Elicitation of Sunflower Resistance to Meloidogyne incognita by Ascorbic and Glutamic Acids: Integrating Direct Nematicidal Activity and Defense Enzyme Induction

Elicitation of Sunflower Resistance to Meloidogyne incognita by Ascorbic and Glutamic Acids: Integrating Direct Nematicidal Activity and Defense Enzyme Induction | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Elicitation of Sunflower Resistance to Meloidogyne incognita by Ascorbic and Glutamic Acids: Integrating Direct Nematicidal Activity and Defense Enzyme Induction Abdulrahman S. Al-Hussein, Sherif M. El-Ganainy, Wael H. Elmenofy, and 3 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8464901/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 10 You are reading this latest preprint version Abstract Root-knot nematode ( Meloidogyne incognita ) causes a significant reduction in the production of sunflower ( Helianthus annuus ) globally. The paper assessed the nematicidal activity of ascorbic acid and glutamic acid and their ability to trigger systemic resistance in sunflower within controlled conditions. Laboratory evaluations proved that both compounds had a considerable effect on suppressing egg hatch and elevating juvenile mortality in a concentration-dependent manner, with ascorbic acid (2 mg/ml) showing the strongest effects. Experiments in greenhouses found that pre and post inoculation foliar application of these acids suppressed nematode root galling and population density, boosted shoot and root biomass and increased the activities of major defense enzymes, such as peroxidase, phenylalanine ammonia-lyase, superoxide dismutase, and ascorbate peroxidase. Pre-inoculation treatments were more efficient, which demonstrates the role of priming in the activation of defense. Our results indicate that ascorbic and glutamic acid have a combination of direct nematicidal activity with induction of host defense systems, which is promising as a safe means of managing nematode infestation in sunflower farming. Root-knot nematode Induce systematic resistance Foliar spray Antioxidant enzymes Nematicidal compounds Sunflower defense Figures Figure 1 1 Introduction Root-knot nematodes ( Meloidogyne spp.) are some of the most destructive plant-parasitic nematodes throughout the world and they result in significant economic losses in various crops (Onkendi et al., 2014 ). Such nematodes cause root galling, interfere with the uptake of water and nutrients, and eventually decrease the vigor and yield of plants (Habteweld et al., 2024 ). Meloidogyne incognita is especially perilous in the tropics and subtropics, resulting in negative economic effects on high-value crops that are under the protection of cultivation, which explains why its control is of high urgency to local producers (Prajapati et al., 2025 ). Control of the root-knot nematodes has been primarily based on the use of chemical nematicides. However, concerns about environmental pollution, health hazards, and restrictions on their usage have increased, particularly over the past years (Forghani and Hajihassani, 2020 ). These concerns have made it necessary to seek effective and environmentally friendly management approaches that will minimize or eliminate the use of synthetic nematicides (Maleita et al., 2023 ). Within these alternatives, plant-derived compounds and resistance-inducing agents have demonstrated high potential in reducing the negative impact of the environment, and at the same time have proven to be effective against nematodes (Barbosa et al., 2024 ). Due to its role in plant antioxidants and defense activation, ascorbic acid (vitamin C) has gained attention (Akram et al., 2017 ; Al-Sayed and Montasser, 1986 ). It plays an important role in the detoxification of reactive oxygen species produced during nematode infection, the regulation of the expression of defense-related genes, and results in an increase in systemic resistance (Singh et al., 2021 ). Ascorbate oxidation triggers other metabolic pathways, such as the phenylpropanoid cascade, which elevates the level of defense enzymes, including peroxidase and phenylalanine ammonia-lyase, which is vital in the defense of nematodes (Singh et al., 2020 ). In a similar manner, glutamic acid serves significant functions in the plant stress responses through its influence on antioxidant enzyme functions and also in the redox balance of the cell (Guo et al., 2017 ). It positively influences growth and photosynthesis during stress and triggers defense mechanisms that lead to a decrease in nematode penetration and reproduction (Hamza et al., 2013 ). Glutamic acid is also used as a precursor in the production of glutathione, a strong antioxidant that is utilized in cellular detoxification (Noctor et al., 2014 ). The use of ascorbic acid and glutamic acid in nematode management programs is a sustainable solution that tries to integrate the direct nematicidal effect of the product with the development of host resistance mechanisms (Abd-Elgawad, 2025 ). This double action is likely to decrease nematode populations and increase plant resistance, which makes the use of BCAs a safer and more effective approach for RKN management (Singh et al., 2021 ). Although there have been improvements in the management of nematodes, the control of M . incognita has been tough because of the limitations of chemical nematicides and the variability of plant resistance. This study is interesting as it deals with natural products (ascorbic and glutamic acids) for their nematicidal effect, in addition to their ability to induce the resistance enzymes under laboratory, greenhouse, and field assays on tomatoes. The aim is to determine the effectiveness of these compounds in suppressing nematode infection, egg hatching, and juvenile viability, and clarify the effect of these compounds in the stimulation of plant defensive enzymes. The research is the combination of in vitro egg hatch tests with biochemical testing of the treated plants, giving a holistic evaluation of dual modes of action of these compounds. This method will help in the sustainable and eco-friendly management of nematodes with possible implications for integrated pest control policies. 2 Materials and Methods 2.1 Propagation and Maintenance of Root-Knot Nematode ( M. incognita ) A pure culture of M . incognita was maintained on susceptible plants of sunflower ( Helianthus annuus cv. Sunspot) that were planted in sterile soils of sandy loam and under greenhouse conditions at 28–32°C. Standard methods were used to collect egg masses from the infected roots, according to the procedure by (Hartman and Sasser, 1985 ). The nematode inoculum was extracted by hatching freshly laid egg masses in aerated distilled water at room temperature and second-stage Juveniles (J2) were collected for use in the studies. 2.2 Laboratory Studies (Survival and Mortality of M. incognita J2) Bioassays involving the assessment of the impact of ascorbic acid and glutamic acid on M . incognita second-stage juveniles (J2) survival and mortality were conducted according to protocols developed by Neeraj et al. )2017) and related studies. Freshly hatched J2s were produced by incubating egg masses collected on infected sunflower roots by extracting them with sodium hypochlorite then hatching in aerated distilled water at 28°C. About 100 of the newly hatched J2s were released in multi-well plates with aqueous solutions of ascorbic acid or glutamic acid at specified concentrations (e.g., 1 mg/ml and 2 mg/ml). Four times were replicated in each treatment. The nematodes were subjected to the test solutions for different times, namely 3, 6, 9, and 12 days, under a dark environment at room temperature to avoid photo-degradation of the compounds. The microscopic observation was used to evaluate juvenile survival at each time point. Mortality was confirmed by probing the nematodes with a fine eyelash or needle, those who did not respond were considered dead. The egg masses and juvenile’s numbers were standardized to obtain the same level of inoculum. The percentages of egg hatching and juvenile mortality were obtained by dividing the number of juveniles hatched and the number of juveniles killed by the initial counts, respectively, and the percentages were used to give quantitative data on the nematicidal effects of the treatments. 2.3 Greenhouse Studies The seeds of sunflower (cv. Sunspot) were surface sterilized in 1% sodium hypochlorite solution for 5 minutes and rinsed in distilled water and then sown individually in plastic pots (15 cm diameter) filled with steam-sterilized loamy sand soil. Following three weeks of growth under controlled conditions, uniform seedlings were chosen and transplanted in separate pots. The study design was a randomized complete block design of four replications per treatment. Treatments involved foliar application of ascorbic acid or glutamic acid at two concentrations (1 mg/ml and 2mg/ml). The applications were done either a week before nematode inoculation (pre-inoculation) or a week after nematode inoculation (post-inoculation), to be able to evaluate the timing effects on the efficacy of the treatments. The control plants were inoculated and not treated. After the foliar application schedule, all plants were inoculated with 1000 freshly hatched second-stage juveniles (J2) of M. incognita per pot in order to cause infection. Plants were kept in a greenhouse under temperature control and day temperatures of between 28–32, relative humidity was maintained at a constant, and the plants were irrigated and fertilized regularly to provide maximum growth conditions. Forty-five days after inoculation, plants were harvested carefully and kept assessing the parameters of nematode infection, the measures of plant growth, and the biochemical tests. 2.4 Growth and Nematode Assessments After harvesting, plants were uprooted with utmost care and roots were washed to clean soil. The number of root galls was scored using a 0–10 scale. The reproduction of nematodes was measured by counting egg masses and the extraction of the nematodes from the soil and roots using modified Baermann funnels (Hooper et al., 1983 ). Shoot and root fresh weights were measured. These parameters were applied to measure the effects of the treatment on plant growth and the effects on nematode infestation. 2.5 Enzyme Activity Assays Samples of leaves and roots were collected immediately after harvest, frozen in liquid nitrogen, and stored at -80°C for enzyme assays. Activities of defense-related enzymes including peroxidase (POX), phenylalanine ammonia-lyase (PAL), superoxide dismutase (SOD), and ascorbate peroxidase (APX), were measured spectrophotometrically following protocols by Pandey et al. )2018) and Singh et al. )2021). Assays were conducted in triplicate, and enzyme activities were expressed per mg protein. 