Therefore, a more detailed understanding of the relationship between plant responses to nutritional status and biotic stresses is essential. Zinc plays a crucial role in the plant response to pests and diseases. Plants naturally absorb high concentrations of metals such as Zn from the substrate as a self-defense mechanism against pathogens and herbivores. Metal ions may activate defense reactions through a signaling pathway or by plant fortification. Several studies have demonstrated that Zn fertilization decreases disease severity of Phytophthora root rot and common leaf spot in alfalfa , peach gummosis in peach and early blight in potato. However, a protective concentration of Zn against certain pathogens can also induce increased susceptibility to another pathogen on the same host. Despite these conflicting reports on Zn and disease resistance and susceptibility, information is lacking on how Zn would affect susceptibility and resistance of watermelon plants against FON and RKN. It is not understood if transcriptome level changes can occur especially in pathways that are related to induced resistance. In the present study, we utilized a hydroponic system to evaluate if varying levels of Zn with or without challenge inoculation with either FON or RKN or both can affect genes in pathways related to induced resistance. A comparative gene expression analysis of high-Zn and low-Zn treated plants under inoculated or RKN or both and non-inoculated treatments was conducted. In non-inoculated plants, the number of DEGs in plants treated with high-Zn was higher than plants treated with low-Zn at both 7 and 11 days post-treatment . At 7 dpt,vertical greenhouse under high-Zn treatment eight DEGs were identified whereas in plants treated with low-Zn treatment six DEGs were obtained. At 11 dpt, there were 333 DEGs in plants treated with high-Zn whereas 14 DEGs in plants treated with low-Zn were identified.

GO terms with corrected p-value less than 0.05 were considered significantly enriched by differentially expressed genes and are shown in Figure 2 . Significantly enriched GO terms were observed only at 11 dpt. In the biological process, movement of cell or subcellular component, micro-tubule-based process and micro-tubule based movement were the significantly enriched terms common in high-Zn treated plants inoculated with FON alone, low-Zn treated plants inoculated with FON alone, high-Zn treated plants inoculated with RKN alone, and low-Zn treated plants inoculated with FON and RKN . In the cellular component, thylakoid part, thylakoid and photo system II were significantly enriched terms in both high-Zn treated plants, with and without inoculation with RKN . In high-Zn treated plants inoculated with FON alone, chromosome, chromosomal part and chromatin were the most enriched terms . Moreover, in molecular function, exopeptidase activity was the only significantly enriched term in low-Zn treated plants . In high-Zn treated plants inoculated with FON alone, transcription factor activity sequence-specific DNA binding, nucleic acid binding transcription factor activity and protein dimerization activity were the most enriched terms. The most enriched term in Steiner treated plant inoculated with FON alone were transcription factor activity sequence-specific DNA binding, nucleic acid binding transcription factor activity and sequence-specific DNA binding. Similarly, tubulin binding, protein complex binding and motor activity were the most enriched terms in low-Zn treated plants inoculated with FON alone. High-Zn treated plants inoculated with RKN alone showed the most enrichment of cytoskeletal protein binding, macromolecular complex binding and protein complex binding. In low-Zn treated plants inoculated with FON and RKN, pyrophosphatase activity, nucleoside-triphosphatase activity and hydrolase activity acting on acKEGG enrichment results showed that DEGs were classified into multiple signaling pathways . Significant KEGG enrichment was observed in plants treated with high-Zn at 7 dpt. At 11 dpt plants in treatments: high-Zn inoculated with FON alone, low-Zn inoculated with FON alone, and high-Zn inoculated with RKN alone, showed significant enrichment.

