**7. Genetic genotype determination of FGSC**

Traditionally, chemotyping of FGSC strains has been carried out using gas chromatography/mass spectroscopy. This method can be time-consuming and expensive. The genome sequences of several FGSC strains have been published. The trichothecene core gene cluster nucleotide sequences of many strains representatives 3-ADON, 15-ADON, and NIV genotypes have also been deposited in the GenBank. The availability of this information makes it possible to reveal the structural features and allowed selection of several primer sets used successfully in PCR experiments for the molecular characterization of the various chemotypes. Molecular genetic assays allow for high throughput screening of large numbers of field isolates.

Lee et al. [34] sequenced the gene cluster for trichothecene biosynthesis from a 15-ADON producer (strain H-11) and a NIV producer (strain 88-1), and sequence polymorphisms within the *Tri7* open reading frame was found between the two strains. Alignment analysis suggesting that the *Tri7* gene of H-11 carried several mutations and an insertion compared to the *Tri7* gene from 88-1, and based on the sequence difference a PCR-based diagnostic method for differentiating DON and NIV producers by polyacrylamide gel electrophoresis was developed.

Lee et al. [35] subsequently sequenced the *Tri13* homolog from DON (strain H-11) and NIV producers (strain 88-1) and found that the gene differs drastically between the two producers, suggesting that the *Tri13* gene could be used for genetic genotype distinction for DON and NIV producers [35, 36]. They further confirmed the roles of the *Tri7* and *Tri13* genes in trichothecene production, and the results suggested that both the *Tri7* and *Tri13* genes are nonfunctional in DON producers [35].

The PCR assays to *Tri7* and *Tri13* genes developed by Lee et al. [34, 35] allowed clear differentiation between DON and NIV genotypes. However, they could not be used to further classify the DON-producing isolates to 3-ADON or 15-ADON producer. Ward et al. [29] examined a 19-kb region of the trichothecene gene cluster that sequenced in 39 strains representing 3-ADON, 15-ADON, and NIV genotypes. They found that Tri-cluster haplotypes group according to genotype rather than by species indicated that 3-ADON, 15-ADON, and NIV genotypes each have a single evolutionary origin. Reciprocally monophyletic groups, corresponding to each of 3-ADON, 15-ADON, and NIV genotypes, were strongly supported in *Tri3*, *Tri11*, and *Tri12* genes trees. Two sets of primers specific to the individual genotypes were designed from *Tri3* and *Tri12* genes. The genotype-specific PCR tests developed by Ward et al. [29] provide a rapid and direct genetic method for distinguishing among 3-ADON, 15-ADON, and NIV producer, this is the first report differentiated these three genotype strains by a PCR method.

The work by Lee et al. [34, 35] and Brown et al. [37] indicated that the genes *Tri13* and *Tri7* from trichothecene biosynthetic cluster are responsible for conversion of DON to NIV (*Tri13* gene) and the *Tri7* gene product modifies NIV by acetylation of C-4 atom hydroxyl to produce 4-ANIV. Based on these results sets of positive-negative PCR assays to *Tri7* and *Tri13* genes for trichothecene determination of FGSC were developed by Chandler et al. [38], and the assays can accurately indicate a DON or NIV genotype in FGSC, *F. culmorum* and *F. cerealis*. The assays

were successfully used to screen isolates from different countries and the genotypespecific assays were able to detect and characterize a wider range of species and haplotypes than previous methods.

By comparing the published sequences for *Tri13* gene from known DON- and NIV- producers, Waalwijk et al. [39] designed a primer pair to discriminate the two genotypes which generated a 234 bp fragment in DON-producers and a fragment of 415 bp in NIV-producers. The *Tri13* primer pair was capable and robust to determine the genotype of strains from *F. culmorum*.

Based on information reported and deposited by Ward et al. [29], three primer sets were designed to the *Tri3* gene by Jennings et al. [40] to allow further differentiation of the DON genotype into either 3-ADON or 15-ADON. Each isolate produces a PCR product with only one of these primer sets but not the other two from *F. culmorum* and FGSC strains [40, 41].

Li et al. [42] found that the intergenic sequences between *Tri5* and *Tri6* genes appear to be mycotoxin genotype-specific, and based on the sequence length polymorphism a generic PCR assay was developed to detect a 300 bp fragment of DON-genotype strains and a 360 bp fragment of NIV-genotypes from FGSC.

Based on the sequences of FGSC described by Lee et al. [34] and Ward et al. [29], a series of PCR assays have been designed to *Tri3* and *Tri7* by Quarta et al. [43], in order to permit specific detection of 3-ADON, 15-ADON, and NIV genotypes, respectively. These primers were subjected to a multiplex PCR assay for the identification of the different genotypes of *Fusarium* strains combined with the primer pair derived from the *Tri5* gene by Bakan et al. [44]. The multiplex PCR was validated on FGSC, *F. cerealis*, *F. culmorum* strains from different European countries, and successfully used to identify the genotype of the *Fusarium* strain contaminating wheat kernels [43, 45].

