International Journal of Food Microbiology 147 (2011) 58–68

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International Journal of Food Microbiology

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Fusarium sibiricum sp. nov, a novel type A trichothecene-producing from northern Asia closely related to F. sporotrichioides and F. langsethiae

Tapani Yli-Mattila a,⁎, Todd J. Ward b, Kerry O'Donnell b, Robert H. Proctor b, Alexey A. Burkin c, Galina P. Kononenko c, Olga P. Gavrilova d, Takayuki Aoki e, Susan P. McCormick b, Tatiana Yu. Gagkaeva d a Molecular Plant Biology, Department of Biochemistry and Food Chemistry, University of Turku, FI-20014 Turku, Finland b Bacterial Foodborne Pathogens and Mycology Research Unit, USDA-ARS, 1815 N. University St., Peoria, IL 61604, USA c Laboratory of Mycotoxicology, All-Russian Research Institute for Veterinary Sanitation, Hygiene and Ecology, Zvenigorodskoe shosse 5, Moscow 123022, Russia d Laboratory of Mycology and Phytopathology, All-Russian Institute of Plant Protection (VIZR), Podbelskogo 3, St. Petersburg-Pushkin, 196608, Pushkin, Russia e NIAS Genbank-Microorganisms Section (MAFF), National Institute of Agrobiological Sciences (NIAS), 2-1-2 Kannondai, Tsukuba, Ibaraki 305-8602, Japan article info abstract

Article history: Production of type A trichothecenes has been reported in the closely related species Fusarium langsethiae and Received 12 November 2010 F. sporotrichioides. Here, we characterized a collection of Fusarium isolates from Siberia and the Russian Far Received in revised form 4 March 2011 East (hereafter Asian isolates) that produce high levels of the type A trichothecene T-2 toxin and are similar in Accepted 7 March 2011 morphology to the type A trichothecene-producing F. langsethiae, and to F. poae which often produces the type B trichothecene nivalenol. The Asian isolates possess unique macroscopic and microscopic characters Keywords: and have a unique TG repeat in the nuclear ribosomal intergenic spacer (IGS rDNA) region. In Asian isolates, Fusarium langsethiae – F. poae the TRI1 TRI16 locus, which determines type A versus type B trichothecene production in other species, is F. sibiricum more similar in organization and sequence to the TRI1–TRI16 locus in F. sporotrichioides and F. langsethiae than F. sporotrichioides to that in F. poae. Phylogenetic analysis of the TRI1 and TRI16 gene coding regions indicates that the genes in Phylogenetic analysis the Asian isolates are more closely related to those of F. sporotrichioides than F. langsethiae. Phylogenetic T-2 analysis of the beta-tubulin, translation elongation factor, RNA polymerase II and phosphate permease gene sequences resolved the Asian isolates into a well-supported sister lineage to F. sporotrichioides,withF. langsethiae forming a sister lineage to F. sporotrichioides and the Asian isolates. The Asian isolates are conspecificwithNorwegian isolate IBT 9959 based on morphological and molecular analyses. In addition, the European F. langsethiae isolates from Finland and Russia were resolved into two distinct subgroups based on analyses of translation elongation factor and IGS rDNA sequences. Nucleotide polymorphisms within the IGS rDNA were used to design PCR primers that successfully differentiated the Asian isolates from F. sporotrichioides and F. langsethiae. Based on these data, we formally propose that the Asian isolates together with Norwegian isolate IBT 9959 comprise a novel phylogenetic species, F. sibiricum, while the two subgroups of F. langsethiae only represent intraspecificgroups. © 2011 Elsevier B.V. All rights reserved.

1. Introduction F. langsethiae, isolates of this were reported as F. poae (Torp and Nirenberg, 2004). However, it is possible to distinguish F. langsethiae The type A trichothecenes T-2 toxin (T-2) and HT-2 toxin (HT-2) are from F. poae as well as other fusaria by morphological differences and among the most toxic metabolites produced by Fusarium species. molecular genetic analyses, including AFLP, RFLP, and DNA sequence- Production of these two mycotoxins has been well documented in two based phylogenetic analysis (Knutsen et al., 2004; Konstantinova and species in particular, F. sporotrichioides and F. langsethiae (Jestoi et al., Yli-Mattila, 2004; Mach et al., 2004; Schmidt et al., 2004a,b; Yli-Mattila 2008; Langseth et al., 1999; Logrieco et al., 1990; Torp and Langseth, et al., 2004a). In addition, F. langsethiae and F. poae differ in production of 1999). Fusarium langsethiae was first reported in Norway in 1999 (Torp trichothecene mycotoxins: isolates of F. langsethiae typically produce and Langseth, 1999) and subsequently found to be common in Europe T-2-like trichothecenes, whereas those of F. poae typically produce (Torp and Nirenberg, 2004). The species was first reported in Finland in nivalenol-like trichothecenes (Jestoi et al., 2008; Thrane et al., 2004; 2001 (Yli-Mattila et al., 2004b) and in the European region of Russia Torp and Langseth, 1999). in 2003 (Gagkaeva et al., 2008). Before the formal description of The nuclear IGS rDNA region together with partial sequences from the translation elongation factor 1-alpha (EF-1α) gene has been shown to be very useful in evolutionary studies of type A trichothecene-producing fusaria (Knutsen et al., 2004; Kristensen et al., 2005; Yli-Mattila et al., ⁎ Corresponding author. Tel.: +358 2 3336587; fax: +358 2 3335549. 2004a). European isolates of F. langsethiae were resolved into two E-mail address: tymat@utu.fi (T. Yli-Mattila). subgroups based on sequence differences in the IGS rDNA region; the two

