Nadine R. Sousa

Ectomycorrhizal fungi as an alternative to the use of chemical fertilisers in nursery production of Pinus pinaster

Addition of fertilisers is a common practice in nursery production of conifer seedlings. The aim of this study was to evaluate whether ectomycorrhizal (ECM) fungi can be an alternative to the use of chemical fertilisers in the nursery production of Pinus pinaster. A greenhouse nursery experiment was conducted by inoculating seedlings obtained from seeds of P. pinaster plus trees with a range of compatible ECM fungi: (1) Thelephora terrestris, (2) Rhizopogon vulgaris, (3) a mixture of Pisolithus tinctorius and Scleroderma citrinum, and (4) a mixture of Suillus bovinus, Laccaria laccata and Lactarius deterrimus, using forest soil as substrate. Plant development was assessed at two levels of N-P-K fertiliser (0 or 600 mg/seedling). Inoculation with a mixture of mycelium from S. bovinus, L. laccata and L. deterrimus and with a mixture of spores of P. tinctorius and S. citrinum improved plant growth and nutrition, without the need of fertiliser. Results indicate that selected ECM fungi can be a beneficial biotechnological tool in nursery production of P. pinaster.

The effect of ectomycorrhizal fungi forming symbiosis with Pinus pinaster seedlings exposed to cadmium

Cadmium is one of the most toxic heavy metals and its accumulation in the upper layers of forest soils affects plants, microorganisms and their interactions. Adequate strategies for the reforestation of metal contaminated sites are of vital importance. The aim of this work was to evaluate the response of Pinus pinaster seedlings to Cd exposure and to assess the effect of inoculation with two selected ectomycorrhizal fungi, Suillus bovinus and Rhizopogon roseolus on that response. Seedlings were exposed to soil contaminated at 15 and 30 mg Cd kg(-1). Shoot biomass of P. pinaster decreased ca. 36% when exposed to 15 mg Cd kg(-1). Overall, colonization by S. bovinus significantly enhanced shoot development up to 30% in contaminated soil while colonization by R. roseolus produced no significant effect at both Cd concentrations tested and significantly increased the level of Cd in the shoots at both Cd concentrations. Metal accumulation in the shoots and roots of non-inoculated and S. bovinus-inoculated seedlings increased at the higher Cd levels whereas R. roseolus-inoculated seedlings were not sensitive to Cd variation in the soil. The results from our research show that inoculation with ECM fungi has a significant impact on metal uptake and development of P. pinaster seedlings; the differential response induced by the two tested species highlights the importance of selecting the appropriate strains for nursery inoculation, and, as such, this biological tool ought to be considered in reforestation processes of heavy metal contaminated areas by woody species.

Mycorrhizal symbiosis affected by different genotypes of Pinus pinaster

Background and aimsHigher growth rate and morphological traits have been the major criteria for selecting trees in breeding programs. The symbiotic associations between P. pinaster and ectomycorrhizal fungi can be an effective approach to enhance plant development. The aim of this work was to assess whether the establishment of mycorrhizal symbiosis at nursery stage was affected by tree breeding.MethodsSeeds of P. pinaster from a clonal population, designed to select for various traits, and from neighboring wild plants were inoculated with compatible ectomycorrhizal fungi: Suillus bovinus, Pisolithus tinctorius or Rhizopogon roseolus, and grown in individual cells containing forest soil, in a commercial forest nursery. Growth and nutritional traits, colonisation parameters and the fungal community established were assessed.ResultsR. roseolus and P. tinctorius were the most efficient isolates in promoting plant development. Inoculated selected saplings had an overall superior development than their wild counterparts, with up to a 4.9-fold in root dry weight and a 13.6-fold increase in the total number of ectomycorrhizal root tips. Differences in fungal community were revealed through the denaturing gradient gel electrophoresis profile of each treatment.ConclusionsThe results from our study suggest that the selected genotype benefits more from the mycorrhizal association and therefore this could be a valuable biotechnological tool for the nursery production of P. pinaster.

Diversity and persistence of ectomycorrhizal fungi and their effect on nursery-inoculated Pinus pinaster in a post-fire plantation in Northern Portugal.

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.

The effect of fungal-bacterial interaction on the phenolic profile of Pinus pinea 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.

Vegetation reflectance spectroscopy for biomonitoring of heavy metal pollution in urban soils

Heavy 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.