2.6 Statistical Analysis Data from egg hatch and juvenile mortality tests were analyzed using one-way analysis of variance (ANOVA) to compare the effects of different concentrations of ascorbic acid and glutamic acid treatments on M. incognita J2 survival. Assumptions of normality and homogeneity of variance were tested prior to ANOVA. Means were separated using the Least Significant Difference (LSD) test at p < 0.05. Data from greenhouse experiments were analyzed employing two-way ANOVA to assess the main influences and interaction effects of the following two factors: treatment concentration (1 mg/ml, 2 mg/ml) and time of application (pre-inoculation vs. post-inoculation). This approach allowed examination of how nematode galling, population densities and plant growth responses differ by these variables and their interaction. When significant effects were identified, LSD tests were used for multiple comparisons of means. Both analyses were performed using SPSS statistical software (version 16). Data transformations including logarithmic or square root, were applied as necessary to meet ANOVA assumptions. 3 Results 3.1 Effect of Ascorbic and Glutamic Acids on M. incognita Egg Hatch and J2 Mortality Table 1 presents the effects of ascorbic acid and glutamic acid on the egg hatch percentage and juvenile mortality of M. incognita after 12 days of exposure to the treatments. The control group had a high hatching rate of eggs, 92.3 ± 2.1%, which suggests healthy uninhibited juvenile emergence and only 6.0 ± 1.2% juvenile mortality. These values bring out the base reproductive capability and survival of the nematodes in the absence of chemical interference. Table 1 Effect of ascorbic and glutamic acids on M. incognita egg hatch and juvenile mortality (%) at 12 days post-treatment. Treatment Concentration (mg/ml) Egg Hatch (%) Mortality (%) Control 0 92.3 ± 2.1a 6.0 ± 1.2d Ascorbic Acid 1 59.6 ± 3.4b 38.9 ± 2.8c 2 29.5 ± 2.9c 62.1 ± 3.0a Glutamic Acid 1 65.4 ± 2.8b 34.4 ± 2.5c 2 42.7 ± 3.2c 49.5 ± 2.7b p-value < 0.001 < 0.001 Values are means ± SE; Different letters within columns indicate significant differences at p < 0.05 by LSD test. The exposure to ascorbic acid had a significant inhibiting effect on the hatching of eggs in a concentration-dependent manner. With 1mg/ml, the hatching of the eggs was low at 59.6 ± 3.4, which was significantly lower than the controls (p < 0.001). At 2 mg/ml, the hatching of the eggs was reduced to 29.5 ± 2.9%, which exhibited pronounced ovicidal activity. Likewise, Juvenile mortality also increased tremendously, i.e., 38.9 ± 2.8% at a concentration of 1 mg/ml and reached 62.1 ± 3.0% at a concentration of 2 mg/ml, depicting the direct larvicidal effects (p < 0.001). Inhibitory effects were also exhibited by glutamic acid, though with a moderate lower extent than ascorbic acid at equal concentrations. The hatching rate was reduced to 65.4 ± 2.8% and 42.7 ± 3.2% at 1 mg/ml and 2 mg/ml, respectively, which were significantly different from that for the control (p < 0.001). Mortality of Juveniles under glutamic acid was 34.4 + 2.5% at 1 mg/ml and 49.5 + 2.7% at 2 mg/ml, which were once again significantly high in comparison with the control mortality (p < 0.001). Finally, the results indicate that ascorbic acid at 2 mg/ml is the most effective in reducing egg hatch and increasing juvenile mortality of M. incognita , followed by glutamic acid, emphasizing their potential utility for nematode management. 3.2 Effect of Pre- and Post-Treatments with Ascorbic and Glutamic Acids on Nematode Infection Parameters Table 2 summarizes the efficacy of pre- and post-inoculation treatments with ascorbic acid and glutamic acid at concentrations of 1 mg/ml and 2 mg/ml in reducing root galling and nematode populations in sunflower plants 45 days after inoculation. The uninoculated control group presented a severe infestation with nematodes with a root gall index of 7.8 ± 0.3 and a population density of nematodes and juveniles comprising 2150 ± 120 eggs and juveniles per gram of root tissue, which indicates a high reproduction rate of nematodes and damage to roots. Table 2 Effect of pre- and post-treatments with ascorbic and glutamic acids on nematode infection parameters in sunflower infected with M. incognita . Treatment Application Time Concentration (mg/ml) Root Gall Index (0–10) Nematode Population (eggs + J2/g root) Control (Inoculated only) - 0 7.8 ± 0.3a 2150 ± 120a Ascorbic Acid Pre-Inoculation 1 4.5 ± 0.4c 1280 ± 90c 2 3.2 ± 0.3d 920 ± 85d Glutamic Acid 1 5.6 ± 0.4b 1450 ± 110b 2 3.9 ± 0.4d 1050 ± 100c Ascorbic Acid Post-Inoculation 1 6.5 ± 0.5a 1800 ± 110a 2 5.1 ± 0.4b 1550 ± 100b Glutamic Acid 1 6.7 ± 0.5a 1850 ± 120a 2 5.5 ± 0.3b 1600 ± 110b p-value < 0.001 < 0.001 Values are means ± SE; Different letters within columns indicate significant differences at p < 0.05 by LSD test. Pre-inoculation treatment with ascorbic acid significantly reduced nematode infection severity in a concentration-dependent manner. At 1 mg/ml, the gall index decreased by 42% to 4.5 ± 0.4, and the nematode population declined by 40% to 1280 ± 90. The higher concentration of 2 mg/ml provided better control, reducing galling by 59% (3.2 ± 0.3) and nematode numbers by 57% (920 ± 85). These reductions were statistically significant compared to the control (p < 0.001). Glutamic acid applied pre-inoculation also significantly suppressed nematode infection, albeit slightly less effectively than ascorbic acid. Gall indices were reduced to 5.6 ± 0.4 and 3.9 ± 0.4 at 1 and 2 mg/ml, representing decreases of 28% and 50%, respectively. Corresponding nematode populations were similarly reduced to 1450 ± 110 and 1050 ± 100 at these concentrations (p < 0.001). Post-inoculation treatments were less effective but still significantly lowered gall formation and nematode densities relative to the control. Ascorbic acid at 1 mg/ml post-inoculation reduced the gall index to 6.5 ± 0.5 and nematode population to 1800 ± 110, while 2 mg/ml reduced them further to 5.1 ± 0.4 and 1550 ± 100, respectively. Glutamic acid at the same concentrations resulted in gall indices of 6.7 ± 0.5 and 5.5 ± 0.3 and nematode populations of 1850 ± 120 and 1600 ± 110, respectively (p < 0.001). Finally, pre-inoculation application of ascorbic acid at 2 mg/ml was most effective in minimizing root galling and nematode populations, confirming its potential as a nematicidal agent promoting resistance against M. incognita in sunflower cultivation. 3.3 Effect of Pre- and Post-Treatments with Ascorbic and Glutamic Acids on Plant Growth Parameters of Sunflower Table 3 presents the influence of pre- and post-inoculation foliar applications of ascorbic acid and glutamic acid on sunflower shoot and root fresh weights 45 days post nematode inoculation. Inoculated control plants exhibited reduced growth with shoot fresh weight of 25.6 ± 1.2 g and root fresh weight of 12.1 ± 0.6 g and this reflects the detrimental effects of M. incognita infection. Table 3 Effect of pre- and post-treatments with ascorbic and glutamic acids on plant growth parameters of sunflower infected with M. incognita . Treatment Application Time Concentration (mg/ml) Shoot Fresh Weight (g) % Increase Shoot FW Root Fresh Weight (g) % Increase Root FW Control (Inoculated only) - - 25.6 ± 1.2c - 12.1 ± 0.6d - Ascorbic Acid Pre-Inoculation 1 29.2 ± 1.0b 14.06 14.3 ± 0.5c 18.18 2 31.0 ± 1.1a 21.09 15.1 ± 0.6b 24.79 Glutamic Acid 1 28.3 ± 1.3b 10.55 14.0 ± 0.7c 15.70 2 29.7 ± 1.1ab 16.02 14.9 ± 0.8b 23.14 Ascorbic Acid Post-Inoculation 1 27.0 ± 1.3c 5.47 13.0 ± 0.5cd 7.44 2 28.5 ± 1.4b 11.33 13.5 ± 0.6c 11.57 Glutamic Acid 1 26.8 ± 1.2c 4.69 13.2 ± 0.5c 9.09 2 27.5 ± 1.3bc 7.42 13.8 ± 0.4bc 14.05 p-value < 0.001 < 0.003 Values are means ± SE; Different letters within columns indicate significant differences at p < 0.05 by LSD test. Pre-inoculation treatment with ascorbic acid significantly enhanced plant growth in a dose-dependent manner. At 1 mg/ml, shoot fresh weight improved by 14.06% to 29.2 ± 1.0 g, and root fresh weight increased by 18.18% to 14.3 ± 0.5 g compared to controls (p < 0.001 and p = 0.003, respectively). Doubling the concentration to 2 mg/ml further boosted growth with a 21.09% increase in shoot weight (31.0 ± 1.1 g) and 24.79% increase in root weight (15.1 ± 0.6 g). Similarly, glutamic acid application pre-inoculation improved shoot and root growth significantly. Shoot fresh weight increased by 10.55% and 16.02% at 1 mg/ml and 2 mg/ml, respectively, while root fresh weight increased by 15.70% and 23.14% (p-values significant). The treatments of post-inoculation showed mild but still significant growth promotion influences. Ascorbic acid at 1 mg/ml increased shoot and root fresh weights by 5.47% and 7.44%, respectively. At 2 mg/ml, increases reached 11.33% (shoot) and 11.57% (root). Glutamic acid post-inoculation treatments yielded similar moderate improvements. Statistical analysis confirmed significant effects of treatment concentration on shoot and root growth (p < 0.001 and p = 0.003, respectively). Application timing also influenced growth responses, with pre-inoculation applications generally more effective, although all treatments demonstrated better growth performance than the inoculated control. 3.4 Effect of Pre- and Post-Treatments with Ascorbic and Glutamic Acids on Defense-Related Enzyme Activity in Sunflower Leaves Table 4 presents the activities of key defense enzymes peroxidase (POX), phenylalanine ammonia-lyase (PAL), superoxide dismutase (SOD), and ascorbate peroxidase (APX) in sunflower leaves following treatment with ascorbic and glutamic acids at two application timings relative to M. incognita inoculation. The inoculated control plants exhibited the lowest enzymatic activities, with POX activity at 14.5 ± 1.2 µmol min⁻¹ mg⁻¹, PAL at 8.6 ± 0.8 µmol cinnamic acid h⁻¹ mg⁻¹, SOD at 25.3 ± 1.5 units mg⁻¹ protein, and APX at 5.8 ± 0.5 µmol min⁻¹ mg⁻¹. Table 4 Activity of defense-related enzymes in sunflower leaves after pre- and post-treatments with ascorbic and glutamic acids (units/mg protein). Treatment Application Time POX (µmol min⁻¹ mg⁻¹) PAL (µmol cinnamic acid h⁻¹ mg⁻¹) SOD (units mg⁻¹ protein) APX (µmol min⁻¹ mg⁻¹) Control (Inoculated) - 14.5 ± 1.2d 8.6 ± 0.8d 25.3 ± 1.5d 5.8 ± 0.5d Ascorbic Acid Pre-Inoculation 33.5 ± 1.8a 19.8 ± 1.2a 52.4 ± 2.7a 15.3 ± 1.0a Post-Inoculation 28.0 ± 1.5b 16.2 ± 1.0b 44.3 ± 2.3b 12.0 ± 0.8b Glutamic Acid Pre-Inoculation 28.3 ± 2.0b 17.1 ± 1.1b 45.7 ± 2.3b 12.6 ± 1.1b Post-Inoculation 24.5 ± 1.7c 14.0 ± 0.9c 38.7 ± 1.8c 10.2 ± 0.7c p-value < 0.001 < 0.001 < 0.001 < 0.001 Values are means ± SE. Different letters within columns indicate significant differences at p < 0.05 (LSD test). Pre-inoculation treatment with ascorbic acid markedly enhanced enzyme activities, with POX activity reaching 33.5 ± 1.8, PAL at 19.8 ± 1.2, SOD at 52.4 ± 2.7, and APX at 15.3 ± 1.0. This is more than a twofold rise relative to levels of control (p < 0.001). The presence of such an induction shows a strong defense system activation of the plant prior to nematode challenge. The enzyme activities were also significantly elevated by post-inoculation applications of ascorbic acid relative to the control, although to a smaller degree as compared to pre-inoculation treatments (POX 28.0 ± 1.5, PAL 16.2 ± 1.0, SOD 44.3 ± 2.3, APX 12.0 ± 0.8). The treatments of glutamic acid similarly promoted defense enzyme activities. Pre-inoculation applications elevated POX, PAL, SOD, and APX activities to 28.3 ± 2.0, 17.1 ± 1.1, 45.7 ± 2.3, and 12.6 ± 1.1, respectively. Post-inoculation treatments produced moderate increases (POX 24.5 ± 1.7, PAL 14.0 ± 0.9, SOD 38.7 ± 1.8, APX 10.2 ± 0.7), all significantly above untreated controls (p < 0.001). The p-values of all the enzyme activities were highly significant, implying strong treatment effects. The increase in the activities of defense-related enzymes noted is associated with enhanced resistance mechanisms in sunflower, leading to the suppression of nematodes as well as a reduction in root damage. The highest and most potent induction of defensive enzymes was recorded following pre-inoculation foliar application of ascorbic acid that indicates its potential use as an effective biological elicitor in nematode management programs. 