TY - JOUR
AU1 - Usman, Muhammad
AU2 - Gulzar, Sehrish
AU3 - Wakil, Waqas
AU4 - Wu, Shaohui
AU5 - Piñero, Jaime C
AU6 - Leskey, Tracy C
AU7 - Nixon, Laura J
AU8 - Oliveira-Hofman, Camila
AU9 - Toews, Michael D
AU1 - Shapiro-Ilan, David
AB - Abstract The objectives of this study were to quantify the virulence of four entomopathogenic fungal species to pupae of Rhagoletis pomonella (Walsh) (Diptera: Tephritidae) and to determine the potential to combine entomopathogenic fungi (EPFs) and entomopathogenic nematodes (EPNs) for biological control of this pest. The four species of EPFs included Beauveria bassiana (strain GHA), Metarhizium brunneum (strain F52), Isaria javanica (wf GA17), and Isaria fumosorosea (Apopka 97 strain). In laboratory assays, all fungi reduced adult emergence but there were no differences between fungal species. Isaria javanica and M. brunneum were examined further in a EPFs and EPNs bioassay that also included the EPNs Steinernema carpocapsae (ALL strain) and S. riobrave (355 strain). All nematodes and fungi were applied either alone or in combination (fungus + nematode). There were no differences between species within the same entomopathogen group (fungi and nematodes). However, the treatment with S. riobrave resulted in lower R. pomonella emergence than either fungal species. The combination of S. riobrave and I. javanica resulted in the lowest R. pomonella emergence (3%) at fourth-week interval, which was significantly lower than any of the single-agent applications, yet virulence of the other three combination treatments was not different from their respective nematode treatments applied alone. Additive interactions were detected for all fungus–nematode combinations. This study suggests that application of entomopathogenic nematodes and fungi could be an effective option to suppress R. pomonella populations. entomopathogenic nematodes, entomopathogenic fungus, additive interaction, Rhagoletis pomonella pupa Fruit flies (Diptera: Tephritidae) are considered to be the most damaging pests of fruit crops worldwide as they deposit eggs under fruit pulp and cause blemishes which ultimately reduce the fruit quality (Weems et al. 2014, Qin et al. 2015, Suckling et al. 2016, Villalobos et al. 2017). Among temperate fruit fly species, the apple maggot, Rhagoletis pomonella (Walsh) (Diptera: Tephritidae), is a direct pest of apple in eastern North America (Dean and Chapman 1973). Rhagoletis pomonella overwinters in the soil (2–5 cm depth) as pupae, and adult emergence normally begins in late June after the accumulation of 482 growing degree days (based 10°C) is reached (Howitt 1993). Upon emergence females reach sexual maturity within 7–10 d. The fertile female lays eggs under the fruit skin and hatching occurs within 5 d. Neonates make tunnels throughout the flesh and feed there until mature at which time they make an exit hole to pupate in the soil (Reissig and Agnello 1996). Most commercial apple markets have zero tolerance for R. pomonella inside the fruit at the time of harvest, but damage is only detected when it exceeds 5% (Reissig and Agnello 1996, CABI 2019). Management of R. pomonella is reliant on applications of broad-spectrum insecticides to the entire orchard. To reduce the risk of insecticide exposure over the large area, an alternative attract and kill strategy may be adopted using a red sphere impregnated with pheromone and covered with Tangle-trap (Prokopy 1968, Reissig et al. 1982, Prokopy et al. 1990, Prokopy and Mason 1996, Bostanian et al. 1999) or baited attracticidal spheres (Green and Wright 2009, Wright et al. 2012, Morrison et al. 2016). A reduction in insecticide reliance to minimize potential negative impact on humans, nontarget organisms, and the environment is desirable. Further, environmental and regulatory concerns, as well as increasing insecticide resistance suggest that alternative nonchemical pest management strategies are needed to manage this pest (Magaña et al. 2007). Microbial control agents may serve as biologically based alternatives to synthetic pesticides (Lacey and Shapiro-Ilan 2008, Lacey et al. 2015). Within the microbial groups, entomopathogenic fungi (EPFs) have an advantage relative to most other entomopathogen groups because the fungi can infect insect hosts via penetration of the insect integument, and thus ingestion is not necessary (Goettel et al. 2005, Lacey and Shapiro-Ilan 2008, Ali et al. 2010). Soil-dwelling stages including late-stage larvae and pupae of fruit flies are an attractive target for Metarhizium spp. (Ekesi et al. 2011, Yousef et al. 2017), because the soil, as the natural habitat for EPFs, provides protection from harmful environmental conditions such as UV radiation (Ekesi et al. 2007; Zimmermann 2007; Vega et al. 2009; Garrido-Jurado et al. 2011a, b, c). While the late-stage larvae and the pupal stages of fruit flies provide an opportunity for targeting with EPF, relatively few previous studies against fruit fly pupae have been conducted (Ekesi et al. 2002, Quesada-Moraga et al. 2006, Goble et al. 2011, Beris et al. 2013, Wilson et al. 2017). There is no published literature on the efficacy of EPFs against R. pomonella. Therefore, we investigated the virulence of EPFs to R. pomonella in this study. We focused on the virulence of three commercial EPF species (B. bassiana, M. brunneum, and I. fumosorosea) to facilitate relevance to the grower, but also included a new strain of I. javanica that showed high levels of virulence to several other insects pests (unpublished data). Another entomopathogen group is entomopathogenic nematodes (EPNs). Heterorhabditis and Steinernema are two EPN genera that are effective biological control agents for use against various soil-dwelling insect pests in different cropping systems (Shapiro-Ilan et al. 2018). EPNs are safe to humans and the environment and can be applied to soil in aqueous suspension using standard agricultural equipment (irrigation systems and sprayers), or by applying nematode-infected insect host cadavers (the cadaver approach has been adopted on a small scale such as in greenhouse or bee-keeping systems) (Shapiro-Ilan et al. 2012). In a previous study, we found S. riobrave and S. carpocapsae to be highly virulent to R. pomonella pupae and capable of significantly reducing adult R. pomonella emergence (Usman et al. 2020). However, none of the EPN treatments achieved complete suppression of the R. pomonella pupal population, and thus other approaches merit investigation. Combining entomopathogenic fungi and nematodes can produce different types of interactions, including additive, synergistic, or antagonistic: additive refers to the impact on the target organism as being the sum of the impact of each one, typically because the agents act independently; synergistic suggests an interaction where the outcome is greater than the sum of the parts such as one agent causing the target organism to become more susceptible to the other; and antagonistic is an interaction where the agents are in competition or interfere with each other (Ansari et al. 2004, 2008; Koppenhöfer and Grewal 2005; Tarasco et al. 2011). EPN and EPF may be able to mutually weaken the host so that the sum of their impacts is synergistic. Alternatively toxins or antibiotic properties of each organism (nematode or fungus) may interfere with the other and thus interactions could be antagonistic. When investigating control of different insect pests, a number of studies demonstrate additivity when combining EPNs and EPF (Barbercheck and Kaya 1990, Shapiro-Ilan et al. 2004, Acevedo et al. 2007, Ansari et al. 2008, Wu et al. 2014), whereas others observed antagonism (Shapiro-Ilan et al. 2004, Ansari et al. 2005, Wu et al. 2014) or synergism (Ansari et al. 2008, Correa-Cuadros et al. 2016, Wakil et al. 2017). Due to zero tolerance of maggots inside the fruit in high-value markets, it may be necessary to deploy both entomopathogen agents collectively to achieve complete suppression of R. pomonella. The objective of this study was to assess the efficacy of different EPF species against pupae of R. pomonella and to investigate the combination of EPF with EPNs. Materials and Methods EPFs Isolates and Their Culturing Four entomopathogenic fungal species including Beauveria bassiana (Balsamo) Vuillemin (GHA strain) (Hypocreales: Cordycipitaceae), Metarhizium brunneum Petch (F52 strain) (Hypocreales: Clavicipitaceae), Isaria javanica (Friedrichs & Bally) Samson & Hywel-Jones (wf GA17 strain) (Hypocreales: Cordycipitaceae), and Isaria fumosorosea Wize (Apopka strain 97) (Hypocreales: Cordycipitaceae) were cultured for use in experiments. Each species was individually reared on PDA plates (100 mm) wrapped with parafilm and incubated at 25°C with a 14:10 (L:D) h photoperiod. The fungi were harvested in 7–10 d with a sterile scalpel and conidia were suspended in a 50-ml conical tube with 30 ml of sterile 0.05% Silwet L-77 solution. The tubes were vortexed for 5 min by the addition of eight glass beads for agitation, and conidial concentration was then quantified with a hemocytometer. Viability of conidia was assessed by spread-plating 0.1 ml of conidial suspension (1 × 106 conidia per ml) on two small SDAY plates (Inglis et al. 2012) and incubated at 25°C with a 14:10 (L:D) h photoperiod. Percentage germination was examined after 16–18 h by placing a sterile cover slip on plates and assessing 200 spores for germination; conidia with germinated tubes at least twice long as propagules were considered viable (Inglis et al. 2012). A total of four counts were made from two plates per fungal species, and the mean germination rate was used to adjust the desirable concentrations of viable conidia used in experiments. Entomopathogenic Nematodes The EPNs tested were Steinernema carpocapsae (Weiser) (ALL strain) (Rhabditida: Steinernematidae) and S. riobrave Cabanillas, Poinar and Raulston (355 strain). Both were cultured in vivo on the last instar of Galleria mellonella L. and collected using the White trap method (Shapiro-Ilan et al. 2016). The EPNs were stored in aqueous suspensions in 250-ml tissue culture flasks at 14°C. Nematodes were stored for less than 2 wk before using in experiments. The nematodes used in this study were from the United States Department of Agriculture (USDA) International Culture Collection held in Byron, GA. Rhagoletis pomonella Colony Pupae of R. pomonella were supplied by the USDA-ARS, Appalachian Fruit Research Station (Kearneysville, WV). Briefly, the rearing methods involved rearing adults in a colony room at 16:8 (L:D) h and 25°C. Females were provided organic ‘Red Delicious’ apples as an oviposition substrate. Apples were exposed to adults for 3–4 d, then removed and suspended over trays of moistened sand for 4 wk at 16:8 (L:D) h, 24°C, and 45% RH. Pupae were removed from sand using water and agitation to float them to the surface of trays. Removed pupae were placed in small plastic cups with tissue to absorb excess moisture and shipped overnight within 2 d of removal from sand to be used in experiments. EPFs Bioassay The objective of this experiment was to compare the acute virulence of entomopathogenic fungus species. The experiment was conducted using methods previously reported by Shapiro-Ilan et al. (2003). The experiment evaluated B. bassiana (GHA strain), M. brunneum (F52 strain), I. javanica (wf GA17 strain), and I. fumosorosea, and was organized in a completely randomized design (CRD). The experimental arena consisted of a lidded 30-ml plastic cup filled with 15 g of autoclaved loamy sand soil (84% sand, 10% silt, 6% clay; 2.8% organic matter; pH 6.1) with 0% soil moisture content. One milliliter of each EPF species with 1 × 107 viable conidia per ml was added to each cup, and subsequently an additional 1.1 ml of distilled water was added to reach soil field capacity (14% soil moisture content). The conidia concentration was selected on the basis of a preliminary bioassay (using three different concentrations, i.e., 1 × 106, 1 × 107, and 1 × 108 viable conidia