Zinc and Boron Soil Applications Affect Athelia rolfsii Stress Response in Sugar Beet (Beta vulgaris L.) Plants

Generation of reactive oxygen species (ROS) constitutes an initial defense approach in plants during pathogen infection. Here, the effects of the two micronutrients, namely, zinc (Zn) and boron (B), on enzymatic and non-enzymatic antioxidant properties, as well as malondialdehyde (MDA) contents in leaves and roots challenged with Athelia rolfsii, which cause root rot disease, were investigated. The findings revealed that Zn and B application to the potting soil alleviated the adverse effect of A. rolfsii on sugar beet plants and increased the chlorophyll content in leaves. The increased enzymatic antioxidant activities such as catalase (CAT), peroxidase (POX), and ascorbate peroxidase (APX), and non-enzymatic antioxidants such as ascorbic acid (AsA) were observed in Zn applied plants compared to both uninoculated and inoculated control plants. A significant rise in CAT activity was noted in both leaves (335.1%) and roots (264.82%) due to the Zn2B1.5 + Ar treatment, in comparison to the inoculated control plants. On the other hand, B did not enhance the activity of any one of them except AsA. Meanwhile, A. rolfsii infection led to the increased accumulation of MDA content both in the leaves and roots of sugar beet plants. Interestingly, reduced MDA content was recorded in leaves and roots treated with both Zn and B. The results of this study demonstrate that both Zn and B played a vital role in A. rofsii tolerance in sugar beet, while Zn enhances antioxidant enzyme activities, B appeared to have a less pronounced effect on modulating the antioxidant system to alleviate the adverse effect of A. rolfsii.


Introduction
Sugar beet (Beta vulgaris L.) holds significant global importance as a cultivated sugar crop, ranking second only to sugarcane [1]. This crop accounts for nearly 30% of the world's sugar production for human consumption [2,3]. Unlike sugarcane, sugar beet has a relatively short growth period, with its mature tap root containing sucrose (15-20%), water (75-76%), non-sugars (2.6%), and pulp (4-6%) [4,5]. Beyond sugar extraction, both the foliage and beet pulp find utility as cattle feed [6]. More recently, sugar beet has gained prominence as a crucial resource for ethanol production as a biofuel [7]. Nonetheless, the production of sugar beet faces significant challenges posed by various diseases. Among toms. Notably, the application of Zn and B in sugar beet plots partially suppressed root and crown rot symptoms in the sugar beet field (unpublished data). Despite sclerotium root rot posing a significant challenge in sugar beet cultivation worldwide, the potential for disease control through the utilization of Zn and B has not previously been investigated. A recent study conducted by Rerhou et al. [28] has shed light on the management of root rot disease in sugar beet by applying micronutrients, including Zn and B. Nevertheless, the precise mechanisms underlying how Zn and B effectively suppress A. rolfsii in sugar beet plants remain undisclosed. It is conceivable that these micronutrients might trigger the activation of the antioxidant system within sugar beet plants, assisting the plants in fortifying their defense mechanisms against A. rolfsii. Consequently, investigating the roles of Zn and B could present a promising avenue to diminish the incidence of root rot disease caused by A. rolfsii, thereby bolstering resistance, and promoting plant growth. The present study evaluated the contributions of Zn and B to the activation of the defense mechanisms in sugar beet plants against A. rolfsii.

Planting Material and Experimental Design
The experiment was conducted at the Agronomy Field Laboratory, Department of Agronomy, Bangladesh Agricultural University, Mymensingh, from November, 2019 to February, 2020. The tropical sugar beet variety PAC-60008 was used for the experiment.

