Introduction

Plants, although sessile organisms can change their physical traits in reaction to environmental stressors including animal attacks and competition from other plants. In their ecosystems, plants interface with a wide variety of herbivorous insects that have different infestation patterns and methods of survival. Many herbivorous insects are able to characteristically differentiate between various host plants or diets, and they will only consume and lay their eggs on superior plants1,2. Disease and insect herbivore attacks, whether innate or intuitive, cause significant metabolic changes in many plants3,4 which plays a critical role in defense mechanisms against a number of living and non-living stressors. Many complex communication pathways that involve various plant harmones, secondary messengers and numerous defensive protein substances are interconnected which collectively allows plants to resist these stressors5.

One of the first biological reactions that occurs frequently in plants as a result of oxidative stress from various components of ecosystem is the quick and temporary generation of reactive oxygen species (ROS)6,7,8. In order to combat this, plants have a robust enzymatic antioxidant resilience network that includes antioxidative enzymes and small molecule antioxidants which eliminates reactive oxygen species (ROS) to shield plant cells from oxidative injury9,10,11. In plant stress endurance, Superoxide dismutase (SOD) which enzymatically reduces superoxide radicals to H2O2, which is then scavenged into H2O and O2 by catalase (CAT) and ascorbate peroxidase (APX) may be the primary safeguard against raised ROS levels12. A common occurrence in reaction to insect infestation is structural and measurable modifications in phenols and an increase in oxidative enzyme activity13. Host plant resistance (HPR) to herbivores, including insects, depends on plant phenols, one of the most common and extensively dispersed families of secondary metabolites14,15. Plants may use the oxidative degradation of phenols, which is facilitated by polyphenol oxidase (PPO) and peroxidase (PO), as a protective mechanism against insects that feed on plants16,17,18,19,20.

A recent invasive pest, the fall armyworm, Spodoptera frugiperda, unarguably, one of the deadliest insect-pest, is a lepidopteran that attacks more than 350 plant species in large numbers with highest affinity for maize21, and it also seriously harms other vegetable crops, cotton, and other sorghum species, as well as essential grasses like rice, sorghum, sugarcane, and wheat22. Although in India, it was predominantly observed in host plants including maize, sugarcane and sorghum, where it resulted in yield losses of up to 57.60 to 58.00% in case of maize23,24. However, the larvae in India’s lab were shown to consume a variety of host species, such as maize, sorghum, cabbage, tomato, peanut, and sugarcane25.

Given the growing threat posed by Spodoptera frugiperda (fall armyworm) across diverse agroecosystems, this study aimed to systematically investigate the biochemical and nutritional responses of five economically important host plants—maize, cabbage, rice, ginger, and brinjal to larval infestation. Emphasis was placed on elucidating host-specific alterations in primary and secondary metabolites and identifying potential resistance-associated biochemical markers that may underpin differential susceptibility to S. frugiperda.

Materials and methods

Experimental site

For the experiment five different host crops of varieties viz., maize (Zea mays var. P 3369), cabbage (Brassica oleracea var. Fire ball 175), rice (Oryza sativa var.IET 4786), ginger (Zingiber officinale var. Gorubathan) and brinjal (Solanum melongena var. Makra) were used. The seed material of these plants were collected from University Research Farm, Gayeshpur and were grown in pots (11.5 × 7.5 × 8 cm; top width × bottom width × height) under insect proof net with fine mesh conditions to exclude other insects in our experimental fields at Central Research Farm (22.95540 N latitude, 88.49610 E longitude), Gayeshpur, Bidhan Chandra Krishi Viswavidyalaya, West Bengal, India and were maintained insecticide free at temperature 29 ± 5 °C, and RH 70 ± 5%. The plants that attained 6–7 leaf stage were used for the experiment.

Insect culture

The nucleus culture of FAW was initiated with larvae collected from maize fields at the farm of Gayeshpur, West Bengal, India. The larvae were reared using petri dish - circular (100 × 50 mm dia) containing the maize (variety P 3396) cut leaf bits and closed with lid and maintained at room temperature. After the adult emergence, the male and female moths were separated and allowed to mate in transparent jars covered with fine muslin cloth and secured with rubber bands. Inside the jars, paper towels were lined as oviposition substrate along with fresh seedling of maize having 5–7 leaves was placed in 250 ml conical flask with water, 10% honey solution soaked on cotton pads was placed inside each jar for moth feeding. Eggs were collected and kept in a petri-dish for hatching. These eggs were used as nucleus culture for mass rearing and newly hatched larvae from eggs were collected and were fed with maize plant leaves. By assuring adequate food supply, all larvae were fed daily with fresh leaves. Healthy neonate larvae of S. frugiperda were used for the experiments.

