Micronutrients Affect Expression of Induced Resistance Genes in Hydroponically Grown Watermelon against Fusarium oxysporum f. sp. niveum and Meloidogyne incognita

The soil-borne pathogens, particularly Fusarium oxysporum f. sp. niveum (FON) and southern root-knot nematode (RKN, Meloidogyne incognita) are the major threats to watermelon production in the southeastern United States. The role of soil micronutrients on induced resistance (IR) to plant diseases is well-documented in soil-based media. However, soil-based media do not allow us to determine the contribution of individual micronutrients in the induction of IR. In this manuscript, we utilized hydroponics-medium to assess the effect of controlled application of micronutrients, including iron (Fe), manganese (Mn), and zinc (Zn) on the expression of important IR genes (PR1, PR5, and NPR1 from salicylic acid (SA) pathway, and VSP, PDF, and LOX genes from jasmonic acid (JA) pathway) in watermelon seedlings upon inoculation with either FON or RKN or both. A subset of micronutrient-treated plants was inoculated (on the eighth day of micronutrient application) with FON and RKN (single or mixed inoculation). The expression of the IR genes in treated and control samples was evaluated using qRT-PCR. Although, significant phenotypic differences were not observed with respect to the severity of wilt symptoms or RKN galling with any of the micronutrient treatments within the 30-day experimental period, differences in the induction of IR genes were considerably noticeable. However, the level of gene expression varied with sampling period, type and concentration of micronutrients applied, and pathogen inoculation. In the absence of pathogens, micronutrient applications on the seventh day, in general, downregulated the expression of the majority of the IR genes. However, pathogen inoculation preferentially either up- or down-regulated the expression levels of the IR genes at three days post-inoculation depending on the type and concentration of micronutrients. The results demonstrated here indicate that micronutrients in watermelon may potentially make watermelon plants susceptible to infection by FON and RKN. However, upon infection the IR genes are significantly up-regulated that they may potentially aid the prevention of further infection via SA- and JA-pathways. This is the first demonstration of the impact of micronutrients affecting IR in watermelon against FON and RKN infection.


Introduction
Watermelon (Citrullus lanatus var. lanatus (Thunb.) Matsum. & Nakai) is one of the most popular fruits in the world, with a total production of approximately 104 million tons [1]. The United States is the seventh-largest watermelon producer globally with 2.18 million tons of annual production and a farm gate value of USD 657 million [2]. The southeastern states including, Florida, Georgia, North Carolina, and South Carolina tions and disease onset in non-infected plant parts of an infected plant [3]. In SA-dependent signaling, PR1 and PR5 are two important markers [39] and the non-expressor pathogenesisrelated gene 1 (NPR1) is a global transcription factor [40,41], which is essential for regulating pathogenesis-related defense responses [42]. Plant defensins (PDFs), vegetative storage proteins (VSPs), and lipoxygenases (LOXs) are key genes involved in the JA-dependent signaling pathway [43][44][45]. The transcriptional activation of these genes represents a critical part of plants' defense machinery against pathogens following pathogen or elicitor perception.
Nutritional status, particularly micronutrients, serves as a factor affecting ISR in plants, and therefore, it can influence crop response to disease [46]. Micronutrients such as divalent metallic ions including iron (Fe), manganese (Mn), and zinc (Zn) are cofactors for a wide range of enzymes and are often essential for the activation of host defense responses following tissue infection [46]. The acquisition of metal ions by plants from their surrounding environment is pivotal for survival and defense against biotic and abiotic stress [47]. The presence of Fe, Mn, and Zn has been reported to promote disease resistance in the host plant [48][49][50]. The effect of soil micronutrients in plant defense genes against pathogens has been studied previously in a variety of crops. In Alium cepa (onions), the incidence of sour skin was found to be correlated with the soil and tissue Cu, Fe, Mn, and Zn concentrations [51]. The subsequent transcriptome analysis indicated upregulation (>5000 fold) of PR1 in onion tissues collected from high soil Cu:Fe concentration ratios compared to low Cu:Fe concentration ratios [52]. A field study in Nicotiana tabacum (tobacco) also displayed a substantial correlation between tomato spotted wilt (TSW) and soil Cu:Fe values [53,54]. A risk model based on this study was developed [55], which successfully predicted TSW risk before planting in 2014 and 2015. Additionally, in a gene expression study, plants from a low-risk zone exhibited a 650-fold increase in the expression of NPR1 compared to samples from a high-risk zone [55]. Similarly, another model can predict the risk of bacterial leaf spot (BLS) on Capsicum annuum (peppers) based on the micronutrient concentrations in the soil prior to pepper planting [54]. The defense genes from the SA pathway in peppers were significantly upregulated in predicted low-risk vs. predicted high-risk BLS sites.
Although the above studies indicated the roles of soil micronutrients in promoting plant defense against pathogens, the specific roles of individual micronutrients in a complexsoil medium are still elusive. Experiments using soil-less medium (e.g., hydroponics) may aid in determining specific roles of individual micronutrients by reducing or negating the interactive effects that may often be present in a soil medium. Considering that FON and RKN are soil-borne pathogens, soil micronutrients may potentially play an important role in host-pathogen interactions during infection. However, limited information is available on this relationship. In this study, we used a hydroponic system to investigate whether Fe, Mn, and Zn (higher and lower than the standard dose) treatments can influence the expression of IR genes in the SA (PR1, NPR1, and PR5) and JA (LOX, VSP, and PDF) pathways in preand post-pathogen infection events (FON or RKN or FON+RKN) in watermelon.

