Diversity in metabolite production by Fusarium langsethiae, Fusarium poae, and Fusarium sporotrichioides

International Journal of Food Microbiology 95 (2004) 257 – 266 www.elsevier.com/locate/ijfoodmicro

Diversity in metabolite production by Fusarium langsethiae, Fusarium poae, and Fusarium sporotrichioides Ulf Thrane a,*, Andreas Adler b, Per-Erik Clasen c, Fabio Galvano d, Wenche Langseth c,1, Hans Lew b, Antonio Logrieco e, Kristian Fog Nielsen a, Alberto Ritieni d a

Center for Process Biotechnology, BioCentrum-DTU, Søltofts Plads 221, Technical University of Denmark, Building 221, Kgs. Lyngby DK-2800, Denmark b Austrian Agency for Health and Food Safety, Agrobiology Linz, Wieningerstrasse 8, Linz A-4020, Austria c Department of Food and Feed Hygiene, National Veterinary Institute, PO Box 8156 Dep, Oslo N-0033, Norway d Dipartimento di Scienza degli Alimenti, Universita` degli Studi di Napoli Federico II, Portici 80055, Italy e Istituto di Scienze delle Produzioni Alimentari, CNR, Bari 70125, Italy

Abstract The production of mycotoxins and other metabolites by 109 strains of Fusarium langsethiae, Fusarium poae, Fusarium sporotrichioides, and F. kyushuense was investigated independently in four laboratories by liquid or gas chromatography analyses of cultural extracts with UV diode array, electron capture, or mass spectrometric detection systems. From the compiled results, it was found that F. langsethiae consistently produced the trichothecenes diacetoxyscirpenol (DAS), T-2 toxin (T-2), HT2 toxin (HT-2), and neosolaniol (NEO) and, to a lesser extent, some additional trichothecene derivatives. F. langsethiae also produced culmorins, chrysogine (CHRYS), aurofusarin (AUF), and enniatin (EN). F. sporotrichioides showed a metabolite profile similar to that of F. langsethiae, while F. poae had a different profile as 41 of 49 strains produced nivalenol (NIV) and other 8-keto trichothecenes, in addition to DAS and derivatives of this metabolite. Only a trace amount of NIV was detected from one strain of F. kyushuense. In summary, all the three core taxa of this joint study were found to produce trichothecenes. Fusarin C (F-C) was not detected from F. langsethiae, but it was produced by F. poae and F. sporotrichioides. Aurofusarin was only detected from a few strains of F. langsethiae, while nearly all strains of F. poae and F. sporotrichioides produced this compound. In contrast, chrysogine was not detected from F. poae, but was produced by the other two taxa. Production of enniatins was scattered among the three main taxa of this study, whereas beauvericin (BEA) was produced by many strains of F. poae and F. sporotrichioides. Only one odd strain of F. langsethiae (IBT 9959) produced beauvericin. However, the status of this strain is uncertain. By a polyphasic approach using species-specific metabolite profiles, the fruity odour of F. poae, and morphological observations, it was concluded that F. langsethiae, F. poae, and F. sporotrichioides should be regarded as three significant taxa at a species level. D 2003 Elsevier B.V. All rights reserved. Keywords: Fusarium; Chemotaxonomy; Mass spectrometry; Trichothecenes; Beauvericin; Enniatins; Chemical diversity

* Corresponding author. Tel.: +45-4525-2630; fax: +45-45884922. E-mail address: [email protected] (U. Thrane). 1 Deceased. 0168-1605/$ - see front matter D 2003 Elsevier B.V. All rights reserved. doi:10.1016/j.ijfoodmicro.2003.12.005

