## 4 Discussion
We isolated N-fixing diazotrophic bacteria from leaves sampled in three collection sites: MP, G, and M. The abundance of N-fixing bacteria varied among sites, with higher MP frequency than G and M. Over half of them (3 out of 5) can solubilize phosphate. At the same time, only 25% of the phosphate solubilizing isolates were N-fixers (Table 1). Molecular analysis indicated that the N-fixing isolates belonged to spore-forming species affiliated with _Bacillaceae_ (Figure 2). [PERSON] et al. [62] reported that spore-forming _Actinobacteria_ and _Firmicutes_ are common in dryland ecosystems. Similar results were reported by [PERSON] et al. [63], who analyzed the phyllosphere microbiome of _Quercus__lix_ L. from Southern Italy. Several authors reported that the composition of a plant microbiome is profoundly influenced by the host plant [64] and, within the same species, by the environment [63] and the plant-plant interaction. As shown by [PERSON] et al. [65], on peanut and maize, and by [PERSON] et al. [66], on alfalfa, Brassica, and fescue, intercropping of different species affects the plant microbiome composition determining an increase in specific taxa, which are not present in monocultures. In agreement, our results on the phenotypic characterization of the isolates from the different sampling sites indicate that plant-plant and plant-environmental interactions contribute to shaping the _P. lentiscus_-associated microbiome. A SynCom comprising the five N-fixing bacteria was used to evaluate their ability to promote plant growth when applied at root (R), leaf (L), or root and leaf (RL) levels. Optimizing inoculation methods is crucial for maximizing the PGPR's beneficial effects on crops. A comparison of PGPR inoculation methods has already been analyzed in cereals and vegetable crops. In maize, although higher compared to non-inoculated plants, foliar application of _Azobacter droococcum_ B002, _Bacillus subtilis_ B004, and _Bacillus megaterium_, individually or as SynComs reduced leaf biomass and grain yield compared to soil application [67]. A similar study on kale foliar and soil applications of _A. clrococcum_, _B. subtilis_, _Priestia megaterium_ (formerly _B megaterium_), and a mixture of _A. clrococcum_ and _B. subtilis_ demonstrated that the application method did not significantly affect above-ground biomass production, but the specific PGPR strain did [68]. In contrast to [PERSON] et al. [68], our results highlight the importance of the inoculation method on leaf vegetables. Inoculation with the SynCom differently affected lettuce biomass production depending on the method of application: an increase in total plant biomass compared to non-inoculated plants was only observed when the SynCom was inoculated in both roots and leaves (RL) compared to non-inoculated and to R- and L-inoculated plants (Figure 3a). The higher total plant biomass resulted from a 33% increase in leaf biomass compared to non-inoculated lettuce (Figure 3c). This can be due to the higher leaf mass per area and number of leaves per plant in RL-inoculated lettuce plants compared to non-inoculated controls (Table 2). The heightened LMA indicates increased leaf density, likely caused by the production of cells characterized by thicker cell walls and a higher proportion of structural tissues, conferring enhanced resistance to physical and chemical stressors [69] or a palisade/spongy ratio modification. The leaf investment in structural components is usually associated with reduced nutrient concentrations and lower rates of both photosynthesis and respiration [70]. While the higher leaf dry weight in RL-inoculated plants did not result in a statistically significant increase in above-ground biomass compared to the non-inoculated group (Figure 3b), the combined effect of increased leaf and root biomass (+31%) accounts for the observed statistically significant difference in total plant biomass.