Potential of Native Entomopathogenic Nematodes (Steinernematidae) as Biological Control Agents of Tetranychus urticae Koch

Abstract

The two-spotted spider mite (Tetranychus urticae Koch) (Acari: Tetranychidae) is one of the most widespread and destructive phytophagous mite species, occurring across all climatic zones worldwide. Currently, the control of spider mites in crop protection relies primarily on chemical acaricides. However, the selection of resistant populations to their active ingredients is reducing their efficacy. The aim of the present study was to assess the susceptibility of T. urticae to a native isolate of entomopathogenic nematodes, Steinernema feltiae Filipjev ZWO21, under laboratory conditions. The experiment was conducted using Petri dishes, each containing 22–28 adult T. urticae. Infective juveniles (IJs) of the nematodes were then applied at a dose of 8000 IJs per dish (±300 IJs per mite). Petri dishes with mites treated with nematodes were placed in a Sanyo incubation chamber at 25 °C and 60% relative humidity. After three days, dead mites were collected from the Petri dishes and dissected, and mortality was subsequently determined. The present study confirmed that the S. feltiae ZWO21 isolate exhibited considerable potential for the biological control of T. urticae, causing 37.5–83.3% (mean 57.0%) mortality in this pest species. Although this result indicates a moderate efficacy when nematodes are applied alone, it also underscores the relevance of further research into their integration with other control strategies, including acaricides, within integrated pest management (IPM) programmes.

1. Introduction

The two-spotted spider mite (Tetranychus urticae Koch) (Acari: Tetranychidae) is one of the most widespread and destructive phytophagous mite species, occurring across all climatic zones worldwide. Due to its exceptionally broad host range, comprising over 1200 plant species across numerous botanical families, it poses a substantial threat to both cultivated and ornamental plants [1]. Particularly susceptible hosts include vegetables, fruit trees and shrubs [2], a variety of arable crops [3,4], and numerous wild plant species [3,5]. Muluken et al. [6] reported a complete destruction of potato crops under field conditions in Ethiopia during 2014/2015. Nyoike and Liburd [7] documented strawberry yield losses ranging from 50% to 80% in north-central Florida, while Jayasinghe and Mallik [8] reported tomato yield reductions of up to 50% in India. In sugar beet, intensive feeding by T. urticae can result in root yield losses of 20–50%, accompanied by a reduction in sugar content of up to 2% [9,10,11].

The biology of T. urticae is characterised by a high reproductive potential and considerable developmental plasticity. This species reproduces via haplodiploidy (arrhenotoky), whereby males arise from unfertilised eggs and females from fertilised ones [12]. The mites’ life cycle comprises several stages, namely, egg, larva, protonymph, deutonymph, and adult form, interspersed with quiescent phases. The development and population dynamics of T. urticae are strongly influenced by temperature, host plant type, and plant health. Under favourable conditions, particularly at temperatures between 25 °C and 30 °C, the entire life cycle may be completed in less than 10 days, and a single female can lay over 100 eggs, leading to rapid population growth during the growing season [13,14]. In Poland, four to six generations may occur on sugar beet between May and October, provided that weather conditions are suitable (temperatures 25–30 °C and low precipitation 0–200 mm) [4]. Peak reproductive activity, and consequently the highest pest pressure, typically occurs in August and September. The mites move short distances actively, but they primarily disperse passively through wind. In cases of food scarcity, immature females form silk ball-dense groups that are carried by the wind to new locations. Females can also enter diapause, a dormant state that enhances their survival under harsh conditions, particularly in colder climates [15].

The harmful effects of the two-spotted spider mite result from its feeding injury, which involves piercing and sucking the contents of leaf cells. This disrupts photosynthesis and impairs the plant’s water balance. Early symptoms of infestation include mosaic-like chlorotic spots and the appearance of characteristic webbing on the underside of the leaves. As the infestation progresses, leaves turn yellow, desiccate, and abscise, while the aerial parts of the plant may become deformed. In severe cases, complete plant dieback can occur. Depending on the crop species, yield losses may reach 50–80% [7], and the quality of plant material can be substantially reduced.