1. Introduction According to Gerlach and Nirenberg (1982), the Fusarium section Sporotrichiella comprises Fusarium

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chlamydosporum, Fusarium tricinctum, Fusarium poae, and two varieties of Fusarium sporotrichioides. The latter three species have been related to observed toxicoses in man and animals (Joffe, 1986). However, due to improper taxonomic concepts in the past, where F. tricinctum was used as a collective taxon for the section Sporotrichiella, many older reports are misapplied to this taxon (Marasas et al., 1984). Updated information on the chemical diversity of Fusarium species has been published by Thrane (2001), and it is widely accepted that the main producers of known mycotoxins within Sporotrichiella are F. sporotrichioides and F. poae. Among the mycotoxins produced by Fusarium, the largest group is the trichothecenes, of which diacetoxyscirpenol (DAS), T-2 toxin (T-2), nivalenol (NIV), fusarenon-X (FX), and deoxynivalenol (DON) are the most important ones of the more than 200 analogues described (Ueno, 1983). Trichothecenes often have been central metabolites in chemotaxonomic analyses of Fusarium (e.g., Fusarium culmorum and Fusarium graminearum) (Thrane, 1990; Miller et al., 1991) as well as of Fusarium sambucinum and related species (Thrane and Hansen, 1995). F. sporotrichioides consistently produces T-2, HT-2 toxin (HT-2), and neosolaniol (NEO) (Marasas et al., 1987; Logrieco et al., 1990; Langseth et al., 1999), in addition to a number of less abundant trichothecenes (Ishii and Ueno, 1981; Corley et al., 1987; Greenhalgh et al., 1988, 1990; Bekele et al., 1991; Fort et al., 1993). Fusarin C (F-C), butenolide, and aurofusarin (AUF) have also been reported from this species (Thrane, 2001). The main trichothecenes produced by F. poae are DAS, monoacetoxyscirpenols, scirpentriol (SCR), NIV, and FX (Pettersson, 1991; Liu et al., 1998; Torp and Langseth, 1999). Other metabolites from F. poae are F-C, AUF, and the volatile gamma-lactopyrone (Thrane, 2001). Recently, Fusarium langsethiae (as ‘powdery F. poae’) was reported to produce T-2, HT2, NEO, traces of DAS, and SCR, and a number of derivatives hereof in addition to other sesquiterpenes, the hydroxyculmorins (CULM) (Torp and Langseth, 1999). The overall profile of trichothecenes produced by F. langsethiae is very similar to that of F. sporotrichioides, whereas the morphology is similar to F. poae (Torp and Langseth, 1999; Torp and Nirenberg, 2004).

Recently, Fusarium kyushuense was described based on reexamination of four trichothecene-producing strains, which have been used widely in toxicological studies (Aoki and O’Donnell, 1998). The trichothecenes produced by this taxon are NIV, FX, and other derivatives, as well as DAS and other trichothecenes (Hedman and Pettersson, 1996; Ueno et al., 1997). Strains of this taxon were originally identified as Fusarium nivale and Fusarium episphaeria. However, they were later reidentified as F. tricinctum and F. sporotrichioides by different authors until Aoki and O’Donnell (1998) described them as F. kyushuense. They did not, however, assign this species to any Fusarium section. Beauvericin (BEA) and enniatin (EN) are well known cyclic hexadepsipeptides with specific cholesterol acyltransferase inhibitor activity (Tomoda et al., 1992). Specifically, BEA is reported toxic to several human cell lines (Logrieco et al., 2002) and can induce apoptosis accompanied by internucleosomal DNA fragmentation (Ojcious et al., 1991). Logrieco et al. (1998) reported that four strains of F. poae isolated from maize were able to produce beauvericin, whereas BEA was not detected in either of two F. sporotrichioides cultures. At present, more than 15 derivatives of EN are known (Visconti et al., 1992; Krause et al., 2001). However, within Fusarium section Sporotrichiella, ennaitins have only been detected in F. tricinctum (sensu stricto) (Burmeister and Plattner, 1987; Herrmann et al., 1996). An unidentified cyclic peptide called ‘swelling factor’ was reported from F. tricinctum (sensu lato) (Burmeister et al., 1981). However, some of the strains were later reidentified as F. sporotrichioides or F. kyushuense (Marasas et al., 1984; Aoki and O’Donnell, 1998). The aim of this study was to determine the chemical diversity of F. sporotrichioides, F. poae, F. kyushuense, and F. langsethiae by chemical analyses of cultural extracts. As each laboratory used different methods, we also wanted to compile individual results to present an overall profile of metabolites produced by the strains involved. The obtained data on the chemical diversity have been used as an integrated part of circumscriptions of fungal species (Frisvad et al., 1998) in the context of the present collaborative project on Fusarium section Sporotrichiella (Torp and Adler, 2004).

