The burrowing nematode (Radopholus similis), stands out due to its prevalence and the substantial threat it poses to banana plants. Severe symptoms are observed in banana plantations, such as root detachment, stem from the destructive feeding habits of these nematodes, resulting in compromised root integrity. This root damage results in delayed shoot development, fruiting, reduced fruit size, bunch weight, and a shortened plant lifespan. Additionally, R. similis infestation triggers the mortality of vascular roots, leaving behind distinct brown-red lesions on larger root surfaces. As these nematodes migrate from the roots to the rhizome, circular black lesions, commonly referred to as blackhead disease, emerge (Brooks 2014).

A cascade of defense responses is triggered upon R. similis infestation on host plant roots, prominently featuring the generation of reactive oxygen species (ROS). These ROS, encompassing superoxide anion (O₂^-), singlet oxygen (¹O₂), hydrogen peroxide (H₂O₂), and hydroxyl radicals (·OH), serve as continual byproducts of diverse metabolic pathways (El-Beltagi et al. 2012). Plants have evolved enzymatic and non-enzymatic defenses, including glutathione (GSH), superoxide dismutase (SOD), and catalase (CAT), to counteract excessive ROS accumulation within their cellular framework. These antioxidants serve as pivotal guardians, detoxifying ROS by neutralizing reactive molecules and converting organic hydroperoxides into less toxic alcohols precisely at sites of heightened ROS production (El-Beltagi et al. 2011). However, under stressful conditions, their accumulation can surpass the antioxidant defense capacity, leading to oxidative stress and damage to all biological molecules. Furthermore, ROS display heightened reactivity towards membrane lipids, proteins, and DNA, contributing to stress-induced damage and swift cellular injury (El-Beltagi et al. 2012). The widespread application of chemical nematicides to combat nematodes presents substantial risks to humans, animals, plants, and the environment, given their non-target effects, hazardous characteristics, and high expenses. As awareness grows regarding the potential dangers posed by chemicals, there is a growing inclination toward exploring biological control methods for managing plant-pathogenic nematodes (Haroon et al. 2018).

Chromolaena odorata is a wild plant indigenous to Central and South America, proliferating in tropical and subtropical regions. Its introduction to Southeast Asia in the 1920s and Africa around the 1940s as a cover crop plantation has led to its global dissemination. The dried leaves and stems of C. odorata consist of ash (11%), crude fat (11%), fiber (15%), moisture (15%), crude protein (18%), and carbohydrates (31%). Active phytochemicals in this plant comprise: Flavonoid aglycones (flavanones, flavonols, and flavones, such as acacetin, chalcones, eupatilin, luteolin, naringenin, kaempferol, quercetin, quercetagetin, and sinensetin), terpenes and terpenoids, essential oils, alkaloids (including pyrrolizidine), saponins and tannins, phenolic acids (ferulic acid and protocatechuic acid, phytoprostane compounds (including chromomoric acid). With a foundation in traditional uses and the plant’s chemical composition, C. odorata demonstrates a spectrum of activities, including antibacterial, anticancer, anticonvulsant, anti-diabetic, antidiarrheal, antifungal, anti-inflammatory, antioxidant, antiparasitic, hemostatic, wound healing, and hepatoprotective effects (Sirinthipaporn and Jiraungkoorskul 2017). Furthermore, C. odorata has varied biotechnological uses, including controlling insects and exhibiting insecticidal properties during the larval stage of the disease-carrying mosquito Aedes aegypti. It is also effective in nematode control, targeting and eliminating second-stage larvae (J2) of nematodes such as Meloidogyne spp., Helicotylenchus spp., and Pratylenchus spp., among others (Kato-Noguchi and Kato 2023). Given the detrimental impact of Radopholus similis nematode invasion on banana crops, this study investigates the potential of Chromolaena odorata extract as a remedy to alleviate these negative consequences. Additionally, the research unveils the complex interplay between nematode invasion, plant defense mechanisms, and oxidative stress. This underscores the importance of comprehending these mechanisms and devising strategies to safeguard plant cells from oxidative damage using C. odorata extract.

Collection plant material and preparation of the extract

Collection plant material: In October 2022, leaves and stems of C. odorata were harvested during the vegetative phase from diverse locations in Cu Chi District, Ho Chi Minh City, Vietnam. Plant identification was based on voucher specimens archived in the herbarium of the Faculty of Biotechnology, Institute of Biotechnology and Food Technology, Ho Chi Minh City University of Industry. After a thorough cleaning, removal of damaged sections, and a 15-day air-drying period in the laboratory shade, the material was finely powdered using an electric commercial grinder (Model FW177, TaisiteLab Sciences Inc., China). The resulting powder was then stored in moisture-resistant bags for subsequent use.

Preparation of aqueous extract: Twenty-five grams of powder were added to a 500 mL glass flask containing 250 mL of distilled water at 80% capacity for water extraction. The flasks were then placed on a BW201 thermostatic shaker (Japan) and agitated for 4 hours at a speed of 500 revolutions per minute. The resulting mixture underwent filtration through a funnel equipped with 100 µm filter paper (No.1) and centrifugation using a Swing-3000 horizontal centrifuge (Germany) for 15 minutes at 1500 revolutions per minute to remove debris. The water from the extraction solution was evaporated using an rotary evaporator (Model RE601B-O, Yamato Scientific Co., Ltd., Japan) at 60°C. The obtained extract from the water extract solution after evaporation was diluted with 25 mL of distilled water. This resulting solution, termed AECO, was designated as the stock solution and stored at 4°C for use in subsequent experiments.

Screening and phytochemical quantification of extracts

Screening phytochemical: This qualitative investigation seeks to identify key chemical treatments (alkaloids, flavonoids, coumarins, tannins, etc.). Characteristic tests rely on precipitation reactions and complex formation, resulting in the development of insoluble and colored compounds. The observed colors are indicative of the appropriate reagent use, such as, the emergence of a green precipitate indicates the presence of tannins in the extract, and the formation of a yellow precipitate signals the presence of flavonoids, the appearance of a deep green color signifies the presence of phenolics, and, in a broader sense, reflects reactions occurring between molecules. The filtration process adhered to established protocols outlined by Tran et al. (2023).

