Wood-boring ambrosia beetles and their offspring obligately depend on cultivated fungi that they maintain within host trees. Stressed trees produce at least two potent antimicrobials, ethanol and acetic acid, which typically suppress the growth of most fungi. Ethanol facilitates the growth of ambrosia beetles' fungal mutualists and thus aids in host colonization; however, acetic acid's effects are unknown. Here, we evaluated the effects of acetic acid on tunneling and offspring production by two exotic ambrosia beetle species, Xylosandrus germanus and Anisandrus maiche. Cut wood stems (i.e., bolts) were infused with water alone, 5% ethanol, or mixtures of 5% ethanol with acetic acid at various dilutions. Infestations by X. germanus and A. maiche resulted in more frass, larvae, pupae, and adults in the combined 5% ethanol-acetic acid treatment, than in the 5% ethanol alone or water control treatments. X-ray micro-computed tomography revealed that bolts infused with this 5% ethanol-acetic acid mixture had larger insect gallery volumes than those with only 5% ethanol, which in turn were larger than the water control. A positive relationship was found between frass ejected by each beetle and gallery volume across all experiments. This study demonstrates that acetic acid and ethanol synergistically increase offspring production while facilitating gallery expansion by the larvae. Understanding how ambrosia beetles utilize stress-induced compounds within host trees to benefit their fungiculture could lead to novel management strategies for these invasive insects.

Certain invasive ambrosia beetles are attracted to and colonize physiologically stressed trees emitting ethanol. Xylosandrus germanus (Blandford) and Xylosandrus crassiusculus (Motschulsky) are attracted to trees emitting ethanol and are 2 of the most damaging invasive ambrosia beetles in North America. Acetic acid has been detected in stressed trees emitting ethanol, but whether it has a role in ambrosia beetle ecology is unknown. Sections of tree stems (bolts) were soaked in various dilutions of acetic acid alone and mixed with a 5% dilution of ethanol, then deployed in a field near a woodland. Bolts soaked in 5% or 10% dilutions of acetic acid were more attractive to X. germanus and X. crassiusculus than bolts soaked in dilutions of 0.1% or 1.0%. Dilutions of acetic acid mixed with 5% ethanol were more attractive to X. germanus and X. crassiusculus than similar dilutions of acetic acid alone or 5% ethanol alone. Furthermore, more offspring were produced in bolts soaked in mixtures of acetic acid and 5% ethanol than either compound alone. This information helps improve our knowledge of the physiological conditions in trees that make them suitable for colonization by X. germanus and X. crassiusculus. Furthermore, the increased attraction of damaging ambrosia beetles to acetic acid + ethanol could be useful for improving baits used for monitoring emergence of X. germanus and X. crassiusculus in spring.

ABSTRACTEnvironmental DNA (eDNA) consists of genetic material shed by living organisms, including those that are deceased, offering a unique opportunity to detect and identify terrestrial insect pests without requiring visual identification. The sweetpotato whitefly, Bemisia argentifolii, and the twospotted spider mite, Tetranychus urticae, are notorious for causing crop losses through virus transmission and direct feeding. Our study aimed to: (1) assess the effectiveness of B. argentifolii literature‐based PCR primers compared to newly developed primers for eDNA amplification, (2) evaluate the sensitivity of conventional PCR (cPCR) and real‐time quantitative PCR (qPCR) for detecting eDNA of B. argentifolii and T. urticae, (3) establish a rapid eDNA processing methodology using the LGC Biosearch Technologies QuickExtract DNA extraction kit and the Qiagen DNeasy Blood and Tissue kit, and (4) test the specificity of the developed primers against non‐target species. B. argentifolii and T. urticae were confined to tomato leaves (Solanum lycopersicum) using clip cages for 24 h, after which eDNA was collected from leaf surfaces using a water spray method, filtered, and processed for DNA amplification. While literature‐based primers showed sufficient sensitivity, their specificity for eDNA applications was inadequate, prompting the design of novel PCR primers for both pest species. Positive eDNA detection was achieved with both amplification methods, with qPCR proving more reliable than cPCR due to the latter's inconsistent performance with positive control samples. We also introduced a rapid eDNA processing approach using the QuickExtract DNA extraction kit, contrasting it with the more conventional Qiagen DNeasy Blood and Tissue kit. We believe that our findings are the first step toward the practical use of eDNA as a highly sensitive, early detection technique.

Ambrosia beetles, particularly invasive species within the tribe Xyleborini, such as Xylosandrus germanus (Blandford, 1894), pose significant threats to various ecosystems and managed habitats worldwide. Monitoring these invaders is vital for effective pest management, typically accomplished through ethanol-baited traps. We compared trap efficacy using denatured ethanol versus absolute ethanol in orchards, tree nurseries, and lumber yards in northeastern Ohio, USA, finding that absolute ethanol traps captured significantly more X. germanus. Analysis revealed acetone, ethanol, and methyl isobutyl ketone in the denatured ethanol, likely impacting trap efficacy. Our study underscores the importance of using pure denatured ethanol without acetone for effective monitoring, especially for X. germanus. Exotic xyleborines dominated trap captures across various habitats, emphasizing the need for tailored pest management strategies. Further research is warranted to explore the chemical ecology of ambrosia beetles and the influence of ethanol impurities on trap effectiveness.

