PCR detection assays for the trichothecene-producing species Fusarium graminearum, Fusarium culmorum, Fusarium poae, Fusarium equiseti and Fusarium sporotrichioides

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Systematic and Applied Microbiology 28 (2005) 562–568

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PCR detection assays for the trichothecene-producing species

Fusarium graminearum, Fusarium culmorum, Fusarium poae,Fusarium equiseti and Fusarium sporotrichioides

Miguel Juradoa, Covadonga Vazquezb, Belen Patinob, M. Teresa Gonzalez-Jaena,�

aDepartment of Genetics, Faculty of Biology, University Complutense of Madrid, Jose Antonio Novais 2, 28040 Madrid, SpainbMicrobiology III, Faculty of Biology, University Complutense of Madrid, Jose Antonio Novais 2, 28040 Madrid, Spain

Received 18 January 2005

Abstract

Contamination of small-grain cereals with the fungal species Fusarium graminearum, F. culmorum, F. poae, F.

sporotrichioides and F. equiseti is an important source of trichothecenes, Zearalenone and other mycotoxins whichcause serious diseases in human and animals. Additionally, these species contribute to Fusarium Head Blight, a diseasewhich produces important losses in cereal yield. Early detection and control of these Fusarium species is crucial toprevent toxins entering the food chain and a useful tool in disease management practices. We describe the developmentof specific PCR assays to F. graminearum, F. culmorum, F. poae, F. sporotrichioides and F. equiseti using DNA frompure fungal cultures as well as from naturally infected wheat seeds, using in this case a rapid and easy protocol forDNA isolation. The specific primers were designed on the basis of IGS sequences (Intergenic Spacer of rDNA), amulticopy region in the genome that permits to enhance the sensitivity of the assay in comparison with PCR assaysbased on single-copy sequences.r 2005 Elsevier GmbH. All rights reserved.

Keywords: Toxins; Fusarium graminearum; F. culmorum; F. equiseti; F. sporotrichioides; F. poae; Wheat; PCR

Introduction

The genus Fusarium includes a diverse group ofwidespread phytopathogenic fungi, including severalspecies able to produce a number of highly toxiccompounds. Among the mycotoxins produced byFusarium, trichothecenes are especially important, asthey are potent inhibitors of eukaryotic protein synthesis[5]. Trichothecenes can cause a wide range of acute and

e front matter r 2005 Elsevier GmbH. All rights reserved.

apm.2005.02.003

ing author. Tel.: +34913 944 830;

4 844.

ess: [email protected] (M. Teresa Gonzalez-Jaen).

chronic effects in humans and animals [4] throughingestion of food and feed prepared from cereal cropscontaminated with the toxins [3]. The effects includeskin inflammation, digestive disorders, tachycardia,oedema, and haemorrhages in several internal organs,haemolytic disorders, impairment of immune responses,and nervous disorders [7]. Chemically, trichothecenesare a large group of sesquiterpenes epoxides, and can becharacterized by the presence (type A trichothecenes) orabsence (type B trichothecenes) of a keto group at the C-8 position. Fusarium graminearum and F. culmorum

(type B trichothecene-producers), F. sporotrichioides, F.

poae and F. equiseti (type A trichothecene-producers)

ARTICLE IN PRESSM. Jurado et al. / Systematic and Applied Microbiology 28 (2005) 562–568 563

are among the main trichothene-producing Fusarium

species. All of these species are common fungalpathogens of cereals, and cause Fusarium head blightin small-grain cereals and ear rot in maize, beingresponsible for losses yearly due to lower yield andquality of the grain. These species may differ inpathogenicity, host preference, geographical distributionand, what is even more important, in their mycotoxinprofiles, including not only thrichothecenes but othertoxins such as zearalenone, zearalenol and beauvericine.Therefore, there is a need for a rapid and preciseidentification of trichothecene-producer species ofFusarium.