Phytochemical quantification: Polyphenols, flavonoids, and tannins play pivotal roles in plant physiology, acting as potent antioxidants to shield against oxidative stress and demonstrating nematocidal properties, thereby fortifying plant defenses against nematode attacks. Precise quantification of these compounds in plant extracts is necessary, and it is executed employing well-established methodologies detailed by Belkhodja et al. (2020). The determination of total polyphenol content involves the Folin-Ciocalteu method, expressing results as milligrams of gallic acid equivalent per gram of dry plant material (mg GAE/g) with the gallic acid standard. Total flavonoid content is quantified using the aluminum chloride (AlCl₃) method, presenting outcomes in milligrams of quercetin equivalent per gram of dry plant material (mg QE/g) with the quercetin standard. Tannin content is determined using the vanillin spectrophotometric method, and the total condensed tannin content is calculated in milligrams of catechin equivalent (mg CE/g) from the derived standard curve. These standardized methodologies offer robust metrics for evaluating concentrations of polyphenols, flavonoids, and tannins in plant extracts, providing valuable insights into their potential roles in promoting plant health and fortifying defenses against nematode-induced damage.

In vitro antioxidant studies of aqueous extract of C. odorata

DPPH radical-scavenging assay: The free radical scavenging activity of the aqueous extract of C. odorata was measured using 1,1-diphenyl-2-picryl hydrazyl (DPPH) according to the method described by Hussen and Endalew (2023). A 0.1 mM DPPH solution was created by dissolving 0.004 g of crystalline DPPH in 100 mL of methanol and storing it at 4°C. A solution was prepared by dissolving 4 mg of the extract in 10 mL of methanol to create a 400 µg/mL stock solution, followed by progressive dilution with methanol to produce concentrated solutions (50, 100, 150, 200, 250, and 300 µg/mL). Two milliliters of extract solution from each concentration were placed in test tubes, and 3 mL of the DPPH solution was added to each tube. After a 30-minute incubation in the dark, absorbance at 517 nm was measured using a UV-Vis spectrophotometer (Genesys 20, Thermo Scientific, USA) in optical density (OD).

Ascorbic acid (AA) served as the reference standard, with an 800 µg/mL stock solution prepared by dissolving 2 mg of ascorbic acid in 2.5 mL of distilled water. Serial dilutions were then made using various concentrated solutions (50, 100, 150, 200, 250, and 300 µg/mL) for the corresponding extract solution, with a 0.1 mM DPPH solution used as the control. The DPPH free radical scavenging ability (DPPH_RSA) was quantified as the percentage of inhibition, calculated using the formula.

$$DPP{H}_{RSA}(\%)=\frac{A_{control}-A_{sample}}{A_{control}}\times 100$$

where A_control: Absorbance of a solution containing DPPH solution and A_sample: Absorbance of the sample in the presence of DPPH solution.

ABTS radical scavenging activity: The scavenging ability of ABTS free radicals by the aqueous extract of C. odorata was assessed following the protocol outlined by Hussen and Endalew (2023). A 7 mM ABTS solution was prepared by dissolving 0.36 g of ABTS salt in 100 mL of distilled water. Additionally, a 2.45 mM potassium persulfate solution was prepared by dissolving 0.066 g of the salt in 100 mL of distilled water. Following this, the ABTS cation radical stock solution was generated by gently mixing 10 mL of the 7 mM ABTS solution with 10 mL of the 2.45 mM potassium persulfate solution. This mixture was stored in darkness at room temperature for 12 hours until the reaction was complete and the absorption stabilized.

The resulting cation radical was further diluted in ethanol (1:1) to achieve an absorbance value of 0.7 at a wavelength of 734 nm using a UV-Vis spectrophotometer model 752N Plus (USA). Five microliters of the extract solution at various concentrations (50, 100, 150, 200, 250, and 300 µg/mL) were then combined with 4000 µL of the ABTS⁺ solution and incubated in the dark for 2 hours at room temperature. Subsequently, the absorbance was measured at 734 nm using a UV-Vis spectrophotometer. As a control, a mixture of 10 mL (7 mM ABTS, 2.45 mM K₂S₂O₈) and 20 mL water for water extraction was used. Ascorbic acid (AA) serves as the reference standard for comparison with the stock solution and is also prepared using the method outlined in the ABTS radical scavenging assay. The percentage of ABTS⁺ free radical scavenging ability (ABTS_RSA) was computed for different concentrations of standards and extracts according to the established formula.

$$ABT{S}_{RSA}(\%)=\frac{A_{control}-A_{sample}}{A_{control}}\times 100$$

where A_control: Absorbance of a solution containing ABTS solution and A_sample: Absorbance of the sample in the presence of ABTS solution.

Experimental design

The original AECO solution was diluted with distilled water at varying ratios (1:30, 1:16, 1:8, 1:4, and 1:2) to achieve concentrations of 780, 1560, 3120, 6250, and 12500 mg DW/L. Five experimental treatments, namely AECO₇₈₀, AECO₁₅₆₀, AECO₃₁₂₀, AECO₆₂₅₀, and AECO₁₂₅₀₀, were established to represent these concentrations. Additionally, two control treatments were included: the fenamiphos treatment at a concentration of 4.65 mg/mL as the positive control and the water treatment as the negative control. The specified concentrations of both the extract and fenamiphos were administered in both in vitro and in vivo models (Nhung and Quoc 2023).

Radopholus similis preparation for bioassays

R. similis populations were collected from Cavendish banana plants in a plantation in Phu My Hung commune, Cu Chi district, Ho Chi Minh City. The isolation and identification of R. similis populations were conducted at the Laboratory of Biotechnology, Institute of Biotechnology and Food Technology, Ho Chi Minh City University of Industry (IUH). Following the protocol outlined by Reise et al. (1987), these populations were cultured and preserved in carrot agar medium, which serves as nutritional support for the growth, development, and reproduction of nematodes.

