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ORIGINAL ARTICLE
Scolytus multistriatus associated with Dutch elm diseaseon the island of Gotland: phenology and communitiesof 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 smallerEuropean elm bark beetle, is a vector for Dutch elm disease(DED) that in the year 2005 invaded the island of Gotland(Sweden). The island possesses the largest population of elm(mainly Ulmus minor Mill.) in northern Europe. The aim ofthis study was to monitor flying periods of S. multistriatusduring three consecutive years and by using high-throughputsequencing to assess communities of vectored fungi.Sampling of the beetles was carried out at two different sitesin Gotland in 2012, 2013, and 2014. In total, 50 pheromonetraps were placed at each site and checked weekly duringJune-August each year. From all sites and years, 177 beetleswere trapped. Among these, 6.2 % were trapped in June,76.8 % in July, and 16.9 % in August (difference significantat p<0.007). Sequencing of ITS rDNA from the beetles
revealed the presence of 1589 fungal taxa, among which vir-ulent DED pathogen Ophiostoma novo-ulmi Brasier was thesecond most common species (9.0 % of all fungal sequences).O. ulmi Buisman, the less virulent DED pathogen, was alsodetected but only in a single beetle, which was sampled in2012 (0.04 % of sequences). There were 13.0 % of the beetlesinfestedwithO. novo-ulmi in 2012, 4.0% in 2013, and 27.7%in 2014.O. novo-ulmi comprised 0.8 % of fungal sequences in2012, 0.002 % in 2013, and 8.2 % in 2014. The study showedthat the proportion of S. multistriatus vectoring O. novo-ulmihas increased in recent years.
Keywords Ophiostoma . Invasive pathogens . Bark beetles .
Disease management . Fungal community .Ulmus
Introduction
Scolytus multistriatus (Scolytinae: Scolytini), the smallerEuropean elm bark beetle, is native to Europe, the MiddleEast, and northern Africa (Bellows et al. 1998), but was intro-duced with elm wood to other areas including North America,New Zealand, and Australia (Brockerhoff et al. 2003; Leeet al. 2009; Parbery and Rumba 1991) and generally occurswithin the areal of host trees (mainly Ulmus spp.). Adults (1.9to 3.1 mm in length) bore through the bark of weakened and/or stressed elms, breed under the bark and produce egg gal-leries in the vascular tissues. Females lay eggs along the egggallery, and larvae tunnel across the vascular tissues awayfrom the egg gallery (Wood 1982). S. multistriatus overwin-ters as larvae under the bark and new adults emerge in thespring or early summer after elm leaves have fully developed.
S. multistriatus is one of the most effective vectors forDutch elm disease (DED) (Santini and Faccoli 2015;Webber 1990) caused by fungi from the genus
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.
* Audrius [email protected]
1 Department of Forest Mycology and Plant Pathology, UppsalaBioCenter, Swedish University of Agricultural Sciences,P.O. Box 7026, SE-75007 Uppsala, Sweden
2 Swedish Forest Agency Gotland District, P.O. Box 1417, SE-62125 Visby, Sweden
3 Ukrainian Research Institute of Forestry and Forest Melioration,Pushkinska str. 86, 61024 Kharkiv, Ukraine
4 Institute of Forest Biology and Silviculture, Aleksandras StulginskisUniversity, Studentu str. 11, LT-53361 Akademija KaunasDistrict, Lithuania
5 Institute of Life Sciences and Technology, Daugavpils University,Vienibas str. 13, LV-5401 Daugavpils, Latvia
Mycol Progress (2016) 15:55 DOI 10.1007/s11557-016-1199-3
Ophiostoma (Ascomycota) (Kirisits 2013), which duringthe last 100 years have destroyed billions of elm treesworldwide (Phillips and Burdekin 1982). DED is a le-thal vascular wilt disease comprised of three distinctfungal pathogens, less virulent O. ulmi, and highly vir-ulent O. novo-ulmi and O. himal-ulmi Brasier &Mehrotra, a species endemic to the western Himalayas(Brasier and Mehrotra 1995). Conidia, which are theinfection source of DED pathogens, are transmitted onthe body surface of the beetles into the tree, and a newgeneration of beetles is only infested if the DED fungusis present in the galleries. Conidia are produced insticky masses that facilitate their attachment and trans-portation by beetles as they emerge from the trees(Ploetz et al. 2013). When DED-infested beetles emergeand fly to feed in the twig crotches of healthy elms,they form grooves in the wood through which the fun-gus enters the twig and spreads within the branch by ayeast-like budding process causing leaves to wilt anddie. This is due to the blockage of the conducting sys-tem subsequent to the formation of tyloses and gels inthe xylem vessels and the production of toxins, andeventually causing the death of a tree (Phillips andBurdekin 1982).