0168-1605/$ – see front matter © 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.ijfoodmicro.2011.03.007 T. Yli-Mattila et al. / International Journal of Food Microbiology 147 (2011) 58–68 59 subgroups can be distinguished from one another by a PCR assay using IGS 5-ml aliquot of the culture was extracted with 2 ml ethyl acetate, and the rDNA subgroup-specific primers (Konstantinova and Yli-Mattila, 2004). extract was analyzed for the presence of trichothecenes using a Hewlett Recently, T-2-producing isolates of Fusarium that are morpholog- Packard 6890 gas chromatograph fitted with a HP-5MS column ically similar to F. langsethiae and F. poae were isolated in Siberia and (30m×0.25mmfilm thickness) and a 5973 mass detector as previously the Russian Far East (Burkin et al., 2008). To clarify the identity of described (McCormick and Alexander, 2002). The presence of T-2 in these Asian isolates, and a morphologically similar Norwegian isolate culture extracts was determined by the occurrence of a metabolite with (IBT 9959; Knutsen et al., 2004; Torp and Nirenberg, 2004; Yli-Mattila thesameretentiontimeandmassspectrumaspurified T-2. et al., 2004a; Yli-Mattila, 2010), we subjected the isolates to multi- locus phylogenetic and phenotypic analyses. The results indicate that 2.4. DNA extraction the Asian isolates together with Norwegian isolate IBT 9959 belong to a distinct phylogenetic species, which is characterized and formally Fusarium isolates were cultured on potato dextrose agar (PDA; described in the present paper. Our results indicate that this novel Scharlau Chemie S.A., Spain) at 25 °C and DNA was extracted with the toxigenic species is more closely related to F. sporotrichioides than to GenElute™ Plant Genomic DNA Kit (Sigma-Aldrich, St. Louis, MO, either F. langsethiae or F. poae. USA) as described by Yli-Mattila et al. (2008) or in broth culture, which was lyophilized prior to DNA extraction, by using a CTAB 2. Materials and methods (hexadecyltrimethylammonium bromide) miniprep as described previously (O'Donnell et al., 1998). 2.1. Fusarium isolates For analysis of the TRI1–TRI16 locus, Fusarium strains were grown in liquid GYEP medium (0.3% glucose, 0.1% yeast extract, and 0.1% Fusarium isolates analyzed in this study listed in Table 1 are stored peptone) for up to four days. The resulting growth was lyophilized, in the Agricultural Research Service Culture Collection (NRRL, Peoria, ground to a powder, and suspended in extraction buffer (200 mM IL, USA) and the VIZR All-Russian Plant Protection Institute collection Tris–Cl pH 8, 250 mM NaCl, 25 mM EDTA pH 8, and 0.5% SDS) at (MFG, St. Petersburg-Pushkin, Russia). Isolate IBT 9959 was kindly ~50 mg per 250 μl buffer. Genomic DNA was purified by mixing the provided by Dr. Ulf Thrane, while Finnish isolates from the years resulting suspension with an equal volume of 1:1 (v/v) TRIS- 2001–2003 were obtained from the Norwegian Veterinary Institute equilibrated phenol (pH 8) and chloroform. The aqueous phase was from Dr. Ralf Kristensen. mixed with 2 volumes sodium iodide solution and 5 μl of UltraBind solution from the UltraClean DNA Purification kit (Mo Bio Laborato- 2.2. Cultural growth rate and phenotypic characterization ries, Inc. Carlsbad, CA, USA). DNA was then purified following instructions provided with the kit. Isolates were cultivated on potato sucrose agar (PSA; Booth, 1971)at 24 °C in complete darkness. All microscopic studies were performed on 2.5. Specific primers used for species and group identification cultures grown on Spezieller Nährstoffarmer Agar (SNA; Nirenberg, 1976)for10–14 days in darkness at 25 °C. Colony growth rate was For identification of F. langsethiae and F. sporotrichioides, primer measured at 15 and 24 °C in Petri dishes 9 cm in diameter containing pairs FlangF3/LanspoR1 and FsporF1/LanspoR1, which are based on 20 ml of PSA. For this test, 5 mm disks were taken from the edge of species-specific RAPD-PCR products of European F. langsethiae and colonies growing on SNA and placed in the center of a PSA dish. The F. sporotrichioides isolates, were used as described by Wilson et al. average mycelial growth rate for all species was based on colony (2004). For identification of F. poae primer pairs Fp82f/r and PoaeIGS/ diameter after 5 days of incubation. Colony diameters were measured CNL12 were used as described by Parry and Nicholson (1996) and using three replicates for each isolate at both temperatures. The length Konstantinova and Yli-Mattila (2004). In addition, primer pairs and width of microconidia were measured with a Axio Imager M1 PfusF/FspoR and PfusF/FlanR were used for the identification of microscope (Carl Zeiss, Germany) using the AxioVision software F. sporotrichioides and F. langsethiae as described by Yli-Mattila et al. package to record the minimum and maximum size including mean (2004b) and Halstensen et al. (2006). The latter primer pair has been value and standard deviations (S.D.). At least 50 conidia for each shown to give false positives for a few F. sporotrichioides isolates, specified number of septa were measured for at least three strains of which have identical ITS sequences with F. langsethiae (Yli-Mattila each species (F. langsethiae MFG 11019, 11020, 11027; Asian isolates et al., 2004a,b). Primers CNL12 and PulvIGSr were used to distinguish MFG 11007, 11008, 11013, 11014; F. sporotrichioides MFG 11017, 11018, two subgroups of European F. langsethiae isolates designated I and II; a 11024, 11026; and F. poae MFG 11043, 11044, 11045). long deletion present within the IGS rDNA region of subgroup II (Konstantinova and Yli-Mattila, 2004), which is absent within 2.3. Mycotoxin analyses subgroup I, distinguishes isolates within these two subgroups.

Fusarium isolates were cultivated for 7 days in a small glass bottle 2.6. DNA sequence analyses (diameter 18 mm) with 1 ml PSA medium at 24 °C with 3–6 replicates. Cultures were extracted with 1 ml of acetonitrile:water Portions of the translation elongation factor 1α (EF-1α), phos- (84:16) and extracts were analyzed for T-2 and diacetoxyscirpenol phate permease (PHO), RNA polymerase II (RPB2), and beta-tubulin (DAS) by indirect competitive ELISA with a detection limit of 4 ppb (TUB2) genes were amplified and sequenced using primers listed in (Burkin et al., 2007; Kononenko et al., 1999). The ELISA for detection Supplementary Table S1. Amplifications were performed in 40 μl of T-2 and DAS is based on polyclonal antibodies and it has been reactions with 1× PCR buffer, 2.0 mM MgSO4, 0.2 mM dNTP's, 0.25 μM qualified to have less than 0.1% cross reactivity with structurally each primer, 0.8 U of Platinum Taq DNA Polymerase High Fidelity related analogs. Analysis of variance (ANOVA) was carried out with (Invitrogen Life Technologies, Carlsbad, CA, USA), and 25 ng of the Microsoft program EXCEL. After ANOVA, comparisons of means genomic DNA. The PCR profile consisted of an initial denaturation at were applied to the experimental data with Fisher's F statistic at a 96 °C (90 s), followed by 40 cycles of 94 °C (30 s), 52 °C (30 s), and significant level of P≤0.05. 68 °C (60 s), followed by 68 °C (5:00 min). The annealing temperature For analysis of trichothecene production by gas chromatography-mass used to amplify RPB2 was 54 °C. The extension time was 45 s for EF-1α spectrometry (GCMS), isolates were grown for seven days in liquid GYEP and 2 min for RPB2. After PCR reactions were purified using medium(5%glucose,0.1%yeastextract,and0.1%peptone)aspreviously MultiScreenHTS PCR 96-well plates (Millipore, Billerica, MA, USA), described (McCormick and Alexander, 2002). After 7 days of incubation, a they were suspended in 60 μl of Milli-Q grade water. Sequencing 60 T. Yli-Mattila et al. / International Journal of Food Microbiology 147 (2011) 58–68

Table 1 Origin of Fusarium isolates used in this study. In the table, the ‘Asian isolates’ are referred to with the new species name F. sibiricum.

