*Article* **Biosolid-Amended Soil Enhances Defense Responses in Tomato Based on Metagenomic Profile and Expression of Pathogenesis-Related Genes**

**Evangelia Stavridou 1,2, Ioannis Giannakis 3, Ioanna Karamichali 1, Nathalie N. Kamou 4, George Lagiotis 1, Panagiotis Madesis 1,5, Christina Emmanouil 6, Athanasios Kungolos 3, Irini Nianiou-Obeidat 2,\* and Anastasia L. Lagopodi 4,\***

	- <sup>3</sup> School of Civil Engineering, Aristotle University of Thessaloniki, 54124 Thessaloniki, Greece; iogianna@civil.auth.gr (I.G.); kungolos@civil.auth.gr (A.K.)
	- <sup>4</sup> Laboratory of Plant Pathology, School of Agriculture, Forestry and Natural Environment, Aristotle University of Thessaloniki, 54124 Thessaloniki, Greece; ngkamou@agro.auth.gr
	- <sup>5</sup> Laboratory of Molecular Biology of Plants, School of Agricultural Sciences, University of Thessaly, 38221 Volos, Greece
	- <sup>6</sup> School of Spatial Planning and Development, Aristotle University of Thessaloniki, 54124 Thessaloniki, Greece; chemmanouil@plandevel.auth.gr
	- **\*** Correspondence: nianiou@agro.auth.gr (I.N.-O.); lagopodi@agro.auth.gr (A.L.L.)

**Abstract:** Biosolid application is an effective strategy, alternative to synthetic chemicals, for enhancing plant growth and performance and improving soil properties. In previous research, biosolid application has shown promising results with respect to tomato resistance against *Fusarium oxysporum* f. sp. *radicis*-*lycopersici* (Forl). Herein, we aimed at elucidating the effect of biosolid application on the plant–microbiome response mechanisms for tomato resistance against Forl at a molecular level. More specifically, plant–microbiome interactions in the presence of biosolid application and the biocontrol mechanism against Forl in tomato were investigated. We examined whether biosolids application in vitro could act as an inhibitor of growth and sporulation of Forl. The effect of biosolid application on the biocontrol of Forl was investigated based on the enhanced plant resistance, measured as expression of pathogen-response genes, and pathogen suppression in the context of soil microbiome diversity, abundance, and predicted functions. The expression of the pathogen-response genes was variably induced in tomato plants in different time points between 12 and 72 h post inoculation in the biosolid-enriched treatments, in the presence or absence of pathogens, indicating activation of defense responses in the plant. This further suggests that biosolid application resulted in a successful priming of tomato plants inducing resistance mechanisms against Forl. Our results have also demonstrated that biosolid application alters microbial diversity and the predicted soil functioning, along with the relative abundance of specific phyla and classes, as a proxy for disease suppression. Overall, the use of biosolid as a sustainable soil amendment had positive effects not only on plant health and protection, but also on growth of non-pathogenic antagonistic microorganisms against Forl in the tomato rhizosphere and thus, on plant–soil microbiome interactions, toward biocontrol of Forl.

**Keywords:** biosolid leachates; sludge; *S. lycopersicum* L.; *Fusarium oxysporum*; PR-related genes; defense-related proteins; soil bacteria communities; 16S sequencing; biocontrol