Five fall armyworm neonates obtained from the mass rearing colony maintained under laboratory conditions were released on the leaf of the host plants (maize, brinjal, ginger, rice and cabbage at 6–7 leaf stage) using a soft hair brush, then the plants were enclosed in a netting cloth. The trial was replicated three times. The remaining leaf portions after feeding (50–60%) were used for the biochemical assays. The insects were removed after feeding and then the infested leaves were collected one week after pest feeding.

Procedure and chemicals

The biochemical parameters viz., the contents of moisture (%), protein (%), phenols (mg GAE/g), and carbohydrates (mg/100 mg) and a few plant defensive enzymes with antioxidative activity viz., SOD, CAT and PO were analyzed by following different procedures in the laboratory which are detailed below. Biochemical composition of the host plant leaves were calculated as per the standard protocols from the leaf samples collected from control plants (fresh leaves of normal, intact and healthy host plants of similar age and conditions without FAW infestation) and the infested plants (seven days after infestation with FAW).

Table 1 List of the chemical used in analysis of biochemical constituents.
Full size table

Moisture content (%)

Using the wet basis method, the moisture content of the host plants’ new leaves was calculated on a percentage basis. Leaf samples with known weights were put in a hot air oven set at 70 degrees Celsius. Every day until the weight of the leaf sample remained consistent, the weight loss was recorded. The following formula is used to estimate the % moisture content:

$${\text{Moisture}}\,{\text{content}}\left( \% \right) = \frac{{{\text{Initial}}\,{\text{weight}}\,{\text{of}}\,{\text{the}}\,{\text{sample}} - {\text{Final}}\,{\text{weight}}\,{\text{of}}\,{\text{the}}\,{\text{dry}}\,{\text{sample}}~}}{{{\text{Initial}}\,{\text{weight}}\,{\text{of}}\,{\text{the}}\,{\text{sample}}}} \times 100$$

Protein (mg/g)

After centrifuging the fresh leaf sample (0.5 g) for 10 min at 5000 rpm, 10 milliliters of sodium phosphate buffer (pH 6.8) was used to extract the soluble protein. A 0.2 ml aliquot of the sample extract was placed in a test tube, and distilled water was added to bring the volume up to 1.0 ml. Reagent C (5 ml), which was made by combining 50 ml of reagent “A” (2% Na2CO3 in 0.1 N NaOH) with 1 ml of reagent “B” (0.5% CuSO4 in 1% potassium sodium tartrate) (Table 1), was added to the sample, and it was then incubated at room temperature (10 min). This was followed by the addition of 0.5 ml of FCR reagent (commercially available Folin Ciocalteu’s reagent diluted with equal quantities of water), thorough mixing, and another half hour of room temperature incubation in a dark chamber. Lowery’s approach was used to estimate the amount of soluble protein by comparing the final solution’s absorbance at 660 nm to a reagent blank and calculating the quantity using a calibration curve made using standard bovine serum albumin26.

Phenols (mg GAE/g)

Total phenol was extracted from the leaf sample (1 g) by macerating with 10 ml of 80% ethanol in a mortar and a pestle and the supernatant was saved after centrifuging it for 20 min at 10,000 rpm. Repeating the same procedure, re-extraction of the residue is done for five times and the pooled supernatant thus obtained was then evaporated to dryness and redissolved in a suitable volume of distilled water. Estimation of total phenol was done by following the standard Folin-Ciocalteau method26 using gallic acid standard.

