Nematicidal Effect of Eupatorium odoratum Linn. Aqueous Extract on Burrowing Nematodes (Radopholus similis) and its Application to Control Toppling Disease on Cavendish Banana (Musa acuminata)

The powdered plant material (1.5 g) was added to 20 mL of distilled water and boiled for 5 min, then allowed to cool and filtered through a thin cotton cloth and filter paper (Whatman No. 1). The volume of water was adjusted to 25 mL to obtain 6% (w/v) extract. The extract was then collected, sterilized using a 0.22 μm Millipore filter (Merck-Millipore, Burlington, MA, USA), and frozen until use. The stock solution was diluted with distilled water at 1 : 2, 1 : 4, 1 : 8, 1 : 16, and 1 : 30. The five dilution ratios corresponded to five concentrations: 12500, 6250, 3120, 1560, 780 mg DW·L⁻¹ (mg dry weight per liter).

A standard phytochemical screening method was used to evaluate the phytochemical profile of EAEO. Chemical reactions and color changes were used as indicators to determine the presence of major chemical groups, such as tannins, saponins, flavonoids, phenols, terpenoids, alkaloids, glycosides, coumarins, and steroids in EAEO (Table 1) (Nascimento et al. 2013; Hodges & Minich 2015; Kumar et al. 2016; Loman & Ju 2017; Wang et al. 2018).

The population of burring nematodes (R. similis) was collected from the roots of Cavendish bananas grown at a banana farm in Phu My Hung commune, Cu Chi district, Ho Chi Minh City, Vietnam. The process of culturing R. similis was carried out in a growth chamber on fresh organic carrots that had been washed and soaked in a 2% sodium hypochlorite solution, following the method described by Koshy and Sosamma (1980). The carrot cores were prepared according to Moody et al. (1973). Centrifuge tubes of 1.5 mL were used to collect and multiply the nematodes. Twenty-five female nematodes were selected under a dissecting microscope and transferred to tubes with 600 μL of 1% ampicillin using a fine needle. After ten minutes, most of the ampicillin solution was removed and replaced with 200 μL of sterile water. Five minutes later, most of the tube water was removed and replaced with 200 μL of antibiotic solution. After a ten-minute rest, 150 μL of the antibiotic solution was removed, and the remaining 50 μL of nematode cyst suspension was pipetted onto the surface of carrot cores. The carrot cores were inoculated with 25 females and maintained in an adjusted growth chamber at 28ºC. After five days, each flask was checked for any contaminating organisms, and contaminated flasks were discarded. After 20 days, nematodes were extracted from carrot tissue as Coolen and D’Herde (1972) described. The nematodes were collected in a 400-mesh sieve. The sieve was washed with sterile water, and the nematodes were recovered. The number of nematodes (including eggs) was estimated by counting them in a counting chamber under a dissecting microscope.

Approximately 800 nematodes of R. similis, second-stage juveniles (J2) were tested for their survival ability after exposure to EAEO for 48 h. Five dilutions of EAEO were prepared corresponding to concentrations of 12500, 6250, 3120, 1560, 780 mg DW·L⁻¹ (EAEO₁₂₅₀₀, EAEO₆₂₅₀, EAEO₃₁₂₀, EAEO₁₅₆₀, and EAEO₇₈₀ treatment, respectively), and two controls (fenamiphos at a concentration of 4.65 mg·mL⁻¹ – positive control, water – negative control). Three replicates were prepared in test tubes for each dilution and maintained at room temperature. Both mobile and immobile nematodes were counted under a microscope. Dead nematodes (immobile and stained with tryptophan) exhibited various peculiar body shapes, such as S-shaped and coiled, with a fluorescent greenish hue indicating tryptophan. The mortality rate was calculated using the following formula (Kesba et al. 2021): Mortality%=Number of death nematode in a treatmentNumber of total tested nematode in the same treatment×100. Mortality\left( \% \right) = {{Number}\,{of}\,{death}\,{nematode}\,{in}\,{a}\,{treatment} \over {Number}\,{of}\,{total}\,{tested}\,{nematode}\,{in}\,{the}\,{same}\,{treatment}} \times 100.