Conventional methods to assess mould presence arelabour and time-consuming, and they are particularlycomplex in Fusarium, since the genus is diverse, presentsintraspecific variability, and conflicting taxonomy [19].Several studies have been recently published on thephylogenetic relationships of those species using com-bined gene genealogies and polyphasic approaches[9,16,18,20]. The results confirmed the monophyleticcharacter of some of those species and the existence oflineages, as in the case of F. graminearum [16] whichshould be taken into account to develop reliablediagnostic methods.

The polymerase chain reaction (PCR) is a rapidand specific method, and its high sensitivity allowsdetection of target DNA molecules in a complexmixture, offering an alternative to microbiologicalconventional procedures in fungal diagnostic. Severalspecies-specific primer assays have been developedto detect some of the trichothecene-producing speciesof Fusarium, although in many cases, the sampleof the strains tested has not taken into account theexistence of lineages or subpopulations within thespecies or other Fusarium species commonly associatedto cereals which apparently are not closely related[6,14,15].

The use of multicopy target sequences to developspecific primers enhances the sensitivity of the assaysince it reduces the amount of DNA template necessaryfor PCR amplification and simplifies detection protocolsof infected plant tissue material. PCR protocols basedon spacers of rDNA, IGS (Intergenic Spacer of rDNAunit) and ITS (Internal Transcribed Spacer of rDNAunit) have been reported for Fusarium species[2,10,11,17].

The objective of this work was to develop specificprimers, and the corresponding PCR assays, to detectthe main trichothecene-producing species of Fusarium

associated to cereals, F. graminearum, F. culmorum,F. equiseti, F. poae and F. sporotrichioides, on the basisof sequences of the IGS region. The IGS region containshigh levels of sequence variability among species of thesame genus and allows differentiation of closelygenetically related species [12,13] in contrast with ITS

region which may have insufficient polymorphism topermit the design of robust assays [15].

Material and methods

Fungal isolates and culture conditions

The strains used in this study are described in Table 1.Cultures were maintained on potato dextrose agarmedium (PDA) (Scharlau Chemie, Barcelona, Spain)at 4 1C and stored as spore suspensions in 15% glycerolat �80 1C. Submerged fungal cultures were obtained byinoculating 100ml Erlenmeyer flasks containing 20ml ofSabouraud liquid medium (Scharlau Chemie, Barcelo-na, Spain) with mycelial disks excised from the marginof 7-day-old PDA cultures. Cultures were incubated ona rotary shaker (150 r.p.m.) at 25 1C for 7 days.Mycelium was filtrated through Whatman paper 1,frozen with liquid nitrogen and kept at �80 1C for DNAextraction.

DNA extraction and sequencing of IGS region

Total genomic DNA from fungal cultures wasextracted using the Genomix DNA Extraction Kit(Talent, Trieste, Italy), according to the manufacturer’sinstructions. The IGS region of nuclear ribosomal DNAfrom Fusarium strains was amplified using PCR primersCNL12 and CNS1, located in the 28S and 18S genes [1].Amplification reactions were carried out in volumes of25ml containing 25 ng of template DNA in 3ml, 1.25ml ofeach primer (20mM), 0.2ml of Taq DNA polymerase(5U/ml), 2.5 ml of 10�PCR buffer, 1 ml of MgCl2(50mM), and 0.25ml of dNTPs (100mM) supplied bythe manufacturer (Ecogen, Barcelona, Spain). PCR wasperformed in a thermocycler (Eppendorf MastercyclerGradient, Eppendorf, Hamburg, Germany) with thefollowing conditions: 1 cycle of 85 s at 94 1C, 35 cycles of35 s at 95 1C (denaturalization), 55 s at 58 1C (annealing),2min at 72 1C (extension), and 1 cycle of 10min at 72 1C.Amplification products were checked by electrophoresison 1% agarose ethidium bromide gels in 40mMTris–acetate and 1.0mM EDTA 1� buffer. PCR pro-ducts generated were purified using the High Pure PCRProduct Purification Kit (Roche, Mannheim, Germany)and cloned into the cloning vector pCR4-TOPO with theTOPO TA Cloning Kit for Sequencing (Invitrogen, UK).Plasmids were sequenced in both directions in the ABI3700 DNA Sequencer in the Sequencing Service Unit ofthe Centro Investigaciones Biologicas, CSIC (Madrid,Spain), and in the Genomic Unit of the UniversidadComplutense of Madrid (Madrid, Spain). The IGSsequences were deposited in the EMBL database underaccession numbers AJ862327, AJ854656, AJ854657,AJ854658, AJ854659 and AJ854660.

