Introduction
The two-spotted spider mite (TSSM), Tetranychus urticae Koch (Acari: Tetranychidae), is one of the more economically important pests of many plants (e.g. tomato, cucumber, cotton, eggplant, bean, raspberry, soybean and others) in different parts of the world (Van Leeuwen et al., Reference Van Leeuwen, Vontas, Tsagkarakou, Dermauw and Tirry2010; Sedaratian et al., Reference Sedaratian, Fathipour and Moharramipour2011; Vergel et al., Reference Vergel, Bustos, Rodríguez and Cantor2011; Khanamani et al., Reference Khanamani, Fathipour and Hajiqanbar2013; Havasi et al., Reference Havasi, Kheradmand, Mosallanejad and Fathipour2018; Azadi-Qoort et al., Reference Azadi-Qoort, Sedaratian-Jahromi, Haghani and Ghane-Jahromi2019). The feeding activities of TSSM usually results in chlorosis, leaf and fruit deformation, and stunted plant growth which can lead to reduced marketability of the final production (Lahai et al., Reference Lahai, Ekanayake and George1998; Dogan et al., Reference Dogan, Hazir, Yildiz, Butt and Cakmak2017).
While chemicals have long represented the basis for control programmes for managing pests, diseases and weeds because of rapid impact and low cost (Zhao, Reference Zhao2000; Stanley and Preetha, Reference Stanley and Preetha2016; Akyazi et al., Reference Akyazi, Soysal, Altunç, Lisle, Hassan and Akyol2018), reliance on these chemical compounds may cause high levels of resistance in pest populations and negatively affect natural enemy populations (Bugeme et al., Reference Bugeme, Knapp, Boga, Ekesi and Maniania2014; Liu et al., Reference Liu, Rao and Vinson2014; Li et al., Reference Li, Zhang, Tian, Liu, Liu, Liu and Wang2017; Međo et al., Reference Međo, Stojnić and Marčić2017). It is well documented that T. urticae can develop resistance to pesticides in a short period of time (Van Leeuwen et al., Reference Van Leeuwen, Vontas, Tsagkarakou, Dermauw and Tirry2010) due to its short life cycle and high reproductive potential (Nauen et al., Reference Nauen, Stumpf, Elbert, Zebitz and Kraus2001). Therefore, to achieve sustainable management of this mite pest, it is so crucial to develop integrated programmes that do not rely only on pesticides.
Predatory mites have been adopted as an effective alternative for managing the TSSM on a variety of plants in different agricultural systems (Song et al., Reference Song, Zheng, Zhang and Li2016; Fathipour et al., Reference Fathipour, Karimi, Farazmand and Talebi2017; Riahi et al., Reference Riahi, Fathipour, Talebi and Mehrabadi2017; Zheng et al., Reference Zheng, Clercq, Song, Li and Zhang2017). The predator Neoseiulus californicus McGregor (Acari: Phytoseiidae), is a well-known effective natural enemy against spider mites, other pest mites and small pest insects (Castagnoli and Simoni, Reference Castagnoli and Simoni2003). As a type II generalist, N. californicus can adapt to fluctuate in prey populations and temperatures, providing stable pest suppression over long periods of time (Castagnoli and Simoni, Reference Castagnoli and Simoni2003; Escudero and Ferragut, Reference Escudero and Ferragut2005; Greco et al., Reference Greco, Liljesthröm, Gugole Ottaviano, Cluigt, Cingolani, Zembo and Sánchez2011). That said, use of this predator alone cannot keep the population of target pests below economic injury levels (Alzoubi and Cobanoglu, Reference Alzoubi and Cobanoglu2007). Accordingly, combined use of this predator with other natural enemies could enhance the efficiency of control strategies (Cock et al., Reference Cock, van Lenteren, Brodeur, Barratt, Bigler, Bolckmans, Cônsoli, Haas, Mason and Parra2010).
