Mycol Progress (2016) 15:55 DOI 10.1007/s11557-016-1199-3

ORIGINAL ARTICLE

Scolytus multistriatus associated with Dutch 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 multistriatus Marsham, the smaller revealed the presence of 1589 fungal taxa, among which vir- European elm bark , is a vector for 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 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. (Scolytinae: ), the smaller European elm , 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 , 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 () (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 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. Sequences that were missing a tag or primer mixed with 1 volume of chloroform by gentle vortexing. were excluded. The primer and sample tags were then After centrifugation for 8 min at 10000 rpm, the super- removed from the sequence, but information on the natant was precipitated with 2 volumes of cold sequence association with the sample was stored as meta- isopropanol, washed with 70 % ethanol and dissolved data. The sequences were then clustered into different taxa in 50 μL TE buffer. Additionally, isolated DNA was pu- using single-linkage clustering based on 98.5 % similarity. rified using JETquick DNA Clean-Up System (Genomed, The most common genotype for clusters was used to represent Löhne, Germany). In each sample, concentration of ge- each taxon. For clusters containing two sequences, a consen- nomic DNA was determined using a ND-1000 spectro- sus sequence was produced. The fungal taxa were taxonomi- photometer (NanoDrop Technologies, Wilmington, DE). cally identified using GenBank (NCBI) database and the Diluted (1–10 ng/μL) genomic DNA samples were am- Blastn algorithm. The criteria used for identification were: plified separately using the primer pair fITS9 (5′- sequence coverage > 80 %; similarity to taxon level 98– GAACGCAGCRAAIIGYGA-3′) (Ihrmark et al. 2012) 100 %, similarity to genus level 94–97 %. Sequences not andITS4(5′-xxxxxxxxTCCTCCGCTTATTGATATGC- matching these criteria were considered unidentified and were 3′) (White et al. 1990) containing 8-bp sample identifi- given unique names, as shown in Supplementary Table 1. cation barcodes denoted by x. Using this primer pair, amplified PCR products were estimated to be between Statistical analyses 280–420bpinsizeandtoincludealargepartofthe 5.8S rRNA gene sequence, complete sequence of the As both qualitative and quantitative data of high-throughput noncoding ITS2 rRNA region, and partial sequence of sequencing was shown to be consistent and highly reproduc- the 28S rRNA gene. The PCR reactions, 50 μLinvol- ible (Porazinska et al. 2010), the number of read counts was ume for each sample, were performed using an Applied used to estimate relative abundance of fungal taxa in the sam- Biosystems 2720 Thermal Cycler (Applied Biosystems, ples. The abundance of S. multistriatus and of DED fungi in Carlsbad, CA) and DreamTaq Green DNA polymerase different sampling years was compared by non-parametric (Thermo Fisher Scientific, Waltham, MA). The PCR cy- chi-squared tests calculated from the actual number of obser- cle parameters consisted of an initial denaturation at vations (Mead and Curnow 1983). As the datasets were sub- 95 °C for 2 min, 27 cycles of denaturation at 95 °C jected to multiple comparisons, confidence limits for p-values for30s,annealingat55°Cfor30sandextensionat of the chi-squared tests were reduced a corresponding number 72 °C for 45 s, followed by a final extension step at of times, as required by the Bonferroni correction (Sokal and 72 °C for 7 min. The PCR products were analysed on Rohlf 1995). The rarefaction analysis was performed using 1 % agarose gels (Agarose D1, Conda, Madrid, Spain) Analytical Rarefaction v.1.3 available at http://www.uga.edu/ under UV using GelDocTM 2000 gel documentation sys- strata/software/index.html. The rarefaction analysis was tem (Bio-Rad laboratories, Berkeley, CA). To purify carried out to reveal the relationship between the cumulative amplicons, they were precipitated in a mixture of 1/10 number of taxa found and the sequencing intensity (Colwell volume3MNaAcand2volumes−20 °C pure ethanol, and Coddington 1994). vortexed for 10 min, incubated for 20 min at −70 °C and centrifuged for 5 min at 13,000 rpm. Supernatant was discarded and dried pellets were dissolved in 30 μL Results Milli-Q water. The concentration of purified PCR prod- ucts was determined using Quant-iT™ dsDNA HS Assay During three sampling years, 177 beetles of Kit (Life Technologies, Carlsbad, CA, USA), and an S. multistriatus were trapped, or on average, 0.59 beetle equimolar mix of all PCR products was used for Ion per year per trap. Information on the number of beetles Torrent sequencing. Construction of the sequencing li- trapped (data pooled from both sites) during each year brary and sequencing using a 316 chip was carried out (2012, 2013, and 2014) and mean temperatures are shown by NGI SciLifeLab (Uppsala, Sweden). in Fig. 1. There were 47.5 % of S. multistriatus trapped in 2012, 21.5 % in 2013, and 31.0 % in 2014, and a chi- Bioinformatics squared test showed that it was significantly higher in 2012 than in 2013 or 2014 (p<0.003), but the number of The sequences generated were subjected to quality control and beetles were not significantly different between 2013 and clustering in the SCATA NGS sequencing pipeline (http:// 2014. In all years, 6.2 % of S. multistriatus were trapped scata.mykopat.slu.se). Quality filtering of the sequences in June, 76.8 % in July, and 16.9 % in August, and it was 55 Page 4 of 8 Mycol Progress (2016) 15:55

