Bulletin OEPP/EPPO Bulletin (2020) 0 (0), 1–5 ISSN 0250-8052. DOI: 10.1111/epp.12660

First record of germanus (Blandford) (Coleoptera: Cur- culionidae, Scolytinae) in the Mediterranean scrubland in Southern Italy, and its co-presence with the co-generic species X. compactus (Eichhoff) and X. crassiusculus (Motschulsky)

M. Contarini1 , A. Vannini2 , F. Giarruzzo2 , M. Faccoli3 , C. Morales-Rodriguez2 , L. Rossini1 and S. Speranza1 1DAFNE, Universita degli Studi della Tuscia, Via S. Camillo de Lellis, 01100, Viterbo, Italy; e-mail: [email protected] 2DIBAF, Universita degli Studi della Tuscia, Via S. Camillo de Lellis, 01100, Viterbo, Italy; e-mail: [email protected] 3DAFNAE, Universita degli Studi di Padova, Viale dell’Universita 16, 35020, Legnaro, Italy

In the Circeo National Park (Italy), a survey assessed the occurrence of the ambrosia Xylosandrus germanus in addition to the conspecifics X. compactus and X. crassiusculus. This is the first report in Europe of the co-presence of three Xylosandrus species, in an evergreen Mediterranean maquis dominated by holm oaks (Quercus ilex L.). Although no damage caused by X. germanus has been observed, the richness of plant species in the Cir- ceo National Park may allow the establishment of this highly polyphagous ambrosia beetle, possibly endangering this native ecosystem. The dominance in the associated fungal com- munity of Ambrosiella grosmanniae and Fusarium spp. reflects a stable symbiotic relation- ship of these taxa with X. germanus.

then spread quickly in other regions of the Italian mainland Introduction (Vannini et al., 2017) and Sicily (Longo & Garzia, 2016), Ambrosia (Coleoptera: , Scolytinae where it attacks twigs and small branches of a large number and Platypodinae) represent a particularly important group of Mediterranean host trees. X. compactus was added to the of alien species, capable of travelling undetected within EPPO Alert List in 2017. In the EPPO region, it is a quaran- many different woody materials and being difficult to find tine pest for Israel (EPPO, 2020). In the last 5 years, the during phytosanitary inspections (Brockerhoff et al., 2006). coexistence of X. compactus and X. crassiusculus was The damage they cause is related to both wood-boring reported in the Circeo National Park (Latium, central Italy), activity and the introduction of associated fungi, which are causing dieback of branches and twigs of many species of the of crucial importance for larval feeding development, as Mediterranean maquis, such as Ceratonia siliqua L., Quercus well as being pathogenic for the host plant. spp. and Arbutus unedo L. (Francardi et al., 2017). In particu- Among ambrosia beetles, the species of the genus lar, a serious outbreak of X. compactus was reported causing Xylosandrus belong to a highly damaging group of Asian severe damage to the Mediterranean maquis in an area that tropical origin. In particular, X. germanus (Blandford), extended for more than 13 hectares on the Circeo Promon- X. crassiusculus (Motschulsky) and X. compactus (Eichhoff) tory, within the stands of the Circeo National Park (Vannini are spreading worldwide, invading also temperate areas of et al., 2017). Here, evergreen shrubs and trees suffered strong southern Europe, including Italy (Galko et al., 2019). The and quick desiccation of the small-diameter branches (up to highly polyphagous X. crassiusculus, attacking more than 3 cm) caused by the wood-boring activities of the beetles and 200 plant hosts (Castrillo et al., 2013), was reported for the their associated fungal pathogens (Vannini et al., 2017). A first time in Europe since 2003 on Turkey oaks (Quercus third species, X. germanus, was first reported in northern cerris L.) growing in mixed woods in Tuscany. In a few Italy in 1992 in stands dominated by chestnuts (Castanea years, the species spread in most of central and north Italy sativa Miller; Bernabo, 2000; Dutto et al., 2018), then in wal- (Pennacchio et al., 2003) also invading the Mediterranean nut plantations (Juglans spp.), in mixed broadleaf stands and maquis ecosystems (Francardi et al., 2017, Gallego et al. in apple orchards (Stergulc et al., 1999). X. germanus is cur- 2017). It is currently not regulated by EPPO countries rently not regulated in the EPPO region, but is a quarantine (EPPO, 2020). X. compactus was reported for the first time pest in the Comite de Sanidad Vegetal del Cono Sur in 2011 in two urban parks in Naples and Portici (Campania, (COSAVE) region (EPPO, 2018). Unlike the two previously South Italy) on holm oaks (Quercus ilex L.) and evergreen described species, X. germanus has, to the authors’ knowl- Mediterranean shrubs (Garonna et al., 2012). X. compactus edge, never been recorded in Mediterranean maquis