Strain no in collections Species and Host plant Origin Year subgroupc NRRLa MFGb

53421 11004 F. sibiricum Siberia, Tuva 2002 53422 11005 F. sibiricum Barley Siberia, Buryatia 2002 53423 11006 F. sibiricum Siberia, Chita 2000 53424 11007 F. sibiricum Barley Siberia, Krasnoyarsk 2000 53425 11008 F. sibiricum Siberia, Krasnoyarsk 2001 53426 11009 F. sibiricum Barley Siberia, Irkutsk 2000 53427 11010 F. sibiricum Barley Siberia, Irkutsk, 2000 53428 11011 F. sibiricum Oat Siberia, Irkutsk 2002 53429 11012 F. sibiricum Oat Far East, Vladivostok 2001 53430 11013 F. sibiricum Oat Far East, Chabarovsk 2003 53431 11014 F. sibiricum Oat Far East, Blagoveschensk 2001 53432 11015 F. sibiricum Wheat Siberia, Irkutsk 2000 53433 11016 F. sibiricum Wheat Siberia, Irkutsk 2000 IBT 9959d F. sibiricum Oat Norway 1998 53436 11019 F. langsethiae, I Barley Russia, Central, Orel 2006 53420 11003 F. langsethiae, II Barley Russia, South-European Krasnodar 2002 53437 11020 F. langsethiae, I Oat Russia, Northwest, Pskov 2007 53538 11021 F. langsethiae, I Oat Russia, Northwest, Kaliningrad 2007 53439 11022 F. langsethiae, I Oat Russia, Northwest, Vologda 2007 53409 11027 F. langsethiae, I Barley Finland, Etelä-Karjala 2001 53419 11028 F. langsethiae, I Oat Finland, Satakunta 2003 53410 11029 F. langsethiae, I Oat Finland, Satakunta 2003 53411 11030 F. langsethiae, II Oat Finland, Satakunta 2003 53412 11031 F. langsethiae, II Wheat Finland, Uusimaa 2003 53413 11032 F. langsethiae, II Wheat Finland, Uusimaa 2003 53414 11033 F. langsethiae, II Wheat Finland, Häme 2003 53415 – F. langsethiae, II Oat Finland, Satakunta 2003 53416 – F. langsethiae, II Oat Finland, Varsinais-Suomi, 2003 53417 11034 F. langsethiae, I Oat Finland, Uusimaa, 2003 53418 11035 F. langsethiae, II Wheat Finland, Etelä-Pohjanmaa 2003 53434 11017 F. sporotrichioides Oat Russia, Far East 2001 53435 11018 F. sporotrichioides Cirsium spp. Russia, Far East 2006 – 11024 F. sporotrichioides Apple fruit Russia, Far East 2006 – 11025 F. sporotrichioides Cirsium spp. Russia, Far East 2006 – 11026 F. sporotrichioides Oat Russia, Far East 2001 – 11039 F. sporotrichioides Barley Russia, Northwest 2003 – 11040 F. sporotrichioides Barley Russia, Northwest 2002 – 11041 F. sporotrichioides Wheat Russia, Central 2004 – 11042 F. sporotrichioides – Russia, Far East 2002 3299 ATCCe 24631 F. sporotrichioides Maize France – 6227 – F. armeniacum Fescue hay USA Missouri – 13440 – F. sporotrichioides – USA Wisconsin – 25474 – F. sporotrichioides Picea abies seed Germany – 25479 – F. sporotrichioides Pinus nigra seed Germany – 26342 – F. sporotrichioides Wheat Japan, Eniwa, Hokkaido – 26343 – F. sporotrichioides Wheat Japan, Tanno, Hokkaido – 26923 – F. sporotrichioides Pisum sativum Japan – 26924 – F. sporotrichioides Barley Denmark – 28446 CBSf 249.61 F. sporotrichioides – Former USSR – 29131 – F. sporotrichioides Oat Germany – 29133 CBS 485.94 F. armeniacum – Australia – 29977 – F. sporotrichioides – former Yugoslavia – 29978 – F. sporotrichioides – Former Yugoslavia – 31970 – F. armeniacum Soil Australia – – 11023 F. poae Oat Russia, Far East 2001 – 11043 F. poae Oat Russia, Northwest 2004 – 11044 F. poae Oat Russia, Northwest 2002 – 11045 F. poae – Russia, Far East 2000 – 11046 F. poae Wheat Russia, Central 2006

a Agricultural Research Service Culture Collection (Peoria, IL, USA). b VIZR, All-Russian Plant Protection Institute culture collection (St. Petersburg-Pushkin, Russia). c Subgroups of F. langsethiae were identified by primer pair CNL12/IGSpulvr and by sequencing of IGS rDNA region. d Culture collection of Biocentrum-DTU-Denmark (Technical University of Denmark, Lyngby, Denmark). e American Type Culture Collection (http://www.lgcstandards-atcc.org/). f CBS-KNAW Fungal Diversity Centre (Centraalbureau voor Schimmelcultures, Utrecht, The Netherlands.

reactions were carried out according to the method of Platt et al. (2007). Evolutionary relationships were inferred using the neighbor-joining Sequencing reactions were purified using BigDye XTerminator (Applied method and the Kimura two-parameter model of molecular evolution Biosystems, Foster City, CA, USA) and analyzed with an ABI 3730 DNA implemented in MEGA version 4.0 (Tamura et al., 2007). Support for Analyzer (Applied Biosystems). DNA sequences were edited and aligned individual nodes was assessed by bootstrap analysis with 1000 with Sequencher (version 4.9, Gene Codes, Ann Arbor, MI, USA). replications (Felsenstein, 1985; Penny and Hendy, 1985). T. Yli-Mattila et al. / International Journal of Food Microbiology 147 (2011) 58–68 61

