Introduction

Tomato (Solanum lycopersicum L.) ranks among the most widely grown vegetables worldwide, spanning a total annual production of more than 180 million tons1. It is the second most economically important crop worldwide. Tomato has a high concentration of antioxidant contents and many essential micro and macro nutrients, as well as vitamins such as A, C and E. These nutrients help combat many diseases of humans, including cancer2. However, tomato productivity is severely threatened by vascular wilt disease caused by a destructive soil-borne fungal pathogen Fusarium oxysporum f. sp. lycopersici (FOL) that colonizes the xylem vessels and results in wilting, chlorosis, and death of the tomato plant. The fungal infection destroys the vascular tissues and interferes with the transport of water and nutrients towards the metabolic factories of the plant cell, thus reducing photosynthetic efficiency and turgidity of the cells. The F. oxysporum can cause yield losses of up to 80% in heavily infested fields, particularly under conducive environmental conditions3. The persistent nature of F. oxysporum in soil, combined with the ability to evolve new races, renders traditional control methods such as crop rotation, chemical fungicides4, and resistant cultivars largely ineffective for long-term5.

In recent years, growing interest has been directed towards exploring the use of endogenous plant regulators and signaling molecules to enhance plant resistance against biotic stress6. Among these, melatonin (N-acetyl-5-methoxytryptamine), a pleiotropic molecule known for its role in circadian rhythm regulation in animals, has emerged as a potent bio-regulator in plants7. Melatonin (MT), a tryptophan-derived compound, was first identified in higher plants in 1995, and the term “phytomelatonin” was introduced in 2004. Melatonin has been detected in various parts of plants, including seeds, roots, shoots, stems, and leaves, with the highest levels observed in the reproductive organs8. A key substrate of the biosynthesis of melatonin is Acetyl-CoA found in mitochondria9. This evolutionarily conserved molecule was found to be vital in many important physiological and biochemical processes related to abiotic and biotic stress response, and growth and development in plants10. Amongst the abiotic stresses, it is reported to have an important role against osmotic stress tolerance11, heat12, salinity13, metal toxicity14, cold15 and photo-oxidation16. Studies have reported the role of melatonin in root development, plant growth, fruiting, primary and secondary metabolism and stress responses17. Melatonin has vast applications in the field of agriculture, acting as a stress regulator, a stimulator and a plant protection agent against insect pests, pathogens and harsh abiotic factors such as salinity, drought, and heavy metal toxicity18. More recently, its role in plant immunity has gained attention, with studies indicating that melatonin may modulate defense responses through antioxidant enzyme activity enhancement, reactive oxygen species (ROS) scavenging, and cross-talk with key defense-related plant hormones, including salicylic acid (SA), jasmonic acid (JA), and ethylene (ET)12. Melatonin application enhances the expression of pathogenesis related proteins (PRP) and restores photosynthetic activity in tomatoes by inducing stress resistance against pathogens19. While against Fusarium oxysporum infection in cucumber, melatonin provided protection by enhancing peroxidase and secondary metabolites production20.

Despite these advances, the mechanistic understanding of how melatonin mediates resistance against vascular wilt pathogens, particularly F. oxysporum, remains limited. In tomato, the regulatory pathways and downstream defense response modulated by melatonin under fungal attack are still not fully elucidated. There is a pressing need to dissect the molecular and physiological basis of melatonin-induced resistance, including its effect on oxidative metabolism, transcriptional reprogramming, and defense gene expression. Moreover, the role of melatonin in maintaining redox homeostasis and its potential to act as a priming agent in pathogen-challenged plants represents exciting, yet underexplored, areas of investigation.

Fungicides and resistant cultivars provide only partial control against Fusarium wilt, since the pathogen develop new virulent races capable of evading plant resistance3. Extensive dependence on synthetic fungicides was never reliable because of residual toxicity and the development of fungicide-resistance. Therefore, there is a growing focus on sustainable strategies that strengthen plant defense responses rather than relying solely on chemical control. Melatonin has emerged as a promising defense-inducing molecule because of its role in maintaining redox homeostasis and activating defense-associated signaling pathways7. However, the precise mechanistic basis of melatonin-mediated resistance against vascular wilt of tomato remains a mystery. Addressing these gaps could contribute to improved melatonin-based strategies for sustainable management of Fusarium wilt in tomato cultivation19.

In this study, the main objective was to evaluate the protective role of exogenously applied melatonin against FOL-induced wilt in tomato plants. The effect of application method (foliar and drenching) as well as source of melatonin (chemical and plant derived) was studied on wilt disease symptoms and the plant growth. We hypothesize that melatonin enhances resistance by modulating antioxidant enzymatic activity. The findings will provide new insight into the role of melatonin as a sustainable tool for the management of vascular wilt disease in tomatoes, thus paving the way for developing novel plant protection strategies that integrate endogenous resistance signaling pathways for improved crop health and productivity.