U. Thrane et al. / International Journal of Food Microbiology 95 (2004) 257–266

2. Materials and methods 2.1. Fungal isolates and growth conditions The origin of the Fusarium strains has been reported by Torp and Adler (2004) as part of the collaborative study ‘Sporotrichiella project’ under EU COST action 835. The strains were shipped to each collaborator directly from a central source (National Veterinary Institute, Oslo, Norway). The strains were grown on Spezieller Na¨hrstoffarmer Agar (SNA) (Nirenberg, 1976) under a 12-h dark/12-h light regime (cool daylight and UV black light) for morphological observations and preparation of inocula for metabolite analyses. Table 1 shows which strains were examined at each laboratory. 2.2. Chemical analysis In Denmark, the strains were analysed by highperformance liquid chromatography with diode array detection (HPLC-DAD) (Smedsgaard, 1997). The strains were inoculated on two different media: potato sucrose agar (PSA) and yeast extract sucrose (YES) agar (Samson et al., 2002). Chromatographic peaks monitored at 225 nm were characterised by their alkylphenone retention time index (RI) and their UV spectrum (200 –600 nm) (Frisvad and Thrane, 1987) and compared to metabolite standards of chrysogine (CHRYS), F-C, and AUF analysed under similar conditions. The same extracts were subsequently analysed for production of trichothecenes, detected as their pentafluoropropionoinyl esters by gas chromatography tandem mass spectrometry (GC-MS/MS) as detailed by Nielsen and Thrane (2001). Detection limits were 10 – 120 pg/Al sample injected for individual trichothecenes. The following standards were obtained from Sigma (St. Louis, MO, USA): T-2, HT-2, T-2 triol (T-2TR), T-2 tetraol (T-2TE), SCR, 15-monoacetoxyscirpenol (15-MAS), NEO, 15-acetyldeoxynivalenol (15-ADON), 3-acetyldeoxynivalenol (3-ADON), DAS, NIV, F-X, and DON. In Norway, strains were inoculated on PSA and YES and analysed for production of trichothecenes and other metabolites by gas chromatography mass spectrometry (GC-MS) of their pentafluoropropionoinyl esters as described in detail by Langseth et al. (1999) and Torp and Langseth (1999). The

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determination of the trichothecenes was only semiquantitative, and concentrations as low as about 0.1 Ag/g (wet weight) could be detected. The toxins were identified by comparing mass spectra with mass spectra obtained from the commercial standards except for 4,15-diacetylnivalenol, 15-diacetylnivalenol, dihydroxy-epiapotrichothecene (EPI-APO), and CULM, which were identified by comparing the mass spectra with the Fusarium metabolite MS database library of the National Veterinary Institute. Where metabolites are referred to by a number, this is the number the MS spectrum of the compound is assigned in this MS database library. Commercial standards of NIV, FX, DON, NEO, DAS, HT-2, and T-2 were obtained as a trichothecene standard mixture from Romer Labs (Washington, USA). SCR, 15MAS, T-2TE, T-2TR, and 3-ADON were obtained from Sigma. In Austria, analysis for in vitro mycotoxin production was carried out according to the method of Lew et al. (1991). Briefly, the Fusarium strains were grown on 50 g of maize kernels in 500-ml Erlenmeyer flasks to which 25 ml of water was added before autoclaving. The substrate was inoculated with pieces of SNA single-spore cultures containing mycelium and spores of the strain being tested. The cultures were incubated in diffuse daylight at 28 jC for 1 week and at 20 jC for 2 weeks. The culture material thus prepared was immediately stored at 20 jC until examined. Uninoculated maize was used as control. Group A trichothecenes (T-2 and HT-2) and group B trichothecenes (DON, 3-ADON, 15-ADON, and NIV) were determined by GC electron capture detection (ECD) of the heptafluorbutyryl (HFB) and trimethylsilyl (TMS) derivatives, respectively as described previously (Scott et al., 1986; Lew et al., 2001). In Italy, analysis for BEA and EN was carried out by culturing the fungal strains on 100 g of autoclaved rice, adjusted to about 45% moisture in 50-ml plastic tubes and inoculated with 0.5 ml of an aqueous suspension containing approximately 107 conidia/ ml. Cultures were incubated at 25 jC for 3 weeks in the dark. This procedure was repeated twice to account for possible variability in toxin production by fungi. The harvested culture material was dried in a forced draft oven at 60 jC for 48 h, finely ground, and stored at 4 jC until use. Controls were treated in the same way, except that they were not inoculated.

