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Article

Ovicidal Effect of Entomopathogenic Fungi on Emerald Ash Borer, Agrilus planipennis Fairmaire, Eggs

by
Sofía Simeto
1,2,*,
Benjamin W. Held
1,
David N. Showalter
3,
Kathryn E. Bushley
4 and
Robert A. Blanchette
1
1
Department of Plant Pathology, University of Minnesota, 495 Borlaug Hall 1991 Upper Buford Circle, St. Paul, MN 55108, USA
2
Programa Nacional de Investigación en Producción Forestal, Instituto Nacional de Investigación Agropecuaria (INIA), Estación Experimental INIA Tacuarembó, Ruta 5 km 386, Tacuarembó CP 45000, Uruguay
3
Oregon Department of Forestry, Salem, OR 97310, USA
4
Agricultural Research Service (ARS), United States Department of Agriculture (USDA), Robert Holley Center, Ithaca, NY 14853, USA
*
Author to whom correspondence should be addressed.
Forests 2024, 15(12), 2170; https://doi.org/10.3390/f15122170
Submission received: 11 November 2024 / Revised: 4 December 2024 / Accepted: 7 December 2024 / Published: 9 December 2024
(This article belongs to the Section Forest Health)
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Abstract

:
The emerald ash borer (EAB) is an invasive beetle that has killed hundreds of millions of ash trees throughout North America since its arrival. The use of entomopathogenic fungi as part of integrated pest management approaches is considered effective against a wide range of insect pests. The aim of this study was to screen and select locally adapted EAB-associated entomopathogenic fungi with ovicidal effect on EAB eggs under laboratory conditions. The pathogenicity of nine fungal strains, previously isolated from EAB galleries, and the commercial Beauveria bassiana strain GHA was tested. Three of these, Akanthomyces muscarius 48-27, Lecanicillium longisporum 66-14 Lecanicillium psalliotae 59-2, and GHA B. bassiana strain consistently showed significant ovicidal effects and a high percentage of inoculum recovery both from eggs and neonate larvae. The high levels of inoculum recovery from neonate larvae demonstrate that, even after emergence, larvae were infected. The possibility of disrupting EAB’s life cycle at the egg stage through microbial control represents a potential management opportunity that should be explored in future field studies. Future work should also study the effect of EPF on neonate larvae survival and performance. To our knowledge, this is the first study to evaluate the effect of entomopathogenic fungi against the egg stage of EAB.

