*2.3. Characterization of Microbial Communities in the Different Soil Substrates*

Based on the rank abundance curves for the top 100 OTUs (Figure S1) an even abundance was observed between the 12 and 72 h (Figure S1A). However, the treatments without the addition of biosolid, i.e., F and C, showed unevenness of abundance in soil bacterial communities due to the large difference in detected organisms, whereas in the treatments where biosolid was added (B and FB), species evenness was more homogeneous (Figure S1B).

Microbial diversity was evaluated using the following indicators: observed richness, Chao1, ACE, Shannon, Simpson, and InvSimpson (Figures 3A and 4B). The alpha diversity

indices were notably increased (or did not significantly change) in soil substrate samples at 72 h, indicating that the shift pattern of the microbiome occurred at a later timepoint (Figure 3A). Respectively, treatments enriched with biosolid, regardless of the presence or absence of Forl inoculum (FB and B, respectively) showed greater microbiome diversity in *α*-diversity indices (Observed richness, Shannon, and Simpson), whilst the treatment inoculated with Forl (F) showed a lower species evenness (Figure 3B), indicating that the relative abundances of species within the community vary in distribution.

To further relate bacterial community variance to the different variables, such as the effect of time post Forl inoculation (F and FB) and biosolid application (B and FB) and the treatments, Canonical Correspondence Analysis was performed (Figure 4). The variation explained by the treatments and timepoints (constrained ordination) was 22.8% and the remaining 77.2% of the variation was explained by the unconstrained ordination (Table 2). From the eigenvalues of the constrained axes, it was observed that 45.65% of the variation is explained by the CCA1 and the 19.1% by CCA2 (Table 3). The factors responsible for most of the explained variation in the bacterial community were the treatments enriched with biosolid (B and FB), which were differentiated from those inoculated with Forl without the addition of biosolid (F) at 72 h (Table 4). Therefore, the addition of biosolid, with or without Forl inoculum, affected differently the bacterial community abundance compared to the control and Forl treatments.

**Table 2.** Proportion of inertia explained by constrained and unconstrained ordination.


**Table 3.** Accumulated constrained eigenvalues.


**Table 4.** Biplot scores for constraining variables.


Composition of bacterial populations at the phylum and class taxonomic levels were evaluated using the β-diversity analysis for each treatment and time point tested. Overall, 38 and 94 different phyla and classes, respectively, were identified across the treatments in the two timepoints, yet relative abundance of only 13 phyla and 23 classes with OTUs representation >1% are shown in Figures 5 and 6, respectively. The most abundant amplicons were identified as *Proteobacteria*, *Bacteroidetes, Actinobateria, Acidobacteria, Gemmatimonadetes, Chloroflexi*, and *Planctomycetes*. The biosolid-enriched treatments (B and FB) were characterized by higher abundance of amplicons identified as *Chloroflexi* and *Bacteroidetes*, which increased with time (Figure 5, Table S1). Moreover, the phylum *Patescibacteria*, was detected only in biosolid-enriched treatments (B and FB) in both time points (Figure 5, Table S1). The phylum *Synergistetes* was initially observed in treatment B at 12 h and 72 h and in FB only at 72 h. In contrast, *Acidobacteria* and *Actinobacteria* were characterized by lower abundance in the biosolid-enriched soils (B and FB) compared to the control (C) and Forl (F) treatments (Figure 5, Table S1).

**Figure 4.** Canonical correspondence analysis (CCA) of the relative variance of OTUs in the soil samples subjected to the different control (C), biosolid−enriched treatment (B), Forl inoculation (F), and Forl inoculation with biosolid application (FB) treatments at 12 and 72 h. The biplots present the effect of biosolid application to the bacterial community abundance in the treatments with or without fungal inoculum at two different timepoints.

**Figure 5.** Classification of bacterial phyla based on relative abundance at rate greater than 1%, for the control (C), biosolid−enriched treatment (B), Forl inoculation (F), and Forl inoculation with biosolid application (FB) treatments, at 12 and 72 h after inoculation with *Fusarium oxysporum* f. sp. *radices-lycopersici*. The different phyla are depicted with different colors. Value is the pooled mean of three replicates.

At the bacterial class level, the treatments enriched with biosolid (B and FB) showed higher relative abundances of *Gammaproteobacteria, Bacteroidia, Anaerolineae, Deltaproteobacteria*, and *Acidimicrobiia* in both time points (Figure 6, Table S2). Interestingly, *Clostridia* were detected solely in biosolid-enriched soils and increased with time. A similar trend was observed for *Saccharimonadia* and *Synergistia*, yet these were not present in FB soils at 12 h. In contrast, the classes *Alphaproteobacteria*, *Actinobacteria*, *Planctomycetacia*, *Verrucomicrobiae*, *Acidobacteriia*, *Phycisphaerae*, *Thermoleophilia*, and *Gemmatimonadetes* were found in higher abundance in control soils (C) and soils with Forl inoculum (F) compared to

biosolid-enriched treatments (Figure 6, Table S2). Unclassified actinobacteria were solely detected in C and F treatments. However, in the C treatment the call abundance was reduced over time and in F treatment increased over time. The class *Blastocatellia* (Subgroup 4) showed higher abundance under C conditions and was reduced with either biosolid application and/or Forl inoculation. Soils in C and F treatments showed higher abundance of *Bacilli*, compared to the soils treated with biosolid (B) (Figure 6, Table S2). However, at 72 h, the abundance of this class increased in C treatment, yet it was reduced in B, F, and FB treatments (Figure 6, Table S2). The class *Oxyphotobacteria* was only present in F treatment and *S0134\_terrestrial\_group* in C treatment at 12 h (Figure 6, Table S2).

**Figure 6.** Classification of bacterial classes based on relative abundance at rate greater than 1%, for the treatments control (C), biosolid−enriched treatment (B), Forl inoculation (F), and Forl inoculation with biosolid application (FB) at 12 and 72 h after inoculation with *Fusarium oxysporum* f. sp. *radicis-lycopersici*. The different classes are depicted with different colors. Value is the pooled mean of three replicates.