Results

Tomato growth assessment

The application of natural and synthetic melatonin was tested for its impact on the growth parameters of tomatoes. A very significant three-way interactive effect of MT × APP × FOL was observed on the growth parameters except plant height. The plant biomass varied significantly between the melatonin treated and control plants. The results of three-way ANOVA are summarized in Table 1.

Table 1 Three-way AVOVA represented as level of significance of the interactions of melatonin (MT), application method (App) and Fusarium oxysporum f. sp. lycopersici (FOL) inoculation.
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Effect of melatonin on tomato shoot and root length and dry weight

Data revealed that the melatonin significantly increased the plant height as compared to control. Maximum shoot length (39.56 cm) was reported in tomato plants that were treated with a combination (chemical and plant extracted) melatonin applied through foliar application in the absence of F. oxysporum inoculum. The minimum shoot length (19.80 cm) was observed in plants inoculated with F. oxysporum without melatonin (Table 2). In the presence of FOL, the minimum reduction in shoot length of plants was observed in foliar application of combination melatonin treatment (Fig. 1; Supplementary Fig. 1). The application of plant-extracted melatonin through root-drench in the absence of F. oxysporum resulted the highest root length (24.92 cm) (Table 2). However, in inoculated plants, roots treated with a combination (chemical + plant-extracted) melatonin through the root-drenching method resulted the highest (24.00 cm) root length. The minimum root length (16.18 cm) was observed in tomato plants inoculated with F. oxysporum without treating with melatonin in control plants. The data indicated a significant increase in root length when plant-extracted melatonin and combination (chemical + plant-extracted) melatonin were applied to tomato plants, with no significant effect of application method (Fig. 2; Supplementary Fig. 2).

The dry root weight of tomato was significantly decreased in infected plants. Uninoculated roots treated with a combination of chemical and plant-extracted melatonin applied through foliar application reported the highest root dry weight (0.61 g). In the presence of FOL, foliar application of plant-extracted melatonin showed the highest (0.49 g) root weight. The absence of melatonin in the control plants infected with FOL had the lowest (0.21 g) root weight. It was observed that chemically synthesized melatonin had less significant impact on growth parameters compared to plant extracted melatonin (Table 2). Among treatments which received melatonin, significantly higher shoot dry weights were observed uniformly across all types of melatonin-based treatments in both inoculated and uninoculated plants. Plants treated with foliar application of combination melatonin exhibited the maximum (1.59 g) shoot dry weight, followed by plants (1.57 g) that were treated with chemical melatonin application via root-drenching method in the absence of a pathogen. The lowest shoot dry weight (0.44 g) was observed in the inoculated plants that were untreated with melatonin (Table 2). Overall, in the presence of a pathogen, plant-extracted and combination (chemical + plant-extracted) melatonin applied through the foliar spray method resulted in higher dry shoot weight as compared to the root drench application method.

Fig. 1
figure 1

Tomato plant infected with Fusarium oxysporum f. sp. lycopersici (A) without melatonin (B) with combination (chemical + plant-extracted) melatonin application through foliar spray method.

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Table 2 Effect of chemical, plant-extracted and combination melatonin applied by root-drench and foliar spray method and Fusarium oxysporum on plant height, shoot dry weight, root length and dry weight of tomato plants.
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Fig. 2
figure 2

Roots of tomato plant (A) plants received no treatment with melatonin, infected with FOL, (B) plant treated with combination (chemical + plant-extracted) melatonin.

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Biochemical parameter assessment

Total chlorophyll (a + b) content

Data reported significant influence of melatonin source, method of application, and presence of F. oxysporum on chlorophyll content of tomato plants. Application of a combination (chemical + plant-extracted) melatonin through foliar spray without a pathogen exhibited the highest chlorophyll levels (0.78 ± 0.01 mg g−1 FW). This was closely followed by inoculated plants treated with chemical melatonin through the root-drench method. In the presence of F. oxysporum, a general decline of chlorophyll content was observed across all treatments, with melatonin-treated plants displaying relatively higher values as compared to pathogen-only control plants. Among infected treatments, plants treated with foliar-applied combination melatonin showed the highest chlorophyll retention (0.70 ± 0.01 mg g−1 FW). The lowest chlorophyll content (0.46 ± 0.01 mg g−1 FW) was observed in infected plants that were not treated with melatonin (Fig. 3).

Fig. 3
figure 3

Effect of chemical, plant-extracted, and combination melatonin applied by root drench or foliar spray, and of Fusarium oxysporum infection, on total chlorophyll content (chlorophyll a + b; mg g−1 FW) of tomato plants. Values represent mean ± SE, recorded 35 days after transplantation. CHR = chemical melatonin (root drench); PER = plant-extracted melatonin (root drench); CHR + PER = combination of both (root drench); CHF = chemical melatonin (foliar); PEF = plant-extracted melatonin (foliar); CHF + PEF = combination of both (foliar). Bars sharing the same letter are not significantly different, while bars with different letters differ significantly according to Tukey’s HSD test (P ≤ 0.05).

