Discussion

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Figure 3.2.11 Principal component analysis (PCA) explaining variation in root architectures associated with ectomycorrhizal and non-ectomycorrhizal Q. ilex seedling growing with different litter regimes. In right side score plot of the variable measured to explain root system architectural changes. Left-side trajectories of associated loadings in each sampling date.

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between symbiotrophism and saprotrophism-pathotrophism again its host (Garbaye et al., 1992; Garbaye, 1994; Frey‐Klett et al., 2007).

Few assumptions could be generated in the first experiment with the attempt to explain mechanisms by which soil microbiota is crucial for the positivity of the ECM symbiosis. However, the increase in growth promoted by the treatment with litter lead us to hypothesize that microbial community act as key to unlock nutrient immobilized in leaf litter (Garbaye, 1994; Dix, 2012). Subsequently, the action operated by the soil microbiome lead the possibility to the ectomycorrhizal basidiomycetes to release its enzymatic arsenal, that is developed for the decomposition of detrimental compounds (Baldrian, 2008; Rytioja et al., 2014). Is acknowl-edged that during microbial succession almost the totality of basidiomycetes occurs at the end of the process of decomposition (Frankland, 1998; Purahong et al., 2016; Bonanomi et al., 2019). This since, whether in some way basidiomycetes has high diversity of enzymatic products and secondary metabolites, on the other hand, they lack in the ability to decompose and adsorb the labile compounds in fresh deposed organic matter (Deacon, 2013). So far, could be assumed that the dependence by the microbial community is because it acts as starter for basidiomycetes action and in turn advantage for symbiotic plants (Berg & Ekbohm; Hiscox et al., 2018).

In the first experiment is observed that ectomycorrhizal symbiosis function well in unsterilized condi-tions. The suggestion raised in that trial provided useful information for the understanding of ECM Symbiosis functioning. The full comprehension of the process still requires deeper investigation but underline the im-portance of a proper microbiome in soil for the development of important processes as such as ECM symbiosis in woodland soil. In a different framework, the second experiment provide us a detailed view on the effect of ECM plants growing in presence of litter (differently decomposed) and the effect on plant root proliferation and strategic expansion. We do not apply root sterilization because already stated the essential need of a struc-tured soil microbiome supporting plant growth. Subsequently, the results of our experiment demonstrated that ECM relationship became more positive in presence of self-litter that presumably is used as nutrient resource and, inversely, when ECM symbiosis is not present, self-litter has a detrimental effect on root proliferation with a more probable phytotoxic effect (DeBell, 1971; Dabral et al., 2018). This is also confirmed by the values reported in plant growing in absence of litter enrichment that in ECM condition appear to be depressed, mean-while without symbiotic inoculum is promoted to positive levels. To date, when comparing fresh and decom-posed self-litter, phytotoxicity became higher at 120 day of decomposition. This effect was similarly demon-strated in work on germination of Q. ilex acorns with decomposed self-litter underlining the presence of a species-specific self-detrimental effect from the release of autotoxic self-DNA (Mazzoleni et al., 2015a;

Mazzoleni et al., 2015c). These evidences allow us to think at a similar effect, that is however present but less effective in ECM seedlings when compared to ECM seedling growing in correspondence of fresh litter.

More interestingly, root development in the four date of sampling showed that almost the totality of seedlings has a decreasing ability to allocate biomass in root. Only exception is made for Non-ECM seedlings growing without litter. This is demonstrated by point of inflection of the trend. Indeed, in ECM plants the inflection points commonly take place at 30 day of growth rather than Non-ECM plant do not showed inflection

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but a continuous negative development from the first date of sampling until the end of the experiment. This suggest that negative condition for root expansion appeared later in ECM plants as it is possible that the phy-totoxic compounds from litter, presents in the first layer of soil, are early degraded by the fungal mycorrhizal partner. We consider this effect on the first layer of soil because of the low effectiveness of the ECM hyphal activity in the lower layer of soil (Dickie et al., 2002; Rosling et al., 2003). Inversely in Non-ECM plants phytotoxicity became early present as probably lacking enzymes produced by the Basidiomycetes acting as chemical degrading agent in the upper layers of soil.

Regarding how ECM symbiosis act in positive way for Q. ilex seedlings and how the energy obtained in the first layer of soil is transferred to root structures, our analysis of the root system evidenced that ECM plants has a wider spread of the adsorbing system with respect to Non-ECM seedling. This strategic adaptation is particularly evident when comparing Non-ECM plants growing in absence of self-litter that evidentially prefer to growth in deeper soil layer with no lateral ramification or genesis of lateral leading roots. Concomi-tantly is observed and increase of fine root density in the main leading roots, overly magnifying in a bifidum herring bone structure. This structural development could be interpreted as structural development triggered by an environmental context where perturbation or agent inducing root structural modification are minimized.

Additionally, the downward exploring behaviour of these root structures could be given to the characteristic evolutionary fitness of Mediterranean Q. ilex that preferentially explore deeper layer of well hydrated soil (David et al., 2007). So far, the gravitropic and water-dependent polarity of root appear to be highly preserved with respect to other plants that, although has certainly this kind of effect, preferentially developed for the expansion of the root system in a wider area. The effect could be interpreted in different way: In the first instance is probable that the structure of the root system undergoes earlier modification by the increase in ramification promoted from ECM roots and because the ramification on ECM plants lead to the production of higher number of leading roots. As consequence by a general self-repulsion and intraindividual competition root system assume a wider arrangement. This could also be supported by the higher level of resourced scav-enged by the ECM symbiosis that can support a more extended and articulated root system. On the other hand, although in minor extent, Non-ECM plants preferentially developed a root system with a preferential wider expansion but only in presence of litter. This, bring us to a consideration that the increased expansion could be generated by the loss of apical dominance in the early stage of root development triggering ramification and dominance of lateral roots. To date, the effect of loss of apical dominance could be assigned to a direct phyto-toxicity effect on root apex becoming sensible to phytotoxic compound released from litter. The explanation of the phenomenology could rely on both the hypotheses but do not explain the whole strategic development of the root system. Indeed, whether ECM plants could produce wider root system because of the increased tenor of resource acquisition, the biases on the assumption coming directly by the ability of the Non-ECM plant to produce a preferential wider root system. On the other hand, the self-litter damaging hypothesis could be partially accepted because of the presence of a wider root system in ECM plant growing in absence of litter.

Conceivably, the two mechanisms hypothesized, could be interpreted as a mixed effect of both the processes of increased ramification in ECM plants and loss of apical dominance by phytotoxicity.

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