Intra-strains Diversity of Expression of Polymorphic Pks4 Gene in Comparison in Zearalenone Production by Fusarium Graminearum during in Vitro Cultivation*

Filamentous fungi belonging to the Fusarium genus are responsible for large economic losses due to their high pathogenicity and toxigenicity. Fusarium sp. may produce variety of mycotoxins, one of them is zearalenone (ZEA). The presence of the PKS4 gene shows the possibility of zearalenone biosynthesis by Fusarium sp. In this study, in four Fusarium graminearum and one Fusarium poae strains the presence of PKS4 genes and ZEA concentrations were determined. The presence of the PKS4 gene was confirmed by classical polymerase chain reaction (PCR) in three of four strains of F. graminearum. One strain with no PKS4 gene detected was found while still producing ZEA. In the present study, a real-time PCR assay has been successfully performed for the relative expression of Fusarium strains based on new designed primers targeting the PKS4 gene involved in ZEA biosyn-thesis. Result shows that P56/4 strain of F. graminearum has the highest mRNA level, in the range of 12, what correlates to the high production of this mycotoxin. In this study, a real-time PCR assay has been successfully developed for the prediction of the production of ZEA by F. graminearum strains by PCR real-time techniques based on primers targeting the gene, PKS4, involved in ZEA biosynthesis. The special significance was pointed to occurring genes polymorphism.


INTRODUCTION
The most common pathogens in agriculture are fungi of the genus Fusarium (Mule & Logrieco, 1997;Leslie & Summerell, 2006).They are the cause of large economic losses due to their high pathogenicity and toxigenicity (O'Neil, 1986).In north-western and eastern European countries, the most common pathogenic Fusarium species are F. graminearum and F. culmorum depending on the climate of the country (Stenglein, 2009).F. graminearum is the most important Fusarium species in Central Europe and in large areas in North America and Asia (Yli-Mattila, 2010).Of 144 barley seed samples coming from 12 administrative regions of Quebec in 2000Quebec in , 2001Quebec in and 2002, F. , F. graminearum was the most common, and accounted for approximately 40% of the infected seed lots, but F. poe was the second most abundant species in 2000and 2002(Stenglein, 2009)).
The Fusarium genus currently includes over 80 species.This number is increasing as studies based upon deoxy-ribonucleic acid (DNA) sequence comparison become more common (Cuomo et al., 2007;Liu et al., 2013).The Fusarium graminearum species complex was previously classified as a single species.Advanced phylogenetic analyses based on GCPSR sequences revealed the complex structure of this group of plant pathogens and the presence of 16 phylogenetic species (Gardiner et al., 2014).
One of the most widespread toxin produced by Fusarium spp. is zearalenone (ZEA).It is classified into the group of non-steroid estrogens or mycoestrogens.The reduced form of zearalenone, zearalenol, has increased estrogenic activity (Edwards et al., 2002;Bennett et al., 2003).F. poae is able to synthesise fusarenon X, as well as trichothecenes, but no ZEA (Leslie et al., 2006).
The genetics and biosynthesis of different fusarins have been the subject of intensive study (Brown, 2004), but the genetics and biosynthesis of ZEA have received comparatively little attention.Ward et al. (2002) confirmed that a PKS genes complex is responsible for biosynthesis of family related enzymes called polyketide (2002), which is responsible for ZEA activity.A search of genomic sequence of Fusarium verticillioides, F. graminearum, F. oxysporum and F. solani identified a total of 58 PKS genes (Brown et al., 2012).
In the EU, the maximum limit value was determined for ZEA as 100 g kg -1 in unprocessed cereals (European Commission Regulation No 1126/2007, Yli-Mattila, 2010).In spite of the dangerous characteristics of different Fusarium species, the genetic and environmental regulation of their mycotoxin, specially ZEA production and phytopathogenicity, is still poorly characterised (Yli-Mattila, 2010).
The methods like high performance liquid chromatography (HPLC), gas chromatography (GC) or liquid chromatography with tandem mass spectrometry (LC-MS/MS) are usually used to detect zearalenone.Because these methods are laborious and time-consuming new, rapid and sensitive methods to detect zearalenone-producing Fusarium strains are needed to prevent food and feed contamination.Many possibilities are concerned with prognostic molecular methods (Suchorzyńska & Misiewicz, 2009;Suchorzyńska, Spera & Misiewicz, 2009).