of Fusarium (hereafter referred to as Asian isolates) were recovered. The Asian isolates were similar in morphology to both the type A trichothecene-producing species F. langsethiae and the type B trichothecene-producing species F. poae. However, on PSA, cultures of the Asian isolates produced more aerial mycelia than F. langsethiae and less aerial mycelia than F. poae. In addition, cultures of the Asian isolates were less pigmented than those of F. langsethiae and F. poae. In PCR assays previously designed to distinguish F. langsethiae, F. poae, or F. sporotrichioides from other species, none of the six primer combinations employed consistently distinguished the Asian isolates and representative strains of F. langsethiae or F. sporotrichioides (Supplementary Table SII). With the primer combination PfusF/FspoR no PCR product was produced by any of the isolates shown in Supplementary Table SII (data not shown). In PCR assays, FsporF1/ LanspoR1 was the only primer combination that consistently distin- Fig. 1. Distribution of Fusarium langsethiae in Europe and Asian T-2 producing isolates in guished between F. langsethiae and F. sporotrichioides, while the Asia. The area of alimentary toxic aleukia syndrome in the southern Ural region during combination FlangF3/LanspoR1 yielded only quantitative differences World War II, where the T-2 producing Fusarium isolates of Yagen and Joffe (1976) were collected, has also been identified. between the two species. However, two primer combinations (Poae IGS/CNL12 and Fp82F/R) that were designed to distinguish F. poae from other species consistently distinguished the Asian isolates from F. poae. For amplification of the IGS rDNA primers CNL12 and CNS1 (Appel Analysis of trichothecene production in PSA cultures revealed that and Gordon, 1996) were used as described by Yli-Mattila et al. (2004a). the Asian isolates could produce the type A trichothecene T-2 as well Amplifications were performed in an MJ Research thermal cycler (PTC- as DAS, which is a biosynthetic precursor of some type A and type B 200, Watertown, MA, USA). The PCR program consisted of 30 cycles trichothecenes (Table 2). This analysis also confirmed that selected following an initial denaturation step at 94 °C for 90 s, DNA denaturation strains of F. langsethiae and F. sporotrichioides included in this study at 92 °C for 30 s, primer annealing at 55 °C for 90 s and primer extension produced relatively high levels of T-2 and lower levels of DAS. The at 72 °C for 90 s, followed by an extension at 72 °C for 3 min. IGS rDNA average T-2 and DAS production was highest in F. langsethiae isolates PCR products were purified as described in Paavanen-Huhtala et al. and highest T-2 and DAS production was found in a F. langsethiae (1999). For sequencing of the IGS region primers IGS1 (Yli-Mattila et al., subgroup II isolate, NRRL 53412, from Finland. In contrast, selected 2004a), PulvIGSR (Konstantinova and Yli-Mattila, 2004), PulvIGSF and strains of F. poae produced low levels of DAS and only trace amounts LansF (Supplementary Table SI) were used (Fig. 1). IGS sequences were of T-2, according the ELISA method employed. Production of T-2 by aligned using Clustal W version 1.83 (Thompson et al., 1994) and edited the Asian isolates was confirmed by GCMS analysis of five isolates visually. The PHYLIP 3.5 package (Felsenstein, 1993) was used for (NRRL 53421, NRRL 53423, NRRL 53424, NRRL 53427, and NRRL phylogenetic analyses with 1000 bootstrap replications as described by 53430) grown in GYEP medium, which has been used previously to Yli-Mattila et al. (2004a). Gaps longer than 2 bp were excluded from the evaluate T-2 production in F. sporotrichioides. GCMS analysis con- IGS data before the NJ analysis. firmed the presence of T-2 (44–173 μg/ml culture) in extracts of PCR amplification of the TRI1 and TRI16 genes utilized the DNA seven-day-old cultures of all five isolates. polymerase, reagents, and concentrations specified for the Platinum PCR Together, these results indicated the Asian isolates were morpho- SuperMix High Fidelity protocol (Invitrogen Life Technologies, Carlsbad, logically similar to but distinct from F. poae and F. langsethiae. The CA). Oliognucleotides for PCR amplification and sequence analysis of Asian isolates could also be distinguished from F. poae by their fragments of the TRI1 and TRI16 codingregionsaswellasthePDB1–TRI1 trichothecene production profile and in some PCR assays. In contrast, and TRI1–TRI16 intergenic regions are shown in Supplementary Table SI the Asian isolates possessed the same trichothecene production and were based on previously described sequences from F. sporotrichioides profile as F. langsethiae and F. sporotrichioides and could not be and F. armeniacum (Proctor et al., 2009). In order to fill in gaps in sequence consistently distinguished from either species using the PCR assays. data, additional primers were designed based on intergenic region sequences generated during the current study. Phylogenetic analysis of 3.2. IGS rDNA sequence analysis TRI1 and TRI16 employed the nucleotide sequence of the entire TRI1 coding region (1803 nucleotides) and 94% of the TRI16 coding region Sequence analysis of the IGS rDNA revealed that the Asian isolates are (nucleotides 86 to 1482). Evolutionary relationships were inferred using closely related to a Norwegian isolate (strain IBT 9959) previously the neighbor-joining method implemented in MEGA (version 4.0) as described above. Support for individual nodes was assessed by bootstrap Table 2 Trichothecene mycotoxin production by isolates of Fusarium examined in this study. analysis with 1000 replications. The Asian isolates are referred to as F. sibiricum. All sequences have been deposited in the NCBI GenBank database a under accession numbers HM744650–HM744693 (EF-1α), HM060272– Species (# of isolates) Mycotoxin production, ppm HM060292 (IGS rDNA), HM744650 –HM744693 (TUB2), T-2 DAS HQ154439–HQ154481 (RPB2), HQ154482–HQ154508 (PHO)and Mean Min–max Mean Min–max HQ594535–HQ594543 (TRI1 and TRI16). DNA sequences from GenBank F. sibiricum (13) 17.4 a 4.0–53.5 0.2 a 0.02–0.5 of F. poae, F. langsethiae and F. sporotrichioides were also used in some F. langsethiae Sg Ib (8) 26.5 b 1.1–50.1 1.6 b 0.1–5.5 phylogenetic analyses. F. langsethiae Sg IIb (6) 38.6 b 12.9–100 2.8 b 0.2–6.7 F. sporotrichioides (9) 21.7 b 0.5–75.9 0.09 a 0–0.4 – – 3. Results F. poae (5) 0.06 c 0 0.3 0.3 c 0 0.9 Values within columns followed by the same letter are not significantly different at 3.1. Initial characterisation of Fusarium isolates P≤0.05. Quantification was done by indirect ELISA with a detection limit of 4 ppb. a Trichothecene production was determined from 7-day-old PDA cultures grown at In surveys of fungi that occur in cereal crops cultivated in southern 23 °C. locations of Siberia and the Russian Far East (Fig. 1), multiple isolates b Subgroups of F. langsethiae were identified by primer pair CNL12/IGSpulvr. 62 T. Yli-Mattila et al. / International Journal of Food Microbiology 147 (2011) 58–68

Fig. 2. IGS rDNA sequence differences in the Asian isolates (Fusarium sibiricum) and the two subgroups of European F. langsethiae as compared to F. sporotrichioides. Primers are shown by horizontal arrows and nucleotide differences compared to F. sporotrichioides are shown by vertical arrows.

