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Article

Acaricidal Efficacy of Abamectin against Tetranychus urticae Populations When Combined with Entomopathogenic Fungi

by
Waqas Wakil
1,2,*,
Maria C. Boukouvala
3,
Nickolas G. Kavallieratos
3,*,
Tahira Riasat
4,
Muhammad Usman Ghazanfar
5 and
Pasco B. Avery
6
1
Department of Entomology, University of Agriculture, Faisalabad 38040, Pakistan
2
Senckenberg German Entomological Institute, D-15374 Müncheberg, Germany
3
Laboratory of Agricultural Zoology and Entomology, Department of Crop Science, Agricultural University of Athens, 75 Iera Odos Str., 11855 Athens, Greece
4
Department of Zoology, Government College University, Faisalabad 38000, Pakistan
5
Department of Plant Pathology, College of Agriculture, Sargodha University, Sargodha 40100, Pakistan
6
Indian River Research and Education Center, Department of Entomology and Nematology, Institute for Agricultural Sciences, University of Florida, Ft. Pierce, FL 34945, USA
*
Authors to whom correspondence should be addressed.
Horticulturae 2024, 10(10), 1019; https://doi.org/10.3390/horticulturae10101019
Submission received: 21 August 2024 / Revised: 8 September 2024 / Accepted: 17 September 2024 / Published: 25 September 2024
(This article belongs to the Collection Non-Chemical Strategies for IPM in Horticulture)
Versions Notes

Abstract

:
Tetranychus urticae (Acari: Tetranychidae) is a widespread and serious mite pest that infests tomato plants and causes economic losses worldwide. We investigated the acaricidal efficacy of two isolates of entomopathogenic fungi (EPF) Metarhizium robertsii (WG-7) and Beauveria bassiana (WG-12) alone and in combination with abamectin when applied topically to tomato leaf discs in the laboratory against T. urticae. We also evaluated the establishment and proliferation of T. urticae mite life stages on tomato plants in the greenhouse after application of each of the above treatments. The combination of abamectin with each EPF caused 100% mortality in T. urticae immatures after 2 days while each EPF or abamectin alone caused moderate mortality, not exceeding 74.2% 3 days post-exposure. Complete (100%) mortality of adults was observed after 5 days in leaf discs treated with M. robertsii plus abamectin whereas B. bassiana plus abamectin caused 100% mortality after 7 days. The mean number of eggs, emerged immatures, and adults were significantly reduced on both sides of the leaves (i.e., abaxial and adaxial sides) after using the combined application of M. robertsii or B. bassiana plus abamectin, compared to abamectin alone and controls. Our results reveal that the acaricidal efficacy of abamectin combined with either EPF was significantly better in managing the T. urticae life stages than either treatment alone under greenhouse conditions.

