https://orcid.org/0009-0005-0719-202X
Figures
Abstract
Beauveria bassiana AAD16, isolated from Allomyrina dichotoma, shows promise as a mycoinsecticide against various coleopterans. However, assessing non-target impacts on beneficial beetles like ladybirds is crucial before commercialization. Here we assessed the compatibility between ladybird beetles and B. bassiana AAD16. The virulence of the AAD16 strain was compared with that of an available commercial strain, B. bassiana GHA, for two developmental stages (adults and 3rd instar larvae) of two coccinellids, Harmonia axyridis Pallas and Chilocorus spp. Say using the topical (1μl) application method. The ST50 for the two life stages of the two ladybird beetles were not different between the two tested fungal strains. Mycosis rates recorded from the dead bodies were also not significant except in the 3rd instar which showed 36 and 63% from AAD16 and GHA strains in H. axyridis, while those of Chilocorus spp. were 40 and 63%, respectively. In adult stage, the mycosis rates of H. axyridis (males and females tested separately) were (20–23) % and (26–30) % from the AAD16 and GHA strains, while those of Chilocorus spp. (unsexed) were 23 and 30%, respectively. AAD16 caused similar rates of mortality in the adult stages of both species. Therefore, we conclude that B. bassiana AAD16 would not increase risk to these beneficial insects compared to a similar pathogen commercialized.
Citation: Hasan MR, Raju MR, Lim UT (2025) Laboratory risk assessment of Beauveria bassiana AAD16 on two species of ladybird beetle. PLoS ONE 20(1): e0317483. https://doi.org/10.1371/journal.pone.0317483
Editor: Vivekanandhan Perumal, Chiang Mai University Faculty of Agriculture, THAILAND
Received: October 5, 2024; Accepted: December 30, 2024; Published: January 30, 2025
Copyright: © 2025 Hasan et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Data Availability: All relevant data are within the manuscript and its Supporting Information files.
Funding: Korea Institute of Planning and Evaluation for Technology in Food, Agriculture, and Forestry (IPET). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing interests: NO authors have competing interests.
Introduction
Beauveria bassiana (Balsamo) Vuillemin formulations serve as sustainable and environmentally safe insecticides, providing an alternative to chemical pesticides for controlling numerous pests in agricultural and forestry systems [1–4]. Many countries have developed and registered commercial products based on B. bassiana, which are now utilized globally for pest control [3, 5–7]. However, new molecular studies show that B. bassiana, once thought to be a general-purpose fungus, is a group of closely related species, each adapted to specific hosts or environments [2, 3]. B. bassiana is a widespread soil-dwelling entomopathogenic fungus, making it particularly effective against organisms with soil-associated life stages, such as overwintering coccinellids, melolonthine scarabs, and caterpillars [8–10]. Additionally, some B. bassiana strains have been isolated from the phylloplane (leaf surfaces) of hedgerow vegetation [11], demonstrating its ecological flexibility.
Strains of B. bassiana have been shown to cause high levels of mortality and infection in some non-target organisms [1], as well as to the targeted pests [12–18]. In contrast, other strains, such as GHA, are less harmful to non-target organisms than to their target pests [1, 19, 20]. The virulence and mycosis rates of B. bassiana differ according to the host species and the enzymatic characteristics of the strains [21].
Harmonia axyridis (Pallas) (Coleoptera: Coccinellidae) is a ladybird beetle native to East Asia that can become invasive on other continents. Due to its environmental resiliency and aggressive predation, it has been a very useful biological control agent for aphids, coccids, and other pests, and it has been widely used in horticulture [22]. However, natural enemies of H. axyridis have also been identified including pathogens, parasites, and parasitoids [23–25]. Despite the presence of these natural enemies of H. axyridis, the success of H. axyridis as a biological control agent of insect pests may imply the existence of biological resistance to them. H. axyridis has a robust immune system, giving it defenses that surpass competing ladybirds [26–30]. The immune system of H. axyridis consists of chemical defenses against a variety of bacteria as well as a wide range of antimicrobial peptides that are the result of multiple gene duplication events [26]. Additionally, H. axyridis possesses strong alkaloid chemical defenses against predators and pathogens, which give it a foul smell and taste.
