ace in soil spiked at 30 mg kg-1 dry soil and 65.8% of Ace in soil spiked at 60 mg kg-1 dry soil. The degradation efficiency of BaA by P. ostreatus was 86.7 and 77.4% in soil spiked with Ace at 30 and 60 mg kg-1 dry soil, respectively. After 15 days of incubation, there were no significant differences in Ace concentration between soil spiked with Ace and soil spiked with Ace + BaA, irrespective of the initial soil concentration of both PAHs. There were also no differences in BaA concentration between soil spiked with BaA and soil spiked with BaA + Ace. Conclusions: The results indicate that the fungal degradation of Ace and BaA was not influenced by the presence of each other's PAH in sandy soil. Bioremediation of soils contaminated with Ace and BaA using P. ostreatus is a promising approach to eliminate these PAHs from the environment.

Cowpea (Vigna unguiculata (L.) Walp.), a plant broadly cultivated for human consumption and animal feed, is among the most nutritious grain legumes. Most of the areas where cowpea is grown are drought-prone, and there is a need to address this issue, with water scarcity becoming a major concern in agriculture. Cowpea is known to form mutualistic associations with nitrogen-fixing (NF) bacteria and arbuscular mycorrhizal (AM) fungi. These beneficial soil microorganisms have the capacity to benefit plants by reducing the effects of environmental stresses, including drought. Our aim was to study the effect of inoculation with Bradyrhizobium elkanii and Rhizophagus irregularis on the growth and grain yield of cowpea under water-deficit conditions. Under moderate water deficit, grain yield was increased by 63%, 55% and 84% in plants inoculated with B. elkanii, R. irregularis and B. elkanii + R. irregularis, respectively. Under severe water deficit, inoculation with B. elkanii and B. elkanii + R. irregularis resulted in grain-yield enhancement of 45% and 42%, respectively. The use of cowpea inoculated with NF bacteria and AM fungi has great potential for sustainable agricultural production under drought conditions.

Genetic, phenotypic and functional variation in a Glomus geosporum isolate obtained from a highly alkaline anthropogenic sediment was investigated. Two cultivation lineages of G. geosporum (BEG199 and BEG211) were created by sub-culturing with or without the stress of the sediment. Genetic variation was assessed on spores from both cultivation lineages in the large sub-unit rRNA gene D2 region using PCR-single strand conformational polymorphism (PCR-SSCP) and sequencing. Phenotypical and functional variation of the cultivation lineages were assessed after inoculation onto Conyza bilbaoana. The sequence diversity obtained in G. geosporum BEG211 was significantly different from that obtained in G. geosporum BEG199. Glomus geosporum BEG199 was more effective than G. geosporum BEG211 in promoting the growth of C. bilbaoana in inert substrate and in increasing its leaf phosphorus concentration when the plant was grown in sediment. After inoculation onto C. bilbaoana, G. geosporum BEG199 colonised the roots to a greater extent, produced a significantly larger number of spores and presented a greater length of extraradical mycelium than G. geosporum BEG211. The results indicate that environmental conditions under which arbuscular mycorrhizal fungi (AMF) are cultivated can influence their effects in host plants. Also, AMF might quickly lose gained-tolerance to environmental stresses when maintained without the selective pressure of those stresses. This study has implications for the production of commercial inoculum of AMF and for the maintenance of AMF cultures. The results indicate that inoculum of AMF should be produced and AMF cultures should be maintained in substrates or media containing the original edaphic stresses.

The effect of two different chelating agents [EDTA and EDDS (S,S-ethylenediaminedissucinic acid)] on Zn tissue accumulation in Solanum nigrum L. grown in a naturally contaminated soil was assessed. Under those conditions, the response of the plant to the inoculation with two different isolates of arbuscular mycorrhizal fungi (AMF) - Glomus claroideum and Glomus intraradices - was also studied. Plants grown in the local contaminated soil (Zn levels of 433 mg kg-1) accumulated up to 1191 mg kg-1 of Zn in the roots, 3747 mg kg-1 in the stems and 3409 mg kg-1 in the leaves. S. nigrum plants grown in the same soil spiked with extra Zn (Zn levels of 964 mg kg-1) accumulated up to 4735, 8267 and 7948 mg Zn kg-1 in the leaves, stems and roots, respectively. The addition of EDTA promoted an increase in the concentration of Zn accumulated by S. nigrum of up to 231% in the leaves, 93% in the stems and 81% in the roots, while EDDS application enhanced the accumulation in leaves, stems and roots up to 140, 124 and 104%, respectively. In the stems, the presence of Zn was predominantly detected in the cortex collenchyma cells, the starch sheath and the internal phloem and xylem parenchyma, and the addition of chelating agents did not seem to have an effect on the localisation of accumulation sites. The devise of a chelate-enhanced phytoextraction strategy, using chelating agents and AMF, is discussed.

