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

Abstract 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.

In the present study we challenged Asian longhorned beetle adults with nine strains of five species of EPF to compare their virulence. These EPFs have all been developed commercially and are registered for use in the United States or could potentially be registered in the United States because they are registered in another country.

Fungal Strains
We tested five fungal species in four genera, all of which have been commercially developed ( Table 1). Some of the strains used in the current study are not registered in the United States, including Beauveria bassiana (Balsamo) (Hypocreales: Cordycipitaceae) strain ERL836, which has been commercialized for thrips management in South Korea, the Lecanicillium muscarium (Petch) Zare & Gams (Hypocreales: Cordycipitaceae) strain Ve-6, used as the active ingredient in Mycotal, and the ESALQ E-9 strain of M. anisopliae sensu lato, which is an active ingredient in Metarril, a biopesticide produced by Koppert Brazil. We tested M. brunneum strain F52 which was also referred to as M. anisopliae sensu lato until 2009 (Bischoff et al. 2009). Strains of B. bassiana (ANT-03 and ERL836), Isaria fumosorosea (Wize, 1904) (Hypocreales: Cordycipitaceae) (formerly Paecilomyces fumosoroseus) (Apopka Strain 97 and FE 9901), M. brunneum (F52), and Metarhizium anisopliae (ESALQ E-9) were unformulated, dry, conidial powders produced using solid-state fermentation by the USDA-ARS in Sidney, MT, following methods outlined in Jaronski and Jackson (2012). Beauveria bassiana (ATCC 74040) and L. muscarium (ARSEF 5128) were obtained from the USDA, ARSEF (Agricultural Research Service Collection of Entomopathogenic Fungal Cultures) in Ithaca, NY and were grown in solid culture for conidial production. Cultures of B. bassiana ATCC 74040 were grown on Sabouraud dextrose agar augmented with 1% yeast extract (SDAY) for 14 d before harvesting conidia. Lecanicillium muscarium was grown on selective media described in Kope et al. (2006) for 14 d before harvesting conidia. Dry conidial powder of B. bassiana strain GHA was provided by LAM International in Butte, MT.
One to two days before bioassays, the viability of strains was determined by spreading a dilute aqueous conidial suspension on Sabouraud dextrose agar. Germinated conidia were counted at 400× magnification 14-18 h after incubation at 25°C; all strains had >90% viability before use in bioassays.

Experimental Insects
Adult Asian longhorned beetles were reared under USDA-APHIS permit in a quarantine facility adjacent to Cornell University, as described in the Supplementary Appendix of Goble et al. (2015). Prior to and during bioassays beetles were held in individual 473ml clear plastic containers (PK16SC, Fabri-Kal, Kalamazoo, MI) in a 25°C:15°C day:night incubator with 14:10 (L:D) h. There was 100% relative humidity inside the sealed containers (i.e., visible condensation on the inner surface of the lid). Striped maple (Acer pensylvanicum L.) twigs with leaves removed were used for food. Beetles used for bioassays were 9-16 d after eclosion to adults (mean ± SE: 12.0 ± 0.1 d) when bioassays were begun.

Bioassays Against Anoplophora glabripennis
We conducted immersion-exposure bioassays using methods developed by Dubois et al. (2008), with slight modifications. To compare fungal strains using a consistent exposure concentration, beetles were submerged for 5 s in suspensions of 1.0 × 10 8 viable conidia per ml in 0.05% Tween. This concentration of conidia is a standard dose recommended by the International Centre of Insect Physiology and Ecology (ICIPE) for immersion-exposure bioassays with larger insects. A 1:1 sex ratio of females:males was used for each treatment. During each bioassay, beetles used for negative controls were exposed only to 0.05% Tween. This host is expensive and time-consuming to rear because it requires at least 9 mo to develop from egg to adult, and this situation limited sample sizes for a few EPF trials. The bioassays were repeated from 2 to 9 times on different dates for each strain. Totals of 22-44 insects were treated with each EPF, except the Metarhizium strains for which additional replicates were conducted. After the initial bioassays with M. anisopliae ESALQ E-9 resulted in short survival times for the exposed Asian longhorned beetle adults, we conducted more replicates with both M. brunneum F52 and M. anisopliae ESALQ E-9 for more detailed analysis of survival times. For exposure, individual Asian longhorned beetle adults were placed in 50-ml tubes containing 11-13 ml of the conidial suspension and the tube was gently shaken for 5 s by hand. After immersion in the conidial suspensions, each adult was directly transferred to a container. Twigs used as food were replaced as necessary each week. Beetles were checked daily for mortality and experiments were ended 28 d after exposure. For two B. bassiana strains (ANT-03 and ERL836), few to no beetles had been killed by 28 d, but beetle behavior had significantly changed (noticeable lethargy and reduced feeding), so monitoring for these two EPF was extended to 42 d total. Dead beetles were kept in sealed containers and incubated at 20-25°C (0:24 [L:D] h) for up to 14 d to evaluate outgrowth by fungi.