1. Introduction

Tetranychus urticae is a major pest negatively affecting a wide variety of crops, including vegetables, fruits, and ornamentals [1,2,3]. It is also a key pest of tomato plants, causing substantial economic losses in both greenhouse and field settings worldwide [4,5], resulting in 40–60% yield losses [6]. This mite species has a high reproductive rate, capable of completing its life cycle in about one week under optimal conditions (i.e., 27 °C and 55–60% relative humidity) [7,8]. One other important aspect of T. urticae rapid population growth deals with its reproduction through arrhenotokous parthenogenesis, where females arise from fertilized eggs while males from non-fertilized eggs [8,9]. A single female can produce male offspring with whom she can then mate (oedipal copulation), leading to a population that includes both males and females. This capability is particularly advantageous in environments where mates are scarce, enabling the mites to establish their populations efficiently [8,10]. For instance, Tuan et al. [10] reported that a population of T. urticae originating from the arrhenotokous group (10 eggs) produced approximately 23,000 individuals after a 50-day interval, while a population initiated from the bisexual group (10 eggs) produced about 2,800,000 individuals. These paradigms express the fast reproductive capacity of a mixed population of T. urticae and the dynamics of arrhenotokous parthenogenesis to produce new infestations. Therefore, because it has a short life cycle, T. urticae causes severe damage to the plant very quickly by piercing plant cells and sucking out their contents, leading to a stippled appearance on leaves and overall plant decline [11,12,13]. Heavy infestations can also cause defoliation, stunted growth, and even plant death, which severely impacts plant yield and quality [13,14].
The control of T. urticae is a crucial challenge due to its ability to quickly develop resistance to numerous acaricides [15,16,17]. The ability to reproduce via arrhenotokous parthenogenesis can lead to increased genetic diversity, accelerating the selection of mite individuals resistant to various single-mode chemical acaricides [18]. Furthermore, the short life cycle, fast reproduction rate, and high number of generations per year further facilitate the quick development and spread of resistance genes within its populations [18,19]. Apart from the above mechanisms, several reports have revealed that widespread and indiscriminate single-mode pesticide applications have resulted in the development of resistance for T. urticae to 96 different active ingredients (a.i.) [20]. Among these is abamectin, as well as several new compounds such as cyenopyrafen and difenazate [21,22,23,24,25,26]. Abamectin, part of the avermectin family, is a natural fermentation product derived from the soil-dwelling actinomycete Streptomyces avermitilis (ex. Burg et al., 1979), Kim and Goodfellow 2002 (Streptomycetales: Streptomycetaceae) [27,28]. Abamectin is a biorational pesticide that has been widely used against insects, nematodes, and mites, including T. urticae, for over 30 years [22,24,29,30,31]. This pesticide has shown high efficacy against T. urticae populations due to its ability to disrupt the mite nervous system, leading to paralysis and death [16,32]. One of the significant advantages of abamectin is its translaminar activity. This property allows the chemical compound to penetrate the leaf tissue and form a reservoir within the leaves. Consequently, the compound can negatively affect mites that are on the underside of leaves or in other hard-to-reach areas, ensuring effective control even when not in direct contact with the chemical [33].
The resistance of T. urticae populations to single-mode chemical acaricides has led to the search for alternative control methods, including the use of entomopathogenic fungi (EPF), which have shown significant potential for the management of this pest [34,35]. The infection process of EPF begins when fungal conidia come in contact with the cuticle of the host. The conidia adhere to the cuticle through hydrophobic interactions and enzymatic activity [36]. The conidia secrete cuticle-degrading enzymes which break down the structural components of the cuticle, thus facilitating penetration via an appressoria with a penetration peg below it [36,37,38]. The fungal hyphae proliferate the cuticle and eventually reach the hemocoel where blastospores or hyphal bodies are produced, spreading throughout the internal body of the host, producing toxins and finally causing its death [39,40]. The use of EPF offers several advantages over chemical acaricides. These fungi are environmentally friendly, compatible with most non-target organisms, and reduce the risk of resistance development to mite populations [41,42,43]. Moreover, they can be integrated into and enhance the effectiveness of pest management programs [44]. Several isolates and strains of EPF Metarhizium robertsii, J.F. Bisch., Rehner & Humber (Hypocreales: Clavicipitaceae), and Beauveria bassiana (Balsamo-Crivelli), Vuillemin (Hypocreales: Cordycipitaceae), have been widely used against T. urticae, providing promising adequate management of this pest [40,45,46].
The combination of pesticides with EPF represents a promising approach to integrated pest management (IPM) strategies to control arthropod pests such as mites [47,48,49,50]. This strategy aims to enhance pest management efficacy, delay the development of resistance, reduce single-mode chemical usage, and minimize environmental impacts [47,48,50]. However, research on the combined activity of M. robertsii or B. bassiana with abamectin against T. urticae remains unknown, despite the potential benefits vs. biological or chemical management strategies alone. This knowledge gap is important and hinders the development of IPM programs that could enhance effectiveness towards the reduction of single-mode pesticides. Thus, the objective of the present study was to evaluate the efficacy of the combination of the EPF M. robertsii and B. bassiana with abamectin compared to each treatment applied alone against immatures and adults of T. urticae under laboratory and greenhouse conditions.

2. Materials and Methods

2.1. Tetranychus urticae Rearing

The colony of T. urticae was obtained from stock population reared on tomato plants (Solanum lycopersicum L.) (Department of Entomology, University of Agriculture, Faisalabad (UAF)). Homogenization [51] and maintenance of this population was carried out in the laboratory for the past 3–4 years in a controlled climate chamber (Philips, Karachi, Pakistan) set at 26 ± 0.1 °C and 65 ± 0.2% relative humidity (RH) with a photoperiod of 16:8 h light:dark (L:D), using daylight lamps [52]. Previous studies noted significant variations in the demographic features of T. urticae when fed on leaf discs versus whole leaves [53]. Therefore, to avoid this discrepancy and accurately evaluate the life stages of T. urticae, a standardized rearing method was used for all bioassays following the protocol of Puspitarini et al. [54]. The individuals used originated from eggs laid 24 h prior to the study [54].