Different species of arbuscular mycorrhizal fungi (AMF) can produce different amounts of extraradical mycelium (ERM) with differing architectures. They also have different efficiencies in gathering phosphate from the soil. These differences in phosphate uptake and ERM length or architecture may contribute to differential growth responses of plants and this may be an important contributor to plant species coexistence. The effects of the development of the ERM of AMF on the coexistence of two co-occurring plant species were investigated in root-free hyphal chambers in a rhizobox experimental unit. The dominant shrub (Salix atrocinerea Brot.) and herbaceous (Conyza bilbaoana J. Rémy) plant species found in a highly alkaline anthropogenic sediment were studied in symbiosis with four native AMF species (Glomus intraradices BEG163, Glomus mosseae BEG198, Glomus geosporum BEG199 and Glomus claroideum BEG210) that were the most abundant members of the AMF community found in the sediment. Different AMF species did not influence total plant productivity (sum of the biomass of C. bilbaoana and S. atrocinerea), but had a great impact on the individual biomass of each plant species. The AMF species with greater extracted ERM lengths (G. mosseae BEG198, G. claroideum BEG210 and the four mixed AMF) preferentially benefited the plant species with a high mycorrhizal dependency (C. bilbaoana), while the AMF species with the smallest ERM length (G. geosporum BEG199) benefited the plant species with a low mycorrhizal dependency (S. atrocinerea). Seed production of C. bilbaoana was only observed in plants inoculated with G. mosseae BEG198, G. claroideum BEG210 or the mixture of the four AMF. Our results show that AMF play an important role in the reproduction of C. bilbaoana coexisting with S. atrocinerea in the alkaline sediment and have the potential to stimulate or completely inhibit seed production. The community composition of native AMF and the length of the mycelium they produce spreading from roots into the surrounding soil can be determinant of the coexistence of naturally co-occurring plant species.

The presence of actinorhizas and arbuscular mycorrhizas may reduce plant stresses caused by adverse soil conditions. A greenhouse experiment was conducted using a sediment with a high pH, resulting from the disposal of waste originated at an acetylene and polyvinylchloride factory, in which Black alder (Alnus glutinosa) seedlings were inoculated either with Glomus intraradices BEG163 (originally isolated from the same sediment), Frankia spp. or both symbionts. After a 6-month growth period, plants inoculated with both symbionts had significantly greater leaf area, shoot height and total biomass when compared with the uninoculated control, the Frankia spp. and the G. intraradices treatments alone. In dual inoculated plants the N and P leaf content was significantly increased. A defoliation experiment was performed to evaluate the stress recovery of A. glutinosa and plants inoculated with both symbionts had a faster leaf regrowth and produced greater numbers of leaves. The dual inoculation resulted in greater numbers of and larger root nodules than when inoculated with Frankia spp. alone. The length and NADH diaphorase activity of the extraradical mycelium of G. intraradices was also significantly greater when dual inoculation was performed. The inoculation with Frankia spp. alone was shown to improve A. glutinosa growth, whereas G. intraradices alone had no positive effect under these environmental conditions. However, when the two symbionts were inoculated together a synergistic effect was observed resulting in a greater benefit for the plants and for both symbionts. The relevance of these findings for the phytorestoration of anthropogenic stressed sediments with high pH is discussed.

Drought can drastically reduce cowpea [Vigna unguiculata (L.) Walp.] biomass and grain yield. The application of plant growth‐promoting rhizobacteria and arbuscular mycorrhizal fungi can confer resistance to plants and reduce the effects of environmental stresses, including drought. Seed coating is a technique which allows the application of minor amounts of microbial inocula. Main effects of the factors inoculation and water regime showed that: severe or moderate water deficit had a general negative impact on cowpea plants; total biomass production, seed weight and seed yield were enhanced in plants inoculated with P. putida; inoculation of R. irregularis significantly increased nitrogen (N) and phosphorus (P) shoot concentrations; and R. irregularis enhanced both chlorophyll b and carotenoids contents, particularly under severe water deficit. Plants inoculated with P. putida + R. irregularis had an increase in shoot P concentration of 85% and 57%, under moderate and severe water deficit, respectively. Singly inoculated P. putida improved potassium shoot concentration by 25% under moderate water deficit. Overall, in terms of agricultural productivity the inoculation of P. putida under water deficit might be promising. Seed coating has the potential to be used as a large‐scale delivery system of beneficial microbial inoculants.