A PCR-RFLP Analysis for the Diagnosis of DAS and T-2 Genotypes in Fusarium goolgardi

Fusarium pathogens and their mycotoxins are considered as the main threats to cereal production and food safety worldwide. However, due to the constant discovery of new Fusarium species especially along with mycotoxin production profile differentiation in certain species, efforts on their species composition, geographical distribution, and chemotype proportion are urgently required. In the Fusarium goolgardi species, two distinct trichothecene (TB) genotype populations have recently been identified. Previous studies have shown that the structural variance of TBs biosynthesized by the two genotypes is attributed to the Tri1 gene. Polymorphisms of Tri1 gene from type A TB-producers were investigated in different Fusarium species in the current study. According to these DNA sequence variations identified in Tri1 gene sequences, a polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) based diagnostic approach for the differentiation of T-2 and 4,15-diacetoxyscirpenol (DAS) genotypes in F. goolgardi was successfully developed. The PCR-RFLP assay will facilitate the studies on geographic distribution, frequency and other aspects of the two genotypes within F. goolgardi species.


INTRODUCTION
Fusarium is one of the most economically destructive fungal genera with many species that cause variety of plant diseases.5][6][7][8][9] As an example in the United States, FHB has caused more than 3 billion dollars losses in crop (include wheat and barley) since 1990s. 1,6[9][10] The fungi from Fusarium genera are also of concern because they can produce variety of mycotoxins such as TBs, a series of non-volatile sesquiterpenes have a common core skeleton.Currently, over two hundred TBs have been found, posing a serious risk to the safety of food and feed. 11Among these Fusarium secondary metabolites, type A and type B TBs are the main contaminants of small grains and the two group compounds have aroused considerable public concern worldwide. 12ype A and type B TBs are mainly characterized by the presence or absence of a ketone functional group at carbon 8 (abbreviated as C-8).In Type A TBs, there is an ester functional group, or a hydroxyl, or no substituent at all at C-8 position of the core TB molecule, and T-2, neosolaniol (NEO), and 4,15-diacetoxyscirpenol (DAS), respectively, are representative of this group (Figure 1). 13,145][16] Fusarium TBs of particular concern are type A groups, which are very poisonous. 17It has been demonstrated that Type A TBs are strong inhibitors of DNA, RNA, and protein synthesis; they can cause immunosuppressive and pathological changes in liver cells. 18,19Furthermore, these toxins can induce DNA fragmentation characteristic of apoptosis and even death. 20,21raditionally, chemotyping of Fusarium strains have been carried out using gas chromatography/mass spectroscopy which are relatively time-consuming and expensive. 22evertheless, PCR based molecular genetic assay approaches (genotype assays) facilitate the quick screening of Fusarium strain toxin potential.4][25][26][27][28] All these methods are based on the polymorphisms of the specific Tri genes which are involved in TB biosynthesis.Genotyping is a useful tool for predicting TB production potential of Fusarium strains, and is commonly used for Fusarium graminearum species complex (FGSC) strain genotyping studies. 22,29,30High-throughput diagnostic approaches for type A TB producers, on the other hand, have yet to be established. 31usarium goolgardi is a new species recently identified and the members of this species can produce type A TBs. 32,33 According to a recent investigation by Rocha LO et al., F. goolgardi populations have at least two TB genotypes: DAS-NEO-T2 genotype (abbreviated as T-2 genotype) and DAS genotype. 33Available data demonstrates that the enzyme encoded by Tri1 is required to catalyze the hydroxylation reaction of type A TBs at C-8 position, and thus it is involved in the biosynthesis of T-2, DAS, and NEO. 17

Tri1 gene sequence analysis
The Tri1 gene nucleotide sequences of different Fusarium species that produce type A TBs were obtained from GenBank database and evaluated for polymorphism.In total, eighteen Tri1 DNA sequences from four Fusarium species, including F. goolgardi (8 strains: RBG5411, 6914, 6915, 5417, 5419, 5420, 5421, and 5422), F. langsethiae (3 strains: NRRL53410, 53417, and 53439), F. sibiricum (2 strains: NRRL53421 and 53427), and F. sporotrichioides (5 strains: NRRL3299, 53434, 26924, 29977, and 29978), were retrieved from the NCBI GenBank and submitted to multiple sequence alignment analysis with Clustal W (version 2.0). 34The species were chosen for their ability to synthesize type A TBs, and all of the strains included in the study had previously been determined by chemical analyses. 33,35,36able contains complete information on all representative sequences.