Statistical Analysis
Median survival times and SEs were calculated for adults receiving each treatment based on Kaplan-Meier survival distribution functions using the PROC LIFETEST statement in SAS 9.4 (SAS Institute Inc. 2019). For multiple comparisons of survival times among different EPF strains for which mortality was recorded, i.e., Metarhizium strains and B. bassiana GHA, the Cox proportional hazards model was performed in SAS 9.4 using the PROC PHREG statement. Contrasts between EPF strains were conducted using least-square means, adjusted with the Bonferroni correction. Controls were excluded from multiple comparisons because none of them died and, as a result, median survival times could not be calculated.
Asian longhorned beetle adults exposed to B. bassiana strains ANT-03 or ERL836 responded noticeably slower and were feeding less 28 d after exposure, but only the ANT-03 strain killed any of the beetles (5.5%) within 28 d (Table 2). Beauveria bassiana strains ANT-03 and ERL836 caused 39.9% and 42.9% mortality, respectively, after 42 d. For those Asian longhorned beetle adults killed Fig. 1. Survival graphs for Asian longhorned beetle adults exposed to Metarhizium isolates and Beauveria bassiana GHA (censored at 28 d). Median survival time was 11.3 ± 0.4 d for beetles exposed to F52, 11.7 ± 0.3 d for ESALQ E-9, and 21.4 ± 0.8 d for GHA. Letters denote significant differences between treatments based on the Cox proportional hazards model. Controls are included for comparison and had no mortality and therefore could not be included in the multiple comparisons. by B. bassiana ANT-03 and ERL836, 87.3% and 85.7% of the cadavers, respectively, had fungal outgrowth.
Both I. fumosorosea strains and L. muscarium Ve-6 did not kill any Asian longhorned beetle adults within 28 d after exposures and no control beetles died in the bioassays (Table 2).

