https://orcid.org/0000-0003-3507-2605
Figures
Abstract
Nutritional status being the first line of defense for host plants, determines their susceptibility or resistance against invading pathogens. In recent years, the applications of plant nutrient related products have been documented as one of the best performers and considered as alternatives or/and supplements in plant disease management compared to traditional chemicals. However, knowledge about application of plant nutrient related products for the management of destructive fungal pathogen Fusarium oxysporum f.sp. capsici and their impact on the components of the antioxidant defense system, especially in chilli plants, still needs to be discovered. Therefore, in this current study, we aimed to evaluate two nutrient fertilizers viz. Krystafeed and Micro Plus at three different concentrations by soil drenching method for their effects against the Fusarium wilt of chilli and investigate the components of the antioxidant defense system of chilli plants. Correlation and computational analysis on the components of antioxidant defense system in various pathways were performed to predict the suitable binding sites of mineral ions. Results indicated that the combination of Krystafeed and Micro Plus was found the most effective with (27.01, 26.59%) disease incidence, followed by Micro Plus (29.56, 32.35%) and Krystafeed (38.21, 41.15%), both in greenhouse and field conditions, respectively. Moreover, the combination of Krystafeed and Micro Plus significantly increased the concentration of SOD (27.53, 108.96)%, POD (37.29, 45.65)%, CAT (19.33, 95.33)%, H2O2 (22.13, 118.98)%, TPC (27.39, 17.37)%, chlorophyll a (21.80, 35.74)%, chlorophyll b (57.57, 18.25)%, total chlorophyll (30.21, 19.83)%, Tocopherol (13.08, 33.66)%, TrxR (5.03, 36.56)%, MDA (13.84, 54.79)%, ascorbate (4.72, 17.28)%, Proline (5.94, 59.31)%, and phytoalexin (Capsidiol) (11.33, 55.08)% in the treated plants of resistant and susceptible chilli varieties, respectively, as compared to the untreated plants. Pearson’s correlation heat-map analysis showed that all the enzymes of antioxidant defense system were found positively correlated with each other. It is concluded that the improvement of crop resistance by the application of plant nutrient related products may be viable alternatives to synthetic chemicals for managing Fusarium wilt disease of chilli and potentially other pathogens.
Citation: Usman M, Atiq M, Rajput NA, Sagheer M, Xia Y (2025) Computational and experimental approaches to explore defense related enzymes conferring resistance in Fusarium infected chilli plants by regulating plant metabolism through nutritional products. PLoS ONE 20(1): e0309738. https://doi.org/10.1371/journal.pone.0309738
Editor: Faiz Ahmad Joyia, University of Agriculture Faisalabad, PAKISTAN
Received: March 17, 2024; Accepted: August 18, 2024; Published: January 14, 2025
Copyright: © 2025 Usman et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Data Availability: All relevant data are within the manuscript and its Supporting information files.
Funding: The author(s) received no specific funding for this work.
Competing interests: The authors have declared that no competing interests exist.
1. Introduction
Chilli pepper (Capsicum annum) is a key crop which fulfills the world’s spice and pharmaceutical needs. Its production is continuously hammering by different fungal, bacterial, and viral pathogens [1]. Among fungal diseases, Fusarium wilt is one of the most destructive diseases, which is caused by Fusarium oxysporum f. sp. capsici (Foc) [2, 3], leading to the reduction in yield from 68 to 71% of 32.3t/ha of world’s average green chilli production [2, 4], with a 44.94% disease incidence based on the previous study [5]. Foc is an asexual homothallic fungus [6], which remains persistent in soil for long time in the form of chlamydospores [7]. This pathogen has more than one hundred plant species as host, which leads towards momentous economic losses [8].
Mineral nutrients (macro and micro) play a crucial role in the life of chilli plants as their deficiency or excess amount have deleterious effects on plants [9], which makes the host plants vulnerable to pathogen attack. Being an integral part of plant metabolism, nutritional deficiency incited various diseases [10] by disturbing plant metabolism and physiology [11]. Excess amounts of nutrients cause toxicity in plants [12], leading towards the accumulation of sugar and carbohydrates. Higher amount of sugar and carbohydrates enhance susceptibility by facilitating the attack of many pathogens, which indicates the direct involvement of mineral nutrients in defense mechanism of host in the form of metabolic regulators and structural components [13]. Nutritional status being one of the first line of defense of the host plants, determines their susceptibility or resistance against invading pathogens [10]. When pathogen attacks, it feed on the nutrients of host plants [14]. Due to this, host plants can’t meet their nutritional requirement and become weaker, and a weak plant can’t properly run its normal physiological and defense functions, including the activation of defense system against invading pathogens.
During normal metabolic process, highly reactive compounds (free radicals) are produced in the form of reactive oxygen species (ROS) in plants [15]. The balanced concentration of these free radicals (ROS) is beneficial for plant’s indispensable processes, such as the activation of defense through intracellular signaling against pathogens [16]. Too high concentration of ROS is lethal for plants; therefore, ROS needs to be stabilized naturally by various antioxidant enzymes (superoxide dismutase, glutathione peroxidase, and catalase) present in plants [17]. Collectively, these biochemical compounds improve plant health and make their considerable contribution in the activation of defense system [18, 19] by producing different types of enzymes and metabolites. Attacks of pathogens cause disturbance or alterations in these free radicals and antioxidant enzymes of plants [20]. These biochemical alterations induced by pathogens lead to the host plant towards abnormality by disturbing physiological processes [21, 22]. The disturbed biochemical profile of plants can be improved by the application of different macro- and micro- nutrients. Nutrients actively function as metabolic regulators, contributing to enhanced plant health and the improvement of the plant’s biochemical profile [23]. Moreover, nutrients stimulate the production of essential secondary metabolites, such as phenolics, alkaloids, and terpenoids. Micronutrients like Se, Mn, and Zn contribute to the synthesis and functioning of superoxide dismutase, glutathione peroxidase, and catalase, while Ca acts as a secondary messenger in signaling pathways. Considering the gravity of the issue, the current study aimed to determine the biochemical compounds in both nutrient product-treated and untreated chili plants. Determination of biochemical compounds is helpful for scientists towards the development of biochemical markers for identifying resistant sources in chilli plants. Moreover, these biochemical analyses assist the scientists and researchers to understand the mechanism of resistance and susceptibility towards pathogens, which is also helpful in the product development to manage the Fusarium wilt of chilli and potentially other pathogens. We hypnotized that mineral nutrients can be very helpful in managing Fusarium wilt disease of chilli by boosting plant defense through their direct impact on various antioxidant defensive enzymes. Therefore, the objective of this present study was to investigate the management of Fusarium wilt of chilli through diverse mineral nutrients by boosting plant defense.
2. Materials and methods
2.1 Material collection
All chemical and reagents used were of analytical grade, for instance, potassium dihydrogen phosphate (KH2PO4), silver nitrate, metaphosphoric acid, Hydrochloric acid, dinitrosalicylic acid, and Sodium Nitrite were purchased from Sigma Chemical Co. (St. Louis, Mo, USA. Ptoassium iodide, dichloroindophenol, and trichloroacetic acid were purchased from Merck (Darmstadt, Germany). Potassium Chloride, Di potassium hydrogen phosphate, and EDTA were purchased from Samchun. Aluminum chloride and Catechin were purchased from PubChem. Acetone, F-C reagent, and Sodium Hydroxide were purchased from Riedel-de Haen (Germany), Oxford lab chem (India), and Merck Millipore (USA), respectively.