per ml) (unpublished data). After inoculation, the soil-filled cup was manually agitated to ensure equal distribution of conidia. After mixing, an individual pupa was buried inside the soil at 3 cm depth. The control group only received the distilled water (2.1 ml) without EPF. Thus, the experiment consisted of total four treatments (four fungal species) and one control group. Each treatment and the control had 10 cups per replicate and there were three replicates per treatment. Lidded cups were placed on trays and bagged with damp paper towel to help retain moisture and incubated at 25°C. We observed the cups daily for adult emergence until 30 d posttreatment and then the experiment was disposed of without observing the nonemerged pupae. Adults that emerged successfully were considered to have survived the fungal treatment. The experiment was repeated in time (thus two complete trials with a total of 60 pupae per treatment or control). EPFs and EPNs Bioassay The two nematode species, S. riobrave and S. carpocapsae, were selected for the EPFs and EPNs bioassay on their performance in a previous study (Usman et al. 2020). Isaria javanica and M. brunneum were selected based on the positive results of the EPFs bioassay described above. The experiment had a total of eight treatments, including M. brunneum alone; I. javanica alone; S. carpocapsae alone; S. riobrave alone; M. brunneum + S. carpocapsae; M. brunneum + S. riobrave; I. javanica + S. carpocapsae; I. javanica + S. riobrave, plus the control. Approximately 200 g of sterile loamy sand soil at 0% moisture (the same soil used in the cup bioassay) was placed in the plastic pots (10.16 cm diameter). For the treatment with EPF-only, approximately 22 ml of distilled water containing 6 ml of conidial suspension (1 × 107 viable conidia per ml) was added to each pot; the soil was then mixed manually for equal distribution of conidia. Then, 10 pupae were then buried at 3 cm depth and at an equal distance (1 cm) from each other in a circular manner. For application of EPN-only treatments, soil was premoistened with approximately 27 ml of distilled water and then buried 10 pupae at 3 cm depth, and finally 1 ml of suspension containing 1,377 IJs (resulting in approximately 27 IJs/cm2) was applied to the soil surface of each pot (distributing the nematodes evenly over the surface). For the combined application treatments, approximately 21 ml of distilled water was added first, followed by 6 ml of conidial suspension (1 × 107 viable conidia per ml); the pot was then shaken manually for equal distribution of conidia. Ten pupae were buried inside the soil (3 cm deep) at an equal distance (1 cm) from each other in a circular manner, and then 1 ml of an EPN species was applied at 1,377 IJs/ml to the soil surface. The control group received 28 ml of distilled water without EPF or EPNs. A 100-mm Petri dish cover lined with a yellow sticky trap was placed on the top of each pot to monitor adult R. pomonella emergence. There were three replicate pots per treatment and control. Pots were put onto a plastic tray and bagged with a damp paper towel to help retain moisture and incubated at 25°C. Cumulative adult emergence from each pot was observed over a 4-wk period. Adult emergence was observed after 1 wk posttreatment and therefore treatment effects were analyzed after 2, 3, and 4 wk (final) postemergence. Adults that emerged were to have survived the treatments (emergence was used as a proxy to indicate survival). The experiment was repeated in time (two full trials). Statistical Analysis Treatment effects were analyzed with a one-way analysis of variance (ANOVA). If the global ANOVA suggested a significant treatment effect (P ≤ 0.05), then responses were further subjected to Tukey’s HSD test (SAS 2002). Data from repeated experiments (trials) were combined when the treatment * trial interaction was not significant but the trial effect was still included in the model. To meet equal variance assumptions, percentage data were arcsine-transformed prior to analysis (Southwood 1978, Steel and Torrie 1980, SAS 2002). Nontransformed means and treatment standard errors are presented in the Results section and associated figures. The fungus–nematode interactions (synergistic, additive, or antagonistic) were determined using observed versus expected values of insect emergence (Shapiro-Ilan et al. 2004). Expected mortality was calculated using formula PE = P0 + (1 − P0) (P1) + (1 − P0) (1 − P1) (P2), where PE is the expected mortality of the combination, P0 is the control mortality, P1 is the mortality from one pathogen treatment applied alone, and P2 is the mortality from the other pathogen applied alone. A chi-square test was applied to the observed and expected results: χ 2 = (L0 − LE)2/LE + (D0 − DE)2/DE, where L0 is the number of living pupae observed, LE the number of living pupae expected, D0 the number of dead pupae observed, and DE the number of dead pupae expected. Interactions were additive if χ 2 < 3.84, antagonistic if χ 2 > 3.84 and PC < PE, and synergistic if χ 2 > 3.84 and PC > PE, where PC is the observed mortality from the combination and PE is the expected mortality from the combination. Results EPFs Bioassay The interaction between treatment and trial effects was not significant (P = 0.13), and so data from both trials were combined. All the fungal isolates reduced adult R. pomonella emergence compared with the control (F = 19.53; df = 4,20; P < 0.0001), yet no significant differences were observed among the species, with adult emergence ranging from 50.0% in I. javanica to 72% in I. fumosorosea (Fig. 1). Numerically, the lowest adult emergence was observed in the I. javanica treatment followed by M. brunneum (Fig. 1), and thus these two species were chosen for further study in the EPFs and EPNs bioassay. Fig. 1. Open in new tabDownload slide Mean (±SEM) percent emergence of Rhagoletis pomonella adults at 30 d posttreatment after exposure of pupae to Mb = Metarhizium brunneum, Ij = Isaria javanica, Bb = Beauveria bassiana, If = Isaria fumosorosea and a control in a laboratory bioassay (n = 30 per treatment). Different letters above bars indicate statistical differences (P ≤ 0.05; Tukey’s HSD test). EPFs and EPNs Bioassay No adult emergence was observed among all the treatment after first week of treatment. After the second, third, and fourth week of treatment, the interactions between the treatment and trial effects were not significant (P = 0.99, 0.95, and P = 0.61, respectively), and therefore data from repeated trials were combined. Two weeks posttreatment, differences between the single-applied and combined application treatments were observed (F = 6.38; df = 8,36; P < 0.0001). Only the treatment combinations involving M. brunneum + S. riobrave and I. javanica + S. riobrave caused significant reductions in adult R. pomonella emergence relative to the control. None of the treatment combinations caused lower emergence than both of their respective single-applied treatments (Fig. 2). Fig. 2. Open in new tabDownload slide Mean (±SEM) percent cumulative emergence of Rhagoletis pomonella adults at 2, 3, and 4 wk after exposure of pupae to Mb = Metarhizium brunneum, Ij = Isaria javanica, Sc = Steinernema carpocapsae, Sr = Steinernema riobrave, Mb + Sc = M. brunneum + S. carpocapsae, Mb + Sr = M. brunneum + S. riobrave, Ij + Sc = I. javanica+ S. carpocapsae, Ij + Sr = I. javanica + S. riobrave in a laboratory bioassay (n = 30 per treatment). Different letters above bars indicate statistical differences (P ≤ 0.05; Tukey’s HSD test). Three weeks posttreatment, differences among the single and combined treatments were observed (F = 24.64; df = 8,36; P < 0.0001). All treatments reduced R. pomonella emergence relative to the control. The combination of I. javanica + S. riobrave was only the treatment to cause lower emergence than either agent when applied alone (Fig. 2). Four weeks posttreatment, differences between single and combined application treatments were observed (F = 25.7; df = 8,36; P < 0.0001). In the singly applied treatments, no differences were observed among species within the same entomopathogen group (fungi and nematodes) (Fig. 2). However, S. riobrave caused lower R. pomonella emergence than either of the fungal species (Fig. 2). The only combination treatment that caused lower R. pomonella emergence than both of its corresponding single treatments was S. riobrave + I. javanica, which induced only 3% adult emergence (Fig. 2). The combination of M. brunneum with S. riobrave or S. carpocapsae had lower emergence than M. brunneum alone but was not different from nematodes alone. The combination of S. carpocapsae and I. javanica was not different from either agent alone. The combination treatment S. riobrave + I. javanica also produced lower emergence than any of the single treatments but was not significantly separated from the other combination treatments (Fig. 2). Additive interactions were detected for all fungus–nematode combinations at all intervals (Table 1). Table 1. Observed versus expected emergence of Rhagoletis pomonella adults when pupae were treated with combinations of Metarhizium brunneum (Mb), Steinernema carpocapsae (Sc), Steinernema riobrave (Sr), or Isaria javanica (Ij) in a laboratory bioassay Treatments . Intervals (weeks) . Observed % not emergeda . Expected % not emerged . Chi-square . Interaction . Mb + Sc Second 68.75 64.45 0.28 Additive Mb + Sr Second 87.50 74.60 2.27 Additive Ij + Sc Second 68.75 75.39 0.58 Additive Ij + Sr Second 100 82.42 3.74 Additive Mb + Sc Third 86.27 83.85 0.06 Additive Mb + Sr Third 94.11 90.31 0.16 Additive Ij + Sc Third 88.23 88.46 0.0006 Additive Ij + Sr Third 100 93.07 0.15 Additive Mb + Sc Fourth 80.00 78.18 0.04 Additive Mb + Sr Fourth 89.09 85.81 0.12 Additive Ij + Sc Fourth 83.63 84.79 0.01 Additive Ij + Sr Fourth 96.36 96.36 0.43 Additive Treatments . Intervals (weeks) . Observed % not emergeda . Expected % not emerged . Chi-square . Interaction . Mb + Sc Second 68.75 64.45 0.28 Additive Mb + Sr Second 87.50 74.60 2.27 Additive Ij + Sc Second 68.75 75.39 0.58 Additive Ij + Sr Second 100 82.42 3.74 Additive Mb + Sc Third 86.27 83.85 0.06 Additive Mb + Sr Third 94.11 90.31 0.16 Additive Ij + Sc Third 88.23 88.46 0.0006 Additive Ij + Sr Third 100 93.07 0.15 Additive Mb + Sc Fourth 80.00 78.18 0.04 Additive Mb + Sr Fourth 89.09 85.81 0.12 Additive Ij + Sc Fourth 83.63 84.79 0.01 Additive Ij + Sr Fourth 96.36 96.36 0.43 Additive aPercentage of R. pomonella that did not emerge from soil as adults relative to the total number of pupae that were originally added to the soil (n = 60). Open in new tab Table 1. Observed versus expected emergence of Rhagoletis pomonella adults when pupae were treated with combinations of Metarhizium brunneum (Mb), Steinernema carpocapsae (Sc), Steinernema riobrave (Sr), or Isaria javanica (Ij) in a laboratory bioassay Treatments . Intervals (weeks) . Observed % not emergeda . Expected % not emerged . Chi-square . Interaction . Mb + Sc Second 68.75 64.45 0.28 Additive Mb + Sr Second 87.50 74.60 2.27 Additive Ij + Sc Second 68.75 75.39 0.58 Additive Ij + Sr Second 100 82.42 3.74 Additive Mb + Sc Third 86.27 83.85 0.06 Additive Mb + Sr Third 94.11 90.31 0.16 Additive Ij + Sc Third 88.23 88.46 0.0006 Additive Ij + Sr Third 100 93.07 0.15 Additive Mb + Sc Fourth 80.00 78.18 0.04 Additive Mb + Sr Fourth 89.09 85.81 0.12 Additive Ij + Sc Fourth 83.63 84.79 0.01 Additive Ij + Sr Fourth 96.36 96.36 0.43 Additive Treatments . Intervals (weeks) . Observed % not emergeda . Expected % not emerged . Chi-square . Interaction . Mb + Sc Second 68.75 64.45 0.28 Additive Mb + Sr Second 87.50 74.60 2.27 Additive Ij + Sc Second 68.75 75.39 0.58 Additive Ij + Sr Second 100 82.42 3.74 Additive Mb + Sc Third 86.27 83.85 0.06 Additive Mb + Sr Third 94.11 90.31 0.16 Additive Ij + Sc Third 88.23 88.46 0.0006 Additive Ij + Sr Third 100 93.07 0.15 Additive Mb + Sc Fourth 80.00 78.18 0.04 Additive Mb + Sr Fourth 89.09 85.81 0.12 Additive Ij + Sc Fourth 83.63 84.79 0.01 