**3. Discussion**

Biosolids have been characterized as organic soil amendments, supplying the soil with nutrients and organic compounds and contributing to soil moisture and aeration [29–31]. However, there is limited research related to the molecular mechanisms associated with biosolid-elicited suppression of soilborne diseases, such as TFFR and the enhanced plant performance. In a previous work, biosolid application in the soil has been shown to enhance tomato growth and reduce the effects of Forl infection [12]. In the present work, a molecular approach was employed to provide further insight into the mechanistic effect of biosolid application in alleviating the negative impact of Forl on tomato plants. To achieve this, gene expression profiles related to plant response against pathogens coupled with 16S metagenome profile analysis were used to determine the genetic diversity and functions of bacterial communities present in the soil substrates and their potential impact on plant–pathogen relationships.

Biosolid application can enhance tomato tolerance against the Forl [12], which was mainly attributed to the indirect beneficial effects of biosolid application on biotic and abiotic factors [31]. Although growth and sporulation of Forl in the presence of biosolids were not studied in planta, it would have been expected that biosolids could suppress the growth and sporulation of Forl, thus reducing its aggressiveness against tomato. In contrast, based on the growth analysis and sporulation of Forl under in vitro conditions, herein, an increase in colony diameter, the number of fungal spores of Forl, and the number of conidia produced per cm2 of colony was observed in response to the increasing concentration of biosolid-PDA leachates, compared to the control. Such positive effect of biosolid leachates on the colony growth and spore production in vitro could be explained by the fact that various biosolid leachates contained significant amounts of calcium (Ca) and magnesium (Mg) [32]. PDA used for the growth of Forl is a universally used medium providing the necessary nutrients for the growth of fungi for laboratory purposes. However, nutritional requirements in macro- and microelements vary among different fungal species [33]. Magnesium is considered a macro element necessary for enzyme activation and ATP metabolism. On the other hand, Ca is generally accepted as a micronutrient required for enzyme activity and membrane structure in fungi [33] and plays a key role in hyphal tip growth [34]. It is assumed that nutrients provided in the leachates exert positive effects on hyphal growth and production of conidia in Forl. However, this calls for further investigation, as the nutritional requirements for the improvement of growth and sporulation has not been studied in this fungus.

Nevertheless, biosolid application in soils induced an upregulation of defense-related genes in tomato plants post Forl inoculation. Increase in transcript levels of defense-related genes indicate activation of the tomato response mechanisms against the pathogen [35–37]. The observed increase in mean transcript levels of *LOX*, *AOC*, and *GLUA* in the biosolidenriched soils (B and FB) as compared to the non-amended soils (C, F), indicated that biosolid application may play a key role in the early (12 and 24 h) activation of the tomato response mechanisms against Forl. Activation of the ethylene (ETH) and jasmonic acid (JA)/ETH signaling pathways even at 72 h in FB treatment may indicate an attempt to limit pathogen progression under the effect of biosolids. JA elicitor is a signaling molecule involved in various plant developmental processes and defense mechanisms [38]. The role of JA pathway in protection against Forl was confirmed in tomato after biochar application [39]. More specifically, biochar application induced upregulation of the pathways and genes associated with plant defense and growth such as JA, yet biosynthesis and signaling of the salicylic acid (SA) pathway was downregulated (Jaiswal et al., 2020). In contrast, herein, genes involved in the SA biosynthesis and signaling pathways, such as *GLUA* and *PR1-a*, were upregulated in the biosolid-enriched treatments (B and FB). However, the *PR1-a* gene was significantly upregulated only under biosolid application (B) at early time points and only at 72 h in both B and FB treatments. PR proteins are elicited in many plant species by the attack of different pathogens. Plants inoculated with Forl (F) did not show any significant upregulation of the analyzed defense-related genes, which is consistent with other studies showing a delayed induction of defense-related gene expression only 2–3 weeks post inoculation [40–42]. This suggests that either Forl needs to be in close association with tomato roots for upregulation of *PR1-a* to take place, that the accumulation of PR proteins requires longer period post inoculation [42], or even that the selected genotype was rather tolerant to Forl [43]. It has been shown that Forl first interacts with tomato roots 48 h after inoculation [1]. In addition, it has been shown that the developmental stage seems to play an important role in the induction of resistance genes [35].