The fungal species Beauveria bassiana (Bals.) Vuill. (Hyp.: Cordycipitaceae) has a wide host range, and several strains have been used as commercial mycoinsecticides (de Faria and Wraight, Reference de Faria and Wraight2007; Zimmermann, Reference Zimmermann2007), and commercial formulations of B. bassiana are used to suppress outbreaks of a wide range of arthropod pests (Wraight et al., Reference Wraight, Carruthers, Jaronski, Bradley, Garza and Galaini-Wraight2000; de Faria and Wraight, Reference de Faria and Wraight2001; Shipp et al., Reference Shipp, Zhang, Hunt and Ferguson2003; Kaoud, Reference Kaoud2010; Steenberg and Kilpinen, Reference Steenberg and Kilpinen2014).
As Integrated Pest Management (IPM) aims to reduce pest populations to tolerable levels (Croft, Reference Croft1990), the possible impacts of sublethal concentrations of chemicals and entomopathogens are poorly understood (Pozzebon and Duso, Reference Pozzebon and Duso2010; Seiedy et al., Reference Seiedy, Saboori and Allahyari2012; Shipp et al., Reference Shipp, Kapongo, Park and Kevan2012). Sublethal effects are determined as physiological and/or behavioural effects on individuals that survive after exposure to a toxic compound at sublethal concentrations/doses (Desneux et al., Reference Desneux, Decourtye and Delpuech2007). Sublethal effects of fungal infection may have significant implications for the population dynamics of the hosts, which finally contributes to the pest status of the target organisms (Arthurs and Thomas, Reference Arthurs and Thomas2000; Blanford and Thomas, Reference Blanford and Thomas2001). Furthermore, demographic toxicological analysis is generally considered to be the best way for measuring the total effects of insecticides on non-target insects (Biondi et al., Reference Biondi, Desneux, Siscaro and Zappalà2012), and life tables have been used to help determine the sublethal effects at the population level (Stark and Banks, Reference Stark and Banks2003; Biondi et al., Reference Biondi, Zappalà, Stark and Desneux2013; Sedaratian et al., Reference Sedaratian, Fathipour, Talaei-Hassanloui and Jurat-Fuentes2013; Nawaz et al., Reference Nawaz, Cai, Jing, Zhou, Mabubu and Hua2017).
The lack of information on the sensitivity of predatory mites to B. bassiana has limited their usage in controlling spider mites (Ullah and Lim, Reference Ullah and Lim2017a, Reference Ullah and Lim2017b). The toxicity effects of various species of fungi (e.g. B. bassiana [strains Bb-032, Bb-053, Bb-057, Bb-086 and ICMP 8701] and Paecilomyces fumosoroseus (Wize)), on life table parameters of phytoseiid mites such as Phytoseiulus persimilis Athis-Henriot, N. californicus, Amblydromalus limonicus Garman & McGregor and Amblyseius largoensis (Muma) (Acari: Phytoseiidae) (Acari: Phytoseiidae) were previously well-documented by several researchers (e.g. Vergel et al., Reference Vergel, Bustos, Rodríguez and Cantor2011; Liu et al., Reference Liu, Zhang, Beggs and Wei2019; de Freitas et al., Reference de Freitas, de Araújo Lira, Jumbo, dos Santos, Rêgo and Teodoro2021). However, no evidence is available regarding the sublethal (LC10, LC20 and LC30) effects of the native strain (TV) of B. bassiana on the biological attributes of N. californicus. The effects of sublethal concentrations of this fungus on the different biological stages were determined in the present study. In this context, we assessed the sublethal concentrations (LC10, LC20 and LC30) of B. bassiana on the life table parameters of N. californicus, in order to provide basic information for sustainable management of T. urticae in different cropping systems.
Materials and methods
Biological materials
Stock colonies of N. californicus were provided by Koppert Biological Systems and reared in the laboratory on Cucumis sativus L. (Cucurbitaceae; cv. Veolla F1). Colonies were fed with immature stages of T. urticae. The TSSM were taken from infested plants (Tehran; Iran) and released on the cucumber plants under conditions of 25 ± 2°C, 60 ± 5% RH and a photoperiod of 16 h light with 8 h darkness. The arenas used for N. californicus rearing were made according to McMurtry and Scriven (Reference McMurtry and Scriven1965). They were stored in a controlled climate room under conditions of 25 ± 2°C and 70 ± 5% RH, and a photoperiod of 16 h light and 8 h darkness.