Pot Preparation, Fertilizer Application and Seed Sowing
To perform the study, soils were collected from the medium-high land of the Agronomy Field Laboratory at BAU. The soil possesses a silty loam texture with a pH 6.9, electrical conductivity (EC) 0.4 dS/m, organic carbon 1.00%, N 0.09%, P 1.60 ppm, K 0.10% meq/100 g soil, Ca 8.30 meq/100 g soil, Mg 3.29 meq/100 g soil, S 2.98 ppm, Zn 0.21 ppm, and B 0.23 ppm. The soil was properly air dried, crushed with a wooden hammer, and thoroughly mixed. It was then cleaned and autoclaved. Finally, an equal amount of soil (10 kg) was used to fill each pot. Inorganic fertilizers were mixed thoroughly with the soil during pot filling [5]. Seeds were directly shown in the pots, with three seedlings in each pot, and kept in a growth room at a temperature of 20-25 • C and 12 h/12 h light/dark photoperiods. Light irrigation was applied every alternate day.

Fungi Culture, Inoculation and Sample Collection
The A. rolfsii isolate BTSB2 was used for inoculation assay [8]. Fungal inoculum was prepared using the technique reported by Figueredo et al. [29]. Briefly, wheat seeds were autoclaved and placed in a 50 mL flask. Approximately, 50 mm agar block from the actively growing A. rolfsii culture were cut and placed into the flask containing wheat seeds. The flask was then incubated for 10-15 days at 25 • C with a 12-h photoperiod. Infected wheat seeds were placed adjacent to the collar region of sugar beet plants 42 days after emergence and leaf and root samples were collected for analysis 7 days after the fungal inoculum application. The survival rate of the inoculated plants was calculated 20 days after the pathogen application and expressed as a percentage.

Determination of Soil-Plant-Analysis Development (SPAD) Value
A transportable SPAD meter (Model SPAD-502, Minolta Crop., Ramsey, NJ, USA) was used to determine the greenness (chlorophyll content) of sugar beet leaves 7 days after pathogen inoculation. Five fully expanded new leaves were selected SPAD values recording of beet plants. The average data were collected from the middle portion of the five leaves per plant between 9:00 to 10.00 a.m. [5].

Enzyme Extraction and Assay
Two plants from each replication (e.g., six plants per treatment) were taken and separated into shoots and roots. The collected leaf and root samples were then used for further analysis. Initially, 1 g of green leaves and roots was thoroughly blended and homogenized in a pre-chilled mortar-pestle, adding 5 mL of Tris-HCl buffer (50 mM, pH 8.0) for CAT and KH 2 PO 4 buffer (50 mM, pH 7.0) for POX and APX analysis. The homogenized samples were collected and centrifuged at 5000 rpm for 20 min at 4 • C. Assay of antioxidant enzymes (CAT, POX, and APX) was conducted using the supernatant.
The CAT activity was determined following the technique described by Aebi [30]. The reaction mixtures comprised Tris-HCl buffer (50 mM, pH 8.0), EDTA (0.25 mM), H 2 O 2 (20 mM), and 100 µL of enzyme extract (supernatant). The H 2 O 2 was added to start the reaction and the CAT activity was measured at 240 nm wavelength.
Activity of POX was measured according to the method described by Nakano and Asada [31]. The reaction buffer solution included KH 2 PO 4 buffer (50 mM, pH 7.0), EDTA (0.1 mM), H 2 O 2 (0.1 mM) and guaiacol (10 mM). The reaction was initiated by adding 50 µL of sample solution to the reaction buffer (950 µL). The activity was determined at a wavelength of 470 nm for 30 sec, using an extinction coefficient 26.6 mM −1 cm −1 .

Estimation of the Ascorbic Acid (AsA) Content
The AsA content was calculated following the method described by Rangana [32]. The plant sample mixture, along with acetic acid (5 mL) and 3% of meta phosphate (5 mL) were titrated against 2,6 dichlorophenol indophenol until the pink color appeared. The concentration was determined in comparison with the standard solution.

Determination of the Malondialdehyde (MDA) Content
Oxidative damage in the leaf and root cells was estimated as the content of total 2-thiobarbituric acid (TBA)-responsive substances and expressed as equivalents of MDA following the method of Cakmak and Horst [33]. The amount of MDA was determined by measuring the absorbance at 532 nm and correcting for non-specific turbidity by subtracting the absorbance at 600 nm. An extinction coefficient of 155 mM −1 cm −1 was applied to calculate the MDA, expressed in µmol g −1 fresh weight [34].