Ambrosia beetles (Coleoptera: Curculionidae: Scolytinae) are among the most successful invaders of trees on a global scale. Exotic species can establish large populations within forested habitats and disperse into tree nurseries and orchards with the potential for substantial economic losses. Our objective was to assess the seasonal dominance of exotic Scolytinae compared to native species by characterizing their flight phenology, abundance and species diversity. Weekly sampling using ethanol-baited traps was conducted within deciduous and coniferous woodlots in Ohio, USA from March/April to September/October in 2014, 2015, 2016, and 2019. Over the course of the study, 16 native and 11 exotic species of Scolytinae were identified. No difference was detected in the number of exotic Scolytinae species or their abundance, Shannon’s index ( H ), and evenness (E h ) between the coniferous vs. deciduous woodlots. On average, initial flight occurred at 188 degree days (DD) for exotic species compared to 273 DD for native species. Seasonal flight duration of exotic species averaged 49 days compared to 10 days for native species. Of the 145,882 total Scolytinae captured over the four years, only 622 were native beetles. Captures of exotic Scolytinae were 341-times greater than native species across the four trapping seasons, including captures of the exotic ambrosia beetle Xylosandrus germanus being 450-times greater than the most common native species Xyloborinus politus . These results provide insight into the invasion success of ambrosia beetles and will aid in predicting and monitoring key species.

Xylosandrus crassiusculus (Motschulsky) and Xylosandrus germanus (Blandford) (Coleoptera: Curculionidae: Scolytinae) are major ambrosia beetle pests in tree nut and fruit orchards and ornamental nurseries in the eastern United States (USA). Ethanol-baited bottle traps and ethanol-infused tree stem sections (i.e., bolts) have been used to monitor ambrosia beetles, but limited studies exist on the influence of ethanol-lure release rate on ambrosia beetle trap captures and bolt attacks. We designed this study to compare low-release (LR) and high-release (HR) ethanol lures in bottle traps for capturing invasive ambrosia beetles. We also compared beetle attacks among bolts pre-soaked in ethanol solutions of low (10%) and high (90%) concentrations and bolts cored and filled with the same low and high ethanol concentrations. In 2022, experiments were conducted in ornamental nurseries and apple, peach, or pecan orchards in five USA states. Higher numbers of X. crassiusculus and X. germanus were captured in bottle traps baited with the HR ethanol lure compared to the LR lure at most of the study sites. More attacks per bolt by X. crassiusculus and X. germanus were observed at most sites on pre-soaked and filled bolts with 90% compared to 10% solutions of ethanol. Bolts soaked in low (10%) ethanol solutions sustained more attacks from both X. crassiusculus and X. germanus than cored bolts filled with low ethanol. These results will assist with monitoring the flight activity of invasive ambrosia beetles within nut, fruit, and ornamental tree crops.

Infestations of ambrosia beetles in the tribe Xyleborini (Coleoptera: Scolytinae) are associated with economic losses to horticultural trees due to branch die-back and tree death. Ethanol is a key attractant used for monitoring flight activity. Trapping experiments were conducted in woodlots in Ohio, USA, and Veneto, Italy, to characterize the effect of ethanol release rate on captures of Anisandrus maiche, Xyleborinus saxesenii, Xylosandrus crassiusculus and Xylosandrus germanus. In Ohio (2019, 2020 and 2021) and Italy (2021), traps were baited with centrifuge tubes that were modified to achieve ethanol release rates of 0.1-13.2 g/day. In Ohio (2022), traps were baited with varying quantities of manufactured lures to achieve release rates of 0.02-1.4 g/day. There was no consistent relationship between ethanol release rate and trap captures for the modified centrifuge tubes. In nine of sixteen analyses, traps baited with the centrifuge tubes releasing ethanol at 1.1 g/day or higher collected more A. maiche, X. saxesenii, X. crassiusculus and X. germanus than traps baited with centrifuge tubes releasing 0.1 g/day. In contrast, the manufactured lures releasing 0.1-1.4 g/day attracted more A. maiche, X. saxesenii, X. crassiusculus and X. germanus than lures releasing 0.02-0.05 g/day. This comprehensive study provides important insights into monitoring tactics for ambrosia beetles along with implications for optimizing ethanol-baited traps as part of a 'push-pull' strategy whereby repellents are used to 'push' beetles away from vulnerable trees and attractants are used to 'pull' them into annihilative traps.