3.5 Percentage Increase in Activity of Defense-Related Enzymes in Sunflower Leaves Relative to Control Figure (1) illustrates the percentage increases in the activities of defense-related enzymes peroxidase (POX), phenylalanine ammonia-lyase (PAL), superoxide dismutase (SOD), and ascorbate peroxidase (APX) in sunflower leaves following foliar application of ascorbic acid and glutamic acid at different timings relative to M. incognita inoculation. The largest increases in enzyme activities were observed by pre-inoculation treatment with ascorbic acid, as POX activity increased by 131.0, PAL increased by 130.2, SOD increased by 107.5, and APX increased by 163.8 relative to untreated inoculated controls. This high induction indicates a high level of priming of the plant defense system, which allows the plant to be more capable of resisting nematode infection. The use of ascorbic acid after inoculation also increased enzyme activities to a significant extent of 93.1% (POX), 88.4% (PAL), 75.5% (SOD), and 106.9% (APX), but to a slightly lesser degree. These findings show that ascorbic acid is able to activate antioxidative defenses even when nematode infestation has begun. Glutamic treatments adhered to the same pattern, with pre-inoculation treatment showing increases of 95.2% (POX), 98.8% (PAL), 80.6% (SOD) and 117.2% (APX), and post-inoculation treatment showing increases of 69.0%, 62.8%, 53.0% and 75.9%, respectively. These results refer ascorbic and glutamic acids promising and green alternatives or supplements to traditional nematicides, which may be used as integrated management to enhance sunflower health and yield. The enhanced enzyme activities are attributed to the increased resistance responses and lesser nematode damage. 4 Discussion The current study demonstrates the effect of ascorbic acid and glutamic acid in inhibiting M. incognita egg hatching, juvenile mortality, reduction in the severity of nematode infection, and promoting host plant resistance in sunflower. The concentration-dependent inhibition of egg hatch and the high mortality of juvenile limnicoles is a direct effect of nematicidal action, likely associated with the disruption of key physiological processes in the nematode (Neeraj et al., 2017 ; Singh et al., 2021 ). Host-parasite interactions between M. incognita and sunflower roots involve complex molecular dialogues where nematodes deploy effectors to suppress plant immune responses and establish feeding sites known as giant cells (Goverse and Smant, 2014 ; Kaloshian and Teixeira, 2019 ; Zhou et al., 2023 ). The ability of ascorbic and glutamic acids to suppress the reproduction of nematodes and galling of roots implies that they interfere not only with the development phase of nematodes but also with the host immune level. Pre-inoculation spray application was better than post-inoculation application, which suggests that systemic defenses can be induced by priming the plant prior to nematode attack. This is consistent with the known paradigm of induced resistance; timely application of the elicitor triggers the activation of the immune system of the plant that results in complex signalling pathways that lead to upregulation of the phenylpropanoid pathway, an oxidative burst, and the orchestrated action of antioxidant enzymes, thus strengthening the plant against parasitic attack (Li et al., 2019 ; Mauch-Mani et al., 2017 ; Pieterse et al., 2014 ). Increased activities of defense-related enzymes, such as peroxidase (POX), phenylalanine ammonia-lyase (PAL), superoxide dismutase (SOD), and ascorbate peroxidase (APX) are biochemical predictors of an active defense state (Singh et al., 2021 ). These enzymes regulate the balance of reactive oxygen species (ROS) and the production of antimicrobial complexes, which are important in limiting the development and settlement of nematodes (Pandey et al., 2018 ). The significant rise in enzyme activities at pre-inoculation ascorbic acid treatment indicates strong elicitor activity, which is in line with earlier findings that vitamins could also serve as stress signals to promote pathogen resistance (Al-Sayed and Montasser, 1986 ). Mechanistically, nematodes sense and react to exudates of host roots with specialized sensory systems to locate sites of infection (Mendy et al., 2017 ). On the other hand, plants sense nematode invasion through pattern recognition receptors, which cause defensive responses, including hypersensitive response and cell wall strengthening processes, which could be enhanced by exogenous application of elicitors, such as ascorbic and glutamic acids (Zhou et al., 2023 ). Furthermore, ascorbic acid antioxidants may preserve host cells against oxidative stress caused by nematode infection, which preserves physiological processes critical to resistance (Singh et al., 2020 ). Co-evolutionary arms race between root-knot nematodes and their host plants involves structural, as well as biochemical adaptations based on the innate immune system of the plant and the arsenal of effector proteins of the nematode (Jones and Dangl, 2006 ; Mitchum et al., 2013 ). Recent research indicates that nematode effectors attack host defense to support the formation of feeding sites, and plants evolve enzymes and secondary metabolites to counter these effectors (Phani et al., 2021 ). Our results indicate that the foliar application of ascorbic and glutamic acids enhances the defensive biochemical armory system of the host plant, which leans towards resistance. 5 Conclusion This paper has effectively shown that foliar applications of ascorbic acid and glutamic acid can greatly reduce the Hatching of M. incognita eggs and the survival of juvenile M. incognita, which results in reduced root galling and decreased nematode populations in sunflower plants. Pre-inoculation treatment with ascorbic acid at 2 mg/ml was the most effective, showing that it is a strong nematicide and can make plants resistant to nematodes. The increase in the activity of enzymes related to defense, including peroxidase, phenylalanine ammonia-lyase, superoxide dismutase, and ascorbate peroxidase, after treatment shows induced systemic resistance as one of the mechanisms behind nematode suppression. These results refer ascorbic and glutamic acids promising and green alternatives or supplements to traditional nematicides, which may be used as integrated management to enhance sunflower health and yield. Further studies are required to optimize the application protocols and to understand the molecular signaling pathways that these compounds stimulate in order to utilize the potential of the compounds in nematode control to the fullest extent. Declarations Funding The authors want to express their gratitude to the Deanship of Scientific Research, Vice Presidency for Graduate Studies and Scientific Research, King Faisal University, Saudi Arabia, for funding this study under the terms of KFU254813. Author Contribution Author contributions Abdulrahman S. Al-Hussein : Methodology, Formal analysis, Data curation. Hosny H. Kesba : Conceptualization, Supervision, Formal analysis, Data curation, Writing – review & editing, Funding. Sherif M. El-Ganainy : Methodology, Formal analysis, Data curation. Wael H. Elmenofy : Supervision, Data curation, Methodology. Ahmed M. Ismail : Conceptualization, Methodology. Hossam S. El-Belatgi : Supervision, Data curation. All authors have reviewed and consented to the final version of the manuscript. Acknowledgement The authors are grateful to Deanship of Scientific Research, Vice Presidency for Graduate Studies and Scientific Research, King Faisal University, Saudi Arabia (KFU254813), for supporting this research work. Data Availability The tables in this research article show all the data that was collected. References Abd-Elgawad, M.M.M., 2025. 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Plant Sci. 11, 557202. https://doi.org/10.3389/fpls.2020.01125 Goverse, A., Smant, G., 2014. The activation and suppression of plant innate immunity by parasitic nematodes. Annu. Rev. Phytopathol. 52, 243–265. https://doi.org/10.1146/annurev-phyto-102313-050118 Guo, Z., Yang, N., Zhu, C., Gan, L., 2017. Exogenously applied poly-γ-glutamic acid alleviates salt stress in wheat seedlings by modulating ion balance and the antioxidant system. Environ. Sci. Pollut. Res. 24, 6592–6598. https://doi.org/10.1007/s11356-016-8295-4 Habteweld, A., Kantor, M., Kantor, C., Handoo, Z., 2024. Understanding the dynamic interactions of root-knot nematodes and their host: role of plant growth promoting bacteria and abiotic factors. Front. Plant Sci. 15, 1377453. https://doi.org/10.3389/fpls.2024.1377453 Hamza, A., Abd El-Kafie, O., Nour El-Deen, A., Abd El-Baset, M., 2013. Improving Carnation Resistance To Root-Knot Nematode Infection Under Greenhouse Conditions. J. Plant Prod. 4, 1159–1168. https://doi.org/10.21608/jpp.2013.73766 Hartman K. M., Sasser, J.N., 1985. Identification of Meloidogyne spesies on the Basis of Differential Host Test and Perineal-Pattern Morphology. An Advanced Treatise on Meloidogyne Volume II: Methodology., in: Barker, K.R., Carter, C.C., Sasser, J.N. (Eds.),. North Carolina State University Graphics, pp. 69–77. Hooper, D.J, Hallman, J, Subbotin, 1983. Methods for Extraction,Processing and Detection of Plant Soil Nematodes in Plant Parasitic Nematodes and Tropical Agriculture. CAB International, pp. 53–80. Jones, J.D.G., Dangl, J.L., 2006. The plant immune system. Nature 444, 323–329. https://doi.org/10.1038/nature05286 Kaloshian, I., Teixeira, M., 2019. Advances in plant_nematode interactions with emphasis on the notorious nematode genus Meloidogyne . Phytopathology 109, 1988–1996. https://doi.org/10.1094/PHYTO-05-19-0163-IA Li, N., Han, X., Feng, D., Yuan, D., Huang, L.J., 2019. Signaling crosstalk between salicylic acid and ethylene/Jasmonate in plant defense: Do we understand what they are whispering? Int. J. Mol. Sci. 20, 671. https://doi.org/10.3390/ijms20030671 Maleita, C., Esteves, I., Ciancio, A., Oka, Y., 2023. Editorial: Sustainable strategies for the management of phytoparasitic nematodes. Front. Plant Sci. 14, 1148261. https://doi.org/10.3389/fpls.2023.1148261 Mauch-Mani, B., Baccelli, I., Luna, E., Flors, V., 2017. Defense Priming: An Adaptive Part of Induced Resistance. Annu. Rev. Plant Biol. 68, 485–512. https://doi.org/10.1146/annurev-arplant-042916-041132 Mendy, B., Wang’ombe, M.W., Radakovic, Z.S., Holbein, J., Ilyas, M., Chopra, D., Holton, N., Zipfel, C., Grundler, F.M.W., Siddique, S., 2017. Arabidopsis leucine-rich repeat receptor–like kinase NILR1 is required for induction of innate immunity to parasitic nematodes. PLoS Pathog. 13, e1006284. https://doi.org/10.1371/journal.ppat.1006284 Mitchum, M.G., Hussey, R.S., Baum, T.J., Wang, X., Elling, A.A., Wubben, M., Davis, E.L., 2013. Nematode effector proteins: An emerging paradigm of parasitism. New Phytol. 