Semiochemical-mediated host selection by Xylosandrus spp. ambrosia beetles (Coleoptera: Curculionidae) attacking horticultural tree crops: a review of basic and applied science

Christopher M. Ranger; Michael E. Reding; Karla Addesso; Matthew Ginzel; Davide Rassati

doi:10.4039/tce.2020.51

Abstract

Exotic 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.

Introduction

Ambrosia beetles (Coleoptera: Curculionidae) represent about 3400 species within the Scolytinae and 1400 species within the Platypodinae (Hulcr et al. Reference Hulcr, Atkinson, Cognato, Jordal, McKenna, Vega and Hofstetter2015). These beetles, and especially those belonging to the tribe Xyleborini, are recognised as extremely successful invaders worldwide (Hulcr and Stelinski Reference Hulcr and Stelinski2017). Around 50% of nonnative scolytines established in North America and Europe are within the tribe Xyleborini (Haack et al. Reference Haack, Rabaglia, Peña and Peña2013; Rassati et al. Reference Rassati, Lieutier, Faccoli, Paine and Lieutier2016b; Gomez et al. Reference Gomez, Rabaglia, Fairbanks and Hulcr2018), including some serious pests of trees growing in natural and managed habitats (Agnello et al. Reference Agnello, Breth, Tee, Cox and Warren2015; Ranger et al. Reference Ranger, Reding, Schultz, Oliver, Frank, Addesso, Chong, Sampson, Werle, Gill and Krause2016; Hulcr and Stelinksi Reference Hulcr and Stelinski2017). Key traits that contribute to the invasion success of xyleborine ambrosia beetles also make them particularly challenging pests to manage. These include a broad host range, cryptic wood-boring behaviour, an association with fungal symbionts, haplodiploid reproduction, and sib-mating (Kirkendall et al. Reference Kirkendall, Wrensch, Ebbert, Wrensch and Ebbert1993; Normark et al. Reference Normark, Jordal and Farrell1999; Dole et al. Reference Dole, Jordal and Cognato2010; Hulcr and Stelinksi Reference Hulcr and Stelinski2017). Adult female xyleborines tunnel into the heartwood of trees to create galleries where they cultivate fungal symbionts and rear offspring (Hulcr and Stelinksi Reference Hulcr and Stelinski2017). Symptoms of infestation include sawdust “toothpicks” and sap emerging from the tunnel entrances, branch and twig dieback, and death of saplings and small trees (Greco and Wright Reference Greco and Wright2015; Ranger et al. Reference Ranger, Reding, Schultz, Oliver, Frank, Addesso, Chong, Sampson, Werle, Gill and Krause2016).

Three species of xyleborine ambrosia beetles are especially problematic in nurseries, orchards, groves, and plantations: these are black twig borer, Xylosandrus compactus (Eichhoff); granulate ambrosia beetle, Xylosandrus crassiusculus (Motschulsky), and black stem borer, Xylosandrus germanus (Blandford) (Chong et al. Reference Chong, Reid and Williamson2009; Greco and Wright Reference Greco and Wright2012, Reference Greco and Wright2015; Agnello et al. Reference Agnello, Breth, Tee, Cox and Warren2015, Reference Agnello, Breth, Tee, Cox, Villani, Ayer, Wallis, Donahue, Combs, Davis and Neal2017; Ranger et al. Reference Ranger, Tobin and Reding2015b, Reference Ranger, Reding, Schultz, Oliver, Frank, Addesso, Chong, Sampson, Werle, Gill and Krause2016). Xylosandrus compactus is native to Asia but has been introduced to parts of Africa, Europe, New Zealand, North America, the Pacific Islands, and South America (Rabaglia et al. Reference Rabaglia, Dole and Cognato2006; Garonna et al. Reference Garonna, Dole, Saracino, Mazzoleni and Cristinzio2012; Gomez et al. Reference Gomez, Rabaglia, Fairbanks and Hulcr2018; CABI 2019a). Since being first reported in Florida in 1941, X. compactus has become established in 13 states in the midwestern, southeastern, and southern United States, as well as in Hawaii (Rabaglia et al. Reference Rabaglia, Dole and Cognato2006, Reference Rabaglia, Cognato, Hoebeke, Johnson, LaBonte, Carter and Vlach2019; Chong et al. Reference Chong, Reid and Williamson2009; Gomez et al. Reference Gomez, Rabaglia, Fairbanks and Hulcr2018). Xylosandrus crassiusculus is native to southeast Asia but is now established in parts of Africa, Central America, North America, Oceania, and South America (Storer et al. Reference Storer, Payton, McDaniel, Jordal and Hulcr2017; CABI 2019b). Since being first reported in South Carolina in 1974, X. crassiusculus is now established in 31 states in the northeastern, mid-Atlantic, southeastern, southern, and midwestern United States, along with Hawaii and the Canadian province of Ontario (Anderson Reference Anderson1974; Rabaglia et al. Reference Rabaglia, Dole and Cognato2006, Reference Rabaglia, Cognato, Hoebeke, Johnson, LaBonte, Carter and Vlach2019; Haack et al. Reference Haack, Rabaglia, Peña and Peña2013; Gomez et al. Reference Gomez, Rabaglia, Fairbanks and Hulcr2018). Xylosandrus germanus is native to southeast Asia but is now established in Europe, Great Britain, and North America (Ito et al. Reference Ito, Kajimura, Hamaguchi, Araya and Lakatos2008; CABI 2019c; Inward Reference Inward2020). Since being first detected in New York in 1932 (Felt Reference Felt1932), X. germanus has spread to 34 states in the northeastern, mid-Atlantic, southeastern, southern, midwestern, and northwestern United States, along with the Canadian provinces of British Columbia, Nova Scotia, Ontario, and Québec (Rabaglia et al. Reference Rabaglia, Dole and Cognato2006, Reference Rabaglia, Cognato, Hoebeke, Johnson, LaBonte, Carter and Vlach2019; Gomez et al. Reference Gomez, Rabaglia, Fairbanks and Hulcr2018; CABI 2019c).