Chilocorus spp. (Say) (Coleoptera: Coccinellidae) is an omnivorous predator of several scale insects, aphids, and mealybugs [31]. It has been reported often as a predator of the pine needle scale (Chionaspis pinifoliae) [32].
The interactions between B. bassiana and coccinellids have been explored from two primary perspectives. The first of these is the study of the mortality of overwintering populations of coccinellids that are naturally exposed B. bassiana in the soil [9, 33–35], while the second study focus has been deaths of non-target coccinellids when exposed to B. bassiana used as biorational pesticides [36–41]. No previous studies have addressed the susceptibility of Chilocorus spp. to any entomopathogenic fungi, and the risk to H. axyridis has only been assessed as having low susceptibility to commercial B. bassiana GHA [27]. Therefore, the objective of this study was to assess the compatibility of adults and 3rd instar larvae of H. axyridis and Chilocorus spp. with B. bassiana AAD16 in comparison to a commercial product based on B. bassiana GHA.
Materials and methods
Insect colonies
Colonies of H. axyridis and Chilocorus spp. originated from adult beetles collected from peach orchards at the experimental fields of Andong National University, Korea (36° 34’ 6.0744’ N latitude and 128° 43’ 45.6852’’ E longitudes). The colonies were housed in onion bags (45×30 cm) in a peach tree naturally infested with aphids (Myzus persicae Sulzer). Onion bags, made from synthetic fabrics, permit ventilation and are used for containing field population of the ladybird species for short period of time. The 3rd instar larvae and adults of both ladybird species were collected as needed from the onion bag for laboratory bioassays.
Fungal pathogens used and preparation of the conidial suspensions tested
Two strains (AAD16 and GHA) of B. bassiana were evaluated in this study. The AAD16 strain was isolated from the scarabaeid beetle Allomyrina dichotoma (L.) [42], while GHA (Botanigard® ES) is a commercially available strain from Laverlam International Cooperation (Butte, Montana, USA) that was grown in our laboratory on Sabouraud Dextrose Agar (SDA) media (Difco™, Sparks, MD, USA) in Petri dishes (60 mm diameter × 15 mm height) maintained in the dark at 25.3 ± 0.1°C and 94.9 ± 0.3% RH for 14 days [43]. Strain AAD16 was also cultured in SDA media in the dark at 25°C. Conidia were collected from the culture surfaces by scraping, and then the conidia were suspended in sterile distilled water with 0.1% Triton X-100 in a 20-ml scintillation vial (240804, Wheaton, Millville, NJ) containing autoclaved Triton X-100 (0.1%) solution (Duksan Pure Chemicals Co. Ltd., Ansan, Republic of Korea). Conidial suspensions were vortexed for at least 2 minutes until it became a homogeneous suspension. The conidial concentration of the suspension was measured using a Neubauer hemocytometer (Marienfeld-Superior, Paul Marienfeld GmbH and Co. KG, Lauda-Königshofen, Germany). Conidial viability was assessed before the bioassay by spreading 100μl of 1×104 CFU/ml suspension on SDA plates. The plates were incubated at 25°C, and the percentage germination was determined after 18 hours for groups of 100 spores by placing a sterile microscope cover slip on each plate under a compound microscope. In all experiments, conidia germination exceeded 95%.
Adult and larval bioassays
Two developmental stages (adults and 3rd instar larvae) of two coccinellids, H. axyridis and Chilocorus spp., were tested using 1×108 conidia/ml concentrations of both B. bassiana AAD16 and GHA strain, using 0.1% Triton X-100 ddH2O as a control. For H. axyridis, the two sexes of adults were tested separately; but for Chilocorus spp., unsexed adults were tested. On the day of the experiment, beetles were collected from the colony and placed in Petri dishes (9 cm dia) sealed with parafilm to prevent escape. The dishes were then placed on a container filled with ice for 10 minutes to reduce beetle activity before the topical application. All bioassays were done with adults and 3rd instar larvae. Beetles were removed from the ice, and 1μl of a treatment was applied using micro syringe onto the dorsal surface of the abdomen. The adult beetles were subsequently placed in sterile 9 cm-dia Petri dishes with 10 beetles/dish. All plates with the same treatment were grouped together and held in desiccators to maintain high relative humidity (4202–0000, Bel-Art Products, Pequannock, New Jersey, USA), while maintained at 25.3 ± 0.1°C and 95.1 ± 3.4% RH in an incubator. Treated larvae were kept in 2 ml Eppendorf tubes without a food source, with a small hole in the lid. Tubes were kept in desiccators (4202–0000, Bel-Art Products, Pequannock, New Jersey, USA) at 25.2 ± 0.1°C and 94.7 ± 0.4% RH inside a growth chamber.
