Accumulation of Fungal Pathogens Infecting the Invasive Spotted Lanternfly, Lycorma delicatula.

In the eastern United States, populations of the invasive spotted lanternfly, Lycorma delicatula, are abundant and spreading. Four species of naturally occurring entomopathogenic fungi have previously been reported as infecting these planthoppers, with two of these causing epizootics. Nymphal- and adult-stage lanternflies in Pennsylvania and New York were surveyed for entomopathogenic fungal infections from October 2021 to November 2023, and assays were conducted to confirm the pathogenicity of species that were potentially pathogenic. Beauveria bassiana was the most abundant pathogen, but we report an additional 15 previously unreported species of entomopathogenic fungi infecting spotted lanternflies, all in the order Hypocreales (Ascomycota). The next most common pathogens were Fusarium fujikuroi and Sarocladium strictum. While infection prevalence by species was often low, probably impacted to some extent by the summer drought in 2022, together these pathogens caused a total of 6.7% mortality. A significant trend was evident over time within a season, with low levels of infection among nymphs and higher infection levels in mid- and late-stage adults, the stages when mating and oviposition occur.

Cryptic diversity and virulence of Beauveria bassiana recovered from Lycorma delicatula (spotted lanternfly) in eastern Pennsylvania.

The entomopathogenic fungus Beauveria bassiana is cosmopolitan and known to infect a variety of sap-sucking pests like aphids, mealybugs, and scales in the order of Hemiptera. In Fall 2017, spotted lanternfly (SLF) adults killed by the fungal entomopathogen B. bassiana were found in Berks County, Pennsylvania. In 2018-2020 we collected SLF and nearby non-target insects killed by Beauveria spp. from 18 field sites in southeastern Pennsylvania. We identified 159 Beauveria isolates from SLF and six isolates from non-targets. Five isolates of B. bassiana and one isolate of B. brongniartii were identified from the non-targets. Based on sequence data from the nuclear B locus (Bloc) intergenic region, all the isolates from SLF were identified as B. bassiana, but there were 20 different strains within this species, grouped into two clades. Three B. bassiana strains (A, B, and L) were found in most field sites and were the most prevalent. Representative isolates for these three strains were used in laboratory bioassays and were compared to a commercial B. bassiana strain (GHA). Strain B was inferior to A, L, and GHA against nymphs; strains A and L had greater efficacy than B and GHA against adults. We also quantified conidial production on SLF cadavers. This paper discusses the diversity of these B. bassiana strains in SLF populations and implications for biological control of this abundant invasive.

Batkoa major infecting the invasive planthopper Lycorma delicatula.

The entomopathogenic fungi Batkoa major and Beauveria bassiana caused co-epizootics in populations of invasive spotted lanternflies, Lycorma delicatula, in 2018 in northeastern North America. Although first described from North America in 1888, the biology and ecology of Batkoa major had not been studied since that time. This entomophthoralean fungus found infecting L. delicatula in 2018 produces conidia and rhizoids similar in appearance to the original description. We conducted laboratory bioassays to investigate infection of different ages and sexes of these planthoppers, inoculating via showered conidia. All nymphs, and male and female adults were susceptible, dying in 4.3-6.7 days. Adult males died more quickly than adult females or fourth instars. Batkoa major grew out of cadavers of adult males more frequently than adult females or fourth instar nymphs. Rhizoids that provide attachment of cadavers to substrates were produced from adult cadavers more frequently than conidia. Resting spores were not observed in vivo or in vitro in the lab, or in the field.

Discovery of two hypocrealean fungi infecting spotted lanternflies, Lycorma delicatula: Metarhizium pemphigi and a novel species, Ophiocordyceps delicatula.

In the eastern United States, populations of the invasive spotted lanternfly, Lycorma delicatula, can be infected by native fungal entomopathogens, including Batkoa major and Beauveria bassiana. In some areas of southeastern Pennsylvania, localized population collapses have been observed in L. delicatula populations to be caused by these pathogens. Two additional fungal pathogens were discovered infecting L. delicatula at low levels, and these were identified as Metarhizium pemphigi and Ophiocordyceps delicatula, a new species that has not been previously described. Therefore, four species of native entomopathogenic fungi have now been documented infecting this abundant, invasive planthopper that is spreading in the United States.