Fig. 1 Bars show relative abundance of Scolytus multistriatus beetles trapped/ collected (data pooled from both sites) and lines show mean temperatures (retrieved from http://luftwebb.smhi.se) during June-August of 2012, 2013, and 2014 on the island of Gotland

significantlyhigherinJulythaninJuneorAugust available from GenBank under accession numbers (p<0.0001), and significantly higher in August than in KP890936 - KP892524). Identification at least to genus level June (p<0.007). was successful for 928 (58.4 %) out of 1589 fungal taxa. The A total of 9,914,812 sequences were generated by Ion most common taxa were Cladosporium sp. 2170_0 (37.9 %), Torrent sequencing from the 177 beetles. Of those, 9,474, O. novo-ulmi (9.0 %), Aureobasidium pullulans (7.5 %), 995 (95.6 %) did not pass quality control and were thus ex- Dioszegia fristingensis (4.9 %), and Cryptococcus wieringae cluded. Clustering of the remaining 439,817 high-quality se- (3.9 %). Information on the 30 most common fungal taxa quences (272 bp on average) resulted in 1764 non-singleton representing 90.1 % of all fungal sequences is shown in the contigs and 2745 singleton contigs, which were excluded Table 1. The remaining 1559 taxa were relatively rare and from the further analyses. Among the non-singletons, 1589 their relative abundances varied between 0.3 % and contigs (90.1 %) represented fungi, 163 (9.2 %) plants, nine 0.00005 % (Supplementary Table 1). (0.5 %) , and three (0.2 %) protists. A plot of fungal In the present study, both DED pathogens, i.e. less virulent taxa vs. the number of sequences resulted in rarefaction curves O. ulmi and virulent O. novo-ulmi, were detected by ITS that reached the asymptote (Fig. 2). There were between two rDNA sequencing of S. multistriatus beetles (Supplementary and 158 fungal taxa detected per individual beetle that com- Table 1). However, O. ulmi was detected in a single (0.6 %) prised 67.6 % Ascomycota, 31.0 % Basidiomycota, 0.7 % beetle while O. novo-ulmi was detected in 79 (44.6 %) beetles Mortierellomycotina, 0.4 % Chytridiomycota, 0.2 % (difference significantly at p<0.0001). O. ulmi was detected at Glomeromycota, and 0.1 % Mucoromycotina (representative Hogrän in 2012, while O. novo-ulmi was detected on both ITS rDNA fungal sequences of all non-singletons are sites and during the entire sampling period (Table 2). The