ª 2020 The Authors. Journal compilation ª 2020 OEPP/EPPO, EPPO Bulletin 0, 1–5 1 2 M. Contarini et al. ecosystems in Italy or in maquis ecosystems elsewhere. fungal community through mass sequencing. Adult

X. germanus is currently present in most of Europe (EPPO, were repeatedly washed in sterile distilled H2O and dried 2018). on filter paper before DNA extraction. Total DNA from a Within the framework of the monitoring activity carried bulk of the 7 adults was extracted using a DNeasy Power- out in Italy, France and Spain by the European LIFE pro- Soil Kit (Qiagen, Hilden, Germany), following the manu- ject SAMFIX (SAving Mediterranean Forest from Invasions facturer’s instructions. The ITS1 region was amplified with of Xylosandrus beetles and associated pathogenic fungi – a dual indexing primer using the tagged primer pair ITS1F LIFE17 NAT/IT/000609) the present paper reports the first (50-xxxxCTYGGTCATTTAGAGGAAGTAA-30) and ITS2 detection of X. germanus in the Mediterranean maquis (5-xxxxGCHRCGTTCTTCATCGDTGC-30), where xxx ecosystem in Europe, and the first report for Europe of the represents the barcoding key. The PCR reaction mixture co-presence of the three Xylosandrus species in the same comprised 12.5 mL of Maxima Hot Start PCR Master Mix environment. (29) (Thermo Fisher Scientific, Waltham, Massachusetts, USA) and 1 mM of each primer in a total volume of 25 mL containing 24 mL of reaction mixture and 1 mL of Materials and methods template. The thermal cycle was an initial denaturation at 94°C for 10 min followed by 30 cycles of 95°C for 40 s, monitoring and collection 60°C for 40 s and 72°C for 1 min, and a final elongation From 6 August until 6 November 2018 four black 9-funnel step of 72°C for 10 min. Amplicons were purified using traps were set up in the Quarto Freddo site located in the Cir- MagJET NGS Cleanup (Thermo Fisher Scientific, Waltham, ceo Promontory of the Circeo National Park (Latium Region, Massachusetts, USA), quantified with a Qubit Quantitation central Italy) at about 160 m above sea level (41° 140 17″ N, kit (Invitrogen, Carlsbad, California, USA) and pooled at 13° 040 49″ E). The area is characterized by the presence of equal concentrations for sequencing. Paired-end sequencing mixed wood stands dominated by Q. ilex associated with a (2 9 300 bp) was carried out on an Illumina MiSeq large number of tree and shrub species typical of the Mediter- sequencer by Eurofins Genomics GmbH (Ebersberg, Ger- ranean maquis. Traps were positioned about 100 m apart many). Data sets were analysed following the pipeline from each other and baited with a dispenser of 5 mL of pure described by Morales-Rodriguez et al. (2019). ethanol and a dispenser of 1 mL of ()-alpha-pinene. Both lures were put separately in polyethylene diffusion bags hung Results at the second funnel located above the trap collection cup. In the collection cups, an insecticide net was placed to kill col- Trapped species lected specimens avoiding their escape. Lures and nets were renewed every 15 days, while trapped insects were collected A total of 283 Xylosandrus adults were collected in the four and brought to the laboratory weekly. Xylosandrus species traps. Three species of Xylosandrus were identified: were determined based on morphological characteristics and X. compactus and X. crassiusculus, previously reported at following published keys (Dole & Cognato, 2010). this site (Vannini et al., 2017, Francardi et al., 2017), and a third species, new for the area, identified as X. germanus (Fig. 1). X. compactus was the most common species Fungal isolation and identification trapped with a total of 266 individuals, followed by Among the adults of X. germanus collected from different X. germanus with 15 specimens and 2 adults of traps and dates between 20 August and 3 September 2018, X. crassiusculus. Results show the emergence peak of seven specimens were used to investigate the associated X. compactus, which occurred in middle September

Fig. 1 (A) Xylosandrus compactus, (B) Xylosandrus crassiusculu and (C) Xylosandrus germanus. Scale bar 0.5 mm.