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Table 1. Fusarium isolates analysed indicating origin, species and the occurrence of PCR amplification product using specific PCR

assays to F. graminearum (FG), F. culmorum (FC), F. equiseti (FE), F. sporotrichioides (FSP) and F. poae (FP)

Isolates Origin Species FG FC FE FSP FP

Fg2 Central Italy F. graminearum + � � � �

Fg4 North Italy F. graminearum + � � � �

Fg5 Central Italy F. graminearum + � � � �

CECT 2150a USA F. graminearum + � � � �

NRRL 28585b Venezuela F. graminearum (lineage 1) + � � � �

NRRL 28436b New Caledonia F. graminearum (lineage 2) + � � � �

NRRL 29020b South Africa F. graminearum (lineage 3) + � � � �

NRRL 29148b Pennsylvania-USA F. graminearum (lineage 4) + � � � �

NRRL 26755b South Africa F. graminearum (lineage 5) � � � � �

NRRL 13818b Japan F. graminearum (lineage 6) + � � � �

NRRL 29169b Kansas-USA F. graminearum (lineage 7) + � � � �

NRRL 29306b New Zealand F. graminearum (lineage 8) + � � � �

CECT 2148a F. culmorum � + � � �

ITEM 6717c Hungary F. culmorum � + � � �

ITEM 6718c Hungary F. culmorum � + � � �

ITEM 628c Yugoslavia F. culmorum � + � � �

ITEM 4335c F. culmorum � + � � �

MUCL 42823d Belgium F. culmorum � + � � �

MUCL 42826d Belgium F. culmorum � + � � �

Feq3 South Italy F. equiseti � � + � �

VI01087e Norway F. equiseti � � + � �

VI01093e Norway F. equiseti � � + � �

VI01096e Norway F. equiseti � � + � �

Eq-U6 South Spain F. equiseti � � + � �

L1-2 South Spain F. equiseti � � + � �

CECT 20166a Russia F. sporotrichioides � � � + �

ITEM 695c USA F. sporotrichioides � � � + �

ITEM 707c Poland F. sporotrichioides � � � + �

ITEM 4596c Russia F. sporotrichioides � � � + �

ITEM 4597c Russia F. sporotrichioides � � � + �

ITEM 550c Poland F. sporotrichioides � � � + �

ITEM 6607c England F. poae � � � � +

ITEM 6606c England F. poae � � � � +

MUCL 6114d Belgium F. poae � � � � +

MUCL 7555d Belgium F. poae � � � � +

MUCL 42824d Belgium F. poae � � � � +

MPA 0999f USA F. verticillioides � � � � �

MPB 3853f F. sacchari � � � � �

MPC 1995f Taiwan F. fujikuroi � � � � �

MPD 4853f F. proliferatum � � � � �

MPE 2192f USA F. subglutinans � � � � �

MPF 4093f F. thapsinum � � � � �

MPG 05111f F. nygamai � � � � �

MPH 69722f South Africa F. circinatum � � � � �

MUCL 42821d Belgium F. tricinctum � � � � �

Fps6 South Italy F. pseudograminearum � � � � �

Av-U3 South Spain F. avenaceum � � � � �

aStrains supplied by Coleccion Espanola de Cultivos Tipo (CECT, Spain).bStrains kindly provided by K. O0Donnell (NCAUR, USA).cStrains kindly provided by A. Moretti (CNR, Italy).dStrains supplied by Belgian Co-ordinated Collections of Micro-organisms (BCCM, Belgium).eStrains kindly provided by M. Torp (NVI, Norway).fStrains from G. fujikuroi mating populations A–H.