The island of Gotland (Sweden) possesses the largestand highly valuable wild population of elms (more thanone million trees that are mainly Ulmus minor) in north-ern Europe, which until recently was not affected byDED (Östbrant et al. 2009). In 2005, however, DEDwas observed in Gotland and in the following years, itrapidly spread in all directions, causing extensive mortal-ity of elm trees (Menkis et al. 2016). Among the elmbark beetles known from Sweden, which includeS. triarmatus Eggers., S. laevis Chapuis, S. rugulosusO.F. Muller, S. pygmaeus F. and S. multistriatus, onlythe latter species occurs in Gotland (Schlyter et al.1987) and is therefore thought to be responsible for thecurrent spread of DED. Interestingly, S. multistriatus hasbeen known in Gotland for decades, which suggests thatuntil 2005 its population on the island was free ofO. novo-ulmi. Although the precise route of disease ar-rival is not known, it was probably brought to the islandwith DED-infested elm wood that would resemble pat-terns of human-mediated spread of DED (Brasier et al.2004) . Howeve r, l i t t l e i s known abou t whenS. multistriatus is most active in Gotland and especiallywhat proportion of those beetles vector conidia of DEDfungi. Moreover, little is known about other fungal spe-cies vectored by S. multistriatus.
The aim of the present study was to monitor the seasonalflying intensity of S. multistriatus and to assess communitiesof vectored fungi at different time periods, particularly focus-ing on DED pathogens.
Materials and methods
Study sites and sampling
Mean temperatures for the study area were retrieved fromhttp://luftwebb.smhi.se. The study sites were at Vallstena(N57°36′, E18°41′) and Hogrän (N57°31′, E18°18′) on theBaltic Sea island of Gotland. The distance between the siteswas ca. 26 km. The site at Vallstena was a mixed forestcomposed of Pinus sylvestris L., Picea abies (L.) Karst.,Betula pendula Roth, Ulmus spp. and Alnus spp. The site atHogrän was a mixture of open fields and forest land withsimilar tree species in admixture as at the Vallstena site.Both sites were characteristic to Gotland in terms oflandscape and trees species composition, and were in theareas characterised by a high incidence of DED. At eachsite, 50 transparent delta traps with a sticky insert(Pherobank, Wijk bij Duurstede, The Netherlands) on thebo t t om and a P188 phe romone lu r e (Syne rgySemiochemcials Corp., Burnaby, Canada) were placed every50m along a transect, which was 2.5 km long. Lures consistedof two semi-permeable plastic pouches containing a mixtureof cubeb oil, 1-hexanol, multistriatin and 4-methyl-3-heptanol. The lure used attracts Scolytus spp. beetles. In thistype of trap, beetles firmly stick to the sticky insert, whichprevents physical contact among different individuals, andprevents cross-contamination with e.g. fungal spores. To setthe traps, two sticks 1.5 m in length were hammered to theground and a trap was fastened to them about 1.2 m above theground. Each trap was labelled and a global positioning sys-tem (GPS) coordinates were recorded in order to set the trapsat the same position each year. Sampling was carried out fromthe beginning of June until the end of August in the years2012, 2013, and 2014. During the sampling period, traps werevisited once a week and sticky inserts with trapped insectswere collected and replacedwith new inserts. Collected insertswere transported the same day to the laboratory and examinedunder Carl Zeiss Stemi 2000-C dissection microscope(Oberkochen, Germany). When the beetles of S. multistriatuswere detected, theywere individually placed into 2-mL screw-cap centrifugation tubes and stored at −20 °C until furtherDNA processing.