Fungus source
A endemic strain (TV) of B. bassiana (soil origin) was cultured on Sabouraud Dexterose Agar (SDA) and maintained in absolute darkness at 25 ± 2°C and 70 ± 5% RH. This strain was obtained from the College of Agriculture and Natural Resource, University of Tehran. Cultures were discarded after sporulation and conidia were obtained (Goettel and Inglis, Reference Goettel and Inglis1997).
Bioassay
In bioassays, the confidence limits of mortality were of 10–90%, and in control treatment adults were treated with distilled water + 0.02% Tween-80. Additionally, 20 adult mites (24 h-old 10 males and 10 females) were located on the treated leaf discs (4 cm diameter) for every concentration by using a soft brush (0.00). Then, the leaf discs of cucumber were sprayed (Posada et al., Reference Posada, Aime, Peterson, Rehner and Vega2007) with B. bassiana and dried for 30 min at room conditions. After that, the Petri dish lids were sealed and transferred to the controlled climate room under conditions of 25 ± 2°C, 70 ± 5% RH and 16 h light and 8 h darkness. According to the results obtained, the sublethal concentrations were prepared with following average levels of fungus: LC10 = 6.76 × 102 ml−1, LC20 = 8.74 × 103 ml−1 and LC30 = 55.38 × 103 ml−1 conidia of entomopathogenic fungus.
Life-table assay
To assess the sublethal effects of B. bassiana on the dynamic performance of N. californicus, 60 adult females were transferred to fresh cucumber leaf discs, each of which was placed on a sponge in a Petri dish. The leaf discs were treated with different dosages of B. bassiana (i.e. distilled water, LC10, LC20 and LC30). After 24 h, surviving female adults were relocated separately on to untreated plants and left for one day to oviposit. After 24 h, one egg was randomly kept in each Petri dish and the other ones were eliminated. In the next step, all retained eggs were daily checked and their developmental time and survivorship, as well as female fecundity, were noted until death of the last mite. During experiments, the leaf discs were replaced by new ones as required.
Data analysis
The mortality data and the LC10, LC20 and LC30 values and their 95% fiducial limits were computed by probit analysis (IBM SPSS, Version, 19.0). Differences among population parameters were contrasted using the paired bootstrap method based on the confidence interval of difference (Akca et al., Reference Akca, Ayvaz, Yazici, Smith and Chi2015). Also, differences between the duration of different life stages were investigated with the Tukey–Kramer procedure (SAS Institute, 2002). The raw data of life table parameters were analysed according to the theory of age-stage, two-sex life table (Chi and Liu, Reference Chi and Liu1985; Chi, Reference Chi1988) by using the computer program of TWOSEX MSChart (Chi, Reference Chi2021). Population parameters and their standard errors were calculated using the bootstrap method with 100,000 replications (Huang and Chi, Reference Huang and Chi2013; Akköprü et al., Reference Akköprü, Atlıhan, Okut and Chi2015).
Results
Survival and fecundity
lx, mx and fxj of N. californicus at sublethal concentrations of B. bassiana are shown in fig. 1. The total lifespan for the predatory mites was 40, 41, 38 and 38 days at control, LC10, LC20 and LC30, respectively (fig. 1). Although the maximum value of mx was 1.28 eggs per female per day for untreated mites, which was on day 19 (fig. 1), the maximum value of mx for mites treated with LC10 treatment was 1.26 eggs per female per day that was observed on day 21 of the life period. However, maximum values of mx for LC20 and LC30 treatments were approximately 1.11 and 1.01 eggs per female per day, respectively, which was on days 18 and 19 (fig. 1).
Figure 1. Age-specific survivorship (lx), age-specific fecundity (mx) and age-stage-specific fecundity (fxj) of Neoseiulus californicus at different concentrations of Beauveria bassiana.