M. Jurado et al. / Systematic and Applied Microbiology 28 (2005) 562–568564

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Development and evaluation of species-specific PCR

primers

DNA sequences were analysed and aligned by Clustalmethod using Dnastar (Lasergene, Madison, WI, USA).Primers were designed on the basis of these DNAalignments. The sequences of the primers and the sizesof the amplicons produced in the respective PCRreactions are shown in Table 2. The relative positionof the primers within the IGS region is indicated inFig. 1. All genomic DNAs used in this work were testedfor suitability for PCR amplification using primersCNL12 and CNS1 [1] in the conditions indicated above.The amplification protocol with primers Fgr-F/Fgc-Rwas 1 cycle of 85 s at 94 1C, 25 cycles of 35 s at 95 1C(denaturalization), 30 s at 53 1C (annealing), 30 s at 72 1C(extension), and 1 cycle of 5min at 72 1C. The sameprogramme was used with primer pairs Fps-F/Fpo-Rand Fps-F/Fsp-R, except for the annealing temperature(61 1C). The program used with primer pairs Feq-F/Feq-R and Fcu-F/Fgc-R was the same described aboveexcept for the number of cycles and the annealingtemperature: 30 cycles and 66 1C, and 20 cycles and54 1C, respectively. In all the cases, amplificationreactions were carried out in volumes of 25 ml containing25 ng of template DNA in 3 ml, 1.25 ml of each primer

Table 2. Oligonucleotide primer sequences and sizes of the PCR p

Species specificity Primer name Primer sequ

F. graminearum Fgr-F 50-GTTGAT

Fgc-R 50-CTCTCA

F. culmorum Fcu-F 50-GACTAT

Fgc-R 50-CTCTCA

F. equiseti Feq-F 50-GGCCTG

Feq-R 50-CGATAC

F. sporotrichioides Fps-F 50-CGCACG

Fsp-R 50-GTCAGA

F. poae Fps-F 50-CGCACG

Fpo-R 50-CAGCGC

IGS+95

FSP ~ 400 bp

IGS-221 IGS+188

IG

FP ~ 400 bp

IGS-221 IGS+179

FE ~ 990 bp

IGS+76

IGS (~2028S

Fig. 1. Relative position on the IGS region of the amplification ban

(FC), F. equiseti (FE), F. sporotrichioides (FSP) and F. poae (FP). T

IGS region. (-) indicate a position within 28S gene, upstream of the

(20 mM), 0.2 ml of Taq DNA polymerase (5U/ml), 2.5 mlof 10�PCR buffer, 1 ml of MgCl2 (50mM), and 0.25 mlof dNTPs (100mM) (Ecogen, Barcelona, Spain). PCRwas performed in a thermocycler (Eppendorf Master-cycler Gradient, Eppendorf, Hamburg, Germany).Amplification products were detected by electrophoresison 1.5% agarose ethidium bromide gels in 40mMTris–acetate and 1.0mM EDTA 1�buffer. Dilutionscontaining from 90 ng to 170 fg of DNA were preparedto test the sensitivity of each primer pair in respectivePCR assays.

Isolation of DNA from infected wheat samples

Wheat samples were collected in different fields in theSouth of Spain [8]. Samples of five spikes were collectedand the seeds with the glumes were separated andpooled. Four grams of each pool of seeds wereincubated in 20ml Sabouraud liquid medium (ScharlauChemie, Barcelona, Spain) supplemented with chlor-amphenicol (0.5%) at 25 1C during 5 days. The liquidmedium was discarded and the seeds were grounded inliquid nitrogen and genomic DNA extracted withGenomix DNA Extraction Kit (Talent, Trieste, Italy)according the manufacturer’s instructions. Total DNA

roducts

ence Product size (bp)

