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- Diverse Arbuscular Mycorrhizal Fungi (AMF) communities colonize plants inhabiting a constructed wetland for wastewater treatmentPublication . Calheiros, Cristina S. C.; Pereira, Sofia I. A.; Franco, Albina R.; Castro, Paula M. L.Constructed wetlands (CWs) are biological wastewater treatment systems that comprise several components where plants and associated organisms play an important role in water depuration. Microbial studies emphasize bacterial dynamics, whereas studies of arbuscular mycorrhizal fungi (AMF) are scarce and the functional role of AMF in aquatic and wetland plants is poorly understood. The aim of this study was to analyze the AMF communities colonizing the roots of Canna indica, Canna flaccida, and Watsonia borbonica inhabiting a CW treating wastewater of a tourism unit. The dynamics of the AMF communities were evaluated by Denaturing Gradient Gel Electrophoresis (DGGE) of 18S rRNA gene amplification products along cold (C) and hot (H) seasons for three consecutive years. DGGE profiles allowed the estimation of AMF species richness (S), and Shannon-Wienner (H) and Pielou (J) indexes, for the different plant species, showing differences between species and along the years. Excised bands from DGGE were analyzed and identified through sequencing for arbuscular mycorrhiza, revealing the presence of AMF strains closely related to Glomus sp., Rhizophagus sp. and Acaulospora sp. genera. Concomitant water quality analyses showed that the system was effective in organic and nutrient removal during the sampling period. Findings from this study suggest that AMF diversity found in the CW is influenced by the water constituents, season, and plant species.
- Bacteria immobilisation on hydroxyapatite surface for heavy metals removalPublication . Piccirillo, C.; Pereira, S. I. A.; Marques, A. P. G. C.; Pullar, R. C.; Tobaldi, D. M.; Pintado, M. E.; Castro, P. M. L.Selected bacterial strains were immobilised on the surface of hydroxyapatite (Ca-10(PO4)(6)(OH)(2) - HAp) of natural origin (fish bones). The capacity of the material, alone and in combination with the bacterial strains to act as heavy metal removers from aqueous streams was assessed. Pseudomonas fluorescens (S3X), Micro bacterium oxydans (EC29) and Cupriavidus sp. (1C2) were chosen based on their resistance to heavy metals and capacity of adsorbing the metals. These systems were tested using solutions of Zn(II), Cd(II) and in solutions containing both metals. A synergistic effect between the strains and HAp, which is effective in removing the target heavy metals on its own, was observed, as the combination of HAp with the bacterial strains led to higher adsorption capacity for both elements. For the solutions containing only one metal the synergistic effect was greater for higher metal concentrations; 1C2 and EC29 were the most effective strains for Zn(II) and Cd(II) respectively, while S3X was less effective. Overall, an almost four-fold increase was observed for the maximum adsorption capacity for Zn(II) when 10 was employed - 0.433 mmol/g in comparison of 0.121 mmol/g for the unmodified HAP. For Cd(II), on the other hand, an almost three-fold increase was registered with EC29 bacterial strain - 0.090 vs 0.036 mmol/g for the unmodified HAp. When the solutions containing both metals were tested, the effect was more marked for lower concentrations.
- Mine land valorization through energy maize production enhanced by the application of plant growth-promoting rhizobacteria and arbuscular mycorrhizal fungiPublication . Moreira, Helena; Pereira, Sofia I. A.; Marques, Ana P. G. C.; Rangel, António O. S. S.; Castro, Paula M. L.The use of heavy metals (HM) contaminated soils to grow energy crops can diminish the negative impact of HM in the environment improving land restoration. The effect of two PGPR (B1—Chryseobacterium humi ECP37T and B2—Pseudomonas reactans EDP28) and an AMF (F—Rhizophagus irregularis) on growth, Cd and Zn accumulation, and nutritional status of energy maize plants grown in a soil collected from an area adjacent to a Portuguese mine was assessed in a greenhouse experiment. Both bacterial strains, especially when co-inoculated with the AMF, acted as plant growth-promoting inoculants, increasing root and shoot biomass as well as shoot elongation. Cadmium was not detected in the maize tissues and a decrease in Zn accumulation was observed for all microbial treatments in aboveground and belowground tissues—with inoculation of maize with AMF and strain B2 leading to maximum reductions in Zn shoot and root accumulation of up to 48 and 43 %, respectively. Although microbial single inoculation generally did not increase N and P levels in maize plants, co-inoculation of the PGPR and the AMF improved substantially P accumulation in roots. The DGGE analysis of the bacterial rhizosphere community showed that the samples inoculated with the AMF clustered apart of those without the AMF and the Shannon-Wiener Index (H′) increased over the course of the experiment when both inoculants were present. This work shows the benefits of combined inoculation of AMF and PGPR for the growth energy maize in metal contaminated soils and their potential for the application in phytomanagement strategies.
