Kök, Şahin ¹ ; Yaşar, İpek ² and Kasap, İsmail ³

¹✉ Çanakkale Onsekiz Mart University, Faculty of Agriculture, Department of Plant Protection, Çanakkale, Türkiye & Çanakkale Onsekiz Mart University, Biodiversity Application and Research Center, Çanakkale, Türkiye.
²Çanakkale Onsekiz Mart University, Faculty of Agriculture, Department of Plant Protection, Çanakkale, Türkiye.
³Çanakkale Onsekiz Mart University, Faculty of Agriculture, Department of Plant Protection, Çanakkale, Türkiye.

2026 - Volume: 66 Issue: 2 pages: 406-413

Original research

Keywords

Abstract

Introduction

The two-spotted spidermite, Tetranychus urticae Koch (Acari: Tetranychidae) (TSSM) is a devastating pests that infests numerous crops and plants worldwide (Migeon and Dorkeld 2024), causing serious economic losses in both greenhouses and fields (Van Leeuwen et al. 2007). Chemical control, primarily with acaricides is the general control strategy of TSSM worldwide. However, mites gain acaricide resistance due to high reproductive rate, short life span and detoxification abilities (Van Leeuwen et al. 2006). TSSM, developed resistance to more than 80 acaricides (Van Leeuwen et al. 2010). The widespread use of pesticides including acaricides has detrimental effects such as disruption of ecological balance (Sharma et al. 2019), environmental pollution (Tang et al. 2021), negative impacts on human health (Zhou et al. 2024), and natural enemies (Schmidt-Jeffris 2023), and residue problems in plants or food (Carvalho 2017). Considering these negative effects of pesticides, there is a need for effective alternative control methods.

Biological control is an effective strategy and environmentally friendly that uses biocontrol agents to control pest mites (Daniels et al. 2023). Among the Phytoseiidae mites (Acari), Phytoseiulus persimilis Athias-Henriot (PP) is a important predator that feeds primarily on the Tetranychus mites and effectively suppresses TSSM on various crops (Çakmak et al. 2005; Tiftikçi et al. 2020). Diatomaceous earths (DE) knowns as the fossilized remains of phytoplankton, which are diatoms that occurred during the Miocene and Eocene periods (Korunić 1998). Diatoms, which are distributed in fresh-water, marine environments and terrestrial ecosystems, are unicellular eukaryotic algae that are characterized by an external skeleton rich in silicon dioxide whose fossilized remains constitute DE (Korunić 2016). DE particles cause mortality in pests through the desiccation, attaching to the cuticle on insect and the abrasion, cuticular micro-wounds (Subramanyam et al. 2000; Vayias et al. 2009). Beyond biological control agents, DE has a significant potential for preventing and controlling numerous arthropod pests (Zeni et al. 2021). DE is advantageous for pest control due to its low toxicity to mammals and the environment, and the absence of known physiological resistance to pest (Vayias et al. 2008). Combining DE with some control methods such as synthetic insecticides, fungal agents and botanical extracts has proven successful (Athanassiou et al. 2008; Yang et al. 2010; Wakil et al. 2021). It is recommended that DE be used as an adjuvant together with other biological control agents rather than alone (Constantinescu-Aruxandei et al. 2020). Laboratory studies have shown that DE causes moderately mortality in predatory mites such as PP (46.4%) and Neoseiulus fallacis Garman (Mesostigmata: Phytoseiidae) (34.3%) (Shah and Appleby 2019).

The synergistic interaction between DE and several biological control agents coupled with its low negative effect on the agent, particularly on PP suggests it potential as a valuable adjuvant of PP in the biological control of TSSM. However, data on the effect of DE on the biological control effectiveness of PP on TSSM are limited. Therefore, this study aimed to investigate the effect of combined and separate application of PP and DE on the biological control of TSSM in the laboratory conditions.

Material and methods

Stock culture of mites

TSSM used in the tests were provided from the stock culture, which has been reared fourteen years on bean plants in a climate chamber (25±2 °C, 65±10% humidity and 16L:8D photoperiod) of the Acarology Laboratory in Çanakkale Onsekiz Mart University in Türkiye. The population of PP was collected from Çanakkale, Northwest of Türkiye and were reared to have a stock culture on TSSM infested plants in the same conditions for about ten years.