The penetration assays of R. similis (J2) into the host plant roots were conducted using the method described by Marin et al. (2000). 75 wells of the 96-well flat bottom microtiter plate (7 mm diameter and 10 mm depth) was filled with 0.6 g of sterile fine sand (pre-autoclaved). Root segments 4 mm long were excised from the elongation zone of banana plants in the greenhouse. A volume of 25 mL of test EAEO, fenamiphos, and water was distributed to each well, before placing a single root segment horizontally in the growth medium, and adding an extra 0.28 g of dry sand. Nematode cysts were extracted from carrot disk cultures, quantified, and resuspended in sterile deionized water at 8,000 nematodes per mL concentration. A 25 mL of the nematode cyst suspension (~200 nematodes) was added to each well. The microtiter plates were covered with plastic lids and placed in a humidity chamber (100% RH at 25 °C) for 24 h. The root pieces were then collected, and the active nematodes were extracted by soaking in 300 mL of sterile deionized water in separate microtiter plate wells. The plates were then incubated in the humidity chamber (100% RH at 25 °C) for 48 h. The nematode extracts were quantified under a compound microscope using phase-contrast illumination. To detect any remaining nematodes, the root segments were soaked in 1% NaOCl for 10 min, rinsed under running tap water for 15 min, and stained for 30 seconds at boiling temperature with acid fuchsin solution in water. The root segments were placed between two microscope slides (25 × 75 mm), and all nematodes were counted. The experiment was designed and analyzed in a completely randomized design with three replicates. The data were transformed with log10 (x + 1) before analysis.

Cavendish banana plants from tissue culture were grown in 414 mL foam cups (top diameter 8.9 cm, height 13.6 cm). A 1 : 1 mixture of sterile coarse river sand and 254 μm sand (autoclaved for one hour at 121 °C) was used as the growth medium. A complete nutrient solution (Hydro Umat F, Vietnam) was supplemented twice a week. Before nematode inoculation, the plants were grown for four weeks at 27 °C and 80% relative humidity (RH) in the greenhouse. Nematode cysts were extracted from carrot disk culture medium, quantified, and resuspended in sterile deionized water (40 cysts per mL). Five milliliters of nematode cyst suspension (~200 nematodes) were added to the base of each plant. The experiment was designed with seven treatments as above. The EAEO and fenamiphos solution were distributed in 40 mL aliquots and applied to the plants at zero and five days after inoculation. The plants were maintained for eight weeks at approximately 27 °C and 80% RH in the greenhouse (Marin et al. 2000).

At the end of the experiment, the plants were excavated, their roots and stems were cleaned to remove any soil particles, and the root necrosis index (RNI) and the fresh weight of roots and corm (RCFW) were determined. To assess the extent of root decay, the RNI was scored according to Bartholomew et al. (2014) on a scale from 0 to 4 [0: no damage; 1: 1–25% of the total root cortex necrotic; 2: 26–50% of the total root cortex necrotic; 3: 51–75% of the total root cortex necrotic; 4: more than 75% of the total root cortex necrotic].

Throughout the research process, the following plant measurements were recorded on a weekly basis: (a) pseudostem length (measured from the lowest leaf point to the base of the pseudostem, cm); (b) pseudostem circumference (girth measured at a point halfway along the pseudostem length, cm); (c) total number of fully opened functional leaves; (d) leaf area (LA, cm²) predicted using regression models: LA = 0.0266 + (L × W × 0.7629) (r = 0.98), where L represents leaf length and W represents leaf width. The third leaf from the top was selected as the reference leaf for measurement (Potdar & Pawar 1991).