Optimizing Application Rates of Metarhizium brunneum (Hypocreales: Clavicipitaceae) Microsclerotia for Infecting the Invasive Asian Longhorned Beetle (Coleoptera: Cerambycidae).

The Asian longhorned beetle (Anoplophora glabripennis [Motschulsky]) is an invasive wood-boring beetle that threatens urban trees and forests in North America and Europe. The entomopathogenic fungus Metarhizium brunneum Petch strain F52 can infect and kill A. glabripennis adults. Products containing this fungus were available for commercial use in the United States but not registered for Asian longhorned beetle. This study tested different formulations and application rates of M. brunneum F52 microsclerotial granules for their potential development for management of A. glabripennis adults. Three application rates of M. brunneum microsclerotial granules relative to a 1× formulation from previous experiments (0.03 g/cm2; 2× = 0.06 g/cm2 and 3× = 0.09 g/cm2) were exposed on tree trunks for 4-wk periods during May-September. Increased application rates had better retention (% of initial g applied) than the 1× rate, rather than greater weathering loss. Microsclerotia at the 2× application produced 5.05 × 106 conidia/cm2, which was 18 times more conidia than the 1× application. Since A. glabripennis is under active eradication, bioassays with adult beetles were carried out in a quarantine laboratory, using the formulation samples from field exposures. The 2× application resulted in faster beetle mortality. The 3× and 2× rates were not significantly different in retention of the formulation, conidial production, or mortality, but 2× produced the most conidia per gram applied (3.92 × 109 conidia/g). An augmented formulation containing 70% M. brunneum by weight, rather than 50%, produced significantly more conidia and faster beetle mortality than the 50% formulation.

Applying a Selection Experiment to Test for Fitness Costs of Bt Resistance in Western Corn Rootworm (Coleoptera: Chrysomelidae) and the Effect of Density on Fitness Costs.

Western corn rootworm, Diabrotica virgifera virgifera LeConte, is a serious pest of corn and is often managed with transgenic corn producing insecticidal toxins from the bacterium Bacillus thuringiensis (Bt). This pest has developed field-evolved resistance to all commercially available Bt traits, beginning with Cry3Bb1 in 2009. Fitness costs may accompany Bt resistance, where individuals with alleles for Bt resistance have reduced fitness on non-Bt corn compared to Bt-susceptible individuals. In conjunction with non-Bt refuges, fitness costs can delay the evolution of Bt resistance. Importantly, ecological factors may affect the presence and magnitude of fitness costs. For western corn rootworm, available data suggest that fitness costs of Bt resistance may be present in some cases. Using two Cry3Bb1-resistant western corn rootworm strains (Hopkinton and Cresco), a fitness-cost experiment was performed by rearing rootworm in the absence of Bt for six generations to test for fitness costs of Cry3Bb1 resistance and the effect of larval rearing density on fitness costs. Fitness costs were detected for both strains; however, strains were still resistant to Cry3Bb1 corn at the end of the experiment. Cresco experienced a greater loss of resistance at low versus high density, but no effect of density was detected in Hopkinton. Our study shows that fitness costs can accompany Bt resistance in western corn rootworm and may be more pronounced under low larval density. Even though fitness costs were present, it appears that rootworm populations may remain resistant to Cry3Bb1 corn for years after resistance has evolved.

Applications of Beauveria bassiana (Hypocreales: Cordycipitaceae) to Control Populations of Spotted Lanternfly (Hemiptera: Fulgoridae), in Semi-Natural Landscapes and on Grapevines.

Spotted lanternfly, Lycorma delicatula (White), is an invasive Asian insect that was initially found in Berks County, Pennsylvania, in 2014. As of early 2020, this pest had been found in five more eastern states and it is expected to continue to expand its geographical range. Lycorma delicatula is highly polyphagous but seems to prefer tree-of-heaven, Ailanthus altissima. However, grape growers in Pennsylvania have reported significant damage and loss of vines caused by L. delicatula adults. In fall 2018, two fungal entomopathogens (Beauveria bassiana and Batkoa major) drove localized collapses in L. delicatula populations in Berks County, Pennsylvania. In 2019, we tested applications of a commercialized mycoinsecticide based on B. bassiana strain GHA on L. delicatula populations in a public park in southeastern Pennsylvania. A single application of B. bassiana reduced fourth instar nymphs by 48% after 14 d. Applications of B. bassiana to L. delicatula adults in the same park resulted in 43% mortality after 14 d. Beauveria bassiana spores remained viable on foliage for 5-7 d after spraying. We also conducted semi-field bioassays with B. bassiana GHA (formulated as BoteGHA and Aprehend) and another mycoinsecticide containing Isaria fumosorosea Apopka Strain 97 against L. delicatula adults feeding on potted grapes. All the mycoinsecticides killed ≥90% of adults after 9 d using direct applications. Aprehend killed 99% of adults after 9 d with exposure to residues on sprayed grapes. These data show that fungal entomopathogens can help to suppress populations of L. delicatula in agroecosystems and natural areas.