199, 879–894. https://doi.org/10.1111/nph.12323 Neeraj, N., R. Goel, S., Kumar, A., Singh, G., K. Madan, V., 2017. Effect of Plant Extracts on Hatching and Mortality of Root-Knot Nematode, Meloidogyne incognita Larvae (in-vitro). Biosci. Biotechnol. Res. Asia 14, 467–471. https://doi.org/10.13005/bbra/2466 Noctor, G., Mhamdi, A., Foyer, C.H., 2014. The roles of reactive oxygen metabolism in drought: Not so cut and dried. Plant Physiol. 164, 1636–1648. https://doi.org/10.1104/pp.113.233478 Onkendi, E.M., Kariuki, G.M., Marais, M., Moleleki, L.N., 2014. The threat of root-knot nematodes ( Meloidogyne spp.) in Africa: A review. Plant Pathol. 63, 727–737. https://doi.org/10.1111/ppa.12202 Pandey, V., Tewari, A.K., Saxena, D., 2018. Activities of Defensive Antioxidant Enzymes and Biochemical Compounds Induced by Bioagents in Indian Mustard Against Alternaria Blight. Proc. Natl. Acad. Sci. India Sect. B - Biol. Sci. 88, 1507–1516. https://doi.org/10.1007/s40011-017-0888-2 Phani, V., Khan, M.R., Dutta, T.K., 2021. Plant-parasitic nematodes as a potential threat to protected agriculture: Current status and management options. Crop Prot. 144, 105573. https://doi.org/10.1016/j.cropro.2021.105573 Pieterse, C.M.J., Zamioudis, C., Berendsen, R.L., Weller, D.M., Van Wees, S.C.M., Bakker, P.A.H.M., 2014. Induced systemic resistance by beneficial microbes. Annu. Rev. Phytopathol. 52, 347–375. https://doi.org/10.1146/annurev-phyto-082712-102340 Prajapati, A.B., Thumar, R., Singh, T., Patel, M.Y., 2025. Estimation of avoidable yield loss caused in capsicum due to root-knot nematodes ( Meloidogyne spp.) in polyhouse. Int. J. Adv. Biochem. Res. 9, 09–11. https://doi.org/10.33545/26174693.2025.v9.i4a.4048 Singh, R.R., Nobleza, N., Demeestere, K., Kyndt, T., 2020. Ascorbate Oxidase Induces Systemic Resistance in Sugar Beet Against Cyst Nematode Heterodera schachtii. Front. Plant Sci. 11, 591715. https://doi.org/10.3389/fpls.2020.591715 Singh, R.R., Pajar, J.A., Audenaert, K., Kyndt, T., 2021. Induced Resistance by Ascorbate Oxidation Involves Potentiating of the Phenylpropanoid Pathway and Improved Rice Tolerance to Parasitic Nematodes. Front. Plant Sci. 12, 713870. https://doi.org/10.3389/fpls.2021.713870 Zhou, J. jing, Zhang, X. ping, Liu, R., Ling, J., Li, Y., Yang, Y. hong, Xie, B. yan, Zhao, J. long, Mao, Z. chuan, 2023. A Meloidogyne incognita effector Minc03329 suppresses plant immunity and promotes parasitism. J. Integr. Agric. 22, 799–811. https://doi.org/10.1016/j.jia.2022.08.117 Additional Declarations No competing interests reported. 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1","display":"","copyAsset":false,"role":"figure","size":70264,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003ePercentage Increase in Activity of Defense-Related Enzymes in Sunflower Leaves Relative to Control.\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"floatimage1.png","url":"https://assets-eu.researchsquare.com/files/rs-8464901/v1/0f63ea4a7d0b09443bcb9a0c.png"},{"id":101298888,"identity":"d03dc797-0e2c-4df1-8c92-a1c4538c6110","added_by":"auto","created_at":"2026-01-28 09:37:07","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1135029,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8464901/v1/dc966d81-2646-494a-a26f-57c50e98d297.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Elicitation of Sunflower Resistance to Meloidogyne incognita by Ascorbic and Glutamic Acids: Integrating Direct Nematicidal Activity and Defense Enzyme Induction","fulltext":[{"header":"1 Introduction","content":"\u003cp\u003eRoot-knot nematodes (\u003cem\u003eMeloidogyne\u003c/em\u003e spp.) are some of the most destructive plant-parasitic\u0026ensp;nematodes throughout the world and they result in significant economic losses in various crops (Onkendi et al., \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2014\u003c/span\u003e). Such nematodes cause root galling, interfere with the uptake of water and nutrients, and eventually decrease the vigor and yield of plants (Habteweld et al., \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). \u003cem\u003eMeloidogyne incognita\u003c/em\u003e is especially perilous in the tropics and subtropics, resulting in negative economic effects on high-value crops that are under the protection of cultivation, which explains why its control is of high urgency to local producers (Prajapati et al., \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2025\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eControl of\u0026ensp;the root-knot nematodes has been primarily based on the use of chemical nematicides. However, concerns about environmental pollution, health hazards, and restrictions on their usage have increased, particularly over the past years (Forghani and Hajihassani, \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). These concerns have made it necessary to seek effective and environmentally friendly management approaches that will minimize or eliminate the use of synthetic nematicides (Maleita et al., \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). Within these alternatives, plant-derived compounds and resistance-inducing agents have demonstrated high potential in reducing the negative impact of the environment, and at the same time have proven to be effective against nematodes (Barbosa et al., \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2024\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eDue to its role in plant antioxidants and defense activation, ascorbic acid (vitamin C) has gained attention (Akram et al., \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2017\u003c/span\u003e; Al-Sayed and Montasser, \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e1986\u003c/span\u003e). It plays an important role in the detoxification of reactive oxygen species produced during nematode infection, the regulation of the expression of defense-related genes, and results in an increase in systemic resistance (Singh et al., \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). Ascorbate oxidation triggers other metabolic pathways, such as the phenylpropanoid cascade, which elevates the level of defense enzymes, including peroxidase and phenylalanine ammonia-lyase, which is vital in the defense of nematodes (Singh et al., \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2020\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eIn a similar manner, glutamic acid serves significant functions in the plant stress responses through its influence on antioxidant enzyme functions and also in the redox balance of the cell (Guo et al., \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). It positively influences growth and photosynthesis during stress and triggers defense mechanisms that lead to a decrease in nematode penetration and reproduction (Hamza et al., \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2013\u003c/span\u003e). Glutamic acid is also used as a precursor in the production of glutathione, a strong antioxidant that is utilized in cellular detoxification (Noctor et al., \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2014\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eThe use of ascorbic acid and glutamic acid in nematode management programs is a sustainable solution that tries to integrate the direct nematicidal effect of the product with the development of host resistance mechanisms (Abd-Elgawad, \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2025\u003c/span\u003e). This double action is likely to decrease nematode populations and increase plant resistance, which makes the use of BCAs a safer and more effective approach for\u0026ensp;RKN management (Singh et al., \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2021\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eAlthough there have been improvements in the management of nematodes, the control of \u003cem\u003eM\u003c/em\u003e. \u003cem\u003eincognita\u003c/em\u003e has been tough because of the limitations of chemical nematicides and the variability of plant resistance. This study is interesting as it deals with natural products (ascorbic and glutamic acids) for\u0026ensp;their nematicidal effect, in addition to their ability to induce the resistance enzymes under laboratory, greenhouse, and field assays on tomatoes. The aim is to determine the effectiveness of these compounds in suppressing nematode infection, egg hatching, and juvenile viability, and clarify the effect of these compounds in the stimulation of plant defensive enzymes.\u003c/p\u003e \u003cp\u003eThe research is the combination of in vitro egg hatch tests with biochemical testing of the treated plants, giving a holistic evaluation of dual modes of action of these compounds. This method will help in the sustainable and eco-friendly management of nematodes with possible implications for integrated\u0026ensp;pest control policies.\u003c/p\u003e"},{"header":"2 Materials and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003e2.1 Propagation and Maintenance of Root-Knot Nematode (\u003cem\u003eM. incognita\u003c/em\u003e)\u003c/h2\u003e \u003cp\u003eA pure culture of \u003cem\u003eM\u003c/em\u003e. \u003cem\u003eincognita\u003c/em\u003e was maintained on susceptible plants of sunflower (\u003cem\u003eHelianthus annuus\u003c/em\u003e cv. Sunspot) that were planted in sterile soils of sandy loam and under greenhouse conditions at 28\u0026ndash;32\u0026deg;C. Standard methods were used to collect egg masses from the infected roots, according to the procedure by (Hartman and Sasser, \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e1985\u003c/span\u003e). The nematode inoculum was extracted by hatching freshly laid egg masses in aerated distilled water at room temperature and\u0026ensp;second-stage Juveniles (J2) were collected for use in the studies.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003e2.2 Laboratory Studies (Survival and Mortality of \u003cem\u003eM. incognita\u003c/em\u003e J2)\u003c/h2\u003e \u003cp\u003eBioassays involving the assessment of the impact of ascorbic acid and glutamic acid on \u003cem\u003eM\u003c/em\u003e. \u003cem\u003eincognita\u003c/em\u003e second-stage juveniles (J2) survival and mortality were conducted according to protocols developed by Neeraj et al. )2017) and related studies. Freshly hatched J2s were produced by incubating egg masses collected on infected sunflower roots by extracting them with sodium hypochlorite then hatching in aerated distilled water at 28\u0026deg;C. About 100 of the newly hatched J2s were released in multi-well plates with aqueous solutions of ascorbic acid or glutamic acid at specified concentrations (e.g., 1 mg/ml and 2 mg/ml). Four times were replicated in each treatment.\u003c/p\u003e \u003cp\u003eThe nematodes were subjected to the test solutions for different times, namely 3, 6, 9, and 12 days, under a dark environment at room temperature to avoid photo-degradation of the compounds. The microscopic observation was used to evaluate juvenile survival at each time point. Mortality was confirmed by probing the nematodes with a fine eyelash or needle, those who did not respond were considered dead. The egg masses and juvenile\u0026rsquo;s numbers were standardized to obtain the same level of inoculum.\u003c/p\u003e \u003cp\u003eThe percentages of egg hatching and juvenile mortality were obtained by dividing the number of juveniles hatched and the number of juveniles killed by the initial counts, respectively, and the percentages were used to give quantitative data on the nematicidal effects of the treatments.