Estimation of sxj represents the probability that an egg will survive to age x and develop to stage j (fig. 2). Obvious overlap in these curves is attributable to the different developmental rates among the individuals. Both female and male individuals of N. californicus survived shorter periods of time with the LC20 and LC30 treatments than the control (fig. 2).
Figure 2. The age-stage-specific survival rate (sxj) of Neoseiulus californicus for control and different concentrations of Beauveria bassiana.
Impact on various life stages and female fecundity
The impact of sublethal concentrations of B. bassiana on the duration of separate life stages of N. californicus is shown in table 1. None of the larvae, protonymphs or deutonymphs of male and female individuals were affected by diverse concentrations of B. bassiana (male: larva, F = 4.86, df = 3, 68, P = 0.0021; protonymph, F = 1.14, df = 3, 68, P = 0.34; deutonymph, F = 2.16, df = 3, 68, P = 0.09; female: larva, F = 0.13, df = 3, 156, P = 0.02; protonymph, F = 0.34, df = 3, 156, P = 0.79; deutonymph, F = 0.17, df = 3, 156, P = 0.91). Egg development period of males and females was not affected by sublethal concentrations (male: df = 3, 68, F = 5.07, P = 0.0030; female: df = 3, 156, F = 5.26, P = 0.0018). When the individuals treated with higher concentrations of B. bassiana, longevity of male (LC20 and LC30) and female (LC20 and LC30) individuals (male: df = 3, 68, F = 22.7, P < 0.0001; female: df = 3, 156, F = 19.37, P < 0.0001), as well as total life span (male: df = 3, 68, F = 45.17, P < 0.0001; female: df = 3, 156, F = 30.29, P < 0.0001) were significantly different from the controls. The longest (31 days) and shortest (25 days) for female longevity as well as total life span were observed in control and LC30 treatments, respectively (table 1). The APOP (adult pre-ovipositional period from adult emergence to the first oviposition) and TPOP (total pre-ovipositional period from egg to the first oviposition) periods were not significantly affected by our experimental treatments (table 2), and TPOP ranged from 8.25 to 8.72 days (F = 2.23; df = 3, 156; P = 0.08). The shortest time for oviposition was 19 days for the mites treated at the highest concentration (F = 8.91; df = 3, 156; P < 0.0001). Similarly, fecundity of females was lowest at LC30 (24 eggs); whereas that of the control treatment was markedly higher (37 eggs) (F = 52.37; df = 3, 156; P < 0.0001).
Table 1. Sublethal effects of Beauveria bassiana on developmental times, adult longevity and total life span (mean ± SE) of Neoseiulus californicus
Means within a row followed by the same letter are not significantly different (Tukey–Kramer, P < 0.05).
Table 2. Mean (±SE) reproductive periods and total fecundity of offspring from females of Neoseiulus californicus at different concentrations of Beauveria bassiana
Means followed by the same letter in the same row are not significantly different (Tukey–Kramer, P < 0.05).
1 APOP = adult pre-ovipositional period (from adult emergence to the first oviposition).
2 TPOP = total pre-ovipositional period (from egg to the first oviposition).
Population parameters
Table 3 displays the population growth characteristics of offspring from the females of N. californicus treated with sublethal concentrations of B. bassiana. The data reveal that the gross reproductive rate (GRR) varied from 17.76 to 24.96 offspring/individual. The lowest values of GRR (17.76 offspring/individual) as well as R 0 (13.87 offspring/individual) were recorded for the mites exposed to the highest concentration (LC30) (table 3). The values of the intrinsic rate of increase (r) and finite rate of increase (λ) at the highest concentration (LC30) were not significantly lower than the lower concentration (LC10) and untreated mites. The mean generation times (T) calculated were 17.86 and 17.38 days for control and LC10 treatments, which were significantly different from the LC20 and LC30 values (16.57 and 16.26 days, respectively) (table 3).