Mortality and mycosis rates (%) were recorded for adult beetles at 24-hour intervals from the time of exposure until 23 days, and for larvae until 15 days. An insect was classified as dead when there was no movement observed following any of three strokes with a fine brush under a stereo-microscope. Insects were categorized as having mycosis by B. bassiana when fungus mycelia were visible on the insects’ integument through a stereo-microscope.
Data analysis
All statistical analyses were conducted using SAS version 9.3 [44]. The median survival time (ST50) per treatment was determined, and treatment values were compared using Kaplan-Meier survival analysis since mortality did not reach 100% in either the treatment or control groups. Subsequently, the survival curves were compared using the Log-rank test at the 95% confidence level. Significant differences among treatments were determined by assessing the 95% confidence interval (CI). The fungal mycosis development rates over time within a strain of the AAD16 and GHA strains were analyzed with repeated measures ANOVA, along with post hoc multiple comparison tests that were analogous to Tukey’s test [45]. Data for mortality and fungal mycosis rates of larval stage between the AAD16 and GHA strains were compared using repeated measures ANOVA.
Result
Adult bioassay
For adult’s male of H. axyridis, the ST50 value of B. bassiana AAD16 was 80.01 h (χ2 = 122.97, df = 19, P < 0.001), which did not differ significantly from that of B. bassiana GHA, which had an ST50 value of 82.30 h (χ2 = 104.12, df = 16, P < 0.001) (Table 1). Survival analysis was carried out with adult males of H. axyridis exposed to entomopathogenic fungi, and survival rates were significantly different among the treatments (χ2 = 6.10, df = 2, P = 0.047) (Fig 1). The mortality rate of males was 100% for AAD16 and GHA, compared to 87% in the control over the entire period of observation (Fig 1). No significant difference in mycosis rates was observed between two fungus treatments (Treatment: F = 34.72, df = 1, 22, P < 0.001; Time: F = 49.10, df = 22, 92, P < 0.001; Interaction: F = 1.04, df = 22, 92, P < 0.431) (Fig 2).
Means followed by the same letter were not significantly different from each other (Tukey studentized range HSD test, α = 0.05).
Means followed by the same letter are not significantly different (Tukey studentized range HSD test, α = 0.05).
For female adults of H. axyridis, there were significant differences in ST50 values between the AAD16 strain (109.91 h, χ2 = 134.95, df = 21, P < 0.001) and the GHA strain (165.33 h, χ2 = 131, df = 21, P < 0.001) (Table 2). The mortality rate of females was 100% for AAD16 and 97% for GHA, compared to 97% in the control over the entire period of observation (Fig 3). Survival analysis for female adults of H. axyridis exposed to entomopathogenic fungi showed no significant differences among treatments (χ2 = 1.99, df = 2, P = 0.368) (Fig 3). The mycosis rate for female adults of H. axyridis showed significant differences between two fungus treatments (Treatment: F = 79.35, df = 1, 22, P < 0.001; Time: F = 9.80, df = 22, 92, P < 0.001; Interaction: F = 1.88, df = 22, 92, P < 0.019) (Fig 4).
Means followed by the same letter are not significantly different (Tukey studentized range HSD test, α = 0.05).
Means followed by the same letter are not significantly different (Tukey studentized range HSD test, α = 0.05).