2.2. Metarhizium robertsii and Beauveria bassiana Isolates

Two EPF isolates obtained from M. robertsii (Mr WG-7) and B. bassiana (Bb WG-12) were used in the bioassays. Mr WG-7 was originally isolated from soil samples taken from forest lands in Lal Sohanra, Bahawalpur, and Bb WG-12 from soil samples taken from a fruit orchard in Chichawatni, Pakistan [55]. Both fungal isolates were previously deposited and maintained at the culture collections at the Microbial Control Laboratory, UAF, in Petri dishes with potato dextrose agar (PDA) (Sigma-Aldrich, Taufkirchen, Germany) stored in a refrigerator at 4 °C [46]. These isolates were sub-cultured in the media. Each fungal isolate was inoculated on Petri dishes (6 cm Ø and 1.5 cm high) containing Sabouraud dextrose agar (SDA) (BD-Difco, Franklin Lakes, NJ, USA) supplemented with 1% yeast (SDAY) and incubated for 14 days at 24 °C in darkness [56,57]. The dishes were sealed with Parafilm (Sigma-Aldrich, Taufkirchen, Germany) and incubated in a growth chamber (MIR-254-PE, Panasonic, Kadoma, Japan) under a 16:8 h L:D photoperiod at 25 ± 0.1 °C for 10 days [46]. Conidia were produced from these dishes 14 days after inoculation. A sterilized glass spatula (Drigalski glass spatula, LaborXing, Shenzhen, China) was used to gently scrape the dry conidia from the SDA medium and transfer the conidial powder to a sterilized 10 mL tube. A portion of the harvested conidia in the tube was suspended in a 50 mL Falcon tube containing a 30 mL sterile Tween 80 solution (0.05%) (Merck, Kenilworth, NJ, USA). Tween 80 was used to decrease the conidia clumping [58]. The sterile Tween 80 solution was prepared using autoclaved H2O. A magnetic stirrer (IRMECO, Lütjensee, Germany) with eight sterile glass beads was used to homogenize the conidial suspension. The suspension was filtered into a 50 mL Falcon tube through a double layer of sterilized cheesecloth to remove fragments of mycelia [59,60]. The stock concentration was quantified using a Neubauer Improved haemocytometer (Marienfeld, Lauda-Königshofen, Germany) with the use of a light microscope (Euromex BB.1152-PLi microscope, Euromex Microscopen, Arnhem, The Netherlands) at 400× magnification.
Conidia viability was measured after inoculation of 0.1 mL of 106 conidia/mL of the conidia suspension on two dishes (6 cm Ø and 1.5 cm high) containing SDA with 1% yeast (SDAY) [46]. Dishes were sealed with Parafilm and incubated for 18 h at 25 ± 0.1 °C under a 14:10 h L:D photoperiod. Sterile slips were utilized to cover the dishes after their incubation. Per fungal isolate, 200 conidia were observed per dish [46]. If the germ tube length was twice the diameter of the conidia, germination was noted as successful [61,62]. This procedure was carried out at 400× magnification (Euromex BB.1152-PLi microscope, Euromex Microscopen, Arnhem, The Netherlands). The viability of conidia was determined to be >93% before initiation of trials.

2.3. Insecticide

The insecticide Shogun EC (Agrow Limited, Lahore, Pakistan) with 1.8% abamectin was used in this study against T. urticae. The label dose is 1.25 mL a.i./L H2O.

2.4. Laboratory Trials

Experiments were conducted in 5 cm Ø and 1.5 cm high plastic Petri dishes (i.e., 3 sub-replicates/trial), each containing a 20 mm Ø tomato (var. “Moneymaker”) leaf disc [63]. This variety was chosen because it is the most promising and widely cultivated by tomato growers in Pakistan [64,65]. The discs were prepared before the spraying using a cork borer. Each tomato leaf disc was arranged abaxial side up on wet tissue paper (Ø  =  3 cm) placed in the dish and surrounded by a ring of moistened cotton swabs to provide moisture according to Wakil et al. [46] and Wu et al. [66]. Groups of 20 one-day-old T. urticae female adults [52] or nymphs were individually transferred to each leaf disc [63]. The discs containing the mites were then sprayed with 1000 μL [46,63] of either isolate of Mr WG-7 or Bb WG-12 at 1 × 107 conidia/mL, or abamectin (Aba) at a concentration of 0.5 mL/L of H2O v/v, utilizing an airbrush (Master Multi-purpose Airbrush, Las Vegas, NM, USA). Control leaf discs were sprayed with 1000 μL of a 0.05% solution of Tween 80 [58,66]. After each spray, the discs were air-dried for approximately 300 s [66] and covered with PVC film made of polyvinyl chloride. This film had fine holes all over the surface for ventilation [46,67]. Regarding the combined application of each EPF isolate with abamectin (i.e., Mr WG-7 + Aba and Bb WG-12 + Aba), two consecutive sprays were performed [68]. These leaf discs were first sprayed with 1000 µL of abamectin and allowed to air dry. After one day, 20 one-day-old T. urticae adults or one-day-old nymphs were transferred to each abamectin-contaminated disc and then topically sprayed with 1000 µL of one of the fungal isolates, corresponding to the respective combinations [68]. The protocol was the same as above once the leaf discs were dried. The replicate-covered dish bioassay was transferred to an incubator at 25 ± 0.1 °C with 60 ± 0.2% RH under a 16:8 h L:D photoperiod [69,70]. Nymphal mortality was observed 1, 2, and 3 days post-spray [70]. Dead adults were recorded 3, 5, and 7 days post-spray [45]. For each exposure interval, different leaf discs with each life stage of the mite were prepared. Mortality was observed using a stereomicroscope (Leica Wild M3B, Heerbrugg, Switzerland). Nymphs or adults were deemed dead if they did not make any movement of their appendages when prodded with a brush made of camel hair [66]. The whole experimental procedure was carried out four separate times in a completely randomized design (CRD) by preparing new leaf discs, new dishes, new mite individuals, and new solutions for each trial (i.e., 3 sub-replicates × 2 life stages × 6 treatments + control × 4 replications).

