Damage to Sweet Chestnut Orchards in Croatia due to Invasive Alien bark beetle Xylosandrus germanus

Black stem borer, Xylosandrus germanus (Coleoptera: Scolytinae), was first recorded in Croatia in 2009. Until now, the insect has been reported only as a part of ambrosia beetles entomofauna in oak stands ( Quercus robur and Quercus petraea ). In 2021, severe attack of ambrosia beetles causing tree decline and mortality have been observed in three orchards of hybrid sweet chestnut ( Castanea sativa x Castenea crenata ). Orchards near Topusko, Marija Bistrica and Vugrovec were inspected for the symptoms. Twenty-seven adult ambrosia beetle specimens were collected from eight samples of damaged sweet chestnut trees, and were identified according to their morphology. All 27 ambrosia beetles were morphologically identified as X. germanus . This is the first record of black stem borer causing damage on cultivated sweet chestnut in Croatia. Considering similar reports from Italy and Slovenia, X. germanus could be regarded as a new, potentially damaging pest in chestnut plantations.


INtrODUCtION
Sweet chestnut (Castanea sativa Mill.) is a valuable tree species used in agriculture.In Europe, sweet chestnut is cultivated in various systems, from semi-natural forest stands to intensive orchards (Prgomet et al. 2013).Sweet chestnut trees cultivated in orchards are mostly hybrids between C. sativa and Asian species Castanea crenata Siebold & Zucc.or Castanea mollissima Blume (Prgomet et al. 2013).Today, several devastating diseases and pests threaten chestnut cultivation.Chestnut blight, caused by Cryphonectria parasitica (Murrill) M.E.Barr, continues to cause damage to native sweet chestnuts in forests, but it also attacks hybrids in orchards (Glavaš 1999, Jurc and Reščič 2013, Prgomet et al. 2013).Ink disease, caused by oomycetes Phytophthora cambivora (Petri) Buisman and Phytophthora cinnamomi Rands, has resurged in Europe at the end of 20 th century (Vannini and Vettraino 2001).During the last decade, newly emerged fruit rot disease caused by Gnomoniopsis smithogilvyi L.A. Shuttlew., E.C.Y.Liew & D.I. Guest has caused substantial yield losses in some European countries (Visentin et al. 2012, Dennert et al. 2015, Ivić and Novak 2018).Oriental chestnut gall wasp (Dryocosmus kuriphilus Yasumatsu) has been considered as a damaging invasive alien chestnut pest in Europe, until the successful introduction of its natural enemy (Avtzis et al. 2019).
Numerous other invasive alien pests and pathogens emerge regularly in Europe, posing risks to European forestry, agriculture and natural environments (Bebber et al. 2014, Panzavolta et al. 2021).Black stem borer, Xylosandrus germanus (Blandford 1894, Coleoptera, Scolytinae), is one of such species.It is an ambrosia beetle originating from Japan (Dzurenko et al. 2021), recorded in Europe for the first time in Germany in 1952 (Groschke 1953).From the establishment period until now, X. germanus has become an economically relevant forest pest (Galko et al. 2019).Damage has been recorded on various broadleaf and conifer species (Graf and Manser 2000, Galko et al. 2019, Hauptman et al. 2019).In 2019, black stem borer was reported to occur in Croatia (Franjević et al. 2019).The insect has been caught in pheromone baited traps for ambrosia beetles, from 2009 to 2011.Its presence has been confirmed in lowland oak stands near Zagreb (Franjević et al. 2019).
While recognized mostly as a forest pest in Europe, X. germanus in the USA is also reported as a destructive pest  (Oliver and Mannion 2001).It shows a potential of polyphagous ambrosia beetles to adapt to different hosts, causing damage to forest, fruit or landscape trees.
In 2021, severe attacks of beetles were observed in three chestnut orchards, near Topusko, Marija Bistrica and Vugrovec (Zagreb).Since such heavy attacks on sweet chestnut have not been previously recorded, beetles were collected from infested trees and identified to the species level.The aim of the research was to identify beetles causing damage in sweet chestnut orchards.