For mixed sex adults of Chilocorus spp., the ST50 value caused by strain AAD16 was 88.96 h (χ2 = 142.70, df = 21, P < 0.001), which was not significantly different from that of the GHA strain, which was 79.31 h (χ2 = 99, df = 12, P < 0.001) (Table 3). The survival analysis conducted on adults of Chilocorus spp. exposed to entomopathogenic fungi showed no significant differences among treatments (χ2 = 1.99, df = 2, P = 0.368) (Fig 5). Mycosis rates were also significant between B. bassiana AAD16 and GHA (Treatment F = 6.00, df = 1, 22, P < 0.001; Time F = 38.77, df = 22, 92, P < 0.016; Interaction F = 0.86, df = 22, 92, P < 0.644) (Fig 6).
Means followed by the same letter are not significantly different (Tukey studentized range HSD test, α = 0.05).
Means followed by the same letter are not significantly different (Tukey studentized range HSD test, α = 0.05).
Larval bioassay for both lady beetle species
The virulence of the two entomopathogenic fungal strains to 3rd instar larvae of H. axyridis was similar, with the ST50 value of AAD16 being 67.88 h (χ2 = 31.09, df = 8, P < 0.001) and that of GHA being 72.10 h (χ2 = 55.82, df = 9, P < 0.001), which were not significantly different (Table 4). Repeated measures ANOVA showed for H. axyridis larvae, there were significant differences among treatments (Treatment F = 16.08, df = 2, 14, P < 0.001; Time F = 191.35, df = 14, 90, P < 0.001; Interaction F = 2.41, df = 28, 90, P = 0.009) (Fig 7). No significant differences in the mycosis rate were observed between two fungus treatments for larvae of H. axyridis (Treatment: F = 0.56, df = 1, 14, P = 0.456; Time: F = 10.97, df = 14, 60, P < 0.001; Interaction: F = 1.08, df = 14, 60, P = 0.397) (Fig 8).
Means followed by the same letter are not significantly different (Tukey studentized range HSD test, α = 0.05).
Means followed by the same letter are not significantly different (Tukey studentized range HSD test, α = 0.05).
For Chilocorus spp. larvae, the ST50 value of AAD16 was 78.88 h (χ2 = 47.53, df = 8, P < 0.001), while that of GHA was 34.20 h (χ2 = 32.68, df = 7, P < 0.001), which differ significantly. (Table 5). The ST50 for both the AAD16 and GHA strains were significantly different from that of the control, which died off more rapidly than the fungal treated beetles (Table 5) (Treatment: F = 22.84, df = 2, 14, P < 0.001; Time: F = 22.27, df = 14, 90, P < 0.001; Interaction: F = 9.08, df = 28, 90, P < 0.001) (Fig 9). There were no significant differences in mycosis rate between two fungus treatments (not including the control, which does not cause mycosis) (Treatment: F = 0.46, df = 1, 14, P = 0.502; Time: F = 23.12, df = 14, 60, P < 0.001; Interaction: F = 0.93, df = 14, 60, P = 0.533) (Figs 10 and 11). The mycosis rate was 63% in B. bassiana AAD16 and 60% in B. bassiana GHA (Fig 10).
Means followed by the same letter are not significantly different (Tukey studentized range HSD test, α = 0.05).
Means followed by the same letter are not significantly different (Tukey studentized range HSD test, α = 0.05).
bassiana strains AAD16 (A, C, E, G, I) and GHA (B, D, F, H, J).
Discussion
The impact of entomopathogenic fungi (EPF) upon arthropod natural enemies, specifically the predaceous Coccinellidae, is not well understood [35]. The process of choosing biological control agents for pest management programs typically involves considering factors such as host range, geographic distribution, fecundity, and voltinism. However, the evaluation of their susceptibility to insect pathogens native to the region is rarely integrated into this decision-making process. Specifically, for the effective establishment of natural enemies in new environmental settings—whether they involve different geographical locations or crop cultivation—it is crucial to assess the potential risk posed by endemic pathogens [46]. EPFs are infrequently found infecting coccinellids (ladybird beetles) under natural conditions [47], and studies have shown that natural fungal infections in these beetles rarely exceed 20% [47, 48]. Among EPFs, B. bassiana stands out as a generalist pathogen capable of infecting both plant-feeding and predatory coccinellids [49, 50].