1. Introduction

The emerald ash borer (EAB), Agrilus planipennis Fairmaire (Coleptera, Buprestidae) is an invasive phloem and wood boring beetle native to eastern Asia that has caused extensive mortality of mature ash trees (Fraxinus L.) throughout North America. Although it was initially detected in North America in 2002 near Detroit, MI, USA [1], it had likely become established in the early 1990s and had begun killing trees by 1997 [2]. Since then, it has killed hundreds of millions of ash trees in urban and rural areas, landscapes and forests in eastern North America [3,4,5], with significant economic and ecological impact [3,6,7,8,9,10,11,12]. It has also been found recently in western North America [13]. It is considered to have the potential to decimate ash as a component of natural forests in North America [3,14,15]. A loss of ash within the EAB’s invasion range is expected to have drastic ecological impacts on forest composition and structure, understory vegetation, wildlife, water, nutrient and carbon cycling, and land use [7,9,10,14,16].
Native North American ash species have limited resistance to EAB [17,18,19] and tree mortality can reach nearly 100% within 3–6 years after establishment in an area [9,20]. Attempts to eradicate the pest in North America were abandoned a few years after its first detection due to its rapid spread [21,22], and integrated pest management strategies (IPM), such as monitoring, quarantine, chemical control, tree removal, and biological control with co-evolved EAB parasitoids have been developed and implemented [4,21]. Recent studies have shown that the implemented biological control approach using parasitoids is starting to show some promising results on sites where they have been released [21,23] and is expected to allow ash regeneration in the aftermath of EAB invasion. Additionally, highly effective systemic insecticides have been identified [24] and are a good management option to protect highly valued landscape ash trees, though costs and environmental concerns prevent this from being a feasible option for natural ash forests [21]. Despite all of this, EAB continues to spread along the ash range in North America and billions of trees are still threatened.
Integrated pest management (IPM) approaches combine a broad range of compatible techniques with the expectation that their combined effect will reduce the impact caused by pests to tolerable levels and with minimal environmental side effects [25,26]. Among the control strategies that aim to directly manipulate insect pest populations, microbial biological control has long been considered a significant component of IPM practices [25,27,28,29]. Microbial biological control agents include entomopathogenic bacteria, fungi, nematodes, and viruses. Among these, entomopathogenic fungi (EPF) are considered to be effective against a wide range of insect pests as they do not rely on the ingestion of their spores to infect their host. Instead, they have several mechanisms by which to penetrate the insect cuticle and can evade their immune system [30,31,32,33]. There are numerous studies that report negative effects on the targeted insect pest after using EPF under field conditions [28,30,34,35,36,37,38,39,40,41]. In the past, EPF have been isolated from mycosed EAB adults, larvae and galleries [42,43,44,45,46]. Some strains, especially those belonging to Beauveria bassiana (Bals.-Criv.) Vuill., have been investigated in order to explore their pathogenicity and virulence against EAB adults and larvae [43,47,48,49,50]. Nonetheless, there are no previous studies that have screened entomopathogenic fungi against EAB eggs. The egg stage of different insect pests has been targeted by other studies whose objective was to evaluate the impact of entomopathogenic fungi on this life stage [51,52,53,54,55,56,57,58].
The emerald ash borer spends most of its life cycle under the bark where the larvae feed on the inner phloem, cambium, and outer xylem, tunnelling characteristic serpentine galleries during one (univoltine) or two (semivoltine) growing seasons [59,60,61]. After this, larvae overwinter as J-shaped pre-pupae until spring when molting into pupa occurs before adults emergence [59,61,62]. After a few weeks of maturation feeding and mating, female adults lay eggs within bark cracks or beneath bark flakes [3,60]. Finally, neonate larvae bore through bark to start feeding on phloem and to make galleries under the bark. Given the cryptic life cycle of EAB under the bark, being able to interfere with the viability of the eggs laid on the exterior of the tree using entomopathogenic fungi could be a potential way to lower the pest population. In a recent study, Held et al. [44] found several entomopathogenic fungi (EPF) species associated with EAB galleries in Minnesota, belonging to the genera Purpureocillium, Beauveria, Clonostachys, Lecanicillium, Akanthomyces, Cordyceps, Microcera, Tolypocladium, and Pochonia [44]. Several authors have reported that isolates of EPF are generally more virulent to the species of insects from which they are originally obtained and that native, locally adapted isolates are more effective than non-native strains [63,64,65,66,67].
The aim of this study was to screen and select locally adapted EAB-associated entomopathogenic fungal strains with ovicidal effect on EAB eggs under laboratory conditions.

2. Materials and Methods

Pathogenicity on EAB eggs was evaluated for 10 fungal strains belonging to Ascomycota. Nine of the fungal strains were previously obtained from EAB galleries by Held et al. [44], and one was the GHA B. bassiana strain (Table 1).

2.1. Inoculum Preparation

In a biosafety cabinet, pure cultures of each fungal strain were transferred from slant tubes to fresh potato dextrose agar media (PDA) (39 g Oxoid™ PDA, 1 L distilled water). Conidia from fifteen-day-old cultures of each strain were harvested by adding 3–4 mL of a sterile 0.02% Tween 20 water solution to each colony, gently scrapping the surface of the colony with a sterile L-shaped plastic spatula, and by recovering the conidial suspension in a sterile glass tube. The conidial suspension was vigorously vortexed and 250 µL of each spore suspension were plated onto 3 plates filled with half-strength PDA (19.5 g Oxoid™ PDA, 7.5 gr Difco™ Bacto agar, 1 L distilled water) and 3 plates with corn meal agar (CMA) (17 g of Oxoid™ CMA, 1 L distilled water). Plates were sealed with Parafilm® and incubated at 23 °C for 15 days. After incubation, aerial conidia were harvested following the methodology previously described. The spore suspension of each fungal strain was collected in a sterile beaker and stirred continuously on a magnetic stirrer while its concentration was measured and adjusted. The concentration of all fungal spore suspensions was adjusted to 1.0 × 108 conidia/mL with a hemocytometer. Each fungal inoculum was used right after its concentration was adjusted. Spore viability was evaluated for every fungal treatment by plating an aliquot of 250 µL of a 10−3 or 10−4 dilution from a serial dilution onto water–agar plates. Two plates per fungal strain were incubated at 23 °C for 24 h, and germination was evaluated for 300 spores following Lopes et al. [68]. The germination percentage was calculated as the total number of germinated conidia/total number of conidia ×100. Conidia were considered viable if the germinative tube reached ¾ of the size of the spore.