Three species of fungi have been reported as mycangial symbionts of X. compactus, including Ambrosiella xylebori Brader (Ceratocystidaceae), Ambrosiella macrospora (Francke-Grossman) Batra, and Fusarium solani (Martius) Saccardo (Nectriaceae) (Bateman et al. Reference Bateman, Šigut, Skelton, Smith and Hulcr2016). Ambrosiella roeperi T.C. Harrington & McNew is the mycangial symbiont of X. crassiusculus (Harrington et al. Reference Harrington, McNew, Mayers, Fraedrich and Reed2014; Mayers et al. Reference Mayers, McNew, Harrington, Roeper, Fraedrich, Biedermann, Castrillo and Reed2015), and Ambrosiella grosmanniae C. Mayers, McNew & T.C. Harrington is the symbiont of X. germanus (Mayers et al. Reference Mayers, McNew, Harrington, Roeper, Fraedrich, Biedermann, Castrillo and Reed2015). A variety of secondary microorganisms can also be transported on the cuticle or in the mycangium, some of which can be tree pathogens (Kinuura Reference Kinuura1995; Dute et al. Reference Dute, Miller, Davis, Woods and McLean2002; Carrillo et al. Reference Carrillo, Duncan, Ploetz, Campbell, Ploetz and Peña2014; Bateman et al. Reference Bateman, Šigut, Skelton, Smith and Hulcr2016; Juzwik et al. Reference Juzwik, McDermott-Kubeczko, Stewart and Ginzel2016; Malacrinò et al. Reference Malacrinò, Rassati, Schena, Mehzabin, Battisti and Palmeri2017).

The goal of this review is to describe the host-selection behaviour of these three Xylosandrus spp., characterise the associated semiochemicals, and assess how such interactions relate to managing these destructive insects of horticultural tree crops. Possible reasons why these pests, which are restricted to dying or dead host substrates in their native habitats, began attacking apparently healthy trees in horticultural production systems following their introduction into North America are also discussed.

Host-selection behaviour

Host range

Influence of host quality

The majority of ambrosia beetle species use dying or dead hosts (Hulcr and Stelinski Reference Hulcr and Stelinski2017), but some species, including X. crassiusculus and X. germanus, preferentially attack stressed or weakened trees (Kühnholz et al. Reference Kühnholz, Borden and Uzunovic2001; Ranger et al. Reference Ranger, Reding, Schultz, Oliver, Frank, Addesso, Chong, Sampson, Werle, Gill and Krause2016). Both X. crassiusculus and X. germanus have been reported to attack apparently healthy trees that show no visible symptoms of stress (Weber and McPherson Reference Weber and McPherson1984). However, physiologically stressed trees can visually appear healthy while emitting stress-associated volatiles (i.e., ethanol) that signal their vulnerable state to generalist ambrosia beetles (Ranger et al. Reference Ranger, Reding, Persad and Herms2010, Reference Ranger, Reding, Schultz and Oliver2013a, Reference Ranger, Tobin and Reding2015b). Studies described herein demonstrate the preference of X. crassiusculus and X. germanus for trees in the early stages of physiological stress over healthy trees. Despite having a broad host species range, X. crassiusculus and X. germanus function more like specialists when influenced by hosts in a weakened condition, such that certain species and individual trees are attacked in a nonrandom fashion within diverse horticultural production systems (Ranger et al. Reference Ranger, Tobin and Reding2015b). Xylosandrus compactus reportedly attacks both apparently healthy and physiologically stressed trees (Masuya Reference Masuya2007; Oliveira et al. Reference Oliveira, Flechtmann and Frizzas2008; Chong et al. Reference Chong, Reid and Williamson2009; Greco and Wright Reference Greco and Wright2012, Reference Greco and Wright2015; Vannini et al. Reference Vannini, Contarini, Faccoli, Valle, Rodriguez, Mazzetto, Guarneri, Vettraino and Speranza2017), but colonisation success has not been compared under controlled conditions to determine the influence of host condition.

In support of anecdotal field observations from ornamental nurseries where poor drainage preceded attacks on flood-intolerant Cornus florida L. (Cornaceae) trees (Ranger et al. Reference Ranger, Reding, Schultz and Oliver2013a, Reference Ranger, Reding, Schultz, Oliver, Frank, Addesso, Chong, Sampson, Werle, Gill and Krause2016), studies under controlled conditions have demonstrated that flood stress predisposes trees to attack by X. crassiusculus and X. germanus. Ott (Reference Ott2007) induced attacks by X. crassiusculus and other ambrosia beetles within one to nine days of initiating flood stress of container-grown Quercus alba Linnaeus (Fagaceae) saplings; notably, the trees appeared healthy as attacks were initiated. Similarly, more X. germanus were attracted to and landed on flood-stressed C. florida trees over nonflooded trees (Ranger et al. Reference Ranger, Reding, Schultz and Oliver2013a). Moreover, X. germanus began landing on C. florida trees within one day of flooding, whereas no attacks occurred on nonflooded control trees. During subsequent free-choice experiments, X. crassiusculus and X. germanus readily distinguished among flooded trees varying in their tolerance of flood stress and preferentially attacked flood-intolerant deciduous tree species over flood-tolerant species (Ranger et al. Reference Ranger, Schultz, Frank, Chong and Reding2015a). When X. crassiusculus and X. germanus were caged to stems of flooded or nonflooded C. florida trees, foundresses established fungal gardens and produced offspring only in stems of flooded trees. Additional studies further support the preference of X. crassiusculus and X. germanus for thin-barked tree species subjected to flood stress, including Cercis canadanesis Linnaeus (Fabaceae), Malus domestica Borkhausen (Rosaceae), and Styrax japonicus Siebold (Styracaceae) (Frank and Ranger Reference Frank and Ranger2016; Ranger et al. Reference Ranger, Reding, Schultz, Oliver, Frank, Addesso, Chong, Sampson, Werle, Gill and Krause2016, Reference Ranger, Werle, Schultz, Addesso, Oliver and Reding2020; Agnello et al. Reference Agnello, Breth, Tee, Cox, Villani, Ayer, Wallis, Donahue, Combs, Davis and Neal2017; Frank et al. Reference Frank, Anderson and Ranger2017; Werle et al. Reference Werle, Ranger, Schultz, Reding, Addesso, Oliver and Sampson2019).