Fig. 2 Rarefaction curve showing the relationship between the cumulative number of fungal taxa and the number of ITS rDNA fungal sequences obtained from 177 beetles of S. multistriatus sampled on the island of Gotland Mycol Progress (2016) 15:55 Page 5 of 8 55

Table 1 List of the 30 most common fungal taxa found in 177 Taxon Reference Sequence Similarity, No. of Frequency of beetles of S. multistriatus sampled sequence length (%)* sequences occurrence, (%) on the island of Gotland Ascomycota Cladosporium sp. 2170_0 HG530747 262 262/262 162589 37.9 (100) Ophiostoma novo-ulmi EF638891 329 327/327 38632 9.0 (100) Aureobasidium pullulans KM388542 268 267/268 (99) 32113 7.5 Cordyceps confragosa KJ529005 274 273/274 (99) 11159 2.6 Epicoccum nigrum KM396372 268 267/268 (99) 7891 1.8 Candida sp. 2170_19 KF057719 212 174/174 6873 1.6 (100) Fusarium tricinctum KM249082 277 277/277 6190 1.4 (100) Alternaria sp. 2170_10 KF728750 271 270/271 (99) 5554 1.3 Candida sp. 2170_12 EU491501 307 292/307 (95) 4858 1.1 Beauveria bassiana KM114549 274 273/274 (99) 4265 1.0 Penicillium kojigenum AM236584 276 275/276 (99) 4241 1.0 Alternaria rosae KF815569 271 270/271 (99) 2879 0.7 Sphaerosporella JQ711781 226 216/226 (96) 2314 0.5 sp. 2170_23 Geosmithia flava KJ513214 287 286/287 (99) 1923 0.4 Botryotinia fuckeliana KJ476441 260 259/260 (99) 1388 0.3 Rachicladosporium KP004448 232 227/232 (98) 1334 0.3 eucalypti Periconia byssoides KC954160 268 267/268 (99) 1264 0.3 Pyrenophora tritici- KM011994 268 267/268 (99) 1201 0.3 repentis All Ascomycota 296668 69.2 Basidiomycota Dioszegia fristingensis EU070927 236 235/236 (99) 20968 4.9 Cryptococcus wieringae KF981864 348 347/348 (99) 16636 3.9 Cryptococcus albidus KJ589643 333 333/333 10385 2.4 (100) Udeniomyces pannonicus AB072229 345 341/342 (99) 8829 2.1 Dioszegia crocea GQ911539 239 239/239 8213 1.9 (100) Cystofilobasidium macerans JX188155 347 346/347 (99) 5692 1.3 Cryptococcus stepposus JX188129 355 354/355 (99) 4311 1.0 Mrakiella aquatica GQ911547 345 344/345 (99) 4102 1.0 Cryptococcus victoriae KM376411 221 221/221 3518 0.8 (100) Sporobolomyces roseus KM376382 319 319/319 2962 0.7 (100) Dioszegia butyracea EU266508 236 236/236 1910 0.4 (100) Melampsora caprearum AY444779 342 340/342 (99) 1788 0.4 All Basidiomycota 89314 20.8

* Sequence similarity column shows base pairs compared between the query sequence and the reference sequence at NCBI database, and the percentage of sequence similarity in the parenthesis proportion of S. multistriatus infested with O. novo-ulmi did 2013, and 27.7 % in 2014 (p<0.002) (Table 2). Relative abun- not differ significantly between two sampling sites. dance of vectored O. novo-ulmi (estimated as a proportion of Differences among years (data pooled from both sites) were all fungal sequences) also differed significantly among the significant, with 13.0 % of beetles infested in 2012, 4.0 % in years, being 0.8 % in 2012, 0.002 % in 2013, and 8.2 % in 55 Page 6 of 8 Mycol Progress (2016) 15:55