2.2 Experimental setup (in vitro)
Moderately susceptible variety of chilli (Hot Shot) plants were grown in germination trays (200 holes trays with 2–2’’ hole size) containing peat moss media (2–2.5 g/hole). After forty days, the seedlings were ready to be transplanted. Plants (4–6 leaf stage) were transferred in pots (17×13Cm) containing formalin sterilized sandy loam soil (60% Sand, 10% Clay, and 30% Silt). After one week, two nutrient fertilizers Krystafeed (Arysta Life Science) (T1), Micro Plus (Dalton™) (T2), and their combinations (T3) were applied by soil drenching method using glass beaker at three different concentrations (Krystafeed @ (C1) 2, (C2) 4, and (C3) 6g/liter/Pot; Micro Plus @ (C1) 0.5, (C2) 0.75, and (C3) 1.0g/liter of water/pot; combination of Krystafeed and Micro Plus @ (C1) 2+0.5, (C2) 4+0.75, and (C3) 6+1.0g/liter/pot, respectively). Control treatment was maintained by applying distilled water on chilli plants. The experiment was performed in three replicates. The spore suspension was prepared from a one-week-old culture exhibiting sporulation and dense growth of Fusarium mycelium. Macro and micro conidia were collected by scraping the fungal mass and dissolving it in distilled water. Spore suspension of Foc was measured and diluted to 1×106 spores/mL with the help of a hemocytometer (SKU:100158). Inoculation was done early in the morning by soil drenching method [24]. For this purpose, 100mL of spore suspension was applied in the root zone or near the base of the plant by digging 2–3 cm holes to provide maximum inoculum pressure. Data regarding disease incidence was recorded by using Moniam and Ismail scale [25]. According to this scale 0 = immure, 1 = 1–20% resistant, 2 = 21–40% moderately resistant, 3 = 41–50% moderately susceptible, 4 = 51–70% susceptible, and 5 = 70–100% highly susceptible.
2.3 Experimental setup (in vivo)
For the evaluation of macro- and micro- nutrients in field, two nutrient products Krystafeed (T1), Micro Plus (T2), and their combinations (T3) (Table 1) were mixed at three different concentrations (Krystafeed @ (C1) 300, (C2) 350, and (C3) 400g/100ft2; Micro Plus @ (C1) 30, (C2) 35, and (C3) 40 g/100ft2; combination of Krystafeed and Micro Plus @ (C1) 300+30, (C2) 350+35, (C3) 400+40 g/100ft2, respectively) in sandy loam soil sterilized with 5% formalin solution. Plants (4–6 leaf stage) of moderately susceptible variety (Hot Shot) of chilli were transferred in field under RCBD by maintaining three replications. Chilli plants were treated with the Foc suspension (1×106 spores/mL of water) by soil drenching method to give them the proper inoculum pressure [24]. The data regarding disease incidence was recorded after one weak interval. Disease incidence data was recorded by following formula:
2.4 Sample preparation for biochemical profiling
For the biochemical profiling, leaf samples were collected from both groups, for example, the treated (most effective/best performed treatment) and untreated (application of distilled water) ones. The leaf samples were collected early in the morning of the 7th day of inoculation from top, middle, and lower portions of the plants (6–8 leaf stage) for the in-vitro experiment. The samples were made dirt free by washing with tap water and then with distilled water. After this, leaves were air dried to remove extra water contents from surface and cut into small pieces (1-2mm). Then 0.5 grams of sample (leaves) was mixed in the extraction buffer (10mM K2HPO4, 10mM KH2PO4, 100mM KCL, and 2mM EDTA) at pH 7 with 1: 2 ratios. The mixture was grinded with the help of sterilized pestles and mortars and filled in Eppendorf tubes (1.5 mL), which was centrifuged (TGL-16A) at 12,000rpm for 10 minutes. Supernatant was taken only and stored at -20°C by using sterilized bottles in refrigerator (PEL, PRGD-145) for further biochemical analysis.
2.4.1 Measurement of the level of superoxide dismutase (SOD).
Reaction mixture was prepared by using different chemicals, including 200μL methionine (C5H11N2S), 100μL NBT (Nitro-blue Tetrazolium), 100μL enzyme extract, potassium phosphate buffer, triton X 500μL, and 800μL distilled water. This reaction mixture was poured into a test tube (10mL) and placed under ultraviolet radiation (light intensity 350 μmol m-2 s-1, UV Transilluminator SKU: E3000) for fifteen minutes. After adding 100μL riboflavin, the absorbance was taken at 560nm by using an ELISA plate reader [26]. The amount of superoxide dismutase was calculated as the quantity of enzyme needed to induce a 50% reduction in Nitroblue Tetrazolium activity.
2.4.2 Test of the activities of Peroxidase (POD) and Catalase (CAT).
To determine peroxidase from chilli leaves, the reaction mixture was prepared by adding 100μL H2O2, 100μL enzyme extract, 800μL potassium phosphate buffer (pH 5), and 20mM guaiacol into leaf extract in a 2mL tube. Absorbance was noted at 470nm by using the spectrophotometer (Hitachi U-2001, model 121–003). The reaction mixture for the determination of CAT was prepared by adding 100μL enzyme extract and 100μL H2O2. Spectrophotometer (Hitachi U-2001, model 121–003) was used to measure the absorbance at 240nm [27]. Peroxidase and catalases were determined by following formulas:
2.4.3 Measurement of H2O2 concentration.
For the determination of H2O2, 0.5g leaf sample was taken and grinded in 0.1% Tri-chloro-acetic acid (C2HCl3O2), using pestles and mortars. This mixture was centrifuged at 12,000 rpm for 15 minutes. Supernatant was separated in the Eppendorf tubes (1.5mL) and pellets were discarded. Supernatant was dissolved in 1.3mL potassium phosphate buffer (pH 7) and 1mLpotassium iodide in a test tube (5mL). The reaction mixture was incubated for 5 min and the absorbance was recorded at 390 nm by using the absorbance reader (BioTek, Model: 800TS). The amount of H2O2 was calculated by following formula [28].
2.4.4 Test of the total phenolic contents.
Ten mL of extraction mixture and 1g of grinded sample was dissolved in test tubes (15mL). This mixture was vortexed and centrifuged for five minutes. Then 100μL supernatant was separated and added into 200μL F-C reagents (10%) and vortexed. 800 μL Na2CO3 was added into it, vortexed, and left for one hour. 200μL solution was loaded in one ELISA plate and absorbance was recorded at 765 nm [29].
2.4.5 Determination of chlorophyll contents.
To determine chlorophyll contents, fresh leaves of chilli were collected, grinded, and extracted in acetone (80% solution). Centrifugation was done at 12,000 rpm for 5 minutes to purify the enzyme extract. The supernatant was separated, and the absorbance was taken by a spectrophotometer (Hitachi U-2001, model 121–003) at 645, 663, and 480 nm [30, 31]. Whereas the chlorophyll contents were determined by following formulas:
2.4.6 Estimation the level of tocopherol (μg/mL).
Estimation of the level of tocopherol was carried out by adding 1 mL of pure ethanol into 4 mL test tubes. 0.2 mL of bathophenanthroline (0.2%) and 0.2 mL of Ferric Chloride FeCl3 (0.001M) was added into ethanol containing test tubes and mixed thoroughly using a vortex mixer (Velp Scientifica SA202A0176 - ZX3). After a pause of 60 seconds, the addition of 0.2 mL phosphoric acid H3PO4 (0.001M) was done. The reaction mixture was mixed thoroughly, and the absorbance was taken at 534 nm with the help of a spectrophotometer [32]. The amount of tocopherol was taken in μg/mL and was calculated by using standard curve.
