**3. Discussion**

*F. oxysporum* f. sp. *radicis-lycopersici* is a wide-spread fungus in the plant rhizosphere which causes Fusarium crown and root rot (FCRR) disease and leads to losses of tomato production even in greenhouses and systems of soil production [35]. There are different management methods for root rot disease of tomato crop using chemical and biological controls [36]. Biological control for pathogenic fungi is the new management trend for reducing the harmful effects of chemicals (fungicides) [37,38]. There are four types of biocontrol management: microorganisms, semi-chemical products, plant-based natural products, or living microorganisms [39–43]. Furthermore, plant EOs are effective biocontrol agents against a variety of pathogenic fungi and bacteria [44–47].

The present investigation revealed the effect of *Mentha spicata* and *M. longifolia* on EOs on root rot disease of tomato infected with *F. oxysporum* both in vitro and in vivo. *M. spicata* and *M. longifolia* EOs had potentiality against *Fusarium*. The order of efficient EOs against *Fusarium* pathogen was: *M. longifolia* > *M. spicata*. The highest antifungal activity was observed in the case of all concentrations of *M. longifolia* EO in agreement with previous studies [48]. The capability of two *Mentha* EOs against fusarium is due to the ability of bioactive chemical molecules to penetrate the fungal cell wall and cytoplasmic membrane and destroy mitochondrial membranes [49]. Plant EOs contributed to loss of rigidity of the hyphal cell wall as well as damaging the cellular enzyme system, resulting in cell death [50,51]. Many other studies reported that the antifungal activity of *M. spicata* EO against *F. oxysporum* and *Aspergillus niger* depends on the chemical constituents: menthol, thymol, and piperitone, individually or in synergic effect [52,53]. Thymol compound activity presented in the malformation of the cellular membrane in addition to inhibition for ATPase activity [43,54]; regarding the effect of thymol and eugenol, they correlated to the ability of thymol compounds' lysis of the external membrane of microorganisms which facilitated the entrance of eugenol to cytoplasm and interacted with protein [44,53]. Krishna Kishore et al. [55] demonstrated the antifungal activity of carvacrol, -terpineol, terpinen-4-ol, and linalool against *Rhizoctonia solani*, *F*. *oxysporum*, *Penicillium digitatum*, *A. niger*, *Alternaria alternate*, and *A. flavus*; these produce an effect against different microbial cells due to the ability of these compounds to penetrate the cell membrane, inactivate the enzyme pathway, and disturb their active transport [56,57].

The biological effect of the studied EOs on physiological parameters of tomato seedling is due to the presence of terpenes, alcohols, and phenolic compounds [55,58–60]. Moreover, the infected plants treated with *M. longifolia* EO showed the highest values of plant height (19.86 cm), shoot fresh weight (13.64 g), shoot dry weight (1.85 g), root fresh weight (1.41 g), and root dry weight (0.14 g). The main components in *M. spicata*, adipic acid 25.82% and piperitone 24.76%, are different than their percentage in *M. spicata* EO as reported by Bayan and Küsek [61]. Chemical constituents for *M. longifolia* EO were d-limonene, menthol, menthyl acetate, linalool, and eugenol, with percentages that differ from those reported from GC–MS analysis conducted by Desam et al. [62]. This difference in the percentage of single constituents for *Mentha* EOs may be due to differences in extraction methods or genetic diversity of these plants [63].

Menthol, menthyl acetate, linalool, and eugenol constituents alter cell permeability for *Fusarium* fungi and cause plasmolysis and cell death [64]. This study recorded collapsing of mycelium hyphae for *F. oxysporum* f. sp. *lycopersici* treated with EOs of *M. spicata and M. longifolia* with potential activity as biological control and therapeutic effect against root rot disease.