Change in Micronutrient Level Influences the Expression of SA-and JA-Genes in Watermelon Seedlings
The transcript levels of six key genes-pathogenesis-related gene 1 (PR1), non-expressor pathogenesis-related gene 1 (NPR1), pathogenesis-related gene 5 (PR5), lipoxygenases (LOX), vegetative storage protein (VSP), and plant defensin (PDF)-which are involved in plant IR, were determined using quantitative real-time reverse transcription-polymerase chain reaction (qRT-PCR). Transcripts were quantified in plants treated with micronutrient treatments at 7 DPT and compared with non-treated control plants in Steiner ( Figure 2). Watermelon plants treated with high Fe for seven days significantly downregulated PDF, PR1, and PR5 genes prior to inoculation. When high and low Fe treated plants were compared, relative expression of the PR1 gene was significantly lower in plants treated with high Fe (Figure 2A). The expression of the PR5 gene was significantly downregulated in low Fe treated plants but expression levels were not significantly different between high and low Fe (Figure 2A). Relative expression of NPR1, PDF, PR1, and PR5 genes was significantly downregulated by the Mn treatment ( Figure 2B). Relative expression of PR1 and VSP genes was significantly higher for high Mn compared to low Mn-treated plants. The expression of NPR1, PDF, PR1, and PR5 genes was significantly downregulated by Zn treatments ( Figure 2C). When high and low Zn treated plants were compared, VSP gene expression was significantly higher in the low Zn treatment compared to the high Zn treatment ( Figure 2C). Primer sequences and PCR conditions for test and reference genes are given in the Table 3. Table 2. Mean concentrations (ppm) of Fe, Mn, and Zn in nutrient solutions. The concentrations of micronutrients (Fe, Mn, Zn) in hydroponic solutions were measured at 0-and 7-days post-treatment for high, low, and Steiner solutions.

