Erwiniaceae bacteria play defensive and nutritional roles in two widespread ambrosia beetles.

Ambrosia beetles are fungal-growing insects excavating galleries deep inside the wood. Their success as invaders increased scientific interest towards them. However, most studies on their microbiota targeted their fungal associates whereas the role of bacterial associates is understudied. To explore the role of abundant microbial associates, we isolated bacteria from active galleries of two widespread ambrosia beetles, Xylosandrus crassiusculus and X. germanus. These isolates were classified within the Erwiniaceae family and through a phylogenetic analysis including isolates from other insects we showed that they clustered with isolates obtained from ambrosia and bark beetles, including Erwinia typographi. The whole genome analysis of the isolate from active galleries of X. crassiusculus suggested that this bacterium plays both a nutritional role, by providing essential amino acids and enzymes for the hydrolysis of plant biomass, and a defensive role, by producing antibiotics. This defensive role was also tested in vitro against fungi, including mutualists, common associates, and parasites. The bacteria inhibited the growth of some of the common associates and parasites but did not affect mutualists. Our study supported the hypothesis of a mutualist role of Erwiniaceae bacteria in ambrosia beetles and highlighed the importance of bacteria in maintaining the symbiosis of their host with nutritional fungi.

Major Pathogens Affecting Carob in the Mediterranean Basin: Current Knowledge and Outlook in Italy.

The main pathogens affecting the carob (Ceratonia siliqua) tree in the Mediterranean basin are described in this overview. The most widespread diseases periodically occurring in carob orchards are powdery mildew (Pseudoidium ceratoniae) and cercospora leaf spot (Pseudocercospora ceratoniae). The causal agents of "black leaf spots" (e.g., Pestalotiopsis, Phyllosticta and Septoria spp.) are responsible for symptoms similar to those previously mentioned for foliar diseases, but are reported in carob orchards at a negligible frequency. Likewise, canker and branch diebacks caused by fungal species belonging to Botryosphaeriaceae are almost never recorded. Among the rots of wood tissues that may compromise old carob specimens, "brown cubical rot" caused by Laetiporus sulphureus is the most widespread and recurrent issue; this pathogen is also well-known for producing edible fruit bodies that are appreciated for pharmaceutical and industrial purposes. On the other hand, "white rots" caused by Fomes and Ganoderma species are less common and reported for the first time in this review. Gall-like protuberances on twigs of uncertain aetiology or tumors on branches associated with Rhizobium radiobacter are described, although these symptoms are seldom detected, as they are also observed for necrotic leaf spots caused by Pseudomonas syringae pv. ciccaronei. A worldwide list of pathogens not yet recorded but at high risk of potential introduction in Italian carob-producing areas is also provided. Finally, concerns related to new phytopathogenic fungi vectored by the invasive Xylosandrus compactus ambrosia beetle are addressed. All the described pathogens could become limiting factors for carob production in the near future, because they could be favored by high-density orchards, the increasing global network of trade exchanges, and the high frequency at which extreme events related to climate change occur globally. Thus, symptoms and signs, causal agents, epidemiology, and, whenever applicable, recommendations for disease prevention and management are provided in this review.

Presence of the causal agent of laurel wilt disease in sassafras-associated insects.

Laurel wilt disease (LWD) is a lethal vascular wilt caused by an exotic ambrosia beetle-fungal complex, the redbay ambrosia beetle, Xyleborus glabratus Eichhoff, and its nutritional symbiont, Harringtonia lauricola (Harr., Fraedrich & Aghayeva) de Beer & Procter. LWD is responsible for the widespread mortality of redbay, Persea borbonia (L.) Spreng., devastating coastal forests in the southeast United States. More recently, LWD is causing mortality of understory sassafras, Sassafras albidum (Nutt.) Nees, in deciduous forests in Kentucky, USA; the biology, epidemiology, and long-term impacts of LWD in deciduous forests are unclear. All North American lauraceous species evaluated have shown susceptibility, and numerous additional ambrosia beetles have demonstrated vector potential, but no studies to date have assessed the presence of H. lauricola in other insects associated with LWD-infected sassafras. We sampled infected sassafras from the leading edge of the LWD range and collected insect associates to evaluate phoretic and internal presence of H. lauricola. We recorded 118 individuals of 38 morphospecies emerging; most were Coleoptera. Of the 48 specimens evaluated for H. lauricola, none tested positive for phoretic presence, but internal presence was evident in the granulate ambrosia beetle, Xylosandrus crassiusculus Motschulsky, and in a hidden snout weevil, Apteromechus ferratus Say. This is the first report of H. lauricola associated with a non-ambrosia beetle and expands our understanding of the vector potential of additional insect species while confirming the role of the granulate ambrosia beetle. These findings contribute to our understanding of LWD epidemiology in sassafras hosts from more northerly latitudes.