The current strategy for controlling T. urticae relies primarily on chemical acaricides, particularly synthetic ones from various chemical classes. However, the efficacy of these compounds is increasingly compromised by the rapid selection of resistant individuals T. urticae populations [16,17,18]. Moreover, the overuse of chemical acaricides poses serious risks, including environmental contamination, food residue concerns, and negative impacts on non-target organisms, notably beneficial predatory mites [19]. Consequently, there is growing interest in alternative pest management approaches, including the use of biopesticides derived from essential oils, plant secondary metabolites, microorganisms and their bioactive compounds (e.g., insecticidal, repellent, or oviposition-deterrent compounds) [20]. Biological control strategies involving natural enemies of spider mites are also being investigated as key components of integrated pest management (IPM) programmes [4,21]. The trend towards reducing the use of synthetic pesticides reflects increasing environmental concerns and the growing commitment to sustainable agriculture. Ongoing research into natural, selective, and effective strategies for controlling T. urticae is crucial for ensuring crop health and safeguarding global food security.

Among biological control agents, entomopathogenic nematodes (EPNs) of the genera Steinernema constitute a particularly important group. These nematodes have long been successfully employed in the biological control of a wide spectrum of insect pests [22,23]. Their high efficacy, environmental safety, and compatibility with integrated pest management programmes have established them as some of the most extensively studied and widely applied biological agents in pest control [24,25]. Entomopathogenic nematodes of the genera Heterorhabditis and Steinernema are insect obligate parasites. These nematodes have a symbiotic relationship with bacteria of the genera Photorhabdus and Xenorhabdus, respectively. Infective juveniles (IJs) enter the host through natural openings such as the mouth, anus, or spiracles, but the IJs of some species can also enter through the cuticle. After penetrating the host’s hemocoel, nematodes release their symbiotic bacteria, which usually kill the host [22,26,27].

Despite the well-documented efficacy of EPNs against insect pests, their potential for controlling other pest groups, including mites, remains poorly understood. Research into the effectiveness of EPNs against organisms from different taxonomic groups, such as phytophagous mites, may offer new opportunities in the field of biological plant protection. Such an approach aligns with the principles of sustainable agriculture, promoting innovative and environmentally safe crop protection strategies. The aim of the present study was to assess the susceptibility of T. urticae to a native isolate ofEPNs, Steinernema feltiae Filipjev ZWO21, under laboratory conditions.

2. Materials and Methods

2.1. Rearing of Tetranychus urticae on Sugar Beet Under Controlled Conditions

The sugar beet plants (Beta vulgaris L. cv. Krajan) from which leaves were obtained were grown in pots under controlled conditions in a phytotron chamber (20 °C ± 2 °C; 16/8 h light/dark photoperiod) until reaching the universal phenological scale–BBCH (Biologische Bundesanstalt, Bundessortenamt und CHemische Industrie) growth stage 12–14.

Populations of T. urticae were maintained under controlled conditions in a phytotron chamber at 25 °C ± 2 °C with a 16/8 h light/dark photoperiod, on sugar beet plants. Leaf discs were cut from uninfested leaves and placed on moist cotton wool in Petri dishes. Leaf disc diameter: usually 2–3 cm, so that they fit well into a standard Petri dish (9 cm in diameter) while providing a sufficiently large photosynthetic surface area to maintain the spider mites. Female mites that had entered the oviposition period within the preceding 48 h were transferred onto the discs and allowed to lay eggs for an additional 24 h. After this period, the females were removed, and the hatching of larvae and their subsequent immobilisation prior to moulting were observed. The developmental progression of juvenile stages was assessed based on morphological features and the presence of exuviae, indicating completion of the developmental stage. This phase lasted approximately two days on average. The full developmental cycle to the adult stage was completed within ten days. The experiment was conducted in 2023 using adult individuals.