Virulence of Commercialized Fungal Entomopathogens Against Asian Longhorned Beetle (Coleoptera: Cerambycidae).

Nine strains of five species of entomopathogenic hypocrealean fungi were tested against adults of the Asian longhorned beetle, Anoplophora glabripennis (Motschulsky). These strains have been developed as commercial biopesticide products in the United States, Brazil, South Korea, or the European Union (EU). Metarhizium anisopliae (Metschnikoff) (Hypocreales: Clavicipitaceae) ESALQ E-9 and Metarhizium brunneum (Petch) F52 (formerly M. anisopliae F52) (Hypocreales: Clavicipitaceae) killed 100% of treated beetles with the shortest survival times. Virulence differed among the five strains of Beauveria bassiana (Balsamo) (Hypocreales: Cordycipitaceae) tested, ranging from 0 to 77.3% mortality within 28 d. Two Isaria fumosorosea (Wize, 1904) (Hypocreales: Cordycipitaceae) (formerly Paecilomyces fumosoroseus) strains and the Lecanicillium muscarium (Petch) Zare & Gams (Hypocreales: Cordycipitaceae) strain used in Mycotal were not pathogenic to A. glabripennis adults. Within the entomopathogenic fungi tested, the Metarhizium strains may be the most appropriate for further evaluation.

Impacts of Metarhizium brunneum F52 infection on the flight performance of Asian longhorned beetles, Anoplophora glabripennis.

The Asian longhorned beetle (ALB), Anoplophora glabripennis, is an invasive wood-borer in North America and Europe that threatens a variety of tree genera, including Acer and Populus. All invasive ALB populations occur in quarantine zones where they are under eradication, a process that is difficult and expensive, requiring extensive surveys and host tree removals. Although ALB has been described as an insect that is typically slow to disperse, some rare individuals that fly longer distances have the potential to start infestations outside of quarantine zones. Biological control using entomopathogenic fungi has been considered as another option for managing ALB infestations. The entomopathogenic fungus Metarhizium brunneum strain F52, registered for commercial use in the United States, is effective at killing ALB adults but information is lacking on how this entomopathogen affects ALB flight behavior before death. Using quarantine-reared ALB, flight mills were used to collect data on flight performance of beetles at multiple time points after infection. Healthy (uninfected) male ALB adults always flew significantly greater distances than females. The maximum observation for total flight distance was a healthy male that flew 10.9 km in 24 hours on a flight mill. ALB adults infected with M. brunneum F52 flew significantly shorter distances compared to healthy adults, starting one week after fungal exposure. Biological control of ALB with this fungal entomopathogen could help to reduce their dispersal in the environment and, thereby, decrease the risk of adults moving outside of quarantine zones.

A pair of native fungal pathogens drives decline of a new invasive herbivore.

Two North American fungal pathogens caused a coepizootic leading to localized collapse of an outbreak population of the newly invasive planthopper pest, the spotted lanternfly (Lycorma delicatula), in the eastern United States. The pathogens partitioned the habitat, with the majority of L. delicatula on tree trunks killed by Batkoa major, while cadavers of L. delicatula killed by Beauveria bassiana were usually on the ground. The future will show whether these pathogens will be drivers in boom-bust cycles or will result in recurrent low population densities of this new invasive species.

Asian longhorned beetle bioassays to evaluate formulation and dose-response effects of Metarhizium microsclerotia.

Asian longhorned beetles (ALB; Anoplophora glabripennis), are invasive wood borers susceptible to Metarhizium brunneum. This fungus can be prepared as dried microsclerotia which, after rehydration, produce infective conidia within weeks. Wood samples coated with formulated microsclerotia were attached to trees in the Ohio USA ALB-eradication zone and collected after 4-week periods. Adult ALB exposed to these samples had 100% mortality. In an experiment comparing formulations with or without humectant hydrogel, hydrogel did not significantly increase mortality of exposed ALB. In a dose-response experiment with 5 application rates, ALB survival decreased with increasing application rate and conidial density.

