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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.

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.

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 .

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.

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.