The possibility to distinguish by a singleplex PCR 3-ADON, 15-ADON, and NIV genotypes was not yet resolved until very recently. Wang et al. [46] developed a *Tri13* based PCR assay and successfully identified the 3-ADON, 15-ADON, and NIV genotypes in FGSC from Asia, Europe, and America. Using the primer pair, specific amplification products of 644, 583, and 859 bp were obtained from isolates producing 3-ADON, 15-ADON, and NIV, respectively. All three types of PCR fragments had different molecular sizes with a smallest difference of 61 bp can be directly differentiated on an agarose gel. The method should be more reliable than other PCR-based assays that show the absence or presence of a PCR fragment since these assays may generate false-negative results. This is a rapid, reliable and cost-effective method for the determination of 3-ADON, 15-ADON, and NIV genotype strains in FGSC.

Recently Suzuki et al. [47] reported a multiplex PCR assay for simultaneous identification of the species and trichothecene genotypes for *F. graminearum sensu stricto* and *F. asiaticum* based on *Tri3* and *Tri6* genes. This approach proved successful for Japanese strains [47].

An alternative method based on *Tri11* polymorphism was developed by Zhang et al. [48] to differentiate 3-ADON, 15-ADON, and NIV genotypes of FGSC strains. Similarly, we presented another multiplex assay based on the single nucleotide polymorphism of *Tri11* gene between strains of different genotype [49]. The assay was also validated on plant material.

Recent work by Kulik [50] and Nielsen et al. [51] to detect and quantify FGSC genotypes in plants/grains were developed based on TaqMan probe set and SYBR green method with *Tri12* gene, respectively.

Due to the toxicological differences between DON and NIV, it is important to monitor the population and determine the chemotypes of strains present in any given geographic region. Mycotoxin producing capability of a certain strain could be established both through biochemical and molecular techniques.

**73**

**Target gene**

*Tri3*

**Primers**

3CON 3D3A 3CON 3D15A 3CON

3NA Tri303F Tri303R Tri315F Tri315R Tri3NivF Tri3NivR Tri3F971 Tri3R1679 Tri3F1325 Tri3R1679

3D15AF

**Sequences (5′ to 3′)**

TGGCAAAGACTGGTTCAC

CGCATTGGCTAACACATG

TGGCAAAGACTGGTTCAC

ACTGACCCAAGCTGCCATC

TGGCAAAGACTGGTTCAC

GTGCACAGAATATACGAGC

GATGGCCGCAAGTGGA

GCCGGACTGCCCTATTG

CTCGCTGAAGTTGGAC

GTAA GTCTATGCTCTCAACG

GACAAC

GGACGTGA(CG)TACT

549

NIV

CTTGGCAA

CCCAG(AG)GCCTCTA

AGAA(AG)GGB

CATCATACTCGC

708

15-ADON

Quarta et al. [43]

TCTGCTG

TT(AG)TAGTTTGCATC

ATT(AG)TAG

GCATTGGCTAACACATGA

TT(AG)TAGTTTGCA

TCATT(AG)TAG

AACTGACCCAAGCTG

420

15-ADON (*F. asiaticum* and *F.* 

Suzuki et al. [47]

*graminearum ss*)

CCATC

354

3-ADON

864

15-ADON

586

3-ADON

Jennings et al. [40, 41]

840

NIV

610

15-ADON

**Fragment size (bp)**

243

**Chemotypes**

3-ADON

**References** Ward et al. [29]

Fusarium graminearum *Species Complex and Trichothecene Genotype*

*DOI: http://dx.doi.org/10.5772/intechopen.89045*


#### Fusarium graminearum *Species Complex and Trichothecene Genotype DOI: http://dx.doi.org/10.5772/intechopen.89045*

*Mycotoxins and Food Safety*

kernels [43, 45].

ful for Japanese strains [47].

was also validated on plant material.

green method with *Tri12* gene, respectively.

haplotypes than previous methods.

mine the genotype of strains from *F. culmorum*.

from *F. culmorum* and FGSC strains [40, 41].

were successfully used to screen isolates from different countries and the genotypespecific assays were able to detect and characterize a wider range of species and

By comparing the published sequences for *Tri13* gene from known DON- and NIV- producers, Waalwijk et al. [39] designed a primer pair to discriminate the two genotypes which generated a 234 bp fragment in DON-producers and a fragment of 415 bp in NIV-producers. The *Tri13* primer pair was capable and robust to deter-