2.4.7 Assessment of the level of thioredoxin reductase.
For the assessment of Thioredoxin Reductase (TrxR), the reaction mixture was prepared by adding 200 μL NADPH, 10 mL ETDA, 5 mL 5,5’-Dithiobis (2-nitrobenzoic acid) (DTNB), and 30 μL enzyme extract into 100 mL of potassium phosphate buffer (pH 7.0). Reaction mixture was divided into two groups. 10 μL of TrxR Inhibitor was added in one group for testing the background enzyme activity; and 10 μL of Assay Buffer was added into 2nd group for estimating the total DTNB reduction. Absorbance of these reaction mixtures was taken at 412 nm [33]. TrxR activity was measured in nmolmin-1ml-1 after preparing TNB standard curve.
Where:
ΔB = TNB amount from TNB standard curve in nmol
T2 = Time of second reading (in min)
T1 = Time of first reading (in min)
V = pretreated sample volume (ml) added into the reaction well
2.4.8 Estimation of the level of Malondialdehyde (MDA).
Cakmak and Horst [34] method was used for the estimation of the level of MDA (Malondialdehyde). For this purpose, 1g of leaf samples were ground in 20 mL of trichloroacetic acid (0.1%). After homogenization, this solution was poured into eppendorf tubes (1.5 mL) and centrifuged at 12000 rpm for 10 minutes. 4 mL of mixed solution of trichloroacetic acid (20%) and thiobarbituric acid (0.5%) was taken and added into 1 mL of supernatant. This solution was heated at 100 °C for 30 minutes in water bath (Model: DK-98-IIA; Dimensions: 355×240×190mm) and then suddenly cooled down on ice bath (Model: CB-200D-IB Electronic Ice Bucket; Dimensions: 190 x 240 x 225 mm). This solution was centrifuged at 12,000 rpm for 10 minutes and supernatant was separated to take the absorbance at 532 & 600 nm. The amount of MDA was determined by using the extinction coefficient of 155/(mM/cm) using the relation shown in equation below, where A is the absorbance.
2.4.9 Estimation of the level of ascorbate.
Estimation of ascorbate level was taken by mixing 100 μL metaphosphoric acid (0.1%), 100 μL enzyme extract, and 1 mL dichloroindophenol into 900 μL distilled water. The solution was mixed well using vortex (Velp Scientifica SA202A0176 - ZX3), 200 μL/sample was loaded on 96 well ELISA plate and absorbance was taken at 520 nm [35]. The amount of ascorbate was taken as μg/mL.
2.4.10 Estimation of proline contents.
For the estimation of proline contents, 0.1 g leaf plant samples were taken and homogenized into 5mL of 5-sulfosalicylic acid (3%). Acid ninhydrin was prepared by dissolving 1.25 g of ninhydrin into 20 mL of phosphoric acid (6 M) and 30 mL of glacial acetic acid. The plant extract was reacted with 2 mL acid ninhydrin and 2 mL glacial acetic acid at 100 °C for 60 minutes. This reaction was ended in ice bath. After the addition of 1 mL toluene, the absorbance was recorded at 520 nm [36]. Proline contents were determined by following equation:
Where:
A520 = Sample absorbance at 520nm absorbance
Slope = Determined by linear regression (expressed as absorbance∙nmol-1)
Volextract = Total volume of the extract
Volaliquot = Volume used in the assay
FW = Amount of plant material extracted (expressed in mg)
2.4.11 Measurement of the phytoalexin (Capsidiol) level.
Color reaction in thin layer chromatography was used to identify the phytoalexin (Capsidiol). Pure capsidiol (known concentrations) was mixed with ethyl acetate and poured into eppendorf tubes (1.5 mL). After the evaporation of ethyl acetate, residue was redissolved into ethanol absolute (0.5 mL) with vertexing. 0.5 mL of vanillin (2%) was gently added into each eppendorf tubes and vortexed carefully. These tubes were incubated for 10 minutes at the room temperature. Color reactions produced by the different amounts of capsidiol were analyzed by taking absorbance at 200–700 nm by using a spectrophotometer [37].
2.5 Statistical analysis
Analysis of variance (ANOVA) was used to test the effects of different treatments on disease incidence of Fusarium wilt and biochemical attributes of chilli plants with the least significant difference (LSD) at p<0.05 using Statistics 8.1 software. Moreover, Pearson correlation analysis between various enzymes of antioxidant defense system was performed in OriginPro 2023.
2.6 Computational analysis of selected enzymes for biochemical metabolism
The complete protein sequences of the superoxide dismutase (NP_001311927.1), peroxidase (AAL35364.1), catalase (NP_001311603.1), chitinase (ACM47315.1), and ribulose-1,5-bisphosphate carboxylase/oxygenase (AID55408.1) metabolic enzymes from Capsicum annuum were obtained from the NCBI GenBank database (https://www.ncbi.nlm.nih.gov). The 3D models of the chosen metabolic enzymes were acquired from the online Alpha Fold Protein Structure Database, a resource jointly developed by EMBL-EBI and DeepMind (https://alphafold.ebi.ac.uk/) [38]. The PDB files for the chosen metabolic enzyme models were accessed, and the 3D representations of these selected metabolic enzymes from the Capsicum annuum were visualized using the freely accessible Discovery Studio software (https://discover.3ds.com/discovery-studio-visualizer-download). The precision of the metabolic enzyme models in the Capsicum annuum was verified using the freely accessible SAVES server available online which has been used to study cytochrome b protein ((https://saves.mbi.ucla.edu/) [39]. The ERRAT server was used to establish the overall quality factor for the non-bonded atomic interactions in all the chosen metabolic enzyme models in the Capsicum annuum (https://servicesn.mbi.ucla.edu/ERRAT/) [40]. The PROCHECK server was employed to predict the stereochemical characteristics of the overall structural geometry of the chosen metabolic enzyme models in the Capsicum annuum with reference to amylase studies in different model organisms (https://servicesn.mbi.ucla.edu/PROCHECK/) [41, 42]. The interactions between proteins and distinct metal ions play a pivotal role in various physiological processes. MIB2 (metal ion-binding) strives to address the constraint by structure-based prediction methods, as numerous proteins still lack resolved structures [43]. Nine metal ions (Ca2+, Cu2+, Fe3+, Mg2+, Mn2+, Zn2+, Ni2+, Co2+, and Ba2+) which have major and minor contributions in plants’ nutrition were extensively docked with five selected metabolic enzymes to find their suitable binding sites [44]. Moreover, MIB2 provides enhanced predictive accuracy and encompasses a broader range of metal ion types (http://combio.life.nctu.edu.tw/MIB2/) [45].
