Our results show that an increase in the inoculum quantity applied can improve the level of T. melanosporum colonization. For the 2018 experiment, this increase happened even in the range 0.8 (with I2 alone or with I3 alone) to 1.70 g fresh truffle per seedling (I1 + I2 + I3), which is commonly used in commercial seedling production (Granetti 2005; Hall et al. 2007; Palazón and Barriuso 2007). However, this increase was barely apparent in the 2015 experiment, agreeing with Palazón et al. (2007) who did not find increases in colonization levels between 1–5 g fresh truffle per seedling with an inoculation method based on a single moment of application, and with Pruett et al. (2008) who could not increase mycorrhizal levels when they applied supplemental inoculation. The fact that different inoculum delivery systems and application moments were combined may be playing a role in increasing mycorrhizal rates. However, as clearly shown in both experiments, the combination of several inoculation methods and the resulting increase of the amount of inoculum applied did not have an additive effect on mycorrhizal colonization.
On the other hand, none of the three inoculation methods applied separately appeared clearly superior to the other. This is particularly meaningful in the case of the radicle inoculation, which spent three to seven times less inoculum. However, the relative effectiveness of these methods may depend on the cultivation conditions of the seedlings and/or the timing of the nursery operations, as suggested by the differences between the 2015 and the 2018 experiments. Interestingly, the inoculation rates obtained with early inoculation methods (I1, I2, I1 + I2) were similar in both experiments, whereas this did not happen in treatments including an inoculation in the pot (Figs. 1, 3; Tables S1, S6). Among the latter, treatments with lower inoculum quantity (I3, I1 + I3) seemed to perform better in the 2015 experiment, whereas those with higher inoculum quantity (I2 + I3, I1 + I2 + I3) seemed to perform better in the 2018 experiment. These could be related to the experiment timing, as the 2018 seedlings had the spring of their second year to develop new fine roots. The stronger effect of I3 along the depth profile of 2018 seedlings seems to support this hypothesis. Percent root colonization is the variable generally used to evaluate truffle-inoculated seedlings, but its dynamics relies on the relative rhythm and timing of fine root formation and fine root colonization by truffle (Andres-Alpuente et al. 2014).
Regarding the different mycorrhization percentages along the root ball, the depth distribution of mycorrhizal levels provided insight into the patterns governing T. melanosporum colonization, with each inoculation method affecting depth distribution differently. In almost all cases the percent root colonization decreased with substrate depth, with the only exception of samples in which there was no I1 or I2 (i.e. treatment wo I1-wo I2 in 2018; Fig. 4a). The I3 inoculation was the only one that showed a significant effect on depth distribution in both experiments. Interestingly, this method not only increased mycorrhizal levels in the lower part of the root system, where the other methods did not act, but also in the middle part (2015) or in all depth (2018), despite the fact that in I3 most inoculated substrate was added in the lower depth.
The other inoculation methods showed less consistent results: the I1 inoculation did not affect the depth distribution in 2015, but showed a positive effect on the upper part of the pot in 2018 (w I1-wo I2 vs. wo I1-wo I2; Fig. 4a); whereas the I2 inoculation did not affect the depth distribution in 2015, but showed a positive effect on the upper and middle parts of the pot in 2018 (wo I1-w I2 vs. wo I1-wo I2; Fig. 4a), coinciding with the depth of the seedling trays. This differs from the findings of Garcia-Barreda et al. (2017), where no significant depth patterns in T. melanosporum colonization were found with the two inoculation method used, both of them applied in the final pot. All this indicates that, separately, I1 and I2 are not effective in achieving high levels of inoculation in the lower part of the root system, which could lead to mycorrhizal levels being more irregular along the depth of the final seedling, at least during the first year in the nursery.
The tested inoculation methods not only improved T. melanosporum colonization levels but also decreased S. brunnea spread on the seedling roots, suggesting that S. brunnea colonization was related to low inoculation levels by the target fungus. This agrees with the pioneer behavior of this fungus, which usually colonizes the roots during late autumn or winter, thus reducing the availability of root tips for the target fungus during the second year in the nursery (Sánchez et al. 2014; Garcia-Barreda et al. 2017). Besides, S. brunnea is able of rapidly fruiting as soon as it establishes its first mycorrhizae, which promotes the rapid spread of the fungus in greenhouses in with batches of different ages are kept together (Meotto and Carraturo 1988; Garcia-Montero et al. 2008). In 2015, the three inoculation methods reduced the occurrence of S. brunnea, whereas in 2018 only I2 did. The results in the 2018 experiment suggest that an early inoculation of the substrate was more effective in controlling the non-desired infection by S. brunnea, although in 2015, with the inoculation treatments applied 2–3 months earlier than in 2018, the three inoculation treatments were effective. The effectiveness of early substrate inoculation could be related to T. melanosporum mycorrhization being spread throughout the roots before the autumn temperature drop and the ensuing period of high and continued moisture.
Regarding to the other contaminating fungus found in the 2015 experiment, our study is the second report of P. constellatio in Spanish nurseries (Sánchez et al. 2020). This fungus is relatively frequent in Italy (Marozzi et al. 2018). However, in Spain we have only found it in two cases, both of them using the same commercial substrate (which is no longer marketed in Spain).
In the context of commercial production of truffle-inoculated seedlings, the sequential application of three inoculations appears as an effective and realistic alternative for the inoculation of Q. ilex seedlings with T. melanosporum (Palazón and Barriuso 2007; Donnini et al. 2014; Murat 2015). This method is based on (i) putting ascospores in contact with roots before the formation of fine roots (Chevalier 2001; Granetti 2005; Palazón and Barriuso 2007; Garcia-Barreda et al. 2017), and (ii) reducing the deficiencies of single methods and the impact of contingencies in the nursery management by distributing the risk among three inoculations. The early inoculations (I1 and I2) showed positive implications in the management of the opportunist S. brunnea, which frequently appears as a serious problem in some nurseries (Sánchez et al. 2014). The third inoculation (I3) showed positive implications in the mycorrhization of roots in the lower depth, which in our experience is frequently the cause of depth irregularity in the mycorrhizal levels of inoculated seedlings, especially during its first year in the nursery. Finally, the transplant of the root ball to the final pot (step from I2 to I3) could also play a role on reducing seedling mortality, which uses to reach 2–10% when nude root transplanting is performed (Palazón and Barriuso 2007).
However, it would be interesting to test whether the mycorrhizal levels of these seedlings are equivalent to those of Q. ilex seedlings inoculated with a single method but the same total amount of inoculum, and whether the sequential inoculation affects the growth and morphology of the plant material. For that matter, it should be considered that in Spain commercial seedlings can be marketed after their first year in the nursery (from about seven months after inoculation) or during their second year (12–19 months after inoculation). For seedlings in their first year, early inoculations could provide some competitive advantage against contaminants colonization. It would also be interesting to investigate how truffle inoculum reaches the fine roots in the radicle inoculation, in order to optimize this method that requires lower amounts of inoculum. Finally, it would also be important to test whether the sequential inoculation could have implications for the relationship between mating types in nursery seedlings. So far, this relationship has only been studied with a single inoculation method applied in the final container, with a tendency for one mating type to dominate over the other from the first to the second year in the nursery (Rubini et al. 2011; Gómez-Molina et al. 2023).