Acibenzolar-S-methyl induces resistance against ambrosia beetle attacks in dogwoods exposed to simulated flood stress.

Xylosandrus spp. ambrosia beetles (Coleoptera: Curculionidae: Scolytinae) are important wood-boring pests of nursery trees weakened by abiotic and biotic stressors. Acibenzolar-S-methyl (ASM), a plant defense elicitor, was tested for inhibiting Xylosandrus spp. tunneling (i.e., attacks) into flood-stressed flowering dogwoods (Cornus florida L. (Cornales: Cornaceae)). Container-grown dogwoods were treated with ASM substrate drench + flooding, ASM foliar spray + flooding, ASM drench + no flooding, ASM foliar + no flooding, no ASM + flooding, or no ASM + no flooding at 3 days before flood stress in a completely randomized design under field conditions. Trees were flooded for 14 days and then drained and watered as needed. Attacks were counted every 2 days for 28 days. Plant tissue samples were collected at 7 and 14 days after flooding to determine ethanol content using solid-phase microextraction-gas chromatography-mass spectrometry. Trees were dissected to determine gallery formation and depth, fungal colonization, and the presence of eggs, larvae, and adults. The highest number of Xylosandrus beetle species attacks were recorded from plants exposed to no ASM + flooding, but attacks were reduced in ASM treated trees (drench or foliar) + flooding. Trees treated with drenches had fewer attacks than foliar sprays. Plants assigned to no flood had the fewest beetle attacks. Moreover, ASM reduced Xylosandrus spp. gallery formation and depth, fungal colonization, and presence of eggs, larvae, and adults. All flooded trees produced ethanol. In conclusion, ASM induced a plant defense response to Xylosandrus spp. tunneling in dogwoods under flood stress conditions.

Effects of permethrin on ambrosia beetles (Coleoptera: Curculionidae: Scolytinae) in ornamental nurseries.

Exotic ambrosia beetles (Coleoptera: Curculionidae: Scolytinae), such as Xylosandrus crassiusculus (Motschulsky), Xylosandrus germanus (Blandford), and Xylosandrus compactus (Eichoff) are serious pests in southeastern ornamental nurseries. Preventative pyrethroid trunk sprays effectively reduce boring damage. However, it is unclear how pyrethroids such as permethrin prevent attack. Thus, the objective was to determine how permethrin-treated bolts interact with invading ambrosia beetles. In 2022, a study with 2 independent trials was conducted in a nursery on red maple (Acer rubrum L.), bolts during March and April, respectively. The treatments were (i) nonbaited, nontreated bolt, (ii) ethanol baited bolt, (iii) nonbaited bolt + glue [painted on bolt], (iv) ethanol baited bolt + glue, (v) ethanol baited bolt + glue + permethrin, (vi) ethanol baited bolt + glue + permethrin + verbenone, and (vii) ethanol baited bolt + glue + verbenone. Ambrosia beetles trapped on glue, beetles which fell into the pail with soap solution under the bolts, and entry holes on bolts were quantified. Permethrin prevented beetle attacks but did not reduce the number of ambrosia beetles landing on the treated bolts. Verbenone reduced ambrosia beetles from landing on the bolts but did not prevent boring into bolts. The numbers of ambrosia beetles in soapy water were not significantly different among treatments. Ambrosia beetles are landing on permethrin-treated bolts but not boring into the bolts, implying that fresh permethrin residues may not be necessary for ambrosia beetle management.

The biological relevance of the FspTF transcription factor, homologous of Bqt4, in Fusarium sp. associated with the ambrosia beetle Xylosandrus morigerus.