3. Results
3.1 Impact of plant nutrients on disease incidence
Among evaluated plant nutrients, the combination of Krystafeed and Micro Plus (T3) exhibited minimum disease incidence under greenhouse and field conditions followed by Micro Plus (T2) and Krystafeed (T1) as compared to the control (Fig 1). In case of interaction between treatments and concentrations (T × C), the combination of Krystafeed and Micro Plus showed minimum disease incidence (29.41, 27.13, 24.51%) at C1, C2 and C3, concentrations respectively, followed by Micro Plus (32.66, 29.6, 26.43%) and Krystafeed (41.12, 37.96, 35.57%) as compared to the control under greenhouse conditions. While under field conditions, the interaction between T × C showed that T3 exhibited significant results with least disease incidence (31.52, 25.62, 22.63%) followed by T2 (35.76, 32.33, 28.98%) and T1 (44.01, 40.81, 38.63%) as compared to the control (Fig 2).
The P-values for treatments (T), concentrations (C), and their interaction (T*C) showed significant statistical differences (p<0.05) among them.
The P-values for treatments (T), concentrations (C), and their interaction (T*C) showed significant statistical differences (p<0.05) among them.
3.2 Impact of plant nutrients on biochemical compounds
3.2.1 Activity of SOD, POD, CAT, and H2O2.
All biochemical enzymes, including superoxide dismutase, peroxidase, catalase, and hydrogen peroxide, were significantly increased in the treated (T) plants of resistant (Bird eye, Sayban, HP-1810 F1) and susceptible (Maxi, BS Hari Rani, Janbaz-F1) varieties of chilli (Fig 3). Application of most effective treatment T3 at C3 significantly (p<0.05) increased levels of the superoxide dismutase (0.37, 0.45, 0.71, 0.56, 0.49, 0.46)μg/g, peroxidase (0.08, 0.08, 0.11, 0.05, 0.05, 0.045)μg/g, catalase (0.44, 0.43, 0.6, 0.62, 0.393, 0.52)μg/g, and hydrogen peroxide (0.34, 0.24, 0.61, 0.56, 0.72, 0.49)μg/g in the treated plants of chilli varieties, such as Maxi, BS Hari Rani, Janbaz-F1, Bird eye, and Sayban, HP-1810 F1, respectively, as compared to the untreated plants (Fig 3).
Small letters define the statistical significance (p<0.05) in different treated (T) and Un-treated (UT) plants. Capital letters indicate significant difference (p<0.05) among the resistant and susceptible varieties of chilli.
3.2.2 Impact on phenolic contents and photosynthetic pigments.
Total phenolic contents, chlorophyll a, chlorophyll b, and total chlorophyll were significantly (p<0.05) increased in the treated plants (T3 at C3 concentration) of resistant varieties (Bird eye, Sayban, HP-1810 F1) by (29.51, 18.48, 34.18%), (18.18, 19.23, 28%), (50, 50, 72.72%) and (29.41, 27.08, 34.14%), respectively, as compared to the untreated plants. While the amount of TPC, Chl a, Chl b, and total Chl was significantly increased in the treated plants of susceptible varieties (Maxi, BS Hari Rani, Janbaz-F1) by (25.44, 15.71, 10.97%), (58.33, 31.25, 17.64%), (22.22, 5.26, 27.27%), and (24, 14.28, 21.21%), respectively as compared to the untreated plants (Fig 4).
Small letters define the statistical significance (p<0.05) in different treated (T) and Un-treated (UT) varieties. Capital letters indicate significant difference (p<0.05) among resistant and susceptible varieties of chilli.
3.2.3 Activity of Tocophherol, TrxR, MDA, ascorbate.
Application of the most effective treatment (T3) significantly (p<0.05) increased tocopherol (2.13, 3.15, 1.69), (3.41, 3, 3.31)μg/mL, thioredoxin reductase (1.21, 1.31, 1.67), (7.03, 6.75, 6.66) mU/mL, malondialdehyde (1.49, 0.82, 1.35), (2.54, 2.01, 1.84) nM, and ascorbate (80, 56, 44), (125, 110, 115)μg/mL in the treated (T) plants as compared to the untreated (UT) plants of resistant (Bird eye, Sayban, HP-1810 F1) and susceptible (Maxi, BS Hari Rani, Janbaz-F1) chilli varieties, respectively (Fig 5).
Small letters define the statistical significance (p<0.05) in different treated (T) and Un-treated (UT) plants of chilli. Capital letters indicate significant differences (p<0.05) among resistant and susceptible varieties of chilli.
3.2.4 Impact on proline and capsidiol contents.
The proline contents of resistant varieties (Bird Eye, Sayban, HP-1810 F1) significantly increased by 5.40%, 3.79%, and 8.64%, respectively, in treated plants compared to untreated ones. Similarly, susceptible varieties (Maxi, BS Hari Rani, Janbaz-F1) showed significant increases of 80.95%, 39.28%, and 57.69%, respectively, in proline content in treated plants compared to untreated plants (Fig 6). The concentration of capsidiol increased by 14.81%, 3.57%, and 15.62% in treated plants of resistant varieties (Bird Eye, Sayban, HP-1810 F1), respectively. In contrast, the increase in treated plants of susceptible varieties (Maxi, BS Hari Rani, Janbaz-F1) was 58.33%, 53.84%, and 53.07%, respectively, compared to untreated plants (Fig 6).
Small letters define the statistical significance (p<0.05) in different treated (T) and Un-treated (UT) varieties. Capital letters indicate significant difference (p<0.05) among resistant and susceptible varieties of chilli.
3.3 Correlation analysis
The Pearson correlation analysis showed that there is a strong correlation exists between various enzymes of antioxidant defense system (Fig 7). Superoxide dismutase was positively correlated with peroxidases (r = 0.99), catalases (r = 0.95), hydrogen peroxide (r = 0.93), total phenolic contents (r = 0.97), total chlorophyll (r = 0.81), chlorophyll a (r = 0.82), chlorophyll b (r = 0.84), α-Tocopherol (r = 0.95), TrxR (r = 0.99), MDA (r = 0.95), ascorbate (r = 0.95), prolin (r = 0.95), and phytoalexin (r = 0.98).
3.4 Computationally exploration of metabolic enzymes
The full-length protein sequences from NCBI database were used to generate 3D model of the superoxide dismutase (NP_001311927.1), peroxidase (AAL35364.1), catalase (NP_001311603.1), chitinase (ACM47315.1), and ribulose-1,5-bisphosphate carboxylase/oxygenase (AID55408.1) metabolic enzymes from Capsicum annuum. The 3D model generated through alphaFold was further validated through ERRAT, Verify3D and PROCHECK (Fig 8a–8e). The overall quality of the 3D models was more than 80%, indicating the proper folding of the selected metabolic enzymes (Table 2). The refined 3D models of selected metabolic enzymes were used for docking through the MIB2 server to explore potential metal ions binding sites of selected (Ca2+, Cu2+, Fe3+, Mg2+, Mn2+, Zn2+, Ni2+, Co2+, and Ba2+) plants-based nutrients. The binding sites of metals ions in superoxide dismutase metabolic enzyme were predicted for Ca2+ (52H, 193H), Cu2+ (158D, 160E, 181N), Fe3+(52H, 100H, 189D, 193H), Mg2+(158D, 160E, 161L, 181N), Mn2+ (52H, 100H, 189D, 193H), Zn2+ (189D, 193H), Ni2+ (52H, 57H), Co2+ (52H, 56H), and Ba2+ (79D, 80S, 81P, 82T) (Fig 9a–9c). Similarly, the metal binding sites of (Ca2+, Cu2+, Fe3+, Mg2+, Mn2+, Zn2+, Ni2+, Co2+, and Ba2+) (S2 File) the superoxide dismutase, peroxidase, catalase, chitinase, and ribulose-1,5-bisphosphate carboxylase/oxygenase metabolic enzymes from Capsicum annuum were enlisted in Table 3.