The influence of drought stress on host selection by pioneering ambrosia beetles is less clear. Anecdotal field observations by Hara and Beardsley (Reference Hara and Beardsley1979) noted that Croton reflexifolius Kunth (Euphorbiaceae) and Acacia koa Gray (Fabaceae) trees stressed by drought and transplanting were especially vulnerable to X. compactus. Reduced sap production associated with drought and transplanting stress has been surmised to influence the host selection and colonisation success of X. compactus. Similarly, Greco and Wright (Reference Greco and Wright2015) observed X. compactus attacking coffee plants stressed by a lack of water and fertiliser. Compared to flood-stressed trees, drought-stressed C. florida were only mildly attractive and sustained few attacks by X. crassiusculus and X. germanus (Ranger and Schultz, unpublished data). Because the growth of ambrosia beetle fungal symbionts within host tree galleries is likely influenced by the moisture content of the tissue, additional studies are warranted to characterise the extent to which drought stress influences host selection and colonisation success.

Low-temperature stress also influences the host-selection behaviour of X. crassiusculus and X. germanus. Anecdotal field observations indicated that stress from low temperatures preceded attacks by X. germanus on Quercus rubra L. (Fagaceae), Acer pseudoplatanus L. (Sapindaceae) (Heidenreich Reference Heidenreich1960), S. japonicus (Meyer Reference Meyer1992), Fagus sylvatica Linnaeus (Fagaceae) (Grégoire et al. Reference Grégoire, Piel, De Proft and Gilbert2001; La Spina et al. Reference La Spina, De Cannière, Dekri and Grégoire2013), Acer palmatum Thunb. (Sapindaceae), Cercis canadensis Linnaeus, Cladrastis kentukea (Dumont de Courset) Rudd (Fabaceae), Liriodendron tulipifera Linnaeus (Magnoliaceae), S. japonicus, and Zelkova serrata (Thunberg) Makino (Ulmaceae) (Ranger et al. Reference Ranger, Schultz, Frank, Chong and Reding2015a). Controlled experiments by La Spina et al. (Reference La Spina, De Cannière, Dekri and Grégoire2013) demonstrated that X. germanus was more attracted to bark tissue of F. sylvatica trees experimentally injured by freezing compared to noninjured tissues. Similarly, X. crassiusculus and X. germanus rapidly began attacking and establishing galleries in container-grown C. canadensis, C. florida, Malus pumila Mill., and S. japonicus trees that were experimentally freeze-stressed, while no attacks occurred on noninjured trees (Ranger et al. Reference Ranger, Schultz, Frank and Reding2019). Attacks on freeze-stressed trees occurred disproportionately on the upper stem and into the canopy, whereas attacks on flood-stressed trees were more prevalent on the lower portion of the stem. This phenomenon is likely a function of a physiological stressor influencing the tissue-specific production of stress-induced volatiles.

Girdling and herbicide damage can also predispose trees to attack by X. crassiusculus and X. germanus. Dodds and Miller (Reference Dodds and Miller2010) attracted X. germanus to trap trees by injecting trunks of Pinus resinosa Aiton (Pinaceae) with the herbicide dicamba. Similarly, Reed et al. (Reference Reed, Juzwik, English and Ginzel2015) induced attacks by X. crassiusculus, X. germanus, and a variety of other ambrosia beetles by first mechanically girdling the trunk of Juglans nigra Linnaeus (Juglandaceae) trees and then treating the wounded tissue with glyphosate. Xylosandrus crassiusculus and X. germanus represented the majority of nearly 17 000 insects reared from logs harvested from these trees (Reed et al. Reference Reed, Juzwik, English and Ginzel2015).

Anecdotal observations indicate that stress caused by fungi and bacteria can predispose trees to attack by Xylosandrus spp. beetles. However, few studies have directly tested this association. Early observations by Buchanan (Reference Buchanan1940, Reference Buchanan1941) indicated that X. germanus was attracted to elm, Ulmus sp., infected with Dutch elm disease, Ceratostomella ulmi (Schwarz) (Ophiostomataceae). Similarly, Fusarium sp. cankers on stems of black walnut, Juglans nigra L. (Kessler Reference Kessler1974; Weber and McPherson Reference Weber and McPherson1985), and tulip poplar, Liriodendron tulipifera L. (Anderson and Hoffard Reference Anderson and Hoffard1978), were associated with attacks by X. germanus. Cultivars of Malus × domestica Borkh. trees infected with fireblight, Erwinia amylovora (Burrill) (Erwiniaceae), were also attacked by X. germanus, and Hall et al. (Reference Hall, Ellis and Ferree1982) proposed the beetles were attracted to the trees due to the infection but were not vectors of the pathogen. Symptomatic J. nigra trees infected with the causal agent of 1000 cankers disease, Geosmithia morbida Kolařík, Freeland, Utley, and Tisserat (Bionectriaceae), and Persea spp. (Lauraceae) infected with laurel wilt, Raffaelea lauricola Harrington & Fraedrich (Ophiostomataceae), were attacked by X. crassiusculus (Juzwik et al. Reference Juzwik, McDermott-Kubeczko, Stewart and Ginzel2016). Recently, Rassati et al. (Reference Rassati, Contarini, Ranger, Cavaletto, Rossini, Speranza, Faccoli and Marini2020) reported that infection of Castanea sativa (Miller) (Fagaceae) logs by chestnut blight (Cryphonectria parasitica (Murrill) Barr) (Cryphonectriaceae) influences host-selection behaviour in the xyleborine Anisandrus dispar F., but not in X. germanus. The combination of root infection by Phytophthora cinnamomi Rands (Peronosporaceae) and flood stress also influences the host-selection behaviour of X. crassiusculus and X. germanus (Addesso et al. Reference Addesso, Baysal-Gurel, Oliver, Ranger and O’Neal2018; Brown et al. Reference Brown, Baysal-Gurel, Oliver and Addesso2019). Future work should focus on understanding the extent to which beetles are attracted to volatiles that may arise from the interaction of pathogens with their woody hosts. Similarly, the impact of abiotic and biotic tree stressors on host selection by X. compactus should be examined under controlled experimental conditions.