Nevertheless, genes associated with the JA and SA biosynthesis and signaling pathways such as *CHI3, AOC, PR1-a*, and *GLUA* were upregulated 2.45-, 5.5-, 3.76-, and 2.45-fold, respectively, at 72 h in biosolid without Forl (B), but not after inoculation with Forl (FB), indicating that the plant's innate defense mechanisms were induced even without the presence of the pathogen. Therefore, such effects imply indirect interactions possibly via the induction of systemic acquired resistance (SAR) [44,45], given that SAR is mediated by pathways that are dependent on, but not only to, SA, JA, and ethylene [46,47]. Overall, the upregulation of genes involved in plant defense and plant growth may indicate defense priming that could explain the significant improvement in plant performance and Forl suppression observed in the presence of biosolid.

Elicitors of biosolid-mediated plant defenses include chemical compounds that are beneficial to the plant along with biosolid-induced microorganisms with potentially direct antagonistic effects towards Forl [4,35,36]. The potential induction of systemic acquired resistance in plants by compost mixtures, used as soil conditioners in plant growth substrates, has been demonstrated in several studies [48,49]. Therefore, it was hypothesized that these indirect plant defense mechanisms could also be induced by other biodegradable and noncomposted materials, such as the anaerobically digested biosolid used in the present study.

Enhanced tomato performance against Forl could also be attributed to different types of interactions between the phytopathogenic fungus and the beneficial to the plant soil microflora, which is induced by biosolid application. Such interactions include competition for nutrients, production of antifungal metabolites, parasitism, and enzymatic hydrolysis of fungal cell walls [50–52]. Although the interaction mechanisms between microbial communities and plants are very complex, intense microbial diversity usually has a beneficial effect on both plant diversity and their growth and productivity [48,53,54]. Most of the microorganisms in the soil and the rhizosphere develop beneficial and mutual symbiotic relationships with the plant and among other microorganisms [55].

The bacterial biodiversity observed in the biosolid-enriched treatments, seems to have had a beneficial effect on both growth of the tomato plants and protection against Forl. The relation of the relative abundance of bacterial groups and pathogen inhibition along with development of disease suppression in soils has been recently demonstrated [16,17]. Herein, four phyla of bacteria were identified in greater abundance in biosolid-enriched soils, namely *Chloroflexi* (class *Anaerolineae*), *Bacteroidetes* (class *Bacteroidia*), *Patescibacteria* (class *Saccharimonadia*), and *Synergistetes* (class *Synergistia*). Interestingly, *Clostridia* (phylum *Firmicutes*), were detected solely in biosolid-enriched treatments and increased with time. Several genera within the phylum *Firmicutes*, have been shown to positively affect disease suppressiveness of soil amendments, such as compost [56]. *Clostridia* occur mainly in the rhizosphere and perform beneficial functions for the plants, such as atmospheric nitrogen fixation, phosphate solubilization and the reduction of Fe3+ to the more readily available

iron form Fe2+ [57]. The higher abundance of *Bacilli* class was increased at 72 h under control conditions, yet it was reduced in B, FB, and F treatments. Bacillus species have exhibited ability for plant growth promotion [58] and have also demonstrated the ability to excrete exopolysaccharides, biosurfactants, and chelating agents, which are important for the remediation of heavy metals from soils [59]. *Bacillus* species have also demonstrated broad functions, especially in various enzymatic activities [60], indicating a possible role in pathogen control [61,62]. Nevertheless, the reduced abundance over time in the biosolidenriched treatments indicate that possibly the presence or absence of mineral fertilization affects the structure of the bacterial community in the soil [63]. A possible explanation could be the acidification of soil by higher NPK content [64] present in the sewage sludge [65] and therefore the biosolid applied herein [12].