GGGTAAAAGTGTG-30 �500

TATACCCTCCG-30

CATTATGCTTGCGAGAG-30 �200

TATACCCTCCG-30

CCGATGCGTC-30 �990

TGAAACCGACCTC-30

TATAGATGGACAAG-30 �400

AGAGACGCATCCGCC-30

TATAGATGGACAAG-30 �400

ACCCCTCAGAGC-30

FG ~ 500 bp

4 IGS+1456

FC ~ 200 bp

IGS+1396 S+1195

IGS+1062

00 bp)18S

ds and primers specific to F. graminearum (FG), F. culmorum

he numbers indicate the position of primers with respect to the

IGS region.

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(25 ng) was used for PCR amplification with specificprimers following the protocols described above.

Results

We have obtained the IGS sequences by cloning andsequencing the IGS region of several strains of those

a) M 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 M

c) M 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 M

e) M 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 M

500 bp5

500 bp5

500 bp

Fig. 2. PCR assays specific to: (a) F. culmorum with primers Fcu-F/F

6717, ITEM 6718, ITEM 628, ITEM 4335, MUCL 42823 and MUC

graminearum Fg2, F. equiseti Feq3, F. sporotrichioides CECT 20166, F

MPD 4853, F. subglutinans MPE 2192, F. circinatum MPH 69722,

negative control (lane 18); DNA marker (M). (b) F. equiseti with p

VI01087, VI01093, VI01096, Eq-U6 and L1-2 (lanes 1–6) and the Fu

CECT 2148, F. sporotrichioides CECT 20166, F. poae ITEM 6607

subglutinans MPE 2192, F. nygamai MPG 05111, F. circinatum MPH

42821; negative control (lane 18); DNA marker (M). (c) F. graminear

strains Fg2, Fg4, CECT 2150, NRRL 28585, NRRL 29148, NRR

culmorum CECT 2148, F. equiseti Feq3, F. sporotrichioides CECT

proliferatum MPD 4853, F. subglutinans MPE 2192, F. nygamai MPG

and F. tricinctum MUCL 42821; negative control (lane 18): DNA m

from F. poae strains ITEM 6607, ITEM 6606, MUCL 6114, MUCL

6–17): F. graminearum Fg2, F. culmorum CECT 2148, F. sporotrichio

3853, F. fujikuroi MPC 1995, F. proliferatum MPD 4853, F. subglut

69722, F. pseudograminearum Fps6 and F. tricinctum MUCL 42

sporotrichioides with primers Fps-F/Fsp-R and DNA from F. sporo

4596, ITEM 4597 and ITEM 550 (lanes 1–6) and the Fusarium strains

equiseti Feq3, F. poae ITEM 6607, F. verticillioides MPA 0999, F. p

MPG 05111, F. circinatum MPH 69722, F. pseudograminearum Fps

DNA marker (M).

species, which are available under the accession numbersindicated in Material and methods, and other closelyrelated Fusarium species. Additionally, some partial IGSsequences available in data bases were also used in thealignments. On the basis of these alignments specificprimers have been designed for the species F. grami-

nearum, F. culmorum, F. sporotrichioides, F. poae and F.

equiseti (Table 2).

b) M 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 M

d) M 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 M

00 bp

00 bp

gc-R and DNA from F. culmorum strains CECT 2148, ITEM

L 42826 (lanes 1–7) and the Fusarium strains (lanes 8–17): F.

. poae ITEM 6607, F. verticillioides MPA 0999, F. proliferatum

F. pseudograminearum Fps6 and F. tricinctum MUCL 42821;

rimers Feq-F/Feq-R and DNA from F. equiseti strains Feq3,

sarium strains (lanes 7–17): F. graminearum Fg2, F. culmorum

, F. verticillioides MPA 0999, F. proliferatum MPD 4853, F.