Transcription factors in phytopathogenic fungi are key players due to their gene expression regulation leading to fungal growth and pathogenicity. The KilA-N family encompasses transcription factors unique to fungi, and the Bqt4 subfamily is included in it and is poorly understood in filamentous fungi. In this study, we evaluated the role in growth and pathogenesis of the homologous of Bqt4, FspTF, in Fusarium sp. isolated from the ambrosia beetle Xylosandrus morigerus through the characterization of a CRISPR/Cas9 edited strain in Fsptf. The phenotypic analysis revealed that TF65-6, the edited strain, modified its mycelia growth and conidia production, exhibited affectation in mycelia and culture pigmentation, and in the response to certain stress conditions. In addition, the plant infection process was compromised. Untargeted metabolomic and transcriptomic analysis, clearly showed that FspTF may regulate secondary metabolism, transmembrane transport, virulence, and diverse metabolic pathways such as lipid metabolism, and signal transduction. These data highlight for the first time the biological relevance of an orthologue of Bqt4 in Fusarium sp. associated with an ambrosia beetle.

The worldwide invasion history of a pest ambrosia beetle inferred using population genomics.

Xylosandrus crassiusculus, a fungus-farming wood borer native to Southeastern Asia, is the most rapidly spreading invasive ambrosia species worldwide. Previous studies focusing on its genetic structure suggested the existence of cryptic genetic variation in this species. Yet, these studies used different genetic markers, focused on different geographical areas and did not include Europe. Our first goal was to determine the worldwide genetic structure of this species based on both mitochondrial and genomic markers. Our second goal was to study X. crassiusculus' invasion history on a global level and identify the origins of the invasion in Europe. We used a COI and RAD sequencing design to characterize 188 and 206 specimens worldwide, building the most comprehensive genetic data set for any ambrosia beetle to date. The results were largely consistent between markers. Two differentiated genetic clusters were invasive, albeit in different regions of the world. The markers were inconsistent only for a few specimens found exclusively in Japan. Mainland USA could have acted as a source for further expansion to Canada and Argentina through stepping stone expansion and bridgehead events. We showed that Europe was only colonized by Cluster II through a complex invasion history including several arrivals from multiple origins in the native area, and possibly including bridgehead from the United States. Our results also suggested that Spain was colonized directly from Italy through intracontinental dispersion. It is unclear whether the mutually exclusive allopatric distribution of the two clusters is due to neutral effects or due to different ecological requirements.

Type and duration of water stress influence host selection and colonization by exotic ambrosia beetles (Coleoptera: Curculionidae).

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.

Molecular Characterization of Native Entomopathogenic Fungi from Ambrosia Beetles in Hazelnut Orchards of Turkey and Evaluation of Their In Vitro Efficacy.

Ambrosia beetles, Anisandrus dispar Fabricius, Xylosandrus germanus Blandford, and Xyleborinus saxesenii Ratzeburg (Coleoptera: Curculionidae: Scolytinae) are among the most significant hazelnut pests in Turkey. The control of these pests is difficult and expensive due to their biology. The present study aimed to isolate entomopathogenic fungi (EPF) from A. dispar, X. germanus, and X. saxesenii individuals that were obtained from the main hazelnut production areas of Turkey, characterize the EPF isolates using internal transcribed spacer (ITS)-DNA sequencing and iPBS profiling, and determine the efficacy of the isolates against A. dispar, X. germanus, and X. saxesenii under laboratory conditions. Phylogenetic analyses based on ITS revealed that the 47 native isolates were Beauveria bassiana (11), B. pseudobassiana (8), Cordyceps fumosorosea (6), Cordyceps farinosa (1), Akanthomyces lecanii (13), Purpureocillium lilacinum (3), Clonostachys rosea (2) and Metarhizium anisopliae (3). For the first time, the primer binding site (PBS) marker system, based on retrotransposons, was used to discriminate successfully among the EPF species. Some isolates of B. bassiana, B. pseudobassiana, C. fumosorosea, A. lecanii, and M. anisopliae caused 100% mortality of the beetle species within 7 to 9 days. The findings of this study indicated that some isolated entomopathogenic fungi provide an essential basis for the development of bioproducts, as well as a promising alternative method for controlling these ambrosia beetles.