DNA isolation, amplification and sequencing
Total DNA was isolated separately from each beetle. Nosurface sterilisation was carried out. Prior to isolation ofDNA, the beetles were freeze-dried at −60 °C for 2 days,and together with glass beads were homogenized for2 min at 5000 rpm using a Fast prep shaker (Precellys24, Bertin Technologies, Rockville, MD). Then, 800 μLof CTAB extraction buffer (3 % cetyltrimethylammoniumbromide, 2 mM EDTA, 150 mM Tris–HCl, 2.6 M NaCl,
55 Page 2 of 8 Mycol Progress (2016) 15:55
pH 8) was added to each tube, followed by incubation at65 °C for 1 h. After centrifugation, the supernatant wastransferred to new 1.5-mL centrifugation tubes and thenmixed with 1 volume of chloroform by gentle vortexing.After centrifugation for 8 min at 10000 rpm, the super-natant was precipitated with 2 volumes of coldisopropanol, washed with 70 % ethanol and dissolvedin 50 μL TE buffer. Additionally, isolated DNA was pu-rified using JETquick DNA Clean-Up System (Genomed,Löhne, Germany). In each sample, concentration of ge-nomic DNA was determined using a ND-1000 spectro-photometer (NanoDrop Technologies, Wilmington, DE).Diluted (1–10 ng/μL) genomic DNA samples were am-plified separately using the primer pair fITS9 (5′-GAACGCAGCRAAIIGYGA-3′) (Ihrmark et al. 2012)and ITS4 (5′-xxxxxxxxTCCTCCGCTTATTGATATGC-3′) (White et al. 1990) containing 8-bp sample identifi-cation barcodes denoted by x. Using this primer pair,amplified PCR products were estimated to be between280–420 bp in size and to include a large part of the5.8S rRNA gene sequence, complete sequence of thenoncoding ITS2 rRNA region, and partial sequence ofthe 28S rRNA gene. The PCR reactions, 50 μL in vol-ume for each sample, were performed using an AppliedBiosystems 2720 Thermal Cycler (Applied Biosystems,Carlsbad, CA) and DreamTaq Green DNA polymerase(Thermo Fisher Scientific, Waltham, MA). The PCR cy-cle parameters consisted of an initial denaturation at95 °C for 2 min, 27 cycles of denaturation at 95 °Cfor 30 s, annealing at 55 °C for 30 s and extension at72 °C for 45 s, followed by a final extension step at72 °C for 7 min. The PCR products were analysed on1 % agarose gels (Agarose D1, Conda, Madrid, Spain)under UV using GelDocTM 2000 gel documentation sys-tem (Bio-Rad laboratories, Berkeley, CA). To purifyamplicons, they were precipitated in a mixture of 1/10volume 3 M NaAc and 2 volumes −20 °C pure ethanol,vortexed for 10 min, incubated for 20 min at −70 °C andcentrifuged for 5 min at 13,000 rpm. Supernatant wasdiscarded and dried pellets were dissolved in 30 μLMilli-Q water. The concentration of purified PCR prod-ucts was determined using Quant-iT™ dsDNA HS AssayKit (Life Technologies, Carlsbad, CA, USA), and anequimolar mix of all PCR products was used for IonTorrent sequencing. Construction of the sequencing li-brary and sequencing using a 316 chip was carried outby NGI SciLifeLab (Uppsala, Sweden).
