The pine nematode is a quarantine migratory endoparasite known to cause severe economic losses in pine forest ecosystems. The present study reviews the nematicidal activity of halogenated indoles against pine nematodes and their mechanism of action. The nematicidal activities of 5-iodoindole and avermectin (positive control) against pine nematodes were similar and high at low concentrations (10 μg/mL). 5-iodoindole reduced fecundity, reproductive activity, embryonic and larval mortality, and locomotor behavior. Molecular interactions of ligands with invertebrate-specific glutamate-gated chloride channel receptors support the notion that 5-iodoindole, like avermectin, binds tightly to the receptor active site. 5-Iodoindole also induced various phenotypic deformations in nematodes, including abnormal organ collapse/shrinkage and increased vacuolization. These results suggest that vacuoles may play a role in nematode methylation-mediated death. Importantly, 5-iodoindole was non-toxic to both plant species (cabbage and radish). Thus, this study demonstrates that iodoindole application under environmental conditions can control pine wilt injury.
Pine wood nematode (Bursaphelenchus xylophilus) belongs to the pine wood nematodes (PWN), migratory endoparasitic nematodes known to cause severe ecological damage to pine forest ecosystems1. Pine wilt disease (PWD) caused by the pine wood nematode is becoming a serious problem on several continents, including Asia and Europe, and in North America, the nematode destroys introduced pine species1,2. Pine tree decline is a major economic problem, and the prospect of its global spread is worrying3. The following pine species are most commonly attacked by the nematode: Pinus densiflora, Pinus sylvestris, Pinus thunbergii, Pinus koraiensis, Pinus thunbergii, Pinus thunbergii, and Pinus radiata4. Pine nematode is a serious disease that can kill pine trees within weeks or months of infection. In addition, pine nematode outbreaks are common in a variety of ecosystems, so persistent infection chains have been established1.
Bursaphelenchus xylophilus is a quarantine plant-parasitic nematode belonging to the superfamily Aphelenchoidea and clade 102.5. The nematode feeds on fungi and reproduces in the wood tissues of pine trees, developing into four different larval stages: L1, L2, L3, L4 and an adult individual1,6. Under conditions of food shortage, the pine nematode passes into a specialized larval stage – dauer, which parasitizes its vector – the pine bark beetle (Monochamus alternatus) and is transferred to healthy pine trees. In healthy hosts, nematodes quickly migrate through plant tissues and feed on parenchymatous cells, which leads to a number of hypersensitivity reactions, pine wilting and death within a year after infection1,7,8.
Biological control of pine nematodes has long been a challenge, with quarantine measures dating back to the 20th century. Current strategies for controlling pine nematodes primarily involve chemical treatments, including wood fumigation and implantation of nematicides into tree trunks. The most commonly used nematicides are avermectin and avermectin benzoate, which belong to the avermectin family. These expensive chemicals are highly effective against many nematode species and are considered environmentally safe9. However, repeated use of these nematicides is expected to create selection pressure that will almost certainly lead to the emergence of resistant pine nematodes, as has been demonstrated for several insect pests, such as Leptinotarsa decemlineata, Plutella xylostella and the nematodes Trichostrongylus colubriformis and Ostertagia circumcincta, which have gradually developed resistance to avermectins10,11,12. Therefore, resistance patterns need to be regularly studied and nematicides screened continuously to find alternative, cost-effective and environmentally friendly measures to control PVD. In recent decades, a number of authors have proposed the use of plant extracts, essential oils and volatiles as nematode control agents13,14,15,16.
We recently demonstrated the nematicidal activity of indole, an intercellular and interkingdom signaling molecule, in Caenorhabditis elegans 17 . Indole is a widespread intracellular signal in microbial ecology, controlling numerous functions that affect microbial physiology, spore formation, plasmid stability, drug resistance, biofilm formation, and virulence 18, 19 . The activity of indole and its derivatives against other pathogenic nematodes has not been studied. In this study, we investigated the nematicidal activity of 34 indoles against pine nematodes and elucidated the mechanism of action of the most potent 5-iodoindole using microscopy, time-lapse photography, and molecular docking experiments, and assessed its toxic effects on plants using a seed germination assay.