2.5. Greenhouse Trials

Tomato seeds of the variety “Moneymaker” were used for the experiments. Seeds were sown in seedling trays (2 seeds per well) and irrigated daily. Three weeks after the sowing of the nursery, 3-L plastic pots containing Sphagnum Peat Moss were used to transplant the seedlings individually (i.e., 1 plant/pot). These pots were then placed in growth chambers at 25 ± 0.1 °C with 65 ± 0.2% RH under a photoperiod of 16:8 h L:D [71]. The plants were watered at 3-day intervals, while after 15 days the fertilizers Actosol (Evyol Group Pvt. Ltd., Lahore, Pakistan), which carries 15% N, 20% P, and 20% K, and Zendal 10% G (Rustam Seed and Agrochemicals, Jullundur Pvt. Ltd., Lahore, Pakistan), a micronutrient which carries 7% Zn and 3% Fe, were placed in the pots [52]. Twenty days later, plants that had uniform growth characteristics were utilized in the bioassays [52]. Three pots with three tomato plants were selected for each treatment or control (i.e., 3 subreplications). Each tomato plant had 10 to 15 leaves. For each tomato plant per pot, 20 female T. urticae adults were released on the three terminal leaflets of the second oldest true leaf using a fine hairbrush. Then, a 15 mL solution of abamectin (1 mL/L H2O v/v) or a conidial suspension of Mr WG-7 or Bb WG-12 (1 × 109 conidia/mL) were separately applied to the plants per pot in the respective group, using a hand-held sprayer (Kissan Ghar, Sargodha, Pakistan). All parts of the plant were sprayed uniformly with one of the EPF suspensions or abamectin solution [66]. Control plants were sprayed with a 15 mL solution of Tween 80 (0.05%) [58,66]. Two consecutive sprays were performed for the combined application of Mr WG-7 or Bb WG-12 with abamectin as described in Section 2.4 [68]. All leaves per plant were cut and observed after 21 days post-initial infestation to monitor overall mite (adults, immatures, eggs) establishment and proliferation on the abaxial and adaxial sides of the leaves [72] using a stereomicroscope (Leica Wild M3B, Heerbrugg, Switzerland). The experiment was replicated four times in a randomized complete block design (RCBD) by preparing new potted plants, new mite individuals, and new solutions each time (i.e., 3 sub-replications × 6 treatments + control × 4 replications).

2.6. Statistical Analysis

All the data were transformed using the formula log (x + 1) to normalize variance prior to conducting statistical analysis [73,74]. Mortality data were computed using Abbott’s formula [75]. For the mortality bioassay experiment, the exposure interval, life stage, and treatment were the main effects. The response variable was mortality. Regarding the data related to the presence of mite individuals per leaf, the position, life stage, and treatment were the main effects of the greenhouse experiments. The presence of mites on leaves was the response variable. All data were analyzed using two-way ANOVA, considering both the main effects and their interactions in all cases. Mortalities observed in the controls were less than 5%. The Tukey (HSD) test was used for mean separation at a level of significance of 5% [76]. All analyses were carried out with the Minitab statistical package [77].

3. Results

3.1. Mortality of Tetranychus urticae Female Adults and Nymphs in the Laboratory

All the main effects/associated interactions concerning the mortality of the mite life stages were significant (Table 1). Mortality of nymphs was very low in the individual application of EPF and Aba ranging from 21.7% to 48.8% after 1 d of exposure in comparison with the combined application of EPF with Aba (Table 2). The combination Mr WG-7 + Aba caused significantly higher nymphal mortality (84.6%) than Bb WG-12 + Aba (72.9%) one day after the application. After 2 d, 100% mortality in T. urticae nymphs was observed in both EPF + Aba combinations, whereas Bb WG-12, Mr WG-7, or Aba alone caused mortality not exceeding 65.4%. Three days later, the Mr WG-7 isolate killed 74.2% of the exposed nymphs, which was significantly different from the percentage of dead nymphs obtained in Bb WG-12 (63.8%) and Aba (48.3%) treatments.
Regarding female adults, mortality was extremely low in individual applications of EPF and Aba, reaching 39.2% after 3 d (Table 2). In the combination treatment Mr WG-7 +Aba, the highest mortality was recorded (76.3%), which differed significantly from the Bb WG-12 + Aba combination (67.1%) and the application of EPF and Aba treatments alone. Complete (100.0%) mortality of adults was noted after 5 days in leaf discs treated with the Mr WG-7 + Aba, whereas Bb WG-12 + Aba caused significantly lower mortality (83.8%) than the other combination treatments. The application of Mr WG-7 alone caused 51.3% mortality, while Bb WG-12 and Aba treatments did not surpass 38.8% after 5 d. After one week, Bb WG-12 + Aba caused 100% mortality, similar to the other combination treatment after 5 d. Concerning the individual applications, significant differences were observed among Mr WG-7 (65.0%), Bb WG-12 (56.7%), and Aba (37.1%).

