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ArticleTitle Cadophora margaritata sp. nov., and other fungi associated with the longhorn Anoplophora glabripennis and carcharias in Finland Article Sub-Title Article CopyRight Springer International Publishing AG, part of Springer Nature (This will be the copyright line in the final PDF) Journal Name Antonie van Leeuwenhoek Corresponding Author Family Name Linnakoski Particle Given Name Riikka Suffix Division Department of Forest Sciences Organization University of Helsinki Address Helsinki, Finland Division Organization Natural Resources Institute Finland (Luke) Address Helsinki, Finland Division Department of Microbiology and Plant Pathology, Forestry and Agricultural Biotechnology Institute (FABI) Organization University of Pretoria Address Pretoria, South Africa Phone +358 29 532 2289 Fax Email [email protected] URL ORCID http://orcid.org/0000-0002-3294-8088

Author Family Name Kasanen Particle Given Name Risto Suffix Division Department of Forest Sciences Organization University of Helsinki Address Helsinki, Finland Phone Fax Email URL ORCID http://orcid.org/0000-0002-9948-2200 Author Family Name Lasarov Particle Given Name Ilmeini Suffix Division School of Forest Sciences Organization University of Eastern Finland Address Joensuu, Finland Phone Fax Email URL ORCID Author Family Name Marttinen Particle Given Name Tiia Suffix Division Department of Forest Sciences Organization University of Helsinki Address Helsinki, Finland Phone Fax Email URL ORCID Author Family Name Oghenekaro Particle Given Name Abbot O. Suffix Division Department of Forest Sciences Organization University of Helsinki Address Helsinki, Finland Division Department of Plant Biology and Biotechnology Organization University of Benin Address Benin City, Nigeria Phone Fax Email URL ORCID Author Family Name Sun Particle Given Name Hui Suffix Division Department of Forest Sciences Organization University of Helsinki Address Helsinki, Finland Phone Fax Email URL ORCID Author Family Name Asiegbu Particle Given Name Fred O. Suffix Division Department of Forest Sciences Organization University of Helsinki Address Helsinki, Finland Phone Fax Email URL ORCID http://orcid.org/0000-0003-0223-7194 Author Family Name Wingfield Particle Given Name Michael J. Suffix Division Department of Microbiology and Plant Pathology, Forestry and Agricultural Biotechnology Institute (FABI) Organization University of Pretoria Address Pretoria, South Africa Phone Fax Email URL ORCID http://orcid.org/0000-0001-9346-2009 Author Family Name Hantula Particle Given Name Jarkko Suffix Division Organization Natural Resources Institute Finland (Luke) Address Helsinki, Finland Phone Fax Email URL ORCID http://orcid.org/0000-0002-1016-0636 Author Family Name Heliövaara Particle Given Name Kari Suffix Division Department of Forest Sciences Organization University of Helsinki Address Helsinki, Finland Phone Fax Email URL ORCID http://orcid.org/0000-0003-0657-8885

Received 25 January 2018 Schedule Revised Accepted 7 June 2018

Abstract Symbiosis with microbes is crucial for survival and development of wood-inhabiting longhorn beetles (Coleoptera: Cerambycidae). Thus, knowledge of the endemic fungal associates of would facilitate risk assessment in cases where a new invasive pest occupies the same ecological niche. However, the diversity of fungi associated with insects remains poorly understood. The aim of this study was to investigate fungi associated with the native large poplar longhorn () and the recently introduced Asian (Anoplophora glabripennis) infesting hardwood trees in Finland. We studied the cultivable fungal associates obtained from tremula colonised by S. carcharias, and Betula pendula and Salix caprea infested by A. glabripennis, and compared these to the samples collected from intact wood material. This study detected a number of plant pathogenic and saprotrophic fungi, and species with known potential for enzymatic degradation of wood components. Phylogenetic analyses of the most commonly encountered fungi isolated from the longhorn beetles revealed an association with fungi residing in the Cadophora–Mollisia species complex. A commonly encountered was Cadophora spadicis, a recently described fungus associated with wood-decay. In addition, a novel species of Cadophora, for which the name Cadophora margaritata sp. nov. is provided, was isolated from the colonised wood. Keywords (separated by '-') 1 New taxon - Alien invasive species - Cadophora sp. - Introduced species - –fungus symbiosis - Longhorn beetles - Vectored pathogen Footnote Information Electronic supplementary material The online version of this article (https://doi.org/10.1007/ s10482-018-1112-y) contains supplementary material, which is available to authorized users. Antonie van Leeuwenhoek https://doi.org/10.1007/s10482-018-1112-y

1 ORIGINAL PAPER

2 Cadophora margaritata sp. nov., and other fungi associated 3 with the longhorn beetles Anoplophora glabripennis 4 and Saperda carcharias in Finland

5 Riikka Linnakoski . Risto Kasanen . Ilmeini Lasarov . Tiia Marttinen . 6 Abbot O. Oghenekaro . Hui Sun . Fred O. Asiegbu . Michael J. Wingfield . 7 Jarkko Hantula . Kari Helio¨vaara Author Proof

8 Received: 25 January 2018 / Accepted: 7 June 2018 9 Ó Springer International Publishing AG, part of Springer Nature 2018

10AQ1 Abstract Symbiosis with microbes is crucial for obtained fromPROOFPROOFPopulus tremula colonised by S. 23 11 survival and development of wood-inhabiting long- carcharias, and Betula pendula and Salix caprea 24 12 horn beetles (Coleoptera: Cerambycidae). Thus, infested by A. glabripennis, and compared these to the 25 13 knowledge of the endemic fungal associates of insects samples collected from intact wood material. This 26 14 would facilitate risk assessment in cases where a new study detected a number of plant pathogenic and 27 15 invasive pest occupies the same ecological niche. saprotrophic fungi, and species with known potential 28 16 However, the diversity of fungi associated with insects for enzymatic degradation of wood components. 29 17 remains poorly understood. The aim of this study was Phylogenetic analyses of the most commonly encoun- 30 18 to investigate fungi associated with the native large tered fungi isolated from the longhorn beetles revealed 31 19 poplar longhorn beetle (Saperda carcharias) and the an association with fungi residing in the Cadophora– 32 20 recently introduced Asian longhorn beetle (Ano- Mollisia species complex. A commonly encountered 33 21 plophora glabripennis) infesting hardwood trees in fungus was Cadophora spadicis, a recently described 34 22 Finland. We studied the cultivable fungal associates fungus associated with wood-decay. In addition, a 35 novel species of Cadophora, for which the name 36 Cadophora margaritata sp. nov. is provided, was 37 A1 Electronic supplementary material The online version of A2 this article (https://doi.org/10.1007/s10482-018-1112-y) con- isolated from the colonised wood. 38 A3 tains supplementary material, which is available to authorized users.