identified as F. langsethiae. The Asian isolates and strain IBT 9959 possess a 3.4. Sequence analysis of TRI1–TRI16 locus TG repeat that is ≥36 bp in length in the IGS sequence, whereas isolates of F. sporotrichioides and both subgroups of F. langsethiae have a shorter TG Analysis of partial sequence from the region spanned by PDB1, TRI1 repeat (8–20 bp) (Fig. 2). In the PCR analysis with IGS primers CNL12 and and TRI16 in four Asian isolates, seven F. langsethiae isolates, and six PulvIGSr (Konstantinova and Yli-Mattila, 2004), the Asian isolates and IBT F. sporotrichioides isolates (five from Europe and one from Asia) revealed 9959 yielded amplicons that were ≥16 bp larger than amplicons from F. that the order and orientation of these genes in F. langsethiae and the sporotrichioides and F. langsethiae subgroup I (Supplementary Fig. S1). It is Asian isolates, formally described herein as F. sibiricum, was the same as noteworthy that the amplicon from F. langsethiae subgroup II was ~100 bp previously described for F. sporotrichioides and F. armeniacum (Proctor smaller than amplicons from isolates of F. langsethiae subgroup I, et al., 2009). Among the isolates examined here, the length of the regardless of geographical origin of the isolates. Phylogenetic analysis TRI1–TRI16 intergenic region varied among and within species, although using the IGS rDNA sequence resolved the Asian isolates as a distinct the length (2607–2609 bp) was relatively uniform among the Asian lineage from F. sporotrichioides and F. langsethiae (data not shown). The isolates examined (Supplementary Fig. SII). The most variation was subgroups of European isolates of F. langsethiae based on IGS sequences observed within F. langsethiae, where the number of bp between the TRI1 (Fig. 2, Supplementary Fig. SI, Table 1) were in agreement with the two and TRI16 stop codons was 758 and 761 in isolates NRRL 53410 and NRRL subgroups resolved using EF-1α sequence data (Fig. 3). Subgroup II was 53411, respectively, and 1404–2299 in the other isolates examined. The more common in Finland than in Russia (Table 1). length (1404–2707 bp) of the TRI1–TRI16 intergenic region also varied markedly in F. sporotrichioides. In contrast, the PDB1–TRI1 intergenic 3.3. Molecular phylogenetics region varied little (959–968 bp) among all isolates examined. Sequence comparisons of the TRI1 and TRI16 coding regions from Phylogenetic analyses of the individual EF-1α, PHO, RPB2 and TUB2 the same subset of isolates revealed that the sequences were and combined partitions differentiated the Asian isolates from relatively uniform among the F. sibiricum and F. langsethiae isolates F. sporotrichioides, although only bootstrap analyses of RPB2 and the but varied considerably among the F. sporotrichioides isolates combined dataset supported their reciprocal monophyly (Fig. 3). examined. Phylogenetic analysis of TRI1 and TRI16 sequences alone European F. langsethiae isolates were resolved into two distinct and in combination resolved sequences from the F. sibiricum isolates subgroups in the EF-1α phylogeny (bootstrap value=88%) (Fig. 3). In a into a well-supported clade; however, isolates of F. sporotrichioides combined phylogenetic analysis of EF-1α, PHO, RPB2andTUB2 sequences, were resolved as a grade ( Fig. 4). In addition, F. langsethiae formed a the Asian isolates formed a well-supported clade with a bootstrap value clade distinct from F. sibiricum and F. sporotrichioides. of 100%, while the clade of F. sporotrichioides isolates was supported by a bootstrap value of 98%. European F. langsethiae isolates formed two distinct subgroups, although only one of these was supported by 3.5. bootstrapping. Although several subgroups were resolved within F. sporotrichioides, no variation was detected within the Asian isolates Species descriptions are provided for the novel species of Fusarium sampled, suggesting that they may represent a clone or clonal lineage represented by the Asian isolates and IBT 9959, and Russian and (Fig. 3). Based on the genealogical exclusivity of the Asian isolates it is Finnish isolates of F. langsethiae for nomenclatural purposes, as well as formally described herein as the novel species F. sibiricum (see below). for illustrating and comparing their morphological features.

Fig. 3. Neighbor-joining phylograms inferred from analysis of individual data partitions and combined data (in the middle). Bootstrap support ≥70% is indicated by nodes. Trees were rooted with sequences from Fusarium graminearum strain PH-1 (NRRL 31084). The individual data partitions consisted of 628 bp (EF-1α), 770 bp (PHO), 1882 bp (RPB2), and 1254 bp (TUB2) unambiguously aligned nucleotides after excluding sites containing gaps or missing data. Similar results were obtained under different models of molecular evolution and when maximum parsimony was used as the optimality criterion. T. Yli-Mattila et al. / International Journal of Food Microbiology 147 (2011) 58–68 63 64 T. Yli-Mattila et al. / International Journal of Food Microbiology 147 (2011) 58–68

