Development of a Generic PCR Detection of 3-Acetyldeoxy-nivalenol-, 15-Acetyldeoxynivalenol- and Nivalenol-Chemotypes of Fusarium graminearum Clade

Fusarium graminearum clade pathogens cause Fusarium head blight (FHB) or scab of wheat and other small cereal grains, producing different kinds of trichothecene mycotoxins that are detrimental to human and domestic animals. Type B trichothecene mycotoxins such as deoxynivalenol, 3-acetyldeoxynivalenol (3-AcDON), 15-acetyldeoxynivalenol (15-AcDON) and nivalenol (NIV) are the principal Fusarium mycotoxins reported in China, as well as in other countries. A genomic polymerase chain reaction (PCR) to predict chemotypes was developed based on the structural gene sequences of Tri13 genes involved in trichothecene mycotoxin biosynthesis pathways. A single pair of primers derived from the Tri13 genes detected a 583 bp fragment from 15-AcDON-chemotypes, a 644 bp fragment from 3-AcDON-chemotypes and an 859 bp fragment from NIV-producing strains. Fusarium strains from China, Nepal, USA and Europe were identified by this method, revealing their mycotoxin chemotypes identical to that obtained by chemical analyses of HPLC or GC/MS and other PCR assays. The mycotoxin chemotype-specific fragments were amplified from a highly variable region located in Tri13 genes with three deletions for 15-AcDON-chemotypes, two deletions for 3-AcDON-chemotypes and no deletion for NIV-producers. This PCR assay generated a single amplicon and thus should be more reliable than other PCR-based assays that showed the absence or presence of a PCR fragment since these assays may generate false-negative results. The results with strains from several different countries as well as from different hosts further indicated that this method should be globally applicable. This is a rapid, reliable and cost-effective method for the identification of type B trichothecene mycotoxin chemotypes in Fusarium species and food safety controls.


Introduction
Fusarium head blight (FHB) or scab of wheat and other small cereal grains caused by Fusarium graminearum clade pathogens is an economically devastating disease worldwide [1]. FHB occurs both in the field and during storage, producing mycotoxins in moldy corn and wheat that are toxic to human and domestic animals [2][3][4][5][6]. Type B trichothecenes (8-ketotrichothecenes) are the principal toxins produced by F. graminearum clade. Based on the chemical structures and the acetylation positions of different 8-ketotrichothecenes, three trichothecene mycotoxin chemotypes have been identified within the type B trichothecene-producing F. graminearum clade: (IA) deoxynivalenol and 3-acetyldeoxynivalenol (3-AcDON), (IB) deoxynivalenol and 15-acetyldeoxynivalenol , and (II) nivalenol and 4-acetylnivalenol (4-AcNIV) [7][8][9][10]. These trichothecene mycotoxins are difficult to detect and thus pose a serious risk to human health. Development of a fast, generic detection for DON, NIV and their acetylated mycotoxins will facilitate the molecular biology study and analysis of those mycotoxins in cereal grains and the derived products for food and livestock to reduce mycotoxin load.
Molecular characterization of trichothecene mycotoxin biosynthesis pathways has revealed the mycotoxin gene clusters and their regulations [5,6,[11][12][13][14][15][16]. The Tri13 gene has been found to be the determinant for the DON-NIV switching in Fusarium, and the Tri7 gene is responsible for acetylation of NIV to produce 4-AcNIV. NIV-producers carry functional Tri7 and Tri13 genes while nonfunctional copies of both genes are present in DON-producers [17][18][19][20]. Comparative analysis of Tri13 gene sequences from different Fusarium fungi revealed that the deletion sequences within Tri13 gene appear to be associated with acetylation position of DON-chemotypes in addition to their association with the DON-NIV switching. Based on these sequences we have designed a pair of primers that allows amplification of 3-AcDON-, 15-AcDON-and NIV-chemotype-specific fragments of different sizes by PCR. With this method all isolates of F. graminearum clade should produce a trichothecene mycotoxin chemotype-specific product, which makes the PCR-based mycotoxin assays more efficient and reliable.
To demonstrate the applicability and reliability of this generic PCR detection, a global collection of F. graminearum clade strains from China, Nepal, USA and different European countries were assayed with this method together with GC/MS analysis. The results indicated that three different mycotoxin chemotypes of F. graminearum clade pathogens can be identified efficiently with this pair of primers, suggesting that this generic PCR detection could be used in the identification of mycotoxin chemotypes and food safety controls.

Fusarium Strains
All the Chinese Fusarium strains were selected from a large collection of Fusarium strains described previously [21]. They were isolated by single-spore isolation from the diseased wheat spikes that were collected from the regions with a known history of FHB epidemics in China. All the foreign Fusarium strains from France, Germany, Italy, Nepal, UK, and USA were obtained from the fungal collection of the John Innes Centre, UK, that were kindly provided by Dr. Paul Nicholson [22]. In total 54 strains of F. graminearum clade were used in this study, and their known chemotypes and detailed information are listed in Table 1.

DNA Extraction
Fusarium strains were grown on sterile glass-membrane paper overlaying potato dextrose (PDA) at 25 °C for 5 days. The mycelium were harvested and ground to fine powder in the presence of liquid nitrogen. Total genomic DNA was extracted using the CTAB method as described by Nicholson et al. [23].