Effects of endophytic entomopathogenic fungi on soybean aphid and identification of Metarhizium isolates from agricultural fields.

Terrestrial plants can harbor endophytic fungi that may induce changes in plant physiology that in turn affect interactions with herbivorous insects. We evaluated whether the application of entomopathogenic fungi Beauveria bassiana and Metarhizium brunneum to soybean seeds could become endophytic and affect interactions with soybean aphid (Aphis glycines Matsumura). It was found that A. glycines population sizes increased on plants with M. brunneum (strain F52) seed inoculum, but no significant effects were shown with analogous treatments with B. bassiana (strain GHA). Fungi recovered from soybean plant tissues indicate that endophytism was established, and that B. bassiana was more prevalent. Metarhizium brunneum was only recovered from stems, but B. bassiana was recovered from stems and leaves. This work confirms that some entomopathogenic fungi can be endophytic in soybean, however, some of these fungi may have a negative effect on the plants by increasing susceptibility of soybean to A. glycines. We also used DNA sequence data to identify species of Metarhizium obtained from agricultural fields in Iowa. Phylogenetic analyses, based on DNA sequence data, found that all isolates were Metarhizium robertsii, which is consistent with past studies indicating a cosmopolitan distribution and wide host range for this species. These results are important for understanding the dynamics of implementing environmentally sustainable measures for the control of pest insects.

Interactions of effects of host plant resistance and seed treatments on soybean aphid (Aphis glycines Matsumura) and soybean cyst nematode (Heterodera glycines Ichinohe).

BACKGROUND: The soybean cyst nematode, Heterodera glycines, and soybean aphid, Aphis glycines, are invasive, widespread and economically important pests of soybean, Glycine max, in North America. Management of these pests relies primarily on use of pesticides and soybean germplasm with genetic resistance. A 3-year field study and complementary greenhouse experiment were conducted to determine the benefits of host plant resistance (HPR) and pesticidal seed treatments for managing pest populations and preserving soybean yield. RESULTS: Host plant resistance significantly decreased the abundance of A. glycines and, in most study sites, suppressed H. glycines. Neonicotinoid seed treatment reduced A. glycines abundance on the cultivar that was susceptible to both aphids and nematodes, but abamectin nematicide seed treatment had no effect on H. glycines populations in the field or greenhouse. CONCLUSION: These results suggest that the seed treatments included in our experiments may suppress pests, but not consistently for all soybean cultivars or study sites. Ultimately, HPR more consistently reduced pest numbers compared with the use of pesticidal seed treatments. The planting of HPR cultivars should be a primary tool for integrated pest management of both soybean pests. © 2017 Society of Chemical Industry.

Evidence of Resistance to Cry34/35Ab1 Corn by Western Corn Rootworm (Coleoptera: Chrysomelidae): Root Injury in the Field and Larval Survival in Plant-Based Bioassays.

Western corn rootworm, Diabrotica virgifera virgifera LeConte (Coleoptera: Chrysomelidae), is a serious pest of corn in the United States, and recent management of western corn rootworm has included planting of Bt corn. Beginning in 2009, western corn rootworm populations with resistance to Cry3Bb1 corn and mCry3A corn were found in Iowa and elsewhere. To date, western corn rootworm populations have remained susceptible to corn producing Bt toxin Cry34/35Ab1. In this study, we used single-plant bioassays to test field populations of western corn rootworm for resistance to Cry34/35Ab1 corn, Cry3Bb1 corn, and mCry3A corn. Bioassays included nine rootworm populations collected from fields where severe injury to Bt corn had been observed and six control populations that had never been exposed to Bt corn. We found incomplete resistance to Cry34/35Ab1 corn among field populations collected from fields where severe injury to corn producing Cry34/35Ab1, either singly or as a pyramid, had been observed. Additionally, resistance to Cry3Bb1 corn and mCry3A corn was found among the majority of populations tested. These first cases of resistance to Cry34/35Ab1 corn, and the presence of resistance to multiple Bt toxins by western corn rootworm, highlight the potential vulnerability of Bt corn to the evolution of resistance by western corn rootworm. The use of more diversified management practices, in addition to insect resistance management, likely will be essential to sustain the viability of Bt corn for management of western corn rootworm.