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Antioxidant activity

Application of melatonin enhanced the antioxidant activity in tomato plants infected with F. oxysporum f. sp. lycopersici. As compared to other treatments, combination melatonin applied to the roots reduced SOD and POD activity by approximately 45.8% and 48%, respectively, indicating enhanced oxidative stress inhibition. Similarly, CAT activity was reduced by 53.8% relative to the infected untreated plants. In healthy plants, melatonin alone slightly elevated antioxidant enzyme levels compared to the untreated healthy control, suggesting a priming effect. Overall, the combination (chemical + plant-extracted) melatonin applied through the root drench method proved most effective in reducing oxidative damage under Fusarium wilt stress, while maintaining balanced antioxidant activity in the absence of a pathogen (Fig. 4).

Fig. 4
figure 4

(a) Effect of chemical, plant-extracted, and combination melatonin applied by root drench or foliar spray, and of Fusarium oxysporum infection, on superoxide dismutase (SOD) activity. (b) Effect on peroxidase (POD) activity. (c) Effect on catalase (CAT) activity. Values are mean ± SE, recorded 35 days after transplantation. Abbreviations used for the treatments were: CHR = chemical melatonin (root drench); PER = plant-extracted melatonin (root drench); CHR + PER = combination of both (root drench); CHF = chemical melatonin (foliar); PEF = plant-extracted melatonin (foliar); CHF + PEF = combination of both (foliar). Bars sharing the same letter are not significantly different, while bars with different letters differ significantly according to Tukey’s HSD test (P ≤ 0.05).

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Disease assessment

The disease incidence (DI), disease severity index (PSI), and disease response (DR) of tomato plants infected with Fusarium wilt were assessed 35 days after inoculation based on the development of symptoms and wilting progression (Table 3). The application of melatonin significantly reduced disease symptoms compared to the infected untreated control (FO), which exhibited the highest DI (100%) and PSI (73.16%), while categorized as highly susceptible (HS). Among treated plants, the foliar combination of chemical and plant-extracted melatonin (CHF + PEF) suppressed disease development, resulting in 10% DI and 3.24% PSI, with highly resistant (HR) response. A similar highly resistant (HR) response was observed in the root-drenched combination melatonin treatment (CHR + PER + FO), with only 10% DI and 1.4% PSI. Plants treated with foliar (PEF + FO) and root-drench (PER + FO) applications of plant-extracted melatonin showed high resistance (HR), recording disease severity of 3.77% and 4.77%, respectively, both with 20% DI. Chemical melatonin, when applied by root drenching (CHR + FO) or foliar spray (CHF + FO), provided moderate protection, showing resistant (R) responses with DI ranging from 30 to 40% and PSI between 10.45% and 16.22%. The data indicated that foliar application of the combination (chemical + plant-extracted) melatonin was the most effective, followed by plant-extracted melatonin, in limiting disease development. In comparison, the absence of melatonin resulted in severe disease progression and maximum wilt symptoms.

Table 3 Effect of root drenching of chemical (CHR) melatonin, plant-extracted (PER) as well as combination (CHR + PER) melatonin treatment and foliar application of chemical (CHF), plant extracted (PEF) and combination (CHF + PEF) melatonin on diseased incidence (DI), percent severity index (PSI) and diseases response (DR) of tomato plants towards FOL.
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Identification of PMTR1 genes

The peptide sequences of putative Phytomelatonin Receptor 1 (PMTR1) protein of different crops, namely tomato (S. lycopersicum L.), potato (S. tuberosum L.), cucumber (Cucumis sativus L.), cotton (Gossypium hirsutum L.), rice (Oryza sativa L.), maize (Zea mays L.), and wheat (Triticum aestivum L.) were retrieved from Phytozome v13 for further analysis (https://phytozome-next.jgi.doe.gov/blast-search). The PF10160 domain was BLAST searched against all crops and after removing the redundant sequences, a total of two sequences of tomato, two sequences of cucumber, five sequences of cotton, three proteins of wheat, one sequence of rice, two sequences of potato and two proteins of maize were identified (Table 4). Molecular weight of the PMTR1 amongst different sequences ranged from 44940.73 kDa (S. lycopersicum PMTR1a) to 15072.97 kDa (G. hirsutum PMTR1e). The approximate isoelectric points (pI) spanned from 4.89 (T. aestivum PMTR1b-c) to 6.88 (G. hirsutum PMTR1e). Gene orientation was observed to be of forward direction for eight sequences belonging to C. sativus, G. hirsutum, T. aestivum and Z. mays and the remaining nine were aligned in a reverse direction belonging to S. lycopersicum, G. hirsutum, T. aestivum, O. sativa and S. tuberosum. The length of coding sequences of mRNA ranged from 403 to 1188 with G. hirsutum showing the lowest length while the highest was observed for S. lycopersicum.