Preparation of diatomaceous earth

In laboratory tests, Turkish diatomaceous earth Detech® (Entoteam Arge Tic. Ltd. Şti.) was used. A 5% concentration of DE was prepared by mixing 50 gr of Detech® in one litre of distilled water.

Laboratory tests

To determine the combined and separate effect of PP and DE on mortality and egg-laying behavior of TSSM, modified Munger cells were used in the laboratory tests. Each cell consisted of a 60x45x3 mm plexiglass plate, a water-saturated cotton layer topped with blotting paper, and a fresh bean leaf. The bean leaf was placed on this wet surface and covered with a plexiglass plate containing a 25 mm diameter circular hole, followed by a perforated acetate film to prevent mite escape and moisture buildup.

The layers were fixed using a small metal clamp. Additionally, cotton was connected to a distilled water source to maintain the freshness of bean leaves. Four treatment groups were established: TSSM+PP; TSSM+DE; TSSM+PP+DE and the control group of TSSM. For the laboratory tests, the predator/prey ratio of 1:10, which showed important successful in suppressing TSSM under field conditions (Tiftikçi et al. 2020), was used. Ten TSSM adult females and one PP adult female were placed in Munger cells using a fine-tipped brush for the group of TSSM+PP. Ten TSSM adult females were placed in the cell and sprayed with 5% DE for the group of TSSM+DE. Ten TSSM adult females and one PP adult female were placed in the cell and a 5% dose of DE was sprayed with an hand spreyer (Botanika Aquatic 155552) for the group of TSSM+PP+DE. Finally, ten TSSM adult females were placed in the cell and sprayed with distilled water for the control group. The females of TSSM and PP used in the tests were selected from among the individuals of one- to three-day-old. The experiments were repeated ten times for all test groups. The test cells were placed in a climate chamber (25±2 °C, 65±10% humidity and 16L:8D photoperiod). Counting was made at 1, 24, 48, 72 and 96 hours after application. Dead-alive individuals of TSSM and the eggs laid by the surviving females were recorded.

Statistical analysis

The mortalities of TSSM in different test groups and counting times were corrected by Abbot's formula (Abbott 1925). The Abbot's formula was as follows:

Abbott Value= [ (X - Y) / (100 - Y)] x 100

where Abbott Value represents the means of corrected mortality (%), X represents the means of the treated mortality (%), and Y represents the means of the control mortality (%).The following formula was used to calculate the number of eggs laid by a single TSSM female in the test groups and counting times.

NE = TNE / NL

where NE represents the number of eggs per a single TSSM female, TNE represents the total number of eggs laid, and NL represents the number of live TSSM females.

Initially, both the means of corrected mortality values (Abbott Values,%) at the same counting hours and the eggs laid by the surviving females of TSSM at the same counting hours were compared using One Way ANOVA. Tukey test was used to determine the differences between the test groups at the counting times. The values were subjected to box-cox transformation before statistical analysis and untransformed means were presented.

Also, statistical evaluation of the data obtained was carried out in the Minitab 17 software using General Linear Model to compare the corrected cumulative mortality (Abbott Value;%) of TSSM in different test groups and counting times, and the mean number of eggs per a single TSSM female in different test groups and counting times.

The statistical model for both the corrected cumulative mortality and the mean number of eggs per a single TSSM were as follows:

Y_ijk= μ + α_i + β_j + αβ_ij + ε_ijk

where Y_ijk is the response (the corrected cumulative mortality of TSSM / the mean number of eggs per a single TSSM female), μ is the overall mean, α_i is the fixed effect of test groups (i = TSSM+PP; TSSM+DE; TSSM+PP+DE and control group), β_j is the fixed effect of the counting time (j = 1, 24, 48, 72 and 96 hours), αβ_ij is the effect of interaction between the test groups and counting time and ε_ijk is the residual error distributed as N (0, σ²).