2.2. Nematodes

A native isolate of S. feltiae ZWO21 was used in the present study. This strain was originally isolated in autumn 2011 from a meadow in the Zwoleńka River valley (Kozienicka Forest, Poland; 51°23′21.7” N, 21°33′38.9” E). Nematodes were identified using both morphological and genetic criteria [28]. The native isolate of S. feltiae ZWO21 is currently maintained in continuous laboratory culture at the Department of Animal Environment Biology (Institute of Animal Sciences, Warsaw University of Life Sciences–SGGW). EPNs were maintained on the larvae of Galleria mellonella L. (Lepidoptera: Pyralidae) in the laboratory, following the technique from Kaya and Stock [29]. The IJs were collected using White traps [30] and kept in water at 4 °C for later use.

2.3. Bioassay

The experiment was conducted under laboratory conditions using Petri dishes (Ø 5 cm) lined with a double layer of moist filter paper Whatman^® (Cytiva, Maidstone, UK) to maintain optimal humidity throughout the duration of the study. Between 22 and 28 adult T. urticae individuals were transferred to each dish, along with sugar beet leaves serving as a food source. In total, 177 T. urticae individuals were used in the experimental trials, distributed across seven Petri dishes (n = 7). Each Petri dish was treated with 2 mL of a suspension of infective juveniles of S. feltiae ZWO21 at a dose of 8000 IJs per dish (±300 IJs per mite). The number of IJs was determined by counting the number of IJs in five droplets (5 μL) for a previously prepared nematode suspension in water. The selected concentrations of nematode suspension were obtained by diluting the solution with tap water or concentrating the suspension through centrifugation [31].

Nematodes were applied evenly to the surface of each Petri dish, and the dishes were then sealed with Parafilm. Prior to nematode application, the physiological condition and viability of the mites were assessed. Observations were carried out using a stereomicroscope (Olympus, magnification ×3–5), which allowed for accurate counting of active individuals on each dish and the exclusion of dead or visibly weakened mites. Petri dishes treated with either nematodes or water (control) were placed in a Sanyo incubation chamber maintained at 25 °C. The average relative humidity in the chamber was approximately 60%.

In the control group, a total of 76 adult T. urticae individuals were used, distributed across three Petri dishes (n = 3) prepared in the same manner as the experimental dishes. Each dish contained between 22 and 28 mites. A volume of distilled water equivalent to that of the nematode suspension used in the experimental treatments was applied to the surface of each control dish. As in the experimental group, the general condition and viability of the mites were assessed prior to the start of the experiment. After three days, dead mites were collected from the Petri dishes and dissected, and mortality (percentage of infected mites in the analysed samples) was determined using an Olympus SZX9 stereomicroscope (magnification ×3–5).

2.4. Statistical Analysis

To evaluate the efficacy of S. feltiae in controlling T. urticae, a comparative analysis was conducted between two independent groups: the experimental group (n = 7) and the control group (n = 3). For each replicate, the percentage of dead individuals was calculated and subsequently used as the dependent variable in the statistical analysis.

To compare the mean mortality of T. urticae between the control group and the group treated with EPNs, an independent samples Student’s t-test was performed. Prior to the analysis, the fundamental assumptions of the test (normality of distribution and homogeneity of variances) were verified. Normality was assessed separately for each group using the Shapiro–Wilk test, which revealed no significant deviations from a normal distribution. Homogeneity of variances was evaluated using Levene’s test, confirming no significant differences between groups.

Additionally, the effect size was estimated using Cohen’s d, calculated based on the pooled standard deviation, together with the corresponding 95% confidence interval. All statistical tests were two-tailed, with the significance level set at α = 0.05. All analyses were performed in the R environment (version 4.1.2; R Core Team, 2021) using the following packages: car (Levene’s test), effectsize (Cohen’s d), dplyr (data aggregation), and ggplot2 and ggpubr (data visualisation).

