Introduction

Muskmelon (Cucumis melo L.) is a widely cultivated cucurbitaceous crop, valued for its sweet and aromatic fruit. It thrives in tropical and subtropical regions, where favorable climatic conditions enable year-round cultivation1. Globally, muskmelon production is estimated at approximately 28.46 million tonnes2, with India ranking third, producing 1.33 million tonnes over an area of 59,000 hectares2. Despite its economic significance, muskmelon production faces significant challenges due to various pests and pathogens, including bacteria, fungi, viruses, and root-knot nematodes. The increasing cultivation of muskmelon has led to concerns regarding plant health, particularly the prevalence of root diseases in major production areas3, with root-knot nematodes being among the most destructive threats.

Root-knot nematodes (Meloidogyne spp.) are responsible for severe yield losses, with damage exceeding 30% in highly susceptible vegetable and fruit crops, including muskmelon4. Among the most common and damaging species affecting muskmelon are M. incognita, M. javanica, and M. arenaria5. These nematodes induce the formation of galls on muskmelon roots, disrupting the uptake of water and nutrients. Their impact on host plants is evident through symptoms such as root swelling, stunted growth, chlorosis, leaf curling, daytime wilting, and overall plant decline6,7,8. Due to their endoparasitic nature, root-knot nematodes persist in the soil for years, making their management challenging once they are established in agricultural fields.

Additionally, nematode-infested roots become highly susceptible to secondary infections by soil-borne pathogens. The mechanical damage caused by nematode penetration alters host physiology, creating entry points for opportunistic fungi such as Fusarium, leading to complex disease interactions that are difficult to manage and result in significant crop losses6,9,10. Considering these challenges, the present study examines the histopathological alterations in muskmelon roots infected by M. incognita using Scanning Electron Microscopy (SEM). Furthermore, this research aims to assess the biochemical changes induced by M. incognita infestation in muskmelon under controlled pot culture conditions.

Results

Histopathological changes caused by M. incognita through SEM analysis

Scanning Electron Microscopy (SEM) analysis of galled muskmelon root sections revealed significant ultrastructural modifications induced by M. incognita infection. Infected roots exhibited severe anatomical distortions, particularly in the vascular system and storage tissues, making it difficult to discern the normal vascular strand organization. SEM observations of healthy muskmelon roots confirmed typical dicot root features, including a uniseriate epidermis, parenchymatous cortex, and well-organized vascular bundles showing clear differentiation of metaxylem, protoxylem, and pith (Fig. 1-a). In contrast, nematode-infested roots displayed extensive structural damage, including epidermal and cortical deformation, vascular tissue disruption, and the presence of irregularly shaped cells and cavities in the cortex (Fig. 1-b).

Fig. 1
figure 1

Scanning Electron Microscope (SEM) images of healthy and infected root sections of Muskmelon. (a) SEM image of Muskmelon root section (Healthy) showing normal cell structures viz., C-cortex, MX-Metaxylem, PX-Protoxylem, Ph-Phloem, P-Pith. (b) SEM image of Muskmelon root section (M. incognita infested) showing distrupted cell structures viz., CD-Cortical distruption, VD-Vascular distruption, GC-Giant cells, IFN-Impression of female nematode. (c) SEM image of Muskmelon root section (M. incognita infested) showing the clear impression of female root-knot nematode (IFN). (d) SEM image of Muskmelon root section (M. incognita infested) showing the cellular ingrowth (CIG). (e) SEM image of Muskmelon root section (M. incognita infested) showing the coalsed cellular (CC) structures in the cortical region. (f) SEM image of Muskmelon root section (M. incognita infested) showing the Xylem wall thickening (XWT) and Giant cells (GC).)

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The vascular region underwent severe alterations, with the epidermal layers ruptured. The stele appeared highly disorganized, forming large cavities due to the deformation of transport elements, which separated it from the cortex. Hypertrophy and hyperplasia of traumatic cells induced by nematode infection reduced the shape and thickness of the affected cortical and stelar regions. Numerous pear-shaped adult female nematodes were observed in the cortical region, embedded within traumatic cells near the root periphery (Fig. 1-c). Additionally, the formation of large, thick-walled giant cells in the damaged parenchymatous tissue was evident, with 4–6 giant cells clustered at feeding sites (Fig. 1-f).

