Validation of a Quick PCR Method Suitable for Direct Sequencing: Identification of Fusarium Fungal Species and Chemotypes for Preventive Approaches in Food Safety

Abstract

Species determination by sequencing and PCR genetic chemotyping, used to determine the toxigenic potential of Fusarium strains, is fundamental for developing preventive strategies in food safety. In this paper, the authors propose and statistically validate a quick protocol for standardizing the procedure of species determination by sequencing of the elongation factor 1-α and multiplex genetic chemotyping using the Tri12 gene, based on fungal growth on Miracloth tissue coupled with microwave extraction. The test was validated on 75 Fusarium culmorum and Fusarium graminearum strains.

Headnote

Summary

Species determination by sequencing and PCR genetic chemotyping, used to determine the toxigenic potential of Fusarium strains, is fundamental for developing preventive strategies in food safety. Here we propose and statistically validate a quick protocol for standardizing the procedure of species determination by sequencing of the elongation factor 1-α and multiplex genetic chemotyping using the Tri12 gene, based on fungal growth on Miracloth tissue coupled with microwave extraction. The test was validated on 75 Fusarium culmorum and Fusarium graminearum strains.

Key words: Fusarium culmorum, Fusarium graminearum, microwave DNA extraction, EF1-^

Introduction

Polymerase chain reaction (PCR) is widely used for the identification of fungal species. Specific primers and sequencing procedures are needed for cross-checking morphological properties (1). More often, specific target sequences allow determining features of the strain that can be useful for its characterization. Genes encoding for dangerous fungal toxins found in food items such as aflatoxins, trichothecenes and fumonisins have already been identified (2). For example, by using primers tar- geting genes involved in the biosynthetic pathway of trichothecenes, it is possible to determine the toxigenic potential of Fusarium strains (3,4) by predicting whether a specific strain will produce deoxynivalenol and its ac- etylated derivates, or nivalenol, which is of significantly higher toxicity for human cell lines (5). Moreover, fungal species determination can be used as a tool for identify- ing potential toxigenic risks linked to the presence of species able to produce toxic compounds. Therefore, PCR is essential for preventive approaches in the area of food safety.

For this purpose, a simple, rapid and reproducible preparation of nucleic acids is required. However, as with plants, fungal nucleic acids are often complicated to ex- tract due to the presence of cell walls and secondary me- tabolites that can inhibit enzymatic reactions, making the DNA extraction process a crucial step. Several proto- cols for fungal DNA extraction have been reported. Many methods rely on a mechanical disruption of the cell by grinding (6,7), which can be preceded by a freezing step in liquid nitrogen (8-11), with the use of a mortar and pestle (12-15) or a bead milling method (16-18). Other methods for breaking up the mycelium include sonica- tion (16) or thermolysis (16,19,20) to extract DNA.

Chemical extraction such as the one using cetyltri- methylammonium bromide (CTAB), phenol, or chloro- form is largely adopted (1,15,16,20-24), as well as enzy- matic extraction (25-27) methods. These methods have gradually been incorporated into or replaced by less cum- bersome procedures such as the use of kits (1,16,19, 28-35) based on membrane filtration or magnetic sepa- ration. In all cited methods, sample preparation, how- ever, is required and even with the use of automatic grinders, the time and the possibility of upscaling the extraction procedures are limited and/or expensive.

Other methods have been developed to simplify the extraction, such as techniques allowing a direct extrac- tion into an Eppendorf tube using a pestle or a mortar (36) from fresh or lyophilized mycelium. For example, Cenis (9) cultivated fungi directly in an Eppendorf tube, which can reduce the risk of contamination.