High concentrations (>1.0 mM) of indole have been previously reported to have a nematicidal effect on nematodes17. Following treatment of B. xylophilus (mixed life stages) with indole or 33 different indole derivatives at 1 mM, mortality of B. xylophilus was measured by counting live and dead nematodes in the control and treated groups. Five indoles exhibited significant nematicidal activity; the survival of the untreated control group was 95 ± 7% after 24 h. Of the 34 indoles tested, 5-iodoindole and 4-fluoroindole at 1 mM caused 100% mortality, whereas 5,6-difluoroindigo, methylindole-7-carboxylate, and 7-iodoindole caused approximately 50% mortality (Table 1).
Effect of 5-iodoindole on vacuole formation and metabolism of pine wood nematode. (A) Effect of avermectin and 5-iodoindole on adult male nematodes, (B) L1 stage nematode eggs and (C) metabolism of B. xylophilus, (i) vacuoles were not observed at 0 h, treatment resulted in (ii) vacuoles, (iii) accumulation of multiple vacuoles, (iv) swelling of vacuoles, (v) fusion of vacuoles and (vi) formation of giant vacuoles. Red arrows indicate swelling of vacuoles, blue arrows indicate fusion of vacuoles and black arrows indicate giant vacuoles. Scale bar = 50 μm.
In addition, this study also described the sequential process of methane-induced death in pine nematodes (Figure 4C). Methanogenic death is a non-apoptotic type of cell death associated with the accumulation of prominent cytoplasmic vacuoles27. The morphological defects observed in pine nematodes appear to be closely related to the mechanism of methane-induced death. Microscopic examination at different times showed that giant vacuoles were formed after 20 h of exposure to 5-iodoindole (0.1 mM). Microscopic vacuoles were observed after 8 h of treatment, and their number increased after 12 h. Several large vacuoles were observed after 14 h. Several fused vacuoles were clearly visible after 12–16 h of treatment, indicating that vacuole fusion is the basis of the methanogenic death mechanism. After 20 hours, several giant vacuoles were found throughout the worm. These observations represent the first report of metuosis in C. elegans .
In 5-iodoindole-treated worms, vacuole aggregation and rupture were also observed (Fig. 5), as evidenced by worm bending and vacuole release into the environment. Vacuole disruption was also observed in the eggshell membrane, which is normally preserved intact by L2 during hatching (Supplementary Fig. S2). These observations support the involvement of fluid accumulation and osmoregulatory failure, as well as reversible cell injury (RCI), in the process of vacuole formation and suppuration (Fig. 5).
Hypothesizing the role of iodine in the observed vacuole formation, we investigated the nematicidal activity of sodium iodide (NaI) and potassium iodide (KI). However, at concentrations (0.1, 0.5 or 1 mM), they did not affect either nematode survival or vacuole formation (Supplementary Fig. S5), although 1 mM KI had a slight nematicidal effect. On the other hand, 7-iodoindole (1 or 2 mM), like 5-iodoindole, induced multiple vacuoles and structural deformations (Supplementary Fig. S6). The two iodoindoles showed similar phenotypic characteristics in pine nematodes, whereas NaI and KI did not. Interestingly, indole did not induce vacuole formation in B. xylophilus at the concentrations tested (data not shown). Thus, the results confirmed that the indole-iodine complex is responsible for the vacuolization and metabolism of B. xylophilus.
Among the indoles tested for nematicidal activity, 5-iodoindole had the highest slip index of -5.89 kcal/mol, followed by 7-iodoindole (-4.48 kcal/mol), 4-fluoroindole (-4.33), and indole (-4.03) ( Figure 6 ). The strong backbone hydrogen bonding of 5-iodoindole to leucine 218 stabilizes its binding, whereas all other indole derivatives bind to serine 260 via side chain hydrogen bonds. Among other modeled iodoindoles, 2-iodoindole has a binding value of -5.248 kcal/mol, which is due to its main hydrogen bond with leucine 218. Other known bindings include 3-iodoindole (-4.3 kcal/mol), 4-iodoindole (-4.0 kcal/mol), and 6-fluoroindole (-2.6 kcal/mol) (Supplementary Figure S8). Most halogenated indoles and indole itself, with the exception of 5-iodoindole and 2-iodoindole, form a bond with serine 260. The fact that hydrogen bonding with leucine 218 is indicative of efficient receptor-ligand binding, as observed for ivermectin (Supplementary Fig. S7), confirms that 5-iodoindole and 2-iodoindole, like ivermectin, bind tightly to the active site of the GluCL receptor via leucine 218 (Fig. 6 and Supplementary Fig. S8). We propose that this binding is required to maintain the open pore structure of the GluCL complex and that by tightly binding to the active site of the GluCL receptor, 5-iodoindole, 2-iodoindole, avermectin and ivermectin thus maintain the ion channel open and allow fluid uptake.
