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- Bioremediation on anthropogenic affected areas: Ectomycorrhizal and plant growth bacteria as promoters of pine establishmentPublication . Franco, Albina R.; Sousa, Nadine R.; Ramos, Miguel A.; Castro, Paula M. L.The recovery of damaged areas due to inadequate farming policies and increased industrial sediment deposition, have contaminated not only soil and surrounding areas but also other natural resources. The potential use of disturbed sites for agriculture and forestry is jeopardised and their remediation is critical and expensive. The utilization of biotechnological tools, such as plant growth promoting bacteria (PGPB) and ectomycorrhizal fungi (ECM) could help remediation of such soils as they can be used as plant facilitators for land recovery. The aim of this study was to assess the potential of PGPB and ECM to enhance the growth of Pinus pinaster in antropogenic sediments and forest soil. Pine seedlings were inoculated with Suillus bovinus, Pisolithus tinctorius and Paxillus involutus, and co-inocualted with Bacillus spp. and Mesorhizobium spp. Plants were harvested after 6 month growth and parametric and nutritional data determined. Results show that P. involutus increased seedling growth(height) in industrial sediments soil, whereas in forest soil, plant performance was higher with S. bovinus. The effect of inoculation on the fungal communityin seedling roots and bacterial rhizosphere was also analysed by PCR-DGGE and differences arose between inoculated and uninoculated soil, indicating that PGPB and ECM may significantly influence the plant growth performance over a period of time. The study shows that PGPB and ECM fungi may be used as a biotechnology tool contributing to the successful plant establishment in disturbed environments.
- Succession dynamics of ectomycorrhizal fungi in inoculated Quercus rubra seedlings – a field studyPublication . Franco, Albina R.; Ramos, Miguel A.; Sousa, Nadine R.; Oliveira, Rui S.; Castro, Paula M. L.Selected ectomycorrhizal (ECM) fungi may have a beneficial effect on the establishment of nursery grown seedlings and may influence their survival on the first years. By increasing seedling capacity to capture nutrients, ECM fungi often contribute to a successful plant establishment. We investigated the performance of pre-inoculated Quercus rubra seedlings on a reforestation site and monitored ECM fungal persistence and succession dynamics between selected ECM fungi and native fungal community. Nursery grown Q. rubra seedlings inoculated with a mixture of ECM fungi (Cenococcum geophilum, Hebeloma crustuliniforme, H. mesophaeum, H. velutipes, Paxillus involutus, Scleroderma citrinum) and non-inoculated control oak seedlings, were transplanted to Serra da Cabreira in Northern Portugal. Two years after planting, the subsistence of the inoculated ECM fungi was assessed using ITS-DGGE. Results demonstrated that inoculation with selected ECM fungi at nursery stage promoted the establishment and improved the growth performance of out planted oak seedlings. C. geophilum was significantly associated with inoculated saplings but other ECM fungi species were also found. Moreover, ECM fungal communities of inoculated and non-inoculated samples are significantly different. Further monitoring is required to increase knowledge on the persistence of ECM fungal communities and their succession dynamics.
- A genotype dependent-response to cadmium contamination in soil is displayed by Pinus pinaster in symbiosis with different mycorrhizal fungiPublication . Sousa, Nadine R.; Ramos, Miguel A.; Marques, Ana P. G. C.; Castro, Paula M. L.Soil contamination with Cd is of primary concern and beneficial soil restoration strategies urge. The aim of this work is to evaluate the response of two different genotypes of Pinus pinaster (wild and selected) to Cd contamination and to assess how inoculation with ectomycorrhizal fungi, Suillus bovinus and Rhizopogon roseolus, influenced each genotype. Seedlings were exposed to soil contaminated at 15 and 30 mg Cd kg−1. Plant growth, mycorrhizal traits and Cd accumulation in different tissues of the plant were determined at harvest. The fungal community was assessed by denaturing gradient gel electrophoresis. At 15 mg Cd kg−1 S. bovinus increased aboveground development in both genotypes. At 30 mg Cd kg−1 non-inoculated wild genotype accumulated more Cd in the shoots (1.7-fold) than the selected genotype; inoculation with R. roseolus decreased Cd concentration in the roots of the selected genotype whereas the opposite occurred in the wild genotype. Cd concentration in the root system was the parameter most influenced by the interaction between the three studied variables. The fungal community established was affected by the Cd concentration in the soil. Results show that different genotypes of P. pinaster react differently to Cd exposure depending on the mycorrhizal association. The importance of considering the combination between plant genotype and its symbiotic partners when aiming at the forestation of degraded land is highlighted.