Based on the discoveries of Goodwin and Lee (37), who showed that it is possible to extract DNA from eukar- yotic cells with microwave radiation, Ferreira and Glass (38) adapted this technique to fungal spores. Direct spore PCR is performed after irradiation of the spores and buf- fer solubilization, a stage of vortexing and of centrifuga- tion. The availability of engineered Taq polymerase has improved the efficiency of direct PCR approaches; there- fore, the implementation of direct PCR methods would facilitate large screening efforts on fungi. Recently, Fata et al. (39) and Borman et al. (40) developed a rapid meth- od for the preparation of total genomic DNA using What- man FTA filter papers (GE Healthcare, Fairfield, CT, USA). The principle is based on the lysis and inactivation of microorganisms, provided by chelators and denaturants present in the Whatman paper, coupled with homoge- neous collection of mycelium on the surface of the pa- per. More recently, Ben Amar et al. (41), developed a di- rect PCR-based procedure for DNA amplification from crude samples or spores in F. culmorum by manual sam- pling of the mycelium.

Fusarium head blight (FHB), mainly caused by Fu- sarium species, is a major disease of small grain cereals. The disease can cause significant problems of yield and quality losses (42). Major concerns arise from the con- tamination of grains with mycotoxins and their impact on human health and animal development (5,43). The prediction of the presence or absence of F. graminearum and F. culmorum, the main producers of mycotoxins in wheat, as well as the determination of chemotypes may play an important role in preventive food safety strate- gies. For this purpose, a cheap, high throughput method is needed.

Here, by combining a homogeneous method of my- celium sampling (simplifying the sample preparation) and a PCR approach, we tested four protocols for their efficacy in multiplexing with four primers and sequenc- ing of PCR products on a large set of Fusarium isolates.

Materials and Methods

Samples

Fusarium samples, deposited in the strain collection of the Centre de Recherche Public - Gabriel Lippmann (Belvaux, Luxembourg), were collected from different wheat fields across Luxembourg in 2011 (Table 1). After isolation as described previously (35), strains were stored at -80 °C in 15 % of glycerol as spore suspensions.

Samples were prepared according to two protocols. First, a potato dextrose agar medium (PDA, 39 g/L) was prepared, sterilized and poured into Petri dishes. To fa- cilitate the sampling of mycelium, 5-mm diameter pieces of four types of materials were placed on the Petri dish before mycelium inoculation: Miracloth (Merck Millipore, Darmstadt, Germany), Wypall L40 (Kimberly-Clark Pro- fessional, Roswell, GA, USA), dialysis membrane (Spec- tra/Por, cellulose, molecular porous membrane tubing, molecular mass cut-off: 12-14 kDa; Spectrum Labora- tories Inc, Rancho Dominguez, CA, USA), and paper (Planet+ plain paper, 80 g/m2; Xerox, Norwalk, CT, USA). Afterwards, Petri dishes were inoculated with 10 ^Lof spore suspension (approx. 1000 spores) and kept at 22 °C in the dark for 5 days (or for 13 days to test the effect of ageing of the mycelium on the extraction method).

In the second procedure used for the Qiagen DNA extraction kit, potato dextrose broth medium (PDB, 24 g/L) was prepared, then 50 mL of it were distributed in Erlenmeyer flasks and sterilized. These were inoculated with 10 ^L of spore suspension and incubated on an or- bital shaker at 150 rpm at 25 °C in the dark. After one week of growth, the mycelium was recovered by filtra- tion and transferred into a 1.5-^L Eppendorf tube. The mycelium was freeze-dried for 24 h (Christ Alpha 2-4 LSC, Marin Christ and Co., Osterode am Harz, Ger- many), immersed in liquid nitrogen and crushed with a mixer mill (Mixer mill MM200, Retsch GmbH and Co., Haan, Germany) at a frequency of 25 Hz (3 times, 20 s). The mycelium was stored at -20 °C.

DNA extraction methods

The following DNA extraction methods were used (Fig. 1): microwave DNA extraction, extraction by a heating step, direct extraction in PCR tubes, mycelium in-tube grinding extraction coupled with microwave ex- traction, and, for comparison, the DNeasy plant Mini Kit extraction (Qiagen, Hilden, Germany).