The aim of this work is to predict pathogenity, through the means of producing ZEA in five Fusarium strains, isolated from wheat seeds, in comparison with real production of this mycotoxin during in vitro cultivations and assessment of efficacy of classical primers.

Materials.
From many isolates from wheat seeds, five strains isolates were chosen as representative examples from five voivoidships of Poland: świętokrzyskie (sample P7/4), opolskie (samples P48/1 and 48/6), śląskie (sample P56/4) and wielkopolskie (sample P63/23).Their localisation was combined with essential wheat areas in Poland.The strains are deposited in the Culture Collection of Industrial Microorganisms, prof.Waclaw Dabrowski Institute of Agricultural and Food Biotechnology, Warsaw.All strains were grown in potato-dextrose agar medium (PDA) plates at 20°C.
Filamentous fungi isolation.Kernels were plated on solid potato-dextrose agar (PDA) medium with the addition of 50% citric acid solution.After a few days of incubation at room temperature, the fragments of single hyphae of strains were transferred onto new plates with PDA plates.This was repeated at least three times to assure their purity.For the isolates, single spore cul-tures were performed according to Leslie & Summerell (2006).
DNA extraction.The mycelium was cultured in PDB liquid medium prior to DNA extraction.DNA extraction from fungal mycelia was carried out as described previously by Liu et al. (2000).In all samples, DNA content was between 200-300 ng l -1 .
Designing primers for real-time PCR reaction.Based on the gene sequence of polyketide synthase PKS4 (GenBank accession no DQ19316) new primers were designed to RT PCR reaction.Products of RT PCR reaction with PKS4 primers were sequenced (Genomed, Warsaw) and deposited (GenBank accession no.LC038084).The obtained PKS4 sequences were also compared with all the sequences from Fusarium ID Database (Geiser et al., 2004), so as to avoid polymorphic sites which gave false negative results in a classic reaction.Sequence specificity of the primers was checked by a blast program (http://www.ncbi.nlm.nih.gov/BLAST/,Altschul et al., 1990).Primers are listed in Table 2. DNA sequences of the EF1alfa gene used as a housekeeping gene (Brunner et al., 2009) were retrieved from GeneBank and aligned using Vector NTI (Lu & Moriyama, 2004).The PKS4_RT_F primer is positioned at the bases 88 to 108, whereas PKS4_RT_R is positioned at the bases 157-179.This set of primers amplified a product of 91bp in F. graminearum.A series of 10-fold dilutions of stock solution were prepared as the templates for generating a standard curve through real-time PCR reactions.Three biological replicates were used.Specificity of the PCR real time reaction.The specificity of PCR real time reaction was tested using Fusarium poae strain 63/23, which has no ability to produce ZEA.
RNA extraction and cDNA reverse transcription.RNA was extracted using the commercially available kit, RNease Plant Mini Kit (Qiagen), and purified from any residual genomic DNA using the RNase-Free DNase Set (Qiagen).The quality and quantity of the extracted RNA was assessed with a NanoDrop Spectrophotometer (Thermo Scientific) and by electrophoresis in 1% agarose (Sigma).The RNA samples were conserved with liquid nitrogen (-196°C).
The extracted RNA was then subjected to reverse transcription (cDNA synthesis) using the QuantiTect Reverse Transcription Kit (Qiagen) with oligo (dT) priming.The purity and yield of cDNA were determined using a NanoDrop Spectrophotometer (Thermo Scientific).The cDNA were stored at -20°C.
Real-time PCR reaction and quantitative analysis of the data.Real-time PCR analysis was performed in Rotor Gene 6000 instrument (Corbett Life Science) using the double-stranded-DNA-specific fluorochrome SYBR Green (Qiagen).PKS4 gene expression analysis was carried out with the QuantiTect SYBR Green PCR Kit (Qiagen), the Maxima SYBR Green qPCR Master Mix (2x) (Thermofisher Scientific).As a reference gene, the EFG gene was used (Brunner et al., 2009).The results of real-time PCR were analysed according to 2-∆∆ method (Livak, 2001).The expression level was described in terms of the cycle threshold value (Ct), which is the number of cycles required to reach a certain fluorescence value (threshold).The data, expressed as Ct, was imported into a Microsoft Excel data sheet for the next, statistical analysis.
Cultures for mycotoxin analysis.The wheat grains were sterilised, cooled and inoculated with spores obtained by growing isolates (Joffe & Palti, 1975) on PDA slants with 10 ml of peptone water.They were then incubated for up to two weeks at room temperature (19-25°C) under varied lighting conditions.Following the dying of the grains, the mycotoxin content was determined.