Geographical Distribution of Three Forest Invasive Beetle Species in Romania.

Ips duplicatus (Sahlberg, 1836), Xylosandrus germanus (Blandford, 1894) and Neoclytus acuminatus (Fabricius, 1775) are invasive species reported in Romania, but their current distribution is poorly known. The research aim was to provide new information on this issue. A survey was conducted over the period 2015-2017 in 82 locations, using flight-interception traps and bottle traps, baited with different attractants. Data obtained in our other unpublished studies were also taken into account. A total of 35,136 I. duplicatus beetles were collected in 30 survey locations. The highest captures were in the log yards of some factories processing logs of Norway spruce (Picea abies (L.) H. Karst.). Considering all known records so far, most of these are in the eastern part of Romania, where an outbreak took place during the years 2005-2014, mainly in spruce stands growing outside their natural range. During the survey, 4259 specimens of X. germanus were collected in 35 locations, but in our other studies the species was found in 13 additional places. It was collected at altitudes of 18-1200 m, and the largest catches were from beech stands, growing at 450-950 m. N. acuminatus was found in only six locations, in the western and southern parts of the country, at low altitudes, in tree stands composed of Fraxinus excelsior L., Quercus spp. and other broadleaf species, as well as in broadleaf log yards. The results suggest that I. duplicatus is established in most parts of the Norway spruce's range, X. germanus is still spreading in the country, with some areas having quite high populations, while N. acuminatus is present only in the warmest regions of the country.

Cerambycid Pheromones 3,2-Hydroxyketones Affect Catches of Some Bark and Ambrosia Beetles (Coleoptera: Curculionidae) in Ethanol-Baited Multiple-Funnel Traps in Southeastern United States.

In 2012-2013, we assessed the interactive effects of the cerambycid pheromones syn-2,3-hexanediol, 3-hydroxyhexan-2-one, and 3-hydroxyoctan-2-one on catches of bark and ambrosia beetles (Coleoptera: Curculionidae) in ethanol-baited multiple-funnel traps in north Georgia and South Carolina. We found that catches for nine of eleven species of ambrosia beetles in ethanol-baited traps were either unaffected or enhanced by the addition of 3,2-hydroxyketones. Similarly catches of five species of bark beetles were either unaffected or enhanced by the addition of 3,2-hydroxyketones. In particular, catches of Xylosandrus crassiusculus (Motschulsky), Cnestus mutilatus (Blandford), and Monarthrum fasciatum (Say) in ethanol-baited traps increased with the addition of 3-hydroxyhexan-2-one and/or 3-hydroxyoctan-2-one. Catches of the bark beetles Hylocurus rudis (LeConte) and Hypothenemus rotundicollis (Eichhoff) were enhanced by the addition of 3-hydroxyhexan-2-one and 3-hydroxyoctan-2-one, respectively. syn-2,3-Hexanediol had no effect on catches of bark and ambrosia beetles in ethanol-baited traps. Our data provide support for the use of ethanol + cerambycid pheromones for targeting non-native species of bark and ambrosia beetles as well as cerambycids in detection programs.

Succession of Ambrosia Beetles Colonizing the Logs of Fallen Alder and Birch Trees.

Ambrosia beetles bore into the xylem of woody plants, reduce timber quality, and can sometimes cause devastating damage to forest ecosystems. The colonization by different beetle species is dependent on host status, from healthy trees to the early stages of wood decay, although the precise factors influencing their host selection are not well known. Classic studies on plant ecology have determined the niches of different plant species in vegetation succession, based on comparisons of successions in different locations using ordination analyses, although the factors influencing the colonization of each species are largely undetermined. In this study, to characterize the succession of ambrosia beetles after tree felling, two Betulaceae tree species, an alder (Alnus hirsuta), and a white birch (Betula platyphylla var. japonica) were felled as bait logs in central Hokkaido, Japan, in 2016. From 2016 to 2018, the bait logs were dissected late in each flying season, and ambrosia beetles were collected from the logs. During the period of monitoring, the beetle colonization in both tree species was mostly concentrated in the first 2 years. We observed similarities in the beetle faunas colonizing the two plant species, and that individual species appeared in the same sequence in the logs of the two plant species, although the temporal patterns of colonization differed. Consequently, significant differences in beetle community compositions in the two host species were detected in each of the first 2 years of the study, whereas the difference in the overall composition of beetle assemblages (=pooled over 3 years) between the two plant species was smaller than that in either 2016 or 2017. We speculated that the differences in the temporal pattern of colonization could be attributable to differences in the rates at which the wood of the two tree species deteriorated. Treptoplatypus severini and Xylosandrus crassiusculus were considered to be early-successional species that commenced log colonization soon after felling, although T. severini has a wide niche and was collected during all 3 years of the study. Conversely, Xyleborinus attenuatus and Heteroborips seriatus were identified as probable late-successional species that showed a preference for older logs.