3.2. Establishment of Tetranychus urticae (Adults, Immatures, and Eggs) on Tomato Plants after Acaricidal Treatments in the Greenhouse

All the main effects/associated interactions concerning the establishment of T. urticae (adults, immatures, and eggs) on tomato plants after acaricidal treatments in the greenhouse were significant (Table 3). The mean number of the recorded adults alive differed significantly among the treatments compared with the control plants on the abaxial side of the leaves 21 d after the initial infestation with 20 adult individuals (Table 4). A significantly lower mean number of adults were found in treated plants with either the combination of Mr WG-7 + Aba (1.0 adult/leaf), Bb WG-12 + Aba (1.4 adult/leaf), or the application of Mr WG-7 (4.8 adult/leaf) alone in comparison with Bb WG-12 (9.0 adult/leaf) and Aba (12.0 adult/leaf), while the highest mean number of adults was observed in control plants (71.2 adult/leaf). Concerning immatures alive on the abaxial side of the leaves, the lowest mean number was recorded in the combined applications of EPF with Aba (i.e., 1.0 and 5.0 immatures/leaf for Mr WG-7 + Aba and Bb WG-12 + Aba, respectively), which differed significantly from the application of EPF alone (17.5 and 28.2 immatures/leaf for Mr WG-7 and Bb WG-12, respectively). Plants treated with Aba had significantly more immatures alive compared to the other treatments, while in control plants, the highest mean number of immatures (i.e., 254.2 immatures/leaf) was recorded. Regarding eggs on the abaxial side of the leaves, a significantly higher mean number was observed in controls (167.4 eggs/leaf) followed by Aba (34.9 eggs/leaf), EPF treatments alone (9.0 and 15.0 eggs/leaf for Mr WG-7 and Bb WG-12, respectively) and EPF + Aba treatment combinations (1.0 and 2.0 eggs/leaf for Mr WG-7 + Aba and Bb WG-12 + Aba, respectively).
A similar trend was observed in the performance of treatments on the adaxial side of the leaves 21 days after the initial infestation with 20 adult individuals (Table 4). Significantly fewer adults were found in both EPF + Aba combination treatments (1.0 and 1.0 adult/leaf for Mr WG-7 + Aba and Bb WG-12 + Aba, respectively) and Mr WG-7 (1.0 adult/leaf) than Aba (5.1 adults/leaf) and the control (8.5 adults/leaf). The mean number of immatures alive on the adaxial side of the leaves was significantly higher in treatments with Aba (21.3 immatures/leaf) than with Bb WG-12 (9.1 immatures/leaf) and the rest treatments, where <1.1 immatures/leaf were found. The control plants had the highest mean number of immatures. The combined application of either EPF with Aba and the application of Mr WG-7 alone significantly reduced the mean number of eggs (<0.9 egg/leaf) on the adaxial side of the leaves in comparison to Bb WG-12 (4.0 eggs/leaf) and Aba (13.0 eggs/leaf). In addition, no significant differences were recorded between abamectin and control plants.