Beauveria bassiana (Balsamo) Vuillemin infects a wide variety of insects, including the green peach aphid, Myzus persicae (Sulzer). Volatiles emitted from B. bassiana can act as semiochemical attractants or repellents, with most responses reported to date resulting in insects avoiding B. bassiana. Since insects can detect 'enemy-specific volatile compounds', we hypothesized the preference behavior of M. persicae would be influenced by volatile emissions from B. bassiana. We conducted Petri dish and Y-tube olfactometer bioassays to characterize the preference of M. persicae to B. bassiana strain GHA. During Petri dish bioassays, more apterous and alate M. persicae were recorded in the vicinity of agar colonized by B. bassiana compared to agar, or Fusarium proliferatum (Matsushima) Nirenberg and Ambrosiella grosmanniae Mayers, McNew, & Harrington as representatives of nonentomopathogenic fungi. Petri dish bioassays also determined that apterous and alate M. persicae preferred filter paper saturated with 1 × 107, 1 × 106, and 1 × 105B. bassiana conidia/ml compared to Tween 80. Y-tube bioassays documented that more apterous and alate M. persicae oriented upwind to volatiles from B. bassiana mycelia compared to agar. Apterous and alate Myzus persicae were also preferentially attracted to 1 × 107 and 1 × 106B. bassiana conidia/ml compared to Tween-80 during Y-tube bioassays. These results complement a previous finding that the mosquito Anopheles stephensi (Diptera: Culicidae) Liston is attracted to volatiles from B. bassiana. Future studies aimed at characterizing the olfactory mechanism leading to the attraction of M. persicae to B. bassiana could aid in optimizing lure-and-kill strategies.

Fungus-farming ambrosia beetles in the tribe Xyleborini tunnel into plants and trees to establish chambers for cultivating their nutritional fungal mutualists and rearing offspring. Some xyleborine ambrosia beetles preferentially infest and perform better in living but weakened trees. Flood stress predisposes horticultural tree crops to infestation, but the impact of drought stress has not been well studied. Our objectives were to compare the effects of flood stress vs. drought stress on host selection and colonization by xyleborine ambrosia beetles and to assess the duration of flooding. Container-grown Cornus florida L. trees were flood stressed using a pot-in-pot system to submerge the roots in water while drought-stressed conditions were imposed by withholding irrigation and precipitation. When experimental trees were held under field conditions for 14 days, 7.5 × more ambrosia beetles landed on stems of the flood-stressed than on the drought-stressed trees. During two additional experiments over 14 and 22 days, ambrosia beetles tunneled into the flood-stressed trees but not the drought-stressed or standard irrigation trees. By simultaneously deploying trees that were flood stressed for varying lengths of time, it was found that more tunnel entrances, and xyleborine adults and offspring were recovered from trees that were flooded for 1–16 days and 7–22 days than from trees that were flooded for 14–29 days and 28–43 days. These results indicate that acute and severe drought stress does not predispose C. florida to infestation, but flood stress and the duration of flooding influence ambrosia beetle host selection and colonization. Understanding the role of host quality on ambrosia beetle preference behavior will assist with predicting the risk of infestation of these opportunistic insects in horticultural tree crops.

The granulate ambrosia beetle (GAB) Xylosandrus crassiusculus , was first formally detected in New Zealand in 2019. Since then, GAB has subsequently been found infesting numerous tree species in the Auckland region. Flight intercept traps baited with ethanol lures were deployed from October 2019 to May 2021 at three sites in the Auckland region to ascertain the phenology of GAB in New Zealand. Two distinct peak flight periods were identified in early and late summer, while a smaller and inconsistent third peak was detected in early autumn. Logistical analysis of GAB captures in the ethanol‐lured traps and degree‐day (DD) accumulation indicated that 90% of flight activity is completed by 800 DD. To assess monitoring tactics, flight intercept traps were baited with three different ethanol lures with varying release rates or ethanol‐soaked or non‐soaked wood bolts from three species of trees. A lure releasing 2 g ethanol per day was most effective at capturing GAB. Ethanol‐soaked bolts were less effective than the lures. Xylosandrus crassiusculus represents a significant risk for shrubs and trees native to New Zealand, as well as commercial horticultural and forestry trees. We recommend using ethanol‐lured panel traps for monitoring purposes.

Ambrosia beetles (Coleoptera; Curculionidae; Scolytinae and Platypodinae) can cause severe damage to trees growing in plant nurseries, orchards and natural forests. Ethanol is emitted by stressed trees and represents an important cue used by ambrosia beetles to locate suitable hosts to infest. Ethanol also favors the growth of ambrosia beetles’ nutritional fungal symbionts and suppresses the growth of antagonistic fungi. An optimal concentration of ethanol in host tissues might maximize fungal growth and offspring production, but it is unclear if this optimal concentration varies among ambrosia beetle species. To investigate this mechanism, we injected five different concentrations of aqueous ethanol solution (5%, 25%, 50%, 75% and 90%) into the stems of container-grown oak trees, Quercus robur L. Modified Falcon tube chambers were used to confine four species of field-collected ambrosia beetles to the injected stems, namely, Anisandrus dispar , Xyleborinus saxesenii , Xylosandrus germanus , and Xylosandrus crassiusculus . Incidence of boring, ejected sawdust, gallery development, and offspring production were then quantified. The incidence of boring generally increased with increasing ethanol concentration for all four Scolytinae species tested. Ejected sawdust and offspring production increased with increasing ethanol concentration up to 90% for A. dispar and X. saxesenii ; by contrast, an increasing trend up to 75% ethanol followed by a decrease at 90% ethanol was associated with X. germanus and X. crassiusculus . Our study highlights the key role of ethanol for ambrosia beetles, and showed that the optimal concentration maximizing colonization and offspring production can vary among species.

