Additional effects of air anions on eco-friendly pest control against Tetranychus urticae and Aphis gossypii

Abstract

Air anions are molecules that have gained one or more additional electrons. While there have been several studies on the application of air anions in various fields such as air pollution, microorganisms, and plants, there has been no reported application to insects. This study explored the effects of air anions on greenhouse pests, Tetranychus urticae and Aphis gossypii, and analyzed their potential as an eco-friendly pest control method. The results showed that air anions exhibited insecticidal and repellent effects on both T. urticae and A. gossypii. Moreover, treatment with air anions for three weeks increased the total phenolic content in kidney beans, and the consumption of these leaves resulted in mortality effects on T. urticae. Additionally, in a greenhouse assay, a three-week air anion treatment on kidney beans resulted in approximately 92.3% control efficacy against T. urticae. In conclusion, air anions negatively impacted T. urticae, suggesting their potential use as an eco-friendly pest control method.

Introduction

Controlled Environment Agriculture (CEA), such as greenhouse, provides ideal conditions for the occurrence and development of pests due to their warm and humid environments¹. Among these pests, the two-spotted spider mite, Tetranychus urticae (Acari: Tetranychidae), and cotton aphid, Aphis gossypii (Hemiptera: Aphididae), stand out as some of the most important agricultural pests^2,3. These pests not only invade a wide range of host crops but also tend to develop insecticide resistance rapidly compared to other pests^4–6. The development of insecticide resistance necessitates the increased use of insecticides, leading to environmental contamination and harm to humans and ecosystems^7,8. For these reasons, Integrated Pest Management (IPM) aims to promote sustainable agricultural practices by significantly reducing the use of synthetic insecticides⁹.

Sustainable agriculture seeks to effectively control pests and diseases while ensuring minimal harm to humans and the environment¹⁰. Within IPM various approaches-including biological, chemical and physical methods-have been explored to achieve this goal. Among these, physical control methods have attracted renewed interest as alternative pest management strategies. Although earlier physical approaches were often considered labor-intensive and inconsistent in efficacy, recent studies have shown that certain physical stimuli, as well as plant physiological responses. These findings highlight the potential of non-chemical physical methods in pest management.

Electroculture, which is the study of how electricity affects crops, involves exposing crops to various electrical stimuli such as electric fields, magnetic fields, and electrostatic fields to observe their responses and study the effects on their growth^11–13. Among them, air anion treatment technology involves the application of air anions, generated from air molecules ionized by electron radiation to crops¹¹. In electroculture, air anion treatment has the advantage of being easier to install, safer, and simpler to maintain compared to other electro-stimulation treatments. Air anion treatment has also been utilized in CEA systems and has been reported to enhance horticultural crop growth and increase yields^10,14.

Preliminary observations suggested that air anion treatment may be associated with reduced pest density. Based on this, the present study aimed to determine whether air anions exhibit insecticidal or repellent activity against greenhouse pests and to examine the relationship between phytochemical changes in treated crops and pest mortality. In addition, the effectiveness of air anions as a pest management approach was evaluated under greenhouse conditions. The findings of this study provide insight into the potential of air anions as an eco-friendly strategy for sustainable pest management.

Materials and methods

Specifications of air anion generator and measurement system

The air anions generator (XHJ-A22F, Shenzhen, China) consists of three parts: a circuit that increases the anion pressure of AC 220 V to DC -3.5∼0.5 KV through oscillation type booster, a carbon brush tip that receives high voltage DC and emits a large amount of electrons at high speed, and a cable for electricity supply (Fig. 1). Notably, the device was configured to minimize collateral gas production, ensuring that ozone emission levels remained below the detection limit (< 0.05 ppm) and within established safety standards throughout the experiment.

Fig. 1.

Air anion generator and its components. The main components include the high-voltage circuit, a power cable, and the carbon brush tip for electron emission.

The target concentrations of air anions were determined based on previous research, which indicated that the effects on plant growth vary depending on whether the anion concentration is higher or lower than 500,000 ions/cm³. Consequently, this concentration (500,000 ions/cm³) was used as the reference level for the present study. Based on this baseline, higher and lower ion concentrations were established to evaluate the effect of different ion density conditions on insect and plant responses. To adjust the air anion levels within the acrylic cages, the number of anion generators was varied, allowing for the controlled increase of ion density to reach the target treatment levels.

