Introduction

Tetranychus urticae Koch (two-spotted spider mite) (Acari: Tetranychidae) is a polyphagous pest that feeds on more than 1100 plant species and varieties, including numerous commercial fruits and vegetables as well as ornamental plants (Migeon and Dorkeld 2010; Atalay and Kumral 2013). This pest has developed resistance to many pesticides. Its high fertility, short life cycle, and haplodiploid reproductive characteristics accelerate the development of resistance (Denholm et al. 1998; Carrière 2003; Van Leeuwen et al. 2010; Dermauw et al. 2012). In addition, these features cause them to reach population levels that cause significant economic losses in cultivated plants (Sato et al. 2007; El-Saiedy and Fahim 2021). Accordingly, it becomes increasingly difficult to control spider mites with only the use of pesticides. The phytophagous feeding feature and short life cycle of this pest allow it to develop resistance to many acaricides after a few applications (Cranham and Helle 1985; Keena and Granett 1990; Devine et al. 2001; Stumpf and Nauen 2001; Sato et al. 2005). Besides, individuals of this species can live individually or in groups. Tetranychus urticae females can establish colonies by arrhenotokous parthenogenetic reproduction and subsequently mate with their own sons (Yano 2008). In addition, T. urticae exhibits various forms of social behavior such as gathering or building a communal web (Saito 1983; Clotuche et al. 2009; Le Goff et al. 2009). Two-spotted spider mite builds complex three-dimensional webs on plant leaves (Saito 1983; Oku et al. 2009). A denser and more protective web provides continuity in group living characteristics, and their control in production areas may become harder as the dense webbing protects them from acaricides and also predators (Le Goff et al. 2010; Clotuche et al. 2011).

Cordyceps (formerly Isaria) fumosorosea (Wize) (Hypocreales: Cordycipitaceae) blastospores have been used extensively in pest management programs around the world. The blastospores are easily produced and require only 6–8 h to germinate (Vega et al. 1999; Avery 2002; Lozano-Contreras et al. 2007; Avery et al. 2009). This entomopathogenic fungus is eco-friendly with minimal impact on beneficial non-target arthropods (Sterk et al. 1995a, b; Avery et al. 2009). Some strains of C. fumosorosea were proven to be pathogenic to multiple mite pests in various habitats and highly virulent against active individuals of T. urticae (Alves et al. 2002; Shi and Feng 2004, 2009; Kim et al. 2008; El-Sharabasy 2015).

Entomopathogenic fungi are important and selective in the control of arthropod species. The inclusion of entomopathogenic fungi in pest control practices is increasing due to environmental awareness and concerns about food safety and pesticide use that lead to an increase in the number of insecticide-resistant species (Rai et al. 2014). Food safety, protection of biodiversity, minimization of pesticide residues, and the safety of non-target organisms are some of the advantages of microbial control agents over conventional chemical pesticides (Shahid et al. 2012). Mycoinsecticides are the first-choice organisms among entomopathogens in various ecosystems. Characteristics of entomopathogenic fungi such as high host specificity, negligible effect on non-target organisms, and easy mass production make them preferred in pest control (Rai et al. 2014; Singh et al. 2017). Entomopathogenic fungi are known to help regulate insect and mite populations through epizootics (Singh et al. 2017). The transfer of conidia from infected individuals to others is crucial as it causes the spread of infections and the onset of epizootics (Lacey et al. 1994; Hesket et al. 2010). Transmission of entomopathogenic fungi to healthy individuals is also achieved with the use of disseminating devices designed for attracting insects and infecting them through entomopathogenic fungi (Baverstock et al. 2010). Arthropods can mediate the horizontal transmission of disease to susceptible hosts either through direct physical contact or indirectly by releasing infective propagules (fungal spores) in the habitat (Lin et al. 2019). Infected individuals remaining in the environment can be beneficial for the spread of fungal pathogens and transmission to healthy conspecifics (Long et al. 2000; Avery et al. 2009, 2010). Reaching dense populations of host individuals increases the possibility of contact between them. Cordyceps fumosorosea has an important potential in the management of T. urticae. This present study aimed to determine the dissemination of this entomopathogenic fungus (Priority®) to uncontaminated (healthy) individuals through infected T. urticae individuals.