In vitro test

Extraction of nematode eggs: Eggs from R. similis-infected banana roots were collected, and root fragments with egg masses were cut into small pieces. These fragments were then introduced into a 500 mL container containing 200 mL of 0.5% Clorox solution (sodium hypochlorite, NaOCl), which was manually agitated vigorously for 4 minutes. This agitation aimed to break down the soft background material surrounding the eggs. Following this, the solution was filtered through two nested sieves, 200-mesh (75 µm) and 500-mesh (25 µm). Eggs retained in the 500-mesh sieve were thoroughly washed from the NaOCl solution with a slow stream of cold running water into a 1 L collection bucket. The root pieces initially placed in the collection bucket were washed twice with water to ensure the removal of any remaining eggs (Haroon et al. 2018).

Evaluating ratios of hatching inhibition: Eggs were collected using the approach outlined by Haroon et al. (2018). A water-based egg suspension was created, consisting of 1 mL of egg suspension (100 ± 10 eggs/mL) and 5 mL of root extract solution. The mixture was then transferred to a petri dish and maintained at room temperature. Each treatment was replicated three times, and petri dishes with 1 mL of egg suspension and 5 mL of distilled water served as controls. Following 24, 48, and 72 hours of exposure, the hatched eggs were quantified using a phase-contrast microscope. The hatching inhibition rate (RHI) was calculated by following the formula (Zaidat et al. 2020):

$$RHI(\%)=\frac{\text{The initial number of eggs - The number of eggs hatched}}{\text{The initial number of eggs}}\times 100$$

Bioassay of R. similis juvenile survival rate: The efficacy of the aqueous extract from C. odorata leaves and stems (AECO) at concentrations of 780, 1560, 3120, 6250, and 12500 mg DW/L (milligrams of dry weight per liter, where 1 mL AECO was mixed with 1 mL nematode juvenile buffer) was assessed over 48 hours. Nematode juvenile buffer (1 mL) with > 100 juvenile nematodes was maintained in an incubator at 24°C, shielded from light, and placed in a 24-well plate. Each treatment was replicated three times. Using a binocular microscope, 100 juvenile nematodes were counted between live and dead samples after 12, 24, and 48 hours of exposure. Confirmation of nematode death, whether in a straight or immobile state, was achieved by stimulating them with a drop of NaOH. The survival rate of juvenile nematodes (SRJ) was calculated using the formula (Zaidat et al. 2020).

$$SRJ(\%)=\frac{\text{Number of living nematodes}}{\text{Total number of initial nematodes}}\times 100$$

In vivo test

Design of in vivo experiment: The in vivo experiment followed the protocol established by Marin et al. (2000) Banana plantlets obtained from tissue culture were individually grown in 2.5-liter foam pots. The substrate was a 1:1 blend of sterile coarse river sand and 254 µm silica sand. A nutrient solution (Chem-Gro, HydroGardens Inc., Colorado Springs, CO) was applied bi-weekly, and water was supplied as necessary. All treatments received consistent fertilizer and water applications. Before inoculation, the plants underwent a one-week acclimatization period, growing at approximately 27°C and 80% relative humidity (RH) in the greenhouse. A mixture of juvenile and adult R. similis was extracted from carrot agar plates, quantified, concentrated, and reconstituted in sterile deionized water (approximately 40 nematodes/mL). Five milliliters of this nematode suspension (approximately 200 nematodes) were introduced into the soil at the base of each plant. The cultivated banana plants were maintained for 9 weeks in a greenhouse with conditions set at around 25 ± 2°C and 80% RH before harvesting.

Quantification of antioxidant compounds in banana roots

Lipid peroxidation determination (MDA contents): Lipid peroxidation products were assessed by measuring thiobarbituric acid reactive substances (TBARS) and quantifying malondialdehyde (MDA), following protocol of El-Beltagi et al. (2011). A 0.5 g root sample was homogenized in 2 mL of 0.1% (w/v) trichloroacetic acid (TCA) and then centrifuged at 12,000 × g for 20 minutes. The resulting supernatant (1 mL) was combined with an equal volume of 10% (w/v) TCA containing 0.5% (w/v) TBARS or without TBARS for the blank sample. The mixture underwent heating at 95 ± 1°C for 30 minutes and subsequent cooling in an ice bath. After centrifugation at 12,000 × g for 15 minutes, surface absorbance was measured at 532 nm and 600 nm. Following the subtraction of nonspecific absorbance (600 nm), MDA concentration was expressed as µmol/g fresh weight.

Determination of total glutathione (GSH): The total glutathione (GSH) synthesis level was assessed following method of El-Beltagi et al. (2012). Root samples (0.5 g) were homogenized in 6% m-phosphoric acid (pH 2.8) with 1 mM EDTA. The resulting mixture was combined with 630 µL of 0.5 M K₂HPO₄ and 25 µL of 5,5’-dithiobis (2-nitrobenzoic acid) at pH 7.0. After 2 minutes, the absorbance at 412 nm was measured. GSH concentration was determined using a standard curve and expressed as µmol/g fresh weight.

Assay of superoxide dismutase (SOD) specific activity: Superoxide dismutase (SOD) activity was assessed by measuring its capacity to inhibit Nitro Blue Tetrazolium (NBT) photoreduction, following the protocol by El-Beltagi et al. (2012). In a 3 mL reaction mixture comprising 50 mM phosphate buffer (pH 7.8), 13 mM methionine, 75 µM NBT, 2 µM riboflavin, 1.0 mM EDTA, and 20 µL of enzyme extract, riboflavin was the final addition. The reaction was initiated by exposing the tubes to a 15W fluorescent lamp placed 30 cm away. After 10 minutes of light exposure, turning off the lamp stopped the reaction, and the tubes were covered with black cloth. Control tubes without light exposure were used as references. Absorbance readings were taken at 560 nm, defining the enzyme unit as the volume of the enzyme extract that corresponded to 50% reaction inhibition.

Catalase (CAT) activity assay: CAT activity was assessed by measuring the degradation of H₂O₂ using method of El-Beltagi et al. (2011). In a 3 mL reaction mixture comprising 50 mM potassium phosphate buffer with a pH of 7.0, 15 mM H₂O₂, and 50 µL of enzyme extract, the reaction was initiated by adding H₂O₂. The reduction in H₂O₂ was monitored by measuring absorbance at 240 nm over 3 minutes using spectrophotometry.