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Table 1 Metabolite profiles of F. poae, F. langsethiae, F. sporotrichioides, and F. kyushuensea Analysed byb

NIV

FX

15-MAS

DAS

SCR

T-2

HT-2

T-2TE

NEO

T-2TR

CULM

CHRYS

AUF

F-C

BEA

EN-B

EN-B1

EN-A1

F. poae BBA 62376 BBA 64321 BBA 64810 BBA 65499 BBA 65613 BBA 67664 BBA 69074 BBA 70744 BBA 70810 IBT 1766 IBT 1876 IBT 2926 IBT 2963 IBT 8452 IBT 9923 IBT 9924 IBT 9925 IBT 9926 IBT 9927 IBT 9928 IBT 9929 IBT 9930 IBT 9931 IBT 9932 IBT 9933 IBT 9934 IBT 9935 IBT 9936 IBT 9937 IBT 9938 IBT 9972 IBT 9973 IBT 9974 IBT 9975 IBT 9976 IBT 9977 IBT 9978 IBT 9979 IBT 9980 IBT 9988

a, a, a, a, a, a, a, a, a, a, c, a, a, a, a, a, a, a, a, a, a, a, a, a, a, a, a, a, a, a, a, a, a, a, a, a, a, a, a, a,

x x x x x x x x x x x x – x x x x x x x x x x – x x x x – x x x x x x x – – x x

x x x x x x x x x x x – – – x x x x x x x x x – x x x x – x x x x x x – – – x x

x – x x x x x x x x x x x x x x x x x x x x x x x x x x x x x x x x x x x – x x

x – x x x x x x x x x x x – x x x x x x x x x x x x x x x x x x x x x – x x x x

– x x x x x x x x x x – – x x x x x – – x – x – x x x x – x x x x x – – – – – x

– – – – – – – – – – – – – – – x – – – x – – – – – – – – – – – – – – x – – – – x

– – – – – – – – – – – – – – – x – – – – – – – – – – – – – – – – – – x – – – – –

– – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – x – – – – – – – –

x – – – x – – – – – – – – – – – – – – x – – – – – – x x – – – – – – – – – – x –

– – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – –

x – – – x x – x x – – x – – x x x x x – x x x – x x x x x x – – – – – – – – – –

– – – – – – – – – – na – – – – – – – – – – – – – – – – – – – – – – – – – – – – –

x x x x x x x x – – na x x x – x x – x – x x x – x x – x x x x x x x x x x x x x

– – x (x)c x x x – – (x) na x x (x) (x) – (x) x x (x) x – – x (x) (x) x x x x x x x (x) (x) (x) x (x) (x) (x)

na x x na na – – x x – – – – – – – – – – – x x x x x x – x x x x – x x x – x x – –

na tr tr na na tr x tr tr tr – – tr tr tr – – – – – x tr tr tr – x – tr tr tr tr tr tr tr tr tr x tr tr tr

na tr tr na na tr tr – tr tr – tr tr tr tr – – – – – tr x – x – – – – – – tr tr x tr tr tr tr tr – –

na – – na na – tr – tr – – – – tr – – x – tr – – – – – – – – – – – – – – tr – – – – tr –