2.2. Experimental Setting and Egg Inoculation

Fresh (1–4 days old) EAB eggs laid on filter paper were provided by the United States Department of Agriculture’s Animal and Plant Health Inspection Service (APHIS), Plant Protection and Quarantine (PPQ) EAB Parasitoid Rearing Facility in Brighton, MI. Individual eggs, laid on filter paper, were examined under a stereomicroscope to discard damaged eggs, and placed in the wells of 24-well plates. Whenever two or more eggs were not able to be separated, they were put together into a well but were evaluated individually. The four corner wells of each plate were filled with a paper towel plug saturated with distilled water to help keep relative humidity high inside the plate. Five plates containing a total of 100 eggs (20 eggs per plate) were randomly assigned to each treatment (Figure 1) (fungal strain, negative control, or standard strain) where a 1 µL droplet of the fungal spore suspension or the water control was applied to each individual egg. A sterile distilled water solution of 0.02% of Tween 20 (used as negative control) and the GHA B. bassiana strain (used as a reference strain and positive control, respectively) were included in each bioassay. The only exception to that were the first two bioassays that did not include the positive control (Table 2). After all the eggs from a plate were inoculated, the lid was misted with distilled water so that it was covered by very fine droplets of water. The plate was then sealed with a double layer of Parafilm®, and all of the plates from the bioassay were incubated at 23 °C within a plastic bin. Eggs within wells were examined under a stereomicroscope, and larval hatching was recorded after 20 to 30 days post-treatment as the hatching time for EAB neonates at room temperature ranges from 12 to 19 days [69,70]. During the bioassay, relative humidity within the plates was kept high by rehydrating the paper towel plugs until saturation and by misting the lid of the plate, as previously described, once every 5–8 days. Each fungal treatment was repeated at least twice on different dates (repetition) to ensure data reproducibility. Finally, before dismantling each bioassay, all non-hatched eggs from a treatment were transferred to PDA plates to evaluate inoculum recovery. Additionally, a maximum of 10 neonate larvae per plate (total of 50 per treatment) were also transferred to PDA media to evaluate potential horizontal transfer from conidia on the eggs to the newly hatched larvae. Both eggs and larvae were surface sterilized by submerging them in a 0.5% bleach solution for 5 s and rinsing them twice in sterile distilled water before transferring them to the media plates (adapted from [71,72]). Identification of each fungal inoculum was undertaken based on colony morphological characteristics.

2.3. Statistical Analysis

Statistical analysis and data visualization were performed using the R statistical software (R core team, v.4.22) [73], R packages lme4 [74], emmeans [75], multcomp [76] and dyplr [77] for data analysis, and tidyverse [78], multcompView [79] and ggplot2 [80] for data visualization.
Data from each experimental repetition were analyzed separately. To analyze the effects of treatments on the response variable “egg hatching” (i.e., did a larva hatch from an egg or not), a generalized linear mixed model (GLMM) was fitted using a logit function for the binomial variable (egg hatching) in which fixed effect was attributed to the treatment (fungal strain or water control) and the effect of the plates was incorporated as a random effect. When the effect of treatments was significant, a multiple pairwise comparison was carried out using Tukey HSD method at 5% significance (p < 0.05) to separate the effect of the fungal strains using the “emmeans” package. Given that all fungal treatments had at least two experimental repetitions (the experiment was repeated on different days), in order to compare results, negative and positive controls from the experimental repetitions were compared separately. This was achieved by fitting a generalized linear mixed model (GLMM) using a logit function for the binomial variable (egg hatching) in which the fixed effect was attributed to the date of the experiment and the effect of the plates was incorporated as a random effect.
Inoculum recovery percentage from eggs was calculated as [(total non-hatched egg with confirmed mycosis/total of plated non-hatched eggs) × 100] and, similarly, for hatched larva as [(total neonate larvae with confirmed mycosis/total of plated neonate larvae) × 100]. This was calculated for each of the five 24-well plates in which each assay was distributed and was presented as a range with the minimum and maximum values of inoculum recovery reached by each fungal treatment. Additionally, a mean inoculum recovery percentage was calculated considering the average result of all plates in all bioassays, in which the fungal treatment was included.