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003e2.3 Greenhouse Studies\u003c/h2\u003e \u003cp\u003eThe seeds of sunflower (cv. Sunspot) were surface sterilized in 1% sodium hypochlorite solution for 5 minutes and rinsed in distilled water and then sown individually in plastic pots (15 cm diameter) filled with steam-sterilized loamy sand soil. Following three weeks of growth under controlled conditions, uniform seedlings were chosen and transplanted in separate pots.\u003c/p\u003e \u003cp\u003eThe study design was a randomized complete block design of four replications per treatment. Treatments involved foliar application of ascorbic acid or glutamic acid at two concentrations (1 mg/ml and 2mg/ml). The applications were done either a week before nematode inoculation (pre-inoculation) or a week after nematode inoculation (post-inoculation), to be able to evaluate the timing effects on the efficacy of the treatments. The control plants were inoculated and not treated.\u003c/p\u003e \u003cp\u003eAfter the foliar application schedule, all plants were inoculated with 1000 freshly hatched second-stage juveniles (J2) of \u003cem\u003eM. incognita\u003c/em\u003e per pot in order to cause infection. Plants were kept in a greenhouse under temperature control and day temperatures of between 28\u0026ndash;32, relative humidity was maintained at a constant, and the plants were irrigated and fertilized regularly to provide maximum growth conditions.\u003c/p\u003e \u003cp\u003eForty-five days after inoculation, plants were harvested carefully and kept assessing the parameters of nematode infection, the measures of plant growth, and the biochemical tests.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003e2.4 Growth and Nematode Assessments\u003c/h2\u003e \u003cp\u003eAfter harvesting, plants were uprooted with utmost care and roots were washed to clean soil. The number of root galls was scored using a 0\u0026ndash;10 scale. The reproduction of nematodes was measured by counting egg masses and the extraction of the nematodes from the soil and roots using modified Baermann funnels (Hooper et al., \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e1983\u003c/span\u003e). Shoot and root fresh weights were measured. These parameters were applied to measure the effects of the treatment on plant growth and the effects on nematode infestation.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003e2.5 Enzyme Activity Assays\u003c/h2\u003e \u003cp\u003eSamples of leaves and roots were collected immediately after harvest, frozen in liquid nitrogen, and stored at -80\u0026deg;C for enzyme assays. Activities of defense-related enzymes including peroxidase (POX), phenylalanine ammonia-lyase (PAL), superoxide dismutase (SOD), and ascorbate peroxidase (APX), were measured spectrophotometrically following protocols by Pandey et al. )2018) and Singh et al. )2021). Assays were conducted in triplicate, and enzyme activities were expressed per mg protein.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003e2.6 Statistical Analysis\u003c/h2\u003e \u003cp\u003eData from egg hatch and juvenile mortality tests were analyzed using one-way analysis of variance (ANOVA) to compare the effects of different concentrations of ascorbic acid and glutamic acid treatments on \u003cem\u003eM. incognita\u003c/em\u003e J2 survival. Assumptions of normality and homogeneity of variance were tested prior to ANOVA. Means were separated using the Least Significant Difference (LSD) test at p\u0026thinsp;\u0026lt;\u0026thinsp;0.05.\u003c/p\u003e \u003cp\u003eData from greenhouse experiments were analyzed employing two-way ANOVA to assess the main influences and interaction effects of the following two factors: treatment concentration (1 mg/ml, 2 mg/ml) and time of application (pre-inoculation vs. post-inoculation). This approach allowed examination of how nematode galling, population densities and plant growth responses differ by these variables and their interaction. When significant effects were identified, LSD tests were used for multiple comparisons of means.\u003c/p\u003e \u003cp\u003eBoth analyses were performed using SPSS statistical software (version 16). Data transformations including logarithmic or square root, were applied as necessary to meet ANOVA assumptions.\u003c/p\u003e \u003c/div\u003e"},{"header":"3 Results","content":"\u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003e3.1 Effect of Ascorbic and Glutamic Acids on \u003cem\u003eM. incognita\u003c/em\u003e Egg Hatch and J2 Mortality\u003c/h2\u003e \u003cp\u003eTable\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e presents the effects of ascorbic acid and glutamic acid on the egg hatch percentage and juvenile mortality of \u003cem\u003eM. incognita\u003c/em\u003e after 12 days of exposure to the treatments. The control group had a high hatching rate of eggs, 92.3\u0026thinsp;\u0026plusmn;\u0026thinsp;2.1%, which suggests healthy uninhibited juvenile emergence and only 6.0\u0026thinsp;\u0026plusmn;\u0026thinsp;1.2% juvenile mortality. These values bring out the base reproductive capability and survival of the nematodes in the absence of chemical interference.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eEffect of ascorbic and glutamic acids on \u003cem\u003eM. incognita\u003c/em\u003e egg hatch and juvenile mortality (%) at 12 days post-treatment.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"4\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eTreatment\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eConcentration (mg/ml)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eEgg Hatch (%)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eMortality (%)\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eControl\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e92.3\u0026thinsp;\u0026plusmn;\u0026thinsp;2.1a\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e6.0\u0026thinsp;\u0026plusmn;\u0026thinsp;1.2d\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eAscorbic Acid\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e59.6\u0026thinsp;\u0026plusmn;\u0026thinsp;3.4b\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e38.9\u0026thinsp;\u0026plusmn;\u0026thinsp;2.8c\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e29.5\u0026thinsp;\u0026plusmn;\u0026thinsp;2.9c\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e62.1\u0026thinsp;\u0026plusmn;\u0026thinsp;3.0a\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eGlutamic Acid\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e65.4\u0026thinsp;\u0026plusmn;\u0026thinsp;2.8b\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e34.4\u0026thinsp;\u0026plusmn;\u0026thinsp;2.5c\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e42.7\u0026thinsp;\u0026plusmn;\u0026thinsp;3.2c\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e49.5\u0026thinsp;\u0026plusmn;\u0026thinsp;2.7b\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ep-value\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u0026lt;\u0026thinsp;0.001\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u0026lt;\u0026thinsp;0.001\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003eValues are means\u0026thinsp;\u0026plusmn;\u0026thinsp;SE; Different letters within columns indicate significant differences at p\u0026thinsp;\u0026lt;\u0026thinsp;0.05 by LSD test.\u003c/p\u003e \u003cp\u003eThe exposure to ascorbic acid had a significant inhibiting effect on the hatching of eggs in a concentration-dependent manner. With 1mg/ml, the hatching of the eggs was low at 59.6\u0026thinsp;\u0026plusmn;\u0026thinsp;3.4, which was significantly lower than the controls (p\u0026thinsp;\u0026lt;\u0026thinsp;0.001). At 2 mg/ml, the hatching of the eggs was reduced to 29.5\u0026thinsp;\u0026plusmn;\u0026thinsp;2.9%, which exhibited pronounced ovicidal activity. Likewise, Juvenile mortality also increased\u0026ensp;tremendously, i.e., 38.9\u0026thinsp;\u0026plusmn;\u0026thinsp;2.8% at a concentration of 1 mg/ml and reached 62.1\u0026thinsp;\u0026plusmn;\u0026thinsp;3.0% at a concentration of 2 mg/ml, depicting the direct larvicidal effects (p\u0026thinsp;\u0026lt;\u0026thinsp;0.001).\u003c/p\u003e \u003cp\u003eInhibitory effects were also exhibited by glutamic acid, though with a moderate lower extent than ascorbic acid at equal concentrations. The hatching rate was reduced to 65.4\u0026thinsp;\u0026plusmn;\u0026thinsp;2.8% and 42.7\u0026thinsp;\u0026plusmn;\u0026thinsp;3.2% at 1 mg/ml and 2 mg/ml, respectively, which were significantly different from that for the control (p\u0026thinsp;\u0026lt;\u0026thinsp;0.001). Mortality of Juveniles under glutamic acid was 34.4\u0026thinsp;+\u0026thinsp;2.5% at 1 mg/ml and 49.5\u0026thinsp;+\u0026thinsp;2.7% at 2 mg/ml, which were once again significantly high in comparison with the control mortality (p\u0026thinsp;\u0026lt;\u0026thinsp;0.001).\u003c/p\u003e \u003cp\u003eFinally, the results indicate that ascorbic acid at 2 mg/ml is the most effective in reducing egg hatch and increasing juvenile mortality of \u003cem\u003eM. incognita\u003c/em\u003e, followed by glutamic acid, emphasizing their potential utility for nematode management.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003e3.2 Effect of Pre- and Post-Treatments with Ascorbic and Glutamic Acids on Nematode Infection Parameters\u003c/h2\u003e \u003cp\u003eTable\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e summarizes the efficacy of pre- and post-inoculation treatments with ascorbic acid and glutamic acid at concentrations of 1 mg/ml and 2 mg/ml in reducing root galling and nematode populations in sunflower plants 45 days after inoculation. The uninoculated control group presented a severe infestation with nematodes with a root gall index of 7.8\u0026thinsp;\u0026plusmn;\u0026thinsp;0.3 and a population density of nematodes and juveniles comprising 2150\u0026thinsp;\u0026plusmn;\u0026thinsp;120 eggs and juveniles per gram of root tissue, which indicates a high reproduction rate of nematodes and damage to roots.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eEffect of pre- and post-treatments with ascorbic and glutamic acids on nematode infection parameters in sunflower infected with \u003cem\u003eM. incognita\u003c/em\u003e.