c, d c, d, b, c, b, c, b, c, b, c, b, c, b, c, b, c, c, d, d, e c, d, c, d, c, d, b, c, b, c, b, c, b, c, b, c, b, c, b, c, b, c, b, c, b, c, b, c, b, c, b, c, b, c, b, c, b, c, b, c, b, c, b, c, b, c, b, c, b, c, b, c, b, c, b, c, b, c,

e d, d d d, d, d, d, e e e e d, d, d, d, d, d, d, d, d, e e e e e e e e e e e e e e e e d,

e

e e e e

e e e e e e e e e

e

U. Thrane et al. / International Journal of Food Microbiology 95 (2004) 257–266

Isolate number

a, a, a, a, a, a, a, a, a,

b, b, b, b, b, b, b, b, b,

c, c, c, c, c, c, c, c, c,

d, d, d, d, d, d, d, d, d,

F. langsethiae IBT 8051 IBT 8052 IBT 9921 IBT 9922 IBT 9951 IBT 9952 IBT 9953 IBT 9954 IBT 9955 IBT 9956 IBT 9957 IBT 9958 IBT 9959 IBT 9960 IBT 9961 IBT 9989 IBT 9990 IBT 40005 IBT 40007 IBT 40008 IBT 40009 IBT 40010 IBT 40011

a, a, a, a, a, a, a, a, a, a, a, a, a, a, a, a, a, a, a, a, a, a, a,

c, d, c, d, b, c, b, c, b, c, b, c, b, c, b, c, b, c, b, c, b, c, b, c, b, c, b, c, b, c, b, c, b, c, b, c, b, c, b, c, b, c, b, c, c, d

e e d, d, d, d, e e e e e e e e d, d, d, d, d, d, d, d,

F. sporotrichioides BBA 62425 a, BBA 62426 a, BBA 64160 a, BBA 69072 a, BBA 69073 a, BBA 70746 a, IBT 1906 a, IBT 1926 c,

b, c, b, c, c, d, c, d, c, d, c, d, c, d, d, e

e e e e e e e

e e e e e e e e e

e e e e

e e e e e e e e

x x – x x x x – –

x x – x x x x – –

x x – x x x x – x

x x x x x x x x x

x x – x x – x – x

– – – – – – – – –

– x – – – – – – –

– – – – – – – – –

– x – – – – – – –

– – – – – – – – –

– – – – x x x – x

– – – – – – – – –

x x x – x x x x x

x – x x – (x) (x) (x) x

x x – – – x x x –

x tr tr – – tr – tr –

tr tr – tr – – – tr tr

– – – – – – – tr –

– – – – – – – – – – – – – – – – – – – – – – –

– – – – – – – – – – – – – – – – – – – – – – –

– – x x x – – – – x – x – – – x x – – x x – –

x x x x x x x x x x x x x x x x x x x x x x x

x – x – – – – – – x – – – – – – – x x x x x x

x x x x x x x x x x x x x x x x x x x x x x x

x x x x x x x x x x x x x x x x x x x x x x x

x – x – x x x x x x x x x – x x x x x x x x x

x x x x x x x x x x x x x x x x x x x x x x x

– – x – x x x x x x x x x x – – – x x x – x –

x x x – x – – x x x x x x x x – – x x x x x x

– – – – x – – x x x x x – x – x x x – – – – x

– x – – – x x – – – – – – – – – – – – x x x –

– – – – – – – – – – – – – – – – – – – – – – –

– – – – – – – – – – – – x – – – – – – – – – na

tr tr tr tr – x x – – tr tr – tr – x x tr tr tr tr tr tr na

– tr tr x – – tr – – – – – tr – tr tr – – – tr tr tr na

– tr – – – – – – – – – – tr – – – – – – – – tr na

– – – – – – – –

– – – – – – – –

x x – x – – x x

x x x x x x x x

– – – – – – x –

x x x x x x x x

x x x x x x x x

x x x x x x x x

x x x x x x x x

x x x – – – x x

– – – – – – x –

– – – – – x – na

x – x x x x x na

(x) – – – – – – na

x x x – – – – x

tr tr – tr tr tr tr tr

tr tr tr – – – – tr

– – tr – – – – – 261

(continued on next page)