Exotic ambrosia beetles (Coleoptera: Curculionidae: Scolytinae) cause considerable damage in ornamental tree nurseries and other tree crops in North America. Ambrosia beetles bore into the xylem of trees to establish gardens of symbiotic fungi, which are the source of nutrition for adults and larvae (Wood 1982, Great Basin Nat. Memoirs. 6: 1–1359). Establishment of fungal gardens is crucial as some species of ambrosia beetles delay oviposition until their symbiotic fungi are growing (French and Roeper 1972, Can. Entomol. 104: 1635–1641; Weber and McPherson 1983, Ann. Entomol. Soc. Amer. 76: 455–462; Peer and Taborsky 2005, Evolution 59: 317–323). Colonization of nursery trees by ambrosia beetles often leads to wilting, stem dieback, or death (Ranger et al. 2016, J. Int. Pest Manag. 7: 1–23). Nursery growers rely on preventive trunk sprays of insecticides to protect trees from ambrosia beetles. However, insecticide sprays were inconsistent at preventing colonization attempts by ambrosia beetles in previous research (Frank and Sadoff 2011, J. Econ. Entomol. 104: 1960–1968; Reding et al. 2013, J. Econ. Entomol. 106: 289–298).Xylosandrus crassiusculus (Motschulsky) and Xylosandrus germanus (Bland-ford) are two of the most damaging ambrosia beetles in ornamental tree nurseries in North America (Oliver and Mannion 2001, Environ. Entomol. 30: 909–918; Fulcher et al. 2012, J. Int. Pest Manag. 3: 1–8; Reding et al. 2013; Ranger et al. 2016). Both species overwinter in galleries in host trees, then in the spring, emerging females search for new hosts to colonize. Xylosandrus crassiusculus and X. germanus preferentially colonize physiologically stressed trees emitting ethanol (Ranger et al. 2021, Can. Entomol. 153: 103–120). Ethanol, which benefits the establishment of fungal gardens and, thus, beetle colonization (Ranger et al. 2018, Proc. Natl. Acad. Sci. USA 115: 4447–4452), is a primary attractant for X. crassiusculus and X. germanus (Oliver and Mannion 2001; Ranger et al. 2021). Woodland habitats adjacent to nurseries are primary sources of X. crassiusculus, X. germanus, and other ambrosia beetles invading nurseries each season (Reding et al. 2015, J. Econ. Entomol. 108: 1947–1953; Werle et al. 2015, Florida Entomol. 98: 884–891). Thus, ethanol-baited traps or ethanol-injected trap trees positioned at the woodland/nursery interface could be an effective tactic for intercepting beetles as they emigrate into nurseries. If not effective as a stand-alone management tool, then the interception tactic could mitigate ambrosia beetle pressure and improve the efficacy of insecticidal trunk sprays.Ethanol-baited traps captured hundreds of X. crassiusculus and X. germanus per trap in previous research (Oliver and Mannion 2001; Reding et al. 2010, J. Environ. Hort. 28: 85–90; Reding et al. 2011, J. Econ. Entomol. 104: 2017–2024). Similarly, X. crassiusculus and X. germanus readily burrowed into trees injected with ethanol, whereas no burrowing was evident in adjacent noninjected trees (Ranger et al. 2010, Agric. For. Entomol. 12: 177–185; Reding et al. 2013). Injecting trees with ethanol to create highly attractive trap-trees might be useful for attracting ambrosia beetles away from crop trees. Every year ornamental tree growers have trees to cull, which could be injected with ethanol to use as trap-trees. Concentrating beetles on injected trap trees, which can later be removed and destroyed, may reduce colonization pressure on saleable trees and improve the efficacy of insecticide treatments.The objectives of the current research were to (a) determine whether using traps to intercept X. germanus and other ambrosia beetles emigrating from adjacent habitats reduces the activity of beetles within nurseries and (b) determine whether ethanol-injected trap-trees reduce colonization attempts by ambrosia beetles on insecticide-treated trees.Interception research was conducted in commercial nurseries in 2015 and 2016. Bottle traps (Reding et al. 2015) baited with ethanol were used for intercepting X. germanus and other ambrosia beetles emigrating into nurseries from adjacent woodlands and for sentinel traps within nurseries. Interception would be considered a successful tactic for managing X. germanus in nurseries if captures in sentinel traps were reduced in nursery plots guarded by interception traps. Traps were placed in nurseries in the spring before the emergence of X. germanus. Interception traps were positioned inside nurseries parallel to and within 1 m of the interface with an adjacent woodland. Sentinel traps were placed within nurseries at various distances from the woodland/nursery interface including 13, 25, 50, and 100 m and within adjacent woodlands at 13 m from the interface (hereafter referred to as the woodland trap). Sentinel traps were positioned at various distances from the woodland/nursery interface to determine whether captures would be influenced by the proximity to intercept traps. Lateral distance between sentinel traps was 25 m. Experiments were set up as randomized complete block designs with nursery as the blocking factor. There were two adjacent plots (100-m width) at each nursery with a sentinel trap of each distance per plot and plots 25 m apart. One plot was guarded by intercept traps, whereas the other plot was unguarded. Intercept traps were rotated to the opposite plot each time traps were checked. Traps were checked at 6- to 15-d intervals, and all Scolytinae individuals were identified to the species level using available keys (Wood 1982; Rabaglia et al. 2006, Ann. Entomol. Soc. Am. 99: 1034–1056).In 2015, the experiment was conducted in four commercial nurseries in northern Ohio, with three nurseries in Lake Co. and one in Wayne Co. Five intercept traps were used for each protected plot. Each intercept trap was positioned to align with a sentinel trap and were 25 m apart for a total of 5 traps guarding a plot. Traps were placed in nurseries on 15 April 2015, and the experiment continued through 23 June 2015. In 2016, the experiment was conducted in five commercial nurseries in Lake Co. In this experiment, intercept traps were positioned 10 m apart with 11 traps guarding each protected plot. Traps were placed in nurseries on 30 March and the experiment ran through 28 June 2016.Captures of X. germanus were log(X+1) transformed for analysis. Transformed data were analyzed by repeated measures analysis of variance using Proc Mixed in SAS (SAS Institute 2013, SAS Institute Inc. Version 9.4. SAS Institute, Cary, NC). For an analysis of interception data, captures in sentinel traps within nurseries (13 m, 25 m, 50 m, and 100 m) were combined (woodland trap data excluded) and captures at each sentinel position including in woodlands were analyzed separately. The presence of intercept traps (present or absent) was the between-subjects factor with captures of X. germanus over time the repeated measure. If significant effects of intercept or intercept × time were detected, captures of X. germanus were compared for each sample date using least square means (lsmeans) with the pdiff option in Proc MIXED (SAS Institute 2013).Captures of X. germanus were compared among the various sentinel trap positions (distance from woodland/nursery interface) using repeated measures analysis of variance (Proc MIXED). In this analysis, the distance from the woodland/nursery interface was the between-subjects factor and captures of X. germanus over time the repeated measure. If significant effects of distance or distance × time were detected, captures of X. germanus were compared for each sample date using lsmeans with the Tukey adjustment and pdiff options in Proc MIXED (SAS Institute 2013).Another experiment was designed to determine whether ethanol-injected trap trees reduced colonization attempts by ambrosia beetles on insecticide-treated trees and improved the efficacy of preventive treatments. The experiment was conducted during spring 2015 and was set up as a completely randomized design in a field adjacent to a woodland. There were eight replications of treatments positioned in a single row parallel to and within 1 m of the woodland, with replications placed 10 m apart. Treatments were paired trees, which included an unsprayed trap-tree and a tree sprayed with bifenthrin (OnyxPro, FMC Corporation, Philadelphia, PA). The trap trees were either high-attraction injected with 50% ethanol or equivalent-attraction injected with 5% ethanol. Sprayed trees in both treatments were injected with 5% ethanol to ensure the attraction of ambrosia beetles. Equivalent attraction trap-trees were included as a nonsprayed control for trees injected with 5% ethanol. Trees within treatments were spaced 0.5 m apart. Experimental trees were injected with 75 ml of ethanol (50% or 5%) using the Arborjet® Tree I.V. Delivery System (Woburn, MA) (Ranger et al. 2010, Reding et al 2013). Experimental trees were red maple (Acer rubrum L.) obtained as a bare root and potted in a soilless substrate in 57-L (#15) containers. Trees were injected with ethanol on 19 May and set up in the field 20 May. Attacks were counted at 3- to 9-d intervals through 18 June. During evaluation, a wax pencil was used to mark tunnel entrances to prevent recounting attacks. A different color pencil was used for each count so excavated beetles could be associated with a specific date. Trees were transported to the lab for the final evaluation where beetles were excavated for identification. Scolytinae individuals were identified to the species level using available keys.Data on total ambrosia beetle colonization attempts (tunnel entrances) and attempts by X. germanus (tunnels with beetles present) were log(X+1) transformed for analysis. Analyses compared total colonization attempts and attempts by X. germanus on bifenthrin-treated trees in relation to the paired trap tree (high versus equivalent attraction). Transformed data (logX+1) were analyzed by repeated measures analysis of variance with trap-tree (high or equivalent attraction) as the between-subjects factor and total attempts or attempts by X. germanus on bifenthrin-treated trees through time as the repeated measures (Proc MIXED, SAS) (SAS Institute 2013). When significant trap-tree or trap-tree × time effects were detected for total colonization attempts or X. germanus attempts, data were compared for each sample date using lsmeans and the pdiff option in Proc MIXED (SAS Institute 2013).During experiments conducted in 2015 and 2016, 22 and 24 species of Scolytinae were captured, respectively. Xylosandrus germanus was the most abundant species accounting for 87% and 85% of the 2,936 and 14,064 Scolytinae captured in 2015 and 2016, respectively. Intercept traps captured 2,522 and 11,914 X. germanus in 2015 (5 traps per plot) and 2016 (11 traps per plot), respectively. However, the presence of intercept traps did not reduce captures of X. germanus in sentinel traps when captures within nurseries were combined or sentinel trap positions analyzed separately (Tables 1, 2). Although not significant, cumulative captures of X. germanus tended to be slightly higher in plots without intercept traps each year. Captures of X. germanus were highest in the woodland traps and decreased as the distance from the nursery/woodland interface increased (F = 4.98; df = 4, 15; P = 0.009, in 2015; F = 28.34; df = 4, 20; P < 0.001, in 2016).Trees sprayed with bifenthrin and paired with high- or equivalent-attraction trap trees had no differences in total colonization attempts (2.6 ± 2.3 versus 1.0 ± 0.9 per tree, respectively) (F = 0.37; df = 1, 63; P = 0.55) or attempts by X. germanus (1.9 ± 1.7 versus 0.4 ± 0.4 per tree, respectively) (F = 0.64; df = 1, 63; P = 0.43). Total colonization attempts were about 7.1 times greater in high- versus equivalent-attraction trap trees (82.8 ± 14.4 versus 11.7 ± 5.9 per tree, respectively), whereas attempts with X. germanus were about 5.7 times greater in high versus equivalent attraction trees (43.9 ± 9.8 versus 7.6 ± 4.0 per tree, respectively).In the current research, interception traps positioned at the woodland/nursery interface captured thousands of ambrosia beetles. However, intercepting large numbers of emigrating X. germanus did not reduce captures within nurseries regardless of trap proximity to the woodland/nursery interface. Addesso et al. (2019, J. Econ. Entomol. 112: 753–762) had similar results with X. crassiusculus; they tried to protect sentinel trees injected with ethanol and ethanol-baited sentinel traps. In their research, sentinel trees and traps were positioned 15 m from a woodland/field interface with various densities of interception traps 5 m from the interface. Werle et al. (2019, J. Appl. Entomol. 21: 168–179) tested a push-pull tactic wherein intercept traps and a repellent were used to protect flood-stressed trees. The traps and repellent did not reduce colonization attempts by X. crassiusculus or X. germanus on flood-stressed trees. In 2015, interception traps were positioned 25 m apart, and a slight numerical reduction in captures of X. germanus occurred in plots guarded by those traps. Consequently, in 2016, more interception traps were added and positioned 10 m apart. We hypothesized that placing traps at 10-m intervals would further reduce captures within guarded plots. We also presumed that placing traps at 10-m intervals around the perimeter of their nurseries would be acceptable to growers. The increased trapping did not lead to a further reduction in captures within the guarded nursery plots. McLean and Borden (1979, J. Econ. Entomol. 72: 165–172) concluded that successful pest suppression by mass trapping is related to pest pressure. Xylosandrus germanus populations may be too high in our area to significantly reduce emigration into nurseries by trapping.In the current research, trap-trees in close proximity (0.5 m) to test trees did not reduce colonization pressure on the test trees. Our results were similar to those of Addesso et al. (2019) who found that trap-trees injected with 50% ethanol did not prevent colonization attempts by ambrosia beetles on unsprayed trees injected with 1% ethanol. Colonization attempts on the high-attraction trap-trees were seven times greater than on equivalent-attraction trap-trees. However, there were no differences in colonization attempts on sprayed trees guarded by high- or equivalent-attraction trap-trees. It is possible there was some “spillover” from the high-attraction trees to the sprayed trees. In previous research, spillover did not occur when injected trees were paired with noninjected trees (Ranger et al. 2010, Reding et al. 2013).In summary, ethanol-baited traps positioned between emergence sites and nurseries were not effective at reducing activity of X. germanus within nurseries. Trap trees injected with high concentrations of ethanol did not reduce pressure from ambrosia beetles on adjacent trees injected with low concentrations of ethanol and sprayed with insecticides.