A4 R. Linnakoski (&) Á R. Kasanen Á T. Marttinen Á A16 I. Lasarov A5 A. O. Oghenekaro Á H. Sun Á F. O. Asiegbu Á A17 School of Forest Sciences, University of Eastern Finland, A6 K. Helio¨vaara A18 Joensuu, Finland A7 Department of Forest Sciences, University of Helsinki, A8 Helsinki, Finland A19 A. O. Oghenekaro A9 e-mail: riikka.linnakoski@luke.fi A20 Department of Plant Biology and Biotechnology, A21 University of Benin, Benin City, Nigeria A10 R. Linnakoski Á J. Hantula A11 Natural Resources InstituteUNCORRECTEDUNCORRECTED Finland (Luke), Helsinki, Finland

A12 R. Linnakoski Á M. J. Wingfield A13 Department of Microbiology and Plant Pathology, A14 Forestry and Agricultural Biotechnology Institute (FABI), A15 University of Pretoria, Pretoria, South Africa

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39 Keywords 1 New taxon Á Alien invasive species Á 2014). However, the diversity of fungi associated with 83 40 Cadophora sp. Á Introduced species Á Insect–fungus most Cerambycidae remains to be investigated. 84 41 symbiosis Á Longhorn beetles Á Vectored pathogen An example of a common but poorly known 85 Cerambycidae is the large poplar longhorn (Saperda 86 carcharias) (Fig. 1). The biology of the insect is not 87 fully understood, and there are no previous reports of 88 42 Introduction its symbiotic fungi. The beetle is considered as the 89 most harmful pest of hybrid poplars and aspen 90 43 A nutrient-poor wood tissue (xylem), lacking readily (Populus tremula) in Finland (Hallaksela 1999; 91 44 available carbohydrates and proteins, is a challenging Va¨lima¨ki and Helio¨vaara 2007). The adult beetle 92 45 niche to be exploited by other organisms. However, feeds on aspen leaves and lays eggs in basal parts of 93 46 cerambycid beetles (Coleoptera: Cerambycidae) are the stems during August–September (Helio¨vaara et al. 94 47 adapted to complete their long lifecycle in wood 2004). The eggs undergo overwintering for 95 48 tissues. In this niche, symbiosis with microbes 10–11 months and the emerging larvae remain in the 96 Author Proof 49 (including filamentous fungi and ) has been tunnels for 2–4 years. During this period, larvae can 97 50 considered to be crucial for their survival and devel- gnaw tunnels up to 1 m long in the xylem. These 98 51 opment (Kukor et al. 1988; Schloss et al. 2006; tunnels are often surrounded by a characteristic dark 99 52 Watanabe and Tokuda 2010; Scully et al. 2013). The brown colour expanding vertically 1–3 m, which 100 53 role of the associated microbes can be considered as prevent the logsPROOFPROOF from being used in the mechanical 101 54 directly nutritional, in which insect larvae cultivate wood industry. Part of the wood material gnawed by 102 55 and feed on the fungi (Francke-Grosmann 1967), or the larvae is transported through their alimentary 103 56 when either ingested fungal enzymes (Kukor and canals. Due to long larval period of development in the 104 57 Martin 1986; Kukor et al. 1988) or endosymbiotic gut tunnels, it is likely that the poplar longhorn has 105 58 microbes contribute to cellulose degradation (Uvarov interactions with a number of fungi during its life 106 59 1929; Kukor et al. 1988;Gru¨nwald et al. 2010; cycle. 107 60 Watanabe and Tokuda 2010; Scully et al. 2013). These The Asian longhorn beetle (Anoplophora 108 61 symbiotic fungi may also have other functional roles, glabripennis) is a serious forest pest native to China 109 62 such as nitrogen and vitamin acquisition (Gibson and (Lingafelter and Hoebeke 2002) (Fig. 2). In its native 110 63 Hunter 2009; Ayayee et al. 2014). range, the beetle is not considered as a pest in natural 111 64 The association of Cerambycidae and fungi was forests. However, the beetle became a common pest in 112 65 recognised relatively long ago (Uvarov 1929; Jurzitza China as a result of widespread planting of susceptible 113 66 1959; Riba 1977; Chararas and Pignal 1981; Nardon poplar (Populus sp.) hybrids (EPPO 2016). As an 114 67 and Grenier 1989). In general, the modified colour and invasive species, A. glabripennis is a highly destruc- 115 68 texture of the wood close to the larval tunnels indicate tive quarantine pest (EPPO 2016). The beetle is 116 69 that symbiotic organisms are commonly involved in capable of infesting a wide range of deciduous trees, 117 70 the diet of xylophagous insects. Many of the wood- including also healthy trees. The Asian longhorned 118 71 inhabiting fungi and insect symbioses have long co- beetle has spread globally mainly in ineffectively 119 72 evolutionary histories (Vega and Blackwell 2005), treated wooden packaging materials originating from 120 73 recently supported by the observations of fungi and China. It was accidentally introduced first into North 121 74 associated organisms successfully passing through the America (Haack et al. 1996; Hu et al. 2009), followed 122 75 insect alimentary tract (Drenkhan et al. 2013, 2016). by several introductions into European countries 123 76 The associations with Cerambycidae have arisen during the past two decades (Tomiczek et al. 2002; 124 77 independently in distantlyUNCORRECTED related fungal genera, He´rard et al. 2006, 2009; Haack et al. 2010), and 125 78 representing examplesUNCORRECTED of convergent evolution (Jones recently into Finland. 126 79 et al. 1999). Cerambycidae are known to harbour a rich In several instances, baseline information on fungal 127 80 assemblage of microbes in terms of taxonomic and associates of insects in their endemic environment is 128 81 functional diversity (Gru¨nwald et al. 2010; Watanabe lacking. This hinders risk assessment measures in 129 82 and Tokuda 2010; Scully et al. 2013; Ayayee et al. cases where a novel forest pest and/or pathogen is 130 introduced. Novel insect–fungal interactions represent 131 123

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PROOFPROOF

Fig. 1 a Aspen (P. tremula) stand in Urjala, Finland, b, c the large poplar longhorn beetle (S. carcharias) adult and larvae, and d brown decay surrounding the beetle gallery on P. tremula

132 an increased risk to forest health (Humble and Allen carcharias in its native host tree and environment, and 144 133 2006; Hulcr and Dunn 2011; Wingfield et al. 2016; with the introduced A. glabripennis infestation in 145 134 Stenlid and Oliva 2016). Examples of devastating tree Finland. Our hypotheses were that the longhorn 146 135 disease problems that have arisen from these associ- beetles are associated with specific fungi, which may 147 136 ations are increasing in number (Lu et al. 2011; improve nutritional quality of the larvae facilitating 148 137 Akbulut and Stamps 2012; Ploetz et al. 2013; Santini larval development inside the wood. Furthermore, we 149 138 and Faccoli 2014; WingfieldUNCORRECTEDUNCORRECTED et al. 2016). Recent hypothesized that differences in fungal species com- 150 139 introductions of plant pathogens (Wingfield et al. positions between intact wood and wood colonized by 151 140 2016) suggest that latent microbial pathogens com- beetles would reflect the specific symbiotic fungi 152 141 monly remain undetected in quarantine inspections. associated with each beetle. In addition, we hypoth- 153 142 The aim of the study was to provide baseline esize that the introduced insect pest would establish 154 143 information on fungal diversity associated with S. interactions with native fungal associates of 155