Methanol:ACN:water mixture (8:46:46) was used as the isocratic mobile phase with the flow rate of 1 mL min -1 .The calibration curve was determined using ZEA standards of the 3.75/18.75/37.50ng mL -1 concentrations (corresponding to concentrations of the analyte in the samples, 10, 50 and 100 µg kg -1 respectively).
Fifty grammes of the sample was ground, mixed with 5 g of NaCl and extracted for two minutes in a homogeniser with 100 mL of 90:10 v/v ACN:water solution.The entire mixture was transferred into a centrifuge tube and centrifuged for 10 minutes at an acceleration of 10 730 × g.Next, 10 mL of the extract, dissolved in 40 mL of water, was treated with ultrasounds in an ultrasonic bath for two minutes, then filtered through a Microfibre Filter (Vicam, Watertown, MA, USA).ZearalaTest ® Immunoafinity Columns (IAC) (Vicam, Milford, USA) were used.
In the case of zearalenone, it is very important to maintain the proportion between organic solvent and water.Zearalenone is not easily soluble in water, therefore, the extraction has to take place with a high proportion of methanol and/or acetonitrile in the extraction mixture.Since too large a proportion of organic solvent in the extract causes elution of the analyte from the IAC columns, the extract was diluted with water to a maximum, with a minimum quantity of organic solvent capable dissolving the analyte.
The dissolved and filtered extract was cleaned up on IAC column: 10 mL of the extract was passed through the column at the 1-2 droplets per second rate.ZEA was eluted with 3 mL of methanol at the 1 droplet per second rate, then evaporated in a stream of nitrogen.Residues dissolved in 2 mL of ACN:methanol:water mixture (46:8:46) were submitted to analyses.
Method recovery rate and precision were tested on some wheat samples spiked with ZEA to the 50 µg kg -1 level.The samples were previously verified as not containing the toxin.The average recovery rate (n=3 samples) was 100.4%.No relative standard deviation (RSD) exceeded 5%.ZEA limit of quantification was 10 µg kg -1 .

RESULTS
Four strains are identified as F. graminearum and one as F. poae.All isolates were checked for cultivation for subsequent mycotoxin assay based on the presence of genes involved in mycotoxin synthesis.In conventional PCR, all F. graminearum strains, except one, gave a 400 bp product.There was no PCR product in the P7/4 (F.graminearum) or in the P63/23 sample (Fusarium poae strain).These samples were subjected to chromatography analysis (Table 3) and these isolates were selected for PKS4 gene expression analysis.The content of ZEA was different in the samples.In the P48/1, P48/6 and P56/4 samples, the content of ZEA was similar in amount, from 89 650 to 94 580 µg kg -1 .In the P/7 sample, the content of ZEA was lower, and lacked mycotoxin in the P63/23 sample (Table 3).
PKS4 gene expression was analysed with real time PCR in the following isolates: P48/1, P48/6, P56/4, due to high mycotoxin concentration, P7/4, due to zearalenone production despite the fact that the PKS4 gene was not detected by conventional PCR, and P63/23, to compare result with a species without the ability to synthesise zearalenone (Fig. 1).The increased PKS4 expression was found to be almost 10-fold in P56/4 and over 6-fold in the P48/6 cultivations.

DISCUSSION
Biochemical and genetic studies of Fusarium sp.conducted by Proctor et al. (1999), Ward et al. (2002) and others led to the identification of the genes responsible for biosynthesis of mycotoxins.These methods allowed for the mechanisms of zearalenone biosynthesis and the significance of the PKS gene complex to be described.The studies revealed the relationship between PKS4 expression and biosynthesis of zearalenone.PKS4 encodes an enzyme that stimulates the expression of another gene, a PKS13 -encoding protein involved in the final stage of zearalenone biosynthesis.This protein catalyses the carbon addition to the final intermediate compound.Studies performed by Lysoe et al. (2006) indicated that PKS4 is necessary for zearalenone biosynthesis.
The complete sequencing of the F. graminearum genome has allowed the identification of distinct regions of high diversity (Trail, 2009).Cuomo et al. (2007) have found that these regions are rich with infection-related genes, which may allow this fungus to adapt rapidly to changing environments or hosts.The availability of its genomic resources will allow the comparative analysis of F. graminearum and F. poae, for understanding the changing of gene clusters (Stenglein, 2009).Genes involved in the production of secondary metabolites and secreted proteins are enriched in non-conserved regions.They found evidence that non-conserved regions are generated by the sequestration of genes from multiple locations (Zhao et al., 2014).