Microwave DNA extraction

Discs of the materials covered with mycelium were removed from the Petri dishes with a sterilized tooth- pick and placed into an Eppendorf tube with 100 ^Lof AE buffer (10 mM Tris-HCl, 0.5 mM EDTA; pH=9.0; Qia- gen). DNA was extracted using microwave irradiation, with a slightly modified protocol of Ferreira and Glass (38). Each tube containing the disc with the mycelium on it and the AE buffer was irradiated in a domestic mi- crowave (Easytronic, M571, Whirlpool, Benton Harbor, MI, USA) with full power (750 W) for 5 min.

Extraction by a heating step

The initial steps of the extraction were carried out according to the same protocol as in the microwave DNA extraction. Once the discs were removed from the Petri dishes, Eppendorf tubes were placed in a heating block at 95 °C for 10 min.

Direct PCR extraction

Samples were also processed in a direct PCR approach using the Phusion® High-Fidelity PCR Master Mix (Thermo Fisher Scientific, Wilmington, DE, USA). One of the discs covered with mycelium was transferred from the Petri dish with a sterilized toothpick and placed into PCR tubes containing 50 ^L of the Master Mix (Thermo Fisher Sci- entific), 2.5 ^L of each primer and 45 ^L of DNAse/ RNAse-free water. The denaturation was extended to 8 min at 98 °C.

Grinding of mycelium and microwave extraction

Aerial mycelium was manually collected from a strain inoculated on PDA, mixed with 180 ^L of AE buffer and ground with a pestle for 1 min (4) in the Eppendorf tube. The mixture was placed in a microwave at 750 W for 5 min. A centrifugation for 30 s at 12 000×g allowed the deposition of mycelia. A volume of 3 ^L of the up- per phase was used for the single and multiplex PCRs carried out as described below.

DNeasy Plant mini kit extraction

Samples were also extracted according to Dubos et al.(35) using the Qiagen DNeasy Plant mini kit (Qia- gen). After DNA extraction, an evaluation of the DNA quality and quantity was done on precast 1 % agarose gel (Bio-Rad Laboratories, Hercules, CA, USA) using ethidium bromide colouration for staining. The gel was run at 110 V for 5 min, then at 80 V for 90 min. Fluores- cent bands, exposed to UV light, were compared relative to a 2-log DNA ladder (0.1-10.0 kb, New England Bio- Labs, Ipswich, MA, USA). Estimation of purity ratio (260/ 280 nm) was calculated using the NanoDrop® ND-1000 (Thermo Fisher Scientific).

Single and multiplex PCR

Single and multiplex PCRs were carried out on the materials obtained from the different sampling and ex- traction procedures. Primers EF1 and EF2 (44) were used to identify the Fusarium species (1). The amplification was optimised using 2× Phusion High-Fidelity PCR Master Mix with high fidelity buffer (Thermo Fisher Scientific). This mix contains a Phusion DNA polymerase, 2× Phu- sion High-Fidelity buffer and 400 ^M of each dNTP and 1.5 mM MgCl2. PCR was performed in a final volume of 50 ^L containing 5 ^L of the supernatant, 25 ^L of the Master Mix, 1.25 ^L of each primer (10 ^M) and 17.5 ^L of DNAse/RNAse-free water.

Gradient PCR was performed on a Biometra TPro- fessional cycler (Göttingen, Germany) to define the opti- mum temperature for annealing, resulting in the final PCR program: 98 °C during 2 min for DNA strand sepa- ration; then 40 cycles of denaturation at 98 °C for 15 s, annealing at 55.2 °C for 20 s, elongation at 72 °C for 20 s; 5 min at 72 °C for the final extension followed by 4 °C until gel loading. A precast 3 % agarose gel (1×32 wells, Bio-Rad Laboratories) was used. The gel was exposed to 110 V for 40 min. Gel pictures were captured using an image analyzer (Ingenius Syngene Bio Imaging, Syngene, Cambridge, UK). Fluorescent bands were compared rela- tive to a MassRuler(TM) Low Range DNA Ladder, ready- -to-use (80-1031 bp, Thermo Fisher Scientific) and to the positive control strain PH1 from the USDA collection. A band between 700 and 800 bp was the evidence of the presence of a Fusarium strain.