A. Three-dimensional ribbon model showed in forest green and surface model showed in cornflower blue color structure of superoxide dismutase enzyme from Capsicum annuum predicted through AlphaFold. Verification of superoxide dismutase metabolic enzyme models through Ramachandran plot. Red color showed the most favored region. The yellow color shows the additional allowed region (a, b, l, p). Generally, the allowed region (~a, ~b, ~l, ~p) is highlighted in light yellow color. The Ramachandran plots were generated through precheck tool. B. Three-dimensional ribbon model showed in forest green and surface model showed in cornflower blue structure of peroxidase enzyme from Capsicum annuum predicted through AlphaFold. Verification of peroxidase metabolic enzyme models through Ramachandran plot. Red color showed the most favored region. The yellow color shows the additional allowed region (a, b, l, p). Generally, the allowed region (~a, ~b, ~l, ~p) is highlighted in light yellow color. The Ramachandran plots were generated through precheck tool. C. Three-dimensional ribbon model showed in forest green and surface model showed in corn cornflower blue structure of catalase enzyme from Capsicum annuum predicted through AlphaFold. Verification of catalase metabolic enzyme models through Ramachandran plot. Red color showed the most favored region. The yellow color shows the additional allowed region (a, b, l, p). Generally, the allowed region (~a, ~b, ~l, ~p) is highlighted in light yellow color. The Ramachandran plots were generated through precheck tool. D. Three-dimensional ribbon model showed in forest green and surface model showed in cornflower blue color structure of chitinase enzyme from Capsicum annuum predicted through AlphaFold. Verification of chitinase metabolic enzyme models through Ramachandran plot. Red color showed the most favored region. The yellow color shows the additional allowed region (a, b, l, p). Generally, the allowed region (~a, ~b, ~l, ~p) is highlighted in light yellow color. The Ramachandran plots were generated through precheck tool. E. Three-dimensional ribbon model showed in forest green and surface model showed in cornflower blue color structure of ribulose-1,5-bisphosphate carboxylase/oxygenase enzyme from Capsicum annuum predicted through AlphaFold. Verification of ribulose-1,5-bisphosphate carboxylase/oxygenase metabolic enzyme models through Ramachandran plot. Red color showed the most favored region. The yellow color shows the additional allowed region (a, b, l, p). Generally, the allowed region (~a, ~b, ~l, ~p) is highlighted in light yellow color. The Ramachandran plots were generated through precheck tool.
(a-c). Ribbon surface model of superoxide dismutase metabolic enzyme with metals ions. The predicted nutritional ions binding site for Ca2+ (52H, 193H), Cu2+ (158D, 160E, 181N), Fe3+ (52H, 100H, 189D, 193H), Mg2+ (158D, 160E, 161L, 181N), Mn2+ (52H, 100H, 189D, 193H), Zn2+ (189D, 193H), Ni2+ (52H, 57H), Co2+ (52H, 56H), and Ba2+ (79D, 80S, 81P, 82T).
4. Discussion
Mineral nutrients have a decisive role in the life of chilli plants as they act as the first line of defense against the invading pathogens by producing various types of enzymes and metabolites [46, 47]. That’s the reason that our study was carried out to induce resistance in chilli plants against Fusarium wilt disease by using macro- and micro- nutrients. In contemporary study, the combination of Krystafeed and Micro Plus (T3) demonstrated significant effects in reducing Fusarium wilt disease of chilli under the greenhouse and field conditions. Results of our study are in line with Bashir et al. [48] For the related study, they evaluated Nutritop (Zn, Br, Fe) and Compound (N, P, K) against the Fusarium wilt of chilli, which concluded that the combination of Compound and Nutritop showed significant inhibition effects towards Fusarium wilt. The previous study had shown that the application of macro- and micro- nutrients decreased the percentage of plant mortality caused by different pathogens by regulating different physiological processes (photosynthesis, respiration, transpiration, and defense) by activating different genes [49], which are responsible for the production of various enzymes, such as Rubisco, Transglutaminases (TGases), hexokinase, phosphofructokinase (PFK), acetyl CoA, α-Amylase, Pyruvic kinase, Superoxide dismutase, Peroxidase, Catalase, chitinase, and polyphenol oxidase.
Nitrogen plays an essential role in defensive primary and secondary metabolism [50] by the production of different enzymes and proteins (antimicrobial proteins (AMPs) and pathogen related proteins (PRPs). In response to the infection, incited by pathogens, these PRPs, AMPs, β-glucanase, and chitinase can degrade dextran or chitin present in the cell wall of fungi [51]. Balanced dose of nitrogen increased the activity of defense related enzymes β-1,3-glucanase (PR-2 protein), chitinase (PR3), and chitosanase, which is positively correlated with plant disease resistance [13, 52]. Another study documented the increased expression of PR4 with the increased nitrogen application [53]. N nutrition can also influence defense via amino acid metabolism and hormone production to affect the downstream defense-related gene expression via transcriptional regulation and nitric oxide (NO) production [13]. The above findings clearly indicated that nitrogen fertilizer have positive effects on disease resistance by expressing various pathogenesis-related proteins and enzymes.
Phosphorus application in plants stimulated the production of enzymes, such as acid phosphatases, alkaline phosphatases, and phosphoenolpyruvate carboxylase, which are involved in various metabolic processes. Moreover, P plays a prime role in the plants to perform various functions like coenzymes NAD, NADP, and ATP [54, 55]. Potassium is a key element which is involved in defense mechanism by regulating stomatal regulation, photosynthesis, protein, cell signaling, cellulose, starch formation, hardening of tissues, enzymatic activation, carbohydrate metabolism, and protein synthesis [56, 57]. As potassium regulates more than 60 enzymes in the plant body for the activation of various physiological processes [56], its deficiency directly or indirectly leads towards abnormal functioning and disease development [58].
Manganese involves in the production of phenolic compounds [59], which stimulates the metabolism of carbohydrate α-ketoglutarate (KG), DNA/ RNA formation, and oxidative phosphorylation [60]. As compared to macronutrients, micronutrients are required by plants in small amounts and assist as the major components of prosthetic groups in metalloproteins (catalyze redox processes by electron transfer (Fe, Mn, Cu, Mo) and activators of various enzymatic reactions (coupling enzymes and substrate to form enzyme-substrate complexes (Fe and Zn), which increase enzymatic reactions by altering the molecular configuration of enzymes (Zn). Moreover, the application of zinc is helpful in reducing disease incidence in infected plants through affecting the resistance indirectly by improving structural integrity and permeability of cell membranes [61].
In our study, the application of macro- and micro- nutrients decreased the disease incidence of Fusarium wilt by increasing superoxide dismutase, peroxidases, catalases, hydrogen peroxide, total phenolic contents, chlorophyll contents (Chlorophyll a, chlorophyll b and total chlorophyll), tocopherol, thioredoxin reductase, malondialdehyde, ascorbate, phytoalexins, and proline. Results of our study are in line with the previous study of Shoaib and Awan [46]. For their study, they concluded that application of macro- and micro- nutrients (N, P, K, Zn, Mg, B) significantly inhibited the fungal diseases by increasing the activity of components of antioxidant defense system (SOD, POD, CAT, TPC, Chlorophyll contents, PAL, and PPO) (Fig 10).