U. Thrane et al. / International Journal of Food Microbiology 95 (2004) 257–266

IBT 9991 IBT 40006 ITEM 2349 ITEM 2350 ITEM 2351 ITEM 2352 ITEM 2353 ITEM 2354 ITEM 2355

262

Table 1 (continued) Analysed byb

NIV

FX

15-MAS

DAS

SCR

T-2

HT-2

T-2TE

NEO

T-2TR

CULM

CHRYS

AUF

F-C

BEA

EN-B

EN-B1

EN-A1

IBT IBT IBT IBT IBT IBT IBT IBT IBT IBT IBT IBT IBT IBT IBT IBT IBT IBT IBT IBT IBT IBT IBT IBT IBT IBT IBT

a, a, a, a, a, a, a, a, a, a, a, a, a, a, a, a, a, a, a, a, a, a, a, a, a, a, a,

– – – – – – – – – – – – – – – – – – – – – – – – – – –

– – – – – – – – – – – – – – – – – – – – – – – – – – –

x x – x – x x x x x – x x x x x – x x x x x – – x x x

x x x x x x x x x x x x x x x x x x x x x x x x x x –

– – x – – – – – – – – x – – x – – x – x x – – – – – –

x x x x x x x x x x x x x x x x x x x x x x x x x x x

x x x x x x x x x x x x x x x x x x x x x x x x x x x

x x x – – x x x – – x x x x x x x – x – – – x x x x x

x x x x x x x x x x x x x x x x x x x x x x x x x x x

– – x x x x x x x x – x x x x x x x x x x x x x – x –

– x x – – – – – – – x – x – – – – – – – – – – – – – –

– – – – – – – – – – – – – – – – – – – – – – – – – – –

x x x x x x x x x x x x x x x x x x x x x x x x x x x

– – x – – – – – – – – – – – – – – – (x) – (x) (x) – – – – –

x x – – x x x – x x x – – x x x x – x – x – – – – – x

tr tr tr tr tr – tr – – tr – – tr – – tr – tr – – – tr tr tr – – tr

– tr – – – – tr – – – – – tr – tr – – – tr tr – – tr tr – – tr

– – tr tr – – – – – – – – tr – – – – – – – – – – tr – – tr

– tr

– –

– –

– –

– –

– –

– –

– –

– –

– –

– –

– –

x –

– –

– –

tr tr

tr tr

– –

1929 8117 8804 9941 9942 9943 9944 9945 9946 9947 9948 9949 9950 9962 9963 9964 9965 9966 9967 9968 9969 9970 9971 40001 40002 40003 40004

F. kyushuense BBA 70812 BBA 70813 a

c, d, c, d, c, d, b, c, b, c, b, c, b, c, b, c, b, c, b, c, b, c, b, c, b, c, b, c, b, c, b, c, b, c, b, c, b, c, b, c, b, c, b, c, b, c, b, c, b, c, b, c, b, c,

e e e e e e e e e e e e e e e e e e e e e e e d, d, d, d,

a, c, d, e a, c, d, e

e e e e

See text for abbreviations of metabolites. Symbols: (x) detected; ( – ) not detected; tr, detected at trace levels; na, not analysed. a = HPLC-DAD at BioCentrum-DTU, Kgs. Lyngby, Denmark; b = GC-MS/MS at BioCentrum-DTU, Kgs. Lyngby, Denmark; c = GC-MS at National Veterinary Institute, Oslo, Norway; d = GC-ECD at Austrian Agency for Health and Food Safety, Agrobiology Linz, Austria; e = HPLC-UV at Universita` degli Studi di Napoli ‘‘Federico II,’’ Parco Gussone Portici, Italy. c Indicates derivatives of F-C detected as only peaks with UV spectra similar to F-C but at a different retention time were detected. b