MAtErIALs AND MEtHODs
In the period from May to July 2021, three sweet chestnut producers observed extensive decline of trees in their orchards.Orchards were located near Topusko (Sisak-Moslavina County, 2.4 ha), Marija Bistrica (Krapina-Zagorje County, 0.7 ha) and Vugrovec (City of Zagreb, 0.2 ha) (Figure 1).Bouche de Betizac, a hybrid between C. sativa and C. crenata, was a dominant cultivar in all three orchards.In Topusko, cultivars Marsol and Maraval were also grown.The orchard in Topusko was four years old, while two smaller orchards in Marija Bistrica and Vugrovec were three years old.All three orchards were in the vicinity of forests.
Orchards were visually inspected during June and July 2021.Symptoms in all three orchards were similar.Numerous trees were obviously dead, not producing any new shoots.Some trees were declining, with dried branches and small, yellow leaves on other parts of the crown.On some trees, reddish patches of cankered tissue were visible on trunks, always with a small round hole in the middle.On others, rusty brown oozing flecks were evident, also with holes in the middle.Numerous holes were regularly noted at the base of the trunk, always accompanied with brownish exudates and oozing flecks.Certain holes on the trunk were filled with sawdust, indicating ambrosia beetles' attack.When outer layers of the bark were removed, numerous holes were evident in the xylem of infested trees (Figure 2).
Trunks of symptomatic trees were cut at nearly soil level to 1-1.5 m up to the trunk.Four trunk samples were collected at Topusko orchard, three samples at Marija Bistrica orchard, and one sample at Vugrovec orchard.Trunks were cut into smaller pieces, transported in plastic bags and analysed in the Centre for Plant Protection, Croatian Agency for Agriculture and Food in Zagreb.In the laboratory, trunks were carefully carved to catch and collect specimens of beetles inside.Some wood pieces were left in sealed plastic bags for two weeks, waiting for beetles to eventually come out of the wood.A total of 27 beetle specimens were collected.
Beetles were identified to the species level based on morphological characteristics of female adults, using classical identification method according to the key provided by Tuncer at al. (2017).Stereomicroscope Olympus SZX 7 was used for morphometry.

rEsULts AND DIsCUssION
All specimens of beetles collected from symptomatic chestnut samples were identified as black stem borer, X. germanus.So far, X. germanus has been reported in Croatia only on oaks (Franjević et al. 2019), and chestnut hybrids are recorded as new hosts of this polyphagous pest.Attacks of X. germanus on cultivated chestnut hybrids have been reported in Slovenia and Italy (Jurc andReščič 2013, Dutto et al. 2018).In Slovenia, an attack was reported in experimental orchard of Bouche de Betizac, Maraval and Marsol varieties near Gorica in 2010 (Jurc and Reščič 2013).It was noted that black stem borer attacked trees from the inoculation site to the 1.3 m height of the trunk.Varieties affected and the patterns of the attacks were very similar to the ones observed in Topusko, Marija Bistrica and Vugrovec orchards.Bouche de Betizac variety has been proven as the most susceptible to X. germanus attacks (Jurc and Reščič 2013).In Italy, severe damage to chestnut plantations in Piedmont has been reported in 2018 (Dutto et al. 2018).Up to 25% tree mortality has been reported (Dutto et al. 2018).Bouche de Betizac variety was predominantly attacked, with up to 40 exit holes per dm 2 of the observed trunk (Dutto et al. 2018).European sweet chestnut has been reported as a natural host of X. germanus in Europe (Galko et al. 2019).However, the possible impact of X. germanus on European sweet chestnuts in forests remains unknown.Infestation of X. germanus on cultivated Chinese chestnut (C.mollissima) has been recorded in the USA (Oliver and Mannion 2001).
Considering the damage observed, as well as experience from Italy (Dutto et al. 2018) and Slovenia (Jurc and Reščić 2013), X. germanus may pose a new threat to sweet chestnut cultivation in Croatia.Along with Cryphonectria parasitica, Phytophthora species and Dryocosmus kuriphilus, this new invasive ambrosia beetle may severely limit the establishment and expansion of sweet chestnut plantations and negatively influence the health of the existing ones.It is also important to monitor the spread of this alien species into natural sweet chestnut forests in Croatia.
Damage to sweet chestnut orchards in Croatia due to black stem borer is a typical example of negative impacts associated with the invasive alien pests entering, establishing and spreading into new areas.Additionally, it is an example of a pest posing risk both to forestry and agriculture.As black stem borer is not regulated as a quarantine pest, it is assumed that its spread and population increase will continue.In such circumstances, efficient methods of X. germanus monitoring and management should be investigated and developed.

CONCLUsIONs
Black stem borer (X.germanus) has been identified as a causal agent of tree mortality in three sweet chestnut orchards in continental Croatia.Hybrid sweet chestnut (C.sativa x C. crenata), cultivated as fruit species, is a new host record for X. germanus in Croatia.Besides being a wellknown forest pest, X. germanus may become an important pest of fruit crops.

Published online: 28 Jan 2024 Damage to Sweet Chestnut Orchards in Croatia due to Invasive Alien bark beetle Xylosandrus germanus 2 SEEFOR
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