SOD plays significant roles against various reactive oxygen species (O2-) and free radicals produced in host plants in response to biotic and abiotic stresses [62, 63]. Moreover, SOD enzyme accumulates H2O2, which diffuses into cells to act as an excellent antioxidant. This accumulated H2O2 is used to perform multiple functions in plant defense against invading pathogens. H2O2 is a very critical component for the synthesis of phytoalexin- defense related compounds, which acts as the retardant at the site of infection by inhibiting pathogen growth, restricts the pathogen spread by triggering programmed cell death (HR), acts as cell to cell signal for the induction of systemic acquired resistance (SAR) [64], and regulates or activates defense related genes [65]. However, the excessive amount of H2O2 is lethal to plants, which can be neutralized by two enzymes named POD and CAT. These two enzymes prevent the accumulation of H2O2 in the cells and converts it into water and oxygen [66].
Total phenolic contents (TPC) protect the major tissues of plants from the lethal effects of ROS and help plants to evade pathogenic infections [67] by minimizing the effects to enzymes, which degrade plant cell wall, inhibit the entry of pathogen secreted enzymes and toxins into plant cells, and stops the flow of nutrients towards pathogens [68]. Proline plays its significant role in plant defense activation by contributing as signaling molecule, antioxidative defense molecule, and metal chelator [69]. Catabolism and biosynthesis of proline produce pyrroline-5-carboxylate (P5C) which plays a significant role in both non-host and R-gene mediated resistance against invading pathogens [70].
Tocopherols are lipid soluble antioxidant molecules, present in plastids and thylakoid membrane, which are responsible for conforming resistance in plants by reducing ROS level in photosynthetic machinery and protect the thylakoid membrane from lipid peroxidation against various biotic stresses [71, 72]. Thioredoxin reductase (TrxR) plays a crucial role in plant defense by maintaining cellular redox homeostasis, regulating gene expression, modulating protein function, and detoxifying reactive oxygen and nitrogen species [73]. Lipid peroxidation is a process which initiates under oxidative stress conditions when reactive oxygen species (ROS) attack polyunsaturated fatty acids in cell membranes, which increase the production of malondialdehyde (MDA). While MDA itself does not have a direct role in plant defense, its accumulation is often used as a marker for oxidative damage and stress in plants [74]. Phytoalexins are low-molecular-weight antimicrobial compounds produced by plants in response to pathogen attack or other stresses [75]. Capsidiol accumulates at the site of infection and acts as a defense molecule by inhibiting the growth and proliferation of the invading pathogens. It directly interferes with pathogen metabolism, disrupts cellular processes, and hampers pathogen survival and reproduction. Our study is supported by the above findings, which clearly justifies the role of various biochemical constituents in plant defense and enhancement of these enzymes through the application of macro- and micro- nutrients, leading towards the activation of defense related responses.
5. Conclusion
This study investigated the role of plant nutrients against Fusarium wilt disease of chilli. Results of our study confirmed that the application of macro- and micro- nutrient significantly reduced the incidence of Fusarium wilt in chilli plants. Moreover, the application of T3 (Krystafeed 400 and Microplus 40 g/100ft2) significantly improved the biochemical constituents (SOD, POD, CAT, H2O2, TrxR, MDA, Ascorbate, Chlorophyll contents, Phenolic contents, and Capsidiol) of chilli plant (S1 File). In conclusion, the application of plant nutrients can be a viable and healthy approach for the management of Fusarium wilt of chilli and potentially other pathogen controls.
Supporting information
S1 File. Raw data of manuscript which was subjected to statistical analysis.
https://doi.org/10.1371/journal.pone.0309738.s001
(XLSX)
S2 File. Supplementary data of enzymes and metal ions.
https://doi.org/10.1371/journal.pone.0309738.s002
(ZIP)
Acknowledgments
We are very much thankful to the scientists and researchers of Molecular Phyto-bacteriology Laboratory for providing research facilities under HEC-NRPU # 9315.
References
- 1. Thangjam B, Chanu WT, Mayanglambam B. Fungal diseases of chilli and their management. AgriCos e-Newslet. 2020; 1: 47–49.
- 2. Gabrekiristos E, Demiyo T. Hot pepper Fusarium wilt (Fusarium oxysporum f. sp. capsici): Epidemics, characteristic features and management options. J Agri Sci. 2020; 12: 347–360.
- 3. Iftikhar S, Bengyella L, Shahid AA, Nawaz K, Anwar W, Ali S. Discovery of succinate dehydrogenase candidate fungicides via lead optimization for effective resistance management of Fusarium oxysporum f. sp. capsici. Biotech. 2021; 12: 102. pmid:35463042
- 4. Demissie S, Megersa G, Meressa BH, Muleta D. Resistance levels of Ethiopian hot pepper (Capsicum spp.) varieties to a pathogenic Fusarium spp. and in vitro antagonistic effect of Trichoderma spp. Arch Phytopathol Plant Prot. 2020; 54: 647–663.
- 5. Mannan MA, Hossain MM, Rahman MM. Survey on the abundance of major insect pests and diseases and their management approaches in different chilli growing areas of Jamalpur region. J Entomol Zool Stud. 2020; 8: 113–116.
- 6. Nasir F. Eco-friendly management of Fusarium wilt disease of chillies caused by Fusarium oxysporum f. sp. capsici. Asian J Emerg Res. 2020; 2: 182–183.
- 7. Atiq M, Adil S, Rajput NA, Sahi ST, Usman M, Iqbal S, et al. Activation of Capsicum annuum L. defense system against Fusarium wilt through plant activators and their impact on horticultural attributes. Int J Phytopathol. 2021; 10: 155–165.
- 8. Edel-Hermann V, Lecomte C. Current status of Fusarium oxysporum formae speciales and races. Phytopathol. 2019; 109: 512–530. pmid:30461350
- 9. Nadeem F, Hanif MA, Majeed MI, Mushtaq Z. Role of macronutrients and micronutrients in the growth and development of plants and prevention of deleterious plant diseases–A comprehensive review. Int J Chem Biochem Sci. 2018; 12: 31–52.
- 10.
Ojha RK, Jha SK. Role of mineral nutrition in management of plant diseases. Farmers’ Prosperity through Improved Agricultural Technologies. 2020; 241–261.
- 11. Koch M, Naumann M, Pawelzik E, Gransee A, Thiel H. The importance of nutrient management for potato production Part I: Plant nutrition and yield. Potato Res. 2020; 63: 97–119.
- 12.
Tiwari S, Patel A, Pandey N, Raju A, Singh M, Prasad SM. Deficiency of essential elements in crop plants. In sustainable solutions for elemental deficiency and excess in crop plants. Springer, Singapore. 2020; 19–52.
- 13. Sun Y, Wang M, Mur LAJ, Shen Q, Guo S. Unraveling the roles of nitrogen nutrition in plant disease defenses. Int J Mol Sci. 2020; 21: 572. pmid:31963138
- 14.
Tanaka A. Interactions between plant pathogens and their implications for crop protection. Department of Forest Mycology and Plant Pathology, Swedish University of Agricultural Sciences, SLU. 2021.
- 15.