PCR products were then purified using the Wizard® SV Gel and PCR Clean-Up System (Promega, Durham, NC, USA) and quantified before PCR labelling. The con- centration of each sample was adjusted to 25 ng/^L. La- belling of PCR was carried out according to the manu- facturer's procedures using the Big Dye Terminator v. 3.1 Cycle Sequencing Kit method (Applied Biosystems, Thermo Fisher Scientific) with EF1 and EF2 primers. PCR conditions were the following: 96 °C for 1 min, 25 cycles of 10 s at 96 °C and 4 min at 60 °C and to finish the cooling at 4 °C until purification. BigDye® XTermi- nator® Purification Kit (Applied Biosystems) was used for eliminating all the non-incorporated bases and the primers.

Sequencing was carried out using an Applied Bio- systems 3130 Genetic Analyzer (Applied Biosystems). Use of the Sequence Scanner v1.0® (Applied Biosystems) and CLC Main Workbench 6® (CLC bio, Mühltal, Ger- many) software allowed to complete the sequence trans- lation from the fluorescence signal intensity to nucleo- tide sequence. Sequencing was performed twice in order to evaluate the technical variability. The sequencing pa- rameters QV20+ and sequence length were investigated.

The chemotyping PCR was performed using the prim- ers 12CON, 12NF, 12-15F and 12-3F (Table 2) (44-46). The amplification was optimised using the Phusion Master Mix (Thermo Fisher Scientific). PCR was performed in a final volume of 50 ^L containing 4 ^L of the supernatant liquid, 25 ^L of the Phusion High-Fidelity PCR Master Mix with HF Buffer (Thermo Fisher Scientific), 1 ^Lof each primer (10 ^M) and 19 ^L of DNAse/RNAse-free water. In all comparative tests among the methods, three strains (Fusarium culmorum 4000, 4016 and 4033) were used.

To test the long-term storage features of the DNA extracted from Miracloth-growing mycelium, samples were stored for 18 months at -20 °C and then used for EF1-^ amplification as described above.

Data analysis

Statistical analyses were performed using SPSS v. 19 software (SPSS Inc., Chicago, IL, USA) and SigmaStat v. 2.03 (Systat Software Inc., Chicago, IL, USA). In order to evaluate the normal distribution, the Kolmogorov-Smir- nov test was applied. Because comparisons were carried out between methods, different strains processed with the same method were considered as replicates to also take into account the biological variability. To assess the purity of the samples, statistical analysis was done on the absorbance ratio at 260/280 nm using one-way anal- ysis of variance in SigmaStat.

To compare the quality of the sequencing results be- tween each modality, two parameters were chosen: the contiguous read length of the sequence and the QV 20+, which is a score of accuracy of sequencing of 99 % or above. The QV 20+ and the contiguous read length are assessed automatically by the Sequencing Analysis soft- ware (Life Technologies, Carlsbad, CA, USA).

First, the effect of the two primers was compared using independent samples for a Mann-Whitney U test. As no significant effect was evident, data of both prim- ers were combined for each treatment. Contiguous length and QV20+ parameters were analysed by the Kruskal- -Wallis test for comparison of the five independent dis- tributions. A corrected p<0.05 (two-sided) was consid- ered as statistically significant.

Results and Discussion

The quick standardized sampling of the microwave extraction procedure was analysed and compared to the other methods. Five different procedures for species iden- tification by PCR were compared. Three isolates (Fusa- rium culmorum 4000, 4016 and 4033) were used to com- pare the methods with each other.