Results

Mortality rates ranged between 0.00 and 73.19±0.72% across all treatments and observational periods (Figure 1). No mortality was observed on TSSM within the first hour in all test groups. After 24 hours, the combined treatment (TSSM+PP+DE) resulted in the highest TSSM mortality (36.25±1.47%), followed by TSSM+PP (28.75±2.26%) and TSSM+DE (10.00±2.43%). Mortality rates after 24 hours were statistically significant in all test groups (F=6.86, df=2, p>0.05). Similar results were observed in all test groups after 48 and 72 hours, respectively (F=14.96, df=2, p>0.05 and F=6.87, df=2, p>0.05).

After 96 hours, TSSM+PP+DE treatment again caused the highest mortality (73.19±0.72%), while TSSM+DE caused the lowest mortality (47.64±0.93%). The cumulative mortalities after 96 hours were statistically significant in all test groups (F=7.27, df=2, p>0.05). The overall effectiveness of the treatments at all counting times, based on the cumulative mortalities, can be ranked as follows: TSSM+PP+DE > TSSM+PP > TSSM+DE (Figure 1). Also, the cumulative mortality of TSSM in the control group was observed 2.5% after 96 hours. On the other hand, no mortality were observed among the the individuals of PP in the group of TSSM+PP+DE at all counting times. The statistical analysis revealed significant interactions between the different test groups and counting times concerning the cumulative corrected mortalies of TSSM. A significant difference was observed for the cumulative corrected mortalies of TSSM across the groups of TSSM+PP, TSSM+DE and TSSM+PP+DE at all counting times (different test groups × counting times interactions: F= 2.69, df= 8, p < 0.05).

The effect of the treatments on fecundutiy of TSSM was also evaluated by counting the number of eggs laid by a single TSSM female and the total number of eggs in each treatment. After one hour, no egg-laying was observed in any of the test groups. Across all subsequent observational time points, the highest number of eggs was in the control group. After 24 hours, the number of eggs per female in TSSM+PP, TSSM+DE and TSSM+PP+DE treatments was calculated as 1.39±0.33, 1.04±0.17 and 0.35±0.25, respectively. After 96 hours, the highest number of eggs per female was recorded in the control group with 9.17±0.19, followed by TSSM+PP, TSSM+DE and TSSM+PP+DE. The number of eggs laid after 96 hours was statistically significant in all test groups (F=9.22, df=3, p>0.05). The total number of eggs laid followed a similar trend. The highest total egg count was recorded in the control group, while the lowest was observed in the TSSM+PP+DE treatment. These differences in total egg counts were also statistically significant (F=53.00, df=3, p>0.05) (Figure 2). The number of eggs laid by surviving females of TSSM at all test groups increased proportionally with the elapsed time. Consedering the fecundity of TSSM female, the statistical analysis revealed significant interactions between the different test groups and counting times. A significant difference was observed for the mean number of eggs per single female across the groups of TSSM+PP, TSSM+DE, TSSM+PP+DE and control at all counting times (different test groups × counting times interactions: F= 6.46, df= 12, p < 0.05).

Discussion

The strategic use of biocontrol agents, both separate and combination is crucial for maximizing the biological control effectiveness and integrated pest management programs against pest mites. Considering the detrimental human health and environment impacts of many conventional pesticides optimizing the use of biocontrol agents is vital. When applied alone, PP has already shown significant success in suppressing the population of TSSM both in laboratory conditions and in fields (Opit et al. 2004; Migeon et al. 2019; Tiftikçi et al. 2020, 2022; Karut et al. 2022; Alpysbayeva et al. 2024). In addition, the combining PP with other biocontrol agents such as some predatory mites, entomopathogenic fungi and DE may further enchance the effectiveness of biological control of pest mites.

DE has demonstrated moderate mortality against TSSM, when applied alone. Our findings, showing 47.64% mortality after 96 hours, align with previous studies. Susurluk and İlktan (2024) reported that DE applied at 5% and 10% concentrations caused the highest mortalities 32.52 and 33.56% after 96 hours, respectively. Similar to this, Shah and Appleby (2019) reported a 24.6% mortality rate of DE applied to TSSM. Also, Başkaya (2020) reported that the average mortality caused by different concentrations of DE on TSSM reached 45.50% after 72 hours. In a study under greenhouse conditions in 2021 and 2022, the general weekly means number of TSSM mobile stages on cucumbers were 40% and 51% lower, respectively, in the application of DE (foliar sprey in the dose of 37.5 gm/L) group compared to the control group (Ammar and Mohamed 2024). The results of our study and the studies presented above demonstrate that DE alone has a moderate effect in suppressing TSSM under both laboratory and greenhouse settings.