ª 2020 The Authors. Journal compilation ª 2020 OEPP/EPPO, EPPO Bulletin 0, 1–5 Xylosandrus spp. in Mediterranean scrubland 3

Weekly catches of Xylosandrus specimens 70

X. compactus 60 X. germanus X. crassiusculus

specimens 50

40

Xylosandrus 30

20 Number of 10

0 g g g p p p p t t t t t v u u u e e e e c c c c c o A A A S S S S O O O O O N 3 0 7 3 0 7 4 1 8 5 2 0 6 1 2 2 0 1 1 2 0 0 1 2 3 0 Fig. 2 Number of specimens of Xylosandrus species trapped weekly in the Circeo National Park.

(Fig. 2). Captures of X. germanus and X. crassiusculus ecosystem, which is already showing signs of the change in were too low for phenology and voltinism assessment. the forest structure. Moreover, the large number of plant species occurring in the Circeo National Park potentially susceptible to be colonized by these three highly polypha- Fungal community of X. germanus gous ambrosia beetles further increases the biological risk From the insect bulk sample analysed, 289 331 reads were for these native ecosystems to be altered by the massive clustered to a 97% similarity level in 164 Operational Tax- presence of Xylosandrus species and their associated fungi. onomic Units (OTUs), among which Ambrosiella In addition, the three Xylosandrus species exploit distinct grossmanniae Mayers, McNew & Harr., Geosmithia pallida ecological niches by colonizing different branch diameters (G. Smith) M. Kolarık, Kubatova & Pazoutova, Fusarium of the host plant, exposing whole forest stands to further spp., Penicillium spp. Cladosporium sp. and Alternaria sp. infestation risk. X. compactus differs from other species as were the most representative taxa, accounting for 42% of it usually colonizes branches smaller than 2–3 cm, even the total reads. though atypical behaviours are sometime reported (Gugli- uzzo et al., 2019), while X. germanus and X. crassiusculus bore galleries in stems larger than 2 cm. Discussion X. germanus is a very polyphagous species, attacking This is the first record of X. germanus presence in Mediter- more than 200 species of trees, including living trees, dying ranean scrubland in Europe and Italy. Previous records in trees or trees which have recently died (Hoffmann, 1941; Europe confined this species to deciduous forests (e.g. Weber & McPherson, 1983; Ranger et al., 2015). Adult Juglans spp., Fagus sylvatica L., Castanea sativa, Quercus females, 2–2.3 mm long, penetrate the wood through entry petraea (Matt.) Liebl) and coniferous trees (CABI, 2020). In holes 1 mm in diameter. Mother galleries widen in brood Italy, X. germanus was reported mainly in the northern part chambers where eggs are laid and larvae develop until the of the peninsula, preferentially associated with chestnut emergence of adults of the new generation (Agnello et al., stands (Rassati et al., 2016; Dutto et al., 2018). Interestingly, 2017). X. germanus completes two generations per year, this association was recently confirmed in spring 2018 in a three in the warmer southern regions of the United States chestnut coppice stand in Viterbo province, central Italy, sug- (Weber & McPherson, 1983). Moreover, it presents hap- gesting the spread of this alien species southward across the loid-diploid parthenogenesis with females and males pro- Italian peninsula (Rassati et al., 2020). duced from fertilized and unfertilized eggs, respectively. Furthermore, the survey activity carried out in the Circeo Female progeny mate with siblings inside the galleries National Park reported, for the first time in Europe, the co- before emergence and dispersal. However, even if very presence of the three Xylosandrus species, X. germanus, rare, mating among non-siblings is known (Keller et al., X. crassiusculus and X. compactus, in a heterogenous stand 2011). The sex ratio is about 10 females per male, but it of evergreen Mediterranean maquis dominated by holm oaks. can vary depending on the host plant as well as brood size Even if X. compactus has a higher population in compar- (Peer & Taborsky, 2004). ison to the other two species, the simultaneous presence of Colonization symptoms are not easy to detect. However, these three introduced alien pests endangers this delicate 3–4 cm long cylinders of woody frass are expelled by