Molecular docking of indole and halogenated indole to GluCL. Binding orientations of (A) indole, (B) 4-fluoroindole, (C) 7-iodoindole, and (D) 5-iodoindole ligands to the active site of GluCL. The protein is represented by a ribbon, and the backbone hydrogen bonds are shown as yellow dotted lines. (A′), (B′), (C′), and (D′) show the interactions of the corresponding ligands with the surrounding amino acid residues, and the side-chain hydrogen bonds are indicated by pink dotted arrows.
Experiments were conducted to evaluate the toxic effect of 5-iodoindole on the germination of cabbage and radish seeds. 5-iodoindole (0.05 or 0.1 mM) or avermectin (10 μg/mL) had little or no effect on initial germination and plantlet emergence (Figure 7). In addition, no significant difference was found between the germination rate of untreated controls and seeds treated with 5-iodoindole or avermectin. The effect on taproot elongation and the number of lateral roots formed was insignificant, although 1 mM (10 times its active concentration) of 5-iodoindole slightly delayed the development of lateral roots. These results indicate that 5-iodoindole is nontoxic to plant cells and does not interfere with plant development processes at the concentrations studied.
Effect of 5-iodoindole on seed germination. Germination, sprouting and lateral rooting of B. oleracea and R. raphanistrum seeds on Murashige and Skoog agar medium with or without avermectin or 5-iodoindole. Germination was recorded after 3 days of incubation at 22°C.
This study reports several cases of nematode killing by indoles. Importantly, this is the first report of iodoindole inducing methylation (a process caused by the accumulation of small vacuoles that gradually merge into giant vacuoles, eventually leading to membrane rupture and death) in pine needles, with iodoindole exhibiting significant nematicidal properties similar to those of the commercial nematicide avermectin.
Indoles have been previously reported to exert multiple signaling functions in prokaryotes and eukaryotes, including biofilm inhibition/formation, bacterial survival, and pathogenicity19,32,33,34. Recently, the potential therapeutic effects of halogenated indoles, indole alkaloids, and semisynthetic indole derivatives have attracted extensive research interest35,36,37. For example, halogenated indoles have been shown to kill persistent Escherichia coli and Staphylococcus aureus cells37. In addition, it is of scientific interest to study the efficacy of halogenated indoles against other species, genera, and kingdoms, and this study is a step toward achieving this goal.
Here, we propose a mechanism for 5-iodoindole-induced lethality in C. elegans based on reversible cell injury (RCI) and methylation (Figures 4C and 5). Edematous changes such as puffiness and vacuolar degeneration are indicators of RCI and methylation, manifested as giant vacuoles in the cytoplasm48,49. RCI interferes with energy production by reducing ATP production, causing failure of the ATPase pump, or disrupting cell membranes and causing a rapid influx of Na+, Ca2+, and water50,51,52. Intracytoplasmic vacuoles arise in animal cells as a result of fluid accumulation in the cytoplasm due to the influx of Ca2+ and water53. Interestingly, this mechanism of cell damage is reversible if the damage is temporary and the cells begin to produce ATP for a certain period of time, but if the damage persists or worsens, the cells die.54 Our observations show that nematodes treated with 5-iodoindole are unable to restore normal biosynthesis after exposure to stress conditions.