The air anion concentrations were monitored using air ion counter (COM-3600 F; COM System Inc., Akishima-city, Tokyo, Japan) based on the Gerdien condenser method. Following the methodology established in previous studies¹⁵, the average values recorded over a 30-minute continuous measurement period were utilized to verify the uniform and stable distribution of anions within the experimental space.

Pests and plants

Tetranychus urticae and Aphis gossypii populations were maintained for more than 3 years on kidney bean and cucumber plants without exposure to any pesticide. In an insect rearing room, plants infested with mites and aphids were kept at 25 ± 2 °C and 45∼60% relative humidity under a 16:8 h (L: D) photoperiod. Kidney bean seeds (Phaseolus vulgaris for. Isackhorangi) and cucumber seeds (Cucumis sativus for. Matgalbakdadagi) obtained from a cheongju seedling nursery were used for the experiment. Seeds were planted in the plant growth chamber, maintained conditions of 200 µmol·m-2·s-1 light intensity, a 16:8 h (L: D) photoperiod, and a temperature of 25 °C with 70% relative humidity. The plant collection and use were in accordance with all the relevant guidelines.

Insecticidal and ovicidal activities

An anion-generation cage was designed to evaluate air anions’ insecticidal and ovicidal activities against T. urticae (Fig. 2A). The anion-generating cage was made by installing an anion generator under the ceiling of an acrylic cage (30 × 30 × 30 cm), and then the concentration was measured using an air ion counter (COM-3600 F; COM System Inc., Akishima-city, Tokyo, Japan). To assess insecticidal activity, a leaf disc (35 mm in diameter) was cut from a kidney bean plant and placed on wet cotton wool in a petri dish (60 mm in diameter). Ten adult mites per three replications were introduced onto the leaf disc. Each plate was placed inside the cage. To assess ovicidal activity, the same method as above was used. Three adult mites were introduced onto the leaf disc the day before the experiment, and the next day, it was confirmed that there were more than 10 eggs, and the adult mites were removed. The anion concentration in each cage was 200,000 ion/cm³, 700,000 ion/cm³, and 1,500,000 ion/cm³, and mortality and hatching rates were examined after 7 days. Mites that did not respond when prodded with a small brush were considered dead. In the negative control group, a mortality rate of less than 10% and a hatching rate of 100% were observed. The following preparation was used to evaluate the insecticidal activity of anion against A. gossypii (Fig. 2B). A leaf disc (60 mm in diameter) was cut from a cucumber plant and placed on agar (1.5%) medium in a petri dish (90 mm in diameter). 20 adult aphids per three replications were introduced onto the leaf disc. An anion generator was installed under the cover after attaching an acrylic pipe (90 × 4 × 180 mm) to a 90 mm petri dish. The cucumber leaf was replaced on the third day due to deterioration during the experiment. Mortality was examined after 5 days. Aphids that did not respond when prodded with a small brush were considered dead. In the negative control group, a mortality rate of less than 10% and a hatching rate of 100% were observed.

Fig. 2.

Bioassay method for evaluating insecticidal activity. (A) T. urticae and (B) A. gossypii.

Repellent activity

To evaluate the repellent activity of anions against T. urticae and A. gossypii, an anion generator was installed under the left ceiling of an acrylic cage (30 × 30 × 30 cm) (Fig. 3A and B). The target air anion concentration was maintained at approximately 500,000 ions/cm³, and the generator was operated continuously throughout the experiment. A partition was constructed in the middle of the cage using a form board to divide the left (treated) and right (untreated) areas. Plants were placed in a vial (2.5 × 7 cm) filled with water, and 2-week-old beans and 3-week-old cucumbers were fixed with cotton. 30 starved adult mites and 20 starved adult aphids (for 4 h) were individually introduced into a vial (2.5 × 7 cm) positioned between the treated and untreated areas. The distance between the plant and the introduction point was 15 cm, and a bridge has been installed to allow movement between the two areas. The distribution of insects was recorded after 48 h of exposure to assess repellency. Repellent activity was calculated using the following equation:

Fig. 3.