SEM analysis also revealed localized cellular degradation at nematode feeding sites, with visible depressions indicating nematode attachment and feeding (Fig. 1-c). Cross-sectional analysis of infected roots displayed cell wall dissolution (Fig. 1-e), cellular ingrowth (Fig. 1-d), and xylem wall thickening (XWT) (Fig. 1-f), likely a consequence of nematode-induced enzymatic activity. The development of reticulate cell wall ingrowths adjacent to xylem elements was particularly notable, with an increased ingrowth density correlating with the extent of giant cell formation (Fig. 1-d). This structural modification potentially impairs vascular function, leading to embolism, where excessive xylem tension results in air and water vapor accumulation, disrupting water transport and causing desiccation in above-ground plant parts.

Furthermore, infected roots exhibited an increased root diameter with an irregular longitudinal cell arrangement, and female nematodes were found adhering to affected areas. The presence of wound-type vascular bundles, characterized by irregular cage-like xylem formations surrounding the infection sites (Fig. 1-f), was also noted. These alterations ultimately contribute to above-ground symptoms, such as stunted growth, chlorosis, and wilting of muskmelon plants. Overall, SEM analysis provided valuable insights into the ultrastructural changes associated with M. incognita infection in muskmelon roots, highlighting critical aspects of host-nematode interactions.

Chlorophyll content

Chlorophyll content, a crucial indicator of plant photosynthetic capacity, demonstrated a significant decline in infected muskmelon plants. Chlorophyll a in the control group averaged 0.1462 mg/g, whereas infection led to a reduction of 66.62%, with a mean value of 0.0488 mg/g. Chlorophyll b followed a similar trend, decreasing by 71.94% from 0.0606 mg/g in the control to 0.0170 mg/g in infected plants. The total chlorophyll content was reduced by 65.36%, from 0.3696 mg/g in the control to 0.1280 mg/g in the infected group. These reductions suggest a compromised photosynthetic efficiency due to nematode infection, potentially affecting the overall health and growth of the plant (Table 1 and Fig. 2-A).

Fig. 2
figure 2

Biochemical analysis of muskmelon under control and nematode-infected conditions. The graphs illustrate variations in (a) Chlorophyll content, (b) Total Phenol content, (c) Peroxidase activity, (d) Polyphenol Oxidase (PPO) activity, (e) Acid Phosphatase activity, and (f) Phenylalanine Ammonia Lyase (PAL) activity. Notable decreases in chlorophyll content and increases in phenol, peroxidase, PPO, acid phosphatase, and PAL activities were observed in infected plants compared to controls, indicating stress responses triggered by nematode infection.

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Effect of M. incognita on biochemical parameters

Root-knot nematode (M. incognita) infection significantly impacts muskmelon plants, causing alterations in key physiological parameters, including chlorophyll content, phenolic compounds, and enzymatic activities. The study presents findings on these parameters in both leaves and roots, revealing notable differences between control and infected plants.

Total phenol content

In contrast to chlorophyll, total phenol content, a key marker of plant defense, increased significantly in response to nematode infection. In leaves, the total phenol content rose by 114.5%, from 0.309 µg/g in the control to 0.663 µg/g in the infected plants. Similarly, root phenol content increased by 48.92%, from 0.531 µg/g to 0.791 µg/g. These increases suggest an induced defense response, likely aimed at mitigating the damage caused by the nematode infection (Table 2 and Fig. 2-B).

Peroxidase activity (PO)

Peroxidase activity, a marker of oxidative stress and defense, was significantly higher in infected muskmelon plants. In leaves, peroxidase activity increased by 51.47%, from 1.657 min− 1 g− 1 in the control to 2.510 absorbance units in the infected group. Root peroxidase activity also showed a 19.18% increase, from 0.907 to 1.081 min− 1 g− 1. This enhanced peroxidase activity indicates the plant’s response to oxidative stress and its role in defending against the nematode invasion (Table 3 and Fig. 2-C).

Polyphenol oxidase (PPO) activity

PPO activity, involved in oxidative defense, also increased significantly in both leaves and roots following nematode infection. Leaf PPO activity increased by 25.91%, from 1.173 min− 1 g− 1 in the control to 1.477 min− 1 g− 1 in infected plants. Root PPO activity showed a more pronounced increase of 36.49%, from 0.970 to 1.324 absorbance units. These results further highlight the plant’s enzymatic defense mechanisms activated during nematode stress (Table 4 and Fig. 2-D).