Stress-induced volatile emissions

The aforementioned abiotic and biotic stressors can induce the production and emission of stress-associated volatiles, and at least some of these may influence the behaviour of Xylosandrus spp. ambrosia beetles. For instance, physiologically stressed trees emit higher amounts of acetaldehyde, acetic acid, acetone, ethane, ethanol, ethylene, and methanol (Kimmerer and Kozlowski Reference Kimmerer and Kozlowski1982; Millar et al. Reference Millar, Zhao, Lanier, O’Callaghan, Griggs, West and Silverstein1986; Kimmerer and MacDonald Reference Kimmerer and MacDonald1987; Holzinger et al. Reference Holzinger, Sandoval-Soto, Rottenberger, Crutzen and Kesselmeier2000; Copolovici and Niinemets Reference Copolovici and Niinemets2010; Ranger et al. Reference Ranger, Reding, Schultz and Oliver2013a, Reference Ranger, Tobin and Reding2015a, Reference Ranger, Tobin and Reding2015b, Reference Ranger, Schultz, Frank and Reding2019). Roots subjected to little or no oxygen, such as those subjected to flooding, will switch from aerobic to anaerobic cellular respiration, which leads to the production of acetaldehyde as an intermediate metabolite (Kimmerer and Kozlowski Reference Kimmerer and Kozlowski1982; Kreuzwieser et al. Reference Kreuzwieser, Scheerer and Rennenberg1999). Acetaldehyde is converted into ethanol, which is then transported from the roots to the stem and leaf tissues and emitted through the epidermis (Kreuzwieser et al. Reference Kreuzwieser, Scheerer and Rennenberg1999). Addesso et al. (Reference Addesso, Baysal-Gurel, Oliver, Ranger and O’Neal2018) observed that ethanol was emitted from C. canadensis trees one day after being flooded. Similarly, higher levels of ethanol were detected three days after flooding within stems of flood-intolerant tree species, including C. canadensis, C. florida, Prunus serrulata Lindley (Rosaceae), and S. japonicus, compared to flood-tolerant tree species, such as Acer saccharinum Linnaeus (Sapindaceae) and Quercus bicolor Willdenow (Fagaceae) (Ranger et al. Reference Ranger, Schultz, Frank, Chong and Reding2015a). Emissions of acetaldehyde, acetic acid, and ethanol were also detected from stems of C. florida at 7 and 14 days after initiating flooding (Ranger et al. Reference Ranger, Reding, Schultz and Oliver2013a).

An inability of cells to take up O₂ due to injury from low-temperature stress can induce the production and emission of ethanol from the bark epidermis (Kimmerer and Kozlowski Reference Kimmerer and Kozlowski1982; Forney et al. Reference Forney, Jordan, Nicholas and DeEll2000; Obenland et al. Reference Obenland, Aung, Bridges and Mackey2003). For instance, ethanol emissions were initiated within 1 day after freeze stress of S. japonicus, peaked at 4 days, and continued to be detected after 19 days (Ranger et al. Reference Ranger, Schultz, Frank and Reding2019). Correspondingly, attacks by X. germanus were initiated within 1 day after experimentally imposing freeze stress on C. florida and peaked by 4–7 days.

Ethanol was demonstrated to be associated with cankers of Phytophthora ramorum Werres et al. (Peronosporaceae) on infected stems of Quercus agrifolia Née (Fagaceae), which then attracted the generalist ambrosia beetle, Xyleborinus saxesenii (Ratzeburg) (Kelsey et al. Reference Kelsey, Beh, Shaw and Manter2013). A similar interaction has not yet been characterised for Xylosandrus spp. However, fireblight infection of M. domestica could induce the emission of ethanol and thereby attract Xylosandrus spp. ambrosia beetles, warranting additional studies as it pertains to rapid apple decline (Agnello et al. Reference Agnello, Breth, Tee, Cox and Warren2015).

Semiochemistry

Attractants

Ethanol is the most studied host-derived kairomone that attracts X. compactus, X. crassiusculus, and X. germanus. During field trapping studies, a positive correlation was demonstrated between ethanol emissions from lures and trap captures of X. germanus (Klimetzek et al. Reference Klimetzek, Köhler, Vité and Kohnle1986). A positive correlation was demonstrated between concentration of ethanol injected into Magnolia virginiana Linneaus (Magnoliaceae) and attacks by X. germanus and other ambrosia beetles (Ranger et al. Reference Ranger, Reding, Schultz and Oliver2012), along with a positive correlation between ethanol concentration within stems of flooded trees and attacks (Ranger et al. Reference Ranger, Schultz, Frank, Chong and Reding2015a). Several studies have demonstrated that ethanol is a long-range attractant for X. compactus (Miller and Rabaglia Reference Miller and Rabaglia2009; Burbano et al. Reference Burbano, Wright, Gillette, Mori, Dudley, Jones and Kaufmann2012) and X. crassiusculus (Miller and Rabaglia Reference Miller and Rabaglia2009; Reding et al. Reference Reding, Schultz, Ranger and Oliver2011; Werle et al. Reference Werle, Ranger, Schultz, Reding, Addesso, Oliver and Sampson2019).

Pressurised injections of ethanol into M. virginiana L. induced more attacks than acetaldehyde, acetone, and methanol under field conditions (Ranger et al. Reference Ranger, Reding, Persad and Herms2010). Ethanol injection into 16 species of deciduous trees from 11 families also induced ambrosia beetle attacks under field conditions (Reding et al. Reference Reding, Ranger, Oliver, Schultz, Youssef and Bray2017). Baiting trees with ethanol-induced attacks on specific trees, however, immediately ceased upon removal of the ethanol cue (Ranger et al. Reference Ranger, Tobin and Reding2015b). Irrigating container-grown trees with solutions of ethanol also induces attacks by X. germanus and other ambrosia beetles (Ranger et al. Reference Ranger, Reding, Schultz and Oliver2012, Reference Ranger, Biedermann, Phuntumart, Beligala, Ghosh, Palmquist, Mueller, Barnett, Schultz, Reding and Benz2018). Stem sections (i.e., bolts) soaked in aqueous solutions of ethanol are readily attacked by X. germanus and other ambrosia beetles when deployed under field conditions (Reding and Ranger Reference Reding and Ranger2019; Rassati et al. Reference Rassati, Contarini, Ranger, Cavaletto, Rossini, Speranza, Faccoli and Marini2020). Ethanol-soaked bolts were generally more attractive to X. germanus than ethanol-baited traps, suggesting that additional host-derived compounds emitted from the bark might enhance the attraction to ethanol (Reding and Ranger Reference Reding and Ranger2019).