69722, F. pseudograminearum Fps6 and F. tricinctum MUCL

um with primers Fgr-F/Fgc-R and DNA from F. graminearum

L 29169 (lanes 1–6) and the Fusarium strains (lanes 7–17): F.

20166, F. poae ITEM 6607, F. verticillioides MPA 0999, F.

05111, F. circinatum MPH 69722, F. pseudograminearum Fps6

arker (M). (d) F. poae with primers Fps-F/Fpo-R and DNA

7555, MUCL 42824 (lanes 1–5) and the Fusarium strains (lanes

ides CECT 20166, F. verticillioides MPA 0999, F. sacchari MPB

inans MPE 2192, F. nygamai MPG 05111, F. circinatum MPH

821; negative control (lane 18): DNA marker (M). (e) F.

trichioides strains CECT 20166, ITEM 695, ITEM 707, ITEM

(lanes 7–17): F. graminearum Fg2, F. culmorum CECT 2148, F.

roliferatum MPD 4853, F. subglutinans MPE 2192, F. nygamai

6 and F. tricinctum MUCL 42821; negative control (lane 18):

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Table 3. Occurrence of F. graminearum (FG), F. culmorum

(FC), F. equiseti (FE), F. sporotrichioides (FSP) and F. poae in

samples of wheat

Sample FG FC FE FSP FP

U6 � � + � �

L1 + + + � �

L3 + � + � �

E1 � + � � �

BE1 � � + � �

BO1 + � � � �

N1 + + + � �

M. Jurado et al. / Systematic and Applied Microbiology 28 (2005) 562–568 567

The specific PCR assays developed have been testedon a diverse sample of Fusarium strains commonlyassociated with cereals which included all the F.

graminearum lineages [16] (Table 1). The PCR primersdeveloped in this study exhibited species-specific resolu-tion (Table 1 and Figs. 2a–e). The five Fusarium speciescould be differentiated from each other as from otherspecies of Fusarium with the respective PCR assay,excluding isolate NRRL 26755, a representative strainof lineage 5 of F. graminearum, which did not producethe band in the corresponding PCR assay for F.

graminearum. Species-specific PCR primers amplified aDNA fragment of the expected size only in the isolatesof Fusarium species for which the primer was originallydesigned (Table 1). In the case of F. sporotrichioides andF. poae a fragment of 400 bp was observed, whereas,fragments of 200, 990 and 500 bp were amplified in thePCR assays for F. culmorum, F. equiseti, and F.

graminearum, respectively (Table 2, Figs. 2a–e). Thedetection limit estimated was 5.5 pg of DNA template inthe PCR reaction carried out with specific primers to F.

graminearum, F. poae, F. equiseti and F. sporotrichioides,and 50 pg with the primer pair specific to F. culmorum

(data not shown).The DNA obtained from wheat seeds was tested with

the primers developed in this work. Three species: F.

graminearum, F. culmorum and F. equiseti were detectedin these samples (Table 3). A conventional method usedfor the isolation of Fusarium strains in the same wheatsamples confirmed the occurrence of the F. graminear-

um, F. culmorum and F. equiseti detected with thespecific primers and the absence of F. sporotrichioides

and F. poae [8].

Discussion

We have developed a set of primers and thecorresponding PCR assays to detect F. graminearum,F. culmorum, F. sporotrichioides, F. poae and F. equiseti.

The assays have been tested on a sample of isolates ofdiverse origin for each species as well as on a range of

Fusarium species that are frequently associated to small-grain cereals. The diverse geographical locations andorigins of the isolates analysed in this work can beconsidered representative of the variability of thesespecies and for the first time, several Spanish isolates [8]were also included in the tests, indicating the widegeographical distribution of those species mainly studiedin Central and Northern Europe. In the case of F.

graminearum, the PCR assay was able to recognize allthe lineages characterized so far except lineage 5 whichdid not amplify with the primers designed for F.

graminearum, however the occurrence of this lineage inEurope and North America has not been reported [16].