The ambrosia beetles Xylosandrus germanus (Blandford) and Xylosandrus crassiusculus (Motschulsky) bore into flood-stressed trees to establish colonies, but the influence of flooding duration on colonization is unknown. This relationship was examined by flooding trees for various time periods and evaluating colonization. In one experiment, X. germanus bored into 20 dogwood (Cornus florida L.) trees during a 3-d flood treatment. Ten trees dissected that season had no offspring present in tunnels; the remaining trees appeared healthy and bloomed the following spring. In another experiment, dogwood trees were flooded for 3 or 7 d and then dissected to assess colonization. The incidence of superficial (short unbranched) and healed (callus tissue in entrance) tunnels was greater in the 3-d trees, while the incidence of tunnels with X. germanus or offspring was greater in the 7-d trees. Four experiments (three in Ohio and one in Virginia) had flood treatments of 0 (nonflooded), 3, 5, 7, and 10 d. Numbers of tunnel entrances, tunnels with X. germanus, and incidence of tunnels with offspring or live foundresses tended to increase as flood duration increased on apple (Malus × domestica Borkh.), dogwood, and redbud (Cercis canadensis L.) in Ohio and redbud in Virginia. Nonflooded trees in Ohio had no boring activity, but ambrosia beetles bored into three nonflooded trees in Virginia. Indicators of unsuccessful colonization, such as superficial tunnels and healing, decreased as flood duration increased. These results suggest tree crops may recover from boring by ambrosia beetles following short-duration flood events, and not necessarily require culling.