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Fig. 2 The Asian longhorned beetle (A. glabripennis) adult and larvae, and brown decayPROOFPROOF surrounding the beetle gallery on B. pendula 156 ecologically and taxonomically closely related insect tunnels with surface disinfected carpenter’s knife and 181 157 species. surgical knife. Control isolations were made from the 182 non-coloured, undamaged distal ends of the logs as 183 described above. All samples were surface disinfected 184 158 Materials and methods in sterile conditions with 70% ethanol (10 s) and 10% 185 sodium hypochlorite (NaClO) (5 s) and finally washed 186 159 Study areas and collection of samples four times with fresh sterile water. The samples were 187 placed onto both water agar (WA: 2% AMRESCOÒ 188 160 Populus tremula trees damaged by S. carcharias were agar, bacteriological grade) and malt extract agar 189 161 collected from a 15-year-old aspen stand in Urjala, (MEA: 2% BactoTM malt extract agar and 1% 190 Ò 162 southwestern Finland (66°000N, 23°290E) in May 2013 AMRESCO agar, bacteriological grade) in 90 mm 191 163 (Fig. 1). The aspen clonal stand 2120/01 was estab- Petri dishes. A subset of unsterilised wood samples 192 164 lished on 25 May 1998 as a part of the aspen clonal was incubated at 25 °C on sterilised filter paper placed 193 165 research programme of the Finnish Forest Research in clean Petri dishes (9 cm). 194 166 Institute. The average diameter at breast height of trees Anoplophora glabripennis-infested trees were 195 167 was 12 cm and their average height was ca. 9.0 ms. detected in Vantaa, Finland (60.3030N, 25.1190E) in 196 168 The stand consisted of both aspen and hybrid aspen, October–November 2015. During the inspections and 197 169 which were planted at intervals of 3 m in 3.0 9 3.0 m eradication measures, a total of 20 Betula pendula and 198 170 blocks. All the five sampled trees were of the same 13 Salix caprea trees showing signs of A. glabripennis 199 171 clone, collected from different blocks. The logs infestation were detected (Fig. 2). The trees were 200 172 including the entire beetle tunnels were cut with a removed by the Finnish Food Safety Authority 201 173 chain saw immediately closed in plastic bags and (Evira), and transported to a quarantine facility for 202 174 stored at ? 4 °C. careful examination and destruction. At the same time, 203 175 The logs (60–90 cmUNCORRECTEDUNCORRECTED of length) were sawn with fungal isolations were made from decayed wood 204 176 table mounted circular saw longitudinally into two surrounding the galleries of A. glabripennis on five B. 205 177 halves in a laboratory for fungal isolations. Two pendula and two S. caprea trees, using a similar 206 178 samples 5 mm 9 10 mm (separated by 5 cm) from sampling scheme as described earlier. Isolations were 207 179 three points (separated by at least 15 cm) were cut also made from the surfaces of five living larvae found 208 180 from the brown-coloured wood adjacent to larval in galleries on S. caprea. Small pieces of wood and the 209

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210 larvae were placed onto the surface of WA and MEA. 45 s of denaturation (95 °C), 45 s of annealing 52 °C, 257 211 The larvae were removed from the plates after and 1 min extension (72 °C). A final extension step 258 212 approximately 15 min. Control isolations were made was 10 min (72 °C). Products were visualised by 259 213 from the non-coloured, undamaged distal ends of the electrophoresis on a 1.5% agarose gel stained with 260 214 logs. ethidium bromide and purified using GenEluteTM PCR 261 215 The cultures obtained from S. carcharias and A. clean-up kit according to the manufacturer’s instruc- 262 216 glabripennis were grown for 1 week in the dark at tions (Sigma-Aldrich, Missouri, USA). 263 217 ? 25 °C and inspected daily. Emerging mycelia was Amplification of the ITS region and purification of 264 218 sub-cultured to obtain pure cultures. Purified fungal the PCR products of fungal samples from A. 265 219 isolates were grown on MEA and grouped based on glabripennis were performed following the same 266 220 their colony and morphological characteristics. Rep- protocols described previously (Linnakoski et al. 267 221 resentative isolates from each group were chosen for 2016). The studied gene region was amplified using 268 222 identification based on DNA sequences. Isolates were the primer pair ITS1-F (Gardes and Bruns 1993) and 269 223 deposited in the Culture Collection (CMW) of the ITS4 (White et al. 1990). The reaction mixture 270 Author Proof 224 Forestry and Agricultural Biotechnology Institute contained 0.15 ll of MyTaqTM DNA Polymerase 271 225 (FABI), University of Pretoria, Pretoria, South Africa, (5 U/ll) (Bioline, Massachusetts, USA), 5 llof 272 226 and the ex-type isolates also in the CBS-KNAW MyTaqTM Reaction Buffer (5 9) containing dNTPs, 273 227 Collections, Westerdijk Fungal Biodiversity Institute, MgCl2 and enhancers, 0.50 ll of each primer (10 mM 274 228 Utrecht, the Netherlands (Table 1). Herbarium spec- stock concentration)PROOFPROOF (Whitehead Scientific Ltd, Cape 275 229 imens were deposited in the Herbarium of the Town, South Africa), and 1 ll fungal genomic DNA. 276 230 University of Turku, Finland (TUR), Finland. Sterile Sabax water was added to adjust the reaction 277 volume to 25 ll. PCR amplifications were performed 278 231 DNA extraction, PCR and sequencing using the following conditions: denaturation at 95 °C 279 for 2 min, followed by 32 cycles of 30 s at 94 °C, 30 s 280 232 Pure cultures for DNA extractions were cultivated on at 55 °C and 1 min at 72 °C, and a final extension at 281 233 MEA plates. DNA was extracted from at least one 72 °C for 7 min. An aliquot of 5 ll of the each PCR 282 234 isolate of each morphological group using the methods product was stained with GelRedTM nucleic acid gel 283 235 described previously (Terhonen et al. 2011; Lin- stain (Biotium, Hayward, CA, USA), run on 2% 284 236 nakoski et al. 2016). Gene regions sequenced in the agarose gel at 60 V along with a 100 bp molecular 285 237 study were the internal transcribed spacer (ITS) marker (Fermentas O0 Gene RulerTM) and visualised 286 238 regions (ITS1 and ITS2) including the 5.8S gene (all with a Gel Doc EZ Imager (Bio-Rad Laboratories 287 239 samples), as well as the partial beta-tubulin gene (only Inc.). Amplified PCR products were cleaned using the 288 240 for the novel species). Exonuclease I—Shrimp Alkaline Phosphatase Clean 289 241 The DNA samples from mycelial cultures obtained Up (Exo-SAP) protocol. 290 242 from S. carcharias were amplified using a PCR The sequencing reactions contained 0.5 llof 291 243 protocol for DyNAzyme DNAÒ polymerase (Finn- BigDyeÒ Terminator v3.1 Ready Reaction mixture 292 244 zymes, Espoo, Finland). A PCR reaction (total volume (Perkin-Elmer Applied Biosystems, Warrington, UK), 293 245 50 ll) contained 30.5 ll of sterile water, 5 llof 2.1 ll of sequencing buffer, 1 ll of either the forward 294 246 DyNAzyme 10 9 buffer, 1 ll of 10 mM dNTP’s, 1 ll or reverse primer (10 mM stock concentration) and 295 247 of 25 mM ITS1-F primer (Gardes and Bruns 1993), 1 ll of purified PCR product. Sterile Sabax water was 296 248 1 ll of 25 mM ITS4 primer (White et al. 1990), 1 llof added to adjust the reaction volume to 12 ll. The 297 Ò 249 50 mM MgCl2, 0.5 ll of DyNAzyme DNA poly- thermal cycling conditions were: 25 cycles of 10 s at 298 250 merase and 10 ll/l of DNAUNCORRECTED template. The partial beta- 96 °C, 5 s at 55 °C and 4 min at 60 °C. Sequencing 299 251 tubulin gene region wasUNCORRECTED amplified using the primer products were then cleaned using ethanol/salt precip- 300 252 pair T10 (O’Donnel and Cigelnik 1997) and Bt2b itation. Sequencing of the isolates from S. carcharias 301 253 (Glass and Donaldson 1995). The PCR reactions were was conducted on the Applied Biosystems (ABI) with 302 254 run with BIOER XP Cycler (BIOER Technology, an ABI 3130xl sequencer (Thermo Fisher Scientific, 303 255 Hangzhou, China). The PCR reaction was started with Casrbad, USA) at the Haartman Institute Sequencing 304 256 4 min of initial denaturation followed by 30 cycles of Unit, University of Helsinki. Sequencing of the 305 123