4. Discussion

Metarhizium robertsii and B. bassiana have shown significant potential for control of T. urticae infestations, offering a biological control method as an alternative to broad spectrum synthetic single-mode chemicals [46,60,78,79]. For example, mortalities >80.0% have been reported for B. bassiana (e.g., isolate UPH-1103) and M. robertsii (isolates Ma106 and Ma110) against T. urticae adults [79,80]. In the current study, treatments with the M. robertsii isolate WG-7 achieved significantly higher mortality to both life stages (immatures and female adults) of T. urticae than the B. bassiana isolate WG-12 and abamectin. Regarding the performance of the two fungal species against T. urticae, previous studies have shown similar results to those of the current investigation. For instance, Elhakim et al. [40] reported that an M. robertsii isolate killed more T. urticae female adults (i.e., 85.00%) than a B. bassiana isolate at the concentration of 1 × 108 conidia/mL after 7 d of spraying. Similarly, an M. robertsii isolate WG-02 was found to be more effective against T. urticae adult females than B. bassiana isolates WG-12 and WG-02 7 days after spraying [46]. One of the main mechanisms through which EPF exert their pathogenicity is protease activity, which plays a key role in the infection process of the host [40,81,82]. For instance, the greater virulence between M. robertsii and B. bassiana isolates against T. urticae was associated with higher protease activity found in M. robertsii compares to the B. bassiana isolate [40].
The combined application of EPF with insecticides has been investigated before this study, offering an important tool in pest control since the risk of insecticide resistance can be reduced [49,83,84]. Our results revealed that the combined application of EPF with abamectin caused complete mortality to both nymphs and adults of T. urticae. On the other hand, the application of M. robertsii or B. bassiana isolates alone resulted in mortality rates of 74.2 and 63.8%, respectively, for nymphs after 3 days of exposure. Similar mortalities were noted for female adults after 7 d of exposure, not exceeding 65.0%. A previous investigation has shown that nymphs were more tolerant to B. bassiana isolate UPH-1103 than the adults [79]. This is related to the short molting stage of the immatures (larvae and nymphs) where spores of EPF are removed with the old cuticle of the host without penetrating the new cuticle [85]. However, in the present research, the tested fungal isolates alone may have low adulticide activity, an issue that merits additional research.
Among treatments, the single application of abamectin was the least effective against T. urticae. For example, 48.3% of nymphs and 37.1% of adults were dead after 3 and 7 d exposure, respectively, differing significantly from all other treatments. Furthermore, significantly more adults, immatures, and eggs were observed in abamectin-treated plants than in plants treated with either of the EPF treatments alone or EPF combined with abamectin. The observed tolerance of T. urticae to abamectin could be attributed to various mechanisms such as increased detoxification and target-site mutations in glutamate-gated chloride channel genes [16,86,87], presenting a significant challenge to effective mite management. In the current study, the most effective treatment against T. urticae was the combination of M. robertsii WG-7 isolate plus abamectin, which provided 100% adult and nymphal mortality after 5 and 2 d, respectively, followed by B. bassiana WG-12 isolate in combination with abamectin, which killed all the exposed adult females and nymphs after 7 and 2 d, respectively. The combination of EPF treatments combined with abamectin acted faster than the EPF treatments or abamectin alone. The delayed activity of EPF against T. urticae alone may be related to the time required for the processes of fungal attachment, penetration, germination, and growth [40,88]. Concerning abamectin, this a.i. has a slow action resulting in a delayed efficacy [89,90,91]. Seyed-Talebi et al. [48] observed increased mortality against the females of T. urticae on plants treated with the combination of B. bassiana and spirodiclofen in comparison to the single application of the EPF treatment and the acaricide.
Tetranychus urticae shows a preference for the adaxial surface of leaves where it feeds and inhabits [92,93,94,95]. In the current study, the potential establishment of T. urticae on both sides of tomato leaves was significantly deterred by the application of EPF treatments or abamectin and their combinations in comparison to the control plants 21 d after infestation, where the abaxial surface of the leaves inhabited a higher number of T. urticae adults, immatures, and eggs than the adaxial surface of the leaves. The most effective treatment was the combination of M. robertsii and abamectin, where there was a significant reduction in the mean number of adults, immatures, and eggs vs. controls. In this study, the efficacies from highest to least were as follows: the application of M. robertsii WG-7 and abamectin > B. bassiana WG-12 and abamectin > M. robertsii WG-7 > B. bassiana WG-12 > abamectin. For instance, Wu et al. [66] recorded a 61.00–72.10% reduction in the number of eggs, immature stages (i.e., larvae and nymphs), and adults one month after spraying with isolate GZGY-1-3 of B. bassiana. Canassa et al. [96] reported a significant suppression in the population of T. urticae in bean plants 35 d after seed inoculation with B. bassiana and M. robertsii alone or in combination with both fungal species. Similar results were obtained by Rasool et al. [60] for both fungal species in tomato plants. Consequently, a reduction of T. urticae populations in agricultural settings after the application of EPF alone or in combination with another acaracide such as abamectin may enhance crop protection and yield.
The results of the current study revealed that the combination of abamectin with M. robertsii WG-7 or B. bassiana WG-12 isolates can be used against T. urticae life stages, since their action was faster, provided increased mortality rates, and kept the parasite population at low levels compared to each treatment applied alone. When two different agents are used in combination, they may act independently or one agent may create enough stress for the pest, enhancing the effectiveness of the second agent [84,97,98]. Further research is needed to investigate the additive or synergistic mechanism of the combination of abamectin with the EPF isolates. Furthermore, more fungal isolates and acaricides should be tested against T. urticae to create a reservoir of toxic combinations to control this pest.

Author Contributions

Conceptualization, W.W. and N.G.K.; methodology, W.W., and N.G.K.; software, W.W., T.R. and M.U.G.; validation, W.W., M.C.B., N.G.K., T.R., and M.U.G.; formal analysis, W.W., M.C.B., N.G.K. and T.R.; investigation, W.W., M.C.B., N.G.K., T.R. and M.U.G.; resources, W.W. and M.U.G.; data curation, W.W., T.R., and M.U.G.; writing—original draft preparation, W.W., M.C.B., N.G.K., T.R., M.U.G. and P.B.A.; writing—review and editing, W.W., M.C.B., N.G.K., T.R., and M.U.G.; visualization, W.W., M.C.B., N.G.K., T.R., M.U.G. and P.B.A.; supervision, W.W.; project administration, W.W.; funding acquisition, W.W. All authors have read and agreed to the published version of the manuscript.

