World Mycotoxin Journal, May 2010; 3 (2): 121-128

WageningenAcademic P u b l i s h e r s

Use of solid phase microextraction coupled to capillary gas chromatography-mass spectrometry for screening Fusarium spp. based on their volatile sesquiterpenes
J.R. Girotti1, I. Malbrn2, G.A. Lori2 and M.P. Jurez1
1Instituto

de Investigaciones Bioqumicas de La Plata (CCT La Plata CONICET -UNLP), Facultad de Ciencias Mdicas, Universidad Nacional de La Plata, 60 y 120, La Plata 1900, Argentina; 2Centro de Investigaciones de Fitopatologa (CIDEFICIC), Facultad de Ciencias Agrarias y Forestales, Universidad Nacional de La Plata, 60 y 119, La Plata 1900, Argentina; mjuarez@isis.unlp.edu.ar Received: 30 September 2009 / Accepted: 29 March 2010 2010 Wageningen Academic Publishers

Abstract
Solid phase microextraction (SPME) coupled to capillary gas chromatography (CGC) and mass spectrometry (MS) was used to evaluate the use of fungal volatiles to discriminate Fusarium species from wheat cultivars in the Argentina pampa region. Monosporic fungal isolates were grown on rice in sealed containers for 1 week and volatile organic compounds (VOC) were sampled for 30 min from the head space by SPME and analysed by CGC and CGCMS. VOC profiles of Fusarium species F. graminearum, F. poae, F. equiseti, F. verticillioides and F. oxysporum were discriminated by comparison of their profiles in the elution zone corresponding to sesquiterpenes. Trichotheceneproducer and non-trichothecene producer Fusarium species were separated by the presence of trichodiene in their VOC fingerprints. Within trichothecene-producers, F. graminearum, F. poae and F. equiseti differed on the structure of their volatile sesquiterpenes. This technique might be also helpful to detect F. graminearum, the major head blight disease-producing fungus in the region.
Keywords: Fusarium, trichodiene, volatile organic compounds, solid phase microextraction

1. Introduction
Fungi included in the genus Fusarium are probably the most important mycotoxin producers worldwide, infecting cereals both in temperate and cold climates. The most relevant toxins are trichothecenes, zearalenone and fumonisins (Desjardins, 2006). Trichothecene toxins contaminate both human foods and animal feeds; their major toxicity mechanism is by inhibition of ribosomal protein synthesis (Desjardins et al., 1993). Many Fusarium species produce trichothecenes; these sesquiterpenes are classified based on the presence or absence of a macrocyclic ester or an ester-ether bridge between C-4 and C-15 (Chu, 1998). The non-macrocyclic components can be in turn classified into four types (A-D). Among them, type-A and type-B are by far well-studied due to their toxicity and frequent occurrence in agricultural products. HT-2, T-2 toxin and diacetoxyscirpenol are the most toxic components

of type A, whereas fusarenon-X, nivalenol (NIV), and deoxinivalenol (DON) are major type B toxins (Bennett and Klich, 2003), DON and NIV appear to aid plant infection (Harris et al., 1999; Proctor et al., 1995, 1997). In the Fusarium genus, F. graminearum and F. culmorum are the most important trichothecene-producing pathogen species infecting cereals. Within these species, three chemotypes were documented based on the chemistry of the trichothecenes produced: (Ia) DON and 3-acetyl DON, (Ib) DON and 15-acetyl DON, especially associated with America, and (II) NIV together with 4-acetyl NIV (Moss and Thrane, 2004). Fusarium species were traditionally separated based on their morphology and specific culture characteristics (Booth, 1971; Leslie and Summerell, 2006; Nelson et al., 1994). However, the taxonomy of this genus remained controversial due to the high degree of variation of these markers (Nelson et al., 1994). Multidisciplinary 121

ISSN 1875-0710 print, ISSN 1875-0796 online, DOI 10.3920/WMJ2009.1182

J.R. Girotti et al.