Materials and methods

Mites were used from a 5-year-old laboratory colony of T. urticae, that was collected from Antalya vegetable greenhouses in 2018 and that has since then been reared on bean plants, Phaseolus vulgaris L. (Fabaceae), and maintained at 25 ± 1 °C, 60–70% RH, and L8:D16 photoperiod. The identification of T. urticae was made using the keys in Bolland et al. (1998) and Jeppson et al. (1975). Bean leaves infested with T. urticae were cut and placed on non-contaminated plants to ensure the production of mites continued. Two commercial pesticides were used in the experiments: the mycopesticide Priority (1.5% C. fumosorosea strain PFs-1, 108 CFU/mL; MRFC [maximum field recommended concentration] = 250 mL/decare) and the acaricide Torpedo (EC 18 g/L abamectin; MFRC = 25 mL/100 L water), obtained from Agrobest (İzmir, Turkey) and Hektaş (Antalya, Turkey), respectively.

The study consisted of four experiments. When bean plants are treated with entomopathogenic fungi (1.5% C. fumosorosea strain PFs-1), the lethal effect on T. urticae females was determined in Petri dish conditions in exp. 1 and on bean plants in the climate room in exp. 2. In these two experiments, sterile distilled water was used as the negative control and abamectin as the positive control. Priority, abamectin, and the water control were sprayed onto leaf discs (6 cm diameter) with an apparatus (glass material that sprays the liquid under pressure at the tip of a motorized insect aspirator), with the ability to mist at 4 atm pressure for 10 s (0.6 mL).

In order to determine the dissemination of the same entomopathogenic fungus by contaminated individuals to conspecific individuals, varying numbers of contaminated individuals (in the range of 1–5 contaminated adults) were transferred to Petri dishes in exp. 3 and bean plants in exp. 4. In these two experiments, 10 uncontaminated females (per replication) were previously transferred to leaf discs prepared in both Petri dishes and pots. For exp. 3 and 4, the entomopathogenic fungus was sprayed with a misting apparatus at 4 atm pressure for 10 s (0.6 mL) onto leaf discs. Then, 20 T. urticae adults were transferred with a fine brush on the bean leaf disc (6 cm diameter) in Petri dishes with 10 replications. After 24 h, the contaminated individuals (a range of 1–5 contaminated females) were used. In exp. 3 and 4, the bean plants were germinated in pots (11.5 cm diameter) containing sterile soil in a climatic chamber.

Petri dishes and pot trials (exp. 1–4) were carried out under climate chamber conditions (25 ± 1 °C, 60 ± 10% RH, and L16:D8 photoperiod). In exp. 1–4, a complete randomized block design was used, and sterile distilled water (dH2O) was applied in the control. The number of deaths was recorded on days 3, 5, and 7 after the applications (DAA). In all experiments, cadavers of T. urticae were placed on potato dextrose agar (PDA) in Priority treatments, and fungal growth was observed in glass Petri dishes (9 cm diameter) by the method of Wraight et al. (1998) with some modifications. All experiments were repeated separately per observation day.

Experiment 1

Exp. 1 was conducted in Petri dishes with bean leaf discs (6 cm diameter) on moist cotton (Fig. 1). Priority, Torpedo, or distilled water were sprayed on leaf discs for 10 s. Afterwards, 10 uncontaminated females were transferred with a brush onto the leaf discs (10 replications).