Table 2 Relative abundance of Scolytus multistriatus beetles infested association between S. multistriatus and DED fungi is well with Ophiostoma novo-ulmi (shown as a proportion of all beetles), and established (Santini and Faccoli 2015). Nevertheless, the relative abundance of vectored O. novo-ulmi (shown as a proportion of all fungal sequences) in different study sites and sampling years forthcoming availability of even more powerful molecular and genomic tools can be expected to provide new insights Sampling year Beetles-infested Vectored O. novo-ulmi into the DED pathosystem and open possibilities for develop- Hogrän Vallstena All Hogrän Vallstena All ment of new control strategies (Bernier et al. 2014). In the present study, despite the use of delta traps that re- 2012 11.7 a 17.5 ab 13.0 1.2 a 0.0 a 0.8 sulted in a relatively small number of trapped beetles of 2013 3.6 b 5.0 a 4.0 0.0 b 0.0 a 0.0 S. multistriatus compared to results using other type of traps 2014 27.0 c 30.0 b 27.7 11.2 c 2.4 b 8.2 (e.g. window traps) (Menkis et al. 2016), delta traps prevented All 42.3 52.5 44.6 12.4 2.4 9.0 cross-contamination among individual beetles (54.8 % of all beetles were not infected by DED), thereby allowing abun- Within columns of respective study site, values followed by the same dance monitoring of the beetles vectoring DED each year. letter are not significantly different However, in order to more precisely monitor the flying inten- sity of S. multistriatus, window traps or Lindgren funnel trap 2014 (p<0.0001) (Table 2). Although several other (Johnson et al. 2008), instead of delta traps, should probably ophiostomatoid fungi have also been detected, these were be used to obtain higher yields of beetles. Although it is ac- identified to the genus level (Supplementary Table 1). knowledged that S. multistriatus may vector Ophiostoma spp. (Ploetz et al. 2013), information on other fungal taxa vectored is scarce. In the present study, the use of high-throughput Discussion sequencing showed that S. multistriatus vectors a highly di- verse fungal community (Supplementary Table 1). The results showed that both O. ulmi and O. novo-ulmi were Furthermore, rarefaction analysis showed that a great majority present in Gotland. However, the occasional occurrence of of fungal taxa was detected (Fig. 2) thereby highlighting the O. ulmi suggests that, as elsewhere, it is being replaced by efficacy of the sequencing method even though only a rela- O. novo-ulmi (Brasier et al. 2004). Brasier et al. (2004)report- tively small proportion of all sequences was of high quality ed that O. novo-ulmi replaced O. ulmi at a relative incidence of and could be used in analyses. The detected richness of fungal about 10 % per year at each location. Taken into consideration taxa was one or two orders of magnitude as compared to that O. novo-ulmi was probably introduced to Gotland ten similar studies, which were based on fungal culturing and/or years ago (Östbrant et al. 2009), O. ulmi should only occa- direct Sanger sequencing (Davydenko et al. 2014; Persson sionally occur or even be completely replaced, which corrob- et al. 2009), showing that our detection method allowed in- orates the results of the present study. Although most of the depth analysis of fungal communities associated with beetles were trapped in 2012, in 2012 a proportion of the S. multistriatus. However, there is increasing evidence that beetles infested with O. novo-ulmi and the abundance of vec- fungal culturing and sequencing methods are both needed, tored inoculum (estimated as a proportion of O. novo-ulmi andshouldberegardedascomplementary,toobtainacom- sequences) was relatively low and further decreased in 2013 plete picture of fungal communities associated with beetles (Table 2). In 2014, however, both of these estimates have (Giordano et al. 2012; Lim et al. 2005). Furthermore, our data sharply increased even when compared to levels observed in corroborates previous observations that fungi from the phy- 2012 (Table 2), showing that association between lum Ascomycota are predominantly associated with the bark S. multistriatus and O. novo-ulmi is very dynamic. It appears beetles (Davydenko et al. 2014; Persson et al. 2009). Among that abundance of the beetles infested with O. novo-ulmi is different bark beetle species, probably the best described are largely dependent on the accuracy of the control measures interactions between the European spruce bark beetle ( implemented. Consequently, until 2014 all DED-infested elms typographus L.) and ophiostomatoid fungi, which, depending were harvested and destroyed each year, which has likely on the fungal species, may have variable effects including resulted in steady decline of the beetles infested with O. antagonism, commensalism or mutualism (Vega and novo-ulmi. In 2014, however, due to administrative issues Blackwell 2005). In the present study, Cladosporium sp. 952 out of 3419 DED-diseased elms were left standing during 2170_0 dominated the fungal community vectored by the entire flying season of S. multistriatus (Inga-Lena S. multistriatus (Table 1). The genus Cladosporium Östbrant, Swedish Forest Agency), which likely resulted in (Ascomycota) includes over 500 different fungal taxa of com- the significant increase of beetles vectoring O. novo-ulmi mon moulds, saprotrophs, and plant and fungal pathogens that (Table 2). The latter shows that the population of S. are all characterised by dark-pigmented mycelium (Domsch multistriatus infested with O. novo-ulmi may recover in a et al. 2007). Among other fungi, yeasts from the genera single flying season. This is not surprising, as mutualistic Dioszegia, Cryptococcus, Udeniomyces, Candida, Mycol Progress (2016) 15:55 Page 7 of 8 55