Holotype: LEP12652, a dried culture, isolated from a grain of oat (Avena sativa). Khabarovsk, the Fast East, Russia, 2003 (#3051/2) by Drs. Ludmila S. Malinovskaya and Elena A. Pirjazeva (Moscow), deposited at the Mycological herbarium VIZR (LEP), St. Petersburg. Isotype: TUR 188557, a dried culture, deposited at the herbarium of the University of Turku. Ex holotype cultures: NRRL 53430=MFG 11013. Isolates studied: NRRL 53421 (=MFG 11004), NRRL 53422 (=MFG 11005), NRRL 53423 (=MFG 11006), NRRL 53424 (=MFG 11007), NRRL 53425 (=MFG 11008), NRRL 53426 (=MFG 11009), NRRL 53427 (=MFG 11010), NRRL 53428 (=MFG 11011), NRRL 53429 (=MFG 11012), NRRL 53430 (=MFG 11013), NRRL 53431 (=MFG 11014), NRRL 53432 (=MFG 11015), and NRRL 53433 (=MFG 11016). Distribution: Siberia, Russian Far East, Norway and Iran. Notes: Fusarium sibiricum can be differentiated morphologically from similar species, i.e. F. langsethiae, F. poae and F. sporotrichioides.At first, F. sibiricum can easily be differentiated from F. sporotrichioides, because macroconidia (i.e., multiseptate sporodochial conidia) and chlamydospores are absent in the former species but present in the Fig. 4. Neighbor-joining phylogram inferred from combined analysis of TRI1 and TRI16 latter. Some strains of F. poae also form macroconidia sparsely under UV sequences (3199 nucleotides). Bootstrap support ≥70% is indicated by nodes based on light (Torp and Nirenberg, 2004), but macroconidia of F. sibiricum were 1000 pseudoreplicates of the data. The tree was rooted with sequences from Fusarium not observed in this study, even when cultured on SNA under near-UV sambucinum strain R-07843 (Proctor et al., 2009). Similar results were obtained using light for 10 days. In addition, frequent production of polyblastic or fi the maximum parsimony method. The isolates identi ed as F. sporotrichioides are “ ” non-monophyletic in this tree. polyphilidic conidia, i.e. so-called mesoconidia by F. sporotrichioides also clearly differentiates it from F. sibiricum. Morphological differen- tiation of F. sibiricum from F. poae and F. langsethiae is difficult. For Fusarium sibiricum Gagkaeva, Burkin, Kononenko, Gavrilova, example, F. sibiricum forms abundant apiculate and globose conidia, and O'Donnell, T. Aoki, et Yli-Mattila, sp. nov. MycoBank MB 519164. F. poae, F. langsethiae and F. sporotrichioides do as well. Size of the 0-septate globose conidia formed by F. sibiricum and F. langsethiae were Coloniae in PSA in quoque die ad 24 °C in proportione 5.9±0.7 mm et 6.4×5.6 μm and 6.2×5.6 μm on average, respectively, and were smaller ad 15 °C in proportione 3.1±0.4 mm crescentes. Mycelium aerium in PSA, than those of F. poae, which were 7.5x6.1 µm on average (Table 3). albidum vel roseo-albidum, parcum vel floccosum, centraliter copiosum, Further, ampulliform monophialides are typically produced by F. poae, plerumque crateriforme cum calvitio in centris coloniarum; reversum and ampulliform and cylindrical monophialides, which are frequently albidum vel cremicolor. Conidiophora in mycelio aerio et in hyphis slightly curved and polyphialides with two loci are formed by F. substrati formantia, erecta vel prostrata, primum simplicia, deinde dense langsethiae.InF. sibiricum, ampulliform and cylindrical, but straight and ramosa in monophialides exeuntia; monophialides ampulliformes, 3.7– erect, monophialides with a noticeable collarette are formed. Moreover 15.0×3.1–4.9 μm, conspicuo collariatae, nonnunquam cylindricae et F. sibiricum forms conidiophores often with a long and thin nodose stipe rectae, 21.3–23.3×3.4–3.9 μm. Conidiophora plerumque longi-stipitata more than 100 μm in length which terminates in a whorl of phialides (N100 μm) et nodosa, in phialides verticillatas exeuntia, cum phialidibus (Table 3, Fig. 5). F. sibiricum is strictly monophialidic, but F. langsethiae brevibus in mycelis aeris formantibus commiscentia. Microconidia rarely forms polyphialides with two conidiogenous loci, that may be apiculata et globosa, ut plurimum 0-septata, raro 1-septata, hyalina, in used as a rare but additional morphological difference distinguishing the massis capitulates copiose formantia; illa 0-septata 4.3–8.2×4.5–7.3 μm. two species. Macroscopically there are some clear differences among the Nec macroconidia nec sporodochia formantia. Chlamydosporae absentes. species. Based on colony morphology, F. sibiricum produces more Sclerotia absentia. Odor nonnumquam dulcis. abundant aerial mycelium than F. langsethiae, particularly in the central Colonies on PSA showing average mycelial growth rates of 5.9± part of colony, which was also reported in the morphological 0.7 mm/day at 24 °C and 3.1±0.4 mm/day at 15 °C, respectively characterization of IBT 9959 (Torp and Nirenberg, 2004). By contrast, (Table 3). Aerial mycelium on PSA sparse to floccose, more abundant most of the F. poae and F. sporotrichioides isolates produced more centrally, often crateriform with bald spot in the very centre of colonies. abundant aerial mycelium than F. sibiricum. All isolates of F. sibiricum Colour of aerial mycelium white, sometimes with a tint of orange grey. studied were less pigmented than the other species being compared. Reverse with white to cream shades. Conidiophores formed in aerial Some isolates of F. langsethiae form cream white to pinkish white aerial mycelium or on running hyphae on the agar, erect or prostrate, at first mycelium and typically produce a pale red to violet pigment in the centre, unbranched, later branched densely terminating with ampulliform often with a bluish tint in reverse. F. langsethiae isolates grew more slowly monophialides measuring 8.3±1.9×3.9±0.5 μm on average and S.D., on PSA (5.1±0.4 mm/day at 24 °C and 2.6±0.3 mm/day at 15 °C) 3.7–15.0×3.1–4.9 μm in total range having a noticeable collarette and compared to F. sibiricum, F. sporotrichioides and F. poae, while no clear rarely with cylindrical, straight monophialides measuring 21.3– difference in growth rate was found between the later three species. 23.3×3.42–3.9 μm in total range. Conidiophores often consist of a long F. sibiricum is phylogenically more closely related to F. sporotrichioides, and nodose stipe (N100 μm) terminating with a whorl of phialides, but is morphologically more similar to F. langsethiae and F. poae.In intermingled with the short monophialides formed directly on the aerial summary, to identify isolates of F. sibiricum morphologically, a precise mycelium (Fig. 5). Polyphialides not observed. Microconidia apiculate and examination and comparison of micro- and macro-morphology is globose, mostly 0-septate, rarely 1-septate, hyaline, formed abundantly in essential. false heads (Fig. 5); 0-septate: measuring 6.4±0.7×5.6±0.6 μmon average and S.D., 4.3–8.2×4.5–7.3 μm in total range. Neither multiseptate Fusarium langsethiae Torp et Nirenberg, Int. Jour. Food Microbiol. macroconidia nor sporodochia formed. Chlamydospores and sclerotial 95: 247-256, 2004 (Table 3). bodies absent. Odour sweetish in some strains. Etymology: The epithet, sibiricum, refers to Siberia in Asia, where Colonies on PSA showing average mycelial growth rates of 5.1± most of the isolates were collected. 0.4mm/dayat24°Cand2.6±0.3mm/dayat15°C,respectively.Arial T. Yli-Mattila et al. / International Journal of Food Microbiology 147 (2011) 58–68 65

Table 3 Comparison of morphological characters observed in the isolates of Fusarium sporotrichioides, F. poae, F. langsethiae and F. sibiricum, studied in the present study, including the F. sibiricum strain IBT 9959.

Characters observed Species

F. sporotrichioides F. poae F. langsethiae F. sibiricum

Profuse mycelial growth on PSAa, colony height ≥3mm + + − + Carmine red pigment in reverse on PSA + ± ± − Radial growth rate (mm/day) on PSA, 24 °C 7.6 ±0.1 6.5 ±0.9 5.1±0.4 5.9±0.7 Maximal growth rate (mm/day) 7.7 7.4 6.3 7.4 Minimal growth rate (mm/day) 7.5 5.2 4.0 3.9 Chlamydospores + −− − Sporodochial conidia (macroconidia) + −− − Average size of 0-septate, globose and apiculate conidia on SNAb (μm) 7.8 ×6.6 7.5 ×6.1 6.4×5.3 6.4×5.6 Polyblastic conidiogenous cells or polyphialides + (two or more loci) − + (two loci) − Unbranched and branched conidophores with monophialides + + + + Ampulliform monophialides − ++ + Bent cylindrical monophialides −−+ − Conidiophores with long stipes (N100 μm) in older cultures −−−+

a Potato-sucrose agar. b Spezieller Nährstoffarmer Agar (Nirenberg, 1976). mycelium on PSA sparse, less than 1–3 mm high. Colour of aerial surface powdery from the production of conidiophores. Conidiophores mycelium cream white to pinkish white. Pigmentation in reverse creamy- unbranched at first, later branched, with bent cylindrical and ampuliform white, pale red and pale violet in center, often with a bluish tint. Colony monophialides bearing a minute but noticeable collarette; cylindrical