Acid phosphatase activity

Acid phosphatase, an enzyme associated with hydrolytic processes and phosphorus metabolism, exhibited significant increases in both leaves and roots of infected muskmelon. Leaf acid phosphatase activity increased by 19.04%, from 1.050 to 1.250 m mol p-nitrophenol mg− 1 protein min− 1, while root activity rose by 25.02%, from 0.951 to 1.189 m mol p-nitrophenol mg− 1 protein min− 1. These increases indicate an enhanced hydrolytic enzyme response as part of the plant’s defense strategy against nematode infection (Table 5 and Fig. 2-E).

Phenylalanine ammonia lyase (PAL) activity

The phenylpropanoid pathway, as evidenced by PAL activity, was also activated in response to nematode infection. In leaves, PAL activity increased by 141.74%, from 1.224 µmol trans-cinnamic acid mg− 1 protein min− 1 to 2.959 µmol. Similarly, root PAL activity increased by 106.64%, from 1.641 to 3.391 µmol. These substantial increases suggest that the plant is activating the phenylpropanoid pathway, which is crucial for producing secondary metabolites that aid in defense (Table 6 and Fig. 2-F).

Table 1 Effect of root-knot nematode M. incognita on chlorophyll (a, b and total) contents in Muskmelon.
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Table 2 Effect of root-knot nematode M. incognita on total phenol content in Muskmelon.
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Table 3 Effect of root-knot nematode M. incognita on peroxidase activity in Muskmelon.
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Table 4 Effect of root-knot nematode M. incognita on polyphenol oxidase activity in Muskmelon.
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Table 5 Effect of root-knot nematode M. incognita on acid phosphatase activity in Muskmelon.
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Table 6 Effect of root-knot nematode Meloidogyne incognita on phenylalanine lyase activity in Muskmelon.
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Discussion

The histopathological analysis of muskmelon roots infected with Meloidogyne incognita using Scanning Electron Microscopy (SEM) revealed extensive tissue disorganization, particularly in the vascular and storage tissues. Cortical deformation, vascular disruption, and irregular cellular structures were prominent, highlighting the nematode’s detrimental effects on the host root system. Pear-shaped female nematodes embedded near the root periphery suggest their exploitation of host tissues to establish feeding sites, a crucial strategy for survival and reproduction. Similar results were observed by Shandeep et al.11 in okra plant infested by M. incognita. These feeding sites disrupt root physiology, impeding nutrient and water uptake, leading to plant decline. Present findings corroborate the findings of Rani and Kumari12 in Mulberry roots infested by M. incognita.

SEM offers detailed topographic insights into structural alterations caused by nematode infection, with a resolution of at least 10 nm13. Nematode-infected root parenchyma cells enlarge, dissociate, and displace around the root surface, preceding the secretion of the gelatinous matrix, likely driven by nematode-induced enzymatic or hormonal changes13. Secondary wall ingrowths within giant cells function as metabolic “sinks,” promoting nutrient accumulation for nematode sustenance. Cytoplasmic contents obscuring inner cell walls were also noted, consistent with previous reports14.

Further supporting the role of SEM in nematology, Uematsu et al.15 documented nematode aggregation on Indian lotus roots, emphasizing structural alterations induced by nematodes. Similarly, Siddiqui et al.16 observed tissue disorganization in the stelar region, making it difficult to identify normal root tissues, aligning with the present study’s findings of severe tissue disruption affecting the endodermis, pericycle, xylem, and phloem. Feeding activity displaces vascular strands, forming abnormal vessel elements in the vascular cylinders, consistent with observations by Krusberg and Nielson17 and Goellner et al.18.

The formation of giant cells in cortical and pericycle regions, coupled with hyperplasia and hypertrophy, corroborates these findings. Giant cells, with thickened cell walls and enhanced metabolic activity, act as specialized feeding sites that deprive the plant of essential nutrients11. The presence of wound-type vascular bundles and xylem wall thickening (XWT) suggests an induced defense mechanism to restrict nematode movement. However, these modifications may also impair vascular function, leading to embolism and reduced water transport, manifesting as chlorosis and wilting11.