Other phyla of anaerobic bacteria, such as *Patescibacteria* and *Synergistetes*, that were observed mainly in the biosolid enriched treatment (B) have been shown to contribute to the degradation of organic matter [66,67]. Bacterial phyla such as *Chloroflexi* (class *Anaerolineae*) thrive under anaerobic conditions and are considered to play an important role in the vital process of photosynthesis [68]. In addition, these bacteria can degrade a large number of organic compounds and producing acetic acid [69,70], which has been shown to stimulate plant growth and inhibit the growth of pathogenic fungi [71,72].

The phylum *Proteobacteria* has a key role in anaerobic digestion by metabolizing volatile fatty acids [73]. In addition, bacteria in this phylum are known to remove a broad range of synthetic, as well as natural organic pollutants [74,75]. Within this phylum, the class *Alphaproteobacteria* were in higher abundance in C and F treatments compared to the biosolid-enriched ones, whereas *Gammaproteobacteria* were in higher abundance in B (increased over time) and FB (decreased over time) treatments. This indicates that different classes of the same phylum are affected differently by the addition of biosolid in the soils and in response to Forl, and further supports the evidence that consortia of beneficial microorganisms, rather than specific taxa, may drive disease suppression and lead to plant protection [76].

Similarly, we observed changes in abundance of *Actinobacteria*, *Firmicutes*, and *Acidobacteria* with time among the different treatments (Table S1). The higher abundance of the classes *Actinobacteria* and *Acidobacteria* have been associated with antagonistic activity toward several phytopathogens, such as *Fusarium* [77], and with suppressiveness in compost [78]. Additionally, high abundance in members of bacterial phyla, such as *Actinobacteria*, *Firmicutes*, and *Acidobacteria*, have been shown to directly antagonize pathogens through various mechanisms [17]. The example of *Firmicutes* abundance being increased over time under control (C) and Forl inoculation with biosolid-enriched soil (FB) treatment, remaining unchanged in the B treatment, and being reduced in the Forl inoculation (F) treatment indicates not only the presence, but also the change of abundance over time is significant. Additionally, *Acidobacteria* decreased in the B, F, and FB treatments and *Actinobacteria* increased in F, decreased in B, and remained unchanged in FB treatments. On the other hand, the abundance of *Actinobacteria*, *Firmicutes*, and *Acidobacteria* in the control treatment, where plants and microorganisms in the rhizosphere were undisturbed, was increased. These observations suggest that: (i) possibly different consortia of beneficial microorganisms, rather than specific species, may provide plant protection against Forl by suppressing the pathogen, which is in accordance with other studies [21,79], and (ii) the relative change (in the concept of increase or decrease) in abundance of these consortia over time may also play a regulatory role in the biocontrol of Forl.

Functional analysis was further performed to acquire information about the potential community functions. Different pathways associated with various aspects of metabolism were activated in biosolid-enriched (B and FB) compared to the non-enriched (C and F) treatments, indicating the impact of biosolid application on various microbial functions. For instance, in the secondary metabolism, vitamin biosynthesis pathway and especially folate biosynthesis pathway were activated in biosolid enriched soils. Potentially, the abundance of nutrients in the biosolid-enriched soil may have enhanced the biosynthesis of plant secondary metabolites, such as folates (reviewed by Kołton et al. [80]). Interestingly, studies have highlighted the importance of folates in inducing plant tolerance to several biotic and abiotic stresses [81,82]. Folates in plants are involved in redox homeostasis, physiological processes, epigenetic regulation, cell proliferation, and mitochondrial respiration, as well as photosynthesis [80,83,84], and have been shown to have antifungal functions [85].