- Metal(loid)-contaminated soils as a source of culturable heterotrophic aerobic bacteria for remediation applicationsPublication . Pires, Carlos; Franco, Albina R.; Pereira, Sofia I. A.; Henriques, Isabel; Correia, António; Magan, Naresh; Castro, Paula M. L.Heavy metal-contaminated soils are a serious environmental problem. Herein, the culturable heterotrophic bacterial community present on two metal(loid)-contaminated sites in the Northern Portugal was investigated. The bacterial counts ranged from 5.96 to 7.69 and 7.04 to 7.51 (log CFUg(-1) soil) in Sites 1 and 3, respectively. The bacterial population was predominantly composed of Firmicutes, Proteobacteria, and Actinobacteria on both sites. The most represented genera in Site 1 were Bacillus (41%) and Pseudomonas (27%), whereas Arthrobacter (21%) and Pseudomonas (13%) were the most represented genera in Site 3. Several bacterial isolates showed tolerance to high concentrations of metal(loid)s, suggesting that both contaminated sites are a valuable source of metal(loid)-tolerant bacteria, which may be further used in bioremediation and/or phytoremediation processes.
- Promotion of sunflower growth under saline water irrigation by the inoculation of beneficial microorganismsPublication . Pereira, Sofia I. A.; Moreira, Helena; Argyras, Konstantinos; Castro, Paula M. L.; Marques, Ana P. G. C.Soil salinization and fresh water scarcity are amongst the main environmental/agricultural problems, with serious consequences to plant productivity. Amelioration with microorganisms can enhance plant performance under salt conditions. The aim of this work was to evaluate the role of beneficial rhizospheric microorganisms on the growth of sunflower plants irrigated with salinized water with particular attention to nutrient balance and biochemical responses. Sunflower seedlings were inoculated with the arbuscular mycorrhizal fungi Rhizophagus irregularis, the rhizobacteria Chryseobacterium humi ECP37T, or the bacterial endophyte Ochrobacterium haematophilum ZR3-5, and with a mixed inocula of those microorganisms. Plant growth, nutrient accumulation and lipid peroxidation in plant tissues, and the activity of soil enzymes, were evaluated. Irrigating sunflower plants with saline water resulted in decreases in growth and negative effects in salt stress markers, however the application of bioinoculants enhanced biomass production and accumulation of K+, Mg2+, Ca2+, N and P, reduced Na+ levels in tissues and increased plant antioxidative response. This study contributes to devise inoculation strategies for sunflower cultivation in areas prone to salinization.
- Assessment of rhizospheric culturable bacteria of Phragmites australis and Juncus effusus from polluted sitesPublication . Pereira, Sofia I. A.; Pires, Carlos; Henriques, Isabel; Correia, António; Magan, Naresh; Castro, Paula M. L.This study aimed at the isolation and characterization of metal(loid)-tolerant bacteria from the rhizosphere of Phragmites australis and Juncus effusus plants growing in two long-term contaminated sites in Northern Portugal. Site 1 had higher contamination than Site 3. Bacteria were isolated using metal(loid)-supplemented (Cd, Zn, and As) media. Isolates were grouped by random amplified polymorphic DNA and identified by 16S rRNA gene sequencing. Strains were also examined for their metal(loid) tolerance. The counts of metal(loid)-tolerant bacteria were higher in Site 1 and ranged between log 7.17 CFU g(-1) soil in As-containing medium and log 7.57 CFU g(-1) soil in Zn-containing medium, while counts at Site 3 varied between log 5.33 CFU g(-1) soil in Cd-containing medium and log 6.97 CFUg(-1) soil in As-containing medium. The composition of bacterial populations varied between locations. In Site 1, the classes Actinobacteria (36%) and Bacilli (24%) were well represented, while in Site 3 strains were mainly affiliated to classes Actinobacteria (35%), 'y-Proteobacteria (35%), and 13-Proteobacteria (12%). The order of metal(loid) toxicity for the isolated strains was Cd > As > Zn. Overall, 10 strains grew at 500 mg Cd L-1, 1000 mg Zn L-1, and 500 mg As L-1, being considered the most metal(loid)-tolerant bacteria. These strains belonged to genera Cupriavidus, Burkholderia, Novosphingobium, Sphingo bacterium, Castellaniella, Mesorhizobium, Chryseobacterium, and Rhodococcus and were mainly retrieved from Site 1. The multiple metal(loid)-tolerant strains isolated in this study have potential to be used in bioremediation/phytoremediation.