Xylosandrus germanus efficiently located and attacked ethanol-injected trees, but rarely landed on adjacent trees not emitting ethanol, and never attacked these neighbouring trees (Ranger et al. Reference Ranger, Tobin and Reding2015b). A spatial analysis of trees attacked by X. germanus within ornamental nurseries revealed that only certain tree species or cultivars, and only certain individuals within these host species or cultivars, were attacked (Ranger et al. Reference Ranger, Schultz, Frank, Chong and Reding2015a). Stress-induced ethanol was present within the tissues of those attacked trees, which further supports the role of ethanol as a long-range attractant that assists X. germanus in efficiently locating vulnerable trees.

Ethanol within host tissues acts as a boring cue for ambrosia beetle species that are attracted to it; Kelsey et al. (Reference Kelsey, Beh, Shaw and Manter2013) reported fourfold more ambrosia beetle attacks on sapwood tissues infused with ethanol compared to the opposite side of the same log which did not receive ethanol treatment. Ethanol within host tissues also appears to be associated with favourable environments for adult foundresses to establish fungal gardens and produce offspring (Ranger et al. Reference Ranger, Biedermann, Phuntumart, Beligala, Ghosh, Palmquist, Mueller, Barnett, Schultz, Reding and Benz2018). Xylosandrus germanus attacked trees baited with ethanol lures, but only superficial tunnels were created, and no fungal gardens or broods were produced unless the living stem tissues contained ethanol introduced through irrigation with ethanol solutions. Ethanol incorporated into growing media promotes the growth of the fungal symbionts of X. germanus (i.e., A. grosmanniae) and X. crassiusculus (i.e., A. roeperi) but inhibits the growth of “weedy” fungal garden competitors, such as Aspergillus sp. (Trichocomaceae) and Penicillium sp. (Trichocomaceae) (Ranger et al. Reference Ranger, Biedermann, Phuntumart, Beligala, Ghosh, Palmquist, Mueller, Barnett, Schultz, Reding and Benz2018).

The presence of ethanol as a potent antimicrobial agent within host tissues may reduce interspecific competition among different species of ambrosia beetles. Rassati et al. (Reference Rassati, Contarini, Ranger, Cavaletto, Rossini, Speranza, Faccoli and Marini2020) demonstrated that ethanol in wood tissue might facilitate niche-partitioning among xyleborine ambrosia beetles. In a field experiment involving ethanol-soaked logs, X. germanus and X. saxesenii were differentially attracted to increasing ethanol concentrations in wood tissues, whereby the number of entry holes decreased with increasing ethanol concentration for X. germanus and increased for X. saxesenii (Rassati et al. Reference Rassati, Contarini, Ranger, Cavaletto, Rossini, Speranza, Faccoli and Marini2020). Ethanol concentration in wood tissues also differentially affected colonisation success of X. germanus and X. saxesenii, in that the number of galleries with brood chambers in ethanol-soaked logs was higher for X. germanus than for X. saxesenii, even though the attack rate was largely higher for the latter species.

A few other host-derived compounds are weak and inconsistent attractants for X. compactus, X. crassiusculus, and X. germanus. Conophthorin, ((E)-(±)-7-methyl-1,6-dioxaspiro[4.5]decane), is associated with the bark of deciduous tree species (Francke et al. Reference Francke, Bartels, Meyer, Schröder, Kohnle, Baader and Vité1995; Byers et al. Reference Byers, Zhang and Schlyter1998; Huber et al. Reference Huber, Gries, Borden and Pierce1999; Huber and Borden Reference Huber and Borden2001; Zhang et al. Reference Zhang, Tolasch, Schlyter and Francke2002), functions as a pheromone component of some bark beetles (Birgersson et al. Reference Birgersson, DeBarr, de Groot, Dalusky, Pierce, Borden, Meyer, Francke, Espelie and Berisford1995; Dallara et al. Reference Dallara, Seybold, Meyer, Tolasch, Francke and Wood2000; DeGroot and DeBarr Reference DeGroot and DeBarr2000), and is produced by symbiotic fungi of bark beetles (Dickschat Reference Dickschat2017; Zhao et al. Reference Zhao, Ganji, Schiebe, Bohman, Weinstein, Krokene, Borg-Karlson and Unelius2019). Conophthorin has been associated with variable effects on Xylosandrus spp., either increasing or decreasing attraction. Kohnle et al. (Reference Kohnle, Densborn, Kölsch, Meyer and Francke1992) demonstrated that conophthorin decreased attraction of X. germanus to ethanol-baited traps, but Dodds and Miller (Reference Dodds and Miller2010) reported that conophthorin enhanced attraction of X. germanus to girdled trap trees. VanDerLaan and Ginzel (Reference VanDerLaan and Ginzel2013) reported that conophthorin enhanced attraction of X. crassiusculus to ethanol but had a negligible effect on X. germanus. Conophthorin in combination with ethanol elicited larger electroantennogram amplitudes than ethanol alone, and conophthorin enhanced attraction of X. germanus to ethanol under field conditions (Ranger et al. Reference Ranger, Gorzlancyk, Addesso, Oliver, Reding, Schultz and Held2014). Conophthorin decreased attraction of X. germanus to ethanol-baited traps deployed in Oregon but had no effect on trap captures of X. germanus in New Hampshire or Michigan (Miller et al. Reference Miller, Dodds, Hoebeke, Poland and Willhite2015). Conophthorin also had no effect on X. compactus and X. crassiusculus during field trapping studies conducted in Georgia (Miller et al. Reference Miller, Dodds, Hoebeke, Poland and Willhite2015).