Change in Micronutrient Level Influences the Expression of SA-and JA-Genes in Watermelon Seedlings
The transcript levels of six key genes-pathogenesis-related gene 1 (PR1), non-expressor pathogenesis-related gene 1 (NPR1), pathogenesis-related gene 5 (PR5), lipoxygenases (LOX), vegetative storage protein (VSP), and plant defensin (PDF)-which are involved in plant IR, were determined using quantitative real-time reverse transcription-polymerase chain reaction (qRT-PCR). Transcripts were quantified in plants treated with micronutrient treatments at 7 DPT and compared with non-treated control plants in Steiner ( Figure 2). Watermelon plants treated with high Fe for seven days significantly downregulated PDF, PR1, and PR5 genes prior to inoculation. When high and low Fe treated plants were compared, relative expression of the PR1 gene was significantly lower in plants treated with high Fe (Figure 2A). The expression of the PR5 gene was significantly downregulated in low Fe treated plants but expression levels were not significantly different between high and low Fe ( Figure 2A). Relative expression of NPR1, PDF, PR1, and PR5 genes was significantly downregulated by the Mn treatment ( Figure 2B). Relative expression of PR1 and VSP genes was significantly higher for high Mn compared to low Mn-treated plants. The expression of NPR1, PDF, PR1, and PR5 genes was significantly downregulated by Zn treatments ( Figure 2C). When high and low Zn treated plants were compared, VSP gene expression was significantly higher in the low Zn treatment compared to the high Zn treatment ( Figure 2C). Primer sequences and PCR conditions for test and reference genes are given in the Table 3.  Watermelon seedlings (cv. Sugar Baby; 3 weeks old) were either treated with Fe or Mn or Zn at high (3X), low (0.5X), or standard concentration (X, Steiner) for 7 days. ß-Actin was used as the reference gene. Plants in the Steiner treatment were considered as non-treated control. Data are the mean fold changes ± SE in gene transcript levels in tissues from micronutrient treated plants relative to tissues from non-treated control plants in Steiner. Letters indicate a significant difference between treatments with the Tukey-Kramer test (p < 0.05).    Figure 3E). RKN inoculation significantly upregulated VSP gene expression in plants treated with low Mn ( Figure 3E). When Mn-treated plants were compared, the relative expression of LOX, PDF, and PR5 genes was higher in plants treated with low Mn with FON inoculation ( Figure 3B). Inoculation of RKN resulted in higher expression of LOX, NPR1, and VSP genes in the same treatment ( Figure 3E). After inoculation of FON and RKN, expression of PR1 and PR5 were again higher in plants treated with low Mn ( Figure 3H).

Differential Expression of SA-and JA-Genes in Zn-Treated Plants Inoculated with FON, RKN, or Both
The PR1 gene was significantly upregulated in plants treated with low Zn in response to co-inoculation with both FON and RKN ( Figure 3I). Co-inoculation with both FON and RKN resulted in significant downregulation of the PR5 gene in plants treated with high Zn ( Figure 3I). When Zn-treated plants were compared, relative expression of the LOX gene was significantly higher in plants treated with high Zn compared to Steiner and low Zn with FON inoculation ( Figure 3C). LOX and PR5 gene expressions were higher in plants treated with high Zn after RKN inoculation ( Figure 3F). However, co-inoculation with both FON and RKN reduced the expression of these genes in the high Zn treatment ( Figure 3I).

FON Recovery and RKN Gall Rating
FON was successfully re-isolated from the tested plants that were previously FONinoculated or co-inoculated with FON and RKN but not from the non-inoculated controls or the plants inoculated with only RKN. Isolates were also examined for FON using traditional PCR as previously described. The PCR assay successfully identified all of the purportedly isolated FON colonies from affected plants as FON  Letters indicate a significant difference between treatments with the Tukey-Kramer test (p < 0.05). Primer sequences and PCR conditions for test and reference genes are given in Table 3.

FON Recovery and RKN Gall Rating
FON was successfully re-isolated from the tested plants that were previously FONinoculated or co-inoculated with FON and RKN but not from the non-inoculated controls or the plants inoculated with only RKN. Isolates were also examined for FON using traditional PCR as previously described. The PCR assay successfully identified all of the purportedly isolated FON colonies from affected plants as FON. In plants colonized by FON, we found no noticeable difference in Fusarium wilt symptoms across micronutrient treatments. Furthermore, there were no differences in root galling between treatments. In ß-Actin was used as the reference gene. Plants in the Steiner treatment were considered as non-treated control. Data are the mean fold changes ± SE in genes transcript levels of tissues from inoculated plants in micronutrient treatments relative to tissues from non-inoculated control plants in Steiner. Letters indicate a significant difference between treatments with the Tukey-Kramer test (p < 0.05). Primer sequences and PCR conditions for test and reference genes are given in Table 3.