Bioinformatics
The sequences generated were subjected to quality control andclustering in the SCATA NGS sequencing pipeline (http://scata.mykopat.slu.se). Quality filtering of the sequences
included the removal of short sequences (<200 bp),sequences with low read quality, primer dimers, andhomopolymers. Sequences that were missing a tag or primerwere excluded. The primer and sample tags were thenremoved from the sequence, but information on thesequence association with the sample was stored as meta-data. The sequences were then clustered into different taxausing single-linkage clustering based on 98.5 % similarity.The most common genotype for clusters was used to representeach taxon. For clusters containing two sequences, a consen-sus sequence was produced. The fungal taxa were taxonomi-cally identified using GenBank (NCBI) database and theBlastn algorithm. The criteria used for identification were:sequence coverage > 80 %; similarity to taxon level 98–100 %, similarity to genus level 94–97 %. Sequences notmatching these criteria were considered unidentified and weregiven unique names, as shown in Supplementary Table 1.
Statistical analyses
As both qualitative and quantitative data of high-throughputsequencing was shown to be consistent and highly reproduc-ible (Porazinska et al. 2010), the number of read counts wasused to estimate relative abundance of fungal taxa in the sam-ples. The abundance of S. multistriatus and of DED fungi indifferent sampling years was compared by non-parametricchi-squared tests calculated from the actual number of obser-vations (Mead and Curnow 1983). As the datasets were sub-jected to multiple comparisons, confidence limits for p-valuesof the chi-squared tests were reduced a corresponding numberof times, as required by the Bonferroni correction (Sokal andRohlf 1995). The rarefaction analysis was performed usingAnalytical Rarefaction v.1.3 available at http://www.uga.edu/strata/software/index.html. The rarefaction analysis wascarried out to reveal the relationship between the cumulativenumber of taxa found and the sequencing intensity (Colwelland Coddington 1994).
Results
Dur i ng t h r ee s amp l i ng yea r s , 177 bee t l e s o fS. multistriatus were trapped, or on average, 0.59 beetleper year per trap. Information on the number of beetlestrapped (data pooled from both sites) during each year(2012, 2013, and 2014) and mean temperatures are shownin Fig. 1. There were 47.5 % of S. multistriatus trapped in2012, 21.5 % in 2013, and 31.0 % in 2014, and a chi-squared test showed that it was significantly higher in2012 than in 2013 or 2014 (p<0.003), but the number ofbeetles were not significantly different between 2013 and2014. In all years, 6.2 % of S. multistriatus were trappedin June, 76.8 % in July, and 16.9 % in August, and it was
Mycol Progress (2016) 15:55 Page 3 of 8 55
significantly higher in July than in June or August(p<0.0001), and significantly higher in August than inJune (p<0.007).
A total of 9,914,812 sequences were generated by IonTorrent sequencing from the 177 beetles. Of those, 9,474,995 (95.6 %) did not pass quality control and were thus ex-cluded. Clustering of the remaining 439,817 high-quality se-quences (272 bp on average) resulted in 1764 non-singletoncontigs and 2745 singleton contigs, which were excludedfrom the further analyses. Among the non-singletons, 1589contigs (90.1 %) represented fungi, 163 (9.2 %) plants, nine(0.5 %) animals, and three (0.2 %) protists. A plot of fungaltaxa vs. the number of sequences resulted in rarefaction curvesthat reached the asymptote (Fig. 2). There were between twoand 158 fungal taxa detected per individual beetle that com-prised 67.6 % Ascomycota, 31.0 % Basidiomycota, 0.7 %Mortierellomycotina, 0.4 % Chytridiomycota, 0.2 %Glomeromycota, and 0.1 % Mucoromycotina (representativeITS rDNA fungal sequences of all non-singletons are
available from GenBank under accession numbersKP890936 - KP892524). Identification at least to genus levelwas successful for 928 (58.4 %) out of 1589 fungal taxa. Themost common taxa were Cladosporium sp. 2170_0 (37.9 %),O. novo-ulmi (9.0 %), Aureobasidium pullulans (7.5 %),Dioszegia fristingensis (4.9 %), and Cryptococcus wieringae(3.9 %). Information on the 30 most common fungal taxarepresenting 90.1 % of all fungal sequences is shown in theTable 1. The remaining 1559 taxa were relatively rare andtheir relative abundances varied between 0.3 % and0.00005 % (Supplementary Table 1).