3. Results

A total of eight different bioassays were performed during this study so that all fungal treatments were tested at least twice against EAB eggs. The viability of spores was considered acceptable for all bioassays as germination rate was over 90%.
During the incubation period, several fungus-treated eggs developed mycelia that covered the whole egg surface (Figure 2B,D). Similarly, some neonate larvae also became infected and developed mycelia (Figure 2C,D).

3.1. Negative and Positive Controls Comparison

Results from the GLMM for the comparison of egg hatching percentage for both positive (GHA reference strain) and negative (water + Tween) controls among bioassays were found to lead to similar results (Figure 3 and Figure 4). This allows a confident comparison of the effect of each fungal strain on egg viability, among bioassays. The reference strain (positive control) included in the “October 2021 I” bioassay did not differ significantly from the negative control, so results from that repetition were not considered for further analysis (Figure S1).

3.2. Ovicidal Effect Evaluation

Results from the GLMM analysis and post hoc Tukey test to contrast the ability of each fungal strain to prevent eggs from hatching were variable, although seven fungal strains significantly prevented EAB eggs from hatching in at least one of the bioassays in which they were included. Tukey test results are summarized in Table 3 and are represented as boxplots of the percentage of unhatched eggs for each bioassay in Figure 5.
The percentage of unhatched eggs for the reference strain B. bassiana GHA was significantly different from negative control in all bioassays, except for “October 2021 I,” which was previously discarded (Table 3, Figure 5). The percentage of unhatched eggs significantly differed from that of the negative control when eggs were treated with A. muscarius 48-27 in all assays, while the percentage of unhatched eggs under A. muscarius 59-16 treatment was significantly different from the control in two bioassays but not in a third assay (Table 3, Figure 5). Unlike the reference GHA strain, B. bassiana 37-9 showed no significant differences when compared with the negative control in any of the bioassays. Beauveria brongniartii 51-13 did not show significant differences in terms of their unhatched egg percentage when compared with control (Table 3, Figure 5). The percentage of unhatched eggs after Cordyceps farinosa 58-18 treatment was significantly higher than the negative control on three bioassays but not on a fourth (Table 3, Figure 5). Lecanicillium longisporum and L. psalliotae showed significant differences when compared with control in all bioassays, while Purpureocillium lilacinum 44-2 had no effect on the percentage of unhatched eggs and P. lilacinum 1-9 showed inconsistent results with no significant differences in two of the bioassays but a significantly higher proportion of unhatched eggs when compared with that of control in one of the bioassays (Table 3, Figure 5).

3.3. Inoculum Recovery

To fulfill Koch’s postulates, inoculum recovery was evaluated for all unhatched eggs and a fraction of the neonate larvae. There are no inoculum recovery results for the “May 2021” bioassay, B. brongniartii 51-13, nor for the negative control in the “Dec 2020” bioassay, in which the recovery of the inoculum failed. The range between minimum and maximum values of inoculum recovery for all treatments and the mean percentage of inoculum recovery are shown in Figure 6 and Figure 7, and Table 4. For each treatment, inoculum recovery results from all bioassays in which that treatment was included are considered. Inoculum recovery for each independent bioassay (separated by date) is shown in Figures S2 and S3 and Table S1. No entomopathogenic fungi were recovered from the negative control in any of the bioassays. All fungal treatments, even those that did not differ significantly from the control, reached a maximum inoculum recovery value of 100% for both egg and larvae, meaning that for at least one of the 24-well plates (out of five per treatment), the inoculum was recovered from all eggs or larvae plated in media culture (Figure 6, Table 4, Figure S3, Table S1). Regarding the mean inoculum recovery percentage, five strains had a mean recovery percentage of 90% or higher for eggs (Figure 7). Similarly, six strains had a mean recovery percentage of 90% or higher for larvae. Even fungal strains that did not show a significant ovicidal effect on eggs, did show high mean inoculum recovery percentages (Figure 7, Table 4, Figure S3, Table S1). This means that the unhatched eggs were effectively colonized by the inoculated fungus in most cases and that the fungi were able to colonize the neonate larva, presumably by contact of the larva with the spores when emerging from the egg.