Statistical Analysis
The statistical analyses were performed using the software package MSTAT-C, and mean differences were assessed using Duncan's Multiple Range Test (DMRT) [35].

Effect of Zn and B on Plant Survival under A. rolfsii Stress
All the plants survived in the uninoculated control (Zn 0 B 0 ), although some yellowing of leaves was observed. On the other hand, A. rolfsii (inoculated control) treated plants showed wilting within 6-10 days after inoculation. Plants died within 20-25 days after inoculation, and infected roots became rotten and covered with mycelial mass ( Figure 1B). However, all the plants treated with Zn and B and whether or not they were inoculated inoculation, and infected roots became rotten and covered with mycelial mass ( Figure 1B). However, all the plants treated with Zn and B and whether or not they were inoculated with A. rolfsii, survived. Nevertheless, the development of lesions in sugar beet roots due to the inoculation of A. rolfsii varied in various combinations of Zn and B treatments (Figure 1).

Effect of Zn and B on Chlorophyll Content (SPAD Value)
The application of Zn and B increased the chlorophyll content in sugar beet leaves. However, A. rolfsii treatment significantly reduced chlorophyll content, with the lowest (18.96) recorded in the inoculated control compared to the uninoculated control (25.03). Elevated chlorophyll content was recorded in plants treated with Zn4B1.5 (69.50) which was 177.67 and 266.56% higher in comparison to the uninoculated and inoculated control, respectively ( Figure 2).

Effect of Zn and B on CAT Activity
The CAT activity in leaves and roots was significantly influenced by various treatments ( Figure 3A). In the leaves of Zn treated plants, increased CAT activity was recorded in both A. rolfsii inoculated and uninoculated plants compared to the uninoculated control (Zn0B0). Interestingly, decreased CAT activity was detected in uninoculated B applied plants but increased in pathogen inoculated plants. The lowest CAT activity was recorded

Effect of Zn and B on Chlorophyll Content (SPAD Value)
The application of Zn and B increased the chlorophyll content in sugar beet leaves. However, A. rolfsii treatment significantly reduced chlorophyll content, with the lowest (18.96) recorded in the inoculated control compared to the uninoculated control (25.03). Elevated chlorophyll content was recorded in plants treated with Zn 4 B 1.5 (69.50) which was 177.67 and 266.56% higher in comparison to the uninoculated and inoculated control, respectively ( Figure 2). inoculation, and infected roots became rotten and covered with mycelial mass ( Figure 1B). However, all the plants treated with Zn and B and whether or not they were inoculated with A. rolfsii, survived. Nevertheless, the development of lesions in sugar beet roots due to the inoculation of A. rolfsii varied in various combinations of Zn and B treatments (Figure 1).

Effect of Zn and B on Chlorophyll Content (SPAD Value)
The application of Zn and B increased the chlorophyll content in sugar beet leaves. However, A. rolfsii treatment significantly reduced chlorophyll content, with the lowest (18.96) recorded in the inoculated control compared to the uninoculated control (25.03). Elevated chlorophyll content was recorded in plants treated with Zn4B1.5 (69.50) which was 177.67 and 266.56% higher in comparison to the uninoculated and inoculated control, respectively ( Figure 2).

Effect of Zn and B on CAT Activity
The CAT activity in leaves and roots was significantly influenced by various treatments ( Figure 3A). In the leaves of Zn treated plants, increased CAT activity was recorded in both A. rolfsii inoculated and uninoculated plants compared to the uninoculated control (Zn0B0). Interestingly, decreased CAT activity was detected in uninoculated B applied plants but increased in pathogen inoculated plants. The lowest CAT activity was recorded