In the present study, both DED pathogens, i.e. less virulentO. ulmi and virulent O. novo-ulmi, were detected by ITSrDNA sequencing of S. multistriatus beetles (SupplementaryTable 1). However, O. ulmi was detected in a single (0.6 %)beetle while O. novo-ulmiwas detected in 79 (44.6 %) beetles(difference significantly at p<0.0001).O. ulmi was detected atHogrän in 2012, while O. novo-ulmi was detected on bothsites and during the entire sampling period (Table 2). The
Fig. 1 Bars show relativeabundance of Scolytusmultistriatus beetles trapped/collected (data pooled from bothsites) and lines show meantemperatures (retrieved fromhttp://luftwebb.smhi.se) duringJune-August of 2012, 2013, and2014 on the island of Gotland
Fig. 2 Rarefaction curveshowing the relationship betweenthe cumulative number of fungaltaxa and the number of ITS rDNAfungal sequences obtained from177 beetles of S. multistriatussampled on the island of Gotland
55 Page 4 of 8 Mycol Progress (2016) 15:55
proportion of S. multistriatus infested with O. novo-ulmi didnot differ significantly between two sampling sites.Differences among years (data pooled from both sites) weresignificant, with 13.0 % of beetles infested in 2012, 4.0 % in
2013, and 27.7 % in 2014 (p<0.002) (Table 2). Relative abun-dance of vectored O. novo-ulmi (estimated as a proportion ofall fungal sequences) also differed significantly among theyears, being 0.8 % in 2012, 0.002 % in 2013, and 8.2 % in
Table 1 List of the 30 mostcommon fungal taxa found in 177beetles of S. multistriatus sampledon the island of Gotland
Taxon Referencesequence
Sequencelength
Similarity,(%)*
No. ofsequences
Frequency ofoccurrence, (%)
Ascomycota
Cladosporium sp. 2170_0 HG530747 262 262/262(100)
162589 37.9
Ophiostoma novo-ulmi EF638891 329 327/327(100)
38632 9.0
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(100)
6873 1.6
Fusarium tricinctum KM249082 277 277/277(100)
6190 1.4
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
Sphaerosporellasp. 2170_23
JQ711781 226 216/226 (96) 2314 0.5
Geosmithia flava KJ513214 287 286/287 (99) 1923 0.4
Botryotinia fuckeliana KJ476441 260 259/260 (99) 1388 0.3
Rachicladosporiumeucalypti
KP004448 232 227/232 (98) 1334 0.3
Periconia byssoides KC954160 268 267/268 (99) 1264 0.3
Pyrenophora tritici-repentis
KM011994 268 267/268 (99) 1201 0.3
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(100)
10385 2.4
Udeniomyces pannonicus AB072229 345 341/342 (99) 8829 2.1
Dioszegia crocea GQ911539 239 239/239(100)
8213 1.9
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(100)
3518 0.8
Sporobolomyces roseus KM376382 319 319/319(100)
2962 0.7
Dioszegia butyracea EU266508 236 236/236(100)
1910 0.4
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 sequenceat NCBI database, and the percentage of sequence similarity in the parenthesis
Mycol Progress (2016) 15:55 Page 5 of 8 55
2014 (p<0.0001) (Table 2). Although several otherophiostomatoid fungi have also been detected, these wereidentified to the genus level (Supplementary Table 1).