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"5\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eTreatment\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eApplication Time\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eConcentration (mg/ml)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eRoot Gall Index (0\u0026ndash;10)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eNematode Population\u003c/p\u003e \u003cp\u003e(eggs\u0026thinsp;+\u0026thinsp;J2/g root)\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eControl\u003c/p\u003e \u003cp\u003e(Inoculated only)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e7.8\u0026thinsp;\u0026plusmn;\u0026thinsp;0.3a\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e2150\u0026thinsp;\u0026plusmn;\u0026thinsp;120a\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eAscorbic Acid\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\" morerows=\"3\" rowspan=\"4\"\u003e \u003cp\u003ePre-Inoculation\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e4.5\u0026thinsp;\u0026plusmn;\u0026thinsp;0.4c\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e1280\u0026thinsp;\u0026plusmn;\u0026thinsp;90c\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e3.2\u0026thinsp;\u0026plusmn;\u0026thinsp;0.3d\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e920\u0026thinsp;\u0026plusmn;\u0026thinsp;85d\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eGlutamic Acid\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e5.6\u0026thinsp;\u0026plusmn;\u0026thinsp;0.4b\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e1450\u0026thinsp;\u0026plusmn;\u0026thinsp;110b\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e3.9\u0026thinsp;\u0026plusmn;\u0026thinsp;0.4d\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e1050\u0026thinsp;\u0026plusmn;\u0026thinsp;100c\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eAscorbic Acid\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\" morerows=\"3\" rowspan=\"4\"\u003e \u003cp\u003ePost-Inoculation\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e6.5\u0026thinsp;\u0026plusmn;\u0026thinsp;0.5a\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e1800\u0026thinsp;\u0026plusmn;\u0026thinsp;110a\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e5.1\u0026thinsp;\u0026plusmn;\u0026thinsp;0.4b\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e1550\u0026thinsp;\u0026plusmn;\u0026thinsp;100b\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eGlutamic Acid\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e6.7\u0026thinsp;\u0026plusmn;\u0026thinsp;0.5a\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e1850\u0026thinsp;\u0026plusmn;\u0026thinsp;120a\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e5.5\u0026thinsp;\u0026plusmn;\u0026thinsp;0.3b\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e1600\u0026thinsp;\u0026plusmn;\u0026thinsp;110b\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ep-value\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u0026lt;\u0026thinsp;0.001\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u0026lt;\u0026thinsp;0.001\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003eValues are means\u0026thinsp;\u0026plusmn;\u0026thinsp;SE; Different letters within columns indicate significant differences at p\u0026thinsp;\u0026lt;\u0026thinsp;0.05 by LSD test.\u003c/p\u003e \u003cp\u003ePre-inoculation treatment with ascorbic acid significantly reduced nematode infection severity in a concentration-dependent manner. At 1 mg/ml, the gall index decreased by 42% to 4.5\u0026thinsp;\u0026plusmn;\u0026thinsp;0.4, and the nematode population declined by 40% to 1280\u0026thinsp;\u0026plusmn;\u0026thinsp;90. The higher concentration of 2 mg/ml provided better control, reducing galling by 59% (3.2\u0026thinsp;\u0026plusmn;\u0026thinsp;0.3) and nematode numbers by 57% (920\u0026thinsp;\u0026plusmn;\u0026thinsp;85). These reductions were statistically significant compared to the control (p\u0026thinsp;\u0026lt;\u0026thinsp;0.001). Glutamic acid applied pre-inoculation also significantly suppressed nematode infection, albeit slightly less effectively than ascorbic acid. Gall indices were reduced to 5.6\u0026thinsp;\u0026plusmn;\u0026thinsp;0.4 and 3.9\u0026thinsp;\u0026plusmn;\u0026thinsp;0.4 at 1 and 2 mg/ml, representing decreases of 28% and 50%, respectively. Corresponding nematode populations were similarly reduced to 1450\u0026thinsp;\u0026plusmn;\u0026thinsp;110 and 1050\u0026thinsp;\u0026plusmn;\u0026thinsp;100 at these concentrations (p\u0026thinsp;\u0026lt;\u0026thinsp;0.001).\u003c/p\u003e \u003cp\u003ePost-inoculation treatments were less effective but still significantly lowered gall formation and nematode densities relative to the control. Ascorbic acid at 1 mg/ml post-inoculation reduced the gall index to 6.5\u0026thinsp;\u0026plusmn;\u0026thinsp;0.5 and nematode population to 1800\u0026thinsp;\u0026plusmn;\u0026thinsp;110, while 2 mg/ml reduced them further to 5.1\u0026thinsp;\u0026plusmn;\u0026thinsp;0.4 and 1550\u0026thinsp;\u0026plusmn;\u0026thinsp;100, respectively. Glutamic acid at the same concentrations resulted in gall indices of 6.7\u0026thinsp;\u0026plusmn;\u0026thinsp;0.5 and 5.5\u0026thinsp;\u0026plusmn;\u0026thinsp;0.3 and nematode populations of 1850\u0026thinsp;\u0026plusmn;\u0026thinsp;120 and 1600\u0026thinsp;\u0026plusmn;\u0026thinsp;110, respectively (p\u0026thinsp;\u0026lt;\u0026thinsp;0.001).\u003c/p\u003e \u003cp\u003eFinally, pre-inoculation application of ascorbic acid at 2 mg/ml was most effective in minimizing root galling and nematode populations, confirming its potential as a nematicidal agent promoting resistance against \u003cem\u003eM. incognita\u003c/em\u003e in sunflower cultivation.\u003c/p\u003e \u003cp\u003e \u003cb\u003e3.3 Effect of Pre- and Post-Treatments with Ascorbic and Glutamic Acids on Plant Growth Parameters of Sunflower\u003c/b\u003e \u003c/p\u003e \u003cp\u003eTable\u0026nbsp;\u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e3\u003c/span\u003e presents the influence of pre- and post-inoculation foliar applications of ascorbic acid and glutamic acid on sunflower shoot and root fresh weights 45 days post nematode inoculation. Inoculated control plants exhibited reduced growth with shoot fresh weight of 25.6\u0026thinsp;\u0026plusmn;\u0026thinsp;1.2 g and root fresh weight of 12.1\u0026thinsp;\u0026plusmn;\u0026thinsp;0.6 g and this reflects the detrimental effects of \u003cem\u003eM. incognita\u003c/em\u003e infection.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab3\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 3\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eEffect of pre- and post-treatments with ascorbic and glutamic acids on plant growth parameters of sunflower infected with \u003cem\u003eM. incognita\u003c/em\u003e.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"7\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eTreatment\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eApplication Time\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eConcentration (mg/ml)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eShoot Fresh Weight (g)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003e% Increase Shoot FW\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003eRoot Fresh Weight (g)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c7\"\u003e \u003cp\u003e% Increase Root FW\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eControl (Inoculated only)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e25.6\u0026thinsp;\u0026plusmn;\u0026thinsp;1.2c\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e12.1\u0026thinsp;\u0026plusmn;\u0026thinsp;0.6d\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eAscorbic Acid\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\" morerows=\"3\" rowspan=\"4\"\u003e \u003cp\u003ePre-Inoculation\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e29.2\u0026thinsp;\u0026plusmn;\u0026thinsp;1.0b\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e14.06\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e14.3\u0026thinsp;\u0026plusmn;\u0026thinsp;0.5c\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e18.18\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e31.0\u0026thinsp;\u0026plusmn;\u0026thinsp;1.1a\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e21.09\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e15.1\u0026thinsp;\u0026plusmn;\u0026thinsp;0.6b\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e24.79\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eGlutamic Acid\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e28.3\u0026thinsp;\u0026plusmn;\u0026thinsp;1.3b\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e10.55\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e14.0\u0026thinsp;\u0026plusmn;\u0026thinsp;0.7c\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e15.70\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e29.7\u0026thinsp;\u0026plusmn;\u0026thinsp;1.1ab\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e16.02\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e14.9\u0026thinsp;\u0026plusmn;\u0026thinsp;0.8b\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e23.14\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eAscorbic Acid\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\" morerows=\"3\" rowspan=\"4\"\u003e \u003cp\u003ePost-Inoculation\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e27.0\u0026thinsp;\u0026plusmn;\u0026thinsp;1.3c\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e5.47\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e13.0\u0026thinsp;\u0026plusmn;\u0026thinsp;0.5cd\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e7.44\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e28.5\u0026thinsp;\u0026plusmn;\u0026thinsp;1.4b\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e11.33\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e13.5\u0026thinsp;\u0026plusmn;\u0026thinsp;0.6c\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e11.57\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eGlutamic Acid\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e26.8\u0026thinsp;\u0026plusmn;\u0026thinsp;1.2c\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e4.69\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e13.2\u0026thinsp;\u0026plusmn;\u0026thinsp;0.5c\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e9.09\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e27.5\u0026thinsp;\u0026plusmn;\u0026thinsp;1.3bc\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e7.42\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e13.8\u0026thinsp;\u0026plusmn;\u0026thinsp;0.4bc\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e14.05\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ep-value\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u0026lt;\u0026thinsp;0.001\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e\u0026lt;\u0026thinsp;0.003\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003eValues are means\u0026thinsp;\u0026plusmn;\u0026thinsp;SE; Different letters within columns indicate significant differences at p\u0026thinsp;\u0026lt;\u0026thinsp;0.05 by LSD test.\u003c/p\u003e \u003cp\u003ePre-inoculation treatment with ascorbic acid significantly enhanced plant growth in a dose-dependent manner. At 1 mg/ml, shoot fresh weight improved by 14.06% to 29.2\u0026thinsp;\u0026plusmn;\u0026thinsp;1.0 g, and root fresh weight increased by 18.18% to 14.3\u0026thinsp;\u0026plusmn;\u0026thinsp;0.5 g compared to controls (p\u0026thinsp;\u0026lt;\u0026thinsp;0.001 and p\u0026thinsp;=\u0026thinsp;0.003, respectively). Doubling the concentration to 2 mg/ml further boosted growth with a 21.09% increase in shoot weight (31.0\u0026thinsp;\u0026plusmn;\u0026thinsp;1.1 g) and 24.79% increase in root weight (15.1\u0026thinsp;\u0026plusmn;\u0026thinsp;0.6 g). Similarly, glutamic acid application pre-inoculation improved shoot and root growth significantly. Shoot fresh weight increased by 10.55% and 16.02% at 1 mg/ml and 2 mg/ml, respectively, while root fresh weight increased by 15.70% and 23.14% (p-values significant).