Effect of Zn and B on CAT Activity
The CAT activity in leaves and roots was significantly influenced by various treatments ( Figure 3A). In the leaves of Zn treated plants, increased CAT activity was recorded in both A. rolfsii inoculated and uninoculated plants compared to the uninoculated control (Zn 0 B 0 ). Interestingly, decreased CAT activity was detected in uninoculated B applied plants but increased in pathogen inoculated plants. The lowest CAT activity was recorded in plants treated only with pathogen (Zn 0 B 0 + Ar), measuring 45.06 µmol min −1 g −1 FW. The highest CAT activity (196.34 µmol min −1 g −1 FW) was recorded for Zn 2 B 1.5 + Ar which was 335.1% higher than inoculated control ( Figure 3A).
In the root, a similar trend in CAT activity was observed. A significant increase in CAT activity (71.98 µmol min −1 g −1 FW) was noticed in the roots of Zn4B0 + Ar treated plants, which was 264.82% higher than the inoculated control plants, followed by Zn2B0 + Ar (68 µmol min −1 g −1 FW), Zn2B1.5 + Ar (61.75 µmol min −1 g −1 FW), Zn2B3 + Ar (58.84 µmol min −1 g −1 FW) and Zn4B3 + Ar (58.09 µmol min −1 g −1 FW) treated plants. Boron treatments did not enhance CAT activity in sugar beet roots. CAT exhibited a substantial decline in its activity in inoculated control (Zn0B0 + Ar) compared to the uninoculated plants ( Figure 3B). Data are presented as treatment mean ± standard error. Data followed by the same letter are not significantly different by DMRT test at p < 0.05.

Effect of Zn and B on POX Activity
The sugar beet plants exhibited a similar pattern of POX activity as CAT in response to the application of Zn and B under A. rolfsii treatment. In both leaves and roots, higher POX activity was detected in plants treated with Zn compared to the uninoculated (Zn0B0) and inoculated (Zn0B0 + Ar) control, while decreased POX activity was detected in plants treated only B but it increased in pathogen inoculated plants. The highest POX activity (1.96 µmol min −1 g −1 FW) was observed in Zn2B1.5 + Ar treated plants, which was 43% higher than inoculated control (1.37 µmol min −1 g −1 FW) in leaves. The lowest POX activity was found in Zn0B3 (0.53 µmol min −1 g −1 FW) ( Figure 4A).
In the roots, the highest POX activity (13.16 µmol min −1 g −1 FW and 12.23 µmol min −1 g −1 FW) was observed in Zn2B0 and Zn2B1.5 treated plants with pathogen inoculation, which Data are presented as treatment mean ± standard error. Data followed by the same letter are not significantly different by DMRT test at p < 0.05.
In the root, a similar trend in CAT activity was observed. A significant increase in CAT activity (71.98 µmol min −1 g −1 FW) was noticed in the roots of Zn 4 B 0 + Ar treated plants, which was 264.82% higher than the inoculated control plants, followed by Zn 2 B 0 + Ar (68 µmol min −1 g −1 FW), Zn 2 B 1.5 + Ar (61.75 µmol min −1 g −1 FW), Zn 2 B 3 + Ar (58.84 µmol min −1 g −1 FW) and Zn 4 B 3 + Ar (58.09 µmol min −1 g −1 FW) treated plants. Boron treatments did not enhance CAT activity in sugar beet roots. CAT exhibited a substantial decline in its activity in inoculated control (Zn 0 B 0 + Ar) compared to the uninoculated plants ( Figure 3B).

Effect of Zn and B on POX Activity
The sugar beet plants exhibited a similar pattern of POX activity as CAT in response to the application of Zn and B under A. rolfsii treatment. In both leaves and roots, higher POX activity was detected in plants treated with Zn compared to the uninoculated (Zn 0 B 0 ) and inoculated (Zn 0 B 0 + Ar) control, while decreased POX activity was detected in plants treated only B but it increased in pathogen inoculated plants. The highest POX activity (1.96 µmol min −1 g −1 FW) was observed in Zn 2 B 1.5 + Ar treated plants, which was 43% higher than inoculated control (1.37 µmol min −1 g −1 FW) in leaves. The lowest POX activity was found in Zn 0 B 3 (0.53 µmol min −1 g −1 FW) ( Figure 4A). , x FOR PEER REVIEW 7 of 15 was 26.05% and 17.14% higher than the inoculated control. The lowest POX activity was recorded in Zn0B1.5 (4.36 µmol min −1 g −1 FW) and Zn0B3 (3.81 µmol min −1 g −1 FW) treated plants ( Figure 4B). Data are presented as treatment mean ± standard error. Data followed by the same letter are not significantly different by DMRT test at p < 0.05.