4. Discussion

During this study, ten fungal strains were tested for their ovicidal effect on EAB eggs and four of them (A. muscarius 48-27, B. bassiana GHA, L. longisporum 66-14 and L. psalliotae 59-2) consistently showed significant ovicidal effects. Those strains also showed a high percentage of inoculum recovery from both surface-sterilized eggs and surface-sterilized neonate larvae. This suggests that the unhatched eggs were effectively colonized by the inoculated fungus but also that the fungi were able to colonize the neonate larva, presumably while in the egg or by contact of the larva with the spores when emerging from the egg. In the case of the inoculated eggs, by reisolating the applied fungus, Koch’s postulates were fulfilled. Fungal infection of neonate larvae was confirmed by high percentages of inoculum recovery and by observation of mycelial growth on larvae. No observations of larval viability or performance were made as the experimental setting was not designed to evaluate pathogenicity and virulence of the fungal treatments on larvae. Other studies have confirmed neonate larvae infection and mortality after the treatment of armyworm eggs with different EPF strains, though the authors did not speculate about the mode of action of the infection process. Additionally, nutritional factors have been implicated to be important modulators of the insect immune response and may thus affect the efficacy of entomopathogenic fungi and other biocontrol agents [81]. Future work should include experimental settings that allow the study of neonate larvae survival after fungal treatment and possible sublethal effects on their performance. Sublethal effects, including prolonged larval development periods, have been previously reported for more advanced EAB larval instars [48].
The experimental setting adjusted for this study allowed the screening and selection of EAB-associated entomopathogenic fungi with an ovicidal effect that could potentially be included in future field applications. While some strains showed significant ovicidal effects every time they were tested, and others consistently had no significant lethal effect on eggs, there were some strains that showed inconsistency in their results regarding their ovicidal ability, as observed in Table 3 and Figure 5. Akanthomyces muscarius 59-16 had significant ovicidal effect two of the three times it was tested. Nonetheless, in November 2021, when it did not have a significant ovicidal effect when compared with the negative control, a high percentage of inoculum was recovered from eggs and larvae (97% and 78%, respectively; Figure S2 and Table S1). Presumably, the fungus was able to infect and colonize the eggs but with a decreased lethal effect. Similarly, C. farinosa 58-18 treatment had a significant lethal effect on eggs in two of the bioassays, but this was not significant in the October 2021 II trial. In this case, the inoculum recovery percentage from eggs was lower for the October 2021 II trial vs. those from the October 2020 and June 2022 bioassays, in which the ovicidal effect was significant (47% vs. 61% and 87%, respectively; Figure S2 and Table S1). Finally, P. lilacinum 1-9 also showed inconsistent results, with no significant differences in two of the bioassays (May 2021 and October 2021 II) but showing a significant ovicidal effect in the June 2022 bioassay. In these cases, inoculum recovery was high for both eggs and larvae in October 2021 II (88% and 98%, respectively) and in June 2021 (92% and 96%, respectively). In all of these cases, we can confirm that the fungal spores were able to colonize the eggs because the inoculum was recovered from surface-disinfected substrates at high percentages; however, in some cases, these fungi were not able to reach a significant ovicidal effect when compared with the negative control. The egg stage of insects is considered to be more resistant to infection by entomopathogenic fungi than other developmental stages [52]. Although there is relatively less published information on egg susceptibility to mycosis [52,82], there are several studies that report an ovicidal effect of EPF on eggs of different insects and arthropod pests [51,52,54,55,58,82,83,84,85,86,87]. Some of these studies have confirmed the propagation of mycelium inside the infected eggs [51,52,83,85] and the development of mycelium and conidia on the egg surface post-inoculation [54,85]. Despite this, there is not a complete understanding of the mechanisms through which the fungal propagules colonize and affect the embryo [51,52,83,85]. Relative humidity has proven to be a key factor for entomopathogenic fungi performance [54,86,88,89,90]. During this study, high relative humidity was reached by the application of water, as a mist, to the inner lid of the 24-cell plates at regular intervals. Nonetheless, the humidity provided may not have been enough for some fungal strains that might be more sensitive to humidity fluctuations. During any screening process aimed at selecting good candidates for a potential field application, regardless of the cause, inconsistent ovicidal results should be a criterion for discarding the isolate.
Virulence among different strains within B. bassiana, A. muscarius and P. lilacinum showed variability, with some strains showing a significant ovicidal effect and others showing no effect. Virulence variation among different strains of the same species is not unusual and has been reported previously for several entomopathogenic fungal genera, including Beauveria, Akanthomyces, and Paecilomyces [87,91,92,93]. By the end of this study, locally adapted EAB-associated entomopathogenic fungal strains were selected for their significant ovicidal effect against EAB eggs under laboratory conditions. According to Goettel et al. (2005) [91], this is of particular importance, as selecting virulent strains is key for biological control applications, given the possible variation in virulence to hosts among strains of the same species. Additionally, the fact that these isolates are locally adapted represents an advantage in moving forward toward field applications at the local scale [87].