Differences in nematode mortality, infection nematode of root segments, root necrosis index, roots and corm fresh weight, and growth parameters were analyzed using ANOVA. Transformed means were reported in figures and tables, and only significant differences (p < 0.05) were considered. A post hoc analysis, such as Tukey’s HSD test, was conducted to examine specific group differences further. All statistical analyses were performed using the statistical software Statgraphics Centurion XIX.

The presence of plant chemicals in EAEO was demonstrated through positive chemical reactions (Table 1), indicated by notable color changes. Phytochemical screening of EAEO revealed the presence of plant chemicals belonging to the following groups: phenols, flavonoids, terpenoids, alkaloids, tannins, and saponins. Glycoside, steroids, and coumarins were absent in the extract.

The nature of the structure and mode of action of most plant chemicals with pesticidal properties has been extensively discussed. The EAEO has been reported to have the ability to increase mortality rates and reduce populations of various insects and parasitic nematodes (Kato-Noguchi & Kato 2023). It is known that extracts from the E. odoratum plant are rich in alkaloids, phenolics, glycosides, and flavonoids. Extracts containing alkaloids have been found to exhibit ovicidal properties against nematode eggs. Alkaloids can affect and paralyze the central nervous system of nematodes, exhibiting strong antibacterial effects. Phenolic compounds interfere with energy production mechanisms by disrupting the process of oxidative phosphorylation and inhibiting glycoprotein on the cell surface of parasites, leading to their death. The nematicidal effect of glycosides is attributed to their function as inhibitors of cholinesterase enzymes, preventing the typical accumulation of nematodes. Flavonoids, as low-molecular-weight secondary metabolites, have diverse functions, including auxin transport inhibition and protection, and are associated with the ability to resist nematodes’ limited movement and migration (Kato-Noguchi & Kato 2023).

The effects of EAEO on the mortality rate of 800 J2 juveniles were tested in a laboratory experiment using five dilutions. Overall, after 48 h of exposure, all doses of EAEO used showed nematicidal activity against R. similis (Table 2). The percentage of mortality rate reduction varied with the degree of extract concentration. However, the first two dilutions, 1 : 2 and 1 : 4 (corresponding to 12500 and 6250 mg DW·L⁻¹, respectively), exhibited high toxicity with 90.1% and 78% mortality rates, respectively. Nevertheless, the mortality rate decreased to approximately 35.3% at the 1 : 30 dilution (780 mg DW·L⁻¹), the lowest mortality rate among the tested doses. However, the number of dead nematodes and the mortality rate in the experimental treatments were significantly higher compared to the water control.

Most R. similis populations thrive best at an average temperature of 25 °C. Their ability to survive in adverse conditions is enhanced by three factors: (i) a wide host range, (ii) a short life cycle that allows for rapid reproduction during favorable periods, and (iii) female reproductive capabilities without the need for males. However, the nematodes become rigid and immobile in plant extracts when exposed to secondary metabolites such as terpenoids, flavonoids, glucosinolates, etc. In vitro experiments have shown that certain common flavonoids exhibit nematicidal activity, with kaempferol inhibiting the hatching of R. similis eggs. In contrast, kaempferol, quercetin, and myricetin all have nematicidal effects and can attract or repel juveniles (J2) (Haegeman et al. 2010). Tannins are a diverse group of polyphenolic compounds, showing strong sensitivity to R. similis at high extract concentrations. Some plant extracts rich in saponins and alkaloids have demonstrated strong nematicidal activity (Kirwa et al. 2018). In the current study, EAEO secondary metabolites significantly increased the mortality rate of R. similis compared to water control. The production of biologically active secondary metabolites by E. odoratum may play an important role in the control of R. similis. It is consistent with the findings of Kato-Noguchi and Kato (2023) regarding the interaction and nematicidal activity of E. odoratum.