Bioassay method for repellent activity. (A) Bioassay for T. urticae, (B) Bioassay for A. gossypii.

where N_c is the number of pests on control plant, while N_t is the number of pests on the treated plant⁸.

Effect of air anions on kidney beans

An air anions generator was installed above transplanted kidney beans to investigate the effects of air anions on kidney beans. Two beans were planted per pot, with five pots per treatment group and five pots per control group. The plants were treated in a growth chamber at 25 ± 2 °C and 45∼60% relative humidity under a 16:8 h (L: D) photoperiod for two weeks. Throughout this two-week experimental period, the air anions were applied continuously for 24 h a day. The air anions generator was mounted on a vertical support stand for installation in the pots. The support in conjunction with the air anions generator was placed in one pot per planted bean seedling and adjusted 11 to 13 cm from the seedling to set 500,000 ions/cm³ concentration. The concentration of air anions was measured using an air ion counter (COM-3600 F; Com System Inc., Akishima-city, Tokyo, Japan). For nutrient management, a full-strength Hoagland solution (EC 1.0 dS/m, pH 5.8) was provided to all plants via sub-irrigation throughout the entire cultivation period.

Growth and photosynthetic rate

After 2 weeks of treatment, the fresh weight of the shoot and root, the number of leaves, and the area of the leaf were measured using an electric scale (LS-3200, ACOM, Pocheon, Korea) and the leaf area meter (LI-3100, LI-COR, Lincoln, NE, USA), respectively. Plant height, stem width and node number were measured using a vernier caliper (NA530-300 S, BLUEBIRD, China). Chlorophyll content was expressed as the average SPAD value of three measurements on a fully expanded leaf per plant using a SPAD meter (SPAD-502; Minolta Corporation, Ltd., Osaka, Japan).

Total phenolic content and antioxidant capacity

To determine changes in the phytochemical content of kidney beans due to treatment with air anions, the total phenolic content and antioxidant capacity were evaluated. Leaf samples were collected to assess the total phenolic content and antioxidant capacity. To evaluate total phenolic content of kidney bean, the collected leaf samples from each treatment were preserved in a -70℃ deep freezer (NF-300SF; NIHON FREEZER CO., Ltd., Tokyo, Japan) to make fresh sample. The total phenolic content of kidney beans was analyzed using the Folin-Ciocalteu method (Anisworth and Gillespie, 2007), with slight modifications. Place the frozen sample in a mortar, add liquid nitrogen and homogenization for one minute. Then add 3 ml of 80% acetone to the mortar and extract after homogenization for one minute. Store extracted acetone in a 4℃ cold chamber for at least 12 hours. For analysis, centrifuge the extracted sample at rcf 3000, 4℃. In a microtube, add 135 µl of distilled water, 750 µl of 10% F.C reagent, 50 µl of sample, and 600 µl of 7.5% Na₂CO₃. For the blank, add 50 µl of acetone. After vortexing, place in a 45℃ water bath for 15 minutes and cool after the reaction. Measure the absorbance of the prepared sample using a 765 nm spectrophotometer. The unit of measurement was expressed as gallic acid (Acros Organics, Geel, Belgium) equivalent (mg) per fresh weight of leaves (GAE mg/shoot FW). To evaluate antioxidant capacity of kidney bean, the collected leaf samples from each treatment were dried for 72 h in a freeze dryer (Alpha 1–4 LSC plus, Martin Christ Co., Osterode, Germany) to make dried sample. The total phenolic content of kidney bean was analyzed using the 2,2’-azino-bis-ethylbenzthoazoline- 6-sulphic acid (ABTS) (Miller and Rice-Evans, 1996) methodologies with slight modifications. Approximately 40 mg of powdered leaf sample for each treatment was mixed with 4 ml of 80% acetone and extracted for 15 min in a sonicator (SK5210HP, KUDOS, Shanghai, China). The extract obtained was stored for 12 h at -20℃. For analysis, the extracted sample was centrifuged at rcf 3000, -20 ° C. Add 900 µl of acetone and 100 µl of sample to a microtube and vortex. Mix 2.5 mM of ABTS stock with 5 mM PBS to which 0.4 g of MnO₂ was added and stirred for 30 min to adjust the absorbance to 0.695–0.705 nm. Then add 100 µl of the diluted sample to a cuvette and measure absorbance after 1 min, vortexing with 1 ml of ABTS + PBS solution. The unit of measurement was expressed as 6-hydroxy-2,5,7,8-tetramethylchromane-2-carboxyl acid (trolox) (Sigma-Aldrich Co., LLC, St. Louis, MO, USA) equivalent antioxidant capacity per unit dry weight (TEAC mM g/DW).