PCR Amplification
PCR reactions were carried out in a volume of 25 μL containing 50 ng DNA template and PCR reagents used were as described by Li et al. [24]. A negative control omitting DNA template was used in every set of reactions. The thermal cycler (Mycycler, Bio-RAD, USA) conditions used were: 94 °C for 4 min, followed by 35 cycles of 94 °C for 1 min, 58 °C for 40 s, 72 °C for 40 s, then a final extension of 72 °C for 6 min. PCR products were separated by electrophoresis on 2 % agarose gels, stained with ethidium bromide and photographed under UV light in the Bio-Imaging system (Bio-RAD, USA).

GC/MS Analysis
The strains were cultured in Petri dishes on the surface of a cellophane membrane laid over the PDA medium. After growth for 4 days in 28 °C, the mycelium was collected and ground as described above for DNA isolation. The powder was dried in electric blast drying oven. Supercritical fluid extraction (SFE) was used to extract mycotoxins. Extraction were analyzed by gas chromatography/mass spectrometry (QP2010, Shimadzu, Japan) as previously described by Maciej et al. [25] with the following modifications. The analysis was performed with a programmed temperature from 120 °C hold for 1 min, then to 280 °C at 20 °C min −1 , and the final temperature being held for 8 min. The helium flow rate was held constant at 1 mL/min. The following ions were used for trichothecene detection: DON, m/z 235 and 422; 3-AcDON, m/z 117 and 392; 15-AcDON, m/z 193 and 392. The first ion in each set was used for quantitative analysis.

Results and Discussion
To investigate the reliability of the Tri13P1 and Tri13P2 primers for the identification of the 3-AcDON-, 15-AcDON-and NIV-chemotypes of F. graminearum clade, twenty six F. graminearum strains from various geographical origins were selected for the PCR assay. The mycotoxin chemotypes of these strains were determined either by HPLC, GC/MS or PCR. For instance, the strain 7015 from China was first determined to produce DON mycotoxin by HPLC [24] and is now identified as a 3-AcDON-producer by GC/MS (Table1). The Chinese strain 5035 was identified as a 15-AcDONproducer by PCR assay [26] as well as GC/MS analysis (Table 1), while RK10 from Nepal produced NIV mycotoxins as revealed by both HPLC and PCR [24] (Table 1). PCR with Tri13P1 and Tri13P2 primers indeed showed that the 15-AcDON-chemotypes yielded a 583 bp fragment, a 644 bp fragment was generated from the 3-AcDON-chemotypes, and the NIV-chemotypes produced an 859 bp fragment. The three different chemotype-specific DNA fragments displayed a distinct profile on an agarose gel that could be easily determined by visual image under UV light (Figure 1).  Table 1.
With this method fifty-four strains from China, Europe, Nepal and USA were assayed and the results indicated that this pair of primers efficiently amplified a DNA fragment for all the strains with a chemotype-specificity. The results from this PCR assay were completely congruent with the previous chemical analyses and PCR identifications (Table 1) [22,24,[26][27][28], indicating the high reliability for this generic PCR detection of three trichothecene mycotoxin chemotypes from F. graminearum clade strains.
The distinct DNA fragments from different chemotype-producing strains generated by this single pair of primers suggested that each chemotype contained a conserved structure within the Tri13 gene sequences. NIV-producers carried an intact, functional Tri13 gene with the region amplified by Tri13P1 and Tri13P2 primers, thus generating an 859 bp fragment (Figure 2-C). However, deletions were present in the Tri13 gene sequences of DON-producing strains. 3-AcDON-chemotypes contained two deletions of 178 bp and 37 bp in length, respectively, that were located in the region spanned by the two primers Tri13P1 and Tri13P2, generating a 644 bp fragment (Figure 2-B producers not only carried these two deletions but also had a third deletion of 61 bp in the region, yielding a 583 bp fragment (Figure 2-A). These structural characters ensure the efficient differentiation among the three chemotypes by this generic detection method.  [30,31]. The current study revealed that the numbers of the deletion within this region of the Tri13 gene were apparently associated with the position of acetylation in DON-mycotoxin producers, which could be used as the molecular distinction between 3-AcDON-and 15-AcDON-chemotypes. Mechanisms involved in the creation of the chemotype-specific sequences in the Tri13 genes during the evolution within F. graminearum clade remain to be investigated.
The generic PCR detection of 3-AcDON-, 15-AcDON-and NIV-chemotypes based on one amplicon should be more reliable than other PCR-based assays that showed the absence or presence of a PCR fragment since these assays may generate false-negative results. The results with strains from several different countries as well as from different hosts further indicated that this method should be globally applicable. This is a rapid, reliable and cost-effective method for the identification of three mycotoxin chemotypes in Fusarium species.

Conclusions
A single pair of primers based on the Tri13 gene sequences of F. graminearum clade was designed that detected a chemotype-specific DNA fragment with different sizes from 3-AcDON-, 15-AcDONand NIV-producers of F. graminearum clade strains. This PCR-based method was applied to assay the Tri13P2 mycotoxin chemotypes of Fusarium strains from different countries and different hosts. The chemotypes revealed with this pair of primers were identical to that obtained by chemical analyses and other PCR-based assays. This generic PCR detection of the type B trichothecene mycotoxin chemotypes apparently appears to be more reliable and accurate than other PCR-based assays that may generate false-negative results based on the presence or absence of a DNA fragment. This is a reliable and cost-effective method for the identification of the trichothecene mycotoxin chemotypes in F. graminearum clade as well as in food and feed safety controls.