Additive Ij + Sr Fourth 96.36 96.36 0.43 Additive aPercentage of R. pomonella that did not emerge from soil as adults relative to the total number of pupae that were originally added to the soil (n = 60). Open in new tab Discussion In the EPFs bioassay, all the fungal species significantly reduced adult R. pomonella emergence compared with the control group. However, we did not detect any significant differences in virulence among the fungal species themselves. Additional research is needed to tease out potential differences in virulence and efficacy among other EPF strains and species for control of R. pomonella. The low to moderate level of virulence we observed among EPF to R. pomonella pupae is consistent with some other studies investigating susceptibility of fruit fly pupae. For example, Beris et al. (2013) reported low susceptibility of the Mediterranean fruit fly, Ceratitis capitata (Wiedemann) (Diptera: Tephritidae) pupae when exposed to different fungal species at the same concentration that we used. The authors concluded the age of the pupae (4–5 d) might have been a reason for low susceptibility. Indeed, adult emergence was increased threefold when pupal age increased from 12 h to 4 d against C. capitata, C. var. rosa jasciventris (Bezzi), and C. cosyra (Walker) (Ekesi et al. 2002), presumably due to hardening of the cuticle that then restricted the sites of fungal penetration. Thus, there may be a relatively narrow window that can be targeted for using EPF against R. pomonella pupae. In our study, pupal age (3-d-old) may also have been a reason for relatively low fungal virulence R. pomonella. The low virulence of EPF renders an opportunity for EPNs to take effect in the combined applications. In our study, S. riobrave was more virulent to R. pomonella pupae than either fungal species. It would be interesting to explore further (with additional species and experimental conditions) if EPNs tend to be more virulent to R. pomonella than EPF. However, it is hard to compare across pathogen groups because one can argue that the measured virulence simply relies on dose (and thus relative virulence can be manipulated). In this study, we standardized the EPN and EPF applications rates based on preliminary data that suggested these rates would produce distinguishing treatment effects (Mbata and Shapiro-Ilan 2005). Alternatively, one could have standardized the rates based on recommended field rates, or on cost of product per unit area (at the recommended rates) (Lacey 1997). We observed additive interactions when EPNs S. riobrave and S. carpocapsae were combined with M. brunneum and I. javanica. Similar to our study, an additive interaction was observed between Heterorhabditis megidis and B. bassiana, and also between H. bacteriophora and M. anisopliae and B. bassiana when applied against the grub Cyclocephala lurida Bland (Coleoptera: Scarabaeidae) (Wu et al. 2014). This is the first study to evaluate combined application of EPF and EPN against R. pomonella. Contrary to our study, antagonistic interactions were observed in several combined applications of EPNs and EPFs against different insect pests (Shapiro-Ilan et al. 2004, Ansari et al. 2005). In some prior studies, synergy has been reported between EPNs and EPFs (Ansari et al. 2006, 2008, 2009). The synergy may arise as one pathogen creates physiological conditions in the host (such host attraction or reducing defenses) that allows the other pathogen to become more successful (Samuels et al. 1988, Gaugler et al. 1994, Ansari et al. 2008). The nature of interaction (synergy, additivity, antagonism) can depend on the pathogen species or strain, and host species as well as timing and rate of applications (simultaneous or staggered) (Koppenhöfer and Grewal 2005, Ansari et al. 2006, Shapiro-Ilan et al. 2018). Therefore, it is conceivable that certain EPN–EPF combinations that we studied or otherwise could be manipulated to achieve synergy. The advantages of applying combined versus single pathogen applications for R. pomonella have yet to be elucidated. Although the combined applications generally produced higher levels of suppression than the EPF-only treatments, only one combination (S. riobrave + I. javanica) was more virulent than the EPN treatments applied alone. Thus, given that interactions were additive (rather than synergistic) it is possible that higher rates of EPN application would be equally efficacious as combined applications. The cost-benefits of each approach would likely be the deciding factor. However, the need to achieve complete suppression (or nearly complete suppression) due to market demand would also play an important role in choosing which microbial control approach to use. The two combination treatments (S. riobrave combined with either M. brunneum or I. Javanica) that caused the most pronounced reduction in R. pomonella survival (lowest emergence) may have potential to substantially reduce damage. In conclusion, the present study revealed that certain single or combined application of EPFs and EPNs has considerable potential to suppress the population of R. pomonella at field condition. Additional studies are needed, including field studies, to determine the optimum approach for leveraging pathogen virulence. Our study represents the first step toward an integrated microbial control program for R. pomonella. In developing a microbial control approach the first step is to determine the biocontrol candidates that show the most promise based on virulence or other properties (Dara et al. 2018). Thus, we have established nematode and fungal candidates for use in microbial control against the target pest. Once candidates are identified they need to be field-tested (which we will tackle in subsequent research). If field results do not provide sufficient results, then the approach can be improved by strain improvement (hybridization or other means), or improving formulation or application technology (Dara et al. 2018, Shapiro-Ilan et al. 2018). Another approach to