(−)-α-Pinene has also exhibited inconsistent effects on the attraction of X. compactus, X. crassiusculus, and X. germanus. (−)-α-Pinene did not enhance the attraction of X. compactus, X. crassiusculus, or X. germanus to ethanol in field trapping studies (Miller and Rabaglia Reference Miller and Rabaglia2009; Gandhi et al. Reference Gandhi, Cognato, Lightle, Mosely, Nielsen and Herms2010; Burbano et al. Reference Burbano, Wright, Gillette, Mori, Dudley, Jones and Kaufmann2012). (−)-α-Pinene enhanced the response of X. germanus to ethanol during trapping studies conducted in 2003, but it had a negligible effect in subsequent trapping studies conducted in 2004 and 2008 (Ranger et al. Reference Ranger, Reding, Gandhi, Oliver, Schultz, Cañas and Herms2011). A lure consisting of ethanol, (−)-α-pinene, (+)-α-pinene, (+)-camphene, (−)-β-pinene, (+)-β-pinene, myrcene, ρ-cymene, limonene, and eucalyptol was prepared based on ratios of the aforementioned compounds emitted from ethanol-injected M. virginiana, but it was not more attractive to X. germanus than ethanol alone (Ranger et al. Reference Ranger, Reding, Schultz and Oliver2012).

Ott (Reference Ott2007) identified volatiles emitted from flood-stressed oak trees, namely, 2-hexen-1-ol, 6-methyl-5-hepten-2-one, ethyl salicylate, nonanal, eugenol, guaiacol, and 1-hexanol. However, a lure composed of these compounds did not enhance the attraction of X. crassiusculus to ethanol alone, nor did they act as attractants when tested singly. Lures composed of eugenol plus ethanol and guaiacol plus ethanol generally caught more X. crassiusculus than ethanol alone, but the results were variable and not significant (Ott Reference Ott2007). Eugenol or α-pinene alone also did not attract more X. compactus than a blank control (Burbano et al. Reference Burbano, Wright, Gillette, Mori, Dudley, Jones and Kaufmann2012). Ethanol plus ginger oil, α-pinene, phoebe oil, or manuka oil also did not attract more X. compactus or X. crassiusculus than ethanol alone (Burbano et al. Reference Burbano, Wright, Gillette, Mori, Dudley, Jones and Kaufmann2012).

Compared to ethanol, other stress-induced and host-derived compounds have generally exhibited negligible effects. A mixture of acetaldehyde, ethanol, and methanol was not more attractive to scolytines (not identified to species level) than ethanol alone was in field trapping studies (Montgomery and Wargo Reference Montgomery and Wargo1983). Acetaldehyde and acetone did not increase trap captures of scolytines when tested individually compared against an ethanol or water control (Montgomery and Wargo Reference Montgomery and Wargo1983). During field trapping studies, ethanol was highly attractive to X. germanus, methanol was slightly attractive, and acetaldehyde and acetone were inactive (Ranger et al. Reference Ranger, Reding, Persad and Herms2010).

Volatiles emitted from the fungal symbionts of Xylosandrus spp. ambrosia beetles are associated with behavioural activity. Hulcr et al. (Reference Hulcr, Mann and Stelinski2011) observed X. crassiusculus was attracted to volatiles emitted by its fungal symbiont, Ambrosiella roeperi (previously Ambrosiella xylebori), during olfactometer studies. Similarly, Egonyu and Torto (Reference Egonyu and Torto2018) observed that X. compactus was attracted to volatiles emitted from its fungal symbiont, Fusarium solani (Mart.) Sacc., during olfactometer studies. A variety of compounds were identified in volatile collections from F. solani, with ethanol, phenethyl alcohol, 3-methyl-1-butanol, and (ϵ)-caryophyllene being among the dominant components. Methyl isovalerate and 2,3-butanediol elicited antennal responses by X. compactus during gas chromatography–electroantennography experiments (Egonyu and Torto Reference Egonyu and Torto2018), but unlike ethanol these compounds were only slightly attractive under field conditions.

There is currently no evidence that X. compactus, X. crassiusculus, X. germanus, or other xyleborines produce a long-range aggregation or sex pheromone that facilitates host-selection processes. Because males are flightless and females either mate before dispersing or produce males (through haplodiploidy) following invasion of the host, there should be little selection pressure for the evolution of a pheromone (Kirkendall et al. Reference Kirkendall, Wrensch, Ebbert, Wrensch and Ebbert1993; Peer and Taborsky Reference Peer and Taborsky2005; Ott Reference Ott2007; Ranger et al. Reference Ranger, Tobin and Reding2015b). Large clusters of X. germanus overwintering within a single gallery in a host tree have been documented (Weber and McPherson Reference Weber and McPherson1984). In one instance, 112 females and 42 males were present in a single gallery, which is more than 1 female could produce, suggesting that beetles probably immigrated from nearby galleries (Weber and McPherson Reference Weber and McPherson1984). Observations by Peer and Taborsky (Reference Peer and Taborsky2005) suggested that short-range chemical communication aids male X. germanus in locating female-occupied galleries.

Repellents

A repellent is a compound that causes directed, oriented movement away from the point source, and determining the extent to which a compound acts as a repellent, in the strict sense, is difficult to assess in field trials. Assessing the influence of a particular compound on baited trap captures or tree attacks does not directly measure the time course of behavioural responses of directed movements by an insect. As such, some compounds termed repellents by researchers and practitioners may have been shown only to inhibit attraction rather than direct movement away from the attractive source (Holighaus and Rohlfs Reference Holighaus and Rohlfs2016). For the purposes of this review, this broader sense of repellent is used.

Several studies have assessed the inhibitory effects of selected compounds on X. compactus, X. crassiusculus, and X. germanus. Currently, the majority of studies have focused on verbenone, [4,6,6-trimethylbicyclo[3.1.1] hept-3-en-2-one], which was first identified from the hindgut of the southern pine beetle, Dendroctonus frontalis Zimmerman, and the western pine beetle, Dendroctonus brevicomis LeConte (Curculionidae) (Renwick Reference Renwick1967). Verbenone has also been isolated from coniferous (Cool and Zavarin Reference Cool and Zavarin1992; Dallara et al. Reference Dallara, Seybold, Meyer, Tolasch, Francke and Wood2000) and deciduous plants (Buttery et al. Reference Buttery, Light, Nam, Merrill and Roitman2000), is emitted by bark beetle fungal symbionts (Cale et al. Reference Cale, Ding, Wang, Rajabzadeh and Erbilgin2019), and is also generated through the autoxidation that occurs when host-produced α-pinene is exposed to the air (Hunt et al. Reference Hunt, Borden, Lindgren and Gries1989).