Table 3. The effect of different treatments of Beauveria bassiana on the population parameters (mean ± SE) of Neoseiulus californicus
Means followed by different letters in the same column are significantly different (paired bootstrap test, P < 0.05).
Discussion
Biological control of insect and mite pests is a basic part of agricultural IPM systems and will reduce our current dependency on chemical pesticides for pest management (Hoy, Reference Hoy2012). We assessed the population dynamics of N. californicus, a predatory mite of T. urticae when treated with sublethal concentrations of the entomopathogenic fungus B. bassiana as this had not been studied previously.
When using phytoseiid predators, it is necessary to evaluate their compatibility with other natural enemies such as pathogens and predators. Most research has indicated that entomopathogenic fungi exhibit high pathogenicity for insect pests and phytophagous mites but no or lower levels of infection in predatory mites (Shi and Feng, Reference Shi and Feng2004; Wekesa et al., Reference Wekesa, Moraes, Knapp and Delalibera2007; Seyed-Talebi et al., Reference Seyed-Talebi, Kheradmand, Talaei-Hassanloui and Talebi-Jahromi2012, Reference Seyed-Talebi, Kheradmand, Talaei-Hassanloui and Talebi-Jahromi2014; Wu et al., Reference Wu, Gao, Zhang, Wang, Xu and Lei2014).
Our findings illustrated that, in terms of changes in development rates of N. californicus individuals, the survival curves at different treatments showed considerable overlap. In this experiment, the predatory mites treated with B. bassiana showed reduced age-specific survival rate (lx) and fecundity (mx). This was in line with the findings of Rashki et al. (Reference Rashki, Kharazi-Pakdel, Allahyari and Van Alphen2009) who studied the impact of B. bassiana (2 × 108 conidia ml−1) on the biological parameters in both sexes of Aphidius matricariae Haliday (Hym.: Braconidae), a parasitoid of the green peach aphid, Myzus persicae (Sulzer) (Hem.: Aphididae).
In our results, fungal treatment had no effect on immature developmental stages (larva, protonymph and deutonymph) which is in accordance with the observations of Zhou et al. (Reference Zhou, Ali and Huang2010) who estimated the effect of sublethal concentrations (1 × 104 and 1 × 108 conidia ml−1) of Isaria fumosorosea (Wize) Brown and Smith on the developmental time of the immature stages of a ladybird beetle Axinoscymnus cardilobus (Ren and Pang) (Col.: Coccinellidae). In contrast, Shang et al. (Reference Shang, Chen and Bai2018) showed that exposure to sublethal concentrations (1 × 108 conidia ml−1) of Acremonium hansfordii (Hyp.: Moniliales) significantly reduced the developmental period of Neoseiulus barkeri (Hughes). Such variations are probably attributable to differing plant nutritional quality of the host plants, and/or morphological or allelochemical features, fungal strain and, in particular, the target experimental species itself (Agrawal, Reference Agrawal2000; Balkema-Boomstra et al., Reference Balkema-Boomstra, Zijlstra, Verstappen, Inggamer, Mercke, Jongsma and Bouwmeester2003; Pietrosiuk et al., Reference Pietrosiuk, Furmanowa, Kropczyńska, Kawka and Wiedenfeld2003).
In our study, longevity and total lifespan of adult females treated with different LCs affected were reduced when compared with those in the control group and for both sexes a declining trend was induced by increasing the fungal concentration. Similar to our findings, Ullah et al. (Reference Ullah, Altaf, Afzal, Arshad, Mehmood, Riaz, Majeed, Ali and Abdullah2019) demonstrated that the entomopathogenic fungus I. fumosorosea strain IF-1712011 × 108 caused a significant reduction in adult longevity of a predatory bug, Rhynocoris marginatus (Fabricius) (Hem.: Reduviidae).