Journal : Medium 10482 Dispatch : 9-6-2018 Pages : 17 Article No. : 1112 h LE h TYPESET MS Code : ANTO-D-18-00032 h44CP h DISK Antonie van Leeuwenhoek -Tubulin b GenBank acc. no. LSU ITS Author Proof 13 Sept 2013 KJ702037 13 Sept 2013 KJ702035 09 Dec 2015 MF188971 09 Dec 2015 MF188970 09 Dec 2015 MF188969 23 Nov 2015 MF188968 13 Aug 2013 KJ702030 20 Sept 2013 KJ702044 20 Sept 2013 KJ702039 01 Aug 201301 Aug MH267288 2013 KJ702027 MH327786 MH203866 23 Nov 2015 MF188973 01 Aug 2013 KJ702019 01 Aug 2013 KJ702022 23 Nov 2015 MH203865 13 Aug 2013 KJ702032 02 Aug 2013 KJ702026 23 Nov 2015 MF188975 isolation wood wood wood wood wood wood wood wood wood wood wood wood wood wood wood wood wood PROOF wood Colonized LarvaeColonized 11 Dec 2015 MF188972 Colonized Colonized Colonized Colonized Colonized Colonized Colonized Colonized Colonized Colonized Colonized Larvae 11 Dec 2015 MF188974 MH327787 Colonized Colonized Colonized Colonized PROOF Colonized tremula pendula colonized wood and intact wood in Finland Betula S. carcharias and glabripennis S. carcharias P. tremula A. glabripennis Salix caprea S. carcharias P. tremula A. glabripennis B. pendula A. glabripennis B. pendula A. glabripennis B. pendula A. glabripennis B. pendula S. carcharias P. tremula S. carcharias P. tremula S. carcharias P. tremula S. carchariasS. carcharias P. tremula P. tremula A. glabripennis B. pendula Saperda carcharias Populus A. glabripennis S. caprea S. carcharias P. tremula Anoplophora S. carcharias P. tremula S. carcharias P. tremula A. glabripennis B. pendula Beetle Host tree Substrate Date of e d A. glabripennis .b Herbarium no TUR207199 TUR207200 c d a CBS144083 CBS144084 CMW51780, N/A CMW49960 N/A CMW49959 CMW49958 CMW49957 CMW49956 N/A N/A CMW51781, CMW49962 N/A N/A 49963 no.

UNCORRECTEDUNCORRECTEDsp. sp. CMW49961 sp. N/A sp. N/A sp. N/A sp. N/A Fungal isolates obtained from longhorn beetles nov. Cadophora margaritata Cadophora spadicis Ascocoryne cylichnium Cladosporium Arthrinium Alternaria Table 1 Species Culture collection Coniothyrium carteri Cosmospora

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Journal : Medium 10482 Dispatch : 9-6-2018 Pages : 17 Article No. : 1112 h LE h TYPESET MS Code : ANTO-D-18-00032 h44CP h DISK Antonie van Leeuwenhoek -Tubulin b GenBank acc. no. LSU ITS Author Proof 23 Nov 2015 MF188978 23 Nov 2015 MF188976 MH327788 23 Nov 2015 MF188979 MH327790 23 Nov 2015 MF188980 23 Nov 2015 MF188981 01 Aug 2013 KJ702025 01 Aug 2013 KJ702024 01 Aug 2013 KJ702017 09 Dec 2015 MF188982 MH327791 09 Dec 2015 MF188983 11 Dec 2015 MF188984 MH327792 11 Dec 2015 MF188985 02 Aug 2013 KJ702020 02 Aug 2013 KJ702018 23 Nov 2015 MF188986 18 Nov 2013 KJ702045 isolation wood wood wood wood wood wood wood wood wood wood wood wood wood wood wood PROOFwood Colonized Colonized Colonized LarvaeIntact wood 18 11 Nov Dec 2013 2015 KJ702040 MF188977 MH327789 Colonized Colonized Colonized Colonized Colonized Colonized Colonized Colonized Intact wood 18 Nov 2013 KJ702042 Intact woodColonized 11 Dec 2015 MH203867 Colonized Colonized Colonized PROOFIntact woodColonized 18 Nov 2013 KJ702041 A. glabripennis B. pendula A. glabripennis B. pendula A. glabripennis B. pendula A. glabripennis S. caprea S. carcharias P. tremula A. glabripennis B. pendula A. glabripennis B. pendula A. glabripennis B. pendula S. carcharias P. tremula S. carcharias P. tremula S. carcharias P. tremula A. glabripennis B. pendula A. glabripennis S. caprea S. carcharias P. tremula A. glabripennis S. caprea A. glabripennis S. caprea S. carcharias P. tremula S. carcharias P. tremula A. glabripennis B. pendula S. carcharias P. tremula S. carcharias P. tremula Beetle Host tree Substrate Date of .b Herbarium no a CMW49967 CMW49965 N/A CMW49968 CMW49969 CMW49970 N/A N/A CMW49971 CMW49972 N/A N/A N/A N/A N/A CMW49973 N/A N/A no. UNCORRECTEDUNCORRECTED hyalina cf. sp. CMW49964 continued sp. CMW49966 sp. N/A Epicoccum Species Culture collection Table 1 Fusarium Mucor Mortierella Mortierella gamsii Mollisia dextrinospora