improving microbial control efficacy is to integrate with other strategies. As discussed, in this study we have explored combinations between EPF and EPN. Future studies are needed to explore combination or integration with chemical insecticides or other strategies. The model that we are following for developing a microbial control strategy can be applicable to various pest management systems. Acknowledgments The authors thank Stacy Byrd for technical assistance. M.U. and S.G. thank the Higher Education Commission (HEC) Islamabad for providing financial support through their ‘International Research Support Initiative Program (IRSIP)’ research grants. We also acknowledge the USDA-National Institute of Food and Agriculture-Crop Protection and Pest Management program award 2018-70006-28890 for funding a portion of the research. References Cited Acevedo , J P , R I Samuels, I R Machado, and C Dolinski. 2007 . Interactions between isolates of the entomopathogenic fungus Metarhizium anisopliae and the entomopathogenic nematode Heterorhabditis bacteriophora JPM4 during infection of the sugar cane borer Diatraea saccharalis (Lepidoptera: Pyralidae) . J. Invertebr. Pathol . 96 : 187 – 192 . Google Scholar Crossref Search ADS PubMed WorldCat Ali , S , Z Huang, and S Ren. 2010 . Production of cuticle degrading enzymes by Isaria fumosorosea and their evaluation as a biological agent against diamondback moth . J. Pest Sci . 83 : 361 – 370 . Google Scholar Crossref Search ADS WorldCat Ansari , M , L Tirry, and M Moens. 2004 . Interaction between Metarhizium anisopliae CLO53 and entomopathogenic nematodes for the control of Hoplia philanthus . Biol. Control . 31 : 172 – 180 . Google Scholar Crossref Search ADS WorldCat Ansari , M , L Tirry, and M Moens. 2005 . Antagonism between entomopathogenic fungi and bacterial symbionts of entomopathogenic nematodes . BioControl . 50 : 465 – 475 . Google Scholar Crossref Search ADS WorldCat Ansari , M A , F A Shah, L Tirry, and M Moens. 2006 . Field trials against Hoplia philanthus (Coleoptera: Scarabaeidae) with a combination of an entomopathogenic nematode and the fungus Metarhizium anisopliae CLO 53 . Biol. Control . 39 : 453 – 459 . Google Scholar Crossref Search ADS WorldCat Ansari , M , F Shah, and T M Butt. 2008 . Combined use of entomopathogenic nematodes and Metarhizium anisopliae as a new approach for black vine weevil, Otiorhynchus sulcatus, control . Entomol. Exp. Appl . 129 : 340 – 347 . Google Scholar Crossref Search ADS WorldCat Ansari , M A , M Evans, and T M Butt. 2009 . Identification of pathogenic strains of entomopathogenic nematodes and fungi for wireworm control . Crop Protect . 28 : 269 – 272 . Google Scholar Crossref Search ADS WorldCat Barbercheck , M , and H Kaya. 1990 . Interactions between Beauveria bassiana and the entomopathogenic nematodes, Steinernema feltiae and Heterorhabditis heliothidis. J. Invertebr. Pathol . 55 : 225 – 234 . Google Scholar Crossref Search ADS WorldCat Beris , E I , D P Papachristos, A Fytrou, S A Antonatos, and D Kontodimas. 2013 . Pathogenicity of three entomopathogenic fungi on pupae and adults of the Mediterranean fruit fly, Ceratitis capitata (Diptera: Tephritidae) . J. Pest Sci . 86 : 275 – 284 . Google Scholar Crossref Search ADS WorldCat Bostanian , N J , C Vincent, G Chouinard, and G Racette. 1999 . Managing apple maggot, Rhagoletis pomonella [Diptera: Tephritidae], by perimeter trapping . Phytoprotection . 80 : 21 – 33 . Google Scholar Crossref Search ADS WorldCat CABI . 2019 . Rhagoletis pomonella (apple maggot). CABI Invasive Species Compendium . https://www.cabi.org/isc/datasheet/47060 (accessed 12 May 2020 ). Correa-Cuadros , J P , A Saenz-Aponte, and M X Rodriguez-Bocanegra. 2016 . In vitro interaction of Metarhizium anisopliae Ma9236 and Beauveria bassiana Bb9205 with Heterorhabditis bacteriophora HNI0100 for the control of Plutella xylostella . Springer Plus . 5 : 2068 . Google Scholar Crossref Search ADS PubMed WorldCat Dara , S K , T A Goble, and D I Shapiro-Ilan. 2018 . Leveraging the cology of invertebrate pathogens in microbial control, pp. 469 – 494 . In A E Hajek and D I Shapiro-Ilan (eds.), Ecology of invertebrate diseases . H John Wiley and Sons , Hoboken, NJ . Google Scholar Google Preview OpenURL Placeholder Text WorldCat COPAC Dean , R W , and P J Chapman. 1973 . Bionomics of apple maggot in eastern New York . Search Agric. (Geneva, NY) . 10 : 1 – 64 . Google Scholar OpenURL Placeholder Text WorldCat Ekesi , S , N K Maniania, and S A Lux. 2002 . Mortality in three African tephritid fruit fly puparia and adults caused by the entomopathogenic fungi, Metarhizium anisopliae and Beauveria bassiana . Biocontrol Sci. Technol . 12 : 7 – 17 . Google Scholar Crossref Search ADS WorldCat Ekesi , S , S Dimbi, and N K Maniania. 2007 . The role of entomopathogenic fungi in the integrated management of fruit flies (Diptera: Tephritidae) with emphasis on species occurring in Africa, pp. 239 – 274 . In S Ekesi and N K Maniania (eds.), Use of entomopathogenic fungi in biological pest management . Research Signpost , Kerala, India . Google Scholar Google Preview OpenURL Placeholder Text WorldCat COPAC Ekesi , S , N K Maniania, and S A Mohamed. 2011 . Efficacy of soil application of Metarhizium anisopliae and the use of GF-120 spinosad bait spray for suppression of Bactrocera invadens (Diptera: Tephritidae) in mango orchards . Biocontrol Sci. Technol . 21 : 299 – 316 . Google Scholar Crossref Search ADS WorldCat Garrido-Jurado , I , F Ruano, M Campos, and E Quesada-Moraga. 2011a . Effects of soil treatments with entomopthogenic fungi on soil dwelling non-target arthropods at a commercial olive orchard . Biol. Control . 59 : 239 – 244 . Google Scholar Crossref Search ADS WorldCat Garrido-Jurado , I , J Torrent, V Barron, A Corpas, and E Quesada-Moraga. 2011b . Soil properties affect the availability, movement, and virulence of entomopathogenic fungi conidia against puparıa of Ceratitis capitata (Diptera: Tephritidae) . Biol. Control . 