Characterization of Two Fusarium solani Species Complex Isolates from the Ambrosia Beetle Xylosandrus morigerus.

Ambrosia beetles are insect vectors of important plant diseases and have been considered as a threat to forest ecosystems, agriculture, and the timber industry. Several factors have been suggested as promoters of the pathogenic behavior of ambrosia beetles; one of them is the nature of the fungal mutualist and its ability to establish an infectious process. In Mexico, Xylosandrus morigerus is an invasive ambrosia beetle that damages many agroecosystems. Herein, two different isolates from the X. morigerus ambrosia beetle belonging to the Fusarium genus are reported. Both isolates belong to the Fusarium solani species complex (FSSC) but not to the Ambrosia Fusarium clade (AFC). The two closely related Fusarium isolates are pathogenic to different forest and agronomic species, and the morphological differences between them and the extracellular protease profile suggest intraspecific variability. This study shows the importance of considering these beetles as vectors of different species of fungal plant pathogens, with some of them even being phylogenetically closely related and having different pathogenic abilities, highlighting the relevance of the fungal mutualist as a factor for the ambrosia complex becoming a pest.

Effects of Trap, and Ethanol Lure Type and Age on Attraction of Ambrosia Beetles (Coleoptera: Curculionidae).

Ambrosia beetles (Xylosandrus spp.) are pests of woody ornamental nurseries and tree nut orchards. Growers use ethanol-infused bolts and ethanol-mediated bottle traps for monitoring ambrosia beetles in the spring. To refine these monitoring tools, we investigated the 1) sensitivity of the commercially available ethanol pouches placed on bolts compared with that of ethanol-infused bolts for ambrosia beetle attacks; and 2) the effect of aging of ethanol pouches on ambrosia beetle captures in bottle traps. Experiments were conducted in ornamental nurseries and pecan orchards in 2019 and 2020. For objective 1, the treatments included a bolt infused with locally purchased ethanol, an ethanol pouch attached to a bolt, and a control (bolt without ethanol). For objective 2, ethanol pouches were initially field-aged for 0, 1, 2, 3, and 4 wk and ambrosia beetle captures were documented weekly for 4 wk. Ethanol pouches older than 5 wk were replaced with fresh pouches at the pecan sites while ethanol pouches at nursery sites were aged up to 8 wk. The ethanol-pouch comparison study showed that the number of ambrosia beetle attacks was significantly greater on ethanol-infused bolt traps than with the ethanol pouches on bolts at the nursery and pecan sites in both years. The age of the ethanol pouches did not affect captures of Xylosandrus crassiusculus (Motschulsky) and X. germanus (Blandford) in bottle traps within 8 wk of deployment. Results provide practical implications into the sensitivity of the different ethanol lure types in association with bolt traps and the longevity of the ethanol pouches for grower use.

Colonization of Trees by Ambrosia Beetles (Coleoptera: Curculionidae: Scolytinae) Is Influenced by Duration of Flood Stress.

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.

Electrophysiological and Behavioral Responses of an Ambrosia Beetle to Volatiles of its Nutritional Fungal Symbiont.

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.

Permethrin Residual Activity Against Ambrosia Beetle (Coleoptera: Curculionidae: Scolytinae) Attacks Following Field Aging and Simulated Rainfall Weathering.