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Journal : Medium 10482 Dispatch : 9-6-2018 Pages : 17 Article No. : 1112 h LE h TYPESET MS Code : ANTO-D-18-00032 h44CP h DISK Antonie van Leeuwenhoek -Tubulin b GenBank acc. no. LSU ITS Author Proof 13 Sept 2013 KJ702036 20 Sept 2013 KJ702038 11 Dec 2015 MF188987 11 Dec 2015 MH203868 01 Aug 2013 KJ702028 20 Sept 2013 KJ702031 01 Aug 2013 KJ702021 13 Aug 2013 KJ702029 13 Aug 2013 KJ702033 20 Sept 2013 KJ702034 01 Aug 2013 KJ702023 isolation wood wood wood wood wood wood wood wood wood wood wood PROOF Colonized Colonized Colonized Colonized Colonized Colonized Colonized Colonized Colonized Colonized Intact wood 18 Nov 2013 KJ702043 Colonized PROOF A. glabripennis S. caprea S. carcharias P. tremula S. carcharias P. tremula A. glabripennis S. caprea S. carcharias P. tremula S. carcharias P. tremula S. carcharias P. tremula S. carcharias P. tremula S. carcharias P. tremula S. carcharias P. tremula S. carcharias P. tremula S. carcharias P. tremula Beetle Host tree Substrate Date of .b Herbarium no a CMW49974 N/A N/A N/A N/A N/A N/A N/A no. species complex UNCORRECTEDUNCORRECTED Mollisia – sp. N/A sp. N/A continued sp. N/A sp. N/A Cadophora TUR Herbarium, Centre for Biodiversity, University of Turku, Finland Ex-paratype CMW, Culture Collection of the Forestry and Agricultural Biotechnology Institute (FABI), University of Pretoria,Ex-type Pretoria, South Africa; CBS, the CBS-KNAW Collections, Holotype Westerdijk Fungal Biodiversity Institute, Utrecht, the Netherlands Phlebia radiata Phialocephala lagerbergii Species Culture collection Table 1 a b c d e Pholiota Trichoderma Tolypocladium Phoma Pseudeurotium bakeri Physalospora scirpi Sarocladium strictum Bold

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311311311311311311 isolates from A. glabripennis was conducted on the (Nylander 2004) based on the Akaike Information 358 312 ABI with an ABI 3500xl sequencer (Thermo Fisher Criterion (AIC) were determined. BI analyses based 359 313 Scientific, Carlsbad, USA) at the DNA Sequencing on a Markov Chain Monte Carlo (MCMC) simulation 360 314 Facility of the Forestry and Agricultural Biotechnol- were carried out with MrBayes v3.2.2 (Ronquist and 361 315 ogy Institute (FABI), University of Pretoria. Huelsenbeck 2003). The MCMC chains were run for 362 five million generations using a sample frequency of 363 316 Sequence analyses and fungal identification 100 (resulting in 50,000 trees). Burn-in values were 364 determined for the respective data sets, and all 365 317 Consensus sequences were assembled with the sampled trees having lower than the burn-in values 366 318 Geneious R6.0.6 (Biomatters Ltd, Auckland, New were discarded. The resulting majority rule consensus 367 319 Zealand), after which initial identification of the trees were viewed with MEGA v.7 (Kumar et al. 368 320 isolates was obtained using a BLAST search in 2016), and post-processed with Adobe Illustrator CC 369 321 GenBank nucleotide database (http://www.ncbi.nlm. 2018 (Adobe, San Jose, USA). 370 322 nih.gov) applying a megablast algorithm and (a) ex- Author Proof 323 cluding uncultured/environmental sample sequences 324 and (b) limiting the search on sequences from type Results 371 325 material (Supplementary Table 1). Identification to 326 the closest genus and species level was made as far as Isolation and identification of fungi 372 327 possible, with caution given to the fact that the ITS PROOFPROOF 328 sequence variability in certain fungal groups is low, Fungi were isolated from all of the collected samples 373 329 and acknowledging possibly misidentified sequences associated with S. carcharias and A. glabripennis 374 330 in GenBank. We considered reliable identification to infestations in Finland. The study resulted in total of 375 331 consist of the BLAST matches that had C 98% 114 fungal isolates. The trimmed consensus sequences 376 332 sequence similarity to ex-type sequences or peer-re- for the ITS gene regions ranged between 493 and 377 333 viewed published studies. For species in the Cado- 574 bp. The sequences obtained in this study were 378 334 phora–Mollisia complex, BLAST searches were also deposited in GenBank and their accession numbers are 379 335 used to retrieve similar sequences, as well as from type presented in the Table 1. 380 336 material to compile data sets for phylogenetic analy- The fungal species richness was higher in the 381 337 sis. Data sets were edited with MEGA v.7 (Kumar colonised parts than in the intact parts of the wood 382 338 et al. 2016). GenBank accession numbers of sequences (Table 2). The most common fungi isolated from both 383 339 are presented in the corresponding phylogenetic trees A. glabripennis and S. carcharias-colonised wood 384 340 (Figs. 3, 4). were members in the Cadophora–Mollisia species 385 341 Cadophora finlandica and Meliniomyces variabilis complex. These fungi were not found in the intact 386 342 were used as outgroups for the ITS data set, and parts of the wood. Only a small number of fungi were 387 343 Cadophora luteo-olivacea and Cadophora malorum detected in both the colonised and intact wood 388 344 for the beta-tubulin. The data sets were aligned using samples, including Epicoccum sp. and Fusarium sp. 389 345 the online version of MAFFT v. 7 (Katoh and Standley Fusarium sp. was the most commonly isolated species 390 346AQ2 2013). Three different phylogenetic methods were in the intact part of P. tremula sample logs. Some 391 347 applied: maximum likelihood (ML), maximum parsi- species were detected only from the intact wood 392 348 mony (MP), and Bayesian inference (BI). ML was including Mortierella hyalina and Sarocladium 393 349 performed in the online version of PhyML 3.0 strictum. 394 350 (Guindon et al. 2010), using automatic model selec- In total, 83 isolates were obtained from S. car- 395 351 tion by SMS (Lefort etUNCORRECTED al. 2017) and Akaike informa- charias in Finland. These represented at least 18 396 352 tion criterion (AIC) (SugiuraUNCORRECTED1978). Confidence levels species of and one species of Basidiomy- 397 353 were estimated with 1000 bootstrap replicates. MP cota (Table 1). The most common species isolated 398 354 analyses were conducted using PAUP v. 4.0b10 from S. carcharias colonised wood was Cadophora 399 355 (Swofford 2002). Gaps and missing data were spadicis, a putatively novel Cadophora species and 400 356 excluded in the MP analyses. For BI analyses, the Pseudeurotium bakeri. The other fungal species were 401 357 best-fitting evolutionary model using MrModeltest 2.3 isolated only occasionally. 402 123

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KJ702035 FINLAND CMW49956 MF188968 FINLAND Cadophora melinii JN689950 LITHUANIA CMW49960 MF188972 FINLAND CMW49957 MF188969 FINLAND CMW49959 MF188971 FINLAND 74/77 Ascomycota sp. MG190586 FINLAND KJ702037 FINLAND Cadophora spadicis CMW49958 MF188970 FINLAND Cadophora sp. KU712222 GERMANY Phialophora dancoi AB190869 JAPAN Cadophora sp. HM116752 NZ Cadophora spadicis KM497030 USA C. spadicis KM497031 USA C. spadicis DQ404351 ITALY T C. spadicis KM497029 USA Exophiala heteromorpha AB190397JAPAN