ª 2020 The Authors. Journal compilation ª 2020 OEPP/EPPO, EPPO Bulletin 0, 1–5 4 M. Contarini et al. adults from the entry holes during the gallery boring in the les scolytes X. compactus et X. crassiusculus. Il s’agit du wood; sometimes, sap pours from the entry holes, staining premier signalement en Europe de la presence conjointe de and wetting the bark. ces trois especes de Xylosandrus, et ce au sein d’un maquis As for other ambrosia beetles, larval development and mediterraneen a feuilles persistantes domine par des ch^enes adult maturation depend on inoculation and growth on the verts (Quercus ilex L.). Bien qu’aucun deg^at cause par X. inner walls of the brood chambers of symbiotic fungi used germanus n’ait ete observe, la richesse des especes as food and carried in special tegumental structures, the vegetales du parc national du Circe pourrait permettre mycangia. In the present study, the fungal community asso- l’etablissement de ce scolyte tres polyphage, presentant un ciated with X. germanus was investigated. The finding of danger eventuel pour cet ecosysteme indigene. La Ambrosiella grosmanniae is consistent with the records in prevalence dominante d’Ambrosiella grosmanniae et de the literature that identify in this species the main symbiont Fusarium spp. au sein de la communaute fongique associee associated with X. germanus (Mayers et al., 2015). The reflete une relation symbiotique stable de ces taxons avec OTU of A. grossmannieae accounted for 7% of the total X. germanus. reads, reflecting the importance of this species for the larval development of the insect. Geosmithia pallida accounted Первое сообщение о Xylosandrus germanus for 14% of the total reads. G. pallida is a species native (Blandford) (Coleoptera: Curculionidae, from Europe accidentally introduced as an alien pathogen Scolytinae) в средиземноморском маквисе на of live oaks in the United States (Lynch et al., 2014). This юге Италии, и о его совместном присутствии shows affinities with a wide range of wood-boring с близкородственными видами X. compactus insects, such as bark beetles and sawyer beetles (Kolarık (Eichhoff) и X. crassiusculus (Motschulsky) et al., 2004). Moreover, it was already reported in associa- tion with X. compactus in the Circeo National Park by Van- В Национальном парке Цирцео (Италия) было nini et al. (2017). Associations among X. germanus and проведено обследование, в ходе которого было оценено Fusarium species were already known, often in presence of присутствие амброзиевого жука Xylosandrus germanus в host dieback and stem cankers (Kessler, 1974; Weber & дополнение к принадлежащим к тому же роду X. McPherson, 1984, 1985; Agnello et al., 2017). compactus и X. crassiusculus. Это первое сообщение для Further studies are already underway (1) to determine the Европы о совместном присутствии трёх видов рода effective damage caused by X. germanus in the Circeo Xylosandrus в вечнозелёном средиземноморском маквисе National Park; (2) to update the list of the potential host с преобладанием каменного дуба (Quercus ilex L.). plants; and (3) to better understand both the relationships Несмотря на то, что не было обнаружено никакого between the three Xylosandrus species and their associa- ущерба, нанесённого X. germanus, разнообразие видов tions with fungi in order to assess the risk of biological растений в Национальном парке Цирцео может invasions in sclerophyllous stands of southern Italy. способствовать акклиматизации этого крайне многоядного амброзиевого жука, что может представлять угрозу для этой аборигенной экосистемы. Acknowledgement Доминирование Ambrosiella grosmanniae и Fusarium spp. The authors are grateful to the Circeo National Park (Italy) в ассоциированных грибных сообществах отражает for the kind support and to Adalgisa Guglielmino for the стабильную симбиотическую связь этих таксонов с X. kind cooperation. This research was co-funded by the LIFE germanus. project (NAT/IT/000609) SAMFIX (SAving Mediterranean Forest from Invasions of Xylosandrus beetles and associated References pathogenic fungi). The research was carried out in the framework of the MIUR (Ministry for Education, Univer- Agnello AM, Breth DI, Tee EM, Cox KD, Villani SM, Ayer KM et al. sity and Research) initiative Department of Excellence (2017) Xylosandrus germanus (Coleoptera: Curculionidae: Scolytinae) (Law 232/2016). occurrence, fungal associations, and management trials in New York apple orchards. Journal of Economic Entomology 110, 2149–2164. 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ª 2020 The Authors. Journal compilation ª 2020 OEPP/EPPO, EPPO Bulletin 0, 1–5