Carbohydrate (mg/100mg)

Using the standard anthrone technique of estimation, the total carbohydrate content of the leaves was determined26. A boiling tube containing 100 mg of leaf material was hydrolyzed with 2.5 N HCl (three hours) in a boiling water bath before cooling down to room temperature. The resultant hydrolysate was produced up to 100 ml and centrifuged after being neutralized with solid Na2CO3 until the effervescence stopped. A sufficient aliquot of the supernatant was collected, and distilled water was added to bring the volume up to 1 ml. The test tubes were then heated in a boiling water bath for eight minutes after adding four milliliters of freshly made anthrone reagent (200 mg of anthrone dissolved in 100 ml of pre-chilled 95% H2SO4) (Table 1). Absorbance was measured after cooling the test tubes, at 630 nm. A calibration curve made using a high-purity glucose standard was used to determine the amount of total carbohydrates, which was then represented as a percentage.

$${\text{The}}\,{\text{amount}}\,{\text{of}}\,{\text{carbohydrate}}\left( {{\text{mg}}/100\,{\text{mg}}} \right){\text{ present}}\,{\text{ in}}\,{\text{ the }}\,{\text{sample}} = ~\frac{{{\text{mg}}\,{\text{of}}\,{\text{glucose}}}}{{{\text{volume}}\,{\text{of}}\,{\text{test}}\,{\text{sample}}}} \times 100$$

Enzymes related to antioxidative defense

Preparations of enzymes extract

With some adjustments, a sample was generated using Nayyar and Gupta’s27 methodology in order to determine the activity of the enzymes under investigation (SOD, CAT, and POD). Each type of host plant’s freshly chopped, preweighed leaf sample was macerated using a 10 ml extraction buffer solution [0.1 M sodium phosphate buffer (pH 7.5) + 0.25% Triton-X detergent + 2% polyvinylpyrrolidone] (Table 1). The supernatant obtained after centrifugation (10,000 rpm) of sample for 30 min was used for testing the enzyme activity.

Superoxide dismutase (SOD) activity

A modified version of Beauchamp and Fridovich’s method28 was used to measure SOD activity based on the extract’s capacity to keep nitro-blue-tetrazolium (NBT) from being photochemically reduced in the riboflavin-light-NBT system. To create a reaction mixture, 4.4–4.0 ml (with a 0.1 ml serial increment) of 0.05 M phosphate buffer (pH 7.8) was added to a series of test tubes that also contained 0.4 ml of 20 mM methionine, 0.4 ml of 1.5 mM NBT, 0.4 ml of 1.5 mM EDTA, and 0.1–0.4 ml of enzyme source (with a 0.1 ml serial increment). Next, 0.4 ml of 75 mM riboflavin was added to each test tube (Table 1). Test tubes were shaken and positioned 30 cm below a light bask with two 15 W fluorescent lamps after riboflavin was added as the final ingredient.

The light was turned on to initiate the reaction, which was then left to run for half an hour. The light was turned off after 30 min to halt the response. At 560 nm, the reaction mixture’s absorbance was measured. At 560 nm, the reaction mixture’s absorbance was measured against reagent blank. The light incubated tube without sample was used as control (A0), producing highest reduction of NBT in absence of enzyme source. Increasing enzyme concentration produced an inverse relationship with NBT reduction and colour development. Superoxide dismutase activity was expressed in Unit (U) defined as amount of sample (mg FW. ml− 1) required to produce 50% inhibition of NBT- formazan formation under this experimental condition.

Catalase (CAT) activity

Following the procedure of Aebi et al.29, CAT activity was ascertained by tracking the rate of H2O2 disappearance at 240 nm (ε240 = 40mM −1 cm −1. 2.6 milliliters of 0.1 M sodium phosphate buffer (pH 7.5), 0.2 milliliters of H2O2 (1%) and 0.2 milliliters of plant extract were formed to form three milliliters of the reaction mixture (Table 1). The reaction mixture’s absorbance was measured using an UV-visible spectrophotometer (240 nm). The Catalase activity (U) was expressed in amount of hydrogen peroxide destroyed/min/g of tissue (fresh weight).

Peroxidase (PO) activity

Lin and Kao30 used the guaiacol oxidation technique to measure peroxidase activity. The reaction process started by addition of 1% (v/v) hydrogen peroxide (0.15 ml) to mixture consisting of 0.15 ml of 4% (v/v) guaiacol, 0.1 M sodium phosphate buffer (2.5 ml, pH 7.5), and 0.20 ml of enzyme extract (Table 1). A spectrophotometer was used to record the increase in absorbance at 470 nm at 30-second intervals and for up to three minutes. Tetraguaiacol’s absorption coefficient (ε470 = 26.6 mM−1 cm−1) at 470 nm was used to compute the POD activity. Peroxidase activity (U) is defined as µ moles of tetraguaiacol produced/min/gram fresh weight of the sample.