Nematode-induced vascular changes play a key role in nutrient supply to the parasite, as giant cells disrupt both cortical and vascular regions19. Abnormal xylem vessels, often disconnected, further impair water and nutrient transport, compounding plant stress20. Robah et al.21 reported that galled roots frequently exhibit distorted xylem vessels, a major symptom of nematode infection. These disruptions contribute to plant stunting and reduced vigor, underscoring the severity of nematode infestation on muskmelon roots.

Biochemical analysis further supports the detrimental effects of M. incognita infection on muskmelon physiology. A significant reduction in chlorophyll content, particularly chlorophyll a and chlorophyll b, indicates impaired photosynthetic capacity, likely due to vascular dysfunction that affects nutrient and water transport and damages chloroplast integrity. This decline in chlorophyll content is consistent with similar observations in nematode-infected ridge gourd and cucumber plants, further emphasizing the commonality of physiological stress induced by nematode infection22,23,24.

In contrast, the observed increase in total phenolic content in infected roots and leaves suggests an induced defense response to the nematode attack. Phenolic compounds play a pivotal role in plant resistance by reinforcing cell walls, inhibiting pathogen penetration, and activating systemic defense pathways25. The significant upregulation of phenolic content in response to infection aligns with previous findings in resistant cultivars, where enhanced phenolic accumulation correlates with reduced nematode proliferation. The present study corroborates the findings of Rizvi and Khan24, Mahapatra and Nayak26, and Nayak and Pandey27.

The elevated enzymatic activities of peroxidase (POX) and polyphenol oxidase (PPO) reinforce the plant’s defensive response. POX is essential for reactive oxygen species (ROS) detoxification and lignin biosynthesis, contributing to cell wall strengthening and nematode resistance25. Similarly, increased PPO activity suggests activation of oxidative defenses that disrupt nematode feeding structures, reducing the nematode’s virulence. The heightened activities of acid phosphatase and phenylalanine ammonia-lyase (PAL) further emphasize an active phenylpropanoid pathway, which is critical for the biosynthesis of secondary metabolites with antimicrobial properties. Notably, the significant increase in PAL activity, particularly in roots, highlights its role in reinforcing structural barriers and limiting nematode colonization25. The biochemical changes in different plants infected by the root-knot nematode M. incognita have been reported by Chakraborty et al.28 in tomatoes, Nikoo et al.29 in tomatoes, Mahapatra and Nayak26, and Ruchi and Singh30 in bitter gourd.

These findings demonstrate the complex interaction between nematode infection and the plant’s physiological responses. While M. incognita induces severe structural and functional damage to muskmelon roots, the plant activates a series of biochemical defense mechanisms in an attempt to mitigate the infestation’s effects. However, the persistent tissue disruption and reduced photosynthetic efficiency ultimately outweigh the plant’s defensive efforts, leading to reduced growth and productivity.

Conclusion

The histopathological and biochemical analyses of muskmelon roots infected with Meloidogyne incognita reveal significant structural and functional disruptions, including cortical deformation, vascular tissue damage, and the formation of giant cells that serve as specialized feeding sites for the nematodes. These alterations are associated with impaired nutrient and water transport, resulting in reduced plant growth and productivity. Despite the plant’s defense mechanisms, such as the increased phenolic content and enzymatic activity, the nematode infestation severely affects the plant’s physiological functions. Integrating resistant cultivars and nematicidal treatments could enhance muskmelon resilience and improve management strategies against root-knot nematodes.

Materials and methods

Experimental location

The study was conducted in a glasshouse at the Department of Studies in Biotechnology, Manasagangotri, University of Mysore, Mysuru, Karnataka (12.3105719° N, 76.6224571° E). Biochemical analysis and histopathological examinations of M. incognita infestations were performed at the Department of Studies in Botany, Manasagangotri, University of Mysore, Mysuru. The glasshouse conditions were maintained at a temperature range of 26–30 °C with a relative humidity of 70–75%.

Nematode inoculum

The root-knot nematode M. incognita used in this study was isolated from infected muskmelon roots. The nematodes were maintained in a glasshouse by culturing them on 14-day-old tomato (Cv. AMBER) seedlings for approximately 45 days. After this period, the nematodes were extracted by washing the roots under running tap water to remove adhering soil particles. The galled roots were then cut into sections and surface-disinfected using a 1% sodium hypochlorite (NaOCl) solution. Any residual NaOCl was eliminated through multiple rinses with sterile distilled water31,32. Egg masses of M. incognita were carefully collected using forceps under a stereo zoom microscope (Labovision AZM 100) and incubated in sterile distilled water for 48 h. After incubation, second-stage juveniles (J2) were extracted and acclimatized for use in subsequent glasshouse experiments.