Author Proof KJ702036 FINLAND

88/82 Phialocephala lagerbergii AB190400 JAPAN P. lagerbergii JN689969 LITHUANIA Cadophora viticola HQ661098 SPAIN Cadophora orientoamericana KM497014 C. orientoamericana KM497017 CadophoraPROOFPROOF orientoamericana 100/ C. orientoamericana KM497015 100 C. orientoamericana KM497018 USA T Cadophora novi-eboraci KM497037 T 89/100 C. novi-eboraci KM497034 Cadophora novi-eboraci C. novi-eboraci KM497036 Mollisia dextrinospora NR _119489 SPAIN T M. dextrinospora HM116746 NZ 85/96 Mollisia dextrinospora CMW49973 MF188986 FINLAND 83/82 71/73 M. dextrinospora MG195556 NZ CBS144083 KJ702027 FINLAND T Cadophora margaritata sp. nov. 98/100 CBS144084 FINLAND Cadophora fastigiata JN689948 C. melinii AB190403 C. melinii KM497033 83/98 C. fastigiata AY249073 SWEDEN T C. melinii NR_111150 SWEDEN T 100/00 Cadophora gregata AY249070 Cadophora gregata C. gregata KP999986 Cadophora malorum KF053555 94/100 C. malorum AY249059 T Cadophora malorum C. malorum AB190402 Cadophora luteo-olivacea EF042108 C. luteo-olivacea AY249066T Cadophora luteo-olivacea 86/91 C. luteo-olivacea KM497043 C. luteo-olivacea KF764524 Meliniomyces variabilis NR_121313 T

100/100 Cadophora finlandica DQ320128 93/98 C. finlandica F486119 T

0.05UNCORRECTEDUNCORRECTED Fig. 3 Phylogenetic tree of species in the Cadophora–Mollisia at the nodes. Posterior probabilities (above 70%) obtained from species complex obtained from ML analyses of the ITS data set. BI are indicated by bold lines at the relevant branching points. Sequences obtained in this study are printed in bold type. *bootstrap values\ 70%. T, ex-type isolates. Scale bar = total Bootstrap support values above 70% for ML/MP are presented nucleotide difference between taxa

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Fig. 4 Phylogenetic tree of 88/87 C. melinii KM497113 species in the Cadophora– 99/100 C. melinii KM497114 Mollisia species complex C. melinii KM497132 T obtained from ML analyses 100/ of the beta-tubulin data set. 100 C. orientoamericana KM497098 Sequences obtained in this 86/98 C. orientoamericana KM497105 study are printed in bold 75/* C. orientoamericana KM497099 T type. Bootstrap support C. fastigiata KM497131 T values above 70% for ML/ C. spadicis KM497136 T MP are presented at the */72 80/91 nodes. Posterior 99/100 C. spadicis KM497111 probabilities (above 70%) 74/* C. spadicis KM497112 obtained from BI are CBS144083 MH327786 Cadophora margaritata sp. nov. 100/100 indicated by bold lines at the C. novi-eboraci KM497135 relevant branching points. *bootstrap values\ 70%. T, 93/100 C. novi-eboraci KM497116 ex-type isolates. Scale 99/100 C. novi-eboraci KM497118 T

Author Proof bar = total nucleotide C. finlandica KM497130 T difference between taxa C. luteo-olivacea KM497129 C. malorum KM497134 T C. luteo-olivacea KM497133 T 0.1 PROOFPROOF

403 Isolates from A. glabripennis in Finland represented The ITS data were sufficient to distinguish between 425 404 at least seven species of Ascomycota and one species of most species included in the data set. However, it did 426 405 Basidiomycota (Table 1). In addition to the Cado- not distinguish between Cadophora melinii and 427 406 phora–Mollisia isolates, other commonly detected Cadophora fastigiata, and species in the Phialo- 428 407 fungi from A. glabripennis-infested wood and larvae cephala lagerbergii clade. Phylogenetic analyses for 429 408 included a Fusarium sp. closely related to an extensively Cadophora–Mollisia confirmed the identities of four 430 409 studied gut symbiont isolate previously derived from an species isolated in this study. These were C. spadicis, 431 410 A. glabripennis larvae (Geib et al. 2012;Herretal. P. lagerbergii, Mollisia dextrinospora, and a puta- 432 411 2016). The other fungi were isolated only occasionally. tively novel species (Fig. 3). The identity of the 433 unknown species was further confirmed by phyloge- 434 412 DNA sequence analysis netic analysis of sequences for the partial beta-tubulin 435 gene region. 436 413 The identities of isolates representing the most com- The aligned beta-tubulin data set for the Cadophora 437 414 monly encountered genera, Cadophora–Mollisia, included 18 taxa and 523 characters (with gaps). The 438 415 were further confirmed by phylogenetic analysis. Data best-fitting substitution model estimated for the data set 439 416 originating from the previous studies by Harrington was GTR ? I ? G. The novel species was clearly 440 417 and McNew (2003) and Travadon et al. (2015) served distinct from the other currently known species of 441 418 as a backbone for the phylogenetic tree construction Cadophora (Fig. 4). The phylogenetic analysis also 442 419 for the Cadophora and allied species. supported the recent phylogenetic placement of Cado- 443 420 The aligned ITS dataUNCORRECTEDUNCORRECTED set included 44 taxa and 575 phora in four separate clades (Travadon et al. 2015). 444 421 characters (with gaps). The best-fitting substitution 422 model estimated for the Cadophora ITS data set was 445 423 GTR ? I ? G. The ML, MP and BI analyses pro- 424 duced trees with similar topologies (Fig. 3). Based on the phylogenetic analyses for ITS region and 446 partial beta-tubulin gene, as well as the morphological 447

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Table 2 Number of fungal isolates obtained from A. glabripennis and S. carcharias in Finland Species Anoplophora glabripennis Saperda carcharias Total Colonized wood Intact wood Colonized wood Intact wood

Alternaria sp. 0 0 4 0 4 Arthrinium sp. 0 0 2 2 4 Ascocoryne cylichnium 00202 Cadophora spadicis 707014 Cadophora margaritata sp. nov. 0 0 10 0 10 Cladosporium sp. 3 0 4 0 7 Coniochaeta sp. 1 0 7 0 8 Coniothyrium carteri 10001 Cosmospora sp. 0 0 7 0 7

Author Proof Epicoccum sp. 2 0 1 1 4 Fusarium sp. 12 1 1 4 18 Mollisia dextrinospora 10001 Mortierella gamsii 00101 Mortierella cf. hyalina 00022PROOFPROOF Mucor sp. 0 0 6 0 6 Phialocephala lagerbergii 00202 Phlebia radiata 10001 Pholiota sp. 0 0 5 0 5 Phoma sp. 0 0 2 0 2 Physalospora scirpi 00101 Pseudeurotium bakeri 0 0 10 0 10 Sarocladium strictum 00022 Tolypocladium sp. 0 1 0 0 1 Trichoderma sp. 0 0 1 0 1 Total 28 2 73 11 114