Discussion
The role of soil micronutrients on IR and plant diseases is well-documented in previous studies [52][53][54][55]. However, the findings from these studies were mostly based on soil-based media. Hence, it is often difficult to deduce what role these individual micronutrients play in IR during pre-or post-inoculation phases with plant pathogens. Additionally, little is known about the function of specific micronutrients in watermelon plant defensive reactions against soil-borne pathogens like FON and RKN. In this study, the influence of the level of micronutrients, Fe, Mn, and Zn, on the expression level of IR genes (PR1, PR5, and NPR1 from SA-pathway, and VSP, PDF, and LOX from JA-pathway) in watermelon leaves pre-or post-inoculation with FON and RKN (single and mixed) in a hydroponics system were monitored through relative gene expression. In order to ascertain that the SAand JA-genes are induced systemically away from the point of contact (roots), leaf samples (instead of root samples) were assayed for monitoring gene expression. The results indicate that micronutrient applications indeed induce SA-and JA-genes in leaves away from the point of contact (root).
The qRT-PCR results demonstrate significant differences in expression of IR genes among treatments and they varied with sampling period, type, and concentration of micronutrients applied, and pathogen-inoculation. We observed that plants treated with Fe, Mn, and Zn at higher and lower doses than those found in standard Steiner solution for seven days demonstrated downregulation of IR genes (PR1, PR5, NPR1, and PDF) (Figure 2A-C). The IR genes were not up-regulated even at 11 days in those treatments ( Figure S1A-C). However, upregulation was observed in some micronutrient-pathogen treatments at three days post-inoculation of the pathogen (equivalent to 11-day post micronutrient treatments). This result is in line with the previous study where no activation of the JA-pathway in the Trichoderma hamatum T382 pre-inoculated Arabidopsis plants was observed without a subsequent infection with Botrytis cinera [68].
At seven days post-treatment, none of the micronutrient treatments resulted in the upregulation of IR genes. However, following pathogen inoculation, tested genes in both SA-and JA-pathways were activated, and at three days post-inoculation (equivalent to 11-days post micronutrient treatments), their levels of expression varied substantially. These observations suggest that the type and concentration of micronutrients in watermelon may potentially influence SA or JA-mediated genes when infected with either FON or RKN or both. We did not observe significant phenotypic differences with respect to the severity of wilt symptoms or RKN galling among the micronutrient treatments within the 30-day experimental period. Although wilt symptoms were not observed, roots were colonized by FON as evident by the consistent recovery of the pathogen on the culture medium along with further confirmation with a PCR assay. It is possible that FON may need a soil system to induce wilt symptoms rather than a hydroponic system, as utilized in this study. Despite the lack of phenotypic differences, significant up-and down-regulation of IR genes among treatments were observed. It is possible that the induction of resistance genes (JA and SA pathways) might not be strong enough to induce phenotypic differences among different micronutrient treatments (type and concentrations).
Although previous reports evaluated the expression of these genes in response to pathogens or micronutrients, none of the studies evaluated these variables in combination. In this study, we provide evidence that watermelon plants respond differentially to distinct micronutrient and pathogen combinations. For example, we observed upregulation in the following micronutrient-pathogen treatments: PDF gene in high Fe treated plants with FON inoculation, VSP gene in low Mn treated plants with RKN inoculation, and PR1 gene in low Zn treated plants with co-inoculation of both FON and RKN. These observations indicate that genes in JA-(PDF and VSP) and SA-pathway (PR1) in watermelon plants respond differentially with respect to different micronutrients and pathogen combination treatments. The PDF and VSP genes are regulated by JA, a key compound of the JA-signaling pathway, and are induced during soil-borne pathogen infection [69,70]. In Bacillus-treated soybean, the upregulation of the VSP gene was observed upon inoculation with Rhizoctonia solani and F. oxysporum [70]. The VSP is present in the host-vegetative tissues and has displayed mutagenic and phosphatase potential against herbivores [71,72]. Small, basic, cysteine-rich peptides known as plant defensins (PDFs) possess antibacterial activity against a variety of microbes [73,74]. The expression of PDF1.2 and nine other genes was observed to be upregulated in Arabidopsis thaliana upon parasitic plant attack by Orobanche ramose [75].
Pathogenesis-related (PR) proteins are induced by products made in the SA-pathway and help defend plants against plant pathogens [76,77]. In the roots of soybean seedlings inoculated with Phytophthora sojae, the upregulation of PR1 and PR2 was connected to limited lesion development [78]. Similarly, high expression of defense-related genes, such as PR1, and greater activities of PR enzymes were found to increase watermelon's resistance to FON in a wheat intercropping system [79].
Induced resistance mediated by several genes governing the SA-and JA-pathways can also be downregulated with specific micronutrient and pathogen combinations. We In the current study, we discovered that high Fe-treated plant leaf tissue had the highest gene expression compared to low-and standard-Fe treated plants (Steiner). Several intrinsic host defense mechanisms require Fe [80]. In Arabidopsis [56,57] and wheat [58], cellular translocation of Fe to infection sites, which coincided with local reactive oxygen species (ROS) formation, revealed direct participation of Fe in the defense response. The biotrophic growth phase of Colletptrichum graminicola was found to be delayed and partially suppressed by maintaining appropriate Fe concentrations in maize [59]. Mn has been shown to induce the production of phenolic compounds and plant-IR mechanisms [60]. It is a cofactor of superoxide dismutase (SOD), which takes part in plant defense against oxidative stress brought on by an increase in the harmful ROS and reactive forms of oxygen (ROV) [61]. Effects of increased Mn availability on disease severity have been found to vary among different plant species with different diseases [62]. In our study, we observed that the relative expression of IR genes was higher in the plants treated with low Mn after pathogen inoculation. Relative expression of defense genes was higher with plants grown in the high Zn treatment in response to inoculation with either FON or RKN. However, expression was higher in plants treated with low Zn after co-inoculation of both FON and RKN. Zn treatment has been reported to reduce symptoms of disease in many cases [63,64,81]. The variability we observed in plants inoculated with FON and RKN might be due to the protective concentration of Zn against one pathogen might have induced increased susceptibility to another pathogen as reported previously [82].
Symptom differences were also not observed among treatments in terms of wilting and gall rating caused by RKN inoculation. The development of symptoms might have been affected by the hydroponics system where the plants had constant access to moisture as the roots were merged in the nutrient solutions.