Abundance of Soil-Borne Entomopathogenic Fungi in Organic and Conventional Fields in the Midwestern USA with an Emphasis on the Effect of Herbicides and Fungicides on Fungal Persistence.

Entomopathogenic fungi (EPF) are widespread in agricultural fields and help suppress crop pests. These natural enemies may be hindered by certain agronomic practices associated with conventional agriculture including the use of pesticides. We tested whether the abundance of EPF differed between organic and conventional fields, and whether specific cropping practices and soil properties were correlated with their abundance. In one year of the survey, soil from organic fields and accompanying margins had significantly more EPF than conventional fields and accompanying margins. Regression analysis revealed that the percentage of silt and the application of organic fertilizer were positively correlated with EPF abundance; but nitrogen concentration, tillage, conventional fields, and margins of conventional fields were negatively correlated with EPF abundance. A greenhouse experiment in which fungicides and herbicides were applied to the soil surface showed no significant effect on EPF. Though organic fields were perceived to be more suitable environments for EPF, abiotic factors and cropping practices such as tillage may have greater impacts on the abundance of EPF. Also, fungicides and herbicides may not be as toxic to soil-borne EPF as originally thought.

Field-evolved resistance by western corn rootworm to multiple Bacillus thuringiensis toxins in transgenic maize.

The widespread planting of crops genetically engineered to produce insecticidal toxins derived from the bacterium Bacillus thuringiensis (Bt) places intense selective pressure on pest populations to evolve resistance. Western corn rootworm is a key pest of maize, and in continuous maize fields it is often managed through planting of Bt maize. During 2009 and 2010, fields were identified in Iowa in which western corn rootworm imposed severe injury to maize producing Bt toxin Cry3Bb1. Subsequent bioassays revealed Cry3Bb1 resistance in these populations. Here, we report that, during 2011, injury to Bt maize in the field expanded to include mCry3A maize in addition to Cry3Bb1 maize and that laboratory analysis of western corn rootworm from these fields found resistance to Cry3Bb1 and mCry3A and cross-resistance between these toxins. Resistance to Bt maize has persisted in Iowa, with both the number of Bt fields identified with severe root injury and the ability western corn rootworm populations to survive on Cry3Bb1 maize increasing between 2009 and 2011. Additionally, Bt maize targeting western corn rootworm does not produce a high dose of Bt toxin, and the magnitude of resistance associated with feeding injury was less than that seen in a high-dose Bt crop. These first cases of resistance by western corn rootworm highlight the vulnerability of Bt maize to further evolution of resistance from this pest and, more broadly, point to the potential of insects to develop resistance rapidly when Bt crops do not achieve a high dose of Bt toxin.

Interactions among Bt maize, entomopathogens, and rootworm species (Coleoptera: Chrysomelidae) in the field: effects on survival, yield, and root injury.

A 2 yr field study was conducted to determine how a blend of entomopathogens interacted with Bt maize to affect mortality of Diabrotica spp. (Coleoptera: Chrysomelidae), root injury to maize (Zea maize L.) and yield. The blend of entomopathogens included two entomopathogenic nematodes, Steinernema carpocapsae Weiser and Heterorhabditis bacteriophora Poinar, and one entomopathogenic fungus, Metarhizium brunneum (Metschnikoff) Sorokin. Bt maize (event DAS59122-7, which produces Bt toxin Cry34/35Ab1) decreased root injury and survival of western corn rootworm (Diabrotica virgifera virgifera LeConte) and northern corn rootworm (Diabrotica barberi Smith & Lawrence) but did not affect yield. During year 1 of the study, when rootworm abundance was high, entomopathogens in combination with Bt maize led to a significant reduction in root injury. In year 2 of the study, when rootworm abundance was lower, entomopathogens significantly decreased injury to non-Bt maize roots, but had no effect on Bt maize roots. Yield was significantly increased by the addition of entomopathogens to the soil. Entomopathogens did not decrease survival of corn rootworm species. The results suggest that soil-borne entomopathogens can complement Bt maize by protecting roots from feeding injury from corn rootworm when pest abundance is high, and can decrease root injury to non-Bt maize when rootworm abundance is low. In addition, this study also showed that the addition of entomopathogens to soil contributed to an overall increase in yield.