The dry weight of the aerial part and the root: Collect banana plants from the pots after the experiment concludes. Separate the aerial parts (stem, leaves) and the roots of the plants. Measure the fresh weight of the aerial and root parts using a precise scale. Subsequently, wash the aerial and root parts with clean water and place them in a dryer at a temperature of 70-80°C until a constant weight is achieved (dry weight). Measure the dry weight of the aerial and root parts using a precise scale (Marin et al. 2000). Calculate the dry weight (DW) using the formula: $DW(g)=FW-\left(FW\times \frac{A}{100}\right)$ With DW as the dry weight, FW as the fresh weight, and A as the moisture in the sample.

Experiment in soil: Pots in the greenhouse were set on benches and kept at a constant temperature of 25 ± 2°C. The R. similis population density in the soil was biweekly assessment by extracting nematodes from 500 mg soil samples per pot, which were then placed in an extraction flask. An equivalent volume of phosphate-buffered saline (PBS) was introduced into the extraction flask. The nematode extraction process employed the centrifugal-flotation and sieving technique, ensuring a uniform interaction between the extraction solution and the soil sample for a specific duration to achieve optimal extraction. The resulting extract solution underwent filtration for debris removal and clarity improvement. The filtered solution was collected and transferred to a sample container. Direct nematode counting was performed by placing a small extract volume (1 mL) on a counting grid and directly counting under a microscope (D’Addabbo et al. 2023). Nematode density per gram (NDG) of soil was determined using the formula:

$$NDG(nematode/g)=\frac{NNE}{VSSP}\times VES$$

The nematode density per gram (NDG) is calculated by dividing the number of nematodes extracted (NNE) by the product of the volume of soil sample collected from each pot (VSSP) and the volume of extraction solution (VES).

Statistical analysis

The experimental setup adhered to a design characterized by complete randomization. In vitro and in vivo test parameters underwent the one-way analysis of variance (ANOVA). Mean comparisons were performed using Tukey’s Honestly Significant Difference (HSD) at a significance level of p < 0.05, facilitated by the Statgraphics Centurion XIX software.

Analyzing and measuring phytochemicals in C. odorata aqueous extract

The qualitative phytochemical analysis of the aqueous extract from C. odorata leaves and stems yielded positive outcomes for various compounds, including polyphenols, flavonoids, terpenoids, steroids, saponins, tannins, and alkaloids, except cardiac glycosides (Table 1). The presence of saponins, indicated by the formation of dense froth, highlighted a substantial presence of saponins in the extract. Positive evaluations confirmed the presence of tannins, flavonoids, and polyphenols in the aqueous extract. The extract demonstrated robust positive results in qualitative tests for alkaloids, steroids, and terpenoids. In terms of quantitative analysis, the botanical constituents were measured, revealing a polyphenol content of 71.84 ± 2.14 mg GAE/g, a flavonoid content of 37.92 ± 2.23 mg QE/g, and a tannin content of 71.84 ± 2.14 mg CE/g (Table 2). These findings underscore the rich phytochemical composition of the C. odorata extract, including notable quantities of polyphenols, flavonoids, and tannins.

Table 1 . Qualitative screening of phytochemicals present in the aqueous extract of C. odorata.

Phytochemicals	Presence in AECO	Phytochemicals	Presence in AECO
Alkaloids	+	Cardiac glycosides	-
Tannins	+	Steroids	-
Saponins	+	Terpenoids	+
Polyphenols	+	Flavonoids	+

AECO, aqueous leaf and stem extract of Chromolaena odorata; +, present; -, absent..

Table 2 . Quantification of flavonoids, alkaloids, and tannins in the aqueous extract of C. odorata.

Sample	Total flavonoid content (mg QE/g)	Total tannin content (mg CE/g)	Total polyphenol content (mg GAE/g)
AECO	37.92 ± 2.23	70.43 ± 1.21	71.84 ± 2.14

AECO, aqueous leaf and stem extract of Chromolaena odorata; GAE, gallic acid equivalents; QE, quercetin equivalents; CE, catechin equivalents..

Antioxidant properties of aqueous extract from C. odorata in in vitro studies

DPPH radical scavenging activity

The DPPH assay was employed to evaluate the antioxidative potential of AECO, relying on the ability of antioxidant compounds to donate atomic or hydrogen electrons to the DPPH radical, transforming it into 1,1-diphenyl-2-picrylhydrazine. The assay, conducted on the aqueous extract of C. odorata leaves and stems across concentrations from 50 to 300 µg/mL (Fig. 1), revealed concentration-dependent free radical scavenging activity. The scavenging activity ranged significantly from 59.67% to 92.13% within the 50 to 300 µg/mL range. In comparison, the standard ascorbic acid (AA) demonstrated activity ranging from 94.56% to 95.75%. These results indicate a noteworthy antioxidative potential of AECO, exhibiting free radical scavenging activity comparable to the standard ascorbic acid.

Figure 1. The 2,2-diphenylpicrylhydrazyl radical-scavenging activity of the aqueous extract derived from Chromolaena odorata leaves and stems was evaluated. The results are presented as the mean ± standard deviation, with letters (a, b, c, d, e, and f) denoting significant differences between treatments (p < 0.05)

ABTS radical scavenging activity

The ABTS radical cation is formed by oxidizing ABTS with potassium persulfate. This cationic radical undergoes a reduction in the presence of antioxidants that provide hydrogen atoms. Fig. 2 illustrates the ABTS scavenging efficiency of the aqueous extract from C. odorata leaves and stems, with the inhibitory percentage increasing proportionally with extract concentration. Comparative assessment against the standard ascorbic acid (AA) shows that AECO reaches its maximum activity at 300 µg/mL. The extract can neutralize 37.26% of ABTS⁺ radicals, while ascorbic acid achieves higher efficacy at 88.47%. These results emphasize the antioxidant potential of C. odorata extract in counteracting ABTS⁺ radicals.