Greenhouse assay

The study was conducted in a greenhouse from 23rd March to 13th April 2024. The experimental site was located at Chungbuk National University, Cheongju, Korea. Two-week-old kidney beans, with two seeds each per pot, were used in this study. For nutritional management, all plants were irrigated with tap water only. We conducted experiments with an anion concentration of 500,000 ions/cm3, comparing treatments with anions to non-treatment conditions. To evaluate the insecticidal effect, ten adult mites were introduced to each pot after the bean seedlings had established. Immediately following the mite introduction, the anion treatment was initiated and applied continuously 24 h a day throughout the experimental period. On the 21st day of anion treatment, the population of mites per pot was surveyed.

Statistical analysis

The bioassay data were collected for corrected mortality using Abbott’s formula. Differences in insecticidal and ovicidal effectiveness of anion concentrations were compared by one-way analysis of variance (ANOVA), followed by Turkey’s test. To evaluate differences between the treated and untreated groups, a t-test was conducted. A pooled t-test was utilized to evaluate repellent activity, while a Satterthwaite t-test was utilized to compare density variations in the greenhouse. All statistical analyses were conducted using SAS software¹⁶.

Results

Toxicity and repellent activities of air anions

The toxicity of air anions was evaluated against adults and eggs T. urticae, and adult of A. gossypii (Table 1). In general, air anions exhibited insecticidal activity on both T. urticae (eggs and adults) and A. gossypii adults. The mortality rates of mite adults increased with the concentration of air anions, with ovicidal activity being highly effective at a concentration of 700,000 ions/cm³. Additionally, a significant difference in hatch rates was observed between the group treated with 700,000 ions/cm³ and the control group (F_2,6 = 5.185, p = 0.028,), analyzed using one-way ANOVA. Furthermore, air anions also demonstrated repellent activity against both T. urticae and A. gossypii (Table 2). The repellency for A. gossypii was calculated at 49.4 ± 13.3%, which was higher than the 44.0 ± 18.1% observed for T. urticae.

Table 1.

Toxicity activity of air anion against T. urticae (7 day) and A. gossypii (5 day).

Species	Concentration	Mortality (%, mean ± SE)	Hatch rate (%, mean ± SE)
T. urticae	0 ion/cm3	0.0 ± 0.0 aa	100.0 ± 0.0 a
	200,000 ion/cm3	23.3 ± 6.7 b	86.7 ± 6.7 ab
	700,000 ion/cm3	33.5 ± 3.7 b	73.3 ± 6.7 b
	1,500,000 ion/cm3	40.0 ± 5.8 b	93.3 ± 3.3 ab
A. gossypii	1,000,000 ion/cm3	44.6 ± 1.7	-

^aMeans followed by the same letters are not significantly different (p < 0.05; Tukey’s studentized range test) (SAS Institute 2019).

Table 2.

Repellent activity (48 h) of air anion against T. urticae and A. gossypii.

Species	Group	No. (Mean ± SE)	Repellency (%, mean ± SE)	t
T. urticae	Untreated	20.8 ± 1.5	44.0 ± 18.1	7.00***
	Treated	8.0 ± 1.0
A. gossypii	Untreated	14.0 ± 0.4	49.4 ± 13.3	11.50***
	Treated	4.8 ± 0.7

Means with ‘***’ are significantly different between treated and untreated by pooled t-test (p = 0.001).