Fig. 1
figure 1

Leaf disc method used in Petri and pot trials

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Experiment 2

In exp. 2, 2-week-old bean plants – with two leaves, in plastic pots (11.5 cm diameter) with sterilized soil mixture (soil + organic matter) – were used in the climatic chamber. For the applications in these plants, a leaf disc area of 6 cm diameter was constructed on each leaf with a Vaseline ring (it was placed around the leaf discs to prevent the escape of T. urticae) (Al-Azzazy et al. 2020) (Fig. 1). One leaf selected on each plant (with two leaves) was evaluated as one replication (total of 10 replications). The distilled water was sprayed on leaf discs selected per plant in the control (with 10 replications).

Experiment 3

The treatments were conducted in Petri dishes (9 cm diameter) in which bean leaf discs were placed on moist cotton. Contaminated females (1–5) were released with a brush to Petri dishes containing 10 non-contaminated individuals. Each Petri dish was one replication (in total 10 replications).

Experiment 4

In this experiment, 24 h after Priority application, contaminated individuals (1–5 females per application) were transferred onto leaves of bean plants in pots (1–5 contaminated females per application). After that, 10 uncontaminated individuals were released to these leaves to determine whether the entomopathogenic fungus could infect healthy individuals. Each plant was a replication and in total 10 replications were made per application.

Statistical analysis

One-way ANOVA was applied to the number of individuals surviving obtained from exp. 1 and 2 followed by Tukey’s honestly significant difference (HSD) test (α = 0.05) (IBM SPSS v.20.0). Mortality rates obtained from exp. 3 and 4 were calculated using Abbott’s formula (Abbott 1925). One-way ANOVA was applied to the data followed by Tukey’s HSD test. The median lethal time (LT50) was calculated using mortality rates in exp. 3 and 4. The independent samples t-test was separately applied to compare the results of the Petri dish and pot trials with each other for mortality rates at each time point. Also correlation and regression analyses were done (α = 0.01). In addition, second order polynomials were used depending on the correlation coefficient appropriate to the obtained mortality rates.

Results

Experiment 1

The ANOVA indicated that treatments had a strong effect on mite survival on each observation day (3 DAA: F2,27 = 4.734, P = 0.017; 5 DAA: F2,27 = 24.927; 7 DAA: F2,27 = 47.499, both P < 0.001). The average number of surviving T. urticae females treated with Priority was not different from that treated with abamectin on 3 and 5 DAA, but it was significantly higher at 7 DAA (Fig. 2). Survival in the water control was higher than in the treatments on all observation days (Fig. 2).

Fig. 2
figure 2

Mean (± SE) number of surviving Tetranychus urticae females treated with a mycopesticide (Priority), abamectin (Torpedo) or distilled water (control) at 3, 5 and 7 days after application (DAA), on bean leaf either in Petri dishes (exp. 1) or on potted plants (exp. 2). Means within a day capped with different letters are significantly different (Tukey’s test: P < 0.005)

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Experiment 2

ANOVA indicated a strong effect of treatments on mite survival on each observation day (3 DAA: F2,27 = 14.289; 5 DAA: F2,27 = 30.465; 7 DAA: F2,27 = 65.498, all P < 0.001). There was no statistical difference between Priority and abamectin on any observation day. The average number of surviving individuals in the control was higher than in the other applications (Fig. 2).

Experiment 3

In exp. 3, it was evaluated whether different numbers of Priority-contaminated T. urticae (1–5) would have an effect on the mortality rate in uncontaminated individuals in Petri dishes. ANOVA indicated strong effects of treatments on mite survival on each observation day (3 DAA: F4,45 = 43.321; 5 DAA: F4,45 = 23.573; 7 DAA: F4,45 = 14.616, all P < 0.001).

At 3 DAA, the mortality rates were 8.2–29.3% in the trial with 1–5 contaminated adults released, respectively. These values were 17.3–44.7% on 5 DAA, and 29.6–56.4% on 7 DAA (Fig. 3). Overall, mortality rates increased with increasing mite density (Fig. 3). The highest mortality rates were achieved in the trials with five contaminated individuals released on all observation days (Fig. 3).