5. Conclusions

Several fungal strains evaluated during this study consistently showed a high ovicidal effect on EAB eggs. As a first round of screening, the selection criterion was the significant ovicidal effect compared with a negative control. In the future, a second round of screening under laboratory conditions appears warranted in order to select a more virulent strain, achieved by determining CL50 and LT50 or by running a Cox regression analysis based on hazard ratio analysis [92,94,95] to compare virulence against strains, before testing the best strains in the field. The selected strains should be included in field assays in the future by applying fungal spore suspensions against ash tree bark, just before the females of EAB begin laying their eggs on bark cracks, to maximize the fungal strains’ performances. Given the cryptic nature of the larval stage of EAB, which lives most of its life cycle under the bark, the possibility of altering the insect´s life cycle by disrupting the egg stage through microbial control represents a potential management opportunity that should be explored. Moreover, inoculum production for future field studies should include strategies to protect fungal spores from environmental conditions [96,97,98,99,100]. Additionally, the optimal time during the day to undertaken the application, the time with the highest RH, should be identified. The high levels of inoculum recovery from neonate larvae observed during the bioassays demonstrate that, even after emergence, larvae were infected with fungal spores. Although the experimental design was not established to evaluate the effect of the fungal treatments on larvae viability, some negative effects on larval survival or performance could be expected. Thus, future bioassays should also focus on evaluating the effect of EPF on neonate larvae. As part of any IPM strategy, the expectations are that each applied practice contributes to the reduction of the impact caused by the targeted pest though their combined effect, thus reaching tolerable pest levels with minimal environmental side effects [25,26]. The microbial control of EAB with entomopathogenic fungi has been explored in the past but none of these past studies focused on egg mortality due to fungi. This study represents the first time that the ovicidal effects of entomopathogenic fungi have been evaluated against EAB. The contribution of this approach to EAB control within an IPM program must now be tested through field evaluations.

Supplementary Materials

The following supporting information can be downloaded at https://www.mdpi.com/article/10.3390/f15122170/s1, Figure S1. Boxplot representing percentage of unhatched eggs from “October 2021 I” bioassay. Same letter and colors represent no statistical differences between treatments from glmm and Tukey post hoc analysis (p < 0.05); Figure S2. Range of percentage of inoculum recovery for each independent bioassay; Figure S3. Mean inoculum recovery percentage for each independent bioassay; Table S1. Mean inoculum recovery percentage from eggs and larvae separated by date.

Author Contributions

Conceptualization: S.S., R.A.B., K.E.B. and B.W.H.; methodology, S.S., D.N.S., K.E.B., R.A.B. and B.W.H.; investigation, S.S. and D.N.S.; software, S.S.; formal analysis, S.S.; resources, S.S., B.W.H. and R.A.B.; data curation, S.S. and R.A.B.; writing—original draft preparation, S.S.; writing—review and editing, S.S., R.A.B., B.W.H., K.E.B. and D.N.S.; visualization, S.S.; supervision, R.A.B.; project administration, R.A.B. and B.W.H.; funding acquisition, R.A.B., B.W.H. and K.E.B. All authors have read and agreed to the published version of the manuscript.

Funding

This research was funded by the Minnesota Invasive and Terrestrial Plant and Pests Center, University of Minnesota, and USDA Hatch project MIN22-089.

Data Availability Statement

The data in this study are available within the article.

Acknowledgments

The authors would like to acknowledge Robert Venette, US Forest Service, and Brian Aukema, University of Minnesota, for their contributions to project improvements and guidance in statistical methodology. The authors specially thank the United States Department of Agriculture’s Animal and Plant Health Inspection Service (APHIS), Plant Protection and Quarantine (PPQ) EAB Parasitoid Rearing Facility in Brighton, MI for the fresh EAB egg provision. The authors also acknowledge Owen Geier, Ada Fitz Axen and Amelia Lochridge for their laboratory assistance.

Conflicts of Interest

The authors declare no conflicts of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript; or in the decision to publish the results.