Discussion
The results showed that both O. ulmi and O. novo-ulmi werepresent in Gotland. However, the occasional occurrence ofO. ulmi suggests that, as elsewhere, it is being replaced byO. novo-ulmi (Brasier et al. 2004). Brasier et al. (2004) report-ed thatO. novo-ulmi replacedO. ulmi at a relative incidence ofabout 10 % per year at each location. Taken into considerationthat O. novo-ulmi was probably introduced to Gotland tenyears ago (Östbrant et al. 2009), O. ulmi should only occa-sionally occur or even be completely replaced, which corrob-orates the results of the present study. Although most of thebeetles were trapped in 2012, in 2012 a proportion of thebeetles infested with O. novo-ulmi and the abundance of vec-tored inoculum (estimated as a proportion of O. novo-ulmisequences) was relatively low and further decreased in 2013(Table 2). In 2014, however, both of these estimates havesharply increased even when compared to levels observed in2012 (Table 2), showing that association betweenS. multistriatus and O. novo-ulmi is very dynamic. It appearsthat abundance of the beetles infested with O. novo-ulmi islargely dependent on the accuracy of the control measuresimplemented. Consequently, until 2014 all DED-infested elmswere harvested and destroyed each year, which has likelyresulted in steady decline of the beetles infested with O.novo-ulmi. In 2014, however, due to administrative issues952 out of 3419 DED-diseased elms were left standing duringthe entire flying season of S. multistriatus (Inga-LenaÖstbrant, Swedish Forest Agency), which likely resulted inthe significant increase of beetles vectoring O. novo-ulmi(Table 2). The latter shows that the population of S.multistriatus infested with O. novo-ulmi may recover in asingle flying season. This is not surprising, as mutualistic
association between S. multistriatus and DED fungi is wellestablished (Santini and Faccoli 2015). Nevertheless, theforthcoming availability of even more powerful molecularand genomic tools can be expected to provide new insightsinto the DED pathosystem and open possibilities for develop-ment of new control strategies (Bernier et al. 2014).
In the present study, despite the use of delta traps that re-sulted in a relatively small number of trapped beetles ofS. multistriatus compared to results using other type of traps(e.g. window traps) (Menkis et al. 2016), delta traps preventedcross-contamination among individual beetles (54.8 % of allbeetles were not infected by DED), thereby allowing abun-dance monitoring of the beetles vectoring DED each year.However, in order to more precisely monitor the flying inten-sity of S. multistriatus, window traps or Lindgren funnel trap(Johnson et al. 2008), instead of delta traps, should probablybe used to obtain higher yields of beetles. Although it is ac-knowledged that S. multistriatusmay vectorOphiostoma spp.(Ploetz et al. 2013), information on other fungal taxa vectoredis scarce. In the present study, the use of high-throughputsequencing showed that S. multistriatus vectors a highly di-verse fungal community (Supplementary Table 1).Furthermore, rarefaction analysis showed that a great majorityof fungal taxa was detected (Fig. 2) thereby highlighting theefficacy of the sequencing method even though only a rela-tively small proportion of all sequences was of high qualityand could be used in analyses. The detected richness of fungaltaxa was one or two orders of magnitude as compared tosimilar studies, which were based on fungal culturing and/ordirect Sanger sequencing (Davydenko et al. 2014; Perssonet al. 2009), showing that our detection method allowed in-depth analysis of fungal communities associated withS. multistriatus. However, there is increasing evidence thatfungal culturing and sequencing methods are both needed,and should be regarded as complementary, to obtain a com-plete picture of fungal communities associated with beetles(Giordano et al. 2012; Lim et al. 2005). Furthermore, our datacorroborates previous observations that fungi from the phy-lum Ascomycota are predominantly associated with the barkbeetles (Davydenko et al. 2014; Persson et al. 2009). Amongdifferent bark beetle species, probably the best described areinteractions between the European spruce bark beetle (Ipstypographus L.) and ophiostomatoid fungi, which, dependingon the fungal species, may have variable effects includingantagonism, commensalism or mutualism (Vega andBlackwell 2005). In the present study, Cladosporium sp.2170_0 dominated the fungal community vectored byS. multistriatus (Table 1). The genus Cladosporium(Ascomycota) includes over 500 different fungal taxa of com-mon moulds, saprotrophs, and plant and fungal pathogens thatare all characterised by dark-pigmented mycelium (Domschet al. 2007). Among other fungi, yeasts from the generaDioszegia, Cryptococcus , Udeniomyces, Candida ,