Effect of air anions on crops

Growth parameters

Air anions treatment tended to decrease the fresh weights of the shoot and roots in kidney beans. Specifically, the fresh weight of the shoot under air anions treatment was decreased by approximately 11.5% compared to the control (Fig. 4A). In contrast, the root fresh weight was significantly reduced by 35.9% compared to the control (Fig. 4B). The number of leaves remained unchanged between the control and air anions treatments, with both having an average of 5 leaves (Fig. 4C). However, the leaf area decreased by approximately 9.1% in the air anions treatment compared to the control (Fig. 4D). Regarding plant height and stem width, both parameters decreased with the treatment with air anions. Plant height was approximately 11.5% lower (Fig. 4E) and stem width was significantly reduced by approximately 10.7% compared to the control (Fig. 4F). However, the number of nodes and the SPAD value (a measure of chlorophyll content) increased significantly under the treatment with air anions. The node number increased by about 17.0% (Fig. 4G) and the SPAD value also increased by approximately 21.0% compared to the control (Fig. 4H).

Fig. 4.

Shoot (A) and root (B) fresh weights, number of leaves (C), leaf area (D), plant height (E), stem width (F), node number (G) and SPAD (H) of kidney bean under air anions treatment and control. The error bars indicate standard errors (n = 10). Significant differences at *p < 0.05 and ***p < 0.001 by t-test.

Total phenolic content and antioxidant capacity

We examined total phenolic content and antioxidant capacity to investigate phytochemical changes in plants induced by the treatment of air anions. Air anions treatment significantly increased the total phenolic content and antioxidant capacity per gram of fresh and dry leaf weight. Specifically, the total phenolic content per gram of fresh leaf increased by approximately 23% compared to the control (Fig. 5A). Furthermore, the antioxidant capacity per gram of dry leaf weight increased significantly by approximately 11. 24% in the treatment with air anions compared to the control (Fig. 5B).

Fig. 5.

Total phenolic content and antioxidant capacity per gram of fresh weight (A) and dry weight (B) of kidney bean under air anions treatment for 14 days. Significant differences at *p < 0.05 and **p < 0.01 by t-test.

Effect of air anion treatment on mortality of T. urticae, and phenol content and antioxidant capacity in kidney beans

To investigate the insecticidal effect of air anion treatment on T. urticae in kidney beans, young kidney bean plants were exposed to air anions in a plant chamber for 14 days. Following this treatment, T. urticae were introduced on cutting leaves, and the number of surviving mites was evaluated on the third day. Compared to the untreated control, the air anion-treated group exhibited a mortality rate ranging from 39.3% to 70.2%, with an average mortality rate of approximately 51.4% (Fig. 6A). A regression analysis examined the relationships between mortality rate, total phenol content, and antioxidant capacity in each treatment group. The results showed a linear relationship between mortality rate and total phenol content with an R² value of more than 0.45 (Fig. 6B). In contrast, there was no linear relationship between mortality rate and antioxidant capacity, indicated by an R² value of 0.1993 (Fig. 6C).

Fig. 6.

Regression analysis of T. urticae mortality with total phenol content and antioxidants capacity in kidney bean under air anions treatment for 14 days. (A) Box plot of T. urticae mortality on kidney beans for 5 days. (B) Regression analysis of total phenol content and mortality rate of T. urticae on kidney beans. (C) Regression analysis of antioxidant capacity and mortality rate of T. urticae on kidney beans.

Greenhouse assay

We examined the variation in T. urticae density in response to air anion treatment (500,000 ions/cm³) in a greenhouse. On day 21 of air anion treatment, a statistically significant difference (p = 0.012) in mite density was observed between the control and air anion groups compared to before treatment (Fig. 7). In the control group, the density of mites increased 29.1-fold to an average of 291.0 mites per plant, while in the air anion group, the density of mites increased only 2.2-fold to an average of 22.3 mites per plant. Thus, air anion treatment showed a control efficacy of 92.3%.

Fig. 7.

Comparison of T. urticae densities in greenhouses by anion treatment. ‘*’ indicates the statistical difference between treated and untreated by Satterthwaite t-test at p ≤ 0.05 (t-value = 4.34).

Discussion

This study aimed to evaluate the potential of air anions as a new eco-friendly pest control method. Air anions, which are air molecules ionized by gaining electrons, have been reported to offer various health benefits to humans and animals^6,7, promote plant development⁵, and inhibit the growth of certain microorganisms^2–4. However, while preliminary experiments investigating the development of greenhouse plants treated with air anions observed an inhibitory effect on pest development, research on the impact of air anions on insects is limited. Therefore, this study sought to investigate the effects of air anions on greenhouse pests and assess their potential applicability as an eco-friendly pest control agent.