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Figure 1. Experimental set-up for egg inoculation. (A) Twenty-four-well plates with four corner wells of each plate filled with a paper towel plug saturated with distilled water. (B) EAB eggs placed individually in each well.
Figure 1. Experimental set-up for egg inoculation. (A) Twenty-four-well plates with four corner wells of each plate filled with a paper towel plug saturated with distilled water. (B) EAB eggs placed individually in each well.
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Figure 2. (A) Larva hatching from healthy egg, negative control treatment. (B) Unhatched egg colonized by Akanthomyces muscarius 48-27. (C) Larva showing signs of mycosis due to Akanthomyces muscarius 48-27. (D) Eggs and larva showing signs of mycosis due to Lecanicillium psalliotae 59-2. (E) Akanthomyces muscarius 48-27 sporulating on larva. (F) Inoculum recovery from Cordyceps farinosa 58-18. Bar = 0.5 cm in (A–C,E) and 1 cm in (D).
Figure 2. (A) Larva hatching from healthy egg, negative control treatment. (B) Unhatched egg colonized by Akanthomyces muscarius 48-27. (C) Larva showing signs of mycosis due to Akanthomyces muscarius 48-27. (D) Eggs and larva showing signs of mycosis due to Lecanicillium psalliotae 59-2. (E) Akanthomyces muscarius 48-27 sporulating on larva. (F) Inoculum recovery from Cordyceps farinosa 58-18. Bar = 0.5 cm in (A–C,E) and 1 cm in (D).
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Figure 3. Boxplot representing the hatching percentage of positive controls among bioassay dates. Different letters represent statistical differences between treatments from the GLMM and from Tukey post hoc analysis (p < 0.05).
Figure 3. Boxplot representing the hatching percentage of positive controls among bioassay dates. Different letters represent statistical differences between treatments from the GLMM and from Tukey post hoc analysis (p < 0.05).
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Figure 4. Boxplot representing the hatching percentage of negative controls among bioassay dates. Different letters represent statistical differences between treatments from the GLMM and Tukey post hoc analysis (p < 0.05).
Figure 4. Boxplot representing the hatching percentage of negative controls among bioassay dates. Different letters represent statistical differences between treatments from the GLMM and Tukey post hoc analysis (p < 0.05).
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Figure 5. Boxplot representing percentage of unhatched eggs among bioassay dates. Different letters and colors represent statistical differences between treatments from GLMM and Tukey post hoc analysis (p < 0.05).
Figure 5. Boxplot representing percentage of unhatched eggs among bioassay dates. Different letters and colors represent statistical differences between treatments from GLMM and Tukey post hoc analysis (p < 0.05).
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Figure 6. Inoculum recovery range for each treatment for both non-hatched eggs and neonate larvae. Minimum and maximum values represented by green and red circles, respectively.
Figure 6. Inoculum recovery range for each treatment for both non-hatched eggs and neonate larvae. Minimum and maximum values represented by green and red circles, respectively.
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Figure 7. Mean inoculum recovery percentage for each treatment.
Figure 7. Mean inoculum recovery percentage for each treatment.
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Table 1. Fungal species and strains ID codes used for egg inoculation studies.
Table 1. Fungal species and strains ID codes used for egg inoculation studies.
Fungal SpeciesStrain IDGenBank
Akanthomyces muscarius (Petch) Spatafora, Kepler and B. ShresthaEAB 48-27MT777299.1
Akanthomyces muscarius (Petch) Spatafora, Kepler and B. ShresthaEAB 59-16PQ578688
Beauveria bassiana (Bals.-Criv.) Vuill.EAB 37-9PQ578689
Beauveria brongniartii (Sacc.) PetchEAB 51-13MT777312.1
Beauveria bassiana (Bals.-Criv.) Vuill.GHA---
