*2.5. qRT-PCR of the Plant Defence System*

qRT-PCR was carried out with mRNA to assess the expression levels of different WRKY transcription factors WRKY1, WRKY4, WRKY33, and WRKY53 (Figures S1–S4) which play an important role in biotic and abiotic tolerance. In addition, qRT-PCR represented the relative expression levels of defense-related proteins such as chitinase (PR3) and defensin (PR12) genes (Figures S5 and S6) in tomato plant roots after two weeks from *Fusarium* inoculation and EOs application, respectively.

**Table 3.** Effects of *M. spicata* and *M. longifolia* EOs on *L. esculentum* seedling growth under Fusarium root rot disease infection.


PH = plant height, SFW = shoot fresh weight, SDW = shoot dry weight, RFW = root fresh weight, RDW = root dry weight, and EOs = essential oils. Different letters indicate significant differences between different treatments at *p* ≤ 0.05. Data are expressed as the mean of three replicates ± SDs.

**Figure 5.** Effects of highest concentrations of *M. spicata* and *M. longifolia* EOs on total chlorophyll content (Chll) in *L. esculentum* seedling under Fusarium root rot disease infection. Bars with different letters indicate significant differences between treatments at *p* ≤ 0.05. Data are expressed as the mean of three replicates ± SDs.

**Figure 6.** Effects of highest concentrations of *M. spicata* and *M. longifolia* EOs on electrical leakage percentage (EL%) in *L. esculentum* seedling leaves under Fusarium root rot disease conditions. Bars with different letters indicate significant differences between treatments at *p* ≤ 0.05. Data are expressed as the mean of three replicates ± SDs.

In our study, the highest expression mRNA level (57.24 ± 0.01) was recorded for chitinase gene in *L. esculentum* plant roots at 1.0% *M. longifolia* under Fusarium infection, followed with (57.16 ± 0.02, 56.4 ± 0.02) of pathogen and *M. spicata* application treatments, respectively, compared with (1.0 ± 0.0) in untreated control treatments. Overall, the WRKY TFs genes (WRKY1, WRKY4, WRKY33, and WRKY53) in tomato seedling roots showed positive expression levels (upregulation) under *M. spicata* and *M. longifolia* treatments and positive control, compared with the untreated control. The highest expression mRNA levels of WRKY transcriptional factors WRKY53 gene (39.233 ± 0.03) represented in tomato roots at 1.0% *M. longifolia* treatment under Fusarium infection condition, followed with (38.12 ± 0.02) in pathogen treatment. Figure 9 shows a hierarchical clustering heat map and the correlation among different treatments and their gene expression. The red color demonstrates the highest correlation, and the blue color demonstrates the lowest correlation.

**Figure 7.** Effects of highest concentrations of *M. spicata* and *M. longifolia* EOs on MDA, TPC, TFC, and PC level in *L. esculentum* seedling under Fusarium root rot disease infection. Bars with different letters indicate significant differences between treatments at *p* ≤ 0.05. Data are expressed as the mean of three replicates ± SDs.

**Figure 8.** Effects of *M. spicata* and *M. longifolia* EOs on SOD, CAT, and APOX activity in *L. esculentum* seedling under Fusarium root rot disease infection. Bars with different letters indicate significant differences between treatments at *p* ≤ 0.05. Data are expressed as the mean of three replicates ± SDs.

**Figure 9.** Hierarchical clustering heat map of relative expression levels of chitinase, defensin genes, and WRKY1, WRKY4, WRKY33, and WRKY53 transcripts in *L. esculentum* seedling under Fusarium injection and application of 1.25% *M. spicata and* 1.0% *M. longifolia* EOs treatments.