To analyze the role of chitinase, defensin, WRKY1, WRKY4, WRKY33, and WRKY53 transcripts in *L. esculentum* plant defense against *F. oxysporum* fungal pathogen, we analyzed their expression after pathogen infection, and pathogen infection and application of 1.25% *M. spicata and* 1.0% *M. longifolia* EOs treatments. The expression results showed over-expression by 57.24 fold and changes in the level of WRKY33 transcription factor were recorded in infected plants treated with 1.0% *M. longfolia*, followed with 57.16 and 56.4 fold changes in pathogen, and 1.25% *M. spicata* treatments, respectively, while minimum expression of WRKY33 2.43 fold was observed with 1.25% *M. spicata* EO compared with control. The expression profile of WRKY35 TF revealed a significant upregulation of 39.23 and 38.12 fold changes at the pathogen-infected plant under 1.0% *M. longfolia* and pathogen treatments, respectively, and the minimum fold change of 3.52 was at 1.25% *M. spicata* EO compared with control. Data obtained in this study showed upregulation in the WRKY4 TF expression of 37.65 and 32.95 change folds at the pathogen-infected plant under 1.0% *M. longfolia* and pathogen treatments, respectively. In comparison, WRKY1 TF expression patterns were 26.35 and 25.17 change folds at the pathogen-infected plant under 1.0% *M. longfolia* and pathogen treatments, respectively, as compared with minimum expression level of 4.23 folds with 1.0% *M. longfolia*. Our results were in agreement with previous studies which reported that *WRKY3* and *WRKY4* encode two structurally similar WRKY proteins, and their expression was responsive to stress conditions. Stress-induced expression of *WRKY4* but not *WRKY3* was further enhanced by pathogen infection. These results strongly suggest that WRKY4 regulates crosstalk between SA and JA/ET-mediated signaling pathways and, as a result, plays opposite roles in resistance to the two different types of microbial pathogens. Interestingly, WRKY proteins such as WRKY4, WRKY33, and redundant WRKY18, WRKY40, and WRKY60 play a positive role in plant resistance to necrotrophic pathogens.

The expression of defense-related genes showed over-expression under pathogen infection conditions and with the pathogen under 1.0% of *M. longfolia* and 1.25% of *M. spicata*. In contrast, chitinase gene was upregulated with 31.25, 29.6, and 27.83 fold changes at pathogen with 1.25% *M. spicata*, pathogenated plants, and then pathogenated with 1.0% *M. longfolia* treatments, respectively. In addition, defensin gene expressed as 18.65, 16.16, and 15.76 fold changes at pathogen with 1.0% *M. longfolia*, pathogen with 1.25% *M. spicata*, and pathogen treatments compared with minimum 2.44 fold expression was recorded at 1.25% *M. spicata* EO treatment.

These results collectively indicate that overexpression of chitinase, defensin, and WRKY transcripts play a positive role in inducing plant resistance against *F. oxysporum* and working toward reducing disease severity. Additionally, WRKY transcripts and PR3, PR12 genes were further enhanced by pathogen infection, and they are already considered as a marker for the plant–microbe interaction.

WRKY family members have diverse regulatory mechanisms; their protein can be effectively combined with W-box elements and bind to acting elements to activate or inhibit the transcription of downstream target genes through the cis-acting mechanism [65]. Thus, WRKY as a transcription factor plays an important role in plant defense in response to attacks by several pathogens. The response works by activating the expression of resistance genes directly or indirectly. It has been reported that WRKY DNA binding proteins bind to the promoter region of Arabidopsis natriuretic peptide receptor 1 (NPR1), which activated the plant defense system [66]. Moreover, WRKY33 activates the plant resistance system against necrotrophic fungi *Alternaria brassicicola* and *Botrytis cinerea* [29], and it can regulate the SAR system in the infected plants and also the PR genes [67,68]. Moreover, the high expression of such transcription factors could regulate the plant pathogen sensitivity to mutants of AtWRKY4, AtWRKY3, and AtWRKY3 WRKY4, increasing the plant susceptibility toward the fungus *B. cinerea*. In contrast, the high expression of the non-mutated AtWRKY4 enhanced the plant's resistance toward the *Pseudomonas syringae* [69]. Many plant WRKY genes are induced by biotrophic and necrotrophic pathogens, including fungi and viruses, through the induction of SA-dependent SAR and PR genes [70,71].