Adult ambrosia beetles (Coleoptera: Curculionidae: Scolytinae) bore into ornamental nursery trees resulting in trunk vascular tissue damage, which can potentially kill trees. Ambrosia beetle exposure to surface-applied insecticides is minimal after internal trunk galleries are formed, so effective management requires insecticide treatments to be applied near the time of infestation or to have residual activity on the bark. Tree trunk sections (bolts) were used to determine the effect of field aging or irrigation (i.e., simulated rainfall weathering) on permethrin residual activity against ambrosia beetles. In all experiments, 30-cm-long bolts from Liriodendron tulipifera L. (Magnoliales: Magnoliaceae) were hollowed and filled with 70% ethanol at field deployment to induce ambrosia beetle attacks over a 2-wk period. To evaluate insecticide residual activity, permethrin was sprayed onto tree bolts at 0, 8, 17, or 24 d before ethanol addition, and then bolts were deployed along a wooded border in fall 2017 and spring 2018. Tree bolts with permethrin residues ≤17 d old had significantly fewer ambrosia beetle attacks than bolts with 24-d-old residues or the non-permethrin-treated control bolts. To evaluate simulated rainfall weathering, permethrin was applied to tree bolts 8 or 22 d before ethanol (spring 2018) or 10 or 24 d before ethanol (fall 2018) with half of the bolts receiving regular irrigation events. Irrigation had no significant effect on permethrin residual activity against ambrosia beetles during either test. This study determined ambrosia beetle control was affected by permethrin residue age more than simulated rainfall weathering, and a reapplication interval of ≤17 d maximized beetle control.

Attraction of Invasive Ambrosia Beetles (Coleoptera: Curculionidae: Scolytinae) to Ethanol-Treated Tree Bolts.

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.

Long-Lasting Insecticide Netting for Protecting Tree Stems from Attack by Ambrosia Beetles (Coleoptera: Curculionidae: Scolytinae).

Ambrosia beetles (Coleoptera: Curculionidae: Scolytinae) are destructive wood-boring insects of horticultural trees. We evaluated long-lasting insecticide netting for protecting stems against ambrosia beetles. Container-grown eastern redbud, Cercis canadensis, trees were flood-stressed to induce ambrosia beetle attacks, and deltamethrin-treated netting was wrapped from the base of the stem vertically to the branch junction. Trees were deployed under field conditions in Ohio, Virginia, Tennessee, and Mississippi with the following treatments: (1) flooded tree; (2) flooded tree with untreated netting; (3) flooded tree with treated 'standard mesh' netting of 24 holes/cm2; (4) flooded tree with treated 'fine mesh' netting of 28 holes/cm2; and/or (5) non-flooded tree. Treated netting reduced attacks compared to untreated netting and/or unprotected trees in Mississippi in 2017, Ohio and Tennessee in 2018, and Virginia in 2017-2018. Inconsistent effects occurred in Mississippi in 2018. Fewer Anisandrus maiche, Xylosandrus germanus, and Xyleborinus saxesenii were dissected from trees deployed in Ohio protected with treated netting compared to untreated netting; trees deployed in other locations were not dissected. These results indicate long-lasting insecticide netting can provide some protection of trees from ambrosia beetle attacks.

Acquisition of fungi from the environment modifies ambrosia beetle mycobiome during invasion.

Microbial symbionts can play critical roles when their host attempts to colonize a new habitat. The lack of symbiont adaptation can in fact hinder the invasion process of their host. This scenario could change if the exotic species are able to acquire microorganisms from the invaded environment. Understanding the ecological factors that influence the take-up of new microorganisms is thus essential to clarify the mechanisms behind biological invasions. In this study, we tested whether different forest habitats influence the structure of the fungal communities associated with ambrosia beetles. We collected individuals of the most widespread exotic (Xylosandrus germanus) and native (Xyleborinus saxesenii) ambrosia beetle species in Europe in several old-growth and restored forests. We characterized the fungal communities associated with both species via metabarcoding. We showed that forest habitat shaped the community of fungi associated with both species, but the effect was stronger for the exotic X. germanus. Our results support the hypothesis that the direct contact with the mycobiome of the invaded environment might lead an exotic species to acquire native fungi. This process is likely favored by the occurrence of a bottleneck effect at the mycobiome level and/or the disruption of the mechanisms sustaining co-evolved insect-fungi symbiosis. Our study contributes to the understanding of the factors affecting insect-microbes interactions, helping to clarify the mechanisms behind biological invasions.