58 : 277 – 285 . Google Scholar Crossref Search ADS WorldCat Garrido-Jurado , I , P Valverde-Gracıa, and E Quesada-Moraga. 2011c . Use of a multiple logistic regression model to determine the effects of soil moisture and temperature on the virulence of entomopathogenic fungi against pre-imaginal Mediterranean fruit fly Ceratitis capitata . Biol. Control . 59 : 366 – 372 . Google Scholar Crossref Search ADS WorldCat Gaugler , R , Y Wang, and J F Campbell. 1994 . Aggressive and evasive behaviors in Popillia japonica (Coleoptera: Scarabaeidae) larvae: defenses against entomopathogenic nematode attack . J. Invertebr. Pathol . 64 : 193 – 199 . Google Scholar Crossref Search ADS WorldCat Goble , T A , J F Dames, M P Hill, and S D Moore. 2011 . Investigation of native isolates of entomopathogenic fungi for the biological control of three citrus pests . Biocontrol Sci. Technol. 21 : 1193 – 1211 . Google Scholar Crossref Search ADS WorldCat Goettel , M S , J Eilenberg, and T R Glare. 2005 . Entomopathogenic fungi and their role in regulation of insect populations, pp. 361 – 406 . In L I Gilbert, K Iatrou, and S Gill (eds.), Comprehensive molecular insect science , vol. 6 . Elsevier/Pergamon Press , Oxford, United Kingdom . Google Scholar Crossref Search ADS Google Preview WorldCat COPAC Green , T A , and S E Wright. 2009 . Speciation, consumers and the market: profit with a conscience, pp. 253 – 83 . In M Aluja, T C Leskey, and C Vincent (eds.), Biorational tree-fruit pest management . CABI International , Cambridge, MA . Google Scholar Crossref Search ADS Google Preview WorldCat COPAC Howitt , A . 1993 . Common tree fruit pests . NCR 63, Michigan State University . http://web1.msue.msu.edu/vanburen/fappmag.htm (accessed June 2006 ). Inglis , G D , J Enkerli, and M S Goettel. 2012 . Laboratory techniques used for entomopathogenic fungi: hypocreales, pp. 189 – 253 . In L A Lacey (ed.), Manual of techniques in invertebrate pathology . Academic Press , London, United Kingdom . Google Scholar Crossref Search ADS Google Preview WorldCat COPAC Koppenhöfer , A , and P Grewal. 2005 . Nematodes as biocontrol agents. Entomology and nematology, pp. 363 – 381 . In P S Grewal, R Ehlers, and D I Shapiro-Ilan (eds.), Compatibility and interactions with agochemicals and other biocontrol agents, chapter 20 . CABI Publishing , Wallingford, Oxfordshire, United Kingdom . Google Scholar Google Preview OpenURL Placeholder Text WorldCat COPAC Lacey , L A (ed.). 1997 . Manual of techniques in invertebrate pathology . Academic Press , London, United Kingdom . Google Scholar Google Preview OpenURL Placeholder Text WorldCat COPAC Lacey , L A , and D I Shapiro-Ilan. 2008 . Microbial control of insect pests in temperate orchard systems: potential for incorporation into IPM . Annu. Rev. Entomol . 53 : 121 – 144 . Google Scholar Crossref Search ADS PubMed WorldCat Lacey , L A , D Grzywacz, D I Shapiro-Ilan, R Frutos, M Brownbridge, and M S Goettel. 2015 . Insect pathogens as biological control agents: back to the future . J. Invertebr. Pathol . 132 : 1 – 41 . Google Scholar Crossref Search ADS PubMed WorldCat Magaña , C , P Hernández-Crespo, F Ortego, and P Castañera. 2007 . Resistance to malathion in field populations of Ceratitis capitata . J. Econ. Entomol . 100 : 1836 – 1843 . Google Scholar Crossref Search ADS PubMed WorldCat Mbata , G N , and D I Shapiro-Ilan. 2005 . Laboratory evaluation of virulence of heterorhabditid nematodes to Plodia interpunctella Hubner (Lepidoptera: Pyralidae) . Environ. Entomol . 34 : 676 – 682 . Google Scholar Crossref Search ADS WorldCat Morrison , W R , 3rd, D H Lee, W H Reissig, D Combs, K Leahy, A Tuttle, D Cooley, and T C Leskey. 2016 . Inclusion of specialist and generalist stimuli in attract-and-kill programs: their relative efficacy in apple maggot fly (Diptera: Tephritidae) pest management . Environ. Entomol . 45 : 974 – 982 . Google Scholar Crossref Search ADS PubMed WorldCat Prokopy , R J , 1968 . Influence of photoperiod, temperature, and food on initiation of diapause in the apple maggot . Can. Entomol . 100 : 318 – 329 . Google Scholar Crossref Search ADS WorldCat Prokopy , R J , and J Mason. 1996 . Behavioral control of apple maggot flies, pp. 555 – 559 . In B Mcpheron and G Steck (eds.), Fruit flies . St. Lucie Press , Delray Beach, FL . Google Scholar Crossref Search ADS Google Preview WorldCat COPAC Prokopy , R J , M Christie, S A Johnson, and M T O’Brien. 1990 . Transitional steps toward second-stage integrated management of arthropod pests of apple in Massachusetts orchards . J. Econ. Entomol . 83 : 2405 – 2410 . Google Scholar Crossref Search ADS WorldCat Qin , Y , D R Paini, C Wang, Y Fang, and Z Li. 2015 . Global establishment risk of economically important fruit fly species (Tephritidae) . PLoS One . 10 : e0116424 . Google Scholar Crossref Search ADS PubMed WorldCat Quesada-Moraga , E , A Ruiz-García, and C Santiago-Alvarez. 2006 . Laboratory evaluation of entomopathogenic fungi Beauveria bassiana and Metarhizium anisopliae against puparia and adults of Ceratitis capitata (Diptera: Tephritidae) . J. Econ. Entomol . 99 : 1955 – 1966 . Google Scholar Crossref Search ADS PubMed WorldCat Reissig , H , and A Agnello. 1996 . Update on pest management and crop development . Scaffolds Fruit J . 5 : 1 – 2 . www.nysaes.cornel.edu/ent/scaffolds/. Google Scholar OpenURL Placeholder Text WorldCat Reissig , W H , B L Fein, and W L Roelofs. 1982 . Field tests of synthetic apple volatiles as apple maggot attractants . Environ. Entomol . 11 : 1294 – 1298 . Google Scholar Crossref Search ADS WorldCat Samuels , R I , A K Charnley, and S E Reynolds. 1988 . The role of destruxins in the pathogenicity of 3 strains of Metarhizium anisopliae for the tobacco hornworm Manduca sexta . Mycopathologia . 104 : 51 – 58 . Google Scholar Crossref Search ADS WorldCat SAS . 