Verbenone interrupts the attraction of several species of bark beetles to conspecific pheromones, host-derived volatiles, and trap trees (Bentz et al. Reference Bentz, Kegley, Gibson and Their2005; Gillette et al. Reference Gillette, Stein, Owen, Webster, Fiddler, Mori and Wood2006). Based on this activity, the effects of verbenone have also been evaluated on ambrosia beetles. Dodds and Miller (Reference Dodds and Miller2010) demonstrated that verbenone emitted from dispensers at a rate of 40 mg/day reduced the attraction of X. germanus to ethanol-baited traps by 67%. Similarly, verbenone emitted from dispensers at 2 mg/day reduced the attraction of X. compactus and X. crassiusculus to ethanol-baited traps (Burbano et al. Reference Burbano, Wright, Gillette, Mori, Dudley, Jones and Kaufmann2012). At release rates of 2 and 50 mg/day, verbenone reduced the attraction of X. germanus to ethanol-baited traps by 98% and 97%, respectively (Ranger et al. Reference Ranger, Tobin, Reding, Bray, Oliver, Schultz, Frank and Persad2013b, Reference Ranger, Gorzlancyk, Addesso, Oliver, Reding, Schultz and Held2014). Traps releasing 32 mg/day verbenone also caught fewer X. crassiusculus and X. germanus than unbaited traps did (VanDerLaan and Ginzel Reference VanDerLaan and Ginzel2013).

Verbenone reduces attraction and attacks on trap trees by Xylosandrus spp., but the results have been inconsistent. For example, a verbenone dispenser (40 mg/day) attached to herbicide-killed Pinus resinosa Aiton trap trees reduced the attraction of X. germanus but was not completely effective at preventing attacks (Dodds and Miller Reference Dodds and Miller2010). Similarly, a verbenone dispenser (50 mg/day) attached to ethanol-injected M. virginiana trap trees reduced ambrosia beetle attacks by 85% compared to trap trees without a verbenone dispenser, but it did not prevent attacks from occurring (Ranger et al. Reference Ranger, Tobin, Reding, Bray, Oliver, Schultz, Frank and Persad2013b). In some instances, attacks on ethanol-injected M. virginiana trees also decreased with an increasing proximity to a verbenone emitter, but attacks still occurred on trap trees and the effect was inconsistent across locations and years. A verbenone (50 mg/day) dispenser attached to flood-stressed apple trees (M. domestica) also resulted in fewer ambrosia beetle galleries containing brood, but the effect was inconsistent and tree attacks still occurred (Agnello et al. Reference Agnello, Breth, Tee, Cox, Villani, Ayer, Wallis, Donahue, Combs, Davis and Neal2017). In contrast, verbenone (50 mg/day) had no effect at reducing attacks on container-grown and flood-stressed Cercis canadensis L., Cornus florida L., and Koelreuteria paniculata Laxmann (Sapindaceae) trees deployed as part of multi-state trials (Werle et al. Reference Werle, Ranger, Schultz, Reding, Addesso, Oliver and Sampson2019). Altogether, these results demonstrate that verbenone can reduce ambrosia beetle attacks on vulnerable trees but has limited promise as a tree protectant.

The monoterpene (+)-limonene is one of the most common components of plant essential oils and is also emitted by a variety of microorganisms (Jongedijk et al. Reference Jongedijk, Cankar, Buchhaupt, Schrader, Bouwmeester and Beekwilder2016). When released at a rate of 600 mg/day, (+)-limonene reduced the attraction of X. crassiusculus to ethanol-baited traps but was less effective than verbenone (Burbano et al. Reference Burbano, Wright, Gillette, Mori, Dudley, Jones and Kaufmann2012). The monoterpene terpinolene released at 266 mg/day reduced captures of X. germanus in ethanol-baited traps by 65% (Ranger et al. Reference Ranger, Gorzlancyk, Addesso, Oliver, Reding, Schultz and Held2014). A combination of verbenone and methyl salicylate was repellent to the redbay ambrosia beetle, Xyleborus glabratus Eichhoff (Curculionidae) (Hughes et al. Reference Hughes, Martini, Kuhns, Colee, Mafra-Neto, Stelinski and Smith2017).

Volatiles produced by deleterious fungal competitors of the beetles’ symbiont could be sources of promising repellent compounds against Xylosandrus spp. During olfactometer studies, X. saxesenii and Xyleborus ferrugineus, but not X. crassiusculus, were repelled by volatiles emitted from Trichoderma sp. (Hypocreaceae) mycelium growing on agar (Hulcr et al. Reference Hulcr, Mann and Stelinski2011).

Applied chemical ecology

Knowledge about the host-selection behaviour and associated semiochemicals of X. compactus, X. crassiusculus, and X. germanus has helped to improve monitoring and management efforts. As discussed, ethanol is the most effective long-range attractant for detecting these Xylosandrus spp. Ethanol-baited traps are effective for monitoring purposes. However, supplementing traps with additional semiochemicals does not appear to offer a cost-effective improvement for the detection of X. compactus, X. crassiusculus, and X. germanus. To induce attacks on trees for monitoring purposes, trap trees can be created by irrigation or injections with ethanol, flood stressing or freeze stressing, or girdling using a combination of mechanical injury and herbicide treatment. Stem sections (i.e., bolts) can also be used for monitoring attacks by soaking bolts in 10% ethanol for 24−48 hours or coring the centre of bolts and filling them with 95% ethanol (Reding and Ranger Reference Reding and Ranger2019).

Deploying repellent dispensers, such as verbenone, among vulnerable trees could be useful for establishing a push–pull management strategy (Pyke et al. Reference Pyke, Rice, Sabine and Zalucki1987; Miller and Cowles Reference Miller and Cowles1990), whereby repellents push beetles away from vulnerable trees and attractants pull beetles into annihilative traps. A push–pull strategy has potential in horticultural production systems, but the strategy has not been widely adopted (Cook et al. Reference Cook, Khan and Pickett2007). Because wooded areas across the perimeter of neighbouring ornamental nurseries can serve as source populations of overwintering Xylosandrus spp. (Ranger et al. Reference Ranger, Tobin, Reding, Bray, Oliver, Schultz, Frank and Persad2013b; Reding et al. Reference Reding, Ranger, Sampson, Werle, Oliver and Schultz2015; Werle et al. Reference Werle, Chong, Sampson, Reding and Adamczyk2015), migration of beetles could potentially be exploited in conjunction with their strong attraction to ethanol. For example, the strategic deployment of ethanol-baited traps or ethanol-infused bolts along the perimeter of production sites, especially sites adjacent to vulnerable trees, could intercept ambrosia beetles before their dispersal into ornamental nurseries.