approaches and the use of molecular tools in phylogenetic studies led to a better consensus, and had a major impact on fungal taxonomy (Leslie and Summerell, 2006; Moss and Thrane, 2004; ODonnell et al., 2000, 2004). The use of metabolite profiling in fungal taxonomy and identification has also been addressed (Ichinoe et al., 1983; Moss and Thrane, 2004). However, the most important application of metabolite fingerprinting is to help understand and detect the contamination of food and animal feeds by mycotoxins (Frisvad et al., 2007, Thrane et al., 2004). Amongst the wide array of available methods to detect Fusarium contamination, most of them are based on the direct detection or quantization of trichothecene mycotoxins; these techniques are time consuming, and usually require solvent extraction steps (Turner et al., 2009). Trichothecene biosynthesis has been extensively studied; the sesquiterpene hydrocarbon trichodiene is known to be a key metabolic intermediate to their formation (Beremand and McCormick, 1992; Demyttenaere et al., 2004; Moss, 1984; Tamm and Breitenstein, 1980). Furthermore, based on volatile organic compounds (VOC) trapping from the head space of fungal cultures and analysis by capillary gas chromatography (CGC), Jelen and co-workers (1995, 1997) proposed trichodiene as a volatile marker for trichothecene formation; trichodiene was usually detected together with other sesquiterpene components, among other volatile metabolites. In the last decade, one of the most popular methods to measure volatile production is the use of head space (HS) solid-phase microextraction (SPME) coupled to CGC (Jelen, 2003; Lord and Pawliszyn, 2000) which has also been used to measure Fusarium volatiles (Demyttenaere et al., 2003). An electronic nose can be also used to predict the VOC and mycotoxin amounts (Olsson, 2000; Presicce et al., 2006), although the gas sensor sensitivity is unknown for different VOC and their combinations, and hence CGC and CGCmass spectrometry (MS) in tandem with an appropriate extraction method are expected to be the most useful approach to detect a wide range of VOC (Magan and Evans, 2000). CGC-MS analysis of volatile secondary metabolites has been used in Penicillium taxonomy (Larsen and Frisvad, 1994). Furthermore, HS-SPME has been proposed as the method of choice for fast screening toxigenic and nontoxigenic Penincillium roqueforti strains (Demyttenaere et al., 2003). This methodology was also suggested as a suitable monitoring technique for the detection of toxigenic species of Fusarium, with trichodiene as the volatile marker for trichothecenes (Demyttenaere et al., 2004). The aim of this study was to evaluate the utility of SPME coupled to CGC and CGC-MS to provide Fusarium VOC profiles useful to differentiate common Fusarium fungal

species isolated from infested wheat cultivar areas in Argentina.