Figure 2. 2,2’-Azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) radical-scavenging activity of the aqueous extract of Chromolaena odorata leaves and stems. The results are presented as the mean ± standard deviation, with letters (a, b, c, d, e, and f) denoting significant differences between treatments (p < 0.05)

In vitro test

The hatching inhibition ratio of R. similis

The results indicate that the aqueous extract from C. odorata leaves and stems exerts a substantial inhibitory effect on the hatching of R. similis eggs (Table 3). Over 24, 48, and 72 hours of exposure, there was a significant reduction in hatching rates, with inhibition rates reaching 26.98%, 55.25%, and 82.92% at 24h, 48h, and 72h, respectively, at an AECO concentration of 12500 mg DW/L (p < 0.05). Notably, there was a pronounced increase (p < 0.05) in inhibition rates with higher extract concentrations, ranging from 780 mg DW/L to 12500 mg DW/L. The results signify that AECO possesses significant inhibitory properties against the hatching of R. similis eggs. The inhibitory effect is dose-dependent, with higher concentrations of the extract demonstrating greater effectiveness in suppressing the hatching process. The substantial reduction in hatching rates across different exposure durations underscores the potential of C. odorata extract as a promising agent for controlling the hatching of R. similis eggs, suggesting its utility as a natural and effective alternative in nematode management strategies.

Table 3 . Effect of AECO treatment on the hatching of Radopholus similis egg.

Treatments	Hatching inhibition ratio of R. similis
	24 h			48 h			72 h
	INI eggs	HAT eggs	RHI (%)	INI eggs	HAT eggs	RHI (%)	INI eggs	HAT eggs	RHI (%)
Water treatment	105.67 ± 2.52ab	83.67 ± 1.53b	20.81 ± 0.46b	23.33 ± 1.53a	13.67 ± 1.53a	41.53 ± 3.43a	10.33 ± 1.53a	3.67 ± 1.15a	65.00 ± 6.01a
Fenamiphos treatment	108.67 ± 1.53b	79.00 ± 2.00a	27.31 ± 0.86e	29.67 ± 1.53d	13.33 ± 1.53a	54.95 ± 5.77c	16.33 ± 1.53c	2.67 ± 1.53a	84.12 ± 8.17c
AECO780 treatment	107.33 ± 2.08ab	84.00 ± 2.00b	21.72 ± 2.11ab	23.33 ± 1.53a	13.00 ± 1.00a	44.01 ± 6.97ab	10.67 ± 0.58a	3.67 ± 0.58a	65.76 ± 3.67ab
AECO1560 treatment	106.33 ± 2.08ab	81.67 ± 0.58b	23.17 ± 1.93bc	24.67 ± 0.58ab	13.67 ± 0.58a	44.61 ± 1.06ab	11.00 ± 1.00a	3.33 ± 0.58a	69.80 ± 3.04ab
AECO3120 treatment	104.67 ± 1.53a	78.67 ± 0.58a	24.83 ± 1.56cd	26.00 ± 1.00bc	13.00 ± 1.00a	49.95 ± 1.93bc	13.33 ± 0.58b	3.33 ± 0.58a	74.91 ± 4.99bc
AECO6250 treatment	106.67 ± 1.53ab	78.67 ± 1.15a	26.25 ± 0.75de	28.00 ± 1.00cd	13.33 ± 0.58a	52.30 ± 3.68c	14.67 ± 0.58bc	3.00 ± 1.00a	79.52 ± 6.72c
AECO12500 treatment	105.00 ± 1.00a	76.67 ± 0.58a	26.98 ± 0.35de	28.33 ± 0.58d	12.67 ± 0.58a	55.25 ± 2.92c	15.67 ± 0.58c	2.67 ± 0.58a	82.92 ± 4.02c

The values are expressed as the mean ± standard deviation, where the letters (a, b, c, d, and e) indicate differences between treatments (p < 0.05). AECO, aqueous leaf and stem extract of Chromolaena odorata; INI eggs, initial number of eggs; HAT eggs, number of eggs hatched; RHI, the hatching inhibition rate..

Bioassay of R. similis juvenile survival rate

The results of the biological assay on the survival rate of juvenile R. similis nematodes provide valuable insights into the impact of C. odorata aqueous extract (AECO) on nematode viability. This bioassay is crucial in evaluating the effectiveness of potential control measures, such as chemical treatments or natural extracts, and provides valuable information for understanding the ability of these interventions to suppress or inhibit the survival and reproduction of R. similis nematodes. Exposure to AECO for 12, 24, and 48 hours resulted in a notable reduction in the survival rate of R. similis juveniles. This reduction exhibited an inverse relationship with both the concentration of the extract and the duration of exposure, as illustrated in Fig. 3. At each of the specified exposure times, namely 12, 24, and 48 hours, there was a significant increase (p < 0.05) in immobility and mortality rates among R. similis juveniles when subjected to AECO concentrations ranging from 780 to 12500 mg DW/L, signifying a substantial difference compared to the water control (p < 0.05). Particularly noteworthy is the 48h exposure time, where the survival rate of R. similis juveniles treated with AECO at 12500 decreased to a mere 10.52%, approaching the effectiveness of fenamiphos treatment (7.68%). This underscores the potent nematocidal activity of AECO, particularly at higher concentrations and longer exposure durations, making it a promising candidate for nematode control strategies, potentially comparable to conventional chemical treatment.

Figure 3. Effect of the aqueous extract of Chromolaena odorata leaves and stems on the survival rate of juvenile Radopholus similis after 12, 24, and 48 h. The results are presented as the mean ± standard deviation, with letters (a, b, c, d, e, and f) denoting significant differences between treatments (p < 0.05)

In vivo test

Quantification of antioxidant compound levels in banana roots

The results depicted in Table 4 provide compelling insights, revealing a significant increase (p < 0.05) in MDA concentrations in roots infected with nematodes (water treatment). This elevation coincides with a substantial reduction in antioxidants such as GSH, CAT, and SOD (p < 0.05). Following treatments with fenamiphos and AECO, a distinct improvement in these parameters is evident. MDA concentrations show a notable decrease across all treatments (p < 0.05), with the most significant decrease was observed in the AECO₁₂₅₀₀ treatment (p < 0.05) and the fenamiphos treatment (p < 0.05). Conversely, the levels of antioxidant compounds in banana roots demonstrate a substantial rise (p < 0.05), with the most pronounced elevation seen in the AECO-treated (12500 mg/kg), nearly equivalent to the standard fenamiphos control treatment. The results suggest that fenamiphos and, notably, AECO, particularly at the highest concentration, exhibit promising effects in alleviating oxidative stress and enhancing the antioxidant defense system in banana roots affected by R. similis nematode infestation.