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Isolate number

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Ten grams of each sample was grounded and homogenized in an Ultraturrax for 3 min with 50 ml of pure methanol (99.5%). Samples were filtered through Whatman No. 4 filter paper, and methanol was removed under reduced pressure. This extraction procedure yielded raw organic extract that was used to quantify BEA, enniatin B (EN-B), enniatin B1 (EN-B1), and enniatin A1 (EN-A1). The extracts were resuspended in 3 ml of methanol and prepurified by C18 column (Varian), which was activated with 3 ml of methanol before the extract was loaded and eluted with 2 ml of methanol. The purified solution was concentrated to 1 ml and then filtered through an Acrodisk filter (pore size, 0.22 Am) before HPLC and 20 Al was loaded onto column. Analyses for BEA and enniatins (B, B1, and A1) were performed as previously described (Monti et al., 2000) with minor modifications. The standards of BEA and enniatin mixture (EN-B 19%, EN-B1 54%, EN-A1 20%, and EN-A 3%). were purchased from Sigma. The detection limit was 0.1 Ag/g for BEA and EN.

3. Results The results from the four laboratories in Austria, Norway, Italy, and Denmark have been transformed into qualitative detection of metabolites and are presented in Table 1. In general, there are no conflicting results from the individual laboratories; however, the quantitative results on trichothecenes vary between the laboratories. There was no trend in the results as high-yielding strains in one laboratory may not be high-yielding strains in another laboratory due to different cultivation methods used by the collaborators (data not shown). All of the 23 strains of F. langsethiae produced the trichothecenes DAS, T-2, HT-2, and NEO, whereas the related derivatives T-2TR and T-2TE have been detected from 15 and 20 strains, respectively. Both SCR and 15-MAS have been detected from nine strains. NIV and FX were not detected from any strains of F. langsethiae. In addition to trichothecenes, 18 strains produced culmorins, 11 strains produced CHRYS, and 6 strains produced the red pigment AUF. Enniatins B, B1, and A1 were detected from 17, 10, and 3 strains, respectively; however, they were detected often at trace levels. Only one strain of F.

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langsethiae (IBT 9959) produced beauvericin and it proved to be the highest amount (555 mg/kg) of all 109 strains examined in this study. No strains of F. langsethiae produced F-C or its derivatives in detectable quantities. The trichothecenes NEO, T-2, and HT-2 were detected from all 35 strains of F. sporotrichioides. DAS was detected from all but one strain, and each of T-2TR and T-2TE was detected by 27 strains. From 26 strains, 15-MAS was detected, whereas SCR has been detected from seven strains. NIV and FX were not detected from any strains of F. sporotrichioides. AUF was detected from all but two strains, and culmorins, F-C, and CHRYS from five, five, and one strain, respectively. The enniatins B, B1, and A1 were detected at trace levels from 21, 13, and 6 strains, respectively, and BEA from 19 strains. F. poae is a good producer of the 8-keto-trichothecenes (type B trichothecenes) NIV and FX, which in this study were found from 41 and 38 of 49 strains examined. Most F. poae strains also produced DAS, 15-MAS, and SCR (46, 45, and 33 strains, respectively). However, metabolites of the T-2 type (T-2, HT-2, and T-2TE) were only found from four, three, and one strain, respectively. T-2TR was not detected. Nearly all F. poae strains produced AUF (40 strains) and F-C (39 strains), and half of strains produced culmorins. BEA was detected from 24 strains of F. poae and the enniatins B, B1, and A1 were detected at trace levels from 33, 26, and 8 strains, respectively. From the two strains of F. kyushuense, only enniatins B and B1 were detected at trace levels and one of the strains also produced AUF while the other produced trace amounts of NIV. All other known metabolites included in this study were not detected.

4. Discussion The compiled data on metabolite production show clearly that F. langsethiae has a profile of known compounds quite similar to that of F. sporotrichioides, as published by Torp and Langseth (1999) using ‘powdery F. poae’ as a temporary epithet for F. langsethiae. Both F. langsethiae and F. sporotrichioides are good producers of T-2 toxin and its derivatives (NEO, HT-2, T-2TR, and T-2TE) and also DAS and 15-MAS. This is to be expected as DAS is co-occurring in many