In the controlled environment agriculture (CEA) setting of a greenhouse, bioassay experiments were designed considering the location of the air anion generator and greenhouse pests (Figs. 2 and 3). Moderate repellent effects were observed in Tetranychus urticae (two-spotted spider mite) and Aphis gossypii (cotton aphid). Notably, in the air anion-treated group, adult A. gossypii exhibited abnormal antennae movements, and more alate adults, which typically emerge under stressful conditions, appeared compared to the untreated group. These findings suggest that air anions may negatively affect T. urticae and A. gossypii, common greenhouse pests.

Air anions generated by the anion generator form an electric field in the air^17,18. Many insects possess sensory receptors capable of detecting electrical signals, triggering avoidance behaviors when encountering such fields^19–21. For example, cockroaches exhibit avoidance behavior upon detecting an electric field, and similar responses have been observed in whiteflies and fruit flies. This may explain the repellent effect of air anions on T. urticae and A. gossypii observed in this study. Moreover, exposure to electric fields has been reported to adversely affect insects, leading to reduced reproductive capacity and energy supply²⁰. Thus, the creation of an electric field by air anions maybe linked to the insecticidal and repellent effects observed.

Electric stimuli, such as air anion treatment, are known to significantly impact plant physiology, although the exact mechanisms are not yet fully understood. Some studies have found that electrical stimuli affect the accumulation of minerals in plants²², and air anion treatment has been reported to increase stomatal conductivity¹⁰. These electrical stimuli-induced changes are hypothesized to enhance plant photosynthesis²³ or alter the plant’s circadian rhythms, thereby improving growth²⁴. However, the effects of air anion treatment vary with concentration and application method, as well as plant species^10,14,15. In our study, air anion treatment significantly reduced the biomass and stem diameter of Phaseolus vulgaris (common bean) while significantly increasing the number of nodes and SPAD values, indicating that air anion treatment had notable effects. Kaimoyo (2008)²⁵ reported that microcurrents stimulate the biosynthesis pathways of secondary metabolites in plants, leading to the synthesis of secondary metabolites. In this study, the microcurrents induced by air anions in P. vulgaris may have stimulated the biosynthetic pathways for phenolic compounds and antioxidants, thereby increasing the total phenolic content and antioxidant capacity of the beans.

Various secondary metabolites synthesized in plants are known to play defensive roles against insects through feeding deterrence and toxic effects^26–28. This study found that air anion treatment increased the total phenolic content and antioxidant capacity of beans, with a moderate positive observed between total phenolic content and the mortality rate of T. urticae. Dáder et al. (2017)²⁹ reported that three weeks of UV-B treatment increased the total phenolic content in Capsicum annuum (pepper), and while it did not affect the life cycle of Myzus persicae (green peach aphid), the establishment of alate aphids, which emerge under stressful conditions, was slower in the treated group compared to the control group.

Consistent with previous observations, our greenhouse bioassay results also showed a lower density of T. urticae in the air anion-treated group. These findings suggest that air anions may contribute to insecticidal and repellent effects on T. urticae, a common greenhouse pest. This study supports the potential of air anions as an eco-friendly pest control approach. Additionally, by investigating the impact of plant physiological changes induced by air anion treatment on pest feeding deterrence and reproductive decline, we provided foundational data for developing more effective pest management strategies. Future research should focus on evaluating the effects of air anions on various greenhouse pests and integrating this method with other eco-friendly pest control methods to establish a comprehensive IPM strategy. These environmentally friendly and sustainable pest control methods are expected to enhance agricultural productivity and contribute to crop protection.

Author contributions

H.-N.K., G.-H.K., K.A.Y. and M.-M.O. conceived and designed the study. D.-H. K and K.S. S performed the experiments and analyzed the data. All authors have read and approved the final manuscript.

Funding

This research was supported by the Regional Innovation System & Education (RISE) program through the (Chungbuk Regional Innovation System & Education Center), funded by the Ministry of Education (MOE) and the Chungcheongbuk-do, Republic of Korea (2026-RISE-11-014-03).

Data availability

The datasets generated during and/or analysed during the current study are available from the corresponding author on reasonable request.

Declarations

Competing interests

The authors declare no competing interests.