3. Results

The results revealed statistically significant differences in T. urticae mortality between the control group and the group treated with EPNs. The mortality of T. urticae following treatment with S. feltiae ZWO21 ranged from 37.5% to 83.3%, whereas in the control group, the percentage of dead individuals remained low, ranging from 8.6% to 13.3% (Figure 1).

The Shapiro–Wilk test indicated no significant deviation from normality in either the treatment group (W = 0.916, p = 0.437) or the control group (W = 0.862, p = 0.273). Levene’s test confirmed homogeneity of variances between the groups (F = 2.38, p = 0.161). The independent samples Student’s t-test revealed a significantly higher mortality of T. urticae in the group treated with S. feltiae ZWO21 compared with the control (t = 4.645, df = 8, p = 0.0017) (Figure 2).

The mean mortality in the experimental group was 57.0% ± 16.7% whereas in the control group it was 10.4% ± 2.56%. The difference between the groups was 46.6 percentage points (SE = 10.03), with a 95% confidence interval ranging from 23.46 to 69.73 percentage points.

The effect size, expressed as Cohen’s d, indicated a very strong impact of the applied biological agent (d = 3.21; 95% CI: 1.11–5.22). This suggests that the observed differences are not only statistically significant but also of substantial practical relevance, confirming the high potential of the S. feltiae isolate for the biological control of T. urticae populations. Despite the relatively small number of replicates, the stability and reliability of the estimate were supported by normality tests, variance analysis, and bootstrap-derived confidence intervals.

Microscopic observations performed using a stereomicroscope confirmed the presence of S. feltiae infective juveniles within the bodies of dead T. urticae individuals. Dissections of mites from the experimental group provided clear evidence of the nematodes’ ability to actively penetrate the mites’ bodies.

4. Discussion

Despite growing awareness of the environmental consequences associated with the use of acaricides are still widely used as the primary method of mite control. Consequently, the search for environmentally safe options, such as effective biological control agents, remains critically important. The present study investigated the potential of EPNs for the control of the two-spotted spider mite. If introduced into the environment as biological formulations, these nematodes would not pose a risk to biodiversity. This is because nematodes are a natural component of soil fauna, and commercial products authorised in Europe are based on native species with a broad geographic distribution [32,33].

The selection of the nematode species for this laboratory study was based on several key considerations. Steinernema feltiae is widely distributed throughout Poland and Europe, which contributes to its ecological safety as a biological control agent [28]. In our previous studies, we demonstrated that S. feltiae is highly effective against a range of insect species [34,35]. This species is also characterised by high biological activity and a strong capacity to adapt to unfavourable environmental conditions, as evidenced by its broad geographical distribution. Moreover, S. feltiae is the most common entomopathogenic nematode species found in Poland and Europe. Its strong adaptability and biological activity [36,37] make this species a promising biological control agent against pests.

An additional factor influencing the selection of this species was its documented efficacy across a broad temperature range, which makes it particularly suitable for use in biological plant protection under temperate climate conditions as well as in greenhouse cultivation systems [38]. In light of these considerations, the S. feltiae isolate was identified as a promising candidate for evaluating its efficacy against T. urticae, despite the fact that mites are not natural hosts of EPNs.

The results of the present study showed that the mean mortality of T. urticae following treatment with S. feltiae ZWO21 was 57.0%, which was substantially higher than the mortality rates reported by Abou El Atta et al. [39]. In their study, three days after the application of Heterorhabditis bacteriophora Poinar and Steinernema carpocapsae (Weiser) at a dose of 2000 IJs, T. urticae mortality reached only 20%, and did not exceed 27% after seven days. Due to the limited literature data on the use of EPNs for the control of T. urticae the results obtained in the present study were compared with those reported for other mite species. The efficacy of EPNs against Rhizoglyphus robini Claparède was investigated by Nermut et al. [40]. In their study, the authors tested several isolates of nematodes belonging to the genera Heterorhabditis and Steinernema, and the highest mortality of R. robini, 30%, was observed following treatment with H. bacteriophora. Despite the smaller body size of T. urticae compared to R. robini, and the larger IJs of S. feltiae relative to H. bacteriophora, the present study achieved nearly twice the mortality. Most studies on the application of EPNs against mites have focused on various tick species (Ixodidae).