- Effect of benfluralin on Pinus pinea seedlings mycorrhized with Pisolithus tinctorius and Suillus bellinii – Study of plant antioxidant responsePublication . Franco, A. R.; Pereira, Sofia I. A.; Castro, PaulaIn this study, Pinus pinea seedlings mycorrhized with selected ectomycorrhizal fungi (ECMF), Pisolithus tinctorius and Suillus bellinii, were exposed to the herbicide benfluralin. Non-mycorrhized P. pinea seedlings and seedlings mycorrhized with ECMF were transferred to benfluralin-spiked soils at levels of 0.165, 1.65 and 16.5 mg kg 1. Plant growth and the fungal role on plant antioxidant response were assessed. In the presence of benfluralin, higher plant growth was observed in mycorrhized plants compared to non-mycorrhized plants, but ECMF colonisation and nutrient uptake were affected by the herbicide. Benfluralin showed no effect on lipid peroxidation in P. pinea seedlings. However, seedlings mycorrhized with S. bellinii showed higher levels of lipid peroxidation when compared to non-mycorrhized ones, both in the presence and absence of benfluralin. The increase of lipid peroxidation could be related to seedling growth induced by the fungus and not to benfluralin toxicity. A similar trend was observed in seedlings mycorrhized with P. tinctorius when exposed to higher benfluralin concentrations, suggesting that the antioxidant response to benfluralin is related not only to fungus species, but also to the level of stress applied in the soil. The higher amount of superoxide dismutase activity in P. pinea seedlings tissues exposed to benfluralin could indicate a plant adaptative response to benfluralin toxicity. Catalase activity showed no increase with benfluralin exposure. Pre-established P. tinctorius mycorrhization conferred root protection and enhanced plant growth in benfluralin spiked soil, inferring that P. tinctorius – P. pinea association could advantageous for plant growth in soils contaminated with pesticides
- Assessment of culturable bacterial endophytic communities colonizing Canna flaccida inhabiting a wastewater treatment constructed wetlandPublication . Calheiros, Cristina Sousa Coutinho; Pereira, Sofia Isabel Almeida; Brix, Hans; Rangel, Antonio Osmaro Santos Silva; Castro, Paula Maria LimaMicroorganisms, including the bacterial populations living inside plant tissues (endophytes), and vegetation are components of constructed wetland (CW) systems playing pivotal roles in the water treatment process and in the ecosystem establishment. The present study focuses on the assessment of the culturable endophytic bacterial communities of Canna flaccida plants, the dominant plant species in a polycultured CW treating wastewater from a tourism facility. Bacterial endophytes were grouped by random amplified polymorphic DNA and identified by 16S ribosomal RNA gene sequencing. From the bacterial isolates, 103 were considered for phylogenetic analysis, falling in 25 genera within the γ-, β-, α-Proteobacteria, Flavobacteria, Sphingobacteria, Actinobacteria and Bacilli classes. Forty-nine percent of the isolates belonged to the Enterobacteriaceae family, suggesting that the plants in CW systems may act as a sink of potential human pathogenic microorganisms; nevertheless their abundance on the plant tissues was reduced from the inlet (62%) to the outlet zone (38%). High diversity in terms of genera was found in C. flaccida tissues, although the number of genera common to both sampling zones was low, which suggests that the processes occurring within the CW, including the water characteristics gradient from the inlet to the outlet (e.g. total suspended solids, organics and fecal loads), had the ability to shape the diversity of the endophytic communities.