Table 4 Accession numbers of the retrieved PMTR1 sequences of different species along with data of the genes and corresponding proteins.
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Phylogenetic analysis

The peptide sequences of putative phytomelatonin receptor 1 (PMTR1) of different crops, namely tomato, potato, cucumber, cotton, rice, maize, and wheat were retrieved from the databanks to construct a Neighbor-Joining phylogenetic tree. The software MEGA 11 was used with default parameters and bootstrap value of 1000 replications, using Pairwise Deletion Method. The tree included two sequences each of tomato, potato, maize, and cucumber; three protein sequences of wheat; five sequences of cotton; while one of rice. All these proteins were arranged in accordance with their sequence similarities and divided into three organized groups (Clade A, Clade B, and Clade C) and one undefined group, as shown in Fig. 5.

Clade A contained three proteins of wheat (TaPMTR1a, TaPMTR1b, and TaPMTR1c), two proteins of maize (ZmPMTR1a and ZmPMTR1b), and one of rice (OsPMTR1a). So, Clade A comprised of six proteins in total spanning across three different crop plants, making it the largest clade of all. Clade B had four proteins, two each from tomato (SlPMTR1a and SlPMTR1b) and potato (StPMTR1a and StPMTR1b). Clade C also contained four proteins in total but only one crop belonged to it i.e., cotton. The proteins of cotton present in this clade are GhPMTR1a, GhPMTR1b, GhPMTR1d, and GhPMTR1e. While the remaining proteins of cotton GhPMTR1c, along with cucumber (CsPMTR1a and CsPMTR1b) were kept into the unclassified group due to their significant difference with the rest of the retrieved proteins.

Fig. 5
figure 5

Phylogenetic tree of the PMTR1 proteins of tomato, potato, cucumber, cotton, rice, maize, and wheat showing three clades and one unidentified group. The number of members in each clade ranged from three to six. Clade A was represented by green color, Clade B by yellow, Clade C by purple, and unidentified group by blue. The members of tomato were marked by violet triangles before the labels.

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Subcellular localization prediction

Subcellular localization of all the retrieved PMTR1 sequences of tomato, potato, cucumber, cotton, rice, maize, and wheat were predicted and visualized in the form of a heatmap (Fig. 6). These proteins exhibited localization in various organelles such as plastids, golgi apparatus, nucleus, cytoplasm, chloroplast, vacuole, endoplasmic reticulum (ER), and extracellular matrix. All of the proteins showed strong localization in plastids and moderate expression in ER. With the exception of GhPMTR1b, all other PMTR1 proteins also displayed localization in vacuole. Only cotton and wheat proteins showed localization in golgi apparatus; cotton in extracellular matrix, while rarely localized outside the cell. PMTR1 proteins of cotton, tomato, potato, and cucumber contained localization signals for nucleus and cytoplasm.

Fig. 6
figure 6

Heatmap showing the predicted subcellular localization signaling of the PMTR1 sequences of different compared species. The strength or frequency of localization signaling is indicated by the gradient of violet shade. Different members showed variable localization for nucleus, cytoplasm, chloroplast, golgi apparatus, and extracellular matrix.

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Analysis of introns and exons

Introns and exons data provides an insight about the splicing sites of a gene and indicates the probability of occurrence of mutations in resultant proteins during gene expression. The number of introns per gene ranged from 2 (GhPMTR1e) to 6 (SlPMTR1a). Among the whole collection, GhPMTR1e had 2 introns per gene; GhPMTR1d, TaPMTR1b, and TaPMTR1c had 4 introns per gene; SlPMTR1a had 6 introns per gene, while the remaining twelve contained 5 introns per gene (Fig. 7).

Fig. 7
figure 7

Intron-exon mapping of different studied PMTR1 genes across the compared species. Green bar represents the upstream and downstream regions, pink bar represents CDS region or exon, and black lines represent the occurrence of intron.

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Protein structure prediction, refinement, and visualization

The Arabidopsis PMTR1 (AtPMTR1) model showed a typical GPCR-like structure, composed of 8 alpha helices, out of which 7 formed a transmembrane-like conformation. The remaining helices, including 2 smaller ones, were positioned at the terminal regions, flanking the core helical bundle. The refined structure included 4,821 atoms, a molecular weight of 34,060.5 u, and a surface area of 35,882.2 Ų. In comparison, the tomato homolog SlPMTR1a exhibited a larger and more open configuration with 9 major helices. Two smaller, broken helices were located near the N-terminal region, while no such structures were observed at the C-terminal. SlPMTR1a was composed of 6,356 atoms with a molecular weight of 44,939.2 u and surface area of 47,671.6 Ų. SlPMTR1b, on the other hand, displayed a compact form with 9 major helices and 2 small internal helices located between the fifth and sixth helices, contributing to a sandwiched configuration. It consisted of 5,462 atoms, a molecular weight of 38,902.9 u, and a surface area of 40,711.0 Å. All three models preserved the core transmembrane-like domains, but with variation in helix orientation and presence of additional minor helices (Fig. 8).