Fusarium strain culture and DNA extraction
Mycelium of Fusarium strains grown on sterile glass-membrane paper overlaying potato dextrose agar, was scraped from the surface cultures and ground to a fine powder in the presence of liquid nitrogen.Homogenized mycelia were suspended in 650 μL pre-heated (65°C) CTAB buffer, mix well and then incubate in a water bath (65°C, 40 min).After incubation, total genomic DNA was extracted as previously reported. 28inally, DNA was diluted to ~50 ng/µL and kept frozen until required.

PCR amplification
Amplifications were performed in a final volume of 20 μL containing 1 × PCR buffer, 0.25 mmol/L of each dNTP, 0.2 mmol/L of primer A-Tri1F and A-Tri1R, and around 50 ng genomic DNA.The reaction was programmed as follows: 94°C 4 min; 30 cycles of 94°C 30 s, 60°C 20 s, 72°C 35 s; and 5 min final extension at 72°C.PCR products (2 μL) were separated through a 1.5% agarose gel electrophoresis and visualized under ultraviolet light after stained with nucleic acid fluorescent stain reagent (Transgen Biotech, Beijing, China).The PCR product sizes were evaluated according to known DNA standards.

Enzyme digestion
The Tri1 amplicon was subsequently digested with BfaI (NEB, Ipswich, MA, USA).The enzyme digestion was carried out in a 20 μL mixture containing 2 μL 10 × rCutSmart buffer, 3 μL Tri1 amplicon, 1 μL BfaI (10 U/μL), and 14 μL water.The digestion reaction was performed in a thermal cycler as previously described. 31mmediately after digestion, the products were detected by electrophoresis on 1.5% agarose gel.The approach is illustrated in Figure 2.

DNA polymorphism of Tri1 gene in Fusarium
In this study, we first compared the sequences of Tri1 homologs from three F. langsethiae (NRRL53410, 53417, 53439), two F. sibiricum (NRRL53421, 53427), and five F. sporotrichioides (NRRL3299, 53434, 26924, 29977, and 29978).The full-length Tri1 DNA sequences of these ten strains are varied from 1857 bp to 1860 bp with four introns, while the coding sequence is 1629 bp in all of them.Differences in DNA length of the intact Tri1 genes are due primarily to one and two nucleotides insertion/deletion in the third and fourth introns, respectively.
The coding sequences of these ten Tri1 DNA sequences were further subjected to alignment assays, and the results indicated that their identity ranged from 96.07% to 100%.The Tri1 coding sequences of F. sibiricum strain   and primer design.Nucleotide sequences of Tri1 gene DNA sequences from four F. goolgardi strains (RBG5411, 5417, 5419, and 5420) with T-2 genotype and four F. goolgardi strains (RBG6914, 6915, 5421, 5422) with DAS genotype were aligned against reference sequences from the abovementioned F. langsethiae, F. sporotrichioides, and F. sibiricum strains (Table ).As shown in Figure 3, Tri1 sequences in F. goolgardi strains with the DAS genotype differed significantly from those in T-2 producers.There is a nonsense mutation occurred at position 868 (C-to-T transition) in the middle of the Tri1 coding region, resulting in a premature stop codon and the failure of normal transcription and translation of Tri1 gene; therefore, it is considered as a pseudo-gene.While the Tri1 sequences from T-2-producing F. goolgardi strains did not exhibit the nonsense mutations, which generally will expressed normally.

