Browsing by Author "Ramos, Miguel A."
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- Benefits of ectomycorrhizal inoculation of tilia tomentosa seedlings on plant growth and vigor depends on substrate pHPublication . Serafim, Cindy; Ramos, Miguel A.; Yilmaz, Tugce; Sousa, Nadine; Castro, Paula M. L.
- 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.
- Combining ectomycorrhizal fungi and bacteria: a powerful tool to improve tree performancePublication . Ramos, Miguel A.; Sousa, Nadine R.; Franco, Albina R.; Dundar, Gonul; Oliveira, Rui S.; Castro, Paula M. L.Ectomycorrhizal fungi are ubiquitous to forest soils and the symbiosis between these microorganisms and plants roots is known to be essential for tree establishment and development, especially in areas of poor soil. Studies have also shown that some rhizosphere bacteria may act as plant stimulating agents and growth promoters. Additionally, bacteria and fungi strongly interact and therefore the analysis of the triangle plant-fungi-bacteria is a vital approach when aiming at the use of microbial inocula to enhance plant performance. To obtain a synergistic combination between microorganisms, a thorough selection is required since bacteria may strongly inhibit fungal growth, and vice versa, incurring in the risk of failed inoculum. In the present work we investigated the use of a dual inoculation system, consisting of a bacterial strain from the genus Mesorhizobium in combination with selected ectomycorrhizal fungi. In vitro tests were performed to assess the dynamic of co-culturing both microorganisms. The inoculum was applied at nursery stage and saplings were then transplanted into the field. Approximately 5000 seedlings were used in this experiment comprising the following species: Quercus suber, Quercus robur, Quercus rubra, Pinus pinaster and Pinus pinea. Seedlings were grown for 9 months in a commercial forest greenhouse and then transplanted to 4 locations in Portugal: Mindelo, Santo Tirso, Vila do Conde and Proença-a-Nova. Samples were taken at the end of nursery phase and 8 months after transplantation. Biometric and mycorrhizal parameters were analyzed and the persistence of the inoculated strains was evaluated. The presence of selected phytohormones was also determined. Results showed that the combined use of fungi-bacteria inoculum can be more effective than the use of each individual microbial partner. The use of ectomycorrhizal fungi and bacteria has great potential in forestry as a biotechnological tool to produce high performance plants at nursery stage and in the field.
- Ectomycorrhizal fungi inocula optimization to aid the health status of trees in the everchanging environment of citiesPublication . Serafim, Cindy; Ramos, Miguel A.; Améglio, Thierry; Castro, Paula M. L.
- Effect of diflubenzuron on the development of Pinus pinaster seedlings inoculated with the ectomycorrhizal fungus Pisolithus tinctoriusPublication . Ramos, Miguel A.; Sousa, Nadine R.; Franco, Albina R.; Costa, Vítor; Oliveira, Rui S.; Castro, Paula M. L.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.
- 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.
- Growth dynamic of dual culture systems comprising ectomycorrhizal fungi and mycorrhiza helper bacteriaPublication . Sousa, Nadine R.; Ramos, Miguel A.; Castro, Paula M. L.Ectomycorrhizal fungi are ubiquitous root symbionts that associate with the majority of forest trees and often have a crucial role on plant survival and growth in impoverished soils. Within the vast community of soil microorganisms, the mycorrhiza helper bacteria (MHB) are recognizably one of the groups that most directly affect fungal growth and mycorrhiza establishment. Although their positive effect on the mycorrhizal partnership has been previously reported, the specificity of such association is high and the dynamics not yet fully understood. Many studies evaluated the impact of bacteria on fungal growth but the reciprocal has received little attention. The present study aimed to evaluate the growth dynamics of co-culturing fungi and bacteria, under both perspectives. Bacillus pumilus was chosen for its capability to promote the growth of Suillus granulatus in a dual culture system with a low nutrient medium. To assess the impact of fungal exudates on bacterial growth, different quantities of fungal culture medium were added to MMN medium containing a Bacillus pumilus inoculum and bacterial growth was monitored. To evaluate the influence of Bacillus pumilus on fungal growth, an experiment was setup where the bacterium was added at two different phases of fungal growth, lag phase and exponential phase. A control without bacteria was also performed. Bacterial growth was monitored through OD readings and fungal growth was assessed through dry weight and ergosterol content. The experience was held for 33 days, with sampling every three days. Triplicates for each treatment were performed. Results from the first experiment showed that fungal exudates influenced the growth of bacteria with a pronounced extension of the lag phase. In the second experiment, fungal growth was completely inhibited when bacteria was added at the fungal lag phase. A different outcome was observed when the bacterium was added in the exponential phase. Results suggest that the regulation of the concentration at which the bacterium is present may be a key factor to optimization of the use of bacteria as MHB.