2002 . SAS software: version 9.1 . SAS Institute , Cary, NC . Google Scholar Google Preview OpenURL Placeholder Text WorldCat COPAC Shapiro-Ilan , D I , W Gardner, J R Fuxa, B W Wood, K Nguyen, B Adams, R A Humber, and M J Hall. 2003 . Survey of entomopathogenic nematodes and fungi endemic to pecan orchards of the southeastern US and their virulence to the pecan weevil (Coleoptera: Curculionidae) . Environ. Entomol . 32 : 187 – 195 . Google Scholar Crossref Search ADS WorldCat Shapiro-Ilan , D I , M Jackson, C C Reilly, and M W Hotchkiss. 2004 . Effects of combining an entomopathogenic fungi or bacterium with entomopathogenic nematodes on mortality of Curculio caryae (Coleoptera: Curculionidae) . Biol. Control . 30 : 119 – 126 . Google Scholar Crossref Search ADS WorldCat Shapiro-Ilan , D I , R Han, and C Dolinksi. 2012 . Entomopathogenic nematode production and application technology . J. Nematol . 44 : 206 – 217 . Google Scholar PubMed OpenURL Placeholder Text WorldCat Shapiro-Ilan , D I , J A Morales-Ramos, and M G Rojas. 2016 . In vivo production of entomopathogenic nematodes, pp. 137 – 58 . In T Glare and M Moran-Diez (eds.), Microbial-based biopesticides – methods and protocols . Human Press , New York . Google Scholar Crossref Search ADS Google Preview WorldCat COPAC Shapiro-Ilan , D I , I Hiltpold, and E E Lewis. 2018 . Ecology of invertebrate pathogens: nematodes, pp. 415 – 440 . In A E Hajek and D I Shapiro-Ilan (eds.), Ecology of invertebrate diseases . John Wiley and Sons , Hoboken, NJ . Google Scholar Google Preview OpenURL Placeholder Text WorldCat COPAC Southwood , T R E . 1978 . Ecological methods: with particular reference to the study of insect populations . Chapman and Hall , London, United Kingdom . Google Scholar Google Preview OpenURL Placeholder Text WorldCat COPAC Steel , R G D , and J H Torrie. 1980 . Principles and procedures of statistics . McGraw-Hill , New York . Google Scholar Google Preview OpenURL Placeholder Text WorldCat COPAC Suckling , D M , J M Kean, L D Stringer, C Cáceres-Barrios, J Hendrichs, J Reyes-Flores, and B C Dominiak. 2016 . Eradication of tephritid fruit fly pest populations: outcomes and prospects . Pest Manag. Sci . 72 : 456 – 465 . Google Scholar Crossref Search ADS PubMed WorldCat Tarasco , E , C Alvarez, O Triggian, and E Quesada-Moraga. 2011 . Laboratory studies on the competition for insect haemocoel between Beauveria bassiana and Steinernema ichnusae recovered in the same ecological niche . Biocontrol Sci. Technol . 21 : 693 – 704 . Google Scholar Crossref Search ADS WorldCat Usman , M , S Gulzar, W Wakil, J C Piñero, T C Leskey, L J Nixon, C Oliveira-Hofman, S Wu, and D Shapiro-Ilan. 2020 . Potential of entomopathogenic nematodes against the pupal stage of the apple maggot Rhagoletis pomonella (Walsh) (Diptera: Tephritidae) . J. Nematol . 52: e2020 - 79 . doi:10.21307/jofnem-2020-079. Google Scholar Crossref Search ADS WorldCat Vega , F E , M S Goettel, M Blackwell, D Chandler, M A Jackson, S Keller, M Koike, N K Maniania, A Monzon, B H Ownley, et al. 2009 . Fungal entomopathogens: new insights on their ecology . Fungal Ecol . 2 : 149 – 159 . Google Scholar Crossref Search ADS WorldCat Villalobos , J , S Flores, P Liedo, and E A Malo. 2017 . Mass trapping is as effective as ground bait sprays for the control of Anastrepha (Diptera: Tephritidae) fruit flies in mango orchards . Pest Manag. Sci . 73 : 2105 – 2110 . Google Scholar Crossref Search ADS PubMed WorldCat Wakil , W , M Yasin, and D Shapiro-Ilan. 2017 . Effects of single and combined applications of entomopathogenic fungi and nematodes against Rhynchophorus ferrugineus (Olivier) . Sci. Rep . 7: 5971 . doi:10.1038/s41598-017-05615-3. Google Scholar Crossref Search ADS PubMed WorldCat Weems , H V , Jr., J B Heppner, T R Fasulo, and J L Nation. 2014 . Caribbean fruit fly (Anastrepha suspensa Loew) (Insecta: Diptera: Tephritidae) . UF/IFAS , Gainesville, FL , Featured Creatures EENY-196, July reviews. Google Scholar Google Preview OpenURL Placeholder Text WorldCat COPAC Wilson , W M , J E Ibarra, A Oropeza, M A Hernández, R A Toledo-Hernández, and J Toledo. 2017 . Infection of Anastrepha ludens (Diptera: Tephritidae) adults during emergence from soil treated with Beauveria bassiana under various texture, humidity, and temperature conditions . Fla. Entomol . 100 : 503 – 508 . Google Scholar Crossref Search ADS WorldCat Wright , S E , T C Leskey, I Jacome, J C Piñero, and R J Prokopy. 2012 . Integration of insecticidal, phagostimulatory, and visual elements of an attract and kill system for apple maggot fly (Diptera: Tephritidae) . J. Econ. Entomol . 105 : 1548 – 1556 . Google Scholar Crossref Search ADS PubMed WorldCat Wu , S , R R Youngman, L T Kok, C A Laub, and D G Pfeiffer. 2014 . Interaction between entomopathogenic nematodes and entomopathogenic fungi applied to third instar southern masked chafer white grubs, Cyclocephala lurida (Coleoptera: Scarabaeidae), under laboratory and greenhouse conditions . Biol. Control. 76 : 65 – 73 . Google Scholar Crossref Search ADS WorldCat Yousef , M , I Garrido-Jurado, M Ruız-Torres, and E Quesada-Moraga. 2017 . Reduction of adult olive fruit fly populations by targeting preimaginals in the soil with the entomopathogenic fungus Metarhizium brunneum . J. Pest Sci . 90 : 345 – 354 . Google Scholar Crossref Search ADS WorldCat Zimmermann , G . 2007 . Review on safety of the entomopathogenic fungi Beauveria bassiana and Beauveria brongniartii . Biocontrol Sci. Technol . 17 : 553 – 596 . Google Scholar Crossref Search ADS WorldCat Published by Oxford University Press on behalf of Entomological Society of America 2020. This work is written by (a) US Government employee(s) and is in the public domain in the US. Published by Oxford University Press on behalf of Entomological Society of America 2020.
TI - Virulence of Entomopathogenic Fungi to Rhagoletis pomonella (Diptera: Tephritidae) and Interactions With Entomopathogenic Nematodes
JF - Journal of Economic Entomology
DO - 10.1093/jee/toaa209
DA - 2020-12-09
UR - https://www.deepdyve.com/lp/oxford-university-press/virulence-of-entomopathogenic-fungi-to-rhagoletis-pomonella-diptera-8ETe406aHT
SP - 2627
EP - 2633
VL - 113
IS - 6
DP - DeepDyve
ER -