- Reforestation of burned stands: the effect of ectomycorrhizal fungi on Pinus pinaster establishmentPublication . Sousa, Nadine R.; Franco, Albina R.; Ramos, Miguel A.; Oliveira, Rui S.; Castro, Paula M. L.The area occupied by Pinus pinaster in Portugal is rapidly diminishing because of forest fires. Ectomycorrhizal fungi form obligate, mutually beneficial associations with P. pinaster which improve plant growth and resistance to adverse conditions. The aim of this work was to assess whether native ectomycorrhizal fungi could be a useful tool in the reforestation of burned areas. The work was conducted in a forest nursery greenhouse, where P. pinaster seedlings were inoculated with compatible ectomycorrhizal fungal isolates: Suillus bovinus, Pisolithus tinctorius, Rhizopogon roseolus, and a mixture of the three fungi, using burned and unburned forest soil as substrate. Inoculation significantly enhanced the growth of P. pinaster, with R. roseolus proving to be the most effective in burned soil, with an 8-fold increase in plant fresh weight. Overall, inoculation stimulated growth most in burned than in unburned soil. This study suggests that inoculation with selected ectomycorrhizal fungi in containerised nurseries can be an advantageous approach for the successful establishment of P. pinaster in burned soil. The obtained results point out to the interest of extending these studies into fire-impacted areas, using ectomycorrhizal fungi as a biological tool.
- Non-invasive monitoring of stress response of urban trees inoculated with EcMPublication . Serafim, Cindy; Ramos, Miguel; Yilmaz, Tugce; Sousa, Nadine; Castro, Paula M. L.
- Vegetation reflectance spectroscopy for biomonitoring of heavy metal pollution in urban soilsPublication . Yu, Kang; Geel, Maarten Van; Ceulemans, Tobias; Geerts, Willem; Ramos, Miguel Marcos; Sousa, Nadine; Castro, Paula M. L.; Somers, BenHeavy metals in urban soils may impose a threat to public health and may negatively affect urban tree viability. Vegetation spectroscopy techniques applied to bio-indicators bring new opportunities to characterize heavy metal contamination, without being constrained by laborious soil sampling and lab-based sample processing. Here we used Tilia tomentosa trees, sampled across three European cities, as bio-indicators i) to investigate the impacts of elevated concentrations of cadmium (Cd) and lead (Pb) on leaf mass per area (LMA), total chlorophyll content (Chl), chlorophyll a to b ratio (Chla:Chlb) and the maximal PSII photochemical efficiency (Fv/Fm); and ii) to evaluate the feasibility of detecting Cd and Pb contamination using leaf reflectance spectra. For the latter, we used a partial-least-squares discriminant analysis (PLS-DA) to train spectral-based models for the classification of Cd and/or Pb contamination. We show that elevated soil Pb concentrations induced a significant decrease in the LMA and Chla:Chlb, with no decrease in Chl. We did not observe pronounced reductions of Fv/Fm due to Cd and Pb contamination. Elevated Cd and Pb concentrations induced contrasting spectral changes in the red-edge (690–740 nm) region, which might be associated with the proportional changes in leaf pigments. PLS-DA models allowed for the classifications of Cd and Pb contamination, with a classification accuracy of 86% (Kappa = 0.48) and 83% (Kappa = 0.66), respectively. PLS-DA models also allowed for the detection of a collective elevation of soil Cd and Pb, with an accuracy of 66% (Kappa = 0.49). This study demonstrates the potential of using reflectance spectroscopy for biomonitoring of heavy metal contamination in urban soils.
- Selection and acclimatization of strains of edible mycorrhizal fungi for improved field persistencePublication . Ramos, Miguel; Sousa, Nadine; Castro, Paula M. L.
- Tree breeding and mycorrhizal symbiosis as important tools in forestation processesPublication . Sousa, N. R.; Franco, A. R.; Ramos, Miguel A.; Oliveira, Rui S.; Castro, Paula M. L.
- The effect of fungal-bacterial interaction on the phenolic profile of Pinus pinea LPublication . Araújo, Giovânia C.; Sousa, Nadine R.; Castro, Paula M. L.Studies on the functional significance of bacteria associated with ectomycorrhizal (ECM) fungi are scarce, as well as information on the metabolism of the host plant when in symbiosis with ECM fungi. Here we intended to evaluate the phenolic profile of seedlings when associated with Bacillus subtilis (B1), Pisolithus tinctorius (Pis) and their combination (PisB1). The interaction between microorganisms was conducted in three stages: (i) in vitro evaluation of fungal/bacterial interaction, (ii) microcosms, (iii) plant transplantation to natural soil. The profile of phenolic compounds was determined at the end of stages (ii) and (iii) and further supplemented with biometric, nutritional and analysis of the ectomycorrhizal community by denaturing gradient gel electrophoresis. In the in vitro compatibility test, B1 inhibited fungal growth at all glucose concentrations tested. In the microcosm, the levels of chlorogenic and p-coumaric acid decreased over time, unlike the protocatechuic acid which tended to increase during 70 days. After transplantation to the soil, the levels of phenolic acids decreased in all treatments, while catechin increased. B. subtilis positively influenced the fungus-plant relationship as was evidenced by higher biomass of seedlings inoculated with the dual inoculum (PisB1), both in the microcosm and soil stages. The presence of the bacteria interfered in the composition of the ECM fungal community installed in Pinus pinea L. in the soil. This leads to infer that B. subtilis may have caused a greater effect on the metabolism of P. pinea, especially in synergy with mycorrhizal fungi, than the action of the isolated fungus.
- The response of Betula pubescens to inoculation with an ectomycorrhizal fungus and a plant growth promoting bacterium is substrate-dependentPublication . Sousa, Nadine R.; Franco, Albina R.; Ramos, Miguel A.; Oliveira, Rui S.; Castro, Paula M. L.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.
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