Preparation of Inoculum of FON and RKN
An isolate of FON was collected from symptomatic watermelon plants in Georgia, U.S., and was identified as FON race two after inoculating them on a set of watermelon differentials as described previously [9,83]. A pure culture of FON was maintained on Potato Dextrose Agar (PDA) (Thermo Fisher Scientific, Waltham, MA, USA) at 25 • C for 7 days. Five mycelial plugs (0.7-cm diameter) were removed from the edge of the colony and were transferred to a 500 mL flask containing 200 mL of potato dextrose broth. Cultures were incubated at 25 • C and 160 rpm on a rotary incubator shaker (Thermo Scientific, Alachua, FL, USA). Microconidia were collected after 2 weeks by pouring the culture through double-layered sterile cheesecloth and using a hemocytometer; the concentration of the microconidia suspension was adjusted to a final concentration of 1 × 10 5 microconidia/mL as described earlier [84]. A population of M. incognita race three (RKN) maintained on eggplant (Solanum melongena L.) was kindly provided by Dr. Richard Davis (USDA-ARS, Tifton, Georgia) and Nematology Lab (Tifton campus). Eggplants were maintained in a greenhouse at 22-30 • C. Plant roots that were infected were cut, cleaned in tap water, and then left in a mist chamber for 2-9 days to allow the eggs to develop. After incubation for 9 days, newly hatched second-stage juveniles (J2) were collected every 3 days. The J2s were also collected from the soil in which the culture was maintained by the sugar centrifugation method [85]. The resulting M. incognita (J2) collected were diluted in tap water to get a final concentration of 6000 J 2 /mL.