- Phytomanagement of metal(loid) - contaminated soils: options, efficiency and valuePublication . Moreira, Helena; Pereira, Sofia I. A.; Mench, Michel; Garbisu, Carlos; Kidd, Petra; Castro, Paula M. L.The growing loss of soil functionality due to contamination by metal(loid)s, alone or in combination with organic pollutants, is a global environmental issue that entails major risks to ecosystems and human health. Consequently, the management and restructuring of large metal(loid)-polluted areas through sustainable nature-based solutions is currently a priority in research programs and legislation worldwide. Over the last few years, phytomanagement has emerged as a promising phytotechnology, focused on the use of plants and associated microorganisms, together with ad hoc site management practices, for an economically viable and ecologically sustainable recovery of contaminated sites. It promotes simultaneously the recovery of soil ecological functions and the decrease of pollutant linkages, while providing economic revenues, e.g. by producing non-food crops for biomass-processing technologies (biofuel and bioenergy sector, ecomaterials, biosourced-chemistry, etc.), thus contributing to the international demand for sustainable and renewable sources of energy and raw materials for the bioeconomy. Potential environmental benefits also include the provision of valuable ecosystem services such as water drainage management, soil erosion deterrence, C sequestration, regulation of nutrient cycles, xenobiotic biodegradation, and metal(loid) stabilization. Phytomanagement relies on the proper selection of (i) plants and (ii) microbial inoculants with the capacity to behave as powerful plant allies, e.g., PGPB: plant growth-promoting bacteria and AMF: arbuscular mycorrhizal fungi. This review gives an up-to-date overview of the main annual, perennial, and woody crops, as well as the most adequate cropping systems, presently used to phytomanage metal(loid)-contaminated soils, and the relevant products and ecosystems services provided by the various phytomanagement options. Suitable bioaugmentation practices with PGPB and AMF are also discussed. Furthermore, we identify the potential interest of phytomanagement for stakeholders and end-users and highlight future opportunities boosted by an effective engagement between environmental protection and economic development. We conclude by presenting the legal and regulatory framework of soil remediation and by discussing prospects for phytotechnologies applications in the future.
- Endophytic culturable bacteria colonizing Lavandula dentata L. plants: Isolation, characterization and evaluation of their plant growth-promoting activitiesPublication . Pereira, S.I.A.; Monteiro, Cristina M.; Vega, A. L.; Castro, Paula M. L.Aromatic plants such as lavender are stirring the attention of many researchers due to their content in bioactive secondary metabolites that can be used in traditional medicine. However, information regarding naturally occurring lavender associated bacterial endophytes (BE) is limited. To the best of our knowledge, this is the first study which aims to assess the phylogenetic diversity of the culturable endophytic bacteria of Lavandula dentata cultivated under organic management and to evaluate their potential as plant growth promoting (PGP) agents. BE were grouped by random amplified polymorphic DNA and identified by 16S ribosomal RNA gene sequencing. Endophytes were further characterized for the ability to produce several PGP substances, like ammonia, siderophores, indol-3-acetic acid, and hydrogen cyanide and for the ability to solubilize phosphate. Plant cell-wall degrading enzymes were also determined. Densities of BE were higher in roots (log 6.39 CFU g−1 fresh weight) than in shoots (log 5.56 CFU g−1 fresh weight). Phylogenetic analysis showed that BE were affiliated to two major groups: -Proteobacteria (50%) and Firmicutes (31.6%) and a small part belonged to - (7.9%) and -Proteobacteria (10.5%), being Pseudomonas and Bacillus the most highly represented genera. Higher bacterial diversity was found in the lavender roots, with endophytes belonging to 6 different genera (Pseudomonas, Variovorax, Rhizobium, Caulobacter, Bacillus and Paenibacillus), than in shoots where only 3 genera (Bacillus, Pseudomonas and Xanthomonas) were found. Overall, BE showed ability to produce extracellular enzymes and multiple PGP traits, suggesting their potential use as efficient bioinoculants in sustainable cultivation of medicinal and aromatic plants.
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