Cordyceps farinosa (Holmsk.) Kepler, B. Shrestha and SpataforaEAB 58-18MT777317.1
Lecanicillium longisporum (Treschew) Zare and W. GamsEAB 66-14MT777366.1
Lecanicillium psalliotae (Treschew) Zare and W. GamsEAB 59-2PQ578690
Purpureocillium lilacinum (Thom) Luangsa-ard, Houbraken, Hywel-Jones and SamsonEAB 1-9PQ578691
Purpureocillium lilacinum (Thom) Luangsa-ard, Houbraken, Hywel-Jones and SamsonEAB 44-2PQ578692
Table 2. Fungal treatments included on each bioassay and date of treatment.
Table 2. Fungal treatments included on each bioassay and date of treatment.
Oct. 2020Dec. 2020May 2021Oct. 2021 IOct. 2021 IINov. 2021Dec. 2021Jun. 2022
A. muscarius 48-27 • ••
A. muscarius 59-16• • •
B. bassiana 37-9 • ••
B. bassiana GHA ••••••
B. brongniartii 51-13 • •
C. farinosa 58-18• • • •
L. longisporum 66-14• • •
L. psalliotae 59-2• •
P. lilacinum 1-9 • • •
P. lilacinum 44-2 • •
Table 3. p-Values and significance of Tukey post hoc comparison of each fungal treatment’s ability to prevent eggs from hatching vs. negative control, for each inoculation assay.
Table 3. p-Values and significance of Tukey post hoc comparison of each fungal treatment’s ability to prevent eggs from hatching vs. negative control, for each inoculation assay.
Fungal TreatmentBioassay Date
p-Value
Oct. 2020Dec. 2020May 2021Oct. 2021 IINov. 2021Dec. 2021Jun. 2022
A. muscarius 48-27 1.59 × 10−6 *** 5.67 × 10−14 ***8.22 × 10−14 ***
A. muscarius 59-16 6.3 × 10−13 *** 0.002 *** 0.188
B. bassiana 37-9 0.833 0.663
B. bassiana GHAn/an/a0.001 ***0.029 *0.024 *5.82 × 10−7 ***7.45 × 10−4 ***
B. brongniartii 51-13 0.753 0.264
C. farinosa 58-18 4.7 × 10−14 *** 3.86 × 10−5 ***0.322 1.21 × 10−4 ***
L. longisporum 66-14 4.85 × 10−14 *** 5.42 × 10−14 ***
L. psalliotae 59-2 3.76 × 10−5 *** 2.9 × 10−14 ***
P. lilacinum 1-9 0.1460.565 7.57 × 10−10 ***
P. lilacinum 44-2 0.753
Significance codes: ‘***’ p < 0.001, ‘*’ p < 0.05.
Table 4. Inoculum recovery percentage for all treatments. min: minimum value of inoculum recovery, max: maximum value of inoculum recovery, and mean: mean value of inoculum recovery.
Table 4. Inoculum recovery percentage for all treatments. min: minimum value of inoculum recovery, max: maximum value of inoculum recovery, and mean: mean value of inoculum recovery.
TreatmentSubstrateMinMaxMean
A. muscarius_48-27egg5810084
A. muscarius_48-27larva8010097
A. muscarius_59-16egg5010090
A. muscarius_59-16larva5010079
B. bassiana_37-9egg2010076
B. bassiana_37-9larva6010092
B. bassiana_GHAegg010075
B. bassiana_GHAlarva5610087
B. brongniartii_51-13egg100100100
B. brongniartii_51-13larva100100100
C. farinosa_58-18egg010066
C. farinosa_58-18larva010063
Controlegg000
Controllarva000
L. longisporum_66-14egg1410072
L. longisporum_66-14larva4010083
L. psalliotae_59-2egg8610097
L. psalliotae_59-2larva100100100
P. lilacinum_1-9egg3810090
P. lilacinum_1-9larva8310097
P. lilacinum_44-2egg100100100
P. lilacinum_44-2larva100100100
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Simeto, S.; Held, B.W.; Showalter, D.N.; Bushley, K.E.; Blanchette, R.A. Ovicidal Effect of Entomopathogenic Fungi on Emerald Ash Borer, Agrilus planipennis Fairmaire, Eggs. Forests 2024, 15, 2170. https://doi.org/10.3390/f15122170

AMA Style

Simeto S, Held BW, Showalter DN, Bushley KE, Blanchette RA. Ovicidal Effect of Entomopathogenic Fungi on Emerald Ash Borer, Agrilus planipennis Fairmaire, Eggs. Forests. 2024; 15(12):2170. https://doi.org/10.3390/f15122170

Chicago/Turabian Style

Simeto, Sofía, Benjamin W. Held, David N. Showalter, Kathryn E. Bushley, and Robert A. Blanchette. 2024. "Ovicidal Effect of Entomopathogenic Fungi on Emerald Ash Borer, Agrilus planipennis Fairmaire, Eggs" Forests 15, no. 12: 2170. https://doi.org/10.3390/f15122170

APA Style

Simeto, S., Held, B. W., Showalter, D. N., Bushley, K. E., & Blanchette, R. A. (2024). Ovicidal Effect of Entomopathogenic Fungi on Emerald Ash Borer, Agrilus planipennis Fairmaire, Eggs. Forests, 15(12), 2170. https://doi.org/10.3390/f15122170

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