Hydroponics Set-Up under Greenhouse Conditions
Two independent greenhouse experiments were designed for use with a hydroponics system to evaluate the effects of micronutrients in watermelon on IR against FON or RKN or co-inoculation of FON and RKN. The greenhouse was maintained at 24 • C and 80% relative humidity throughout the experiment. A small layer of vermiculite was placed on top of the watermelon seeds (cv. Sugar Baby) after they were planted into sheets of 2.5 cm 2 /cell Rockwool cubes (Grodan Inc., Hedehusene, Denmark). Whenever needed, water was used to keep the sheet moist. Following germination, seedlings received water as needed, and a single application of a specially formulated Steiner universal fertilizer solution (Table 1). For this manuscript, we refer to "the modified Steiner universal nutrient solution" [65] as a "Steiner solution". On Rockwool, seedlings were kept for three weeks after which, they were transferred to plastic containers (43 cm W × 30 cm D × 19 cm H, Reynolds Consumer Products, Inc., Louisville, KY, USA) with appropriate micronutrient treatments in addition to the Steiner solution (Table 1) Table 1. The micronutrient treatments were prepared in 8 L of deionized (dH 2 O) water in a separate plastic container. For the hydroponic system, a Styrofoam tray with holes at the bottom was used to facilitate direct access of roots to the micronutrient suspension. Three seedlings in rockwools were placed equidistantly on the Styrofoam plate, which was then placed on the top of the plastic container containing micronutrient treatment (Figure 1). A 15.24 cm aquarium air stone with plastic tubes (0.3 mm diameter) and an air pump (Pentair Aquatic Eco-Systems Inc., Apopka, FL, USA) were used to aerate the solutions (Figure 1). The volume of plastic containers was kept at its initial (8 L) level by adding dH 2 O every two days. Seedlings were treated with different micronutrient treatments for 7 days (Table 1). The micronutrient treatments were either infected with FON or RKN or co-inoculated with both at the 8-day post-treatment (DPT). A 1 ml suspension containing 1 × 10 5 microconidia/mL was pipetted onto the rockwool cubes at the base of the watermelon seedling as part of the FON inoculation process. A 1 ml slurry containing 6000 active RKN J2s was pipetted similarly for nematode inoculation. A similar inoculation strategy was used and the inocula were applied concurrently for treatments that were inoculated with both FON and RKN.
Plants were maintained in nutrient solution for 23 days post-inoculation with a total experimental duration of 30 days (7 days pre-inoculation + 23 days post-inoculation) at 28 • C mean greenhouse temperature. The pH of the initial solutions was approximately 5.5 to 6 and did not change significantly during the experiment. The replicate of each micronutrient treatment was comprised of a plastic container with three seedlings each (Figure 1). Three replicates per treatment (a total of 9 seedlings per treatment) were used in an experiment and two independent experiments were conducted. A completely random design was used. The schematic depiction of the timeframe and treatments is shown in Figure 1.

Mineral Analysis
Samples of nutrient solution (20 mL) were collected from each container at 7-DPT and stored at 4 • C. Concentrations of Fe, Mn, and Zn (mg·L −1 ) were assessed using inductively coupled plasma atomic emission spectroscopy (ICP-AES) at the Waters Agricultural Laboratories (Camilla, GA, USA) for three replicates for each treatment.

Relative Expression of SA-and JA-Genes in Watermelon Seedlings Grown in Specific Micronutrient Solutions
In order to ascertain that the SA-and JA-genes are induced systemically away from the point of contact (roots), leaf samples instead of root samples were assayed for monitoring gene expression. Leaf samples were collected to determine the relative expression of SAand JA-genes in watermelon seedlings grown in various micronutrient treatments before inoculation. Using a pair of sterile scissors (for each cut), 7-DPT, leaf samples (n = 3 per replicate/treatment) were obtained by cutting the third or fourth leaf from the apex of the stem/vine from hydroponically grown watermelon seedling under various treatments. Samples were snap-frozen using liquid nitrogen and then moved to a −80 • C freezer at the UGA Tifton Campus laboratory until they were required for additional analysis. Using the manufacturer's instructions, total RNA was isolated from 100 mg of powdered leaf tissue after leaves were ground into a coarse powder under liquid nitrogen (RNeasy Plant Mini Kit, Qiagen, CA, USA) and NanoDropTM Lite (Thermo Scientific, Wilmington, DE, USA) was used to measure concentrations. The A260/A280 absorbance ratio, which was between 2.1 and 2.2 for all samples and indicated that the RNA was devoid of protein contamination, served as evidence of the RNA's high quality [86].
For later usage, total RNA samples were aliquoted and kept at −80 • C. The iScript cDNA synthesis kit (Bio-Rad Laboratories Inc., Hercules, CA, USA) was used to reversetranscribe 500 ng of total RNA to synthesize first-strand cDNA (Bio-Rad Laboratories Inc., Hercules, CA, USA). Utilizing SsoFast EvaGreen Supermix (Bio-Rad Laboratories Inc., Hercules, CA, USA), specific primer pairs for the marker genes, and a Smart Cycler System (Cepheid, Hercules, CA, USA), qRT-PCR was carried out ( Table 3). The primers and qRT-PCR parameters for PR1 and PR5 genes were the same as described earlier [66]. The list of genes, primer sequences, and qRT-PCR parameters used in this study are listed in Table 3. These genes were selected based on their involvement in plant defense response mechanisms [68][69][70][71][72][75][76][77][78][79]. For real-time PCR, the resulting first-strand cDNA was diluted (1:10) and the specific genes were amplified from the diluted cDNA (5 ng) using SsoFast EvaGreen Supermix (Bio-Rad Laboratories Inc.) and specific primers. The ß-Actin gene was used as an internal control. The forward primer, 5 -CCATGTATGTTGCCATCCAG-3 , and reverse primer, 3 -GGATAGCATGGGGTAGAGCA-5 were used for ß-Actin as described previously [67]. Plants in the Steiner treatment were considered as non-treated control. The 2 −∆∆CT method [87,88] was used to calculate the relative fold changes of the target genes. Relative gene expression was then compared for each treated (n = 48; 24 biological replicates and 2 technical replicates) and non-treated (n = 12; 6 biological replicates and 2 technical replicates) plants. The effects of micronutrient treatment and pathogen inoculation alone or in combination on the relative expression of SA-and JA-induction pathway genes in watermelon seedlings were investigated. Samples were harvested at 3 days post-inoculation (DPI) by cutting the third or fourth leaf from the apex of the stem with a pair of sterile scissors, yielding leaf samples (n = 3 per replicate/treatment) (equivalent to 11 DPT). Non-inoculated plants grown on the Steiner treatment were considered as the non-treated control. From each replicate, two technical replicates per sample were utilized for gene expression anal-ysis. Further procedures spanning from the extraction of RNA to the determination of relative gene expression were performed in the same manner as mentioned above.