The fungus-farming ambrosia beetle Xylosandrus germanus (Blandford) uses a pouch-like structure (i.e., mycangium) to transport spores of its nutritional fungal mutualist. Our current study sought to identify reference genes necessary for future transcriptome analyses aimed at characterizing gene expression within the mycangium. Complementary DNA was synthesized using selected tissue types from laboratory-reared and field-collected X. germanus consisting of the whole body, head + thorax, deflated or inflated mycangium + scutellum, inflated mycangium, and thorax + abdomen. Quantitative reverse-transcription PCR reactions were performed using primers for 28S ribosomal RNA (28S rRNA), arginine kinase (AK), carbamoyl-phosphate synthetase 2-aspartate transcarbamylase-dihydroorotase (CAD), mitochondrial cytochrome oxidase 1 (CO1), and elongation factor-1α (EF1α). Reference gene stability was analyzed using GeNorm, NormFinder, BestKeeper, ΔCt, and a comprehensive final ranking by RefFinder. The gene CO1 was identified as the primary reference gene since it was generally ranked in first or second position among the tissue types containing the mycangium. Reference gene AK was identified as a secondary reference gene. In contrast, EF1α was generally ranked in the last or penultimate place. Identification of two stable reference genes will aid in normalizing the expression of target genes for subsequent gene expression studies of X. germanus' mycangium.