Peroxisome and glycosaminoglycan degradation were significantly enriched with two and one DEGs, respectively, in plants treated with high-Zn at 7 dpt. Two pathways, plant hormone signal transduction and plant-pathogen interaction, were significantly enriched in high-Zn treated plants inoculated with FON alone. In plants treated with low-Zn that were inoculated with FON, alpha-linolenic acid metabolism, valine, leucine and isoleucine biosynthesis and linoleic acid metabolism were significantly enriched. Metabolic pathways, carbon fixation in photosynthetic organisms, photosynthesis, pentose phosphate pathway, starch and sucrose metabolism, ascorbate and aldarate metabolism, carbon metabolism, photosynthesis-antenna proteins and carotenoid biosynthesis were enriched in high-Zn treated plants inoculated with RKN alone.FON was re-isolated from 100% of the plants that were inoculated with FON or coinoculated with RKN but not from non-inoculated Steiner control or plants only inoculated with RKN. Isolates were tested for FON by conventional PCR as described earlier. One hundred percent of the putatively isolated FON colonies from infected plants were confirmed as FON using the PCR assay. No difference in plants being colonized by FON for all treatments and no Fusarium wilt symptoms were observed. Also, no differences in root gall formation between treatments were observed, except for a decrease in total nematode counts in plants inoculated with both FON and RKN under high zinc treatment . Galls were present only on plants inoculated with RKN or RKN co-inoculated with FON but were absent on non-inoculated or plants only inoculated with FON.study, a whole transcriptome analysis was performed using RNA-seq in inoculated and non-inoculated watermelon plants in response to high- and low-Zn levels. We found a higher number of DEGs in high-Zn than in low-Zntreated plants that were not inoculated; also in plants that were inoculated with FON or RKN alone. However, the effect of high-Zn was not consistent in a co-inoculated system; low-Zn treated plants inoculated with FON and RKN had higher DEGs than high-Zn treated plants inoculated with FON and RKN.

These findings indicate that high-Zn treatment can alter differential expression of genes in watermelon, but the level of expression is not consistent across individual or co-inoculated pathosystems. We identified that the expression level of many genes associated with the phytohormone signaling pathway and the MAPK signaling pathway was affected in highZn treated plants, particularly in plants inoculated with FON or RKN alone. As the goal of this study was to evaluate the systemic response of watermelon away from the point of inoculation/infection, leaf tissues instead of root tissues were used for assessments. Within the time frame of the greenhouse study, we did not observe any wilting in plants inoculated with FON, RKN or both. However, fungal isolation and confirmation from the hypocotyl region confirmed the roots were indeed coloniHormone signaling pathways play significant roles in regulating interactions between plants and microorganisms. We observed an increased expression of JA pathway genes: JAZ and MYC2 in high-Zn treated plants inoculated with FON or RKN alone. The core signal transduction mechanism of JA signaling comprises JAZ and MYC. Specific JAZ/TFs are generated by JAZ and different TFs that explicitly regulate many downstream responses. The JAZ-MYC module triggers the plant defense response against pathogen infection by increasing the concentration of defense compounds such as indole alkaloids, terpenoid phytoalexins and often through airborne signals. Gallego et al. also found that surplus Zn can potentiate plant defense responses, especially in the synthesis of JA and it’s signaling pathway, thus improving plant resistance in Arabidopsis thaliana against Alternaria brassicicola. In addition,vertical grow towers we observed an increased expression of NPR1 in high-Zn treated plants inoculated with RKN. NPR1 plays an integral part in the efficacy of the plant defense response. It activates PR gene expression by recruiting TGA transcription factors. Arabidopsis NPR1 mutants showed decreased PR gene expression and increased susceptibility to pathogens. Also, the ERF1/2 gene involved in ethylene signaling was upregulated in high-Zn treated plants inoculated with RKN. Activation of ERF genes is known to enhance plant disease resistance. This could mean that high-Zn concentration in watermelon plants modulated induced resistance against FON and RKN. It is further justified by the number of genes involved in synthesis and regulation of SA, JA, abscisic acid, auxin, brassinosteroid, and cytokinin that were significantly affected in high-Zn treated plants inoculated with FON or RKN alone. Plant MAPKs participate in plant growth, development, and responses to endogenous and environmental cues. Studies have shown that MAPKs could be activated by external sensors for cellular reactions. A study conducted by Bi and Zhou demonstrated that several pathogen-secreted effectors inhibit the MAPK cascade, which confirms the involvement of MAPKs in plant-pathogenic interactions and their role in plant response to pathogen invasion. Our study demonstrated that many genes in the MAPK pathway were significantly affected in high-Zn treated plants inoculated with FON or RKN alone. The genes that were significantly upregulated include WRKY33, RbohD, MYC2, and ACS6. Genes in the WRKY family have been confirmed to perform important regulatory functions to modulate pathogen-triggered cellular responses in a variety of plants, and most WRKY factors participate in the salicylic acid signaling pathway.