Fig. 3
figure 3

Mean (± SE) mortality rates (%) of Tetranychus urticae females treated with 1–5 mycopesticide-contaminated individuals at 3, 5 and 7 days after application (DAA), on bean leaf either in Petri dishes (exp. 3) or on potted plants (exp. 4). Means within a day capped with different letters are significantly different (Tukey’s test: P < 0.005)

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Experiment 4

In exp. 4, it was investigated whether different numbers of Priority-contaminated T. urticae (1–5) would have an effect on the mortality rate of uncontaminated individuals on potted plants. Here, ANOVA indicated strong effects of treatments on mite survival on 5 and 7 days after application (5 DAA: F4,45 = 134.720; 7 DAA: F4,45 = 29.957, both P < 0.001), but not on 3 DAA (F4,45 = 1.336, P = 0.27).

At 3 DAA, the mortality rates were 8.2–11.7% in the trial with 1–5 contaminated adults released, at 5 DAA these values were 10.9–40% and at 7 DAA 14.6–40.7% (Fig. 3). Also here, overall mortality rates increased with increasing mite density (Fig. 3).

The LT50, a measure of the virulence of the entomopathogenic fungus, was calculated separately for experiments 3 and 4. In exp. 3, the LT50 decreased from 10.49 to 5.94 days, with 1 and 5 contaminated individuals released in Petri dishes, respectively (Fig. 4). In exp. 4, the LT50 decreased from 29.38 to 7.50 days, with 1 and 5 contaminated individuals released on potted plants, respectively (Fig. 4).

Fig. 4
figure 4

Median lethal time (LT50) of adult Tetranychus urticae females with 1–5 mycopesticide-contaminated individuals released in Petri dishes (exp. 3) or potted plants (exp. 4)

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Comparison of Petri dish and pot trials (experiments 3 and 4)

Mortality rates in Petri dish and pot experiments, with 1–5 contaminated individuals, were compared per observation day. When 1 contaminated individual was released, mortality rates in Petri dishes and pot trials were the same on 3 and 5 DAA, but higher in Petri dishes on 7 DAA (Fig. 5). Almost all other comparisons – 2–5 contaminated individuals released, 3–7 DAA – indicated higher mortality rates in Petri dishes than in pot trials; only at 5 DAA, the differences in mortality rate were not significantly different with 3 and 5 contaminated individuals released (Fig. 5).

Fig. 5
figure 5

Mean (± SE) mortality rates (%) of Tetranychus urticae in Petri dish vs. potted plant trials, compared at 3, 5 and 7 days after application (DAA). Mortality rates within a day and within a mite density marked with different letters are significantly different (t-test: P < 0.01; equations of the fitted lines are based on correlation and regression analyses)

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Discussion

One of the major disadvantages of continuous chemical control against T. urticae is the fact that it gains resistance to pesticides over time (Herron and Rophail 1998; Van Leeuwen et al. 2004). In order to overcome the resistance problem, it is preferred to use alternative control methods in pest management (Yeşilayer 2018). Auto-dissemination of entomopathogenic fungi has an important role in integrated pest management. This strategy can be used to disseminate microbial organisms against pests that can coexist in the same habitat with natural enemies (Vega et al. 2000). Auto-dissemination can be provided using devices containing both entomopathogens and semiochemicals; pests enter the device, get infected by the pathogen, exit from the device, and transfer the inoculum (Vega et al. 2007; Baverstock et al. 2010; Lacey et al. 2015; Gonzalez et al. 2016). Dispersal of conidia of entomopathogenic fungi with devices has been reported to be simple, economical, and effective (Maniania and Ekesi 2013). Klein and Lacey (1999) and Maniania (2002) determined 95 and 100% infection in Popilia japonica Newman and Glossina fuscipes fuscipes Newstead, respectively, by conidia of Metarhizium anisopliae Petch.