2. Materials and methods

Fungal cultures
Fusarium species were obtained from wheat grains collected from different localities of Buenos Aires province (3354S 6057W to 3452S 5758W). Wheat grains were plated on potato dextrose agar medium (PDA) 2% (w/v) added with 250 mg/l of chloramphenicol and 600 mg/l of pentachloronitrobenzene (PCNB 75% wettable powder) and incubated at 25 C. A single spore culture was obtained from each isolate, subcultured on PDA and maintained on vaseline at 4 C. Three species were selected among trichothecene-producing fungi usually detected in wheat and corn cultivated areas; F. graminearum, Fusarium poae and Fusarium equiseti. F. graminearum produces type B trichothecene mycotoxins such as DON and NIV (Ward et al., 2002). F. poae produces type A trichothecenes, being DAS the predominant although some isolates were reported to synthesise low levels of T-2 and HT-2 toxin, and type B trichothecenes (NIV) (Thrane et al., 2004). Like F. poae, F. equiseti produces both type A (DAS) and type B (NIV) trichothecenes (Kosiac et al., 2004). Fusarium verticillioides and Fusarium oxysporum were selected within non-trichothecene producing toxigenic species. For taxonomic identification, the isolates were placed on carnation leaf agar (CLA), incubated under fluorescent light at 22 C for 7 to 14 days, and identified based on cultural features, micromorphology, and conidial ontogeny, according to the criteria of Booth (1971), Nelson et al. (1994), and Leslie and Summerell (2006). Complementary identification was performed for F. verticillioides and F. graminearum. For the former, mating population was determined by crossing the strain with the tester strains ITEM 3621 and ITEM 3622 belonging to mating population A, following the method of Klittich and Leslie (1988) with the standard tester as female parent and the uncharacterised strain as male parent. Tester strains were supplied by A. Logrieco (ISPA, Bari, Italy). Every seven days, cultures were checked for the appearance of perithecia and asci or ascospores. For F. graminearum, species-specific PCR was performed with the Fg16NF (ACA GAT GAC AAG ATT CAG CGA CA) and Fg16NR (TTC TTT GAC ATC TGT TCA ACC CA) primer pair according to Nicholson et al. (1998). Isolates used in this study were F. graminearum: UM.CIDEFI. Fg0039, UM.CIDEFI.Fg0042, UM.CIDEFI.Fg0053 and UM.CIDEFI.Fg0048; F. verticillioides: UM.CIDEFI. Fv0321, UM.CIDEFI.Fv2001, UM.CIDEFI.Fv2002, and World Mycotoxin Journal 3 (2)

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UM.CIDEFI.Fv1996; F. oxysporum: UM.CIDEFI.Fo0099, UM.CIDEFI.Fo1005, UM.CIDEFI.Fo1021, and UM.CIDEFI. Fo1145; F. poae: UM.CIDEFI.Fp0885, UM.CIDEFI.Fp0931, UM.CIDEFI.Fp0957, and UM.CIDEFI.Fp1080; F. equiseti: UM.CIDEFI.Fe1142, UM.CIDEFI.Fe1144, UM.CIDEFI. Fe1256, and UM.CIDEFI.Fe0341, from the Mycology Collection of CIDEFI. For VOC analyses, flasks of 500 ml glass with an aluminum perforated lid sealed with a rubber septum (Red septa 11 mm for 6890 GC, Agilent, Santa Clara, CA, USA) containing sterile rice (80% RH) as substrate were inoculated with a PDA agar plug containing mycelium from 7 day old monosporic cultures. Non-inoculated flasks were used as controls. Flasks were maintained at 26 C in the dark for 7 days. Four strains of each species were analysed by CGC, with 4 replicates per strain.

Volatile organic compounds measurement

VOC were sampled by HS-SPME for 30 min employing a 65 m film thickness polydimethylsiloxane/ divinylbenzene fiber (PDMS/DVB) (Supelco, Bellefonte, PA, USA). Fibers were previously conditioned according to manufacturer instructions, and reconditioned before each analysis. Analyses were performed using a Hewlett Packard 6890 gas chromatograph employing a non polar DB-5 capillary

column (30m length, 0.32 mm i.d., 0.25 m film thickness) (J&W, Folsom, CA, USA). The injector was operated in the splitless mode at 250 C, the oven temperature was programmed (40 C for 2 min, 10 C/min to 200 C, 15 C/min to 250 C, with a holding time of 5 min at the final temperature). The flame ionisation detector (FID) temperature was set at 280 C. CGC-MS analysis was performed on a HP 5975C VL Agilent mass selective detector (MSD) coupled to the CGC equipment. The MSD was set in the electron impact mode and operated at 70 eV, with the transfer line at 320 C, the ionisation chamber at 230 C, and the quadrupole set at 150 C. VOC were tentatively identified by interpretation of their mass spectral fragmentation; spectra were also compared to data from MS libraries (NIST/EPA/NIH Mass Spectral Database, 2005; Adams, 2007) as well as with spectra and Kovats retention index (KI) (Kovats, 1965) values previously reported (Demyttenaere et al., 2004; Jelen et al., 1997). Trichodiene relative abundance was estimated as the ratio between the corresponding peak area and the sum of total VOC peak areas between 13 and 16 min.