Data analysis

SPSS professional statistics version 26 (SPSS Inc. USA) was used to statistically analyze the data and create the graphs. Shapiro wilk test was used to test Normality of data. Analysis of Variance (ANOVA) was used to evaluate the parametric data, and the Duncan Multiple Range Test (DMRT) was used to separate the treatment means at a 5% probability level. The differences in the concentration of biochemical parameters between S. frugiperda infested and normal healthy uninfested host plants were analysed and compared statistically using paired t-test at p < 0.05.Bartlett Chi-square test was used for equality of variances.

Results

Biochemical basis of host resistance against S. frugiperda

Protein content (mg/g)

Before FAW infestation (control), rice had the greatest protein level among the host plants (2.63 ± 0.18 mg/g) and ginger had the lowest protein content (1.17 ± 0.03 mg/g) (F = 20.384, P = 0.000), however post FAW infestation, brinjal and ginger had the highest and lowest protein contents in the line of 2.72 ± 0.17 and 1.21 ± 0.03 mg/g, respectively (F = 52.574, P = 0.000). A significant rise in protein content for brinjal (t = 18.196, P = 0.003) post FAW infestation (2.72 ± 0.17 mg/g) was recorded when compared to healthy control plant (1.50 ± 0.12 mg/g) (Table 2; Fig. 1).

Phenol content (mg GAE/g)

The rice crop had the greatest phenol content of the host plants in the study’s control group (1.42 ± 0.07 mg GAE/g), while cabbage had the lowest (0.67 ± 0.03 mg GAE/g) (F = 10.637, P = 0.0013) (Table 2). Post FAW-infestation, phenol levels were high in rice crop (1.78 ± 0.03 mg GAE/g) (F = 6.037, P = 0.0098) in comparison to control (1.42 ± 0.07 mg GAE/g) among all the host plants under study. All host plants showed an increase in phenol content following FAW infestation except maize, but significant difference was observed only for cabbage (0.67 ± 0.03 mg GAE/g, 0.81 ± 0.03 mg GAE/g) (t = 30.160, P = 0.001) (Fig. 1).

Carbohydrate content (mg/100 mg)

The highest concentration of carbohydrates was found in rice (19.42 ± 1.43 mg/100 mg), while the lowest concentrations were found in cabbage (5.94 ± 0.14 mg/100 mg), maize (7.19 ± 0.14 mg/100 mg), and brinjal (6.88 ± 0.15 mg/100 mg), all of which had significantly equal carbohydrate contents in control plants (F = 73.175, P = 0.000) (Table 2). One week after FAW infestation with the exception of brinjal (31.63 ± 2.94 mg/100 mg), the carbohydrate content of all the other crops were statistically on par with each other (F = 3.409, P = 0.053). Post FAW infestation, a substantial increase in carbohydrate content was seen in all host crops, ginger (t = 12.258, P = 0.007), cabbage (t = 13.986, P = 0.005), maize (t = 5.562, P = 0.031), brinjal (t = 8.864, P = 0.012) and rice (t = 21.615, P = 0.002) (Fig. 1).

Moisture content (%)

The host plant’s moisture contents were greatest in the case of cabbage (94.50 ± 0.29 and 93.75 ± 0.14%) and lowest in the case of rice (78.75 ± 0.43 and 77.50 ± 0.87%), respectively both in control (F = 186.79, P = 0.000) and infested leaves (F = 199.98, P = 0.000). The respective values in maize leaf were 92.25 ± 0.43 and 91.00 ± 0.29%. After FAW infestation, a negligible drop in moisture content was noticed, although it was not statistically significant (p > 0.05) (Table 2; Fig. 1). The higher level of water content in maize and cabbage expedite the higher feeding preference of S. frugiperda. Higher relative water content facilitates feeding preference of herbivores31.