Primer design
T h e a l i g n m e n t a l l o w e d d e s i g n i n g a p a i r o f p r i m e rs , A -Tr i 1 F (5′-GTAGCAAATCACCTACGCAGAT-3′) and A-Tri1R (5′-GAGGACGCATTCTCGTATATCT-3′).The expected amplicon size is 628 bp for all the type A TB-producing Fusarium strains, including F. sporotrichioides, F. goolgardi, F. sibiricum, and F. langsethiae.

PCR-RFLP approach for genotyping of F. goolgardi
A PCR-RFLP protocol targeting on the Tri1 gene variations was designed to distinguish between DAS and T-2 genotypes (Figure 2).Due to a single-nucleotide mutation, the fragment amplified with A-Tri1F/R from the F. goolgardi DAS strains have been digested into two different fragments of varying sizes by BfaI, MaeI, MthZI, and RmaI (the four restriction endonucleases recognize the same specific nucleotide sequence "CTAG").However, the amplicons from all the other strains, including the F. goolgardi T-2 strains, can't be digested by any of these restriction endonucleases.
To evaluate the reliability of the PCR-RFLP approach for identifying DAS and T-2 genotype strains, strains from F. goolgardi, F. langsethiae, F. sibiricum, and F. sporotrichioides were tested.TBs produced by these strains have been reported previously. 33,35,36As shown in Figure 4, after digestion with BfaI, the amplicons from F. goolgardi DAS strains were cut into two fragments with different sizes, 147 bp and 481 bp, respectively, in length.On the other hand, a single 628 bp band was observed from the amplicons of T-2 producers.The unique DNA profiles obtained from DAS and T-2 genotype strains generated by PCR-RFLP assays suggest that this method can be used for differentiation of these two type A TB producers in F. goolgardi populations.Furthermore, according to our analyses, the molecular genotyping detection method developed in the current study may also can be extended to identify DAS and T-2 genotypes in other species, such as F. langsethiae, F. sibiricum, and F. sporotrichioides.