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T-2 producers because DAS biosynthetically is at a side branch of the T-2 toxin pathway (Desjardins et al., 1993). On the other hand, DAS and 15-MAS are among the major type A trichothecenes of F. poae and only a few strains of this taxon can produce T-2 and its derivatives. Torp and Langseth (1999) speculated that all T-2-producing strains of F. poae could be F. langsethiae. However, the present polyphasic collaborative study shows that some strains of F. poae sensu stricto can produce T-2 in low amounts. Currently, the detailed regulation of T-2 production by F. poae is unknown. In addition, our results show that of these three taxa, only F. poae is able to produce the type B trichothecenes NIVand FX, which is in agreement with previous reports (Pettersson, 1991; Liu et al., 1998; Torp and Langseth, 1999). We did not detect any trichothecenes from the extype strain of F. kyushuense BBA 70812 = Fn-2, which is supposed to be the origin of the other strain BBA 70813 (Marasas et al., 1984; Aoki and O’Donnell, 1998). From the latter strain, however, trace amounts of NIV were detected, which is in agreement with several reports (Marasas et al., 1984; Hedman and Pettersson, 1996; Ueno et al., 1997) that used the original strain number Fn-2B = NRRL 6490 (Aoki and O’Donnell, 1998). From these reports, it is known that F. kyushuense can produce NIV and FX but also other trichothecenes in low amounts. However, according to Hedman and Pettersson (1996), the growth conditions have a major impact on the yield of trichothecenes from this taxon. Qualitative differences in metabolite profiles other than the trichothecenes are that F-C was not detected from F. langsethiae, while it was produced by F. poae and F. sporotrichioides, and that CHRYS was not detected from F. poae, while it was produced by the two other taxa. It should be noted that only in one F. sporotrichioides culture could CHRYS be detected. However, it has been detected in other cultures (Thrane, unpublished). The typical red pigment of many Fusaria, AUF, was only detected from a few strains of F. langsethiae, whereas nearly all strains of F. poae and F. sporotrichioides produced this compound. This is in agreement with cultural observations that many of the F. langsethiae cultures are palecoloured or light rose-coloured (Torp and Nirenberg, 2004). Production of enniatins is scattered among the three main taxa of this study, whereas beauvericin is

produced by many strains of F. poae and F. sporotrichioides and only by one strain of F. langsethiae (IBT 9959). Torp and Langseth (1999), who labelled this strain as 9822-219-1F, noted that the strain was an intermediate between F. poae and F. langsethiae. By the extended chemical data of this compiled study, IBT 9959 is judged to be F. langsethiae; however, the status of this strain has been discussed at several COST 835 meetings (unpublished). Future examinations of this strain may change its present identity. The two strains of F. sporotrichioides var. minus, BBA 62425 and BBA 62426, which were included in this study, could not be differentiated from F. sporotrichioides sensu stricto by the chemical data. From an overall evaluation of the strains by morphology and cultural characteristics, it is concluded that this variety of F. sporotrichioides should be reduced to the core taxon. In addition to Table 1, other characters of differentiation should be used, for example, morphological observations, but also another chemical character, the fruity smell, which only is recognised from cultures of F. poae. This observation has not been confirmed by any analytical detection system. Following the concept of Frisvad et al. (1998), for polyphasic fungal taxonomy using metabolite profiles, F. langsethiae seems to be more similar to F. sporotrichioides than to F. poae as reported elsewhere (Torp and Langseth, 1999). However, it should be noted that for the present study, only known metabolites have been used with a focus on the trichothecenes and, in this context, they only represent one biosynthetic pathway. There is a lot more information in the chemical data than Table 1 indicates. At an early stage of this study, the chromatographic data matrices from the HPLCDAD analyses were subjected to image analysis (Nielsen et al., 1998, 1999). These preliminary results (data not shown) using only part of the isolates showed clustering into species by this technique, where the entire chromatographic data set is used, without any subjective evaluation (identification) of peaks. In conclusion, F. langsethiae, F. poae, and F. sporotrichioides are three good taxa, with speciesspecific metabolite profiles. However, future efforts should be taken to determine a broader range of metabolites produced by these species. The status of F. kyushuense is still unclear until more freshly isolated strains have been examined.

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