Funding

This research was partly funded by Project 3244 of the Higher Education Commission, Islamabad, Pakistan.

Data Availability Statement

The data are contained within the article.

Conflicts of Interest

The authors declare no conflicts of interest.

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Table 1. ANOVA parameters for mortality of Tetranychus urticae on tomato leaf discs treated with Beauveria bassiana WG-12 and Metarhizium robertsii WG-7 isolates or abamectin, and the respective paired combinations of entomopathogenic fungi with abamectin (Total df = 359).
Table 1. ANOVA parameters for mortality of Tetranychus urticae on tomato leaf discs treated with Beauveria bassiana WG-12 and Metarhizium robertsii WG-7 isolates or abamectin, and the respective paired combinations of entomopathogenic fungi with abamectin (Total df = 359).
SourcedfFp
Interval of time (Interval)2729.2<0.01
Life stage (Stage)1220.0<0.01
Treatment41865.3<0.01
Interval × stage28.1<0.01
Interval × treatment810.3<0.01
Stage × treatment47.5<0.01
Interval × stage × treatment84.5<0.01
Table 2. Mortality (mean% ± SE) of Tetranychus urticae nymphs and female adults on tomato leaf discs after the application of Beauveria bassiana (Bb) WG-12, Metarhizium robertsii (Mr) WG-7, or abamectin (Aba), and the respective paired combinations of entomopathogenic fungi with abamectin (Bb WG-12 + Aba and Mr WG-7 + Aba) at different intervals in days. Within each row, means with the same uppercase letter are not significantly different (in all cases, df = 2, 35, Tukey HSD test at p < 0.05). Within each column, means followed by the same lowercase letter are not significantly different (in all cases, df = 4, 59, Tukey HSD test at p < 0.05).
Table 2. Mortality (mean% ± SE) of Tetranychus urticae nymphs and female adults on tomato leaf discs after the application of Beauveria bassiana (Bb) WG-12, Metarhizium robertsii (Mr) WG-7, or abamectin (Aba), and the respective paired combinations of entomopathogenic fungi with abamectin (Bb WG-12 + Aba and Mr WG-7 + Aba) at different intervals in days. Within each row, means with the same uppercase letter are not significantly different (in all cases, df = 2, 35, Tukey HSD test at p < 0.05). Within each column, means followed by the same lowercase letter are not significantly different (in all cases, df = 4, 59, Tukey HSD test at p < 0.05).
Life StageTreatmentIntervals (Days)Fp
123
NymphsBb WG-1236.3 ± 1.4 Cd51.7 ± 2.9 Bc63.8 ± 1.4 Ac73.1<0.01
Mr WG-748.8 ± 1.1 Cc65.4 ± 2.0 Bb74.2 ± 2.2 Ab50.0<0.01
Aba21.7 ± 1.3 Ce35.8 ± 2.0 Bd48.3 ± 1.4 Ad68.8<0.01
Bb WG-12 + Aba72.9 ± 1.3 Bb100.0 ± 0.0 Aa100.0 ± 0.0 Aa434.0<0.01
Mr WG-7 + Aba84.6 ± 1.1 Ba100.0 ± 0.0 Aa100.0 ± 0.0 Aa181.1<0.01
F431.0347.0292.3
p<0.01<0.01<0.01
357
Female adultsBb WG-1223.3 ± 1.8 Cd38.8 ± 2.1 Bd56.7 ± 2.2 Ac69.3<0.01
Mr WG-739.2 ± 1.5 Cc51.3 ± 2.1 Bc65.0 ± 2.5 Ab37.9<0.01
Aba16.3 ± 1.6 Ce24.2 ± 1.6 Be37.1 ± 1.7 Ad40.9<0.01
Bb WG-12 + Aba67.1 ± 2.0 Cb83.8 ± 1.6 Bb100.0 ± 0.0 Aa122.1<0.01
Mr WG-7 + Aba76.3 ± 1.4 Ba100.0 ± 0.0 Aa100.0 ± 0.0 Aa291.0<0.01
F250.1353.0275.1
p<0.01<0.01<0.01
Table 3. ANOVA parameters for the number of Tetranychus urticae alive on tomato leaves after treatment with Beauveria bassiana, Metarhizium robertsii, or abamectin, and the respective paired combinations of entomopathogenic fungi with abamectin (Total df = 431).
Table 3. ANOVA parameters for the number of Tetranychus urticae alive on tomato leaves after treatment with Beauveria bassiana, Metarhizium robertsii, or abamectin, and the respective paired combinations of entomopathogenic fungi with abamectin (Total df = 431).
SourcedfFp
Leaf side (abaxial or adaxial)13618.0<0.01
Life stage2784.3<0.01
Treatment53041.5<0.01