- Impact of bacterial inoculation of Quercus suber on mycorrhization by Suillus species at early stages of tree developmentPublication . Ramos, Miguel A.; Sousa, Nadine R.; Franco, Albina R.; Dundar, Gonul; Oliveira, Rui S.; Castro, Paula M. L.Portugal accounts for approximately 50% of the world cork production. Quercus suber is the second most abundant tree in Continental Portugal, covering currently 737 thousand hectares and representing 23% of the forest area. This species is of paramount importance to the country both ecologically and economically. As many other forest species, Q. suber forms symbiotic associations with ectomycorrhizal (ECM) fungi with the hyphae acting as fine extensions of the plant root system allowing it to capture water and nutrients in exchange for photosynthetic carbohydrates. The use of bacteria as inoculum also has great potential in forestry, whether through their capability to act as plant growth promoters (PGPB) or/and as mycohhizal helper bacteria (MHB). The latter have a direct impact in promoting fungal symbiosis and could possibly represent the third link in the mycorrhizal partnership. However, the association between all vertices of the triangle (plant-fungi-bacteria) is highly specific and needs to be further explored when aiming at the use of microbial inocula in the tuning of mycorrhization at nursery stage. The aim of this work was to study the impact of inoculating Q. suber with a fungus belonging to the genus Suillus and a bacterial strain belonging to the genus Mesorhizobium, proven to be PGPB in preliminary studies. The experiment was conducted in a forest nursery in Amarante. After 9 months, the impact of inoculating with each microorganism individually was compared to that of the use of the combined inoculum. Biometric parameters were determined (shoot height, primary and secondary root length, shoot and root dry weight) and mycorrhizal parameters were assessed (percentage of ECM colonisation and expansion rate). The presence of selected phytohormones was also determined by HPLC. Overall, inoculated plants had a superior performance than the non-inoculated controls, in the majority of the parameters studied. The use of the combined inoculum was more successful than any other treatment in promoting shoot height and led to the highest concentration of indoleacetic acid (IAA). The results obtained suggest that the use of bacteria in the mycorrhization of Q. suber may positively affect plant in its early stage of development.
- Inoculation with ectomycorrhizal fungi affects pinus pinaster performance under cadmium exposurePublication . Sousa, Nadine R.; Ramos, Miguel A.; Marques, Ana P. G. C.; Castro, Paula M. L.Afforestation of contaminated sites can represent a valuable approach to restore degraded ecosystems. Studies on the response of woody species to heavy metal contamination in soil are scarce compared to crop species. Cadmium is one of the most toxic heavy metals and its hazardous effects are well known. The aim of this work was to evaluate Pinus pinaster performance on Cd contaminated soil (15 and 30 mg Cd kg-1) and determine whether inoculation with two ectomycorrhizal fungi, Suillus bovinus and Rhizopogon roseolus influenced such response. Regarding non-inoculated seedlings, Cd exposure led to a lower shoot biomass and metal accumulation on the root system was proportional to its concentration in the soil. Inoculation with S. bovinus was the most favorable treatment for P. pinaster development by enhancing shoot development up to 1.3-fold in contaminated soil. Inoculation with R. roseolus increased Cd concentration in the shoots with no significant effect in any of the biometric traits studied. Metal accumulation on the shoots and roots of P. pinaster seedlings was significantly affected by the interaction between mycorrhizal inoculation and the Cd concentration to which the seedlings were exposed. Results from this study show that inoculation with selected ECM fungi can influence the performance of P. pinaster under Cd exposure and that this biotechnological tool could be of great value for plant establishment in contaminated sites.
- Mycelia inactivation processes – maintaining the flexibility and strength of mycelium-based biocompositesPublication . Oliveira, Ana T.; Ramos, Miguel A.; Castro, Paula M. L.The current trend towards a sustainable and circular economy proposes the development and use of bio-based materials from renewable resources. Mycelium-based biocomposites (MBB), which consists of defragmented lignocellulosic particles linked by dense mycelium, are an ecological and innovative solution to replace petroleum-based products. MBB have shown advantageous properties, such as acoustic insulation, fire resistance, and the absence of harmful synthetic chemical components. These properties are the basis for the production and use of MBB for a wide range of applications, including paper, textiles, foams for packaging material, vehicle parts, and electronic equipment packaging materials. Briefly, MBB production is achieved through the sterilization the biomass, inoculation and incubation with selected fungi, homogenization, and interruption of fungal growth (or inactivation). Most MBB go through a heating treatment to inactivate the mycelia. However, that treatment results in rigid biocomposites with low flexibility. This work investigated the performance of alternative inactivation methods aiming to achieve flexible but sturdy MBB. Low temperature treatments and CuSO4 were tested in biocomposites obtained from two fungi strains. The inactivation efficiency was evaluated through a cell viability assay, MTT assay. The physical properties of the resulting biocomposites were also assessed. Spraying MBB with a CuSO4 solution did not efficiently inactivate the fungi. Although the low temperatures seemed to have inactivated the fungi two days after treatment (ca. 1% cell viability), 15 days later the mycelia resumed growth again (34% cell viability). Therefore, those treatments did not efficiently inactivate the fungi but left them in a latent dormancy state. None of the tested methods compromised the biocomposites’ flexibility features. Further studies need to be conducted to identify inactivation methods that allow the production of MBB with a more diverse range of physical characteristics to expand their application potential.
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