Fig. 5. Fusarium sibiricum cultured on SNA in the dark (A–E, from NRRL 53430=MFG11013; F, from IBT 9959). A. Ampulliform and cylindrical monophialides formed directly on the aerial mycelium. B–C. Conidiophores with long and nodose stipe, with branches terminating in monophialides. D. Densely branched, relatively short and stout conidiophores terminating in ampuliform monophialides. E–F. False heads with apiculate and globose conidia. Scale bars=20 μm. 66 T. Yli-Mattila et al. / International Journal of Food Microbiology 147 (2011) 58–68 phialides 20–25×2.5–4 μm relatively longer and more slender compared expression studies of the F. graminearum and F. sporotrichioides TRI1 with ampuliform phialides 3–15×3–5 μm. Microconidia abundantly orthologues in F. verticillioides; and 2) gene deletion studies of TRI1 and formed, hyaline, apiculate and globose, very rarely pyriform, mostly TRI16 in F. graminearum and F. sporotrichioides (Brown et al., 2003; 0-septate, rarely 1-septate, formed in persistent false heads. 0-septate: McCormick et al., 2004, 2006; Meek et al., 2003; Peplow et al., 2003). measuring 6.2±0.7×5.6±0.6 μm on average and S.D., 4.3–8.4×4.1– The results using specific primers on F. sibiricum in the present study 7.7 μm in total range. Macroconidia, sporodochia, chlamydospores and match those obtained with F. langsethiae in Tehran province by Kachuei sclerotial bodies absent (Table 3). Odour absent. et al. (2009). In both cases the isolates (except for isolate NRRL 53427) Isolates studied: NRRL 53409 (=MFG 11027), NRRL 53410 (=MFG gave a positive reaction with primers FsporF1/LanspoR1 designed for 11029), NRRL 53411 (=MFG 11030), NRRL 53412 (=MFG 11031), European F. sporotrichioides isolates, while they did not give a clear NRRL 53413 (=MFG 11032), NRRL 53414 (=MFG 11033), NRRL 53415, positive signal with primers FlangF3/LanspoR1, designed for European NRRL 53416, NRRL 53417 (=MFG 11034), NRRL 53418 (=MFG 11035), F. langsethiae isolates (Wilson et al., 2004). Thus, primers based only on NRRL 53419 (=MFG 11028), NRRL 53420 (=MFG 11003), NRRL 53436 European isolates may be unreliable for species identification outside (=MFG 11019), NRRL 53437 (=MFG 11020), NRRL 53538 (=MFG Europe. According to previous results (Yli-Mattila et al., 2004a), and 11021), and NRRL 53439 (=MFG 11022). results of the present study, ITS rDNA sequences and primers based on ITS Distribution: Russia, Finland, Norway, Austria, Czech Republic, sequences cannot be used for reliable identification of F. sporotrichioides, Denmark, England, The Netherlands, and other European countries. F. sibiricum and F. langsethiae. Note: Morphology of F. langsethiae isolates from Russia and Finland In the present study IGS rDNA, TUB2, EF-1α, PHO, RPB2, TRI1 and examined in the current study (Table 3) agreed well with the description TRI16 sequences were evaluated separately and in combination for of the species by Torp and Nirenberg (2004). Two phylogenetic subgroups their ability to resolve phylogenetic relationships of F. sporotrichioides, were recovered among the isolates of F. langsethiae analyzed in the European F. langsethiae and F. sibiricum. IGS rDNA and EF-1α current study but they were indistinguishable morphologically. Morpho- sequences were the most informative sequences in resolving the logically F. langsethiae is closely related to F. sibiricum, but differentiation two European subgroups of F. langsethiae, while bootstrap analyses of of F. langsethiae and F. sibiricum was possible based on macroscopic and the PHO, RPB2, TRI1 and TRI16 partitions provided strong support for microscopic characters, as described above. the genealogical exclusivity of F. sibiricum. Given the non-monophyly of F. sporotrichioides and the high nucleotide variation observed, 4. Discussion especially within the TRI1 and TRI16 sequences, further studies including a more extensive collection may reveal cryptic species The first isolate of Fusarium sibiricum (IBT9959) was recovered in within this morphospecies. 1998 in Europe and reported as an intermediate between F. langsethiae F. sporotrichioides isolates were significantly different morphologically and F. sporotrichioides (Knutsen et al., 2004; Thrane et al., 2004; Torp and from the other species included in this study. Cultural characters that Nirenberg, 2004; Yli-Mattila et al., 2004a); a second isolate of this distinguish F. sibiricum and F. poae include less abundant and non- species was discovered in Iran in 2007 and reported as F. cf. langsethiae pigmented aerial mycelium and odour in the latter species (Table 3). It (Kachuei et al., 2009). The remaining F. sibiricum isolates were recovered was also possible to identify F. sibiricum isolates based on macroscopic and in 2000–2003 in Siberia and the Russian Far East, where they were microscopic characters, whereas no morphological differences were originally identified as F. poae producing high levels (5–2000 ppm) of found between the two European subgroups of F. langsethiae. Unlike T-2 in autoclaved rice (Burkin et al., 2008)asdescribedbyKononenko European F. langsethiae, F. sporotrichioides and some F. poae the F. sibiricum et al. (1999). In the present study 13 single-spore isolates derived from isolates did not produce pigment on PSA in the dark. European the Siberian and Russian Far Eastern isolates together with IBT 9959 F. langsethiae isolates grew more slowly on PSA at 24 °C and 15 °C as were resolved as a phylogenetically and morphologically distinct compared to F. sporotrichioides and F. poae, while no clear difference in species, F. sibiricum. growth rate was found between F. sibiricum, F. sporotrichioides and F. poae. Reports concerning the ability of F. poae to produce type A Ampulliform monophialides are typical for F. poae. Ampulliform and trichothecenes, such as HT-2, T-2 and neosolaniol, are contradictory bent, cylindrical phialides, sometimes with polyphialides with 2 loci (Bottalico and Perrone, 2002; Somma et al., 2010; Thrane et al., 2004; were typically produced by F. langsethiae,whileF. sibiricum formed Torp and Langseth, 1999; Yagen and Joffe, 1976). Based on our results, branched conidiophores with ampulliform and cylindrical but more these contradictory reports may be due to the misidentification of F. typically straight monophialides with a clearly visible collarette. It is langsethiae, which is morphologically similar to F. poae (Torp and noteworthy that the stability of microconidia in false heads differs Langseth, 1999; Torp and Nirenberg, 2004; Yli-Mattila et al., 2004a). among the species. These heads in F. sporotrichioides are very easily In the present work only trace amounts of T-2 were found in F. poae destroyed, while in F. sibiricum the false heads are more stable than in with a very sensitive ELISA method, which confirms the results of F. poae, but less stable than in F. langsethiae. There were only a few Vogelgsang et al. (2007), Jestoi et al. (2008) and Somma et al. (2010).In differences in microscopic characters between F. poae and F. sibiricum. a previous analysis of F. poae,theTRI16 gene, which is essential for T-2 Under the culture conditions employed macroconidia were not formed production, was found to be truncated and therefore most likely in F. poae, however, macroconidia are produced sparsely in this species nonfunctional (Brown et al., 2003; Peplow et al., 2003; Proctor et al., under near-UV light (Torp and Nirenberg, 2004). Also in F. sibiricum no 2009). Although TRI16 has not been examined in the strains of F. macroconidia were formed within 10 days on SNA under near-UV light poae analyzed in the current study, truncation of the gene