From the results, it was clear that the potting mix containing composted soil amendment based on mowing and pruning (Mix 2) had excellent performance in terms of survival of the seedlings (90%) and percentage of mycorrhization (60%), resulting from a statistically significant increase in the root system's growth. Instead, potting Mix 1 showed higher mortality (50%) than the control and Mix 2, although the mycorrhization and root growth did not statistically differ from those of the control. Probably, some substances may have had phytotoxic or growth-slowing effects on the Q. pubescens seedlings. In fact, Mx 1, unlike Mix 2, is defined as mixed compost as it contains organic matter coming not only from green waste but also from municipal waste. The aerobic fermentation of mixed compost containing municipal waste might lead to an excess of phytotoxic substances that could have a negative impact on seedlings’ growth (Niccolò and Eugenio 2013). Obviously, these adverse effects can be simply avoided with accurate monitoring of the maturation phase. On the other hand, the results of the chemical-physical analyses of the mixed substrates used in the present study highlighted that the two compost-based amendments present high/very high values of almost all the mineral nutrients concerning the control one, which is used as a routine mycorrhization substrate devoted to the production of mycorrhizal seedlings in Umbria region. Therefore, contrary to expectations, the shoot and root growth performances and percentages of mycorrhization of the two compost-based amendments, were equal to or higher than those of the control mix. On the other hand, all the substrates are characterized by suitable parameters for the fructification of T. melanosporum (Chevalier and Sourzat 2012), such as the content of organic matter, exchangeable calcium, nitrogen, magnesium, and potassium. In addition, also the range of pH values is considered optimal for black truffle colonization (7.5–8.2) (Bustan et al. 2006) (Table 1). Our results could be based on some differences registered in the micronutrient content among the studied substrates. First, the boron content of Mix 1 and 2 is very high with possible phytotoxic effects, especially for Mix 1, which may have been the cause of its higher mortality. Conversely, these potentially harmful effects were not detected both on the aboveground and belowground allocation (Tables 2 and 3). Indeed, the shoot and root dry weight did not significantly differ among the potting mixes. According to Brdar-Jokanovic (2020), the requirements for boron differ significantly between plant species and genotypes within a species. Boron is a mineral element that is employed by plants in the metabolism of phenolic acids and the biosynthesis of lignin, which is directly related to the preservation of cell walls (Hu et al. 1996). In this trial, Q. pubescens seedlings grown on Mix 2, characterized by an optimal pH value for black truffle and an elevated boron content, showed the highest development of the root apparatus (number of root tips and forks, and total root length) combined with a significantly higher mycorrhization rate (Table 3). This could be in line with the evidence reported by Lehto et al. (2010), regarding the positive effect of increased boron availability on mycorrhizal symbiosis both in arbuscular and ectomycorrhizal plants. Indeed, the positive influence of increased boron availability is known on the formation of ectomycorrhizas (Mitchell et al. 1986; Möttönen et al. 2001). For these reasons, the presence of boron for plant growth and mycorrhizal occurrence is crucial as its deficiency is common in more than eighty countries in the world (Shorrocks 1997) especially in sandy soils and soils with an alkaline pH where this element is more easily leached, due to the rainfall affecting auxin transport (Bairu et al. 2009; Gupta et al. 2013). Second, regarding the sodium content, it is worth noting that the two compost-based amendments present higher sodium levels than the control one, even if not enough to be considered sodic soils (Marschner 2011). In this respect, Mix 1 and Mix 2 (Table 1) present an Exchangeable Sodium Percentage (ESP) of 4.8% and 5.2%, respectively. However, this aspect did not modify the capacity of T. melanosporum to colonize the plants of Q. pubescens which showed significantly higher levels of mycorrhizal colonization in Mix 2 compared to the control. Indeed, also Bai et al. (2021) reported that the salt tolerance of Quercus spp. seems to be improved by ectomycorrhizal inoculation. In addition, the alkaline pH of Mix 2 should have enhanced the mycorrhizal symbiosis relieving the sodium effect. Third, the values of phosphorus are very high for both the compost-based amendments, but this aspect did not influence the growth and mycorrhization of the plants. Instead, the low level of phosphorus in the control could be related to the high rate of mycorrhization, considering that T. melanosporum is very efficient to increase phosphorus uptake. This tendency has been observed in Mediterranean environments where limy forest soils are typically lacking in this mineral element (Nuṅez et al. 2006).
Furthermore, the 2 potting mixes with organic matter coming from different sources tested for the mycorrhization experiments (Fig. 1) highlighted that, after spore slurry inoculation with T. melanosporum, ECMs were formed on all potting mixes, matching, and exceeding the quality of standards of European producers (Reyna et al. 2000). In fact, it has been observed that the addition of organic matter in the form of fermented municipal compost and green organic wastes has a good impact on the ability of mycorrhizal fungi to proliferate and colonize (Lunt and Hedger 2003). For example, Pereira et al. (2013) used pine bark compost to evaluate the efficacy of the controlled inoculation of Quercus cerris and Quercus robur with Tuber melanosporum, revealing that with small pH adjustments these substrates are particularly effective. From the results was also evident that, as reported in Table 2, Q. pubescens seedlings inoculated with T. melanosporum grown on compost-based potting mixes, had greater biomass than those grown in the control mix. A wide number of studies have demonstrated that mycorrhizal symbiosis enhances nutrient transport between plant symbionts and soil fungi, resulting in growth benefits for the partners (Zhang et al. 2022; Álvarez-Lafuente et al. 2018; Liese et al. 2017). Oliveira et al. (2010) performed an ectomycorrhizal inoculation of Q. ilex with Cenococcum geophilum, Hebeloma mesophaeum, and Paxillus involutus grown in 3 traditional substrates used for the mycorrhization: composted pine bark, peat-based compost, and forest soil. According to their results, peat-based compost was the superior growth substrate for Q. ilex seedlings inoculated with H. mesophaeum (Oliveira et al. 2010). The incorporation of the organic amendment was also reported by Ríncon et al. (2006) to improve by over 20% the shoot growth of Pinus halepensis, and more than 43% the volume and dry biomass. In field trials, P. halepensis and other plants and shrub species have achieved comparable results using different methods of soil fertilization including composted urban waste and organic additives. (Román et al. 2003; Caravaca et al. 2005).
Our findings revealed that the positive effect of the green compost Mix 2 on the percentage of mycorrhization arises from a more developed root system (Table 3), indicating that the greater the number of tips the plant has, the more ECMs can form. The beneficial impact of ECM inoculation on root plant system development, found in this study, may result from increased nutritional status or stimulated plant hormones production by the plant and/or the ectomycorrhizal fungi to promote the mutualistic association (Abdulsalam et al. 2021). All these compounds can favorably influence root growth parameters (Barker and Tagu 2000; Chalot et al. 2002; Luo et al. 2009). In fact, although there was not a distinct separation of groups, certain individuals of Mix 2 tended to split from the two other treatments along the first principal component (PC1), according to the results of the multiparametric analysis of the principal components (PCA, Fig. 2). The fine-tuning of Mix 2 might be improved in the future to recognize a more distinct separation of the treatment, where factors such the number of root tips and forks, root length and volume, and root area accounted for more variation along PC1. Interestingly, the advantages of using potting Mix 2 are many: we know its composition perfectly; it is a waste substrate that costs little, is environmentally sustainable, and can offer excellent mycorrhization performances with T. melanosporum. In conclusion, the results reported, indicate that inoculation with T. melanosporum spore-slurry and the use of alternative, cheaper, and reused materials are suitable nursery practices to optimize the inoculation of ectomycorrhizal Q. pubescens plants in controlled conditions.