In our work, B. bassiana treatments failed to influence pre and total-ovipositional periods of N. californicus, but decreased the ovipositional period and total fecundity in a concentration-dependent manner, demonstrating that the potential of treated mites for population recovery would be slow. This reduction could be related to: (i) fungal colonisation of tissues such as fat body (source of vitellogeins) and ovaries (Blay and Yuval, Reference Blay and Yuval1999) and (ii) fungal toxin production disrupting cellular and humeral immune reactions (Quesada-Moraga et al., Reference Quesada-Moraga, Santos-Quirós, Valverde-García and Santiago-Alvarez2004). These results are in agreement with another study that focused on the sublethal effect (LC30) of B. bassiana ICMP 8701 on the tomato/potato psyllid (TPP), Bactericera cockerelli (Šulc) (Hem.: Triozidae) (fecundity range = 200–334 offspring/individual) (Liu et al., Reference Liu, Zhang, Beggs, Paderes, Zou and Wei2020). In contrast, Ullah and Lim (Reference Ullah and Lim2017a) and Shang et al. (Reference Shang, Chen and Bai2018) demonstrated that B. bassiana and A. hansfordii had no significant effects on the oviposition period of P. persimilis and N. barkeri, respectively.
As noted earlier for duration of developmental stages, this may again be related to predator species, or/and EPF strains. Demographical assessments have been considered as useful for investigating the dynamics of insect populations in an ecotoxicological framework, and life tables remain a useful ecological tool for evaluating the effectiveness of pest control through natural enemies and host plant resistance (Gao and Yang, Reference Gao and Yang2015).
In the current study, it was found that population parameters such as the net reproductive rate (R 0), gross reproductive rate (GRR) and mean generation time (T) in the treated (LC20 and LC30) individuals of N. californicus attacking fungal-infected prey were significantly lower than control ones. Certainly, the r value is the most significant parameter for monitoring the trend of population growth (Carey, Reference Carey1993; Moscardini et al., Reference Moscardini, da Costa Gontijo, Carvalho, de Oliveira, Maia and Silva2013). The current project showed that exposure of N. californicus to B. bassiana had no obvious effects on the intrinsic and finite rates of increase. Estimate r and λ considering the survival and reproduction data for all females, referred to as true calculation (Ganjisaffar et al., Reference Ganjisaffar, Fathipour and Kamali2011). Our findings are in agreement with B. bassiana treatment (3 × 105 conidia ml−1) on the predatory bug, Andrallus spinidens Fabricius (Hem.: Pentatomidae) where r and λ values were not significantly affected (Gholamzadeh-Chitgar et al., Reference Gholamzadeh-Chitgar, Hajizadeh, Ghadamyari, Karimi-Malati and Hoda2017). Also, population parameters of the whitefly predator, Serangium japonicum Chapin (Col.: Coccinellidae) were not affected by Verticillium lecanii (Z.) at concentrations of 1 × 104, 1 × 105, 1 × 106, 1 × 107 and 1 × 108 conidia ml−1 (Fatiha et al., Reference Fatiha, Huang, Ren and Ali2008). To further confirm our data, Xu et al. (Reference Xu, Ali, Huang, Zhou, Afzal and Bashir2009) showed no significant effects of V. lecanii (1 × 103–107 conidia ml−1) on the life table parameters of Axinoscymnus cardilobus Pang and Ren (Col.: Coccinellidae).
Greenhouse and field studies of the concurrent usage of EPF with some species of phytoseiid mites conclude that the use of predatory mites is compatible with treatments with EPF for plant pest species (Vergel et al., Reference Vergel, Bustos, Rodríguez and Cantor2011; Midthassel et al., Reference Midthassel, Leather, Wright and Baxter2016; Wu et al., Reference Wu, Gao, Smagghe, Xu and Lei2016; Saito and Brownbridge, Reference Saito and Brownbridge2018). However, combined usage may not always lead to excellent pest control when compared to using these agents on their own. Despite the negative effects of B. bassiana on some biological parameters (e.g. GRR, R 0, T, longevity, total life-span and fecundity) of N. californicus under certain conditions, more precise assessments are needed to assess the impact of this fungus on insect and mite pests and its compatibility with other natural enemies under greenhouse and field conditions. Such studies would help to identify and evaluate entomopathogenic fungi for developing biological pest control programmes.