Effect of Zn and B on APX Activity
The APX activity in sugar beet leaves and roots is significantly affected by various treatments. Pathogen inoculation increased the APX activity in both Zn and B treated plants. However, a decreased APX activity was recorded in B applied plants compared to both inoculated and uninoculated control plants. In leaves, the highest APX activity (0.86 µmol min −1 g −1 FW) was recorded for Zn2B0 + Ar, which was 109.75% higher than inoculated control, whereas the lowest APX activity was recorded in Zn0B1.5 (0.37 µmol min −1 g −1 FW) ( Figure 5A).
Similarly, in root samples, the APX activity also showed significant increase when treated with Zn. The B treated plants showed decreased APX activity, but it significantly increased in pathogen inoculated plants. The highest APX activity was recorded for Zn2B1.5 + Ar (13.27 µmol min −1 g −1 FW) treated plants, which was 131.58% higher than the inoculated control. The lowest APX activity was recorded in Zn0B1.5 (4.71 µmol min −1 g −1 FW), which was statistically similar to the Z0B3 treatment ( Figure 5B). Data are presented as treatment mean ± standard error. Data followed by the same letter are not significantly different by DMRT test at p < 0.05.

Effect of Zn and B on APX Activity
The APX activity in sugar beet leaves and roots is significantly affected by various treatments. Pathogen inoculation increased the APX activity in both Zn and B treated plants. However, a decreased APX activity was recorded in B applied plants compared to both inoculated and uninoculated control plants. In leaves, the highest APX activity (0.86 µmol min −1 g −1 FW) was recorded for Zn 2 B 0 + Ar, which was 109.75% higher than inoculated control, whereas the lowest APX activity was recorded in Zn 0 B 1.5 (0.37 µmol min −1 g −1 FW) ( Figure 5A). rolfsii. Data are presented as treatment mean ± standard error. Data followed by the same letter are not significantly different by DMRT test at p < 0.05.

Effect of Zn and B on AsA Concentration
The AsA levels of sugar beet leaves and roots showed significant variation among the treatments ( Figure 6). An increase in AsA levels was noticed in Zn and B treated plants, both in leaves and roots, with a few exceptions. The highest AsA concentration (8.62% mg) was recorded in the leaves of A. rolfsii inoculated plant treated with Zn and B (Zn4B3 + Ar), which was 30.60% higher than the inoculated control (Z0B0). The AsA content in Zn4B3 + Ar was statistically identical to the treatments of Z4B1.5, Zn4B1.5 + Ar, Zn2B3, Zn0B3, and Zn2B0 in leaves ( Figure 6A).
An increased AsA concentration was recorded in the roots of inoculated control compared to the uninoculated control (Zn0B0) in Zn treated plants. Interestingly, a decreased AsA concentration was observed in the roots that were only treated with B ( Figure 6B). The highest AsA concentration (4.1% mg) was observed in Zn4B0 + Ar, which was on par with Zn4B3, while the lowest was in Zn0B3 (1.99% mg). Similarly, in root samples, the APX activity also showed significant increase when treated with Zn. The B treated plants showed decreased APX activity, but it significantly increased in pathogen inoculated plants. The highest APX activity was recorded for Zn 2 B 1.5 + Ar (13.27 µmol min −1 g −1 FW) treated plants, which was 131.58% higher than the inoculated control. The lowest APX activity was recorded in Zn 0 B 1.5 (4.71 µmol min −1 g −1 FW), which was statistically similar to the Z 0 B 3 treatment ( Figure 5B).

