Mycol Progress (2016) 15:55 DOI 10.1007/s11557-016-1199-3 ORIGINAL ARTICLE Scolytus multistriatus associated with Dutch elm disease on the island of Gotland: phenology and communities of vectored fungi Audrius Menkis1 & Inga-Lena Östbrant2 & Kateryna Davydenko3 & Remigijus Bakys4 & Maksims Balalaikins5 & Rimvydas Vasaitis1 Received: 16 February 2016 /Revised: 28 April 2016 /Accepted: 13 May 2016 # German Mycological Society and Springer-Verlag Berlin Heidelberg 2016 Abstract Scolytus multistriatus Marsham, the smaller revealed the presence of 1589 fungal taxa, among which vir- European elm bark beetle, is a vector for Dutch elm disease ulent DED pathogen Ophiostoma novo-ulmi Brasier was the (DED) that in the year 2005 invaded the island of Gotland second most common species (9.0 % of all fungal sequences). (Sweden). The island possesses the largest population of elm O. ulmi Buisman, the less virulent DED pathogen, was also (mainly Ulmus minor Mill.) in northern Europe. The aim of detected but only in a single beetle, which was sampled in this study was to monitor flying periods of S. multistriatus 2012 (0.04 % of sequences). There were 13.0 % of the beetles during three consecutive years and by using high-throughput infested with O. novo-ulmi in 2012, 4.0 % in 2013, and 27.7 % sequencing to assess communities of vectored fungi. in 2014. O. novo-ulmi comprised 0.8 % of fungal sequences in Sampling of the beetles was carried out at two different sites 2012, 0.002 % in 2013, and 8.2 % in 2014. The study showed in Gotland in 2012, 2013, and 2014. In total, 50 pheromone that the proportion of S. multistriatus vectoring O. novo-ulmi traps were placed at each site and checked weekly during has increased in recent years. June-August each year. From all sites and years, 177 beetles were trapped. Among these, 6.2 % were trapped in June, Keywords Ophiostoma . Invasive pathogens . Bark beetles . 76.8 % in July, and 16.9 % in August (difference significant Disease management . Fungal community . Ulmus at p<0.007). Sequencing of ITS rDNA from the beetles Introduction Electronic supplementary material The online version of this article (doi:10.1007/s11557-016-1199-3) contains supplementary material, which is available to authorized users. Scolytus multistriatus (Scolytinae: Scolytini), the smaller European elm bark beetle, is native to Europe, the Middle * Audrius Menkis East, and northern Africa (Bellows et al. 1998), but was intro- [email protected] duced with elm wood to other areas including North America, New Zealand, and Australia (Brockerhoff et al. 2003; Lee et al. 2009; Parbery and Rumba 1991) and generally occurs 1 Department of Forest Mycology and Plant Pathology, Uppsala BioCenter, Swedish University of Agricultural Sciences, within the areal of host trees (mainly Ulmus spp.). Adults (1.9 P.O. Box 7026, SE-75007 Uppsala, Sweden to 3.1 mm in length) bore through the bark of weakened and/ 2 Swedish Forest Agency Gotland District, P.O. Box 1417, SE-621 or stressed elms, breed under the bark and produce egg gal- 25 Visby, Sweden leries in the vascular tissues. Females lay eggs along the egg 3 Ukrainian Research Institute of Forestry and Forest Melioration, gallery, and larvae tunnel across the vascular tissues away Pushkinska str. 86, 61024 Kharkiv, Ukraine from the egg gallery (Wood 1982). S. multistriatus overwin- 4 Institute of Forest Biology and Silviculture, Aleksandras Stulginskis ters as larvae under the bark and new adults emerge in the University, Studentu str. 11, LT-53361 Akademija Kaunas spring or early summer after elm leaves have fully developed. District, Lithuania S. multistriatus is one of the most effective vectors for 5 Institute of Life Sciences and Technology, Daugavpils University, Dutch elm disease (DED) (Santini and Faccoli 2015; Vienibas str. 13, LV-5401 Daugavpils, Latvia Webber 1990) caused by fungi from the genus 55 Page 2 of 8 Mycol Progress (2016) 15:55 Ophiostoma (Ascomycota) (Kirisits 2013), which during Materials and methods the last 100 years have destroyed billions of elm trees worldwide (Phillips and Burdekin 1982). DED is a le- Study sites and sampling thal vascular wilt disease comprised of three distinct fungal pathogens, less virulent O. ulmi, and highly vir- Mean temperatures for the study area were retrieved from ulent O. novo-ulmi and O. himal-ulmi Brasier & http://luftwebb.smhi.se. The study sites were at Vallstena Mehrotra, a species endemic to the western Himalayas (N57°36′, E18°41′) and Hogrän (N57°31′, E18°18′) on the (Brasier and Mehrotra 1995). Conidia, which are the Baltic Sea island of Gotland. The distance between the sites infection source of DED pathogens, are transmitted on was ca. 26 km. The site at Vallstena was a mixed forest the body surface of the beetles into the tree, and a new composed of Pinus sylvestris L., Picea abies (L.) Karst., generationofbeetlesisonlyinfestediftheDEDfungus Betula pendula Roth, Ulmus spp. and Alnus spp. The site at is present in the galleries. Conidia are produced in Hogrän was a mixture of open fields and forest land with sticky masses that facilitate their attachment and trans- similar tree species in admixture as at the Vallstena site. portation by beetles as theyemergefromthetrees Both sites were characteristic to Gotland in terms of (Ploetz et al. 2013). When DED-infested beetles emerge landscape and trees species composition, and were in the and fly to feed in the twig crotches of healthy elms, areas characterised by a high incidence of DED. At each they form grooves in the wood through which the fun- site, 50 transparent delta traps with a sticky insert gus enters the twig and spreads within the branch by a (Pherobank, Wijk bij Duurstede, The Netherlands) on the yeast-like budding process causing leaves to wilt and bottom and a P188 pheromone lure (Synergy die. This is due to the blockage of the conducting sys- Semiochemcials Corp., Burnaby, Canada) were placed every tem subsequent to the formation of tyloses and gels in 50 m along a transect, which was 2.5 km long. Lures consisted the xylem vessels and the production of toxins, and of two semi-permeable plastic pouches containing a mixture eventually causing the death of a tree (Phillips and of cubeb oil, 1-hexanol, multistriatin and 4-methyl-3- Burdekin 1982). heptanol. The lure used attracts Scolytus spp. beetles. In this The island of Gotland (Sweden) possesses the largest type of trap, beetles firmly stick to the sticky insert, which and highly valuable wild population of elms (more than prevents physical contact among different individuals, and one million trees that are mainly Ulmus minor)innorth- prevents cross-contamination with e.g. fungal spores. To set ern Europe, which until recently was not affected by the traps, two sticks 1.5 m in length were hammered to the DED (Östbrant et al. 2009). In 2005, however, DED ground and a trap was fastened to them about 1.2 m above the was observed in Gotland and in the following years, it ground. Each trap was labelled and a global positioning sys- rapidly spread in all directions, causing extensive mortal- tem (GPS) coordinates were recorded in order to set the traps ity of elm trees (Menkis et al. 2016). Among the elm at the same position each year. Sampling was carried out from bark beetles known from Sweden, which include the beginning of June until the end of August in the years S. triarmatus Eggers., S. laevis Chapuis, S. rugulosus 2012, 2013, and 2014. During the sampling period, traps were O.F. Muller, S. pygmaeus F. and S. multistriatus,only visited once a week and sticky inserts with trapped insects the latter species occurs in Gotland (Schlyter et al. were collected and replaced with new inserts. Collected inserts 1987) and is therefore thought to be responsible for the were transported the same day to the laboratory and examined current spread of DED. Interestingly, S. multistriatus has under Carl Zeiss Stemi 2000-C dissection microscope been known in Gotland for decades, which suggests that (Oberkochen, Germany). When the beetles of S. multistriatus until 2005 its population on the island was free of were detected, they were individually placed into 2-mL screw- O. novo-ulmi. Although the precise route of disease ar- cap centrifugation tubes and stored at −20 °C until further rival is not known, it was probably brought to the island DNA processing. with DED-infested elm wood that would resemble pat- terns of human-mediated spread of DED (Brasier et al. DNA isolation, amplification and sequencing 2004). However, little is known about when S. multistriatus is most active in Gotland and especially Total DNA was isolated separately from each beetle. No what proportion of those beetles vector conidia of DED surface sterilisation was carried out. Prior to isolation of fungi. Moreover, little is known about other fungal spe- DNA, the beetles were freeze-dried at −60 °C for 2 days, cies vectored by S. multistriatus. and together with glass beads were homogenized for The aim of the present study was to monitor the seasonal 2 min at 5000 rpm using a Fast prep shaker (Precellys flying intensity of S. multistriatus and to assess communities 24, Bertin Technologies, Rockville, MD). Then, 800 μL of vectored fungi at different time periods, particularly focus- of CTAB extraction buffer (3 % cetyltrimethylammonium ing on DED pathogens. bromide, 2 mM EDTA, 150 mM Tris–HCl, 2.6 M NaCl, Mycol Progress (2016) 15:55 Page 3 of 8 55 pH 8) was added to each tube, followed by incubation at included the removal of short sequences (<200 bp), 65 °C for 1 h. After centrifugation, the supernatant was sequences with low read quality, primer dimers, and transferred to new 1.5-mL centrifugation tubes and then homopolymers.