Table 4 . Effect of AECO on the levels of antioxidant compounds in banana roots parasitized with Radopholus similis nematodes.

Treatments	MDA (µmol/g)	GSH (µmol/g)	SOD (unit/mg protein)	CAT (unit/mg protein)
Water treatment	21.06 ± 2.42e	4.09 ± 1.17a	71.45 ± 4.56a	15.61 ± 1.28a
Fenamiphos treatment	4.37 ± 0.63a	19.35 ± 1.09g	168.08 ± 18.45f	69.16 ± 2.78f
AECO780 treatment	16.14 ± 2.13d	7.22 ± 0.23b	86.04 ± 5.21ab	20.54 ± 1.23b
AECO1560 treatment	13.54 ± 1.28c	9.69 ± 0.69c	101.15 ± 9.78bc	23.57 ± 1.08c
AECO3120 treatment	11.09 ± 1.17bc	11.82 ± 1.14d	122.19 ± 12.49cd	27.57 ± 1.27d
AECO6250 treatment	9.07 ± 1.15b	13.68 ± 1.18e	137.98 ± 15.43de	30.48 ± 2.17d
AECO12500 treatment	6.32 ± 0.62a	17.42 ± 1.27f	159.57 ± 15.36ef	57.39 ± 1.16e

The values are expressed as mean ± standard deviation, where the letters (a, b, c, d, e, f, and g) indicate differences between treatments (p < 0.05). AECO, aqueous leaf and stem extract of Chromolaena odorata; MDA, malondialdehyde; GSH, glutathione; SOD, superoxide dismutase; and CAT, catalase..

The aerial part and root dry weights

The outcomes from Table 5 distinctly illustrate the significant impact of various treatment methods on the dry biomass weight of different parts of banana plants. The aerial part biomass weight of banana plants exhibits a noteworthy variance among experimental treatments (p < 0.05), with the lowest recorded in the water treatment (5.64 ± 0.44 g) (p < 0.05) and the highest in the AECO₁₂₅₀₀ treatment (15.38 ± 0.13 g) (p < 0.05), closely comparable to the fenamiphos treatment (17.33 ± 0.13 g). This suggests that employing C. odorata extract at a concentration of 12500 mg/kg (AECO₁₂₅₀₀) has positively influenced the aerial part growth of banana plants. Particularly, the dry biomass weight of the roots also demonstrates a marked change (29.32 ± 0.15 g) when treated with AECO₁₂₅₀₀ compared to the control treatment (17.33 ± 0.47 g) (p < 0.05), concurrently exhibiting a substantial increase nearly equivalent to the fenamiphos treatment. This outcome suggests that AECO, especially at a concentration of 12500 mg/kg, can significantly contribute to the root growth of banana plants. These findings provide crucial insights into the variations in biomass weight across different plant parts under the influence of diverse treatment methods, enhancing our understanding of growth efficiency and their ecological impact on banana plants.

Table 5 . Effect of AECO on the aerial part and root dry weights of banana plants parasitized with Radopholus similis.

Parameters	Water treatment	Fenamiphos treatment	AECO780 treatment	AECO1560 treatment	AECO3120 treatment	AECO6250 treatment	AECO12500 treatment
Aerial part dry weight (g)	5.64 ± 0.44a	17.33 ± 0.13g	6.37 ± 0.16b	8.59 ± 0.2c	10.45 ± 0.16d	12.86 ± 0.13e	15.38 ± 0.13f
Root dry weight (g)	17.33 ± 0.47a	30.73 ± 0.89g	19.18 ± 0.42b	21.35 ± 0.36c	23.98 ± 0.45d	26.51 ± 0.41e	29.32 ± 0.15f

The values are expressed as the mean ± standard deviation, where the letters (a, b, c, d, e, f, and g) indicate differences between treatments (p < 0.05). AECO, aqueous leaf and stem extract of Chromolaena odorata..

Soil experiment

The investigation into nematode density in the soil of banana pots infected with R. similis primarily aims to assess the extent and impact of R. similis nematode infestation on the soil and plant cultivation environment. Results from Fig. 4 reveal a substantial reduction in R. similis nematode density in the soil of banana pots following treatment with C. odorata extract (AECO). Nematode density markedly decreased in experimental treatments using AECO compared to the negative control treatment (water treatment) (25.12 ± 0.39 nematode/g), especially at higher doses such as AECO₃₁₂₀ mg/kg, AECO₆₂₅₀ mg/kg, and AECO₁₂₅₀₀ mg/kg (16.26 ± 0.3, 13.34 ± 0.28, and 9.2 ± 0.29 nematode/g, respectively) (p < 0.05).

Figure 4. Effect of soil treatment using the aqueous extract of Chromolaena odorata leaves and stems on the population density of Radopholus similis nematodes in banana plants in pots. The results are presented as the mean ± standard deviation, with letters (a, b, c, d, and e) denoting significant differences between treatments (p < 0.05)

This indicates the nematode-suppressing capability of C. odorata extract. The decrease in nematode density tends to increase as the AECO dosage rises, with the highest dose (12500 mg/kg) displaying the most potent effectiveness in nematode control. Results imply that AECO could be an effective alternative, even a potential replacement, for traditional chemical agents in nematode control. Notably, nematode density in the treatment with the highest AECO dose (12500 mg/kg) is nearly equivalent to that of the fenamiphos-treated (8.14 ± 0.36 nematode/g), a conventional chemical phytosanitary nematode control.

The recent strategy in nematode management focuses on specifically addressing the capability to decrease the population of plant-parasitic nematodes in the soil using natural extracts from various plant species. These methods avoid disrupting the natural biological equilibrium. Globally, the utilization of resistant plants or their by-products is a common practice to alleviate the risks linked with conventional chemical nematicides (Sikder and Vestergård 2020).