Peroxidase activity (PO)

The highest and lowest peroxidase activity post FAW infestation was found in maize (486.60 ± 0.41 U, 314.07 ± 1.08 U) and rice (119.47 ± 1.57 U, 233.07 ± 0.96 U), respectively (F = 561.757, P = 0.000) compared to healthy controls. Among the control host plants, brinjal had the highest (403.53 ± 0.56 U) whereas ginger had the lowest (68.67 ± 0.58 U) activity, respectively (Table 2). There was a considerable rise in peroxidase activity in maize, cabbage, and ginger (P = 0.000) in FAW infested plants, however a significant drop was seen in case of brinjal (t = 7.861, P = 0.016) and rice (t = 176.69, P = 0.000) following infestation (Fig. 2).

Catalase activity (CAT)

Maize had the greatest catalase activity (0.97 ± 0.03 U), followed by ginger (0.69 ± 0.03 U), among the control plants (F = 34.056, P = 0.000), but rice and cabbage had the highest and lowest catalase activities (1.63 ± 0.02 U and 0.39 ± 0.03 U, respectively) (F = 269.921, P = 0.000) after one week of FAW infestation. When the same host plants under control and FAW infestation were examined, a substantial rise in catalase activity was seen in the rice (t = 31.668, P = 0.001) and brinjal (t = 4.223, P = 0.042) crops and a substantial decrease in catalase activity was recorded in maize (t = 12.203, P = 0.007) (Table 2; Fig. 2).

Superoxide dismutase activity (SOD)

Superoxide dismutase (SOD) activity increased significantly in infested plants (F = 843.939, P = 0.000) of ginger (72.55, 29.50 U), brinjal (40.20, 33.44 U), and rice (10.28 ± 0.05, 6.86 ± 0.03 U) compared to their respective healthy controls (F = 235.544, P = 0.000) (Table 2). Except for maize and cabbage, host plants ginger (t = 65.454, P = 0.000), brinjal (t = 27.403, P = 0.001) and rice (t = 66.646, P = 0.000) showed a significant increase in SOD activity after infestation (p < 0.01) when infested plants were compared with uninfested controls (Fig. 2).

Fig. 1
Fig. 1
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Quantitative changes in (A) Protein, (B) Phenols, (C) Carbohydrate and (D) Moisture contents (Mean ± SE) in host plants due to S. frugiperda pest feeding.

Fig. 2
Fig. 2
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Difference in activities of various enzymes E. Peroxidase (PO), F. Catalase (CAT), G. Superoxide dismutase (SOD) (Mean ± SE) in host plants due to S. frugiperda pest feeding. Significant differences (P < 0.05 and P < 0.01) are indicated by asterisks *, ** respectively, (paired t-test), between the control and infested plant.

Discussion

The formation of antagonistic metabolites in plants through morphological, chemical, and biological alterations is the most crucial barrier against insect attack. Since these antioxidant molecules participate in the plant’s defense mechanisms against insect herbivory, most people view them as protective substances32.In the present study, we investigated the biochemical and oxidative stress responses of maize (P 3369), cabbage (Fire Ball 175), rice (IET 4786), ginger (Gorubathan), and brinjal (Makra) following insect herbivory, with a particular emphasis on quantifying alterations in primary metabolite levels and the activities of reactive oxygen species (ROS)-scavenging enzymes.

Post FAW infestation, a substantial increase in carbohydrate content was seen in all host crops. This phenomenon was also observed by Yasur et al.33, who reported that, while sucking pests, borer attacks, and mechanical damage reduced carbohydrate content, chewing insect damage increased it, highlighting the role of tissue loss severity in modulating carbohydrate levels. Thus, the observed post-herbivory increase in carbohydrate levels might likely represents a strategic shift in the plant’s resource allocation toward survival and defense synthesis of defense-related compounds such as secondary metabolites and structural barriers (e.g., lignin and callose).

In case of protein a significant increase was observed in Brinjal crop under the study, the role of proteins in plant-induced resistance has been comprehensively studied3,5. Similar results were recorded Pieris rapae feeding on Brassica nigra34. This rise protein content might be due to the activation of defense-related proteins, such as proteinase inhibitors, which limit herbivore feeding by disrupting digestion35, improving its ability to withstand further pest attacks.