would (data not shown). Based on macro- and microscopic characters IBT 9959 explain why the strains did not produce high levels of T-2. The isolate, from Norway, appeared to be conspecificwithF. sibiricum, which organization and genomic context of the trichothecene biosynthetic is supported by the morphological and molecular data in the present locus TRI1-TRI16 is similar in F. sibiricum, F. sporotrichioides,andF. study and previous investigations (Konstantinova and Yli-Mattila, 2004; langsethiae, but markedly different from the organization described Knutsen et al., 2004; Torp and Nirenberg, 2004; Yli-Mattila et al., 2004a). for F. poae (Proctor et al., 2009). F. sibiricum isolates are able to produce high levels of T-2 and they The similarity of the F. sibiricum and F. langsethiae TRI1 and TRI16 also produce DAS, whereas the F. poae isolates examined here were sequences to the F. sporotrichioides sequences is consistent with nonproducers or produced only trace amounts of T-2 or DAS. This is in similar trichothecene chemotypes of the three species. It is the accordance with the results of several published studies (Jestoi et al., F. sporotorichioides-like TRI1 and TRI16 orthologues that determine 2008; Langseth et al., 1999; Somma et al., 2010; and Thrane et al., 2004). that type A trichothecenes, such as T-2 and HT-2, are produced rather Further studies are required by GCMS to confirm the trace amounts of than type B trichothecenes. This has been shown by 1) heterologous T-2 produced by some F. poae isolates and to determine whether any T. Yli-Mattila et al. / International Journal of Food Microbiology 147 (2011) 58–68 67 differences in T-2 or DAS production exist between subgroups I and II of Halstensen, A.S., Nordby, K.-C., Klemsdal, S.S., Eilen, O., Clasen, P.-E., Eduard, W., 2006. Toxigenic Fusarium spp. as determinants of trichothecene mycotoxins in settled F. langsethiae. Studies will also be conducted to determine whether the grain dust. Journal of Occupational and Environmental Hygiene 3, 651–659. TRI16 gene, which is required for T-2 synthesis, is present in any F. poae Jestoi, M., Paavanen-Huhtala, S., Parikka, P., Yli-Mattila, T., 2008. In vitro and in vivo isolate producing trace amounts of T-2. mycotoxin production of Fusarium species isolated from Finnish grains. Archives of α Phytopathology and Plant Protection 41, 545–558. The identical EF-1 , PHO, RPB2 and TUB2 sequences in F. sibiricum Joffe, A.Z., 1986. Fusarium Species: Their Biology and Toxicology. J. Wiley & Sons, New York. suggest that the isolates sampled represent a clone or clonal lineage. Kachuei, R., Yadegari, M.H., Rezaie, S., Allameh, A., Safaie, N., Zaini, F., Yazd, F.K., 2009. However, variation was found within the F. sibiricum isolates Investigation of stored mycoflora, reporting the Fusarium cf. langsethiae in three – morphologically and based on mycotoxin production and lengths of provinces of Iran during 2007. Annals of Microbiology 59, 383 390. Knutsen, A.K., Torp, M., Holst-Jensen, A., 2004. Phylogenetic analyses of the Fusarium the TG repeats in the IGS rDNA region. poae, Fusarium sporotrichioides and Fusarium langsethiae species complex based on According to the present data, F. langsethiae may be restricted to partial sequences of the translation elongation factor-1 alpha gene. International – Europe and Western Siberia (O. Gavrilova and T. Gagkaeva, unpublished Journal of Food Microbiology 95, 287 295. Kononenko, G.P., Burkin, A.A., Soboleva, N.A., Zotova, E.V., 1999. Enzyme immunoassay results). It is unknown whether F. langsethiae and F. sibiricum are present for determination of T-2 toxin in contaminated grain. Applied Biochemistry and in the Orenburg region near southern Ural, where outbreaks of Microbiology 35 (4), 411–416. alimentary toxic aleukia (ATA) were reported during World War II Konstantinova, P., Yli-Mattila, T., 2004. IGS-RFLP analysis and development of molecular markers for identification of Fusarium poae, Fusarium langsethiae, (see Fig. 1; Joffe, 1986; Sarkisov, 1954). ATA outbreaks, which already Fusarium sporotrichioides and Fusarium kyushuense. International Journal of Food started before the war in different parts of the former Soviet Union, have Microbiology 95, 321–331. been attributed to T-2 contamination caused by F. sporotrichioides. Kristensen, R., Torp, M., Kosiak, B., Holst-Jensen, A., 2005. Phylogeny and toxigenic potential is correlated in Fusarium species as revealed by partial translation However, if F. sibiricum and/or F. langsethiae occur in the Orenburg elongation factor 1 alpha gene sequences. Mycological Research 109, 173–186. region, they too may have contributed to the outbreaks of ATA; also the Langseth, W., Bernhoft, A., Rundberget, T., Kosiak, B., Gareis, M., 1999. Mycotoxin T-2-producing F. poae isolates from this region identified by Yagen and production and cytotoxicity of Fusarium strains isolated from Norwegian cereals. Mycopathologia 144, 103–113. Joffe (1976) may belong to these species or even to F. sporotrichioides Logrieco, A., Chelkowski, J., Bottalico, A., Visconti, A., 1990. Further data on specific due to the unreliable morphological identification. In conclusion, the trichothecene production by Fusarium section Sporotrichiella strains. Mycological results indicate that F. sibiricum is phylogenetically distinct from Research 94, 587–589. F. langsethiae and more closely related to F. sporotrichioides, even though Mach, R.L., Kullnig-Gradinger, C.M., Farnleitner, A.H., Reischer, G., Adler, A., Kubicek, C.P., 2004. SpecificdetectionofFusarium langsethiae and related species by DGGE and isolates of F. sibiricum are morphologically more similar to F. poae and ARMS-PCR of a β-tubulin (tub1) gene fragment. International Journal of Food F. langsethiae. Microbiology 95, 333–339. Supplementary materials related to this article can be found online McCormick, S.P., Alexander, N.J., 2002. Fusarium Tri8 encodes a trichothecene C-3 esterase. Applied and Environmental Microbiology 68, 2959–2964. at doi:10.1016/j.ijfoodmicro.2011.03.007. McCormick, S.P., Harris, L.J., Alexander, N.J., Ouellet, T., Saparno, A., Allard, S., Desjardins, A.E., 2004. Tri1 in Fusarium graminearum encodes a P450 oxygenase. Applied and Environmental Microbiology 70, 2044–2051. Acknowledgements McCormick, S.P., Alexander, N.J., Proctor, R.H., 2006. Heterologous expression of two trichothecene P450 genes in Fusarium verticillioides. Canadian Journal of Microbi- The visits of Dr. T. Yli-Mattila to the All-Russian Plant Protection ology 52, 220–226. Institute and to the USDA-ARS, Peoria, IL and the visits of Drs. T. Gagkaeva Meek, I.B., Peplow, A.W., Ake, C., Phillips, T.D., Beremand, M.N., 2003. Tri1 encodes the cytochrome P450 monooxygenase for C-8 hydroxylation during trichothecene and O. Gavrilova to the University of Turku were supported financially by biosynthesis in Fusarium sporotrichioides and resides upstream of another new Tri the Academy of Finland (no. 126917 and 131957) and the Nordic network gene. Applied and Environmental Microbiology 69, 1607–1613. project New Emerging Mycotoxins and Secondary Metabolites in Nirenberg, H.I., 1976. Untersuchungen über die morphologische und biologische Differenzierung in der Fusarium-Sektion Liseola. Mitteilungen aus der Biologischen Toxigenic Fungi of Northern Europe (project 090014), which was funded Bundesanstalt Fur Land- und Forstwirtschaft (Berlin-Dahlem) 169, 1–117. by the Nordic Research Board. We thank Ludmila S. Malinovskaya and O'Donnell, K., Cigelnik, E., Nirenberg, H.I., 1998. Molecular systematics and phylogeo- Elena A. 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