Research has shown zearalenone production in Fusarium graminearum strains in which the PKS4 gene was not detected.As presented in studies performed by Lysoe et al. (2006), PKS4 is necessary for zearalenone biosynthesis, which was proven by DNA fragment insertion into the PKS4 gene resulting in stopped production of mycotoxin.However, the exact zearalenone synthesis gene mechanism is still not known.Gaffoor & Trail (2006) has proposed a possible ZEA production path in which the PKS4 encoded enzyme catalyses the first 10 carbon additions, and after that reduced moiety is subsequently transferred to the PKS13 encoded enzyme, where the remaining three rounds of C2 additions are catalysed.These results show the crucial role of the PKS4 gene in ZEA synthesis.
With regard to the P7/4 isolate, no amplicon for the PKS4 gene was detected in classical PCR reaction using PKS4F and PKS4R primers, although expression of this gene was observed using different, shorter PKS4_RT_F and PKS4_RT_R primers.The zearalenone concentration in this case was 37 700 µg kg -1 , relatively low, which confirm some structural changes in gene sequences, comparing to the rest F. graminearum strains.In the remaining isolates of Fusarium graminearum, mycotoxin content was approximately 90 000 µg kg -1 .Similar findings were observed previously by Stepien et al. (2012) in a few isolates of Fusarium equiseti, which indicates the possible polymorphisms of the PKS4 gene and significant intra-specific diversity.Some authors thought that the diversity and distribution of PKSs was attributed to gene duplication, divergence and differential gene loss rather than horizontal gene transfer (HGT) (Kroken et al., 2003), but the analysis of Brown et al. (2012) suggested that HGT could be responsible for significant PKS diversity.It is also taken into consideration that the PKS13 and PKS4 genes functions together as a multiprotein complex.In addition, genes involved in the production of secondary metabolites and secreted proteins are enriched in non- conserved regions, suggesting that these regions could also be important for adaptations to new environments, including adaptation to new hosts.Zhao et al. (2014) found evidence that non-conserved regions are generated by sequestration of genes from multiple locations.Gene relocations may lead to clustering of genes with similar expression patterns or similar biological functions, which was clearly exemplified by the PKS2 gene cluster (Zhao et al., 2014).Baturo-Ciesniewska & Suchorzyńska (2011) confirmed that primers, recommended in the literature, for the Fusarium species were not completely effective.It was shown that from among the four primer pairs enabling the identification of F. culmorum, and therefore also to establish its presence in the material, only primers Fc01F/ Fc01R seem to be fully effective in the case of Polish strains (Baturo-Ciesniewska & Suchorzyńska, 2011).
In our study, lack of the product of PCR was probably due its polymorphism or changes in genome.With regard to the P63/23 isolate (F.poae), molecular analysis indicated the absence of PKS4 genes, and the expression analysis confirmed the lack of PKS4 expression, which was reflected by no observed zearalenone production.The low content of this mycotoxin was detected in cultures inoculated with P7/4, isolate P48/1, and almost three times higher than in the P56/4 and P48/6 isolates.However, no correlation was found between the PKS4 expression level and the quantity of zearalenone produced by the particular Fusarium spp.isolate.
A pathogen's toxicity is associated with high variations in virulence traits, the spread of isolates with specific attributes into new regions, the ability to produce three types of mycotoxins as well as fungicide resistance (Chen & Zhou, 2009;Yli-Mattila et al., 2009;Yli-Mattila, 2010;Zhang et al., 2012;Sploti et al., 2014).
This observation is a challenge and warrants further studies in this subject, especially on the impact of genes polymorphisms on mycotoxin production and pathogenicity of filamentous fungi (Baldwin et al., 2010).
Production of zearalenone was also observed when neither the PKS4 nor the PKS13 gene was detected.In our results, expression of PKS4 gene is observed with specially designated, new, shorter starters, avoiding polymorphic sites.This observation is a challenge and warrants further studies in this subject, especially on the impact of polymorphisms on mycotoxin production and pathogenicity of filamentous fungi.

Table 3 . Molecular data from the evaluation of Fusarium spp. iso- lates selected for in vitro cultivation for mycotoxin analysis
Figure 1.Comparison of PKS4 gene expression in five isolates of Fusarium spp.Error bars indicate standard deviations.PKS4 gene expression in Fusarium graminearum