To date, only a few studies have assessed behavioural manipulation strategies for reducing the pressure of Xylosandrus spp. on vulnerable trees. Werle et al. (Reference Werle, Ranger, Schultz, Reding, Addesso, Oliver and Sampson2019) assessed the capability of verbenone dispensers as a “push” component and a perimeter of ethanol-baited traps as a “pull” component for reducing attacks on flood-stressed trees. Unexpectedly, verbenone dispensers emitting 50 mg/day and positioned among three to four flood-stressed trees failed to reduce ambrosia beetle attacks during trials conducted in Mississippi, Ohio, and Virginia. Traps baited with ethanol lures (65 mg/day) and positioned at 10 m intervals around the perimeter of the flooded trees intercepted enough ambrosia beetles to reduce attacks in some locations and years, but the effects were inconsistent and not sufficiently efficacious. Similarly, Addesso et al. (Reference Addesso, Oliver, Youssef, O’Neal, Ranger, Reding, Schultz and Werle2019) evaluated interception strategies consisting of ethanol-injected trap trees and ethanol-baited traps. Ethanol-injected trap trees failed to reduce attacks on neighbouring, less attractive trees, and varying densities of perimeter traps baited with ethanol lures (65 mg/day) failed to protect ethanol-injected trap trees.

Ethanol-based interception strategies might be improved if lures with a release rate higher than the 65 mg/day tested by Werle et al. (Reference Werle, Ranger, Schultz, Reding, Addesso, Oliver and Sampson2019) and Addesso et al. (Reference Addesso, Oliver, Youssef, O’Neal, Ranger, Reding, Schultz and Werle2019) are used. A positive correlation exists between ethanol release rate and trap captures or attacks by Xylosandrus spp. (Klimetzek et al. Reference Klimetzek, Köhler, Vité and Kohnle1986; Ranger et al. Reference Ranger, Reding, Schultz and Oliver2012, Reference Ranger, Tobin and Reding2015b), although this pattern was not evident for X. germanus when ethanol-soaked logs were used (Rassati et al. Reference Rassati, Contarini, Ranger, Cavaletto, Rossini, Speranza, Faccoli and Marini2020). Additional studies are warranted because the optimal release rate of ethanol needs to be established for intercepting these Xylosandrus spp., and may vary depending on the species. For example, lures emitting 2 g/day attracted more scolytines than higher release rates did (Montgomery and Wargo Reference Montgomery and Wargo1983).

Maintaining host vigour to reduce the risk of stress-induced volatile emissions is currently the most reliable semiochemical-based management strategy for scolytine ambrosia beetles. Knowledge about the role of flood stress on host selection by Xylosandrus spp. ambrosia beetles has led to improved management tactics. Frank and Ranger (Reference Frank and Ranger2016) demonstrated that a 50% media moisture level for container substrates is a threshold above which trees become vulnerable to attack. However, media moisture levels of container-grown trees in ornamental nurseries during spring months are often 70−90%, thereby increasing the risk for attacks by X. crassiusculus, X. germanus, and other ambrosia beetles.

Conclusions

A broad range of plant and tree species can be attacked by the three Xylosandrus spp. addressed in this review, whereas host-physiological status plays an important role during host selection, especially for X. crassiusculus and X. germanus. Thin-barked deciduous trees in the early stages of physiological stress can appear healthy and asymptomatic while emitting ethanol that functions as a long-range kairomone. Despite a broad host range, dispersing Xylosandrus adults exhibit an efficient capability of locating and preferentially attacking trees emitting ethanol, even within diverse horticultural landscapes.

There is no evidence that X. crassiusculus and X. germanus have generally switched to attacking and colonising healthy trees in nonnative habitats where the beetles have been introduced. Instead, stressed trees within horticultural production sites represent opportunistic hosts for dispersing females. Attacked trees can be spatially clustered within rows in horticultural production systems, a phenomenon that is often attributed to poor site conditions (e.g., low-lying areas prone to flooding) or microclimate effects (e.g., frost pockets). Neighbouring trees not emitting ethanol represent poor hosts for X. crassiusculus and X. germanus and are not typically attacked under experimental conditions. Indeed, foundresses prefer host tissues containing ethanol to initiate tunnelling as this substrate may represent a chemically favourable niche that promotes colonisation success.

Compared to what is known about X. crassiusculus and X. germanus, considerably less is known about the role of host quality during host selection by X. compactus. Nonetheless, dispersing X. compactus females are responsive to ethanol. Attacks by X. compactus have been reported on both apparently healthy and stressed trees, warranting controlled studies to assess the influence of experimentally imposed abiotic stressors on host selection and colonisation.

Similarly, the authors of this review are unaware of studies in the literature to assess the impact of biotic stressors, particularly tree pathogens, on host selection by all three of these Xylosandrus spp. Understanding these interactions is critical for sustainably managing these destructive insects in horticultural cropping systems.

This review’s authors believe that certain semiochemicals may in future be used in xyleborine management if deployed in the context of a push–pull strategy. Verbenone inhibits the attraction of Xylosandrus spp. females to ethanol. However, verbenone alone has limited prospects for adoption as a management tool because growers generally have a low or zero tolerance for attacks. Applying a perimeter of ethanol-baited traps around vulnerable trees has shown some promise for intercepting dispersing ambrosia beetles, but additional studies are needed to increase trap captures. Nonetheless, the aggressiveness with which trees emitting ethanol are attacked, particularly by X. crassiusculus and X. germanus, indicates the importance of maintaining host vigour as the first step in an integrated pest management programme for these insects.

Acknowledgement(s)

We dedicate this review to Steven Seybold (USFS) and thank Brian Sullivan (USFS) and an anonymous reviewer for helping to improve the quality of the manuscript. This review was supported in part by the United States Department of Agriculture Floriculture and Nursery Research Initiative, Horticultural Research Institute, and the United States Department of Agriculture–Agriculture Research Service (USDA-ARS) National Program 305 (Project 5082-21000-018-00D).

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