In the realm of plant extracts, secondary metabolites such as flavonoids, alkaloids, phenolics, terpenoids, saponins, tannins, and steroids manifest potent antioxidant attributes, alleviating the detrimental impacts of reactive oxygen species (ROS). Together, these compounds orchestrate a robust and all-encompassing antioxidant defense mechanism within plants, curtailing the repercussions of ROS triggered by nematode-induced infections. Flavonoids and alkaloids obstruct reactive oxygen species generation by hindering enzymes linked to oxidative pathways, such as lipoxygenase and cyclooxygenase. Phenolic compounds engage directly with ROS, establishing a defensive barrier against their assault on cellular frameworks. Terpenoids distinctly reduce ROS levels by providing electrons or hydrogens to stabilize various ROS variants. Select saponins activate the synthesis of antioxidant enzymes like superoxide dismutase (SOD) and catalase (CAT), establishing conducive circumstances for ROS eradication. Tannins and steroids contribute to the preservation of cell membrane stability, amplifying the efficacy of antioxidant enzymes, countering free radicals, and shielding cells from harm (Ciampi et al. 2020). Moreover, bioactive compounds present in plants hold promise as agents for combatting and deterring nematodes. Phenolics, flavonoids, and tannins assume pivotal roles in shielding plants from oxidative stress and thwarting nematode infestations. Phenolics, serving as potent antioxidants, assist plants in combating oxidative stress by nullifying reactive oxygen species (ROS). They furnish electrons to stabilize free radicals, thereby averting cell damage induced by oxidative reactions. Some phenolic compounds also exhibit nematocidal properties, disrupting the physiological processes of nematodes and impeding their growth. Flavonoids contribute to the comprehensive antioxidant defense system by scavenging free radicals and inhibiting lipid peroxidation processes. They are instrumental in protecting plants from oxidative damage attributable to diverse environmental pollutants. Certain flavonoids showcase nematocidal traits, influencing the behavior, reproduction, and development of nematodes. Tannins exhibit robust antioxidant attributes, proficiently obstructing free radicals and thwarting oxidative damage to cellular components. They play a contributory role in the overall antioxidant defense system of plants. Additionally, tannins can function as nematode repellents, shaping nematode behavior and diminishing their capacity to infect plant roots (Vijayaraghavan et al. 2018).

Plants rich in phenolic compounds, flavonoids, tannins, and similar constituents have attracted considerable attention for their diverse physiological benefits, encompassing activities such as scavenging free radicals, anti-mutagenic, anti-cancer, and anti-inflammatory properties. According to Adebiyi et al. (2017), the antioxidant efficacy of phenolics predominantly stems from their redox attributes, functioning as reducing agents, hydrogen donors, single oxygen quenchers, and potential metal chelators. The ABTS⁺, assay served as a tool to showcase the antioxidant potential of the test samples. AECO demonstrated a notable ABTS⁺ free radical scavenging capacity, peaking at 37.26% in this study, underscoring its proficiency in eliminating ABTS radicals. In this investigation, the DPPH radical scavenging activity exhibited an upward trend with increasing extract concentration. This pattern suggests an augmented ability to supply hydrogen ions, resulting in a lighter solution, proportionally correlated with the quantity of electrons obtained. Hence, it can be deduced that AECO engages in DPPH scavenging by converting free radicals into corresponding hydrazine through its hydro-ion-supplying capability. The demonstrated potential of the aqueous extract of C. odorata leaves and stems to neutralize various free radicals implies its promising therapeutic role in mitigating the impact of oxidative stress.

All developmental stages of R. similis take place within the root system. The nematode infects near or at the root tips and tunnels through the root system, periodically extracting cellular contents for sustenance. Females deposit eggs within the roots or in the surrounding soil. While parthenogenetic reproduction is possible, in instances where males are absent or if females don’t mate within 50 days post-molt into adults, they transition to hermaphroditic reproduction. This reproductive strategy facilitates a swift population increase following the invasion of a new host (Jones et al. 2013). The utilization of plant extracts to manage plant-parasitic nematodes has seen increased interest in recent years (Sikder and Vestergård 2020). The aqueous extract of C. odorata leaves and stems has undergone testing, revealing both antagonistic and nematocidal effects against burrowing nematodes harmful to banana plants (Tran et al. 2023). Consequently, AECO may contain natural nematocidal agents capable of impeding the hatching of nematode eggs. The chemical compounds within AECO establish a comprehensive defense mechanism against the egg-hatching of R. similis nematodes. Phenolics interact with the nematode’s oxidative processes, disrupting the regular egg-hatching sequence. Alkaloids impede the physiological processes and growth of nematode eggs. Flavonoids interfere with free radical reactions, disrupting the typical development of nematode eggs. Tannins bind to nematode proteins, influencing the structural integrity of nematode eggs. Saponins hinder the synthesis of enzymes crucial to the hatching process, creating adverse conditions for egg development. Terpenoids supply electrons or hydrogens to stabilize diverse oxidative reactions, potentially upsetting the necessary oxidative equilibrium for regular egg hatching (Sikder and Vestergård 2020).

R. similis juveniles exhibit a slender morphology with a body structure comprising a cuticle, esophagus, and nerve ring. These juveniles display both passive and active movement within the root zone to locate suitable feeding sites, demonstrating chemotaxis in response to chemical signals for host selection. Operating as plant-parasitic nematodes, R. similis juveniles feed on plant cells, particularly within root tissues, using their stylet to puncture plant cells and release cell contents for nutrition. The juvenile stage is highly responsive to environmental factors, including temperature, humidity, soil conditions, and chemicals, exhibiting adaptive behaviors such as soil migration in response to environmental cues (Haegeman et al. 2010). In vitro assessments highlight the robust efficacy of the aqueous extract of C. odorata leaves and stems on R. similis juveniles. AECO, rich in secondary metabolites like phenolics, alkaloids, flavonoids, tannins, saponins, and terpenoids, exerts diverse effects by disrupting physiological processes, causing damage to cuticular and nerve ring structures, and impeding the feeding capacity of R. similis juveniles. Fenamiphos inhibits cholinesterase enzymes, resulting in delayed nerve conduction, ultimately leading to mortality and increased immobility rates in R. similis juveniles. The compelling evidence of the efficacy of both AECO and fenamiphos in inducing mortality in juvenile R. similis is a noteworthy achievement of this study.

