The application of microbial products as natural biocontrol agents for inducing systemic resistance against plant viral infections represents a promising strategy for sustainable and eco-friendly agricultural applications. Under greenhouse conditions, the efficacy of the culture filtrate of Bacillus subtilis strain HA1 (Acc# OM286889) for protecting tomato plants from Tobacco mosaic virus (TMV) infection was assessed. The results showed that the dual foliar application of this culture filtrate (HA1-CF) 24 h before and 24 h after TMV inoculation was the most effective treatment for enhancing tomato plant development, with substantial improvements in shoot and root parameters. Furthermore, compared to non-treated plants, HA1-CF-treated tomato had a significant increase in total phenolic and flavonoid contents of up to 27% and 50%, respectively. In addition, a considerable increase in the activities of reactive oxygen species scavenging enzymes (PPO, SOD, and POX) and a significant decrease in non-enzymatic oxidative stress markers (H2O2 and MDA) were reported. In comparison to untreated control plants, all HA1-CF-treated plants showed a significant reduction in TMV accumulation in systemically infected tomato leaves, up to a 91% reduction at 15 dpi. The qRT-PCR results confirmed that HA1-CF stimulated the transcription of several defense-related tomato genes (PR-1, PAL, CHS, and HQT), pointing to their potential role in induced resistance against TMV. GC–MS analysis showed that phenol, 2,4-bis (1,1-dimethylethyl)-, Pyrrolo [1,2-a] pyrazine-1,4-dione, hexahydro-3-(2-methylpropyl)- and eicosane are the primary ingredient compounds in the HA1-CF ethyl acetate extract, suggesting that these molecules take part in stimulating induced systemic resistance in tomato plants. Our results imply that HA1-CF is a potential resistance inducer to control plant viral infections, a plant growth promoter, and a source of bioactive compounds for sustainable disease management.

Potato virus Y (PVY) is one of the most harmful phytopathogens. It causes big problems for potatoes and other important crops around the world. Nanoclays have been extensively studied for various biomedical applications. However, reports on their interactions with phytopathogens, particularly viral infections, are still limited. In this study, the protective activity of Egyptian nanoclay (CE) and standard nanoclay (CS) against PVY was evaluated on potato (Solanum tuberosum L.) plants. Their physicochemical and morphological properties were examined with scanning electron microscopy (SEM), transmission electron microscopy (TEM), Fourier-transform infrared spectroscopy (FTIR), and energy dispersive spectrometer (EDS). SEM and TEM analyses revealed that CE has a spherical and hexagonal structure ranging from 20 to 80 nm in size, while CS has boulder-like and tubular structures of about 320 nm in size. FTIR and EDS showed that both nanoclay types have different functional groups and contain many vital plant nutrients that are necessary for every stage and process of the plant, including development, productivity, and metabolism. Under greenhouse conditions, a 1% nanoclay foliar application enhanced potato growth, reduced disease symptoms, and reduced PVY accumulation levels compared with non-treated plants. Significant increases in levels of antioxidant enzymes (PPO and POX) and considerable decreases in oxidative stress markers (MDA and H2O2) were also reported. Moreover, a significant increase in the transcriptional levels of defense-related genes (PAL-1, PR-5, and CHI-2) was observed. All experiment and analysis results indicate that the CE type is more effective than the CS type against PVY infection. Based on these results, the foliar applications of nanoclay could be used to manage plant viral infections in a way that is both effective and environmentally friendly. To our knowledge, this is the first report of the antiviral activity of the foliar application of nanoclay against PVY infection.