The methylation phenotype induced by 5-iodoindole in B. xylophilus may be due to the presence of iodine and its molecular distribution, since 7-iodoindole had less inhibitory effect on B. xylophilus than 5-iodoindole (Table 1 and Supplementary Figure S6). These results are partially consistent with the studies of Maltese et al. (2014), who reported that translocation of the pyridyl nitrogen moiety in indole from the para- to the meta-position abolished vacuolization, growth inhibition, and cytotoxicity in U251 cells, suggesting that the interaction of the molecule with a specific active site in the protein is critical27,44,45. The interactions between indole or halogenated indoles and GluCL receptors observed in this study also support this notion, as 5- and 2-iodoindole were found to bind to GluCL receptors more strongly than the other indoles examined (Figure 6 and Supplementary Figure S8). The iodine at the second or fifth position of the indole was found to bind to leucine 218 of the GluCL receptor via backbone hydrogen bonds, whereas other halogenated indoles and indole itself form weak side-chain hydrogen bonds with serine 260 (Figure 6). We therefore speculate that the localization of the halogen plays an important role in the induction of vacuolar degeneration, whereas the tight binding of 5-iodoindole keeps the ion channel open, thereby allowing rapid fluid influx and vacuole rupture. However, the detailed mechanism of action of 5-iodoindole remains to be determined.
Before practical application of 5-iodoindole, its toxic effect on plants should be analyzed. Our seed germination experiments showed that 5-iodoindole had no negative effect on seed germination or subsequent development processes at the concentrations studied (Figure 7). Thus, this study provides a basis for the use of 5-iodoindole in the ecological environment to control the harmfulness of pine nematodes to pine trees.
Previous reports have demonstrated that indole-based therapy represents a potential approach to address the problem of antibiotic resistance and cancer progression55. In addition, indoles possess antibacterial, anticancer, antioxidant, anti-inflammatory, antidiabetic, antiviral, antiproliferative and antituberculosis activities and may serve as a promising basis for drug development56,57. This study suggests for the first time the potential use of iodine as an antiparasitic and anthelmintic agent.
Avermectin was discovered three decades ago and won the Nobel Prize in 2015, and its use as an anthelmintic is still actively ongoing. However, due to the rapid development of resistance to avermectins in nematodes and insect pests, an alternative, low-cost, and environmentally friendly strategy is needed to control PWN infection in pine trees. This study also reports the mechanism by which 5-iodoindole kills pine nematodes and that 5-iodoindole has low toxicity to plant cells, which opens up good prospects for its future commercial application.
All experiments were approved by the Ethics Committee of Yeungnam University, Gyeongsan, Korea, and the methods were performed in accordance with the guidelines of the Ethics Committee of Yeungnam University.
Egg incubation experiments were performed using established procedures43. To assess hatching rates (HR), 1-day-old adult nematodes (approximately 100 females and 100 males) were transferred to Petri dishes containing the fungus and allowed to grow for 24 h. Eggs were then isolated and treated with 5-iodoindole (0.05 mM and 0.1 mM) or avermectin (10 μg/ml) as a suspension in sterile distilled water. These suspensions (500 μl; approximately 100 eggs) were transferred to the wells of a 24-well tissue culture plate and incubated at 22 °C. L2 counts were made after 24 h of incubation but were considered dead if the cells did not move when stimulated with a fine platinum wire. This experiment was conducted in two stages, each with six repetitions. The data from both experiments were combined and presented. The percentage of HR is calculated as follows:
Larval mortality was assessed using previously developed procedures. Nematode eggs were collected and embryos were synchronized by hatching in sterile distilled water to generate L2 stage larvae. Synchronized larvae (approximately 500 nematodes) were treated with 5-iodoindole (0.05 mM and 0.1 mM) or avermectin (10 μg/ml) and reared on B. cinerea Petri plates. After 48 h of incubation at 22 °C, nematodes were collected in sterile distilled water and examined for the presence of L2, L3, and L4 stages. The presence of L3 and L4 stages indicated larval transformation, whereas the presence of the L2 stage indicated no transformation. Images were acquired using the iRiS™ Digital Cell Imaging System. This experiment was conducted in two stages, each with six repetitions. The data from both experiments were combined and presented.
The toxicity of 5-iodoindole and avermectin to seeds was assessed using germination tests on Murashige and Skoog agar plates.62 B. oleracea and R. raphanistrum seeds were first soaked in sterile distilled water for one day, washed with 1 ml 100% ethanol, sterilized with 1 ml 50% commercial bleach (3% sodium hypochlorite) for 15 min, and washed five times with 1 ml sterile water. The sterilized seeds were then pressed onto germination agar plates containing 0.86 g/l (0.2X) Murashige and Skoog medium and 0.7% bacteriological agar with or without 5-iodoindole or avermectin. The plates were then incubated at 22 °C, and images were taken after 3 days of incubation. This experiment was conducted in two stages, each of which had six repetitions.
Post time: Feb-26-2025