Fig. 8
figure 8

Predicted 3D structures of PMTR1 proteins. (A) AtPMTR1 from Arabidopsis thaliana (B) SlPMTR1a from Solanum lycopersicum (C) SlPMTR1b from S. lycopersicum.

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Discussion

Melatonin, a well-known endogenous components of plant kingdom, however, its role as resistance inducer against phyto-pathogens is yet under-explored21. This study presents, for the first time, the application of both chemically and plant extracted melatonin to analyze the development of F. oxysporum f. sp. lycopersici in tomato plants. The reduction in disease, accompanied by modulation of CAT, SOD and POD activity, suggested the melatonin contribution to enhanced oxidative stress tolerance in the infected plants. Previously, in crops such as wheat and cucumber, melatonin application improved biotic stress resistance through scavenging of ROS and enhanced antioxidant activity20. These protective effects may not only be due to antioxidant capacity but could also result from influence of melatonin on defense signaling pathways, including the activation of MAPK cascades and nitric oxide production, as observed by Arnao and Hernández-Ruiz17.

Our results showed that the effect of melatonin, particularly when applied as a combination of chemical and plant-extracted solution, significantly improved plant growth, biochemical responses, and disease resistance. Melatonin has been reported to enhance CO2 assimilation rate, along with suppression of abscisic acid involved in leaf senescence, and to improve biomass yield. The growth promoting effect of melatonin has been attributed to the auxin like properties of melatonin. Wang22 also reported upregulation of growth hormones in melatonin treated tomato plants which resulted in enhanced growth. The impact of melatonin on growth-related parameters and chlorophyll suggested that melatonin based enhancement of the photosynthetic pigments likely contributed towards improved plant growth, thus emphasizing the growth-promoting effects of melatonin in plants coping with wilt stress23. Foliar application of melatonin primarily enhanced shoot growth, in addition to promoting localized or systemic resistance via signaling from upper plant parts, whereas root drenching improved root health and facilitated broader systemic movement of melatonin through vascular tissues (Fig. 9). These differences likely reflect variations in uptake dynamics, translocation efficiency, and receptor-level recognition. Previous reports stated that root drenching ensures more uniform internal distribution, while foliar application might result in rapid but localized defense activation17. Future studies could explore these dynamics in detail using labelled melatonin or transcriptomic profiling to track downstream responses.

Fig. 9
figure 9

Summary of melatonin-mediated effects on tomato plant under Fusarium wilt stress, highlighting enhanced plant growth, reduced oxidative enzyme activity, and improved disease resistance under combination melatonin treatment.

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Chlorophyll preservation is as a major physiological indicator of melatonin-mediated defense24. Fusarium disrupts chlorophyll biosynthesis, affecting photosynthesis and plant vigor25. Significant chlorophyll degradation in infected plants indicates pathogen-induced stress, often associated with oxidative damage26. However, melatonin application mitigate the chlorophyll degradation27 by preventing photo-oxidative stress and membrane lipid peroxidation28. Additionally, the melatonin receptor CAND2/PMTR1, shown to regulate mitochondrial gene expression in response to oxidative stress contributing to redox balance and membrane integrity29. Melatonin might influence the transcription of chlorophyll biosynthesis or senescence-related genes, as suggested in other studies30,31,32,33. Furthermore, melatonin application strongly affects antioxidant enzyme activity. Infected plants showed increased activities of superoxide dismutase (SOD), peroxidase (POD), and catalase (CAT). Compared to pathogen-only controls, the combined melatonin treatment (chemical + plant-extracted) applied through foliar spray reduced PSI by over 95% and DI by 90%. This reduction was accompanied by a ~ 52% increase in chlorophyll content and a significant suppression of antioxidant enzyme activities, indicating strong resistance induction and effective stress mitigation.

The melatonin application may influence the physiological and biochemical responses of tomato plants under F. oxysporum infection34. However, the negative correlations between growth traits and antioxidant enzymes point to an inverse relationship between oxidative stress and plant vigor. Higher POD, SOD, and CAT activities were associated with reduced shoot and root development, indicating that elevated antioxidant activity is a response to greater stress35. Our data highlights that in treatments where antioxidant levels were high, plants were still experiencing significant biotic stress, likely from FOL infection36.