Phenolic compounds are one of the numerous antioxidant chemicals that are thought of as secondary metabolites36 which mainly involve in deterring herbivores through toxicity, feeding inhibition, or oxidative stress induction. Increase in phenols was observed in all the host plants under study except Maize which is highly susceptible to S. frugiperda. In susceptible host plants, a post-infestation decline in phenol content may be due an inadequate defense response or insufficient biosynthesis in response to sustained herbivore pressure. Similarly, El-Zohri et al.37 showed that S. exigua infestation significantly decreased the bound and free phenols content in infested maize leaves at all time periods (2 h, 2days, and 1 week) in comparision to uninfested controls. Yasur et al.33 also reported increased phenol levels in Ricinus communis leaves infested by Achaea janata and Spodoptera litura. A decrease in moisture content post FAW infestation in all the host plants under study might be due to Physical tissue damage, increased transpiration, or plants may redirect resources (including water) toward the synthesis of defense compounds against herbivory.

The findings of our analysis clearly suggest that S. frugiperda attack has altered the oxidative state of all the host plants. A substantial increase in activity of Peroxidase was noted in Ginger, Cabbage and Maize plants through this study. Similar results were observed in resistant soybean cultivars following infestation by bean leaf beetles and three-cornered alfalfa leafhoppers by Felton et al.38 and S. litura feeding on Ipomoea batatas (sweet potato)39. The increased peroxidase activity may result from the plant reinforcing its cell walls through protein cross-linking and lignification, making the tissue more resistant to chewing and digestion by the insect.

A significant decrease in Catalase activity was observed in Maize and increase was noted in Brinjal and Rice post FAW infestation compared to normal healthy plants. Similarly, Yang et al.40 reported that the Catalase activity initially decreased significantly following infestation but subsequently increased, returning to levels comparable to the control. El-Zohri et al.37 reported a sustained and significant increase in catalase activity throughout the entire post-infestation period following Spodoptera exigua attack as seen in case of Brinjal and Rice.

Table 2 Analysis of protein, phenols, carbohydrate, moisture, peroxidase, catalase and superoxide dismutase (mean ± standard error) of five host plants.
Full size table

In the study, an increase in the activity of SOD was observed in Ginger, Brinjal and Rice plants and a decrease was recorded in Cabbage and Maize. Similarly in maize, El-Zohri et al.37 showed that post S. exigua infestation, the activity response of SOD decreased as infestation duration increased. Herbivory by Spodoptera litura larvae has been shown to elicit a significant upregulation of superoxide dismutase (SOD) activity in Ipomoea batatas, relative to uninfested control plants39. Comparable increases in SOD activity have been documented in Ricinus communis following infestation by the capsule borer Dichocrocis punctiferalis and the semilooper Achaea janata, indicating a conserved oxidative stress response to lepidopteran herbivory across different host species33 as seen in the current study for Ginger, Brinjal and Rice, high SOD activity has also been associated with enhanced plant resistance to a range of abiotic stresses41,42.

These enzymes have been utilized as a marker for resistance cultivars, such as those in alfalfa against Aphis medicaginis Koch, resistant turf grass against chinch bug, and rice cultivars against rice strip virus43,44,45. As a result, it’s possible that the rise of proteins, phenols, carbohydrates, catalase, and SOD is related to brinjal’s FAW resistance. Resistance to FAW can be increased and its bases diversified by using large-scale hybridization with resistant host plants that have high biochemical component activity. Plant resistance in insect-plant interactions is clarified by our results, which show the diverse defensive responses of the various host plants to FAW. Additionally, using this knowledge, enzyme markers that detect FAW resistance and/or susceptibility in various plants can be created. Additionally, by comparing the gene expression of resistant and vulnerable plant species, this study will assist uncover genes implicated in the resistance and advance our understanding of the molecular processes leading to plant resistance to insects.

Conclusions

There were notable differences in biochemical components under study due to herbivory by S. frugiperda among the assessed host plants. This study underscores the pivotal role of biochemical and oxidative stress responses in shaping host plant resistance to S. frugiperda. The observed induction of antioxidant enzymes and shifts in primary metabolites highlight a coordinated defense strategy that varies across crops, with brinjal (Makra) showing strong constitutive defenses and maize (P3369) exhibiting rapid inducible responses. These findings advance our understanding of plant–insect interactions and support the use of biochemical traits as reliable markers for breeding pest-resistant cultivars, contributing to more sustainable and resilient crop protection strategies.