DISCUSSION
TBs' structure diversity is caused by the variability of TB biosynthesis gene functions.3][24][25][26][27][28][29] As we know, Tri1 and Tri16 genes are critical for the hydroxylation reaction and its immediate acylation of the TB skeleton structure at C-8 position in the biosynthetic pathway of T-2, respectively.However, at C-8 of the DAS, there are just two hydrogen atoms, and no further modification occurred.Thus, compared with T-2 producers, there is probably a nonfunctional Tri1 gene or at least the gene can't be expressed normally in Fusarium strains which can only produce DAS toxin.
The occurrence of nonsense mutation in the coding region of Tri1 gene from F. goolgardi strains with a DAS genotype renders the gene non-functional, thus leading to the production of TB homologues without C-8 modifications. 17,33he findings also show that other polymorphisms may have occurred in the DAS lineage, possibly resulting in Tri1 gene dysfunction.The precise scenario that resulted in these mutations in the Tri1 gene is difficult to pinpoint; nonetheless, such changes can be exploited to distinguish between DAS and T-2 producers in the Fusarium genera.
6][37] However, the discovery of two different genotypes, DAS and T-2 types, within F. goolgardi strains has just been reported.The results from Rocha LO et al. demonstrated that some strains of F. goolgardi produced DAS and NEO in addition to T-2, while other strains produced only DAS. 33 In the current study, a PCR-based approach was developed for the diagnosis of DAS and T-2 genotypes in F. goolgardi.In contrast to T-2 genotypes, a nonsense mutation occurred at position 868 (C-to-T transition) in the Tri1 gene of the DAS genotype F. goolgardi strains.This mutation resulted in the emergence of a new enzyme cleavage site, C*TAG (* denotes the cleavage site), which can be recognized by restriction endonucleases BfaI, MaeI, MthZI, and RmaI.Although a small number of Fusarium strains were evaluated with our PCR-RFLP method, findings reported in Rocha LO et al. provide significant support to our results. 33ifferent genotyping methods are extensively applied in species identification, molecular marker screening, and genetic diversity analysis.These fingerprinting techniques consider information distributed over the whole genome of an organism and generally permit discrimination of Fusarium strains with different phenotypes like different species or different TB profiles.Among these techniques, RFLP is famous for its robustness, reproducibility, and reliability. 38ecently, four sets PCR-RFLP diagnostic methods for the differentiation of type B TB-and NXproducing FGSC were successfully developed by us relying on polymorphisms identified in Tri1 sequences. 31As far as we know, though, this is the first report on the genotyping analysis of T-2 and DAS genotypes in F. goolgardi.Further investigations are needed to determine the fungi's temporal and spatial distribution.The PCR-based RFLP assay developed in this work will make easier and faster the investigations on temporal and spatial distribution of DAS and T-2 populations.
Fusarium goolgardi is a recently identified Fusarium species. 32Therefore, further research will be necessary to track and monitor the two genotypes' geographic distribution and spread as well as assess any potential variations in their competitive capacities, including pathogenicity and environmental adaptability.Moreover, the divergence of TB genotype within the F. goolgardi species also highlights the need to re-evaluate genotype and population diversity of the economically destructive fungal pathogens in Fusarium genera throughout different agricultural ecosystems worldwide.
On the other hand, subsequent identification of mutations (premature stop codons and frameshift) in Tri16 coding region in F. goolgardi strains carrying the DAS genotype give rise to new questions and perspectives that whether DAS genotype strains contain a functional Tri16 gene exist or not in nature. 33If so, what kind of TBs produced by such strains?It is still unclear whether strains that primarily produce NEO or coexistences of DAS and NEO, however, lack T-2 toxin exist or not. 17Taking this into account, we recently conducted a comprehensive and systematic analysis of the advances on type A TB biosynthesis in Fusarium species, with respective to their biosynthetic pathway, gene evolution and other concepts, and the potential biosynthetic pathways for NEO and DAS toxins were proposed. 17ased on the previous findings and current questions, further research is needed to discover the biochemical alterations or genetic basis changes in TB biosynthesis and related molecular regulation mechanisms.The settlement of these issues will undoubtedly give greater impetus to the understanding of evolution of secondary metabolites biosynthesis in Fusarium species, and provide new insights on the control and prevention of mycotoxins in food safety.

CONCLUSION
Identification of Fusarium strain toxin potential is essential with respect to food safety issues due to TBs' different toxicological effects.Previously, two distinct TB genotype groups, namely T-2 genotype and DAS genotype, were found in the species of F. goolgardi.In this study, the polymorphisms of Tri1 gene from the two kinds of TB producers were investigated.A PCR-RFLP assay method was successfully developed to distinguish T-2 and DAS producers within F. goolgardi.This promising diagnostic method can be used for high-throughput genotype analysis of F. goolgardi strains as a step forward for plant disease management, TBs assessment and control in agriculture.Besides, this molecular diagnostic technology will facilitate the studies on its epidemiology, host range, and geographical distribution of the novel species.
,33  Current study focused on: (1) the polymorphisms of Tri1 gene from type A TB producers; (2) development of molecular genotyping methods for the diagnosis of T-2 and DAS genotypes in F. goolgardi based on their Tri1 gene polymorphisms.

Figure 2 .
Figure 2. A schematic diagram of the PCR-RFLP analysis for the diagnosis of DAS and T-2 genotypes

Figure 3 .
Figure 3. Alignment analysis of the nucleotides and corresponding protein sequences of partial Tri1.The conserved nucleotides and amino acids are indicated above and under the panel by * and †, respectively.The nonsense mutation sites in the CDS position 868 (C to T) of the F. goolgardi DAS strains indicated with green background, and the corresponding stop codon is represented by a solid circle (referred to strain NRRL29978)

Table .
Species, strains, genotypes, and sequence accession numbers of Fusarium Tri1 genes