Ali MA, Fahad S, Haider I, Ahmed N, Ahmad S, Hussain S, et al. Oxidative stress and antioxidant defense in plants exposed to metal/metalloid toxicity. In: Reactive Oxygen, Nitrogen and Sulfur Species in Plants: Production, Metabolism, Signaling and Defense Mechanisms. Wiley Publishing Company, New Jersey, USA. 2019; 353–370.
- 16. Juan CA, Lastra P, Plou FJ, Pérez-Lebeña E. The chemistry of reactive oxygen species (ROS) revisited: outlining their role in biological macromolecules (DNA, lipids and proteins) and induced pathologies. Int J Mol Sci. 2021; 22: 4642. pmid:33924958
- 17. Kapoor D, Singh S, Kumar V, Romero R, Prasad P, Singh J. Antioxidant enzymes regulation in plants in reference to reactive oxygen species (ROS) and reactive nitrogen species (RNS). Plant Gene. 2019; 19: 100182.
- 18. Usman M, Atiq M, Rajput NA, Sahi ST, Shad M, Lili N, et al. Efficacy of green synthesized silver based nanomaterials against early blight of tomato caused by Alternaria solani. Gesunde Pflanzen. 2023 Dec 7:1–7.
- 19. Chandrawat B, Siddiqui A, Bhati S, Saharan V. Bioagents: A source for initiation of defense enzymes in chilli infected with root-knot nematode, Meloidogyne incognita. J Entomol Zool Stud. 2020; 8: 1684–1688.
- 20. Chhabra R, Kaur S, Vij L, Gaur K. Exploring physiological and biochemical factors governing plant pathogen interaction: A review. Int J Curr Microbiol App Sci. 2020; 9: 1650–1666.
- 21. Tahir ZB, Atiq M, Rajput NA, Akram A, Arif AM, Iqbal S, et al. Determination of biochemical baseline of resistance against bacterial leaf spot of chilli after application of plant defense activators. J Glob Innov Agri Sci. 2023; 11: 61–67.
- 22. Atiq M, Fatima T, Rajput NA, Usman M, Kachelo GA, Ahmad U, et al. Moringa olefra plant based Copper and Silver nanoparticles and its antibacterial activity towards leaf spot of chilli caused by Xanthomonas campestris pv. vesicatoria. Int J Phytopathol. 2023; 12: 63–72.
- 23. Singhal RK, Fahad S, Kumar P, Choyal P, Javed T, Jinger D, et al. Beneficial elements: New Players in improving nutrient use efficiency and abiotic stress tolerance. Plant Growth Reg. 2023; 100: 237–265.
- 24. Maitlo SA, Syed RN, Rustamani MA, Khuhro RD, Lodhi AM. Influence of inoculation methods and inoculum levels on the aggressiveness of Fusarium oxysporum f. sp. ciceris on chickpea and plant growth. Int J Agr Biol. 2016; 18: 31–36.
- 25. Monaim AMF, Ismail ME. The use of antioxidants to control root rot and wilt diseases of pepper. Not Sci Biol. 2010; 2: 46–55.
- 26. Giannopolitis CN, Ries SK. Superoxide Dismutases. Plant Physiol. 1977; 59: 309–314. pmid:16659839
- 27. Chance B, Maehly AC. [136] Assay of catalases and peroxidases. Meth Enzymol. 1955; 2: 764–775.
- 28. Velikova V, Yordanov I, Edreva A. Oxidative stress and some antioxidant systems in acid rain-treated bean plants. Plant Sci. 2000; 151: 59–66.
- 29. Julkunen-Tiitto R. Phenolic constituents in the leaves of northern willows: methods for the analysis of certain phenolics. J Agri Food Chem. 1985; 33: 213–217.
- 30. Iqbal M, Haq Z, Malik A, Ayoub CM, Jamil Y, Nisar J. Presowing seed magnetic field stimulation: a good option to enhance bitter gourd germination, seedling growth and yield characteristics. Biocatal Agri Biotechnol. 2016; 5: 30–37.
- 31. Haq Z, Iqbal M, Jamil Y, Anwar H, Younis A, Arif M, et al. Magnetically treated water irrigation effect on turnip seed germination, seedling growth and enzymatic activities. Info Pro Agr. 2016; 3: 99–106.
- 32. Kayden HJ, Chow CK, Bjornson LK. Spectrophotometric method for determination of tocopherol in red blood cells. J Lipid Res. 1973; 14: 533–540. pmid:4729970
- 33.
Arnér ES, Zhong L, Holmgren A. Preparation and assay of mammalian thioredoxin and thioredoxin reductase. In Methods in enzymology. Elsevier Publishers, Amsterdam, Netherlands. 1999; 226–239.
- 34. Cakmak I, Horst WJ. Effect of aluminum on lipid peroxidation, superoxide dismutase, catalase, and peroxidase activities in root tips of soybean (Glycine max) Physiology. Plants. 1991; 83: 463–468.
- 35. Tabart J, Kevers C, Evers D, Dommes J. Ascorbic acid, phenolic acid, flavonoid, and carotenoid profiles of selected extracts from Ribes nigrum. J Agr Food Chem. 2011; 59: 4763–4770.
- 36. Ünyayar S, Keleþ Y, Ünal E. Proline and ABA levels in two sunflower genotypes subjected to water stress. Bul J Plant Physiol. 2004; 30: 34–47.
- 37. Chávez-Moctezuma MP, Lozoya-Gloria E. Biosynthesis of the sesquiterpenic phytoalexin capsidiol in elicited root cultures of chili pepper (Capsicum annuum). Plant Cell Rep. 1996; 15: 360–366. pmid:24178358
- 38. Jumper J, Evans R, Pritzel A, Green T, Figurnov M, Ronneberger O, et al. Highly accurate protein structure prediction with AlphaFold. Nat. 2021; 596: 583–589.
- 39. Shad M, Hussain N, Usman M, Akhtar MW, Sajjad M. Exploration of computational approaches to predict the structural features and recent trends in α-amylase production for industrial applications. Biotechnol Bioeng. 2023; 120: 2092–2116. pmid:37475649
- 40. Colovos C, Yeates TO. Verification of protein structures: patterns of non-bonded atomic interactions. Pro Sci. 1993; 2: 1511–1519. pmid:8401235
- 41. Laskowski RA, MacArthur MW, Moss DS, Thornton JM. PROCHECK: a program to check the stereochemical quality of protein structures. J App Crystal. 1993; 26: 283–291.
- 42. Shad M, Usman M, Gardner QA. Structural-Functional characterization of cytochrome b in bc1 and b6 f complexes along with polymorphic analysis. Pak J Zool. 2023; 55: 975–983.
- 43. Shad M, Nazir A, Usman M, Akhtar MW, Sajjad M. Investigating the effect of SUMO fusion on solubility and stability of amylase-catalytic domain from Pyrococcus abyssi. Int J Biol Macromol. 2024; 266: 131310.
- 44. Shad M, Sajjad M, Gardner QA, Ahmad S, Akhtar MW. Structural engineering and truncation of α-amylase from the hyperthermophilic archaeon Methanocaldococcus jannaschii. Int J Biol Macromol. 2024; 256: 128387. pmid:38000593
- 45. Lu CH, Chen CC, Yu CS, Liu YY, Liu JJ, Wei ST, et al. MIB2: metal ion-binding site prediction and modeling server. Bioinfor. 2022; 38: 4428–4429. pmid:35904542
- 46. Shoaib A, Awan ZA. Mineral fertilizers improve defense related responses and reduce early blight disease in tomato (Solanum lycopersicum L.). J Plant Pathol. 2021; 103: 217–229.