3. Results
Figure 1 shows the characteristic chromatographic profile of F. graminearum VOC. Trichodiene and other sesquiterpenes, together with minor amounts of short

2e+07 1.8e+07 1.6e+07 1.4e+07 Abundance 1.2e+07 1e+07 0.8e+07 0.6e+07 0.4e+07 0.2e+07 0 2 4

2 1 5 10 34 7 6 9 8 11

12

13.0

13.5

14.0

14.5

15.0

15.5

16.0

10

12

14

16

Time (min)

Figure 1. Total ion chromatogram of the volatile compounds released by Fusarium graminearum, after SPME extraction. The inset figure shows the amplified zone corresponding to peaks eluting between 13 and 16 min. Tentatively identified sesquiterpenes with KI values between parentheses: (1) longifolene (1415); (2) barbatene (1420); (3) thujopsene (1439); (4) isobazzanene (1444); (5) (E)--farnesene (1453); (6) -santelene (1463); (7) 10-epi--acoradiene (1473); (8) -chamigrene (1480); (9) aryl-curcumene (1484); (10) -himachalene (1509); (11) -bisabolene (1513); (12) trichodiene (1533). World Mycotoxin Journal 3 (2) 123

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chain alcohols, esters, ketones, and hydrocarbons were evident. The sesquiterpene fraction eluted between 13 and 16 min (Figure 1 inset); compounds exhibited the characteristic mass spectra for C15H24 hydrocarbons, with molecular ion m/z 204. The major fungal volatile detected in F. graminearum eluted with KI 1533 and a fragmentation pattern (m/z 109 (100), 108 (46.6), 67 (33.9), 93 (30.1), 81 (17.7), 55 (11.4), 41 (13.4) 121 (8.7) and 204 (2.6)) agreeing with the reported spectrum of trichodiene (Jelen et al., 1997; Demyttenaere et al., 2004). Figure 2A shows Fusarium VOC fingerprints between 13 and 16 min, for the 5 species studied. The presence of the marker peak trichodiene was detected in F. poae, F. equiseti and F. graminearum. No trichodiene was detected both in F. verticillioides and F. oxysporum isolates. Among the trichodiene producers, significantly different (P<0.001) relative amounts were detected in trichodiene. Figure 2B shows the relative abundance of major peaks. Four strains of each species were analysed by CGC, with 4 replicates per strain; numerical values are shown in Table 1.

In F. poae and F. equiseti (type A, B trichothecenesproducing strains), unknown components eluting with KI values 1420, 1439 and 1463 were structurally different than barbatene (KI 1420), thujopsene (KI 1439), and -santelene (KI 1463), detected in F. graminearum (type B trichothecene-producing strains) (Figure 2A). In addition, one of the 2 major peaks of these species (Table 1), eluting with KI 1444 (m/z 161 (100), 105 (25.5), 119 (17.9), 91 (12.5), 93 (12.2) and 204 (1.9)), did not show a good match with library data for sesquiterpenes, and is different from isobazzanene (KI 1444) detected in F. graminearum. In the experimental conditions, F. poae and F. graminearum isolates produced much larger amounts of VOC than the other species, as shown by the intensity response (y axis). Figure 3 shows the mass spectra of major unknown components.