High mortality rates of Hyalomma dromedarii Koch were reported by Albogami [41], who tested five isolates of EPNs: three Heterorhabditis indica Poinar, Karunakar and David isolates, H. bacteriophora, and S. feltiae, applied at various doses. Application of S. feltiae at a dose of 300 IJs per female tick resulted in 50% mortality within 72 h post-treatment. In the present study, the application of the same dose yielded a comparable mortality in T. urticae. The highest efficacy in Albogami’s [41] study was observed for H. bacteriophora, which caused 100% tick mortality within three days.

Such high efficacy may be associated with the presence of a tooth-like projection at the anterior tip of IJs, which facilitates penetration through the host cuticle. A similarly high effectiveness of H. bacteriophora against Rhipicephalus microplus Canestrini was reported in laboratory studies by Filgueiras et al. [42], where mortality rates ranged from 97.5% to 98.4%. Comparable mortality levels, exceeding 70%, were also observed under field conditions. The study by El Roby et al. [43] further confirmed the high virulence of Heterorhabditis isolates; however, the authors also reported significant mortality of Boophilus annulatus Stiles and Hassall (86.7%) following treatment with Steinernema isolates, indicating that both genera may be effective in tick control.

Based on previous studies on the use of EPNs in the control of mites, it can be assumed that these organisms hold considerable potential for further evaluation against T. urticae. The search for new native strains and species constitutes a key and promising strategy, capitalising on natural variation in virulence, environmental tolerance, and other adaptive traits. The use of locally adapted isolates is not only effective but also reduces ecological risk, which is crucial for sustainable plant protection practices [44]. Numerous studies have confirmed that native isolates are better adapted to local environmental conditions, thereby enhancing their efficacy under field conditions.

The results obtained in the present study, a 57% mortality of T. urticae, indicate that the use of S. feltiae isolate alone may not be sufficient for effective control of this pest. However, the combined use of EPNs and acaricides may offer a more effective strategy for T. urticae management, while simultaneously reducing reliance on chemical pesticides.

The results obtained in the present study indicate high virulence of the symbiotic bacterium Xenorhabdus nematophilus; however, a major limitation to achieving greater mortality may be the difficulty IJs encounter in penetrating the mite body. A promising direction for future research could involve evaluating the efficacy of the symbiotic bacteria themselves. Such an approach could overcome key limitations associated with nematode-based applications, including desiccation sensitivity, limited mobility in the environment, and difficulties in host penetration. Particularly innovative and potentially groundbreaking is the use of cell-free supernatants (CFS) derived from these symbiotic bacteria. Studies by Eroglu et al. [45] and Priya et al. [46] have demonstrated high, though variable, mortality of T. urticae following CFS application, depending on the bacterial species used.

In this context, particular attention should be given to the potential use of cell-free supernatants obtained from the symbiotic bacteria of the S. feltiae ZWO21 isolate, which demonstrated high efficacy (57%) against T. urticae under experimental conditions. This result suggests that such supernatants may offer a highly effective alternative to the use of whole nematodes in mite control strategies.

5. Conclusions

The present study confirms that the S.feltiae ZWO21 isolate exhibits considerable potential in the biological control of T. urticae, causing 37.5–83.3% (mean 57.0%) mortality of this pest. Although this result indicates a moderate level of efficacy when nematodes are applied alone, it also underscores the relevance of further research into their integration with other control strategies, including acaricides, within integrated pest management frameworks. Of particular scientific interest is the potential of the symbiotic bacterium X. nematophilus, whose high virulence may be harnessed in the form of CFS, thereby overcoming key limitations associated with the biology of the nematodes themselves. In light of the growing need to reduce reliance on chemical plant protection products, the development of effective, environmentally safe biological formulations based on native EPN strains and their symbiotic bacteria represents a promising and forward-looking direction for sustainable pest management research.