The menaquinone (Vitamin K2) biosynthesis pathway was also activated in B and BF treatments. Menaquinones are involved in bacterial electron transport and in sensing environmental changes such as alterations in redox state; they have also been implicated in sporulation and proper regulation of cytochrome formation in all Gram-positive bacteria and anaerobically respiring Gram-negative bacteria [86–88]. In plants, vitamin K functions as a priming agent against biotic and abiotic stresses given its redox properties. Menadione (pro-vitamin K) was found to induce resistance by priming in Arabidopsis against the virulent strain *Pseudomonas syringae* pv. tomato DC3000, with more than two-fold PR1 expression in MSB-pretreated plants as compared to non-treated plants [89]. Similarly, in our study, an increase in PR1-a expression by 3.76-fold was observed 72 h in the B treatment, but not in the BF treatment, indicating the potential priming effect of biosolid in tomato plants.

The autotrophic CO2 fixation pathway found in the biosolid-enriched treatments indicated the presence of autotrophic microorganisms, which contribute significantly to CO2 fixation in the soil carbon sink of agricultural soils [90]. Other pathways activated in the biosolid-enriched treatments include the carbohydrate biosynthesis (gluconeogenesis) indicative of sugar synthesis [91], which is the primary source of energy for all eukaryotic organisms [92]. Specifically, in plants, they are involved in most metabolic and signaling pathways controlling growth, development, and fitness [92]. Additionally, in the biosolidenriched treatments, sequences were attributed to the nitrogen metabolism pathway which, according to Jacoby et al. [93], when active in the rhizosphere, is an indicator that the microbiome plays an important role in mediating plant nutrition. Recent studies have shown that amino acids play a key role in plant root growth and microbial colonization, symbiotic interactions, and pathogenesis in the rhizosphere [94]. Amino acids are considered a key intermediary in the soil nitrogen cycle, and function as carbon and nitrogen sources for both microorganisms and plants, in synthesis and regulation of auxin activity and biofilm formation and disassembly [95]. Therefore, the nucleic acid processing for protein synthesis pathway, which was found active in biosolid-enriched soils, could be an indicator of intrinsic amino acid biosynthesis.

Control and Forl treatments shared common pathways that were active under such conditions including: aerobic respiration and the related TCA cycle pathways, important processes in the global carbon cycle and of crucial importance in the partitioning of energy in soil [96]; the tetrapyrrole biosynthesis (TBS) and heme b biosynthesis pathways, important for oxidative and energy metabolism in a variety of biological functions, such as gas transport, respiration, and nitrite and sulphite reduction [97]; and also in chlorophyll synthesis in plants and algae [98]. In S-adenosyl-L-methionine (SAM) biosynthesis, SAM functions as a methyl donor and plays a key role in antibiotic production [99], and inhibits sporulation and cellular differentiation in *Streptomyces* spp., *Bacillus subtilis*, and *Saccharomyces cerevisiae* [100]. Other functions detected include the sugar nucleotide biosynthesis and sugar degradation (galactose). It has been previously shown that galactose metabolism plays a central role in biofilm formation by *B. subtilis* and other bacteria [101]. Nevertheless, Forl inoculation treatment also showed higher abundance of sequences attributed to Carboxylate (sugar acid) and secondary metabolism (sugar derivative; sulfoquinovose) degradation. Sulfoquinovose biosynthesis is largely conserved within plants, algae, and photosynthetic bacteria and plays a major role in the global biogeochemical sulfur cycle by serving as a sulfur reservoir that can be mobilized in the early stages of sulfur starvation [102]. Nevertheless, a sulfoglycolytic pathway is being employed by a diverse collection of bacterial species, such as γ-*Proteobacteria*, as well as α- and β-*Proteobacteria* [103].

Overall, based on the results of this research, the biosolid application seems to result in a successful priming of tomato plants inducing resistance mechanisms against Forl. This effect was also associated with the microbiome diversity in the biosolid-enriched treatments and the changes in abundance with time in response to Forl. Organic amendments, such as green manures, stable manures, and composts, have long been recognized to facilitate biological control within the context of bacterial communities [104]. Microbe-microbe associations and microbe–plant interactions are important in the context of pathogen inhibition via direct antagonism and mediating processes involved in nutrient dynamics [105,106]. Therefore, the use of biosolid as a soil amendment had a positive effect not only on plant health, but also on the bacterial diversity, relative abundance and predicted soil functioning, toward enhancing tomato resistance against Forl.