\u003c/p\u003e \u003cp\u003eThe treatments of post-inoculation showed mild but still significant growth promotion influences. Ascorbic acid at 1 mg/ml increased shoot and root fresh weights by 5.47% and 7.44%, respectively. At 2 mg/ml, increases reached 11.33% (shoot) and 11.57% (root). Glutamic acid post-inoculation treatments yielded similar moderate improvements.\u003c/p\u003e \u003cp\u003eStatistical analysis confirmed significant effects of treatment concentration on shoot and root growth (p\u0026thinsp;\u0026lt;\u0026thinsp;0.001 and p\u0026thinsp;=\u0026thinsp;0.003, respectively). Application timing also influenced growth responses, with pre-inoculation applications generally more effective, although all treatments demonstrated better growth performance than the inoculated control.\u003c/p\u003e \u003cp\u003e \u003cb\u003e3.4 Effect of Pre- and Post-Treatments with Ascorbic and Glutamic Acids on Defense-Related Enzyme Activity in Sunflower Leaves\u003c/b\u003e \u003c/p\u003e \u003cp\u003eTable\u0026nbsp;\u003cspan refid=\"Tab4\" class=\"InternalRef\"\u003e4\u003c/span\u003e presents the activities of key defense enzymes peroxidase (POX), phenylalanine ammonia-lyase (PAL), superoxide dismutase (SOD), and ascorbate peroxidase (APX) in sunflower leaves following treatment with ascorbic and glutamic acids at two application timings relative to \u003cem\u003eM. incognita\u003c/em\u003e inoculation. The inoculated control plants exhibited the lowest enzymatic activities, with POX activity at 14.5\u0026thinsp;\u0026plusmn;\u0026thinsp;1.2 \u0026micro;mol min⁻\u0026sup1; mg⁻\u0026sup1;, PAL at 8.6\u0026thinsp;\u0026plusmn;\u0026thinsp;0.8 \u0026micro;mol cinnamic acid h⁻\u0026sup1; mg⁻\u0026sup1;, SOD at 25.3\u0026thinsp;\u0026plusmn;\u0026thinsp;1.5 units mg⁻\u0026sup1; protein, and APX at 5.8\u0026thinsp;\u0026plusmn;\u0026thinsp;0.5 \u0026micro;mol min⁻\u0026sup1; mg⁻\u0026sup1;.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab4\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 4\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eActivity of defense-related enzymes in sunflower leaves after pre- and post-treatments with ascorbic and glutamic acids (units/mg protein).\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"6\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eTreatment\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eApplication Time\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003ePOX\u003c/p\u003e \u003cp\u003e(\u0026micro;mol min⁻\u0026sup1; mg⁻\u0026sup1;)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003ePAL\u003c/p\u003e \u003cp\u003e(\u0026micro;mol cinnamic acid h⁻\u0026sup1; mg⁻\u0026sup1;)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eSOD\u003c/p\u003e \u003cp\u003e(units mg⁻\u0026sup1; protein)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003eAPX\u003c/p\u003e \u003cp\u003e(\u0026micro;mol min⁻\u0026sup1; mg⁻\u0026sup1;)\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eControl (Inoculated)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e14.5\u0026thinsp;\u0026plusmn;\u0026thinsp;1.2d\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e8.6\u0026thinsp;\u0026plusmn;\u0026thinsp;0.8d\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e25.3\u0026thinsp;\u0026plusmn;\u0026thinsp;1.5d\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e5.8\u0026thinsp;\u0026plusmn;\u0026thinsp;0.5d\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eAscorbic Acid\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003ePre-Inoculation\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e33.5\u0026thinsp;\u0026plusmn;\u0026thinsp;1.8a\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e19.8\u0026thinsp;\u0026plusmn;\u0026thinsp;1.2a\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e52.4\u0026thinsp;\u0026plusmn;\u0026thinsp;2.7a\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e15.3\u0026thinsp;\u0026plusmn;\u0026thinsp;1.0a\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003ePost-Inoculation\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e28.0\u0026thinsp;\u0026plusmn;\u0026thinsp;1.5b\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e16.2\u0026thinsp;\u0026plusmn;\u0026thinsp;1.0b\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e44.3\u0026thinsp;\u0026plusmn;\u0026thinsp;2.3b\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e12.0\u0026thinsp;\u0026plusmn;\u0026thinsp;0.8b\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eGlutamic Acid\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003ePre-Inoculation\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e28.3\u0026thinsp;\u0026plusmn;\u0026thinsp;2.0b\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e17.1\u0026thinsp;\u0026plusmn;\u0026thinsp;1.1b\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e45.7\u0026thinsp;\u0026plusmn;\u0026thinsp;2.3b\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e12.6\u0026thinsp;\u0026plusmn;\u0026thinsp;1.1b\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003ePost-Inoculation\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e24.5\u0026thinsp;\u0026plusmn;\u0026thinsp;1.7c\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e14.0\u0026thinsp;\u0026plusmn;\u0026thinsp;0.9c\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e38.7\u0026thinsp;\u0026plusmn;\u0026thinsp;1.8c\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e10.2\u0026thinsp;\u0026plusmn;\u0026thinsp;0.7c\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ep-value\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u0026lt;\u0026thinsp;0.001\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u0026lt;\u0026thinsp;0.001\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u0026lt;\u0026thinsp;0.001\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e\u0026lt;\u0026thinsp;0.001\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003eValues are means\u0026thinsp;\u0026plusmn;\u0026thinsp;SE. Different letters within columns indicate significant differences at p\u0026thinsp;\u0026lt;\u0026thinsp;0.05 (LSD test).\u003c/p\u003e \u003cp\u003ePre-inoculation treatment with ascorbic acid markedly enhanced enzyme activities, with POX activity reaching 33.5\u0026thinsp;\u0026plusmn;\u0026thinsp;1.8, PAL at 19.8\u0026thinsp;\u0026plusmn;\u0026thinsp;1.2, SOD at 52.4\u0026thinsp;\u0026plusmn;\u0026thinsp;2.7, and APX at 15.3\u0026thinsp;\u0026plusmn;\u0026thinsp;1.0. This is more than a twofold rise relative to levels of control (p\u0026thinsp;\u0026lt;\u0026thinsp;0.001). The presence of such an induction shows a strong defense system activation of the plant prior to nematode challenge.\u003c/p\u003e \u003cp\u003eThe enzyme activities were also significantly elevated by post-inoculation applications of ascorbic acid relative to the control, although to a smaller degree as compared to pre-inoculation treatments (POX 28.0\u0026thinsp;\u0026plusmn;\u0026thinsp;1.5, PAL 16.2\u0026thinsp;\u0026plusmn;\u0026thinsp;1.0, SOD 44.3\u0026thinsp;\u0026plusmn;\u0026thinsp;2.3, APX 12.0\u0026thinsp;\u0026plusmn;\u0026thinsp;0.8). The treatments of glutamic acid similarly promoted defense enzyme activities. Pre-inoculation applications elevated POX, PAL, SOD, and APX activities to 28.3\u0026thinsp;\u0026plusmn;\u0026thinsp;2.0, 17.1\u0026thinsp;\u0026plusmn;\u0026thinsp;1.1, 45.7\u0026thinsp;\u0026plusmn;\u0026thinsp;2.3, and 12.6\u0026thinsp;\u0026plusmn;\u0026thinsp;1.1, respectively. Post-inoculation treatments produced moderate increases (POX 24.5\u0026thinsp;\u0026plusmn;\u0026thinsp;1.7, PAL 14.0\u0026thinsp;\u0026plusmn;\u0026thinsp;0.9, SOD 38.7\u0026thinsp;\u0026plusmn;\u0026thinsp;1.8, APX 10.2\u0026thinsp;\u0026plusmn;\u0026thinsp;0.7), all significantly above untreated controls (p\u0026thinsp;\u0026lt;\u0026thinsp;0.001).\u003c/p\u003e \u003cp\u003eThe p-values of all the enzyme activities were highly significant, implying strong treatment effects. The increase in the activities of defense-related enzymes noted is associated with enhanced resistance mechanisms in sunflower, leading to the suppression of nematodes as well as a reduction in root damage. The highest and most potent induction of defensive enzymes was recorded following pre-inoculation foliar application of ascorbic acid that indicates its potential use as an effective biological elicitor in nematode management programs.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003e3.5 Percentage Increase in Activity of Defense-Related Enzymes in Sunflower Leaves Relative to Control\u003c/h2\u003e \u003cp\u003eFigure (1) illustrates the percentage increases in the activities of defense-related enzymes peroxidase (POX), phenylalanine ammonia-lyase (PAL), superoxide dismutase (SOD), and ascorbate peroxidase (APX) in sunflower leaves following foliar application of ascorbic acid and glutamic acid at different timings relative to \u003cem\u003eM. incognita\u003c/em\u003e inoculation.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThe largest increases in enzyme activities were observed by pre-inoculation treatment with ascorbic acid, as POX activity increased by 131.0, PAL increased by 130.2, SOD increased by 107.5, and APX increased by 163.8 relative to untreated inoculated controls. This high induction indicates a high level of priming of the plant defense system, which allows the plant to be more capable of resisting nematode infection.\u003c/p\u003e \u003cp\u003eThe use of ascorbic acid after inoculation also increased enzyme activities to a significant extent of 93.1% (POX), 88.4% (PAL), 75.5% (SOD), and 106.9% (APX), but to a slightly lesser degree. These findings show that ascorbic acid is able to activate antioxidative defenses even when nematode infestation has begun. Glutamic treatments adhered to the same pattern, with pre-inoculation treatment showing increases of 95.2% (POX), 98.8% (PAL), 80.6% (SOD) and 117.2% (APX), and post-inoculation treatment showing increases of 69.0%, 62.8%, 53.0% and 75.9%, respectively.\u003c/p\u003e \u003cp\u003eThese results refer ascorbic and glutamic acids promising and green alternatives or supplements to traditional nematicides, which may be used as integrated management to enhance sunflower health and yield. The enhanced enzyme activities are attributed to the increased resistance responses and lesser nematode damage.\u003c/p\u003e \u003c/div\u003e"},{"header":"4 Discussion","content":"\u003cp\u003eThe current study demonstrates the effect of ascorbic acid and glutamic acid in\u0026ensp;inhibiting \u003cem\u003eM. incognita\u003c/em\u003e egg hatching, juvenile mortality, reduction in the severity of nematode infection, and promoting host plant resistance in sunflower. The concentration-dependent inhibition of egg hatch and the high mortality of juvenile limnicoles is a direct effect of nematicidal action, likely associated with the disruption of key physiological processes in the nematode (Neeraj et al., \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2017\u003c/span\u003e; Singh et al., \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2021\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eHost-parasite interactions between M. incognita and sunflower roots involve complex molecular dialogues where nematodes deploy effectors to suppress plant immune responses and establish feeding sites known as giant cells (Goverse and Smant, \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2014\u003c/span\u003e; Kaloshian and Teixeira, \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Zhou et al., \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). The ability of ascorbic and glutamic acids to suppress the reproduction of nematodes and galling of roots implies that they interfere not only with the development phase of nematodes but also with the host immune level.