FON Recovery and RKN Gall Rating
No visible symptoms of wilting in the plants were observed throughout the course of the experiment. In order to confirm the infection status, we used two approaches-(i) isolation of the pathogen from the inoculated and control samples, and (ii) PCR validation of infection in the inoculated and control samples. For the first approach, one plant from each container (n = 3 containers/treatment), either inoculated with FON alone or in combination with RKN and non-inoculated plants in the Steiner solution, was tested for the presence of FON at the conclusion of the experiment. Using a pair of sterile scissors, stem pieces were cut into 0.5-cm-long pieces from the base of the main stem of each plant. After being surface disinfested with 0.6% sodium hypochlorite for 1.5 min, the stem pieces were rinsed in sterile water, and then they were put on a semi-selective peptone pentachloronitrobenzene agar medium [89]. Following a 7-day incubation period at 25 • C, plates were examined under a microscope to identify fungal isolates based on morphological characteristics [90]. FON status was then validated using a PCR assay with the FON-specific primers Fon-1 and Fon-2 [91]. PCR confirmatory assays and morphological analysis were used to identify the percentage of plants that were infected with FON. The 10 cm of roots closest to the plant's base that were inoculated with RKN were taken for gall assessment. Roots were washed after the removal of the rockwool plugs, and the number of galls was visually assessed using a 0-5 galling index (GI) as follows: 0-0 galls, 1-2 galls, 3-10 galls, 11-30 galls, 31-100 galls, and >100 galls [92].

Statistical Analysis
To normalize the data distribution before analysis, data were log-transformed (log10 (x + 1)) wherever appropriate. Untransformed arithmetic means are presented. Data from two trials were combined after ensuring no significant interaction of treatment was present at p ≤ 0.05. Experimental data were statistically analyzed using one-way Analysis of Variance (ANOVA) tool, and the Tukey-Kramer test was used to compare treatment effects at p < 0.05 level in SAS 9.4®.

Conclusions
The findings in this study suggest a possible association between the availability of specific nutrients and the induction of SA-and JA-genes in watermelon against FON and RKN. Importantly, the induction of SA-and JA-genes in leaves away from the point of contact (root) demonstrate the systemic induction of these genes. This also indicates a po-tential crosstalk between pathways affected by intracellular nutrient concentrations, in-cluding micronutrient homeostasis, and pathways governed by IR against FON and RKN infection, which may directly affect IR responses in watermelon. A detailed study is es-sential to understand how nutrient concentrations in substrate or leaf tissue of watermelon affect the expression of IR genes, and also if FON and RKN induce the plant response by itself or in the micronutrient treatment-mediated manner.

Informed Consent Statement: Not applicable.
Data Availability Statement: Not applicable.