RbohD is responsible for ROS production, which is involved in regulation of immune function against various pathogens . As mentioned earlier, MYC2 a triggers defense response via the JA pathway. Similarly, ACS regulates synthesis of ethylene, which plays a positive role in host resistance against fungal and bacterial pathogens. In summary, we observed that a high-Zn level affects plant–pathogen interaction in watermelon by regulating many crucial plant defense pathways . Multiple genes in several hormone signaling pathways, including SA, JA, ethylene, abscisic acid, auxin, brassinosteroid and cytokinin were significantly affected. However, this effect was limited to FON-only and RKN-only inoculated systems. In the FON and RKN coinoculated system, the induction of resistance in gall formation was observed; however, changes in DEGs were not observed. It is well known that FON and RKN together can be more severe to watermelon plants and it is possible that such severe interactions might counteract induced host-resistance with altered response in presence of high zinc levels. Pathogens are also known to evolve strategies to counteract Zn-related plant defense. However, plant infection was confirmed at the end of this experiment by the recovery of the fungus and morphological and molecular identification for FON and by presence of galls for RKN in all inoculated plants. Significant differences in terms of disease severity were not observed among inoculated treatments except for the reduced nematode counts when FON and RKN are inoculated simultaneously in high-Zn treated plants compared with Steiner treated plants with both pathogens inoculated. Future research is required to understand the transcriptional and translational basis of this observation. Further study isalso required to optimize the level of Zn that can successfully reduce disease severity at the field level based on the effect of Zn ions on natural rhizospheral microbial diversity in natural field conditions. To our knowledge, this is the first study to analyze the effect of Zn level on watermelon in relation to FON or RKN or both infection under controlled hydroponics conditions.Visual symptoms of wilting were not observed in plants throughout the experiment. At the end of the experiment, one plant from each container , inoculated with FON alone, or co-inoculated with RKN and non-inoculated plants in the Steiner solution, were checked for the presence of FON. Stem pieces were cut from the base of the main stem of each plant using a pair of scissors sterilized by dipping in 70% ethanol. Stem pieces were surface-disinfested for 1.5 min with 0.6% sodium hypochlorite, rinsed in sterile water and placed on a semi-selective peptone pentachloronitrobenzene agar medium. Plates were incubated at 25 ◦C for 7 days. Fungal isolates were then microscopically identified based on morphological criteria and further were confirmed as FON by PCR assay with FON specific primers Fon-1/Fon-2. Percentage of plants infested with FON as determined by morphological and PCR confirmatory assays were recorded. For plants inoculated with RKN, 10 cm of roots closest to the base of plants were sampled and evaluated, as galls were observed only in this portion. Rockwool plugs were removed from the root system, and roots were washed and rated visually for the presence of galls as described earlier. Mean number of RKN adults for each treatment . The effects of Zn application and pathogen exposure on RKN adult numbers in watermelon roots were also analyzed as described above.Due to the widespread use of PPCP/EDCs, their incomplete removal during wastewater treatment, and the introduction of waste materials to the environment, PPCP/EDCs are detected in a variety of environmental matrices , including surface water, soil, groundwater, plant tissue, and soil biota .