Phytomelatonin Receptor 1 (PMTR1) is a receptor protein involved in the mediation of many MT-related activities by using H2O2 and Ca2+ as signaling molecules18. PMTR1 is said to have a role against biotic and abiotic stresses. PMTR1/CAND2 receptors induce the production of ROS and the expression of stress- related mitochondrial genes in A. thaliana under photo-oxidative stress29,37. To explore molecular aspect, PMTR1 domain analysis was conducted across several Fusarium-susceptible crops. Phylogenetic analysis grouped PMTR1 proteins into Clade A (Poaceae), Clade B (Solanaceae), and Clade C (Malvaceae), each showing distinct subcellular localization patterns indicative of specialized signaling roles. Clade A proteins predominantly localized in plastids, vacuoles, and ER; Clade B in nucleus and cytoplasm; and Clade C exhibited extracellular localization, potentially mediating intercellular defense signaling. These patterns suggested that PMTR1 functionality may be tailored to species-specific stress responses. Clade B includes tomatoes, also had the highest number of introns and longest gene lengths, possibly enhancing regulatory complexity and adaptability. This comparative genomic insight supports the idea that melatonin signaling is both conserved and diversified, allowing tailored responses in different crop families.

The structural variations of PMTR1 could explain the differential effects observed under the application of plant-derived, synthetic, and combined melatonin sources. Receptor conformation may influence how each treatment modulates melatonin perception and downstream signaling38, particularly under FOL stress. Additionally, the variation between foliar and root-drenching methods may also relate to tissue-specific expression or localization of PMTR1 homologs with differing structural traits. Overall, the structural modelling suggested that receptor diversity at the molecular level could be one of the underlying reasons for variability in plant growth and defense responses under melatonin treatment.

Crosstalk between melatonin and the hormones likely contributed to systemic acquired resistance (SAR) and induced systemic resistance (ISR). Exogenous melatonin application activates PRP expression as well salicylic acid (SA), ethylene (ET) and Jasmonic acid (JA) upregulation, thus limiting the susceptibility to the diseases in plants33. Studies till date revealed that the melatonin induce the activation of plant defense related genes mainly (SA and JA) and their combined action impart disease resistance39,40. Importantly, melatonin’s interaction with defense-related hormones further signifies its role in biotic stress resistance. Investigating hormone profiles and downstream transcriptional networks would offer a more comprehensive view of melatonin’s integrative role in plant immunity41.

The natural origin of the phytomelatonin performed comparably to synthetic melatonin, supporting its use as a sustainable, eco-friendly solution in plant protection42. With increasing environmental concerns about agrochemicals, parthenium-based phytomelatonin emerged as a practical alternative. Overall, present study demonstrated the protective impact of melatonin against Fusarium wilt development in tomatoes. Melatonin application controls antioxidant systems, preserve chlorophyll, enhances growth, influencing hormonal and receptor-mediated signaling pathways to help boost resistance in tomatoes.

Conclusion

The reduction in fusarium-induced wilt in tomatoes in response to melatonin was attributed to enhanced stress tolerance in infected plants. The protective effect of melatonin may not only be limited to antioxidant capacity but could also result from the influence of melatonin on defense signaling pathways, including the activation of MAPK cascades and scavenging of ROS. However, further studies are required to understand the mechanism of action of melatonin at the biochemical and molecular levels. In-silico analysis of PMTR1 across crops further supports melatonin’s role in biotic stress tolerance, with species-specific regulatory patterns. As research on melatonin in plant defense is still emerging, further exploration of its molecular pathways could significantly advance our knowledge of sustainable disease management strategies.

Materials and methods

Plant material, inoculum preparation and growth conditions

Fusarium oxysporum f. sp. lycopersici was acquired from The First Fungal Culture Bank of Pakistan (FCBP), Department of Plant Pathology, University of the Punjab Lahore (FCBP Acc. No. FCBP-PTF-1573). The isolate had been previously confirmed to be a highly virulent strain through pathogenicity testing43. The FOL isolates were cultured on Potato Dextrose Agar (PDA) media plates at 25 ± 2 °C. The 14-day old mature Fusarium plates were harvested for inoculum preparation. The fungal mycelial growth was gently scraped, suspended in autoclaved distilled water, and filtered through sterile muslin cloth to remove mycelial debris. The conidial suspension was then adjusted to 1 × 106 cfu/mL using hemocytometer for inoculation44. Certified tomato seeds (Solanum lycopersicum, cv Rio grande) were surface-sterilized using sodium hypochlorite (NaOCl, 5.0%) for 3 min and washed with distilled water. Two-weeks-old healthy seedlings of tomato were uprooted and roots were immersed in fungal suspension for 2 min and transplanted in sterilized earthen pots containing autoclaved soil. The experiment was conducted in a glasshouse maintained at 28 °C under natural daylight with the relative humidity of 60 to 70%. Pots were arranged in randomized design in greenhouse benches and watered regularly to maintain optimum moisture45.