- 47. Cabot C, Martos S, Llugany M, Gallego B, Tolrà R, Poschenrieder C. A role for zinc in plant defense against pathogens and herbivores. Front Plant Sci. 2019; 10: 1171. pmid:31649687
- 48. Bashir MR, Atiq M, Mohsin M, Raheel M, Abbas W. Management of Fusarium wilt of chilli caused by Fusarium oxysporum f. sp. capsici through nutritional amendments under greenhouse conditions. Int J Biosci. 2017; 10: 185–191.
- 49.
Pramudyani L, Sabur A, Yasin M, Yani W. Red chili performance in acid upland applied with biofertilizer. In IOP Conference Series: Earth Environ Sci. 2021; 648: 012163.
- 50. Mur LA, Simpson C, Kumari A, Gupta AK, Gupta KJ. Moving nitrogen to the center of plant defense against pathogens. Ann Bot. 2017; 119: 703–709. pmid:27594647
- 51.
Dar AS, Al Galil AFM. Biodegradation, biosynthesis, isolation, and applications of chitin and chitosan. In Handbook of biodegradable materials (pp. 1–42). Springer International Publishing, Cham, Switzerland. 2022; pp. 1–42.
- 52. Jin X, Hao N, Jiao F, Yang Y, Wang D, Xu C, et al. The effect of nitrogen supply on potato yield, tuber size and pathogen resistance in Solanum tuberosum exposed to Phytophthora infestans. African J Agri Res. 2014; 9: 657–2663.
- 53. Vega A, Canessa P, Hoppe G, Retamal I, Moyano TC, Canales J, et al. Transcriptome analysis reveals regulatory networks underlying differential susceptibility to Botrytis cinerea in response to nitrogen availability in Solanum lycopersicum. Front Plant Sci. 2015; 6: 911. pmid:26583019
- 54. Lovio-Fragoso JP, de Jesús-Campos D, López-Elías JA, Medina-Juárez LA, Fimbres-Olivarría D, Hayano-Kanashiro C. Biochemical and molecular aspects of phosphorus limitation in diatoms and their relationship with biomolecule accumulation. Biol. 2021; 10: 565. pmid:34206287
- 55. Delgado M, Henríquez-Castillo C, Zuniga-Feest A, Sepúlveda F, Hasbún R, Hanna P, et al. Cluster roots of Embothrium coccineum modify their metabolism and show differential gene expression in response to phosphorus supply. Plant Physiol Biochem. 2021; 161: 191–199. pmid:33621863
- 56. Johnson R, Vishwakarma K, Hossen MS, Kumar V, Shackira AM, Puthur JT, et al. Potassium in plants: Growth regulation, signaling, and environmental stress tolerance. Plant Physiol Biochem. 2022; 172: 56–69. pmid:35032888
- 57. Oosterhuis DM, Loka DA, Kawakami EM, Pettigrew WT. The physiology of potassium in crop production. Adv Agron. 2014; 126: 203–233.
- 58. Hasanuzzaman M, Bhuyan MB, Nahar K, Hossain MS, Mahmud JA, Hossen MS, et al. Potassium: a vital regulator of plant responses and tolerance to abiotic stresses. Agron. 2018; 8: 31–39.
- 59. Farzadfar S, Zarinkamar F, Hojati M. Magnesium and manganese affect photosynthesis, essential oil composition and phenolic compounds of Tanacetum parthenium. Plant Physiol Biochem. 2017; 112: 207–217. pmid:28092849
- 60.
Poon L, Johannink P, Appanna VP, Appanna VD. Metabolic reprogramming triggered by abiotic stress: A treasure-trove of bio-based technologies. In Microbial Diversity in the Genomic Era. Elsvier, NY, US. 2024; pp. 217–232.
- 61. Machado PP, Steiner F, Zuffo AM, Machado RA. Could the supply of boron and zinc improve resistance of potato to early blight. Potato Res. 2018; 61: 169–182.
- 62. Stephenie S, Chang YP, Gnanasekaran A, Esa NM, Gnanaraj C. An insight on superoxide dismutase (SOD) from plants for mammalian health enhancement. J Fun Foods. 2020; 68:103917.
- 63. Malik L, Atiq M, Rajput NA, Usman M, Akram A, Jabbar A, et al. Induction of resistance in mungbean against cercospora leaf spot through plant defense activators. Agri Sci J. 2023; 5: 28–34.
- 64. Nasir F, Atiq M, Mubeen I. Biochemical base of resistance against Fusarium wilt of chillies and its management through plant activators. Asian J Emerg Sci. 2020; 2: 174–175.
- 65.
Štolfa Čamagajevac I, Špoljarić Maronić D, Žuna Pfeiffer T, Bek N, Lončarić Z. Nitric oxide and hydrogen peroxide in plant response to biotic stress. In: Nitric Oxide and Hydrogen Peroxide Signaling in Higher Plants. Springer, New York. 2019; 221–243.
- 66. Rajput VD, Singh RK, Verma KK, Sharma L, Quiroz-Figueroa FR, Meena M, et al. Recent developments in enzymatic antioxidant defense mechanism in plants with special reference to abiotic stress. Biol. 2021; 10: 267. pmid:33810535
- 67. Kumar S, Abedin MM, Singh AK, Das S. Role of phenolic compounds in plant-defensive mechanisms. Plant Phenol Sus Agr. 2020; 1: 517–532.
- 68.
Pratyusha S. Phenolic compounds in the plant development and defense: an overview. Plant stress physiology-perspectives in agriculture. 2022; pp.125–140.
- 69. Ghosh UK, Islam NM, Siddiqui NM, Cao X, Khan MAR. Proline, a multifaceted signaling molecule in plant responses to abiotic stress: understanding the physiological mechanisms. Plant Biol. 2022; 24: 227–239. pmid:34796604
- 70. Dubrovna OV, Mykhalska SI, Komisarenko AG. Using proline metabolism genes in plant genetic engineering. Cytol Genet. 2022; 56: 361–378.
- 71.
Ghosh UK, Hossain MS, Islam MN, Khan MAR. Role of tocopherol in conferring abiotic stress tolerance in plants. In Antioxidant Defense in Plants: molecular basis of regulation. 2022; pp. 215–233. Singapore: Springer Nature Singapore.
- 72. Cela J, Tweed JK, Sivakumaran A, Lee MR, Mur LA, Munné-Bosch S. An altered tocopherol composition in chloroplasts reduces plant resistance to Botrytis cinerea. Plant Physiol Biochem. 2018; 127: 200–210. pmid:29609176
- 73. He L, He T, Farrar S, Ji L, Liu T, Ma X. Antioxidants maintain cellular redox homeostasis by elimination of reactive oxygen species. Cell Physiol Biochem. 2017; 44: 532–553. pmid:29145191
- 74. Morales M, Munné-Bosch S. Malondialdehyde: facts and artifacts. Plant Physiol. 2018; 180: 1246–1250. pmid:31253746
- 75.
Tiku AR. Antimicrobial compounds (phytoanticipins and phytoalexins) and their role in plant defense. Co-Evolution of Secondary Metabolites, Springer, New York, USA. 2020; 845–868.