R. similis nematodes, upon infiltrating banana root cells, instigate an excessive generation of reactive oxygen species (ROS). The subsequent cellular discordance with the nematode is characterized by the formation of diverse reactive oxygen species, triggering lipid peroxidation and ensuing cell demise, as evidenced by heightened malondialdehyde (MDA) levels in banana roots (El-Beltagi et al. 2012). The escalated MDA production post-nematode infestation results in a notable depletion of antioxidants, including glutathione (GSH) and catalase (CAT). Furthermore, R. similis incursion induces the production of superoxide anion (O^2-) within banana plants, influencing the activity of superoxide dismutase (SOD) and contributing to diminished SOD levels (El-Beltagi et al. 2011). The phytochemicals in AECO showcase beneficial effects by curtailing MDA levels (indicative of cellular damage) and elevating GSH, CAT, and SOD levels in banana roots. This augmentation of antioxidant prowess serves to mitigate cellular damage and reduce MDA levels, a byproduct of lipid cell damage. The plant compounds in the extract also stimulate the synthesis or regeneration of GSH and CAT, expediting the breakdown of oxidative compounds and shielding cells from contamination, thereby averting the accumulation of oxidative substances within cells. Additionally, AECO propels the production of SOD, a pivotal enzyme that decomposes superoxide into hydrogen peroxide and oxygen, mitigating cellular damage. Therefore, the heightened MDA concentrations in nematode-infected roots under water treatment signal a substantial oxidative stress response, likely stemming from nematode-induced damage. This is corroborated by the concurrent decline in antioxidants (GSH, CAT, and SOD), indicating a compromised defense mechanism against oxidative stress in nematode-affected banana roots. Post-treatment with fenamiphos and AECO reveals a notable alleviation in these parameters, with MDA concentrations experiencing a marked reduction across all treatments. The most significant decline is observed in the AECO₁₂₅₀₀ treatment, highlighting its potent protective effect. The substantial increase in antioxidant compounds in banana roots after treatments, especially in the AECO-treated (12500 mg/kg), suggests a restoration or enhancement of the antioxidant defense system. This noteworthy elevation, nearly equivalent to the standard fenamiphos control, indicates that AECO, particularly at the highest concentration, holds promise in effectively mitigating nematode-induced oxidative stress and enhancing the overall resilience of banana plants.

The assessment of dry biomass weight in the aerial part and root parts of banana plants aims to evaluate the distribution and expression of plant mass in distinct sections, offering insights into growth dynamics. Biomass variations serve as indicators of overall plant performance, aiding the understanding of growth patterns at different developmental stages. The measurement of the aerial part of biomass provides valuable information on resource allocation and energy utilization, contributing to a comprehensive understanding of plant growth processes (Patrick et al. 2010). The substantial reduction in dry biomass weight observed in both the aerial part and root components of banana plants subjected to water treatment highlights the severity of R. similis nematode infestation and its detrimental impact on plant development. The notable decline in the aerial part of biomass may signify disruptions in photosynthesis and energy conversion, as nematodes extract nutrients, impairing the plant’s capacity to absorb and convert essential elements from the soil and air. The considerable decrease in root biomass further indicates significant damage to the root system, diminishing the plant’s ability to absorb water and nutrients, resulting in energy depletion and compromised overall plant health. Conversely, the gradual increase in dry weight in both the aerial part and root portions of banana plants treated with AECO, especially at the highest dose (12500 mg/kg), closely resembling the fenamiphos treatment, suggests the positive impact of C. odorata extract (AECO) in stabilizing and potentially enhancing banana plant growth affected by R. similis nematodes. The rise in the aerial part biomass, particularly at the highest AECO dose, implies the extract’s potential to supply nutrients and support the photosynthesis process, mitigating adverse effects caused by R. similis nematodes. The augmentation in root biomass weight, especially in the treatment with the highest AECO dose, implies the extract’s ability to fortify the health and nutrient absorption efficiency of the root system, potentially enhancing the plant’s resilience to nematode impact. Consequently, the increased biomass in both the aerial part and root sections of AECO-treated serves as positive indicators of the extract’s inhibitory effects on the adverse impact of R. similis nematodes, concurrently providing nutritional support and stimulating overall plant development.

Examining nematode density in the soil is paramount for understanding the infection status, evaluating the effectiveness of control measures, and creating favorable conditions for managing the cropping environment in cases of R. similis nematode infestation. Controlling nematode density in the soil helps assess the level of R. similis nematode infection, the biological health of the soil, and the threat to crops and soil ecology. A comprehensive understanding of soil nematode density provides crucial insights for effective soil management and the development of nematode control strategies in the cropping environment (D’Addabbo et al. 2023). The findings highlight AECO’s potential to decrease R. similis nematode density, especially at the highest dose (12500 mg/kg), showcasing its efficacy compared to the chemical nematode control fenamiphos. This highlights the promising role of AECO as a viable option for nematode control, offering a potential alternative that is both sustainable and environmentally friendly compared to traditional chemical treatment methods.

The investigation into the antioxidant properties of the aqueous extract from C. odorata leaves and stems revealed a significant DPPH scavenging activity, concentration-dependent. The ABTS assay further confirmed its potent antioxidant capability by achieving maximum inhibition of ABTS⁺ radicals at 300 µg/mL. In vitro assessments demonstrated AECO’s pronounced inhibitory effects on R. similis egg hatching, inversely correlating with extract concentration and exposure time. Moreover, AECO effectively reduced the survival rate of R. similis juveniles. In vivo experiments illustrated a slight reduction in MDA concentrations in nematode-infected roots with AECO treatment. The antioxidant compounds in roots (GSH, CAT, SOD) exhibited a significant increase. The substantial increase in the aerial part and root biomass highlighted the growth-promoting effects of AECO. Additionally, the significant reduction in R. similis nematode density in soil, with the highest efficacy at 12500 mg/kg, emphasized the potent control potential of AECO. In summary, the aqueous extract from C. odorata leaves and stems exhibits promising antioxidant properties and effective nematocidal activity against R. similis, showcasing its potential for organic and sustainable pest management in Cavendish banana cultivation.