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Key message Despite the fact that the technique of application of bioinoculants improved the quality ofQuercus suberL. seedlings produced in nurseries, these benefits are dependent on the ecological conditions of the site and the composition of the applied inoculum, which interferes with the profile of the local fungal community. Context Quercus suber L. plays a key ecological and socio-economical role in the Iberian Peninsula. Symbiotic ectomycorrhizal fungi-ECM are crucial partners of several tree species, and assessing the efficacy of bioinoculants at nursery stage helps devising tools to increase plant resilience. Aims The aim of this study was to compare the effects of two inocula formulations of mixed ECM fungi and bacteria on the quality of seedlings produced in two forest nurseries, differing in environmental conditions and forest embedment. Methods Quercus suber L. seedlings were inoculated with a commercial product containing Pisolithus tinctorius (Pers) Coker & Couch, Scleroderma sp., and six bacterial species and with a non-commercial fungal and bacterial dual inoculum (Suillus granulatus (L.) Roussel + Mesorhizobium sp.). Biometric and nutritional parameters and morphological quality indexes were determined on seedlings. The ECM community was assessed by denaturing gradient gel electrophoresis and cloning-sequencing. Results In both nurseries, the seedling quality index in inoculated was up to 2-fold higher than in non-inoculated seedlings. Plant biomass differed significantly among nurseries. The inoculum influenced the profile of the fungal community. S. granulatus and P. tinctorius persisted for 6 months in the inoculated seedlings. Conclusion The nursery ecosystem influenced plant growth. Inoculation treatments increased plant performance; however, the dual inoculum resulted in more consistent improvements of Q. suber at nursery stage, highlighting the importance of inocula selection.

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 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.

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.