Laser-guided variable-rate intelligent spray technology is anticipated to reduce pesticide use in production of crops and safeguard the environment. However, the ability of this technology to effectively control insect pests and diseases of crops must be validated before it becomes part of integrated pest management programs. Abilities of three different intelligent sprayers were tested to control pest insects and plant diseases at one fruit farm and two ornamental nurseries in Ohio during three consecutive growing seasons. The same sprayers with disabled intelligent functions were used as conventional constant-rate applications for comparisons. Test crops were apple (Malus pumila), peach (Prunus persica), blueberry (Vaccinium sect. Cyanococcus), black raspberry (Rubus occidentalis), crabapple (Malus sp.), maple (Acer sp.), birch (Betula sp.), and dogwood (Cornus florida). There were five insects and six diseases total involved in the investigations in the fruit farm and two nurseries. The field tests showed the intelligent spray applications reduced pesticide and foliar fertilizer use by ≈30% to 65% on average during the 3-year experiments. At the same time, intelligent spray technology was similar or more effective than conventional spray technology when controlling insects and diseases on a variety of crops. These results demonstrated that intelligent spray technology was environmentally friendly and more effective for control of insect and disease pests in fruit farms and ornamental tree nurseries.

Infestations of fungus gnats (Diptera: Sciaridae) can reduce the production of oyster mushrooms (Pleurotus spp.) grown as food crops within controlled environments. The objectives of this study were to assess the efficacy of Bacillus thuringiensis var. israelensis (Bti) and Steinernema feltiae against fungus gnat larvae. A bioassay was developed, whereby pasteurized straw was inoculated with Pleurotus columbinus and treated with Bti (Gnatrol®), S. feltiae (Nemashield®), or water. Fungus gnats (Lycoriella sp.) were released into each bioassay container for ovipositing onto the straw, thereby exposing the F1 larvae to treated or untreated substrate. Sticky cards within the containers entrapped fungus gnats emerging from the substrate as an indicator of larval survivorship. Following three bioassays, fewer fungus gnats emerged from straw treated with Bti compared to S. feltiae and the water control. Three additional bioassays using Pleurotus ostreatus also demonstrated that fewer fungus gnats emerged from straw treated with Bti compared to S. feltiae and the untreated control. Steinernema feltiae was generally ineffective. Monitoring substrate weight in the bioassay containers over time indicated that Bti and S. feltiae did not impede colonization by P. ostreatus. Incorporating Bti into straw substrate is a promising approach for managing fungus gnats infesting Pleurotus spp.