Separate analyses were conducted on nematodes recovered from disease-infected roots and nematodes remaining in the roots after a 48-hour incubation in EAEO, fenamiphos, and water. In both analyses, all treatments significantly reduced nematode infection in the roots compared to the water control (Fig. 1). The number of live nematodes inside the roots for the EAEO treatments at 780 and 1560 mg DW·L⁻¹ (31.3 ± 4 and 27.7 ± 4.2, respectively) was higher than the EAEO at 12500 mg DW·L⁻¹ and the fenamiphos treatments (14.7 ± 3.5 and 11.7 ± 2.5, respectively). However, the number of live nematodes in these treatments was still significantly lower than in the water control group (Fig. 1A). The highest number of nematodes escaping from the roots was observed in the treatment with EAEO at 780 mg DW·L⁻¹ (36.7 ± 2.5), which was significantly lower than in the water control group (78.7 ± 4.7). EAEO at 12500 and 6250 mg DW·L⁻¹ (18.7 ± 4.5 and 22.7 ± 3.2, respectively) provided nematode control similar to fenamiphos (21.7 ± 3.5) (Fig. 1B).

Figure 1.

All developmental stages of R. similis can be found inside banana roots. The nematodes puncture the root epidermal cells with a sharp stylet, digesting and extracting cell contents as they penetrate the roots. Invasion typically occurs at the tips of soft, nonwoody, lateral roots or in the region of the feeding site. Once inside the root, the nematode feeds and reproduces. With its rudimentary stylet, the male cannot penetrate the root or cause any damage to the root at any level, while the females and second-stage juveniles (J2) feed on the root and cause significant damage. The nematodes only leave the root and move away when the root decays or their density becomes too high (Haegeman et al. 2010). However, plants possess a complex defense system to resist invading agents, including constitutive mechanisms, preformed structures, and inducible immune responses that occur upon recognition of the invading factors. The immune and constitutive defense systems against nematodes rely on the presence of plant secondary metabolites with antinematode activity (Sato et al. 2019). Particularly in roots, leaves, and stems, a variety of plant secondary metabolites, such as phenolic compounds, terpenoids, saponins, benzoxazinoids, organosulfur compounds, alkaloids, and glucosinolates are present and released, acting as attractants, repellents, stimulants, or nematode inhibitors (Sikder & Vestergård 2020). After a 48-hour incubation in EAEO, fenamiphos, and water, the number of surviving nematodes inside the roots and some nematodes emerging from the roots showed variations among the treatments. The number of nematodes remaining in and emerging from EAEO-treated roots was significantly lower compared to the negative control, and roots treated with high concentrations of the extract exhibited similar efficacy to those treated with fenamiphos. These results once again affirm the role of EAEO in controlling R. similis.

Several limitations have been identified in the experimental study that should be considered and addressed in future research endeavors. The relatively narrow range of dilutions used in the conducted experiments can be viewed as a limitation, and the short exposure time of only 48 hours following EAEO contact may have impacted our understanding of EAEO’s effects. Additionally, the exclusive focus on the R. similis, without extending the investigation to other nematode species, presents a limitation that should be overcome. Notably, the ecological implications of using EAEO for nematode control should have been addressed in depth, warranting further examination in future studies. Future research directions should encompass a more extensive series of experiments to establish a detailed dose–response relationship. Such investigations will aid in determining the optimal dosage for effective and economically viable nematode control. Concurrently, research into the persistence and potential development of drug resistance by nematodes under EAEO conditions is essential. Expanding the scope beyond R. similis, experiments on various nematode species are warranted to evaluate the broad-spectrum efficacy of EAEO. Furthermore, delving into the molecular mechanisms through which EAEO impacts nematodes represents a crucial research avenue. Gaining insights into these mechanisms can facilitate the optimization of EAEO’s application. Lastly, the environmental impact of EAEO usage, encompassing soil health, effects on nontarget organisms, and the overall dynamics of ecosystems, should be rigorously evaluated. This approach will ensure that using EAEO as a nematode control method is positive and sustainable in the long term.