**1. Introduction**

The fungus *Fusarium oxysporum* f.sp. *radicis*-*lycopersici* (Forl) is a destructive pathogen limiting crop productivity and causing significant losses in commercial tomato production

**Citation:** Stavridou, E.; Giannakis, I.; Karamichali, I.; Kamou, N.N.; Lagiotis, G.; Madesis, P.; Emmanouil, C.; Kungolos, A.; Nianiou-Obeidat, I.; Lagopodi, A.L. Biosolid-Amended Soil Enhances Defense Responses in Tomato Based on Metagenomic Profile and Expression of Pathogenesis-Related Genes. *Plants* **2021**, *10*, 2789. https:// doi.org/10.3390/plants10122789

Academic Editors: Carlos Agustí-Brisach and Eugenio Llorens

Received: 29 November 2021 Accepted: 13 December 2021 Published: 16 December 2021

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**Copyright:** © 2021 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

worldwide [1,2]. Forl is a saprotrophic soilborne pathogen causing tomato foot and root rot disease (TFFR) [2] by intense colonization of the root hair zone and especially the crown of the plant [1]. Due to the resistant nature of all formae speciales of *F. oxysporum*, this fungus is extremely difficult to control with synthetic fungicides [2]. Furthermore, synthetic fungicides may also affect beneficial soil microbiota and may accumulate in the food chain. Therefore, developing alternative, more efficient methods to control Forl is necessary [3,4]. To date, several methods have been used to limit the spread of Forl in field, and greenhouse conditions, such as the use of resistant tomato hybrids and rootstocks, as well as soil disinfection [5], which is a rather expensive practice as it must be repeatedly applied, leading in turn to the increase in hazardous inputs in agriculture.

Alternative methods have been proven to trigger mechanisms of disease control in plants, such as the addition of beneficial antagonistic microorganisms applied in tomato rhizosphere [6], or of a suitable soil conditioner containing beneficial microorganisms, such as green waste compost mixtures or sewage sludge [7–9]. Additionally, soil properties and improvement of plant vitality and growth conditions have also been considered as an important factor for altering plant rhizosphere microorganism composition and thus strengthening plant defense [10]. Studies have shown that fertilization with calcium-rich soil amendments, may enhance soil fertility, strengthen the plant defense systems and thus, suppress pathogen infections [11]. This is facilitated mainly through changes in the soil properties, such as the increase in the soil pH, which favors soil microorganisms (actinomycetes and bacteria) that thrive at such pH values and are competitive with Forl.

Recently, Giannakis et al. [12] reported that sludge-based biosolids enhance tomato growth and reduce TFFR severity. Biosolids may benefit the plant's health by improving the soil properties and enhancing the diversity of the rhizosphere bacterial community [13,14]. Soil microbiota play a key role in soil suppressiveness [15], and studies have shown that they are involved in disease suppression [16,17]. Therefore, microbial community abundance, richness, evenness, and diversity have been identified as key factors involved in community functioning, soil health, and plant productivity [8]. Studies have shown that changes in the rhizosphere microbial community may affect the plant's resistance to different formae speciales of *F. oxysporum* [18–20]. Additionally, there is growing evidence that greater disease suppression is induced by a consortium of plant beneficial bacteria rather than individual strains [21]. However, identifying specific bacterial species and the underlying response mechanisms triggered by the application of biosolids against TFFR have yet to be determined. Hence, further insight into the interactions between the pathogen and the potentially suppressive soil microbiota will provide further insight into the mechanisms underlying the plant–microbe interactions against Forl and how biosolid application may impact this relationship.

Considering that *F. oxysporum* challenges not only tomato, but also several Solanaceae species of great agronomic and economic importance, the interaction between tomato and *Fusarium* has been extensively studied as a model pathosystem for disease resistance response [22]. It is therefore important to further investigate the effects of soil amendment methods on plant–pathogen systems and especially their effect on plant resistance mechanisms through monitoring gene regulation and microbiome diversity in the rhizosphere. The defense system of plants against pathogens involves regulation of gene expression, activation of signaling pathways, hormone balancing, and synthesis of defensive metabolites [23]. An investigation of the transcriptomic profile of tomato plants infected with *F. oxysporum* f. sp. *lycopersici* has revealed up-regulated gene expression related to plant response mechanisms and plant–pathogen interactions, mainly associated with maintenance of cellular structures and homeostasis [24]. Recently, Kamou et al. [4] reported induction of defense gene expression in tomato challenged with Forl. More specifically, salicylic acid (SA)-related genes such as *PR-1a* and *GLUA*, together with jasmonic acid (JA)-related ones such as the *CHI3*, were overexpressed in the presence of the pathogen but also after inoculation with beneficial *P. chlororaphis* ToZa7, revealing induction of variable defense mechanisms. Interestingly, studies have demonstrated the important role of the defenserelated phytohormones SA and JA as modulators of the rhizosphere microbiome assembly of plants, such as *Phaseolus vulgaris* and *Arabidopsis thaliana* [25,26]. Moreover, plant's ability to recruit a community of beneficial microbiota and exploit protective rhizosphere processes to their advantage is genotype dependent [27,28].

As a follow-up to the report of Giannakis et al. [12] on the beneficial effects of sludgebased biosolids on tomato growth and TFFR severity, in the present work, we have investigated the plant–pathogen interactions at a molecular level and how they are affected by the addition of biosolids in the soil. More specifically, it was examined whether biosolids: (i) could act in vitro as an inhibitor of growth and sporulation of Forl, (ii) could induce gene expression related to plant response against pathogens in tomato, and (iii) would provide a beneficial substrate for the growth of non-pathogenic antagonistic microorganisms against Forl in the tomato rhizosphere. To achieve this, the relative expression analysis of genes related to defense mechanisms in tomato was analyzed; 16S sequencing analysis of the soil substrates was also performed to determine the genetic diversity and functions of microbial (bacteria and archaea) communities present in the soil substrates, which may have a beneficial effect to the plant and/or suppress the pathogen. This work may elucidate the mechanisms through which biosolid addition enhances plant resistance against pathogens. Furthermore, it is expected to contribute to deciphering the effect of biosolids on soil microbial community to sustainably suppress TFFR disease in tomato crops.