Leaf side × life stage2363.4<0.01
Leaf side × treatment51968.7<0.01
Life stage × treatment10331.4<0.01
Leaf side × life stage × treatment10193.7<0.01
Table 4. Mean (±SE) number of Tetranychus urticae alive life adults, immatures, and eggs on the abaxial (bottom) and adaxial (top) side of the tomato leaves 21 days after the application of Beauveria bassiana (Bb) WG-12, Metarhizium robertsii (Mr) WG-7, or abamectin (Aba) and control, and the respective paired combinations of entomopathogenic fungi with abamectin (Bb WG-12 + Aba and Mr WG-7 + Aba) per leaf side. Within each row, means with the same uppercase letter are not significantly different (in all cases, df = 2, 35, Tukey HSD test at p = 0.05). Within each column, means followed by the same lowercase letter are not significantly different (in all cases, df = 5, 71, Tukey HSD test at p = 0.05).
Table 4. Mean (±SE) number of Tetranychus urticae alive life adults, immatures, and eggs on the abaxial (bottom) and adaxial (top) side of the tomato leaves 21 days after the application of Beauveria bassiana (Bb) WG-12, Metarhizium robertsii (Mr) WG-7, or abamectin (Aba) and control, and the respective paired combinations of entomopathogenic fungi with abamectin (Bb WG-12 + Aba and Mr WG-7 + Aba) per leaf side. Within each row, means with the same uppercase letter are not significantly different (in all cases, df = 2, 35, Tukey HSD test at p = 0.05). Within each column, means followed by the same lowercase letter are not significantly different (in all cases, df = 5, 71, Tukey HSD test at p = 0.05).
Leaf SideTreatmentNumber of Tetranychus urticae Alive/LeafFp
AdultsImmaturesEggs
Abaxial (Bottom)Bb WG-129.1 ± 1.3 Bb28.2 ± 2.7 Ac15.0 ± 1.1 Bc28.7<0.01
Mr WG-74.8 ± 0.8 Bc17.5 ± 1.8 Ac9.0 ± 1.3 Bcd23.4<0.01
Aba12.0 ± 0.4 Cb46.1 ± 3.6 Ab34.9 ± 1.3 Bb61.9<0.01
Bb WG-12 + Aba1.4 ± 0.4 Bc5.0 ± 1.0 Ad2.0 ± 0.6 Bde7.32<0.01
Mr WG-7 + Aba1.0 ± 0.6 Ac1.0 ± 0.5 Ad1.0 ± 0.6 Ae0.00.99
Control71.2 ± 1.7 Ca254.2 ± 5.1 Aa167.4 ± 3.7 Ba594.1<0.01
F763.11132.51316.3
p<0.01<0.01<0.01
Adaxial (Top)Bb WG-122.2 ± 0.6 Bbc9.1 ± 0.6 Ac4.0 ± 0.5 Bb36.8<0.01
Mr WG-71.0 ± 0.6 Ac1.1 ± 0.6 Ad0.9 ± 0.4 Ac0.120.88
Aba5.1 ± 0.8 Cb21.3 ± 1.6 Ab13.0 ± 0.8 Ba51.2<0.01
Bb WG-12 + Aba1.0 ± 0.6 Ac1.0 ± 0.6 Ad0.9 ± 0.6 Ac0.160.84
Mr WG-7 + Aba1.0 ± 0.6 Ac0.8 ± 0.4 Ad0.6 ± 0.3 Ac0.10.87
Control8.5 ± 0.7 Ca34.1 ± 1.8 Aa15.2 ± 0.9 Ba116.0<0.01
F21.6163.0116.1
p<0.01<0.01<0.01
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Wakil, W.; Boukouvala, M.C.; Kavallieratos, N.G.; Riasat, T.; Ghazanfar, M.U.; Avery, P.B. Acaricidal Efficacy of Abamectin against Tetranychus urticae Populations When Combined with Entomopathogenic Fungi. Horticulturae 2024, 10, 1019. https://doi.org/10.3390/horticulturae10101019

AMA Style

Wakil W, Boukouvala MC, Kavallieratos NG, Riasat T, Ghazanfar MU, Avery PB. Acaricidal Efficacy of Abamectin against Tetranychus urticae Populations When Combined with Entomopathogenic Fungi. Horticulturae. 2024; 10(10):1019. https://doi.org/10.3390/horticulturae10101019

Chicago/Turabian Style

Wakil, Waqas, Maria C. Boukouvala, Nickolas G. Kavallieratos, Tahira Riasat, Muhammad Usman Ghazanfar, and Pasco B. Avery. 2024. "Acaricidal Efficacy of Abamectin against Tetranychus urticae Populations When Combined with Entomopathogenic Fungi" Horticulturae 10, no. 10: 1019. https://doi.org/10.3390/horticulturae10101019

APA Style

Wakil, W., Boukouvala, M. C., Kavallieratos, N. G., Riasat, T., Ghazanfar, M. U., & Avery, P. B. (2024). Acaricidal Efficacy of Abamectin against Tetranychus urticae Populations When Combined with Entomopathogenic Fungi. Horticulturae, 10(10), 1019. https://doi.org/10.3390/horticulturae10101019

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