Based on information reported and deposited by Ward et al. [29], three primer

Li et al. [42] found that the intergenic sequences between *Tri5* and *Tri6* genes appear to be mycotoxin genotype-specific, and based on the sequence length polymorphism a generic PCR assay was developed to detect a 300 bp fragment of DON-genotype strains and a 360 bp fragment of NIV-genotypes from FGSC. Based on the sequences of FGSC described by Lee et al. [34] and Ward et al. [29], a series of PCR assays have been designed to *Tri3* and *Tri7* by Quarta et al. [43], in order to permit specific detection of 3-ADON, 15-ADON, and NIV genotypes, respectively. These primers were subjected to a multiplex PCR assay for the identification of the different genotypes of *Fusarium* strains combined with the primer pair derived from the *Tri5* gene by Bakan et al. [44]. The multiplex PCR was validated on FGSC, *F. cerealis*, *F. culmorum* strains from different European countries, and successfully used to identify the genotype of the *Fusarium* strain contaminating wheat

The possibility to distinguish by a singleplex PCR 3-ADON, 15-ADON, and NIV genotypes was not yet resolved until very recently. Wang et al. [46] developed a *Tri13* based PCR assay and successfully identified the 3-ADON, 15-ADON, and NIV genotypes in FGSC from Asia, Europe, and America. Using the primer pair, specific amplification products of 644, 583, and 859 bp were obtained from isolates producing 3-ADON, 15-ADON, and NIV, respectively. All three types of PCR fragments had different molecular sizes with a smallest difference of 61 bp can be directly differentiated on an agarose gel. The method should be more reliable than other PCR-based assays that show the absence or presence of a PCR fragment since these assays may generate false-negative results. This is a rapid, reliable and cost-effective method for the determination of 3-ADON, 15-ADON, and NIV genotype strains in FGSC. Recently Suzuki et al. [47] reported a multiplex PCR assay for simultaneous identification of the species and trichothecene genotypes for *F. graminearum sensu stricto* and *F. asiaticum* based on *Tri3* and *Tri6* genes. This approach proved success-

An alternative method based on *Tri11* polymorphism was developed by Zhang et al. [48] to differentiate 3-ADON, 15-ADON, and NIV genotypes of FGSC strains. Similarly, we presented another multiplex assay based on the single nucleotide polymorphism of *Tri11* gene between strains of different genotype [49]. The assay

Recent work by Kulik [50] and Nielsen et al. [51] to detect and quantify FGSC genotypes in plants/grains were developed based on TaqMan probe set and SYBR

Due to the toxicological differences between DON and NIV, it is important to monitor the population and determine the chemotypes of strains present in any given geographic region. Mycotoxin producing capability of a certain strain could be established both through biochemical and molecular techniques.

sets were designed to the *Tri3* gene by Jennings et al. [40] to allow further differentiation of the DON genotype into either 3-ADON or 15-ADON. Each isolate produces a PCR product with only one of these primer sets but not the other two

**72**


**75**

**Target gene**

**Primers** Tri7DON

Tri7F Tri7NIV MinusTri7F MinusTri7R

Tri7F340 Tri7R965

3D11

11R 15D11

11R N11 11R

*Tri11*

**Sequences (5′ to 3′)**

GTGCTAATATTGT

GCTAATATTGTGC

TGCGTGGCAATAT

CTTCTTCTA GGTTCAAGTAAC

GTTCGACAATAG

TGGATGAATGAC

TTGAGTTGACA

AAAGCCTTCATT

CACAGCC ATCGTGTACAAG

GTTTAC G TTCAAGTAACGT

TCGACAAT

GCAAGTCTGGC

342

3-ADON

Zhang et al. [48]

GAGGCC

TCAAAGGCCAG

AGCAACCC

AAGTATGGTCC

424

15-ADON

AGTTGTCCGTATT

TCAAAGGCCAG

AGCAACCC

CTTGTCAGGCGG

643

NIV

CACAGTAG

TCAAAGGCCAGA

GCAACCC

625

NIV

Quarta et al. [43]

483

3-ADON

465

NIV

**Fragment size (bp)**

**Chemotypes**

**References**

Fusarium graminearum *Species Complex and Trichothecene Genotype*

*DOI: http://dx.doi.org/10.5772/intechopen.89045*


#### Fusarium graminearum *Species Complex and Trichothecene Genotype DOI: http://dx.doi.org/10.5772/intechopen.89045*

*Mycotoxins and Food Safety*

Li et al. [42]

**74**

**Target gene** *Tri5- Tri6* intergenic

region

*Tri6*

ToxP1 ToxP2 6A3AF 6A3AR 6G3AF 6G3AR

6CNF 6ANR GzTri7/f1 GzTri7/r1

Tri7F Tri7R Tri7F

*Tri7*

**Primers** 3D15AR

**Sequences (5′ to 3′)**

CTTCTGTCCCTTCG

AACGGA

GCCGTGGGG(AG)TAA

300

DON

AAGTCAAA

TGACAAGTCCGGTC

360

NIV

GCACTAGCA

CCAAGACTT(GT)GTT

1100

DON (*F. asiaticum*)