Effect of Zn and B on AsA Concentration
The AsA levels of sugar beet leaves and roots showed significant variation among the treatments (Figure 6). An increase in AsA levels was noticed in Zn and B treated plants, both in leaves and roots, with a few exceptions. The highest AsA concentration (8.62% mg) was recorded in the leaves of A. rolfsii inoculated plant treated with Zn and B (Zn 4 B 3 + Ar), which was 30.60% higher than the inoculated control (Z 0 B 0 ). The AsA content in Zn 4 B 3 + Ar was statistically identical to the treatments of Z 4 B 1.5 , Zn 4 B 1.5 + Ar, Zn 2 B 3 , Zn 0 B 3 , and Zn 2 B 0 in leaves ( Figure 6A).
An increased AsA concentration was recorded in the roots of inoculated control compared to the uninoculated control (Zn 0 B 0 ) in Zn treated plants. Interestingly, a decreased AsA concentration was observed in the roots that were only treated with B ( Figure 6B). The highest AsA concentration (4.1% mg) was observed in Zn 4 B 0 + Ar, which was on par with Zn 4 B 3 , while the lowest was in Zn 0 B 3 (1.99% mg).

Effect of Zn and B on MDA Concentration
The increase in MDA is a sign of membrane impairment at the cellular level, and under this stress condition, lipid peroxidation is expressed [36]. The amount of MDA in sugar beet leaves and roots was significantly influenced by various treatments. The MDA content increased in plants inoculated with Ar alone compared to the uninoculated control but decreased in plants treated with Zn and B, with or without Ar (Figure 7). In leaves, the MDA concentration (0.0167 µmol g −1 FW) in inoculated control (Zn0B0 +Ar) was 35.77% higher than the uninoculated control (0.0123 µmol g −1 FW). Similarly, in roots, a higher MDA concentration (0.137 µmol g −1 FW) was recorded in inoculated control (Zn0B0 + Ar), while a lower amount was found in Zn2B3 (0.0036 µmol g −1 FW) treated plants. However, plants treated with Zn and B significantly decreased the MDA contents both in leaves and roots, even after the inoculation of the pathogen (Figure 7A,B).

Effect of Zn and B on MDA Concentration
The increase in MDA is a sign of membrane impairment at the cellular level, and under this stress condition, lipid peroxidation is expressed [36]. The amount of MDA in sugar beet leaves and roots was significantly influenced by various treatments. The MDA content increased in plants inoculated with Ar alone compared to the uninoculated control but decreased in plants treated with Zn and B, with or without Ar (Figure 7). In leaves, the MDA concentration (0.0167 µmol g −1 FW) in inoculated control (Zn 0 B 0 +Ar) was 35.77% higher than the uninoculated control (0.0123 µmol g −1 FW). Similarly, in roots, a higher MDA concentration (0.137 µmol g −1 FW) was recorded in inoculated control (Zn 0 B 0 + Ar), while a lower amount was found in Zn 2 B 3 (0.0036 µmol g −1 FW) treated plants. However, plants treated with Zn and B significantly decreased the MDA contents both in leaves and roots, even after the inoculation of the pathogen (Figure 7A,B). 11, x FOR PEER REVIEW 10 of 15

Discussion
Antioxidative systems in plants generally provide a defense mechanism against the harmful effects of ROS generated during interaction with pathogens [37][38][39][40]. Antioxidant enzymes such as CAT, APX, and POX play important roles in detoxifying ROS. Several studies have suggested that micronutrients suppress pathogen growth either by direct inhibition or by modulating the antioxidant protection mechanism [41][42][43]. In this paper, we demonstrated the differential changes in antioxidant enzymes in sugar beet plants resulting from the application of Zn and B during A. rolfsii treatment. The results obtained from this study support the idea that Zn and B participate in a protective activity in sugar beet plants against A. rolfsii by modulating antioxidant enzymes.
Micronutrients such as Zn and B are considered essential factors for normal plant growth and for defending plants against hostile environments [17][18][19][20]25,26]. In this study, we observed that A. rolfsii causes root rot symptoms in plants and leads to the death of entire plants ( Figure 1); while Zn and B alleviate the adverse effects of A. rolfsii and enhance the growth of sugar beet plants. Moreover, leaf chlorophyll content was reduced by the inoculation of A. rolfsii, but it significantly increased in Zn and B treated plants ( Figure  2). Pathogen stress reduces the photosynthetic rate in plants due to damage to the photosynthetic mechanism, which can interrupt the food producing system [27,29]. Zn and B treatment significantly increases the leaf chlorophyll content, enriching sugar beet growth due to enhanced photosynthetic activity under pathogen stress. These results are in agreement with Noman et al. [44] and Aydin et al. [23], who found that Zn and B application significantly increase chlorophyll content in radish and tomato plants. Numerous studies advocated that the appropriate dose of Zn and B improve growth and production in various crops, including rice, wheat, maize, and chickpea [45][46][47][48].