448 characteristics and ecology, the isolates of one of the excluding conidia, (1.4–)2.1–3(–3.5) lm wide 465 449 Cadophora sp. isolated formed a distinct lineage that (Fig. 5a, b). Conidiogenous cells terminal or lateral, 466 450 represents a novel taxon. This is described as follows mono- or poly-phialidic, hyaline (or paler 467 451 with measurements presented as (min–)(mean brown), (8.9–)12.3–22(–29.2) lm long, slightly swol- 468 452 - SD)–(mean ? SD)(–max)’. len at base, (1.6–)1.9–2.9(–3.6) lm at the widest part, 469 453 Cadophora margaritata R. Linnakoski, I. Lasarov & tapering significantly towards collarette; collarettes 470 454 A.O. Oghenekaro sp. nov. (Fig. 5). MycoBank: MB cup-shaped, (1.3–)2–3(–3.7) lm wide and (0.8–) 471 455 825227. 1–1.9(–2.4) lm long (Fig. 5b). Conidia aseptate, 472 456 Etymology: The name is derived from the Latin hyaline, ovoid, truncate at base (1.9–)2.5–3.7(–4.4) 473 457 word ‘margaritata’’ for ‘beaded’’ or ‘‘pearls’’ and long 9 (1.3–)1.4–2.2(–3.2) lm wide, aggregating 474 458 refers to beaded appearance of conidial mass. into globose (‘beaded appearance’) of conidial mass. 475 459 Sexual state: Not observed.UNCORRECTEDUNCORRECTEDCulture characteristics: Colonies on 2% MEA oliva- 476 460 Asexual state: Aerial mycelium on MEA consisted ceous black (Fig. 5d), mycelium superficial on agar, 477 461 of pale brown, tuberculate, septate hyphae up to (1.2– aerial mycelium sparse. Culture radial growth rate 478 462 )1.5–2.3(–3.2) lm diam. Conidiophores arising from 3.1 mm/d (± 0.3) at 22 °C. 479 463 aerial hyphae mostly branched (2–12 branches), pale Specimens examined: FINLAND, Western Finland 480 464 brown, septate, (93–)114.8–243(–295) lm long Province: Urjala, isolated from Saperda carcharias 481

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PROOFPROOF

Fig. 5 Morphological characteristics of the C. margaritata (CMW 51780 = CBS 144083). a Conidiophores, b conidiogenous cells, c conidia, d culture on MEA. Scale bars a–c 10 lm

482 infested Populus tremula, collector Tiia Marttinen, 01 plant pathogens, saprobes and species with known 496 483 August 2013. Holotype dried specimen TUR 207199 potential to cause wood degradation in the galleries of 497 484 (http://mus.utu.fi/TFU.207199), ex-holotype living the S. carcharias on P. tremula. In total 20 fungal 498 485 culture CMW 51780 = CBS 144083; Paratype dried species residing in two phyla were identified, the most 499 486 specimen TUR 207200 (http://mus.utu.fi/TFU. common detected in the beetle-colonised wood were 500 487 207200), ex-paratype living culture CMW species of Cadophora and allied species. This study 501 488 51781 = CBS 144084. also identified fungi associated with material collected 502 489 Host tree: Populus tremula. from A. glabripennis material retained in quarantine in 503 490 Insect vector: Saperda carcharias. Finland. At least eight fungal species were detected, 504 the most common of these were species of Cadophora 505 and Fusarium. While A. glabripennis is known to 506 491 Discussion UNCORRECTEDUNCORRECTEDharbour endosymbiotic fungi (Scully et al. 507 2013, 2014), these results are amongst the first to 508 492 This study contributes to limited investigations on provide evidence of wood-inhabiting fungi associated 509 493 mycobiota associated with cerambycid beetles, and is with A. glabripennis in its introduced range. It was 510 494 the first to report fungi associated with the large poplar also noteworthy that the introduced pest shares a 511 495 longhorn (S. carcharias). We isolated a number of

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512 number of fungal associates with the native species (Greenleaf and Korf 1980), and it could have a similar 561 513 (Table 1). role in wood decay in its association with the longhorn 562 514 One of the intriguing findings in this study was the beetles. The identity of the fourth species belonging in 563 515 strong presence of a number of Cadophora and allied the Cadophora–Mollisia species complex found in 564 516 species existing in association with the longhorn this study, a fungus closely related to Phialocephala 565 517 beetles. This study is the first to reveal an association lagerbergii could not be resolved due to lack of 566 518 of these fungi with insects. Four Cadophora and allied available type sequence data. 567 519 species were identified, including the novel species C. Fusarium is a highly diverse cosmopolitan genus 568 520 margaritata described in. Cadophora species were that includes plant and human pathogens, saprobes and 569 521 originally described as fungi causing blue stain of endophytes. Therefore, it is not surprising that these 570 522 lumber (Lagerberg et al. 1927). These fungi are now fungi have also been found as symbionts of ceramby- 571 523 known to reside in two distinct genera Phialophora cid and ambrosia beetles (Geib et al. 2012; Morales- 572 524 (Chaetothyriales, Eurotiomycetes) and Cadophora Ramos et al. 2000; Kasson et al. 2013). The presence 573 525 (Helotiales, Leotiomycetes) (Gams 2000; Harrington of these fungi with A. glabripennis in the present study 574 Author Proof 526 and McNew 2003). Cadophora has received little is perhaps not surprising but their consistent occur- 575 527 attention in the past but recent reports implicating rence suggests that they may be relevant to A. 576 528 species as potential grapevine pathogens in various glabripennis. This is supported by the fact that they 577 529 countries have changed this view (Halleen et al. 2007; have previously been found consistently associated 578 530 Casieri et al. 2009; Gramaje et al. 2011; Navarrete with the pest inPROOFPROOF other areas and in different host trees 579 531 et al. 2011;U´ rbez-Torres et al. 2014). Cadophora (Geib et al. 2012). In this association, species of 580 532 spadicis detected in the present study is one of the Fusarium have been considered as internal gut sym- 581 533 recently described species reported from wood-decay bionts supporting cellulose degradation and larval 582 534 of grapevine in North America (Travadon et al. 2015). fitness of A. glabripennis (Scully et al. 2013, 2014). 583 535 A previous study has also reported that Cadophora Species in this group are also symbionts of ambrosia 584 536 species are able to cause dark staining and extensive beetles (Morales-Ramos et al. 2000; Kasson et al. 585 537 soft rot in Betula and Populus wood in vitro (Blanch- 2013). However, in this study Fusarium spp. were also 586 538 ette et al. 2004). Based on past findings, it is detected as the most common isolates in healthy parts 587 539 reasonable to assume that Cadophora species reported of P. tremula wood, indicating their role as endophytes 588 540 here play some role in wood degradation close to the on aspen. 589 541 larval tunnels of Cerambycidae in general. An Aureobasidium sp. was commonly isolated in 590 542 The novel species described in this study, is this study from P. tremula. Aureobasidium species 591 543 characterised by production of phialides with hyaline appear to be a common associate of aspen P. tremula 592 544 collarettes, morphological structures typical for Cado- (Santamarı´a and Diez 2005), with probable fitness 593 545 phora and allied phialide-producing fungi. An exam- effects. Albrectsen et al. (2010) reported that aspen 594 546 ple is Mollisia dextrinospora, which has been reported clonal stands, where Aureobasidium sp. was present, 595 547 to have a cadophora-like asexual stage (Greenleaf and were more resistant to browsing by herbivores. 596 548 Korf 1980). As shown in the present study and also in Another commonly detected fungus was Pseudeu- 597 549 previous phylogenetic analyses (Crous et al. 2003; rotium bakeri in the S. carcharias colonised wood. 598 550 Harrington and McNew 2003; Day et al. 2012;Pa¨rtel The fungus has an ability to utilize lignin residues left 599 551 2016), several species of Cadophora and Mollisia are from brown rot and humus (Ran et al. 2016). 600 552 closely related. Correct identification of these species Pseudeurotium bakeri has also been found as a root- 601 553 is hindered by the occurrence of numerous misiden- endophyte of the common grass, Dactylis glomerata 602 554 tified entries in nucleotideUNCORRECTED sequence databases (Pa¨rtel (Sa´nchez Ma´rquez et al. 2007). The other fungi were 603 555 2016). Consequently, MollisiaUNCORRECTEDappears to be a hetero- detected less commonly. These included species of 604 556 geneous assemblage of fungi that requires taxonomic Alternaria, Cladosporium, Epicoccum, and Sarocla- 605 557 treatment. The Mollisia isolate found in this study dium. These genera include well-know saprobes and 606 558 grouped with the ex-type strain of M. dextrinospora pathogens of taxonomically diverse plant species. 607 559 and its identity seems clear. The species was originally Therefore, finding them as part of the mycobiota 608 560 described from decayed Acacia wood in Madeira colonizing hardwood trees was not surprising. 609 123