Raising of muskmelon plants

Muskmelon (Cv. Arka Siri) seeds were procured from the Indian Institute of Horticultural Sciences, Hesaraghatta, Bengaluru, India (560089). The seeds were surface-sterilized by immersing them in a 0.5% sodium hypochlorite (NaOCl) solution for 15 min, followed by thorough rinsing with distilled water. The sterilized seeds were then sown in germination trays. One week after germination, seedlings were transplanted into earthen pots (2 kg capacity) filled with a sterilized potting mixture consisting of 70% sand, 29% silt, and 1% organic matter. Each pot initially contained three seedlings, which were later thinned to a single plant per pot after seven days of establishment.

Nematode inoculation and maintenance of muskmelon plants

Freshly hatched second-stage juveniles (J2s) of M. incognita were used to inoculate muskmelon plants grown in 2 kg earthen pots. Inoculation was carried out by creating four holes around the root zone of each plant, allowing direct nematode application. To facilitate nematode settlement near the plant roots, the pots were left undisturbed without watering for 24 h. Plants without nematode inoculation served as controls. The experiment followed a completely randomized design (CRD) with three replications. The plants were maintained in a glasshouse at the Department of Studies in Biotechnology, Manasagangotri, University of Mysore, Mysuru (570006), under controlled conditions: a temperature range of 26–30 °C, relative humidity of 70–75%, and a 12-hour photoperiod. Watering was done as needed to support plant growth and ensure optimal experimental conditions22.

Histopathological studies

Preparation of root-sections

Both healthy and M. incognita-infected root samples were collected to examine histopathological changes. The selected roots were thoroughly washed under running water to remove adhering soil particles, ensuring high-quality sections. After cleaning, the roots were cut into small segments (1–2 cm in length) using a sterile blade. The root samples were then fixed in TAF solution for 24 h and examined under a stereo zoom microscope33. Selected roots, both with and without galls, were processed through an alcohol series for dehydration. Transverse sections of the roots were made via the hand sectioning method. Transverse sections were analyzed under a binocular microscope to identify the most suitable samples for scanning electron microscopy (SEM) analysis22. The SEM examination was conducted to investigate the ultrastructural changes in M. incognita-infected muskmelon roots.

Scanning electron microscopic (SEM) studies

For Scanning Electron Microscopy (SEM), smaller transverse sections of M. incognita-infested and healthy (control) muskmelon roots were fixed in 2.5% glutaraldehyde prepared in 0.2 M cacodylate buffer (pH 7.2) for 2 h. The samples were then washed with cacodylate buffer, followed by double-distilled water, and subsequently dehydrated using an ethanol-acetone series. The dehydrated sections were mounted onto copper stubs using double-sided adhesive tape and coated with a 20 nm layer of gold using a sputter coater (EMS-550). The specimens were then examined under a Scanning Electron Microscope (EVO MA 15, Carl Zeiss, Germany) to observe ultrastructural changes11,12,13,34. The SEM analysis was conducted at the Institution of Excellence (IOE), Vijnana Bhavana, Manasagangotri, University of Mysore, Mysuru – 570006.

Estimation of chlorophyll content

The effect of M. incognita on the chlorophyll content of muskmelon was determined using the method described by Arnon35. One gram of finely cut fresh leaves (both healthy and nematode-infested) was ground with 20–40 mL of 80% acetone. The resulting mixture was then centrifuged at 5000–10000 rpm for 5 min. The supernatant was carefully transferred to a new container, and the centrifugation process was repeated until the residue became colorless. The absorbance of the solution was measured at 645 nm and 663 nm against an acetone blank using a Systronics UV-VIS 118 spectrophotometer. The amounts of chlorophyll a, chlorophyll b, and total chlorophyll were calculated in mg/g fresh weight using the formula provided by Arnon35.