Mrakiella,andSporobolomyces were very common (Table 1). Conclusions Similarly, a number of different yeasts were reported previ- ously, which let to suggestion on a very long association be- This study demonstrated that S. multistriatus exhibits highest tween some yeasts and bark beetles (Giordano et al. 2012; flying intensity during July each year, and that the proportion Persson et al. 2009). Fungi from the genus Geosmithia were of the beetles vectoring O. novo-ulmi has increased in recent also detected (Table 1, Supplementary Table 1). While years. Geosmithia is known to develop stable symbioses with differ- ent bark beetle species (Kolarik and Jankowiak 2013; Kolarik Acknowledgments We thank Diem Nguyen at the Dept. of Forest et al. 2008), the results of the present study expand knowledge Mycology and Plant Pathology, SLU, for language revision and Karin Wågström at the Swedish Forest Agency for help with the field work. The on the host, ecology, and distribution in Europe. The detected financial support is gratefully acknowledged from Foundation Oscar and fungi also included a number of entomopathogens, among Lili Lamms Minne, Carl Tryggers Foundation, the Swedish Research which Beauveria bassiana (Bals.-Criv.) Vuill. and Council Formas, and the EU Life+ Nature Elmias (LIFE12 NAT/SE/ Paecilomyces fumosoroseus (Wize) A.H.S.Br. & G.Sm. were 001139) project. shown to infect larvae of S. multistriatus more efficiently than Compliance with ethical standards other fungi tested (Houle et al. 1987). Interestingly, recently described ubiquitous soil fungi of the genus Conflict of interest The authors declare that they have no conflicts of Archaeorhizomyces (Menkis et al. 2014; Rosling et al. 2011) interest. were also detected (Supplementary Table 1). Although repro- duction structures and dispersal strategy of these fungi are largely unknown, the current observation in beetles provides new insights into their biology and ecology. Taken together, References the study demonstrated that S. multistriatus vectors different functional groups of fungi and that some of these may have a Bartels JM, Lanier GN (1974) Emergence and mating in Scolytus direct negative effect on the itself and on colonised elm multistriatus (Coleoptera, Scolytidae). Ann Entomol Soc Am 67: 365–370 trees. Bellows TS, Meisenbacher C, Reardon RC (1998) European elm bark The flying intensity of S. multistriatus in Gotland var- beetle biological control. Paper presented at the Biological control of ied among different years (Fig. 1). Bartels and Lanier forest pests of the western United States: a review and – – (1974)showedthatS. multistriatus did not emerge from recommendations. USDA Forest Service, FHTET 96 21, The University of Georgia, and Southern Forest Insect Work the trees when the temperature was at or below 20 °C. In Conference. 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