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References 659 610 Unlike for example, scolytine beetles (Curculion- 658 611 idae: Scolytinae), very little is known regarding the 612 relationships between cerambycid beetles and fungi. Akbulut S, Stamps WT (2012) Insect vectors of the pinewood 660 Mono- 661 Adults of these insects never enter the wood, and any nematode: a review of the biology and ecology of 613 chamus species. For Pathol 42:89–99. https://doi.org/10. 662 614 organisms such as fungi carried on their bodies would 1111/j.1439-0329.2011.00733.x 663 615 need to be associated with oviposition. While these Albrectsen BR, Bjo¨rke´n L, Varad A, Hagner A˚ , Wedin M, 664 616 insects are well-known to transmit nematodes such as Karlsson J, Jansson S (2010) Endophytic fungi in European 665 666 the devastating pine pathogen Bursaphelenchus xylo- aspen (Populus tremula) leaves—diversity, detection, and 617 a suggested correlation with herbivory resistance. Fungal 667 618 philus (pine wood nematode) during oviposition Divers 41:17–28 668 619AQ3 (Wingfield 1987; Akbulut and Stamps 2012), a fixed Ayayee P, Rosa C, Ferry JG, Felton GW, Saunders M, Hoover K 669 620 association between them and fungi is not known. In (2014) Gut microbes contribute to nitrogen provisioning in a 670 wood-feeding cerambycid. Environ Entomol 43:903–912. 671 AQ4 this regard, Wingfield et al. (2016) suggested that 621 https://doi.org/10.1603/EN14045 672 622 mites carried on the bodies of Cerambycidae could be Blanchette RA, Held BW, Jurgens JA, McNew DL, Harrington 673 623 implicated in the transmission of fungi found in their TC, Duncan SM, Farrell RL (2004) Wood-destroying soft 674 Author Proof 624 galleries. The same situation could be true also for S. rot fungi in the historic expedition huts of Antarctica. Appl 675 676 carcharias and A. glabripennis, but further investiga- Environ Microbiol 3:1328–1335 625 Casieri L, Hofstetter V, Viret O, Gindro K (2009) Fungal com- 677 626 tions would be needed to confirm it. munities living in the wood of different cultivars of young 678 627 Species diversity of wood colonised by the insects Vitis vinifera plants. Phytopathol Mediterr 48:73–83. https:// 679 628 was higher than intact wood highlighting the impor- doi.org/10.14601/Phytopathol_Mediterr-2876PROOFPROOF 680 ´ ˆ 681 tance of larval tunnel for fungal diversity. We detected Chararas C, Pignal M-C (1981) Etude du role de deux levures 629 isole´es dans le tube digestif de Phoracantha semipunctata, 682 630 a number of pathogenic fungi capable of killing living Cole´opte`re Cerambycidae xylophage spe´cifique des Euca- 683 631 cells, although many of these are considered as weak lyptus. Comptes Rendus de l’Acade´mie des Sciences de 684 632 and opportunistic pathogens or endophytes. We also Paris 292:109–112 685 686 found several species with known potential of enzy- Crous PW, Groenewald JZ, Gams W (2003) Eyspot of cereals 633 revisited: ITS phylogeny reveals new species relationships. 687 634 matic degradation of wood components. The avail- Eur J Plant Pathol 109:841–850. https://doi.org/10.1023/A: 688 635 ability of such fungi closely associated with the insect 1026111030426 689 636 suggests that fungal enzymes ingested by the larvae Day MJ, Hall JC, Currah RS (2012) Phialide arrangement and 690 691 play an in key role in improved nutritional quality of character evolution in the helotialean anamorph genera 637 Cadophora and Phialocephala. Mycologia 104:371–381. 692 638 the longhorn beetles. The study shows that cerambycid https://doi.org/10.3852/11-059 693 639 beetles are associated with a diverse microbial Drenkhan T, Sibul I, Kasanen R, Vainio EJ (2013) Viruses of 694 640 assemblage, which can participate in wood degrada- Heterobasidion parviporum persist within their fungal host 695 Hylobius 696 tion by larvae either killing or breaking down host during passage through the alimentary tract of 641 abietis. For Pathol 43:317–323 697 642 tissue. Drenkhan T, Kasanen R, Vainio EJ (2016) Phlebiopsis gigantea 698 and associated viruses survive passing through the diges- 699 643 Acknowledgements This study was financially supported by tive tract of Hylobius abietis. Biocontrol Sci Technol 700 644 the University of Helsinki (RL); the Academy of Finland 26:320–330 701 645 (FOA); the members of the Tree Protection Co-operative EPPO (2016) Data sheets on quarantine pests—Anoplophora AQ5702 646 Programme (TPCP) and the THRIP initiative of the glabripennis. http://www.eppo.int/QUARANTINE/data_ 703 647 Department of Trade and Industry (RL, MJW), South Africa. sheets/insects/ANOLGL_ds.pdf 704 648 We acknowledge the staff at the Finnish Food Safety Authority Francke-Grosmann H (1967) Ectosymbiosis in wood-inhabiting 705 649 (Evira) for their support to this study. insects. Symbiosis 2:141–205 706 Gams W (2000) Phialophora and some similar morphologically 707 650 Funding This study was funded by the University of Helsinki little-differentiated anamorphs of divergent ascomycetes. 708 651 (RL); the Academy of Finland (FOA), the members of the Tree Stud Mycol 45:187–199 709 652 Protection Co-operative Programme (TPCP) and the THRIP Gardes M, Bruns TD (1993) ITS primers with enhanced speci- 710 653 initiative of the DepartmentUNCORRECTEDUNCORRECTED of Trade and Industry (RL, MJW), ficity for basidiomycetes—application to the identification 711 654 South Africa. of mycorrhiza and rusts. Mol Ecol 2:113–118. https://doi. 712 org/10.1111/j.1365-294X.1993.tb00005.x 713 655 Compliance with ethical standards Geib SM, Scully ED, del Mar Jimene-Gasco M, Carlson JE, 714 Tien M, Hoover K (2012) Phylogenetic analysis of 715 656 Conflict of interest The authors declare that have no conflict Fusarium solani associated with the Asian longhorned 716 657 of interest.

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