Detection limit of IGS amplification product, definedas the clearly visible product on agarose gels containingethidium bromide, had not been previously estimated inFusarium. However, detection limit for the ITS region ofthe rDNA, that would be present in the same number ofcopies than IGS region, has been estimated between 1and 10 pg of DNA template in Fusarium [2]. We foundsimilar detection levels (5.5 pg) with the sets of primersdesigned for F. graminearum, F. sporotrichioides, F. poae

and F. equiseti.However, the detection limit of PCR assays developed

for F. culmorum is lower, because of the need ofreducing the number of cycles in PCR assay to avoid acrossed-reaction with F. graminearum. Even so, thesensitivity of all of our PCR assays based on IGSsequences was, therefore, higher than primers based onsingle copy gene, estimated between 0.1 and 1 ng ofDNA template per reaction [2].

The PCR assays tested on DNA from pure culturesdescribed in this work can also be used in DNA isolatedfrom wheat infected seeds using a protocol which allowsto process a high number of samples and to reduce thetime of analysis in comparison with conventionalmethods [8]. In the wheat samples analysed in thiswork, we were able to detect three of the species tested:F. graminearum, F. culmorum and F. equiseti (Table 3),and, therefore, to predict the toxins which are probablypresent in the wheat seeds of the fields analysed.

We can conclude that the PCR assays described inthis work provides a useful tool for rapid and sensitivedetection of the main trichothecene-producing Fusarium

species which can be readily used to enhance theefficiency of disease control and prevention managementpractices and to assess the quality of raw material to beprocessed into food and feed products.

Acknowledgements

This work was supported by the MCYT (AGL2001/2974/C05/5) and the CAM (CAM07G/0007/20031). M.Jurado was supported by pre-doctoral fellowship by theMCYT.

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References

[1] D.J. Appel, T.R. Gordon, Intraspecific variation within

populations of Fusarium oxysporum based on RFLP

analysis of the intergenic spacer region of the rDNA, Exp.

Mycol. 19 (1995) 120–128.

[2] B.H. Bluhm, J.E. Flaherty, M.A. Cousin, C.P. Woloshuk,

Multiplex polymerase chain reaction assay for the

differential detection of trichothecene- and fumonisin-

producing species of Fusarium in cornmeal, J. Food Prot.

65 (2002) 1955–1961.

[3] D.W. Brown, P.S. McCormick, N.J. Alexander, R.H.

Proctor, A.E. Desjardins, Inactivation of a cytochrome

P-450 is a determinant of trichothecene diversity in

Fusarium species, Fungal Genet. Biol. 36 (2002) 224–233.

[4] J.P.F. D’ Mello, C.M. Placinta, A.M.C. MacDonald,

Fusarium mycotoxins: a review of global implications for

animal health, welfare and productivity, Anim. Feed Sci.

Technol. 80 (1999) 183–205.

[5] A.E. Desjardins, T.M. Hohn, S.P. McCormick, Tri-

chothecene biosyntesis in Fusarium species: chemistry,

genetics, and significance, Microbiol. Rev. 1993 (1993)

595–604.

[6] S.G. Edwards, J. O’Callaghan, A.D.W. Dobson, PCR-

based detection and quantification of mycotoxigenic

fungi, Mycol. Res. 106 (2002) 1005–1025.

[7] IARC: IARC Monographs on the Evaluation of Carci-

nogenic risks to Humans, vol 56. Some Naturally

Occurring Substances: Food Items and Constituents,

Heterocyclic Aromatic Amines and Mycotoxins, Interna-

tional Agency for Research on Cancer, Lyon (1993)

397–444, 445–466, 467–488.

[8] M. Jurado, C. Vazquez, E. Lopez-Errasquin, B. Patino,

M.T. Gonzalez-Jaen, Analysis of the occurrence of

Fusarium species in Spanish cereals by PCR assays,

Proceedings of the Second International Symposium

on Fusarium Head Blight, Orlando, FL, USA, 2004,

pp. 460–464.