Discussion
Metarhizium anisopliae ESALQ E-9 and M. brunneum F52 killed 100% of exposed beetles and exhibited higher virulence than other EPF strains in the current study. Both EPF species are native to North America (Rehner and Kepler 2017). The results with M. anisopliae ESALQ E-9 are encouraging, but this strain, while commercial in Brazil, is not currently registered for use in the United States. Metarhizium anisopliae ESALQ E-9 has been studied for management of cattle ticks, spittlebugs, and the sugarcane borer, Diatraea saccharalis (Fabricius) (Lepidoptera: Crambidae), in Brazil, but has never before been tested against Asian longhorned beetle (Rossoni et al. 2014, Camargo et al. 2016. Metarhizium brunneum F52 already is registered for commercial sale in the United States. Not all the EPF strains tested were pathogenic to Asian longhorned beetle adults. In general, the B. bassiana strains tested were less virulent to Asian longhorned beetle than Metarhizium strains. Bioassays by Dubois et al. (2008) compared six different strains of B. bassiana, including strain GHA. In agreement with the results from the present study, Dubois et al. (2008) reported that Asian longhorned beetle adults treated with B. bassiana strains had longer survival times than those exposed to different strains of Metarhizium. Furthermore, Asian strains of other Beauveria species (Beauveria brongniartii (Sacc.) Petch (ARSEF 6412), Beauveria amorpha (Höhn.) Minnis, S.A. Rehner & Humber (ARSEF 6827; formerly reported as B. brongniartii), and Beauveria asiatica S.A. Rehner & Humber (NBL 851; reported as B. brongniartii)) were more effective at killing Asian longhorned beetle than strains of B. bassiana (Dubois et al. 2008). At the time of the Dubois et al. (2008) study, B. brongniartii was not known as being endemic to North America (Rehner and Buckley 2005). No mycoinsecticides have been developed in North America using B. brongniartii as an active ingredient, although B. asiatica NBL 851 is used as the active ingredient in the Biolisa-Kamikiri (Idemitsu Kosan Co. Ltd., Tokyo, Japan) product sold in Japan for control of longhorned beetles (Faria andWraight 2007, Rehner et al. 2011). After it was proven that B. brongniartii inhabits soils in North America (Rehner et al. 2011), Goble et al. (2014) conducted bioassays against Asian longhorned beetle adults, using methods similar to those in the current study, and found that M. brunneum F52 and B. asiatica (NBL 851) were more effective at killing Asian longhorned beetle adults than strains of B. brongniartii (ARSEF 6215 and ARSEF 10279) isolated from North American soils.
The two commercially developed strains of I. fumosorosea tested were not capable of infecting and killing Asian longhorned beetle adults. Commercially available mycoinsecticides containing strains of I. fumosorosea are typically used for sap-sucking pests (Chow et al. 2018), but we included this species in the present study because it has been reported infecting coleopteran pests like the citrus root weevil, Pachnaeus litus (Germar) (Coleoptera: Curculionidae) , yellowmargined leaf beetle, Microtheca ochroloma (Stål) (Coleoptera: Chrysomelidae) (Montemayor et al. 2016), and ambrosia beetles (Kushiyev et al. 2018). While screening EPF isolates for virulence against Asian longhorned beetle, Dubois et al. (2008) included a species of the same genus, Isaria farinosa (Holmsk.) Fr. (ARSEF 8411) (reported as Paecilomyces farinosus (Wize) A.H.S. Br. & G. Sm.), in the study because some Asian longhorned beetles in rearing had been killed by this EPF. However, no strains of this species are currently registered in any EPF products and, while it killed Asian longhorned beetle adults, the median survival time was 24.5 d (Dubois et al. 2008). Therefore, this strain of I. farinosa was not considered effective against Asian longhorned beetle when compared to strains of Metarhizium and Beauveria.
Mycoinsecticides that contain Lecanicillium longisporum (Petch) Zare & W. Gams (ARSEF 5126), L. muscarium (ARSEF 5128), and L. lecanii (formerly Verticillium lecanii) (Zimm.) Zare & W. Gams are available in the EU but not in North America. We tested L. muscarium against Asian longhorned beetle because using similar bioassay methods on the white pine weevil, Pissodes strobi (Peck) (Coleoptera: Curculionidae), with suspensions of 1.0 × 10 7 viable conidia per ml, Kope et al. (2006) found that L. longisporum (Vertalec) and L. muscarium (Mycotal) caused median survival times of 9.1 ± 0.7 d and 7.9 ± 0.6 d, respectively. In addition, isolates of L. muscarium have been collected from great spruce bark beetles Dendroctonus micans (Kugelann) (Coleoptera: Curculionidae, Scolytinae) (Tanyeli et al. 2010). We found that L. muscarium did not kill any Asian longhorned beetle adults within 28 d and a limited assay with the strain of L. longisporum used in Vertalec (now discontinued by Koppert [its manufacturer]) produced the same results (E.H.C., unpublished data). Bioassays were censored at 28 d. Letters next to the median survival times denote significant differences among treatments based on the Cox proportional hazards model. Fungal outgrowth refers to the % of cadavers with conidial outgrowth from the respective fungus used in the bioassay. a Controls were always included in bioassays; hence, the large number of insects that were used.
The labels and descriptions of EPF biopesticides include target pests (Table 1), but the potential host range is not always complete or well understood. For example, the EPF products using M. brunneum F52 and B. bassiana GHA mention beetles and weevils as target pests and both EPF strains killed Asian longhorned beetle adults. In contrast, the B. bassiana strains ATCC 74040 and ANT-03 also include beetles and weevils as target pests, but both of these EPF strains were less effective or failed to kill Asian longhorned beetle. Metarhizium anisopliae ESALQ E-9 is not promoted as killing beetles but this was one of the best strains tested against Asian longhorned beetle. The results in the present study demonstrate how some EPF are virulent to pests that are excluded from a biopesticide label, e.g., M. anisopliae ESALQ E-9 infecting Asian longhorned beetle.
Based on the results of the current study, EPFs in the genus Metarhizium, including M. brunneum F52 and M. anisopliae ESALQ E-9, could be recommended for further development as a tool for biological control of Asian longhorned beetle (Goble et al. 2016b). However, because F52 is already approved for commercial sale and is registered for use in the United States, eradication programs could consider this EPF among their tools. Certainly, field trials are needed with this fungus, or potentially other species or strains, to demonstrate practicality within an eradication program.