DE exhibits limited negative effects on many natural enemies of crop pests, especially Phytoseiidae mites (Zeni et al. 2021), suggesting its potential compatibility with biological control of strategies. Several studies have reported low mortality of Phytoseiid mites such as PP following treatment with DE (Shah and Appleby 2019; Mahmoud et al. 2024). This selective toxicity, with moderate TSSM mortality and low PP mortality, suggests that DE could complement TSSM biological control efforts using PP. No study has investigated the combined use of PP with diatomaceous earth in the biological control of TSSM. Our study demonstrates that the highest mortality rate of TSSM occured in combined treatments of PP and DE and it was statistically significant compared to the test group where PP alone. We believe that this result will contribute to studies investigating the effectiveness of PP on TSSM and will fill an important data gap.

Assessing the fecundity of TSSM in the presence of different biological control agents is important for understanding their reproductivity and in estimating population size. This study revealed the total number of eggs and the number of eggs laid per female in the presence of PP and DE and, their change according to the test groups. Our results clearly showed that the number of eggs laid by a single TSSM female and the total number of eggs laid were lower in the test groups with PP and DE separate and combined than in the control group, and the difference was statistically significant.

Although many studies have observed the effect of the presence or predation of PP on the number of eggs laid per female TSSM, there are very few studies on the number of eggs laid per female TSSM in both diatomaceous earth alone and the combination PP + DE applications. In a study, Moghadasi et al. (2019) reported a significant decrease in the number of eggs laid per female in the presence of PP both with direct application and exposure to PP volatiles. Similarly, Škaloudová et al. (2007) reported that increasing predation risk by introduction of a caged PP and by predator cues on the leaf disc decreased the fecundity of TSSM. Based on the results of above studies, the statistically significant decrease in the number of eggs laid per female TSSM observed in the TSSM+PP test group compared to the control group in our study can be attributed to the presence or predation of PP.

Inert dusts such as diatomous earth have a reducing effect on the fecundity of TSSM. Our laboratory results demonstrate this phenomenon. Also, the results of Shah and Appleby (2019) and Susurluk and İlktan (2024) reported that the application of DE reduced egg-laying per female TSSM compared to control groups. Although we have very limited data to understand the mechanism by which DE affects the fecundity of pest mites, it is a known fact that DE reduces the progeny reproduction and the number of eggs laid of many pests (Kemabonta and Anikwe 2010; Alkan et al. 2019). Decreased reproduction may be related to the stress caused by the inert dusts or to the reduction of both chemical and physical stimuli. Our study corroborates above findings, demonstrating that both PP and DE, when applied separately, significantly suppress TSSM egg-laying. Furthermore, our results highlight the synergistic effect of the combined treatment including PP and DE, showing an even greater reduction in egg-laying per female than either agent applied alone. This decrease stems from both the direct effect of DE on the fecundity of TSSM and the indirect effect of PP such as presence and predation risk. On the other hand, the fact that PP prefers the egg stage of TSSM for feeding than other stages may play a significant role in the decrease in the counting number of eggs of TSSM. Many studies showed that PP and other Phytoseiids prefer the egg stage of mites during the preying period (Moghadasi et al. 2013; Khodayari et al. 2016; Jyothis and Ramani 2019).

This laboratory study demonstrates that DE can significantly enhance the efficacy of PP in the biological control of TSSM. The combined application of PP and DE resulted in both higher TSSM mortality and a substantial reduction in egg-laying (both per female and total). Our results highlight the benefits of combining different biological control agents to improve biological control success. Future studies, including both green house and field trials, need to be carried out under both greenhouse conditions and fields, will be crucial for developing more effective TSSM management strategies and minimizing reliance on conventional pesticides.

Acknowledgements

This study was supported by Çanakkale Onsekiz Mart University The Scientific Research Coordination Unit, Project number: FHD-2023-4556.

Declaration of interest statement

No potential conflict of interest was reported by the author(s).

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