[9] A.K. Knutsen, M. Torp, A. Holst-Jensen, Phylogenetic

analyses of the Fusarium poae, Fusarium sporotrichioides

and Fusarium langsethiae species complex based on partial

sequences of the translation elongation factor-1 alpha

gene, Int. J. Food Microbiol. 95 (2004) 287–295.

[10] P. Konstantinova, T. Yli-Mattila, IGS–RFLP analysis

and development of molecular markers for identification

of Fusarium poae, Fusarium langsethiae, Fusarium sporo-

trichioides and Fusarium kyushuense, Int. J. Food Micro-

biol. 95 (2004) 321–331.

[11] T. Kulik, G. Fordonski, A. Pszczolkowska, K. Plodzien,

M. Lapinski, Development of PCR assay based on ITS2

rDNA polymorphism for the detection and differentia-

tion of Fusarium sporotrichioides, FEMS Microbiol. Lett.

239 (2004) 181–186.

[12] S. Mirete, B. Patino, C. Vazquez, M. Jimenez, M.J.

Hinojo, C. Soldevilla, M.T. Gonzalez-Jaen, Fumonisin

production by Gibberella fujikuroi strains from Pinus

species, Int. J. Food Microbiol. 89 (2003) 213–221.

[13] S. Mirete, M.G. Vazquez, M. Jurado, M.T. Gonzalez-

Jaen, Differentiation of Fusarium verticillioides from

banana fruits by IGS and EF-1a sequence analyses,

Eur. J. Plant Pathol. 110 (2004) 515–523.

[14] P. Nicholson, D.R. Simpson, G. Weston, H.N. Rezanoor,

A.K. Lees, D.W. Parry, D. Joyce, Detection and

quantification of Fusarium culmorum and Fusarium

graminearum in cereals using PCR assays, Physiol. Mol.

Plant Pathol. 53 (1998) 17–37.

[15] P. Nicholson, D.R. Simpson, A.H. Wilson, E. Chandler,

M. Thomsett, Detection and differentiation of trichothe-

cene and eniantin-producing Fusarium species on small-

grain cereals, Eur. J. Plant Pathol. 110 (2004) 503–514.

[16] K. O’Donnell, H.C. Kistler, B.K. Tacke, H.H. Casper,

Gene genealogies reveal global phylogeographic structure

and reproductive isolation among lineages of Fusarium

graminearum, the fungus causing wheat scab, Proc. Natl.

Acad. Sci. USA 97 (2000) 7905–7910.

[17] B. Patino, S. Mirete, M.T. Gonzalez-Jaen, G. Mule, M.T.

Rodrıguez, C. Vazquez, PCR Detection assay of fumo-

nisin-producing Fusarium verticillioides strains, J. Food

Prot. 67 (2004) 1278–1283.

[18] H. Schmidt, A. Adler, A. Holst-Jensen, S.S. Klemsdal, A.

Logrieco, R.L. Mach, H.I. Nirenberg, U. Thrane, M.

Torp, R.F. Vogel, T. Yli-Mattila, L. Niessen, An

integrated taxonomic study of Fusarium langsethiae,

Fusarium poae and Fusarium sporotrichioides based on

the use of composite datasets, Int. J. Food Microbiol. 95

(2004) 341–349.

[19] K.A. Seifert, C.A. Levesque, Phylogeny and molecular

diagnosis of mycotoxigenic fungi, Eur. J. Plant Pathol.

110 (2004) 449–471.

[20] T. Yli-Mattila, R.L. Mach, I.A. Alekhina, S.A. Bulat, S.

Koskinen, C.M. Kullnig-Gradinger, C.P. Kubicek, S.S.

Klemsdal, Phylogenetic relationship of Fusarium lang-

sethiae to Fusarium poae and Fusarium sporotrichioides as

inferred by IGS, ITS, ß-tubulin sequences and UP-PCR

hybridization analysis, Int. J. Food Microbiol. 95 (2004)

267–285.