Discussion
Antioxidative systems in plants generally provide a defense mechanism against the harmful effects of ROS generated during interaction with pathogens [37][38][39][40]. Antioxidant enzymes such as CAT, APX, and POX play important roles in detoxifying ROS. Several studies have suggested that micronutrients suppress pathogen growth either by direct inhibition or by modulating the antioxidant protection mechanism [41][42][43]. In this paper, we demonstrated the differential changes in antioxidant enzymes in sugar beet plants resulting from the application of Zn and B during A. rolfsii treatment. The results obtained from this study support the idea that Zn and B participate in a protective activity in sugar beet plants against A. rolfsii by modulating antioxidant enzymes.
Micronutrients such as Zn and B are considered essential factors for normal plant growth and for defending plants against hostile environments [17][18][19][20]25,26]. In this study, we observed that A. rolfsii causes root rot symptoms in plants and leads to the death of entire plants ( Figure 1); while Zn and B alleviate the adverse effects of A. rolfsii and enhance the growth of sugar beet plants. Moreover, leaf chlorophyll content was reduced by the inoculation of A. rolfsii, but it significantly increased in Zn and B treated plants ( Figure 2). Pathogen stress reduces the photosynthetic rate in plants due to damage to the photosynthetic mechanism, which can interrupt the food producing system [27,29]. Zn and B treatment significantly increases the leaf chlorophyll content, enriching sugar beet growth due to enhanced photosynthetic activity under pathogen stress. These results are in agreement with Noman et al. [44] and Aydin et al. [23], who found that Zn and B application significantly increase chlorophyll content in radish and tomato plants. Numerous studies advocated that the appropriate dose of Zn and B improve growth and production in various crops, including rice, wheat, maize, and chickpea [45][46][47][48].
Upon pathogen recognition, plants initiate the production of elevated levels of ROS. These ROS can damage the cellular membrane lipids of the plants, resulting in subsequent membrane permeability [49]. Membrane lipids are important targets of ROS due to their unsaturated nature, which is involved in the accumulation of MDA; this factor is used to determine the degree of oxidative stress damage. In the study, the application of Zn and B apparently decreased the MDA content in plants, thus improving the comparative membrane permeability of sugar beet plants and enhancing their survival rate under pathogen challenges (Figures 1 and 7). Several studies have reported the role of Zn and B in decreasing the MDA content under various stresses. Zn is an essential element for all metabolic processes, including photosynthesis. Tufail et al. [50] reported that Zn application in rice plants enhances photosynthesis, which might improve the integrity of the cell membrane. On the other hand, B acts as a signal molecule that modulates the expression of enzymes needed for cell wall synthesis and assembly [40,51]. These findings from this study can be correlated with the findings that Zn and B prevent the peroxidation of membrane constituents, thus stabilizing the cell membrane by altering the expression of cell wall synthesis genes or enhancing the metabolic process in the sugar beet plant.
We observed an elevation in CAT, APX, and POX activity in Zn treated plants compared to both uninoculated and inoculated plants. CAT activity was notably higher in Zn-treated plants both before and after inoculation of A. rolfsii. However, in the inoculated control group (A. rolfsii treated plant), CAT activity exhibited a decrease compared to the uninoculated control (Zn 0 B 0 ). It has been suggested that CAT might suppress biotrophic pathogens while encouraging necrotrophic pathogens through host-pathogen interaction [39,52]. The reduction in CAT activity in inoculated control plants likely contributes to the facilitation of pathogen infection. Conversely, the elevated CAT activity observed in Zn-treated plants, in contrast to untreated plants, underscores their enhanced ability to detoxify H 2 O 2 . Other studies have also highlighted the role of CAT in combating various fungal pathogens in important crops such as lettuce, oilseed rape, non-heading Chinese cabbage, and sunflower [37,39,[53][54][55].