4. Discussion
Volatile metabolite profiles have already been associated to fungal toxigenicity, as shown in aflatoxigenic strains of Aspergillus flavus (Zeringue Jr. et al., 1993). Using purge and trap extraction, trichodiene was proposed as

Table 1. Relative abundance of major sesquiterpenes detected in Fusarium verticillioides, Fusarium oxysporum, Fusarium poae and Fusarium equiseti. Four strains of each species were analysed by CGC and CGC-MS (n=4).
KI1 1415 1420 1425 1429 1434 1439 1444 1453 1463 1473 1480 1481 1482 1484 1490 1501 1506 1509 1513 1533
1 KI:

F. verticillioides2 2.371.03 0.000.00 3.570.58 8.421.00 0.831.10 11.151.33 8.140.94 2.461.03 1.350.83 1.290.71 0.000.00 4.500.66 2.441.38 2.563.24 7.281.76 0.690.91 0.000.00 2.131.10 7.002.03 0.000.00

F. oxysporum 0.480.67 2.631.97 1.931.17 10.613.15 4.343.18 7.863.33 5.482.05 2.961.16 0.971.03 31.217.42 0.000.00 6.443.99 8.368.47 1.401.57 2.813.06 0.920.67 0.610.54 8.476.72 1.451.07 1.081.52

F. poae 0.660.32 1.070.48 7.605.93 29.0516.40 4.858.17 4.800.68 19.9210.89 0.451.42 7.982.68 6.841.25 1.541.13 1.901.17 1.310.92 0.791.12 0.380.43 2.621.39 1.270.58 1.410.53 3.945.23 1.391.26

F. equiseti 0.380.35 1.230.92 0.000.00 29.476.47 0.000.00 3.810.97 29.6118.73 5.551.92 0.000.00 10.956.45 1.950.85 0.810.69 1.111.31 1.821.74 0.000.00 1.450.61 1.280.71 1.370.35 0.000.00 8.887.40

Kovatz Indices. correspond to mean values SD of major components. Peaks with KI 1415, 1453, 1473, 1480, 1484, 1513, and 1533 correspond to peaks detailed in Figure 1, except for an unknown peak eluting with KI 1533 in F. oxysporum. In the four species, peaks eluting with KI 1420, 1439, 1444, and 1463 shared a different structure than those with same KI in F. graminearum. The fragmentation pattern of unknown peaks of KI 1425, 1429, 1434, 1481, 1482, 1490, 1501, and 1506 is shown in Figure 3.
2 Numbers

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A pA 500 Detector response F. graminearum 400 300 200 100 0 pA 50 Detector response F. verticillioides 45 40 35 30 25 pA 120 Detector response F. oxysporum
KI 1501 KI 1473 KI 1539 KI 1533

60 50 Relative abundance 40 30 20 10 0 50 Relative abundance 40 30 20 10 0 50 Relative abundance 40 30 20 10 0

100 80 60 40 20
KI 1429

KI 1490

pA 1000 Detector response 800 600 400 200 0 pA 70 Detector response 60 50 40 30 20 13 13.5

KI 1444

60 Relative abundance Relative abundance

KI 1481

50 40 30 20 10 0

F. poae

KI 1463 KI 1481

50
KI 1425

40 30 20 10 0 1420 1440 1460 1480 1500 1520 1540 KI value

F. equiseti

KI 1434

14

14.5 Time (min)

15

15.5

min

Figure 2. Volatile compounds of Fusarium spp. (A) Representative capillary gas chromatographic fingerprints. KI values of major peaks are marked. (B) Relative abundance of major peaks ( SD). Four strains of each species were analysed by CGC, with 4 replicates per strain. Numerical values are shown in Table 1. World Mycotoxin Journal 3 (2) 125

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136.1 121.1 93.0 107.1

119.1

KI 1425
105.1

KI 1429 Abundance

Abundance

161.1

55.1 149.1 161.1173.1 189.1 30 40 50 60 70 80 90 100 110 120 130 140 150 160 170 180 190 200 119.1

41.1

79.0

204.2

91.1 41.0 77.0

136.1

204.2

55.1 175.1 189.1 30 40 50 60 70 80 90 100 110 120 130 140 150 160 170 180 190 200 161.1

KI 1434
105.1 119.1

KI 1439 Abundance
105.1 91.0 133.1 147.1 204.2

Abundance

41.1 55.1

69.0

204.2 175.1 189.2 30 40 50 60 70 80 90 100 110 120 130 140 150 160 170 180 190 200 55.0 77.0 133.0 147.2 161.1