Table 2 Relative abundance of Scolytus multistriatus beetles infestedwith Ophiostoma novo-ulmi (shown as a proportion of all beetles), andrelative abundance of vectoredO. novo-ulmi (shown as a proportion of allfungal sequences) in different study sites and sampling years
Sampling year Beetles-infested Vectored O. novo-ulmi
Hogrän Vallstena All Hogrän Vallstena All
2012 11.7 a 17.5 ab 13.0 1.2 a 0.0 a 0.8
2013 3.6 b 5.0 a 4.0 0.0 b 0.0 a 0.0
2014 27.0 c 30.0 b 27.7 11.2 c 2.4 b 8.2
All 42.3 52.5 44.6 12.4 2.4 9.0
Within columns of respective study site, values followed by the sameletter are not significantly different
55 Page 6 of 8 Mycol Progress (2016) 15:55
Mrakiella, and Sporobolomyceswere very common (Table 1).Similarly, a number of different yeasts were reported previ-ously, which let to suggestion on a very long association be-tween some yeasts and bark beetles (Giordano et al. 2012;Persson et al. 2009). Fungi from the genus Geosmithia werealso detected (Table 1, Supplementary Table 1). WhileGeosmithia is known to develop stable symbioses with differ-ent bark beetle species (Kolarik and Jankowiak 2013; Kolariket al. 2008), the results of the present study expand knowledgeon the host, ecology, and distribution in Europe. The detectedfungi also included a number of entomopathogens, amongwhich Beauveria bassiana (Bals.-Criv.) Vuill. andPaecilomyces fumosoroseus (Wize) A.H.S.Br. & G.Sm. wereshown to infect larvae of S. multistriatusmore efficiently thanother fungi tested (Houle et al. 1987). Interestingly, recentlyde s c r i b ed ub i qu i t ou s s o i l f ung i o f t h e g enu sArchaeorhizomyces (Menkis et al. 2014; Rosling et al. 2011)were also detected (Supplementary Table 1). Although repro-duction structures and dispersal strategy of these fungi arelargely unknown, the current observation in beetles providesnew insights into their biology and ecology. Taken together,the study demonstrated that S. multistriatus vectors differentfunctional groups of fungi and that some of these may have adirect negative effect on the insect itself and on colonised elmtrees.
The flying intensity of S. multistriatus in Gotland var-ied among different years (Fig. 1). Bartels and Lanier(1974) showed that S. multistriatus did not emerge fromthe trees when the temperature was at or below 20 °C. InGotland, a majority of the beetles were trapped each yearat or above 16 °C (Fig. 1), suggesting a certain tempera-ture specificity of S. multistriatus in Gotland but at thesame time corroborating a finding by Bartels and Lanier(1974) that the activity of S. multistriatus is temperaturedependent. Besides, attractiveness of the bark beetles tothe traps is not increased by a combination of differentbark beetle attractants (Wang et al. 2014) or different sup-plementary chemicals (Edde et al. 2011). Taken together,this may suggest that flying intensity of the beetles inGotland is mainly influenced by the environmental condi-tions of each year. Within a year, however, flying intensi-ty of S. multistriatus was more or less consistent, beinghighest in July, then in August, and lowest in June(Fig. 1). This information is of key practical importance,demonstrating that in case harvesting and destruction ofDED-diseased elms is not completed before the beginningof the flying season of S. multistriatus, it should be con-tinued and completed before July, which will result inonly minor release of the beetles vectoring DED.Moreover, it demonstrates that an extensive use of thepheromone traps alone was shown to have little or noreduction effect on the population of S. multistriatus(Paine et al. 1984).
Conclusions
This study demonstrated that S. multistriatus exhibits highestflying intensity during July each year, and that the proportionof the beetles vectoring O. novo-ulmi has increased in recentyears.
Acknowledgments We thank Diem Nguyen at the Dept. of ForestMycology and Plant Pathology, SLU, for language revision and KarinWågström at the Swedish Forest Agency for help with the field work. Thefinancial support is gratefully acknowledged from Foundation Oscar andLili Lamms Minne, Carl Tryggers Foundation, the Swedish ResearchCouncil Formas, and the EU Life+ Nature Elmias (LIFE12 NAT/SE/001139) project.
Compliance with ethical standards
Conflict of interest The authors declare that they have no conflicts ofinterest.
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