Suzuki et al. [47]

(AC)CCCGAA

GCAATCTTTAGAGTG

CCGAC

T(AG)TCCCATCCCAT

330

DON (*F. graminearum ss*)

CAAGGCT

AACAAGTGGTTCTT

CGGAGT

CAAGCAAATGCCC

660

NIV (*F. asiaticum*)

GTATCCC

CGCAACAATATCA

ATGGCTGTGCTA

GGCTTTACGACTC

173–327

15-ADON

Lee et al. [34]

CTCAACAATGG

AGAGCCCTGCGAA

161

NIV

AG(CT)ACTGGTGC

TGCGTGGCAATATC

458–535

DON

Chandler et al. [38]

TTCTTCTA

TGTGGAAGCCGCAGA

TGCGTGGCAATAT

CTTCTTCTA

436 381–445

NIV

DON

**Fragment size (bp)**

**Chemotypes**

**References**


**77**

**Target gene**

**Primers** GzTri13/p2

Tri13F Tri13R Tri13F Tri13DONR

Tri13NIVF

Tri13R Tri13F Tri13R Tri13P1 Tri13P2

**Table 1.**

*Primers designed for genetic genotyping of FGSC so far.*

**Sequences (5′ to 3′)**

GTG(AG)T(AG)TCCCA

GGATCTGCGTGTC

TACGTGAAACAT

TGTTGGC GGTGTCCCAGGA

TCTGCG CATCATGAGACTTGT

(GT)C(AG)AGTTTGGG

GCTAGATCGATT

GTTGCATTGAG

CCAAATCCGAA

AACCGCAG TTGAAAGCTCC

AATGTCGTG

CATCATGAGACTTGT

799

DON

(GT)C(AG)AGTTTGGG

TTGAAAGCTCC

1075

NIV

AATGTCGTG

CTC(CG)ACCGCATC

583

15-ADON

Wang et al. [46]

GAAGA(CG)TCTC

GAA(CG)GTCGCA

644 859

NIV

3-ADON

(AG)GACCTTGTTTC

312

NIV

282

DON

415

NIV

234

DON

Waalwijk et al. [39]

**Fragment size (bp)**

760

**Chemotypes**

NIV

**References**

Fusarium graminearum *Species Complex and Trichothecene Genotype*

*DOI: http://dx.doi.org/10.5772/intechopen.89045*

Chandler et al. [38]

#### *Mycotoxins and Food Safety*


### Fusarium graminearum *Species Complex and Trichothecene Genotype DOI: http://dx.doi.org/10.5772/intechopen.89045*

*Mycotoxins and Food Safety*

**76**

**Target gene**

**Primers** Tri11-CON

Tri11-

3AcDON

Tri11-

CON

Tri11-

15AcDON

Tri11-

CON

Tri11-

NIV

12CON

12-3F 12CON 12-15F 12CON

12NF

> *Tri13*

GzTri13/p1

*Tri12*

**Sequences (5′ to 3′)**

GACTGCTCATGG

AGACGCTG

TCCTCATGCTCG

GTGGACTCG

GACTGCTCATGG

279

15-ADON

AGACGCTG

TGGTCCAGTTG

TCCGTATT

GACTGCTCATG

497

NIV

GAGACGCTG

GTAGGTTCCAT

TGCTTGTTC

CATGAGCATGG

410

3-ADON

Ward et al. [29]

TGATGTC

CTTTGGCAAGC

CCGTGCA

CATGAGCATGG

670

15-ADON

TGATGTC

TACAGCGGTCG

CAACTTC

CATGAGCATGG

840

NIV

TGATGTC

TCTCCTCGTTG

TATCTGG

AATACTA(CA)AAG(CT)

470

DON

Kim et al. [36]

CTAG(GT)ACGACGC

**Fragment size (bp)**

334

**Chemotypes**

3-ADON

**References**

Wang et al. [49]

**Table 1.**

*Primers designed for genetic genotyping of FGSC so far.*

The biochemical approach involves the incubation and extraction of mycotoxins, the methods being complicated and time consuming. The molecular techniques are based on detection of specific gene by using specific primers. All these molecular methods developed for genotype analysis are based on nucleotide diversity of trichothecene synthesis genes. Chemotype characterization has been extensively used to characterize FGSC for their toxigenic potential [52]. The information about the genetic genotyping methods developed so far, such as targeted gene, primer name, primer sequence, and amplification fragment sizes are summarized in **Table 1**.

More effective and accuracy genetic methods are needed. We are doing genomic sequencing of FGSC strains with different trichothecene genotypes, and we believe some new molecular genetic methods will be developed based on the genomic data.