41.1

91.0

161.1

175.1 189.1 30 40 50 60 70 80 90 100 110 120 130 140 150 160 170 180 190 200 119.1

KI 1444 Abundance Abundance

93.0

KI 1481

105.1

91.0 77.0 133.1 146.1 41.0 55.0 175.1 189.1 204.2 30 40 50 60 70 80 90 100 110 120 130 140 150 160 170 180 190 200 119.1

119.1

41.0 55.0

79.0 106.0 134.1 147.1 161.1

204.1

189.1 175.1 30 40 50 60 70 80 90 100 110 120 130 140 150 160 170 180 190 200 119.1 93.0

KI 1482 Abundance

KI 1490

Abundance

105.0 91.0 41.0 55.1 77.0

132.0 145.1 161.1 204.2

41.0 55.1

77.0

106.0 161.1

175.2 189.1

189.1

204.2

30 40 50 60 70 80 90 100 110 120 130 140 150 160 170 180 190 200 161.1

132.9 175.2 147.1 30 40 50 60 70 80 90 100 110 120 130 140 150 160 170 180 190 200 69.1 93.0

KI 1501 Abundance
105.1

KI 1506 Abundance
41.1

147.1 175.1 189.2 30 40 50 60 70 80 90 100 110 120 130 140 150 160 170 180 190 200

41.1 55.1

91.0 77.0

119.1

133.1

204.2

119.1 106.0 55.0 134.1 147.1

161.1 175.1 189.1

204.2

30 40 50 60 70 80 90 100 110 120 130 140 150 160 170 180 190 200

Figure 3. Mass spectra of major unknown peaks detected in Fusarium verticillioides, Fusarium oxysporum, Fusarium poae and Fusarium equiseti.

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a contamination marker to differentiate trichotheceneproducing from non-trichothecene producing isolates of Fusarium sambucinum, easily distinguishable based in their chromatographic fingerprints (Jelen et al., 1995, 1997). Later on, HS-SPME combined to CGC-MS was proposed for monitoring trichothecene production by F. sambucinum, F. sporotrichioides and F. graminearum (Demyttenaere et al., 2004). In our study, the fungal population of Fusarium isolated from wheat grains was clearly separated based on the presence (F. graminearum, F. poae, F. equiseti) or absence (F. oxysporum and F. verticillioides) of trichodiene. Within trichothecenes producers, F. poae and F. equiseti were separated from F. graminearum by their relative amounts of trichodiene (peak 12); this compound was the predominant peak in F. graminearum. Furthermore, sesquiterpene major peaks, other than trichodiene, were structurally different in both groups. F. poae isolate produced significantly larger amounts of sesquiterpenes (3-fold) than F. equiseti. Both isolates of non-trichothecene producing fungi (F. verticillioides and F. oxysporum) released relatively smaller amounts of volatiles with a similar sesquiterpene pattern; the major peak was tentatively identified as 10-epi--acoradiene. The sesquiterpene pattern is shown to help detecting trichothecene producing Fusarium spp., and to differentiate members of this group. In particular F. graminearum, the major responsible for Fusarium Head Blight in the Argentina Pampa region, with no reports on non-toxigenic isolates. This study provides further evidence of the use of SPME-CGC analyses of Fusarium VOC. Although there is a previous report on trichodiene detection in naturally infected grain using an electronic nose (Perkowski et al., 2008); this is the first study of Fusarium VOC, obtained from fungi collected from naturally infected wheat cultivars, measured by SPMECGC. Additional studies should be addressed to establish the optimal measurement conditions to detect mycotoxin producing fungi in stored grains. Further studies should be addressed in order to evaluate the potential of SPME-CGC analysis of Fusarium VOC in chemotaxonomic studies.