\u003c/p\u003e \u003cp\u003ePre-inoculation spray application was better than post-inoculation application, which suggests that systemic defenses can be induced by priming the plant prior to nematode attack. This is consistent with the known paradigm of induced resistance; timely application of the elicitor triggers the activation of the immune system of the plant that results in complex signalling pathways that lead to upregulation of the phenylpropanoid pathway, an oxidative burst, and the orchestrated action of antioxidant enzymes, thus strengthening the plant against parasitic attack (Li et al., \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Mauch-Mani et al., \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2017\u003c/span\u003e; Pieterse et al., \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2014\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eIncreased activities of defense-related enzymes, such as peroxidase (POX), phenylalanine ammonia-lyase (PAL), superoxide dismutase (SOD), and ascorbate peroxidase (APX) are biochemical predictors of an active defense state (Singh et al., \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). These enzymes regulate the balance of reactive oxygen species (ROS) and the production of antimicrobial complexes, which are important in limiting the development and settlement of nematodes (Pandey et al., \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). The significant rise in enzyme activities at pre-inoculation ascorbic acid treatment indicates strong elicitor activity, which is in line with earlier findings that vitamins could also serve as stress signals to promote pathogen resistance (Al-Sayed and Montasser, \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e1986\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eMechanistically, nematodes sense and react to exudates of host roots with specialized sensory systems to locate sites of infection (Mendy et al., \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). On the other hand, plants sense nematode invasion through pattern recognition receptors, which cause defensive responses, including hypersensitive response and cell wall strengthening processes, which could be enhanced by exogenous application of elicitors, such as ascorbic and glutamic acids (Zhou et al., \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). Furthermore, ascorbic acid antioxidants may preserve host cells against oxidative stress caused by nematode infection, which preserves physiological processes critical to resistance (Singh et al., \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2020\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eCo-evolutionary arms race between root-knot nematodes and their host plants involves structural, as well as biochemical adaptations based on the innate immune system of the plant and the arsenal of effector proteins of the nematode (Jones and Dangl, \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2006\u003c/span\u003e; Mitchum et al., \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2013\u003c/span\u003e). Recent research indicates that nematode effectors attack host defense to support the formation of feeding sites, and plants evolve enzymes and secondary metabolites to counter these effectors (Phani et al., \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). Our results indicate that the foliar application of ascorbic and glutamic acids enhances the defensive biochemical armory system of the host plant, which leans towards resistance.\u003c/p\u003e"},{"header":"5 Conclusion","content":"\u003cp\u003eThis paper has effectively shown that foliar applications of ascorbic acid and glutamic acid can greatly reduce the Hatching of \u003cem\u003eM. incognita\u003c/em\u003e eggs and the survival of juvenile M. incognita, which results in reduced root galling and decreased nematode populations in sunflower plants. Pre-inoculation treatment with ascorbic acid at 2 mg/ml was the most effective, showing that it is a strong nematicide and can make plants resistant to nematodes. The increase in the activity of enzymes related to defense, including peroxidase, phenylalanine ammonia-lyase, superoxide dismutase, and ascorbate peroxidase, after treatment shows induced systemic resistance as one of the mechanisms behind nematode suppression. These results refer ascorbic and glutamic acids promising and green alternatives or supplements to traditional nematicides, which may be used as integrated management to enhance sunflower health and yield. Further studies are required to optimize the application protocols and to understand the molecular signaling pathways that these compounds stimulate in order to utilize the potential of the compounds in nematode control to the fullest extent.\u003c/p\u003e"},{"header":"Declarations","content":"\u003ch2\u003eFunding\u003c/h2\u003e \u003cp\u003eThe authors want to express their gratitude to the Deanship of Scientific Research, Vice Presidency for Graduate Studies and Scientific Research, King Faisal University, Saudi Arabia, for funding this study under the terms of KFU254813.\u003c/p\u003e\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eAuthor contributions Abdulrahman S. Al-Hussein : Methodology, Formal analysis, Data curation. Hosny H. Kesba : Conceptualization, Supervision, Formal analysis, Data curation, Writing \u0026ndash; review \u0026amp; editing, Funding. Sherif M. El-Ganainy : Methodology, Formal analysis, Data curation. Wael H. Elmenofy : Supervision, Data curation, Methodology. Ahmed M. Ismail : Conceptualization, Methodology. Hossam S. El-Belatgi : Supervision, Data curation. All authors have reviewed and consented to the final version of the manuscript.\u003c/p\u003e\u003ch2\u003eAcknowledgement\u003c/h2\u003e\u003cp\u003eThe authors are grateful to Deanship of Scientific Research, Vice Presidency for Graduate Studies and Scientific Research, King Faisal University, Saudi Arabia (KFU254813), for supporting this research work.\u003c/p\u003e\u003ch2\u003eData Availability\u003c/h2\u003e\u003cp\u003eThe tables in this research article show all the data that was collected.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eAbd-Elgawad, M.M.M., 2025. Integrated Nematode Management Strategies: Optimization of Combined Nematicidal and Multi-Functional Inputs. 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Agric. 22, 799\u0026ndash;811. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.jia.2022.08.117\u003c/span\u003e\u003cspan address=\"10.1016/j.jia.2022.08.117\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"journal-of-the-saudi-society-of-agricultural-sciences","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"","sideBox":"Learn more about [Journal of the Saudi Society of Agricultural Sciences](https://link.springer.com/journal/44447)","snPcode":"44447","submissionUrl":"https://submission.springernature.com/new-submission/44447/3","title":"Journal of the Saudi Society of Agricultural Sciences","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Springer Open","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Root-knot nematode, Induce systematic resistance, Foliar spray, Antioxidant enzymes, Nematicidal compounds, Sunflower defense","lastPublishedDoi":"10.21203/rs.3.rs-8464901/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-8464901/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eRoot-knot nematode (\u003cem\u003eMeloidogyne incognita\u003c/em\u003e) causes a significant reduction in the production of sunflower (\u003cem\u003eHelianthus annuus\u003c/em\u003e) globally. The paper assessed the nematicidal activity of ascorbic acid and glutamic acid and their ability to trigger systemic resistance in sunflower within controlled conditions. Laboratory evaluations proved that both compounds had a considerable effect on suppressing egg hatch and elevating juvenile mortality in a concentration-dependent manner, with ascorbic acid (2 mg/ml) showing the strongest effects. Experiments in greenhouses found that pre and post inoculation foliar application of these acids suppressed nematode root galling and population density, boosted shoot and root biomass and increased the activities of major defense enzymes, such as peroxidase, phenylalanine ammonia-lyase, superoxide dismutase, and ascorbate peroxidase. Pre-inoculation treatments were more efficient, which demonstrates the role of priming in the activation of defense. Our results indicate that ascorbic and glutamic acid have a combination of direct nematicidal activity with induction of host defense systems, which is promising as a safe means of managing nematode infestation in sunflower farming.\u003c/p\u003e","manuscriptTitle":"Elicitation of Sunflower Resistance to Meloidogyne incognita by Ascorbic and Glutamic Acids: Integrating Direct Nematicidal Activity and Defense Enzyme Induction","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-01-23 20:10:22","doi":"10.21203/rs.3.rs-8464901/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2026-02-19T07:39:14+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-02-18T18:38:05+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"29791900822005559451251347729721355021","date":"2026-02-05T13:34:58+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-02-05T08:43:49+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"40714244005950417720690387098916079734","date":"2026-01-22T04:08:55+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"302156471351855677935750852423010055624","date":"2026-01-21T09:33:39+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2026-01-21T08:46:53+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2026-01-11T09:50:56+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2026-01-09T05:40:40+00:00","index":"","fulltext":""},{"type":"submitted","content":"Journal of the Saudi Society of Agricultural Sciences","date":"2026-01-02T23:32:15+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"journal-of-the-saudi-society-of-agricultural-sciences","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"","sideBox":"Learn more about [Journal of the Saudi Society of Agricultural Sciences](https://link.springer.com/journal/44447)","snPcode":"44447","submissionUrl":"https://submission.springernature.com/new-submission/44447/3","title":"Journal of the Saudi Society of Agricultural Sciences","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Springer Open","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"a678f8d0-df1f-4682-813d-c2e4210910f8","owner":[],"postedDate":"January 23rd, 2026","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[],"tags":[],"updatedAt":"2026-03-31T05:53:57+00:00","versionOfRecord":[],"versionCreatedAt":"2026-01-23 20:10:22","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-8464901","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-8464901","identity":"rs-8464901","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}