An insect’s or mite’s behavioral response to a fungal pathogen has an important impact on the entomopathogenic fungus’ efficacy. In the literature, the effects of insect and mite behavior on the preference of fungal pathogens have been studied (Baverstock et al. 2010; Parker et al. 2011; Vezilier et al. 2015; Zélé et al. 2020). However, the effectiveness of entomopathogens was associated with the contact between the pest and the entomopathogenic fungus (Wraight et al. 2001; FAO/IAEA 2019). A 3-min contact of M. brunneum (GranMet®) with an adult P. japonica was sufficient to infect the insect and eventually kill it (Benvenuti et al. 2019). In the current study, individuals considered contaminated were also exposed to entomopathogenic fungus (1.5% C. fumosorosea) for 24 h.

An increase in the number of contaminated T. urticae adults in this study showed a significant effect on the mortality rate of healthy adults in Petri dishes as well as on potted plants. Yet, on the potted plants it took a bit longer for this effect to become significant, and overall the mortality rate was higher in the Petri dishes than on the potted plants – for instance, the mortality rate was 56.4% on day 7 when five contaminated individuals were released into Petri dishes, whereas this value was 40.7% on potted plants. Amjad et al. (2012) determined a mortality rate of 79% of adult T. urticae females on day 8 after direct application of C. fumosorosea (n32) at 108 conidia/mL.

The virulence of an entomopathogenic fungus can be determined by the LT50, the average lethal time from exposure to the pathogen to the death of an infected insect (FAO/IAEA 2019). The virulence of each entomopathogenic fungus species depends on the host from which the fungus is isolated and the susceptibility of the target for which it is evaluated (Hajek and St. Leger 1994; De la Rosa et al. 2002; Roberts and Leger 2004; Rehner 2005; Meyling and Eilenberg 2007; Zélé et al. 2020). Differences between entomopathogenic fungal strains and their effects on pest species may result from host–pathogen relationships (Lecuona 1996; FAO/IAEA 2019). In the present study, the LT50s obtained at all observation times in the pot trials were higher than in the Petri dishes. In addition, it was determined that the LT50 decreased as the number of contaminated individuals increased in both experiments 3 and 4. Amjad et al. (2012) reported that the LT50 of C. fumosorosea (n32) was 4.6 days in 108 conidia/mL in Petri dishes. In this study, the LT50 value was determined as 5.9 days when five contaminated individuals were released into Petri dishes. In another study, the LT50 for T. urticae adults treated with Beauveria bassiana Bb101, another entomopathogenic fungus, has been detected as 81.8 h (3.4 days) (Saranya et al. 2013). Hence, the LT50 values in the current study were found to be close to those reported in other studies.

Similar to this study, Demirözer (2019) investigated the dissemination of Fusarium subglutinans 12 A through females of Frankliniella occidentalis Pergande. Mortality rates were 39.1, 51.8, 51.6, 48.2, and 50.1% on day 8 in Petri dishes in which, respectively, 1–5 inoculated thrips were released together with 10 uninoculated females. In the present study, mortality rates were 29.6–56.4% on day 7 in Petri dishes in which 1–5 T. urticae adults were contaminated with C. fumosorosea strain PFs-1. In addition, Demirözer (2019) found that the mortality rate on day 7 remained < 30% in cell cage treatments where five inoculated females were used. In the pot trial of the current study, the mortality rate was 40.7% on day 7 after release of five contaminated adults. In both studies, the mortality rates in pot trials were lower than in Petri dish trials.

In the current study, the disseminating ability of individuals exposed to an entomopathogenic fungus to uninfected individuals of the same species was evaluated. As the number of contaminated individuals increased, the mortality rate also increased. Thus, the effectiveness of biological control may increase with the occurrence of indirect contamination from infected to non-contaminated individuals. This study may encourage similar studies on other harmful arthropods that have a high probability of contact with each other.