*2.1. Growth and Sporulation of Fusarium oxysporum f. sp. radicis-lycopersici*

Colony diameter (cm) of Forl increased in the different mixtures of biosolid leachates with PDA compared to the control (Figure 1A). This increase was greater with elevating leachate concentration. As such, the concentrations of 5 and 10% leachate showed the largest colony diameter (an increase of 40% was noted in relation to control) followed by the 2% leachate, where an increase of 28% in colony diameter was noted in relation to control. Sporulation was also significantly affected by the biosolid leachate; the number of fungal spores increased significantly at 10% concentration of leachate (Figure 1B). Hence, the number of conidia produced per cm2 of colony was increased with the increase in the leachate concentration (Table 1).

**Figure 1.** Growth and sporulation of *Fusarium oxysporum* f. sp. *radicis-lycopersici* (Forl) at 0, 2, 5, and 10% biosolid leachate concentrations in PDA. (**A**) Mean colony diameter; (**B**) Mean sporulation. Different letters indicate statistically significant differences between the treatments according to Tukey's post hoc test (*p* < 0.05).


**Table 1.** Mean number of *Fusarium oxysporum* f. sp. *radicis-lycopersici* conidia produced per cm<sup>2</sup> of colony at different biosolid leachate–PDA concentrations in PDA. Different letters indicate statistically significant differences between the treatments according to Tukey's post hoc test (*p* < 0.05).

#### *2.2. Gene Expression Analysis*

Expression patterns of defense-related genes (*GLUA*, *CHI3*, *PR1-a*, *LOX*, and *AOC)* following application of biosolid and inoculation with Forl were analyzed using RT-qPCR (Figure 2). The induction patterns were evaluated in tomato plants at 12, 24, 48, and 72 h after inoculation. As internal control the reference gene *β-actin* was used. A significant upregulation of *LOX* was observed at 12 h in response to Forl inoculation and the addition of biosolid (FB) as compared to the treatments C, F, and B (Figure 2A). Nevertheless, in the later time points, *LOX* was not significantly induced (Figure 2B–D). Interestingly, at 12 h an increase in the *AOC* expression levels was also observed after application of biosolid and Forl inoculation (FB), yet this was not significant in comparison to the other treatments (Figure 2A). However, at 12 h, no induction in *PR1-a*, *GLUA*, and *CHI3* was observed for the different treatments in relation to the control (C) (Figure 2A). At 24 h after inoculation, only *GLUA* was significantly induced in the FB treatment, whilst the *PR1-a* gene was significantly overexpressed only under biosolid application (B), yet a non-significant increase in the expression levels was also observed under FB treatment (Figure 3B). *AOC* was significantly induced at 48 and 72 h after biosolid application (Figure 2C,D). At 72 h (Figure 2D) overexpression of 2.45-, 5.5-, 3.76-, and 2.45-fold was observed for *CHI3*, *AOC*, *PR1-a*, and *GLUA*, respectively, under biosolid application (B), but not after inoculation with Forl (FB) when compared with the untreated control.

**Figure 2.** (**A**–**D**) Relative gene expression analyses of defense-related genes *GLUA*, *CHI3*, *PR1-a*, *LOX*, and *AOC*, in tomato

plant leaves in (**A**) 12 h, (**B**) 24 h, (**C**) 48 h, and (**D**) 72 h post inoculation in control (C), biosolid−enriched treatment (B), Forl inoculation (F), and Forl inoculation with biosolid application (FB). Different letters indicate statistically significant differences between the treatments according to Tukey's post hoc test (*p* < 0.05).

**Figure 3.** Boxplot demonstrating the range and the distribution of the of OTUs, evaluated using the α-diversity indices observed richness, Chao1, ACE, Shannon, Simpson, and InvSimpson, (**A**) 12 and 72 h after inoculation with *Fusarium oxysporum* f. sp. *radicis-lycopersici*; (**B**) for the treatments for the treatments control (C), biosolid−enriched treatment (B), Forl inoculation (F), and Forl inoculation with biosolid application (FB). Values represent the pooled mean of three replicates.