Interspecific interaction among natural enemies is one of the important factors determining effectiveness of biological control programs [51]. However, the interactions between B. bassiana and coccinellids are rarely known, although the interactions between EPFs and some other types of natural enemies (parasitoids and predators) have been studied [52–55]. Applying the EPFs that are compatible with other natural enemies could result in higher control efficacy without increasing risk to non-target organisms and with reduced use of conventional insecticides [56].
In the present study, we investigated the susceptibility of H. axyridis and Chilocorus spp. lady beetles exposed to two EPFs (B. bassiana AAD16 and GHA strain) under laboratory conditions. We found significant differences in ST50 values between the AAD16 and GHA strains when tested against adult females of H. axyridis. However, there was no significant difference observed in adult males of H. axyridis and Chilocorus spp. during the 23 days of our trial (Tables 1–3).
Although the level of difference in mortality between the two ladybird species within a fungal strain was not significant, it is certainly worth further exploration. Several studies have illustrated the physiological susceptibility of coccinellids to B. bassiana [19]. Cottrell and Shapiro-Ilan [19] specifically emphasized the differing susceptibility of a native and an exotic coccinellids to an isolate of B. bassiana originating from the native coccinellid. The exotic coccinellid, H. axyridis, demonstrated lower susceptibility to B. bassiana compared to the indigenous coccinellid, Olla v-nigrum Mulsant (Coleoptera: Coccinellidae). Cagan and Uhlik [57] demonstrated that B. bassiana strains isolated from Ostrinia nubilalis were pathogenic to Coccinella septempunctata and Propylea quatuordecimpunctata. However, they suggested that field susceptibility of these ladybirds would likely be lower due to reduced contact with the pathogen in natural settings. Our findings showed that neither the coccinellids we tested were affected in either mortality or mycosis by the EPF strains assessed here.
H. axyridis has shown great resistance to entomopathogenic nematodes and the entomopathogenic fungi B. bassiana [26]. The resistance of H. axyridis to B. bassiana involves potential mechanisms such as melanin production at infection sites [58] or defensive chemicals [59–61]. Other insects facing pathogen-rich environments, such as rat-tailed maggots of the drone fly thriving in polluted aquatic habitats like liquid manure storage pits [62], and the burying beetle Nicrophorus vespilloides Herbst, which feeds and reproduces on cadavers [63], also exhibit heightened pathogen resistance attributed to a diverse array of antimicrobial peptides (AMPs). Nevertheless, both fungi we tested were more pathogenic to the larval stage than the adult in both species of lady beetles.
For successful integration of EPFs into integrated pest management (IPM) programs, mycoinsecticides must be virulent to the target pests [42, 64, 65] but have only minimal negative effects on non-target organisms [66]. Indeed, the non-target effect of different EPFs on natural enemies has been assessed by many previous studies. Ullah et al. [67] tested the virulence of Isaria fumosorosea Wize and B. bassiana against a reduviid predator and reported no significant impact upon predation and survival rate.
Huang et al. [68] reported that various concentrations of B. bassiana had no significant effect on biological parameters of the coccinellid Prynocaria cogener (Billberg). Similarly, in other laboratory investigations, B. bassiana was found not to be pathogenic to some beneficial arthropods, including Apis mellifera L., Chrysoperla rufilabris Burmeister, Orius insidiosus Say, Hippodamia convergens Guérin-Méneville, H. axyridis, and Coleomegilla maculata De Geer [69]. Harwood et al. [70] reported very low infection rates of Hesperomyces virescens Thaxter on coccinellids.
Until now, no entomopathogenic fungus has been studied to determine its effect on Chilocorus spp. B. bassiana AAD16 was isolated from A. dichotoma, it is comparatively less harmful against both species of coccinellids tested compared to the commercial B. bassiana GHA. Therefore, the two predatory coccinellids would be compatible with the AAD16 strain. In the future, the mechanism underlying the compatibility of these two coccinellid beetles with B. bassiana AAD16 and GHA should be determined, and in addition, those compatibilities should be verified under field conditions.
Supporting information
S1 Data. Raw data_male H. axyridis, raw data_female H. axyridis, raw data_adult Chilocorus spp., raw data_H. axyridis larvae, raw data_Chilocorus spp. larvae.
https://doi.org/10.1371/journal.pone.0317483.s001
(XLSX)
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