Tobacco mosaic virus (TMV) is a major pathogen affecting tomato plants worldwide. The efficacy of silver nanoparticles (Ag-NPs) mediated by Punica granatum biowaste peel extract in mitigating the negative impact of TMV infection on tomato growth and oxidative stress was investigated through scanning electron microscopy (SEM), transmission electron microscopy (TEM), UV-Visible (UV-Vis) spectrophotometer, X-ray Diffraction (XRD), dynamic light scattering (DLS), zeta potential, energy-dispersive X-ray spectroscopy (EDX), and Fourier-transform infrared spectra (FTIR). Results of SEM analysis of green Ag-NPs revealed the presence of condensed spherical or round NPs with diameters ranging between 61 and 97 nm. TEM confirmed the SEM results and showed round-shaped Ag-NPs with an average size of 33.37 ± 12.7 nm. The elemental analysis (EDX) of prepared Ag-NPs revealed the presence of elemental Ag as a major peak (64.43%) at 3–3.5 KeV. The FTIR revealed several functional groups on the prepared Ag-NPs, for which three treatment strategies for Ag-NP applications were evaluated in the greenhouse study and compared to inoculated TMV and control plants: pre-infection treatment (TB), post-infection treatment (TA), and dual treatment (TD). The results showed that the TD strategy is the most effective in improving tomato growth and reducing viral replication, whereas all Ag-NP treatments (TB, TA, and TD) were found to significantly increase expression of the pathogenesis-related (PR) genes PR-1 and PR-2, as well as polyphenolic compounds, HQT, and C4H genes compared to control plants. In contrast, the flavonoid content of tomato plants was not affected by the viral infection, while the phenolic content was significantly reduced in the TMV group. Furthermore, TMV infection led to a significant increase in oxidative stress markers MDA and H2O2, as well as a reduction in the enzymatic activity of the antioxidants PPO, SOD, and POX. Our results clearly showed that the application of Ag-NPs on TMV-infected plants reduces virus accumulation, delays viral replication in all treatments, and greatly enhances the expression of the CHS gene involved in flavonoid biosynthesis. Overall, these findings suggest that treatment with Ag-NPs may be an effective strategy to mitigate the negative impact of TMV infection on tomato plants.

In this study, we used RT-qPCR to examine how PR genes were expressed in model tomato (Solanum lycopersicum L.) plants that had been infected with TMV or CMV. Under greenhouse conditions, the indirect ELISA data showed that both viruses were detected for the first time at 6 dpi. Then, the levels of accumulation increased very quickly, reaching a peak of 15 dpi. During the course of the study (1–15 dpi), the Delta CT, NormFinder, BestKeeper, and GeNorm software tools revealed that the β-actin gene was the most informative reference gene in the virally infected tomato tissues. For both the TMV- and CMV-infected tomato plants, the transcriptional expression levels of most tested genes changed between activation and repression, especially in the first 12 dpi. Compared to mock-inoculated plants, the expression levels of PR-1 were induced at all time intervals except at 8 dpi for CMV and at 6, 7, and 8 dpi for TMV infection. Conversely, the greater activation and accumulation of both viruses were associated with the greater up-regulation of PR-2 at 8 dpi, with relative expression levels of 7.28- and 5.84-fold for TMV and CMV, respectively. The up-regulated expression of PR-3, PR-4, and PR-7 was shown at 4 dpi. In contrast, the PR-5 gene was inhibited in TMV at 1 dpi until 9 dpi, and the induction of this gene at 10 dpi increased by 1.72-fold, but PR-5 was observed to up-regulate the expression of CMV at 1 dpi. This study provides the first valuable information on the comparative transcriptional levels of these tomato genes between TMV and CMV infections.

Plant diseases significantly reduce crop yields, threatening food security and agricultural sustainability. Fungi are the most destructive type of phytopathogen, and they are responsible for major yield losses in some of the most crucial crops grown across the world. In this study, a fungus isolate was detected from infected tomato plants and molecularly identified as Pythium aphanidermatum (GenBank accession number MW725032). This fungus caused damping-off disease and was shown to be pathogenic. Moreover, the expression of five pathogenesis-related genes, namely PR-1, PR-2, PR-3, PR-4, and PR-5, was quantitatively evaluated under the inoculation of tomato with P. aphanidermatum. The quantitative polymerase chain reaction (qPCR) showed that the expression levels of PR-1, PR-2, and PR-5 genes went up significantly at 5 days post-inoculation (dpi). The expression of the PR-1 gene also increased the variably, which reached its highest value at 20 dpi, with a reported relative expression level 6.34-fold higher than that of the control. At 15 dpi, PR-2 and PR-5 increased the most, while PR-1, PR-3, and PR-5 also increased noticeably at 20 dpi. On the contrary, PR-4 gene expression significantly decreased after inoculation, at all time intervals. Regarding PR-5 gene expression, the data showed a variable change in PR-5 gene expression at a different sample collection period. Still, it was highly expressed at 15 dpi and reached 3.99-fold, followed by 20 dpi, where the increasing percentage reached 3.70-fold, relative to the untreated control. The HPLC analysis indicated that the total concentration of all detected polyphenolic compounds was 3858 µg/g and 3202.2 µg/g in control and infected plant leaves, respectively. Moreover, the HPLC results concluded that Pythium infection decreased phenolic acids, such as chlorogenic and ellagic acids, which correlated with the infection–plant complex process. Based on the results, P. aphanidermatum could be a biotic stress pathogen that causes the expression of pathogen-related genes and stops the regulation of defensin phenolic compounds.