The POX is a well-known pathogenesis-associated protein that helps cell wall lignification and suberization, creating a physical barrier against invading pathogens for localized control [34,39]. Treatment with Zn increased the POX activity in both inoculated and uninoculated plants. Similarly, APX activity and AsA content were also increased in Zn treated plants. Since Zn application significantly enhanced POD, APX, and AsA activity and reducing MDA content, the increased survival rate of sugar beet plants under A. rolfsii stress could be attributed to the increased CAT, POD, APX, and AsA activity, which mitigates the harmful effects of ROS generated during pathogen infection. These results are consistent with other studies that Zn amendments or supplementation enhance the activities of various defense enzymes in plants, triggering systemic resistance against pathogens [17,18,20,25,27]. Zn plays a crucial role as a structural element in zinc finger proteins, and several studies suggest that these proteins regulate the expression of key ROS-scavenging enzymes such as CAT, SOD, POD, and APX, thereby imparting resistance to stressors [56]. Consequently, it is plausible to hypothesize that Zn treatment in inoculated plants likely activates zinc finger proteins, which subsequently stimulate a cascade of defense enzymes, thus fortifying the plant's defenses against A. rolfsii.
Boron is another crucial micronutrient for plant growth and development. Interestingly, an increase in CAT, POX, and APX activity was observed in plants grown in boron-deficient soil (uninoculated control plants) compared to soil amended with B. This observation aligns with findings by Song et al. [57] and Wang et al. [58] where they noted that either excessive B or B deficiency induces stress in plants, consequently, leading to heightened activity of antioxidant enzymes in sugar beet plants. Optimum B doses, however, appear to mitigate the oxidative stress brought about by B-related stress. Nevertheless, the inoculation of A. rolfsii only slightly enhanced the activity of CAT, POX, and APX in plants grown in B-amended soil, in comparison to both uninoculated and inoculated control plants. The results of the study suggest that B aids in maintaining ROS homeostasis under conditions of B-deficiency by regulating the antioxidant enzyme activity. However, it does not seem to suppress the growth of A. rolfsii through the enhancement of antioxidant defense mechanisms. Boron is an integral part of cell wall structure, and B-deficiency leads to a decrease in the mechanical strength of the cell wall by altering cellulose, pectin, and lignin biosynthesis [51]. Several studies have indicated that the application of optimum doses of B in plants enhances the integrity of cell membranes and cell wall, which a significant factor is contributing to increased resilience during pathogen infection [24,25]. It is possible that B might promote the stability and rigidity of the cell wall structure, thus inhibiting the pathogen invasion in sugar beet plants resulting in a higher survival rate. On the other hand, B has been reported as an antifungal agent that directly inhibits the growth of numerous pathogenic fungi [40,41]. Furthermore, recent research has also proposed that both Zn and B suppress fungal growth by enhancing the endophytic biocontrol bacterial community thereby enhancing the plant growth [59]. Hence, ensuring proper plant nutrition is one of the essential approaches for the sustainable management of plant disease.

Conclusions
This study is the first to describe the regulation of ROS in the sugar beet-A. rolfsii pathosystem through the application of Zn and B. It is evident that both Zn and B help to alleviate the adverse effects of A. rolfsii in sugar beet plants and increase chlorophyll content. Both Zn and B play distinct roles in modifying the antioxidant defense response within sugar beet plants when faced with A. rolfsii infection. Zn has shown the ability to enhance the activity of antioxidant enzymes, whereas B appeared to have less pronounced effects on modulating the antioxidant system to mitigate the detrimental impacts triggered by A. rolfsii. However, a more comprehensive study is needed to explore the antifungal effects of Zn and B on A. rolfsii, examine the regulation of other antioxidant enzymes, and investigate the modulation of beneficial microorganisms in soil by the application of Zn and B in sugar beet cultivation system. This, in turn, could help to mitigate the detrimental impacts of A. rolfsii on sugar beet plants.