Beremand, M.N. and McCormick, S.P., 1992. Biosynthesis and regulation of trichothecene production by Fusarium species. In: Bhatnagar, D., Lilehoj, E.B. and Arora, D.K. (eds.) Handbook of applied mycology: mycotoxins in ecological systems. Marcel Dekker Inc., New York, NY, USA, pp. 360-389. Booth, C., 1971. The genus Fusarium. Commonwealth Mycological Institute, Kew, UK. 237 pp. Chu, F.S., 1998. Mycotoxins-occurrence and toxic effect. In: Sadler, M., Strain J.J. and Caballero B. (eds.) Encyclopedia of human nutrition. Academic Press, New York, NY, USA, pp. 858-869. Demyttenaere, J.C.R., Moria, R.M. and Sandra, P., 2003. Monitoring and fast detection of mycotoxin-producing fungi based on headspace solid-phase microextraction and headspace sorptive extraction of the volatile metabolites. Journal of Chromatography A 985: 127-135. Demyttenaere, J.C.R., Moria, R.M., De Kimpe, N. and Sandra, P., 2004. Use of headspace solid-phase microextraction and headspace sorptive extraction for the detection of the volatile metabolites produced by toxigenic Fusarium species. Journal of Chromatography A 1027: 147-154. Desjardins, A.E., 2006. Fusarium mycotoxins. In: Desjardins, A.E. (ed.) Fusarium mycotoxins. Chemistry, genetics and biology. The American Phytopathological Society, St. Paul, MN, USA, pp. 11-138. Desjardins, A.E., Honh, T.M. and McCormick, S.P., 1993. Trichothecene biosynthesis in Fusarium species: chemistry, genetics, and significance. Microbiological Reviews 57: 595-604. Frisvad, J.C., Andersen, B. and Thrane, U., 2007. The use of secondary metabolite profiling in chemotaxonomy of filamentous fungi. Mycological Research 112: 231-240. Harris, J.L., Desjardins, A.E., Plattner, R.D., Nicholson, P., Butler, G., Young, J.C., Weston, G., Proctor, R.H. and Hohm, T.M., 1999. Possible role of trichothecene mycotoxins in virulence of Fusarium graminearum on maize. Plant Disease 83: 954-960. Ichinoe, M., Kurata, H., Sugiura, Y. and Ueno, Y., 1983. Chemotaxonomy of Gibberella zeae with special reference to production of trichothecenes and zearalenone. Applied and Environmental Microbiology 46: 1364-1369. Jelen, H.H., 2003. Use of solid phase microextration (SPME) for profiling fungal volatile metabolites. Letters in Applied Microbiology 36: 263-267. Jelen, H.H., Latus-Zietkiewicz, D., Wasowicz, E. and Kaminski, E., 1997. Trichodiene as a volatile marker for trichothecenes biosynthesis. Journal of Microbiology Methods 31: 45-49. Jelen, H.H., Mirocha, C.J., Wasowicz, E. and Kaminski, E., 1995. Production of volatile sesquiterpenes by Fusarium sambucinum strains with different abilities to synthesize trichothecenes. Applied and Environmental Microbiology 61: 3815-3820. Klittich, C.J.R. and Leslie, J.F., 1988. Nitrate reduction mutant of Fusarium moniliforme (Gibberella fujikuroi). Genetics 118: 417-423. Kosiak, B., Torp, M., Skjerve, E. and Andersen, B., 2004. Alternaria and Fusarium in Norwegian grains of reduced quality a matched pair sample study. International Journal of Food Microbiology 93: 51-62. Kovats, E., 1965. Gas chromatographic characterization of organic substances in the retetion index system. Advances in Chromatography 1: 229-247.

Acknowledgements
This work was supported by the National Agency for Science and Technology Promotion in Argentina (PICT 2004-20-25479). M.P. Jurez is a member of CONICET Researchers Career. G.A. Lori is a member of the CIC Researchers Career.

References
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