Ambrosia beetles (Coleoptera: Scolytinae) cultivate their fungal symbiont within host substrates as the sole source of nutrition on which the larvae and adults must feed. To investigate a possible role for semiochemicals in this interaction, we characterized electrophysiological and behavioral responses of Xylosandrus germanus to volatiles associated with its fungal symbiont Ambrosiella grosmanniae . During still-air walking bioassays, X. germanus exhibited an arrestment response to volatiles of A. grosmanniae , but not antagonistic fungi Beauveria bassiana , Metarhizium brunneum , Trichoderma harzianum , the plant pathogen Fusarium proliferatum , or malt extract agar. Solid phase microextraction-gas chromatography-mass spectrometry identified 2-ethyl-1-hexanol, 2-phenylethanol, methyl benzoate and 3-methyl-1-butanol in emissions from A. grosmanniae ; the latter two compounds were also detected in emissions from B. bassiana . Concentration-responses using electroantennography documented weak depolarizations to A. grosmanniae fungal volatiles, unlike the comparatively strong response to ethanol. When tested singly in walking bioassays, volatiles identified from A. grosmanniae elicited relatively weak arrestment responses, unlike the responses to ethanol. Xylosandrus germanus also exhibited weak or no long-range attraction to the fungal volatiles when tested singly during field trials in 2016–2018. None of the fungal volatiles enhanced attraction of X. germanus to ethanol when tested singly; in contrast, 2-phenylethanol and 3-methyl-1-butanol consistently reduced attraction to ethanol. Volatiles emitted by A. grosmanniae may represent short-range olfactory cues that could aid in distinguishing their nutritional fungal symbiont from other fungi, but these compounds are not likely to be useful as long-range attractants for improving detection or mass trapping tactics.

BACKGROUND Helicoverpa zea is managed with foliar applications of chlorantraniliprole in cotton varieties that do not express the Vip3Aa19 toxin in the US Cotton Belt. Foliar insecticides and Bt could interact to influence larval susceptibility. Therefore, it has been suggested that chlorantraniliprole can be used as a tool for Bt resistance management. We designed field and laboratory studies to test the hypothesis that the interaction of Bt toxin and chlorantraniliprole application would result in lower H. zea larval survival when compared to the individual effect of Bt or chlorantraniliprole alone. We also tested for these interactions over time, since chlorantraniliprole residual has not been studied in cotton. RESULTS Results from two field experiments and two laboratory experiments were similar. We found no interactions with Bt and chlorantraniliprole using data not corrected for natural mortality in untreated plots, indicating that these factors did not interact to influence survival. Moreover, we found that Bt and chlorantraniliprole did not interact to influence larval weight and instar. Chlorantraniliprole had lethal and sublethal effects on H. zea larval growth parameters feeding on cotton leaves up to 22 days after application, the final time period that we tested. Finally, concentration of chlorantraniliprole in the leaf was associated with larval survival for the duration of this study, but not larval growth or instar. CONCLUSION Our findings complement the recommendation to use chlorantraniliprole for managing H. zea in cotton, given its long-residual effects. However, the utility of chlorantraniliprole as a Bt-resistance management tool for H. zea remains unclear.

AbstractExotic ambrosia beetles (Curculionidae: Scolytinae) in the tribe Xyleborini include destructive pests of trees growing in horticultural cropping systems. Three species are especially problematic: Xylosandrus compactus (Eichhoff), Xylosandrus crassiusculus (Motschulsky), and Xylosandrus germanus (Blandford). Due to similarities in their host tree interactions, this mini-review focuses on these three species with the goal of describing their host-selection behaviour, characterising associated semiochemicals, and assessing how these interactions relate to their management. All three of these Xylosandrus spp. attack a broad range of trees and shrubs. Physiologically stressed trees are preferentially attacked by X. crassiusculus and X. germanus, but the influence of stress on host selection by X. compactus is less clear. Ethanol is emitted from weakened trees in response to a variety of stressors, and it represents an important attractant for all three species. Other host-derived compounds tested are inconsistent or inactive. Verbenone inhibits attraction to ethanol, but the effect is inconsistent and does not prevent attacks. Integrating repellents and attractants into a push–pull management strategy has been ineffective for reducing attacks but could be optimised further. Overall, maintaining host vigour and minimising stress-induced ethanol are keys for managing these insects, particularly X. crassiusculus and X. germanus.

Ethanol-treated bolts (tree stem sections) have potential as monitoring and pesticide screening tools for ambrosia beetles (Coleoptera: Curculionidae: Scolytinae). Bolts were infused with ethanol by immersing them for at least 24 h. Attacks on ethanol-treated bolts by Xylosandrus species were compared with captures in ethanol-baited traps. Bolts infused in ethanol were usually as attractive or more attractive to Xylosandrus germanus (Blandford) than ethanol-baited bottle traps. Xylosandrus crassiusculus (Motschulsky) were more attracted to bolts than trap in some experiments, but numbers were low and differences were usually not significant. Two techniques for treating bolts with ethanol were compared. Attraction of ambrosia beetles to ethanol-infused bolts were compared with bolts with a drilled cavity filled with ethanol. Drilled bolts filled with ethanol were attractive to X. germanus and were reliably attacked, but numbers of beetles were often lower than in traps and infused bolts. Aged and fresh ethanol-infused bolts were compared with evaluate residual attractiveness. Bolts aged 7 d usually had fewer X. germanus than fresh bolts and traps, and bolts aged 14 d had no beetles. Ethanol-infused bolts from different species of trees were compared. Xylosandrus germanus attacked all species tested with more attacks usually in red maple (Acer rubrum L.). Anisandrus maiche Stark was attracted to ethanol-infused bolts indicating it may attack trees emitting ethanol. Bolts attracted fewer nontarget species than traps, but residual attraction was much less. The selectivity of ethanol-treated bolts for Xylosandrus species should make them useful for monitoring and screening pesticides against those species.