Preparation of melatonin

A 4 mM solution of synthetic melatonin was prepared by dissolving 46.4 mg of melatonin powder (Sigma-Aldrich) in 50 mL of distilled water46. For the plant-derived melatonin, fresh leaves of Parthenium hysterophorus were collected from the experimental fields of the Faculty of Agricultural Sciences, University of the Punjab, Lahore. The leaves were thoroughly sterilized, air-dried, and ground into a fine powder using liquid nitrogen. The powder (10 g) was suspended in 50 mL of a methanol: water (1:1) mixture and ultrasonicated for 20 min at room temperature. The homogenized mixture was centrifuged using an Eppendorf Centrifuge 5418 R (Eppendorf AG, Germany) at 4000 × g for 15 min, and the resulting supernatant was collected and used as the plant-derived melatonin extract42. The combination treatment was prepared by mixing the synthetic melatonin solution and plant extract in a 1:1 ratio, following previous studies exploring synergistic effects of melatonin and botanical extracts47. Each plant received 100 µL of a 4 mM melatonin solution, applied either as a root drench or as a foliar spray, depending on treatment.

Experimental design

The experiment setup included two controls (a healthy control without FOL and an infected control with FOL). The melatonin treatments utilized three variations: (i) synthetic (chemical) source, (ii) plant-extracted, and (iii) 1:1 combination/mixture of both chemical and plant-extracted melatonin. Each source was applied using two different application methods: (a) by root drenching, and (b) by foliar spraying, both in the presence and absence of FOL. As per the treatment plan, each plant was treated with 100µL of 4 mM Melatonin48. Foliar spraying was carried out on both the upper and lower surfaces of the leaves, while root drenching was applied near the root zone. Overall, the experiment comprised of 14 treatments, while each treatment consisted of 2 replicates and each replicate represent 15 plants. The foliar method was used alongside the root drench to assess both localized and systemic effects of exogenous melatonin in mitigating fusarium wilt. Figure 10 illustrates the experimental design in a thematic diagram.

Fig. 10
figure 10

Experimental design illustrating different treatments and application methods of melatonin. (A) Tomato leaves sprayed with parthenium extracted melatonin, (B) leaves sprayed with parthenium extracted melatonin and inoculated with FOL, (C) leaves sprayed with chemical melatonin, (D) leaves sprayed with chemical melatonin and inoculated with FOL, (E) leaves sprayed with combination melatonin, (F) leaves sprayed with combination melatonin and inoculated with FOL, (G) tomato roots treated with parthenium extracted melatonin, (H) roots treated with parthenium extracted melatonin and inoculated with FOL, (I) roots treated with chemical melatonin, (J) roots treated with chemical melatonin and inoculated with FOL, (K) roots treated with combination melatonin, (L) leaves sprayed with combination melatonin and inoculated with FOL (M) heathy untreated plants, (N) infected plants without melatonin treatment.

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Plant growth assessment

Tomato plants were harvested 40 days post inoculation and assessed for agronomic growth parameters such as height and weight of the upper and lower plant parts, separately. The stem and roots of tomato were dried in the oven at 70 °C till constant weight to measure dry weights49.

Disease assessment

Infected tomato plants were visually examined 40 days post inoculation for Fusarium wilt symptoms on the leaves and stem using a disease rating scale ranging from zero to five (Table 5). The disease percentage severity index (PSI) in the infected plants was calculated using the formula PSI= (∑(rating × number of observations)/Total plants number × highest rating) × 100.

Table 5 Disease rating scale for fusarium wilt of tomato caused by Fusarium oxysporum f. sp. lycopersici45.
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Estimation of chlorophyll content

Total Chlorophyll content(chlorophyll a + b) was measured following the method of Wellburn50. Fresh leaf tissues (0.1 g) was grounded in 3 mL of 80% acetone, and the extract was centrifuged at 12,000 rpm for 5 min. The absorbance of the supernatant was recorded at 645 nm and 663 nm for chlorophyll a and chlorophyll b, respectively, using a spectrophotometer (Shimadzu UV-1800, Shimadzu Corp., Japan). Chlorophyll concentrations were first obtained as µg mL−1 of extract using the standard equations and then converted to mg g−1 fresh weight (FW) based on the extraction volume (5 mL) and tissue mass (0.1 g)51.

Determination of biochemical contents of tomato plants

For biochemical assays, the upper most leaves were collected 20 days post inoculation (dpi). Samples were immediately frozen in liquid nitrogen, stored at − 80 °C, and later used for the SOD, POD and CAT enzyme activity assay.

Superoxide dismutase (SOD) activity

SOD activity was measured by the nitro blue tetrazolium (NBT) reduction method as described by Giannopolitis and Ries52. The reaction mixture contained 50 mM phosphate buffer (pH 7.8), 13 mM methionine, 75 µM NBT, 0.1 mM EDTA, and 2 µM riboflavin. The mixture was exposed to light for 10 min, and the absorbance was read at 560 nm52.