Biochemical studies

Estimation of total phenols

One gram of plant tissue (leaf and root samples were processed separately) was crushed in 10 mL of 20% ethanol. The mixture was spun in a centrifuge at 10,000 rpm for 15 min. The clear liquid (supernatant) collected after centrifugation was used as the enzyme extract. For the test, 0.2 mL of this extract was mixed with 3.5 mL of distilled water and 0.5 mL of Folin-Ciocalteau reagent. After waiting for 5 min, 1 mL of 20% sodium carbonate was added. After 30 min, the absorbance was read at 660 nm using a Systronics UV-VIS 118 spectrophotometer. The amount of enzyme activity was calculated and expressed as milligrams per gram of plant tissue, based on the method by Malick and Singh36.

Estimation of peroxidase (PO)

One gram of plant tissue (leaf and root samples handled separately) was ground in 10 mL of 0.1 M phosphate buffer with a pH of 6.5. This mixture was then centrifuged at 10,000 rpm for 15 min. The clear liquid (supernatant) collected was used as the enzyme extract. For the test, 3 mL of 0.05 M pyrogallol solution was added to a test tube along with 0.1 mL of the enzyme extract. After that, 0.5 mL of 1% hydrogen peroxide was added. The absorbance change was measured immediately at 430 nm every minute using a Systronics UV-VIS 118 spectrophotometer. The enzyme activity was calculated based on how quickly the absorbance increased, and results were expressed as the rate of change per minute per milligram of protein or tissue, following the method described by Rad et al.37.

Estimation of polyphenol oxidase (PPO)

One gram of plant tissue (leaf and root samples were treated separately) was blended with 5 mL of chilled 0.1 M sodium phosphate buffer (pH 7.0). The mixture was then centrifuged at 10,000 rpm for 20 min, and the clear liquid (supernatant) obtained was used as the enzyme extract. For the enzyme activity test, 1.5 mL of 0.1 M sodium phosphate buffer (pH 6.5) was mixed with 200 µL of the enzyme extract. The reaction was started by adding 200 µL of 0.001 M catechol. The change in absorbance was measured at 495 nm using a Systronics UV-VIS 118 spectrophotometer, with readings taken every 30 s for 5 min. The enzyme activity was calculated and reported as milligrams per gram of protein per hour, following the method of Mayer et al.38.

Estimation of acid phosphatase

One gram of fresh plant tissue (leaf and root samples handled separately) was crushed and mixed with 10 mL of cold 50 mM citrate buffer (pH 5.3). The mixture was then centrifuged at 10,000 rpm for 10 min, and the clear liquid (supernatant) obtained was used as the enzyme extract. For the enzyme test, 3 mL of the substrate solution was first incubated at 37 °C for 5 min. After that, 0.5 mL of the enzyme extract was added, mixed thoroughly, and incubated again at 37 °C for 15 min. Once the reaction was complete, 0.5 mL of this mixture was combined with 9.5 mL of sodium hydroxide. The absorbance was then measured at 405 nm using a Systronics UV-VIS 118 spectrophotometer. Enzyme activity was calculated and reported as the amount of p-nitrophenol released per minute per milligram of protein, based on the method described by Dickerson et al.39.

Estimation of phenylalanine ammonia lyase (PAL)

One gram of fresh plant tissue (leaf and root samples handled separately) was crushed and mixed with 10 mL of 25 mM borate-HCl buffer (pH 8.8) containing 5 mM mercaptoethanol (0.04 mL/L). The mixture was then centrifuged at 12,000 rpm for 20 min. The clear liquid (supernatant) obtained was used as the enzyme extract. In a test tube, 0.5 mL of borate buffer was combined with 0.2 mL of the enzyme extract and 1.3 mL of distilled water. The reaction was started by adding 1 mL of L-phenylalanine solution, and the mixture was incubated at 32 °C for 30 to 60 min. To stop the reaction, 0.5 mL of 1 M trichloroacetic acid was added. The absorbance was then read at 290 nm using a Systronics UV-VIS 118 spectrophotometer. Enzyme activity was calculated as micrograms of product formed per gram of protein per minute, following the method described by Dickerson et al.39.

Statistical analysis

Each experiment was conducted in triplicate and replicated three times. The data generated from the various experiments in the present study were analyzed using Analysis of Variance (ANOVA). The results were processed using OPSTST software, available online at CCSHAU, Hisar (www.hua.ac.in). The percentage increase and decrease in the biochemical parameters and chlorophyll indices compared to the control were calculated using the formulas provided by Irshad et al.40 and Mukhtar et al.41.