Peroxidase (POD) activity

POD activity was determined using the guaiacol oxidation method53. The reaction mixture consisted of 50 mM phosphate buffer (pH 6.0), 20 mM guaiacol, and 10 mM H₂O₂. The enzyme extract was added to initiate the reaction, and the increase in absorbance was recorded at 470 nm for 3 min.

Catalase (CAT) activity

CAT activity was measured based on the decomposition of H₂O₂ monitored at 240 nm following the method of Aebi (1984). The reaction mixture consisted of 50 mM phosphate buffer and 10 mM H₂O₂54. The reaction was initiated by adding the enzyme extract, and the decrease in absorbance was measured for 3 min.

In-silico analysis, database search and retrieval of PMTR1 genes

Comparative analysis of PMTR1 gene in tomato was performed with other economically important crops affected by F. oxysporum namely Potato, Cucumber, Cotton, Rice, Maize and Wheat. The Pfam sequence (PF10160) of Arabidopsis thaliana (http://pfam.xfam.org/) was used to retrieve sequences of the targeted crops from Phytozome 13 using BLAST-P (Protein-basic local alignment search tool)55. The sequences were renamed in accordance with their similarity level from the reference sequence such that the most similar ones were at the start. The name started with the initials of the scientific name of the organism (e.g., Sl for S. lycopersicum), followed by the protein name (PMTR1), differentiated by “a, b, c” etc., such as SlPMTR1a, SlPMTR1b.

Determination of physiochemical properties and subcellular localization prediction

To determine the physiochemical details of the retrieved protein sequences, ExPasy ProtParam tool (http://web.expasy.org/protparam/) was used. The information regarding the protein’s length, theoretical p1, molecular weight etc., was obtained from ExPasy, whereas the other information regarding the chromosome number, its location and orientation as well as the length of the CDS (mRNA) sequence were retrieved from Phytozome v13 for every compared species56.

To predict the location of each protein within a cell after the expression of the corresponding gene, an online tool WoLF PSORT (https://wolfpsort.hgc.jp/) was used57. The peptide sequences of all the crops were added in FASTA format while keeping the parameters to default settings. For visualization, a Heat Map was created using an R Studio Bioconductor Package “heatmap”.

Analysis of gene structure and phylogenetic analysis

To investigate the arrangement of Introns and Exons in all the retrieved genes of the compared species, genomic and CDS sequences (retrieved from Phytozome) were used. Gene Structure Display Server (GSDS v2.0) (https://gsds.gao-lab.org.php) was used to predict the arrangement of genes58. The genomic and CDS sequences were added there and intron-exon graph was generated under default parameters.

Comparative Multiple Sequence Alignment and Phylogenetic Analysis of the targeted species was performed using MEGA 11, the sequences were aligned using ClustalW version 2.1, under default parameters. The phylogenetic tree was constructed using the Neighbor Joining (NJ) method with Bootstrap value set at 1000 replicates with Pairwise Deletion Method59. The constructed tree was then visualized and modified using Interactive Tree of Life (iTol) (https://itol.embl.de/).

Protein structure prediction, refinement, and visualization

Three-dimensional (3D) structural models of the PMTR1 protein sequences of tomato and arabidopsis were predicted using the trRosetta Server (https://yanglab.qd.sdu.edu.cn/trRosetta/) developed by the Yang Lab60. For each protein sequence, a structure prediction job was submitted to the trRosetta server using default parameters. From the generated structural models, the top-ranked model (model 1) was selected for further refinement and visualization.

To improve the geometry and minimize steric clashes in the predicted structures, the top-ranked models from trRosetta were refined using UCSF Chimera61. The structure files in PDB format were loaded into Chimera, and the structure minimization tool was applied using the default settings: 100 steps of steepest descent followed by 10 steps of conjugate gradient minimization. The “AMBER ff14SB force field” was used for energy calculations. The refined structures were then saved in PDB format for downstream visualization.

The refined protein structures were visualized using PyMOL (The PyMOL Molecular Graphics System, Version 2.5 Schrödinger, LLC). Each structure was rendered using the cartoon representation and color-coded for clarity. Visualization settings such as ray tracing and background color were adjusted to generate publication-quality images. The refined models were exported as high-resolution PNG images using the “ray” and “png” commands in PyMOL.

Statistical analysis

Data analysis and statistical calculations were performed using IBM SPSS Statistics 27. Physiological and biochemical data were subjected to Three-Way ANOVA (Analysis of variance) with melatonin source (chemical, plant-extracted, and combination), method of application (foliar and/or root-drenching) and pathogen treatment (presence or absence of F. oxysporum f. sp. lycopersici) as independent factors. Post-hoc comparisons were conducted using Tukey’s HSD test at P ≤ 0.05. Data were presented as mean values of replicates, while assumptions of normality and homogeneity of variance were tested prior to analysis.