Only three of the methods extracted DNA and al- lowed to consistently amplify the elongation factor (EF) 1 alpha gene from Fusarium species as confirmed by aga- rose gel electrophoresis (Fig. 1): DNeasy Plant Mini Kit, microwave, and mycelium grinding with pestle plus mi- crowave (Fig. 2). Indeed, extraction by a heating step and the use of direct PCR with Phusion Master Mix without further processing were not able to amplify the EF con- sistently (data not shown). This contrasts with the result of Ben Amar et al. (41), who successfully applied direct PCR to characterise a number of F. culmorum strains. This may be due to two factors: the Taq polymerase and Master Mix combination differed from the present con- ditions (41), and the mycelium sampling which could play a role in the efficiency of PCR reaction. We observed that the most difficult aspect of direct PCR is that re- producibility varies according to the amount of myce- lium collected and the strain used. For this reason, we developed a quick and cheap method of growing colo- nies over a standardised defined area of material that can be easily transferred to PCR tubes, and we validated the assay on a large number of samples. We also observed that microwave processing increased the reproducibility of the assay and for this reason we focused on the opti- mal method able to combine the standardised extraction and the speed in the process.

We therefore compared the different materials used for collecting the mycelium from the plate with a stan- dard kit procedure (Qiagen kit) and with a previously developed method based on manual grinding in Eppen- dorf tubes (4). No statistically significant difference (p= 0.333) among Miracloth, paper, Wypall, dialysis mem- brane and Qiagen was found with respect to the purity of the sample as tested by the 260/280 absorbance ratio (Table 3).

When the DNA was then used for EF1/EF2 amplifi- cation, it was evident that the paper modality was work- ing less efficiently (Fig. 2), suggesting a potential increase of inhibitory effects that could be linked to the more in- tense colouring of the mycelium on the substrate. The grinding method performed well in PCR amplification with a lower efficiency of amplification of one isolate (Fig. 2). This difference may be explained by the collec- tion method that did not allow a sampling with a homo- geneous amount of mycelium, further suggesting the advantage of a defined area of mycelium to obtain con- sistent results.

In order to compare the efficacy of the method on old colonies, a PCR was performed comparing five- and thirteen-day-old colonies. Strains of F. culmorum (4043, 4044, 4056 and 4057) were used and DNA 260/280 val- ues were obtained by using the NanoDrop® ND-1000 (Thermo Fisher Scientific). The purity of the extract of the older colonies was slightly lower (1.73±0.15 at 5 days and 1.44±0.07 at 13 days), suggesting that the amount of pigments may increase with the age of the colony (p= 0.004).

Nonetheless, no evident difference of the PCR effi- ciency of the EF1-a could be observed comparing the extraction of 5- and 13-day-old colonies (Fig. 3a). Storing, similarly, did not influence the ability to efficiently am- plify EF1-a, as 1.5 years of storage of samples allowed good quality amplification (Fig. 3b).

As the method using the Miracloth filter paper was efficient and allowed to identify all the 75 strains as listed in Table 1, the same extraction was used for deter- mining the chemotype, by using primers developed on Tri12. The procedure included multiplexing 4 primers, requiring therefore a good DNA quality to be performed. Indeed, chemotypes could be distinguished by using DNA amplification (Fig. 4).

To evaluate the possibility to perform sequencing re- actions on the quickly extracted mycelium, sequencing was performed on the different EF products using EF1 and EF2 primers obtained from the strains Fusarium cul- morum 4000, 4016 and 4033.

A few differences could be detected among the meth- ods; the Qiagen kit had a better QV20+ (Table 4) and was significantly different from the membrane (p=0.0001), the Miracloth (p=0.025) and the paper (p=0.0001) (Table 5) extraction methods. Concerning the length of the se- quence, Wypall was significantly different from Qiagen kit (p=0.012) and paper (p=0.024), but did not differ from the other two methods (Table 5).

Despite the higher intensity of the sequencing signal obtained with the Qiagen kit, sequences obtained using Miracloth, Wypall and dialysis membrane were readable and allowed the identification of the sequences without mistakes by a BLAST search in the Fusarium database (47), according to Geiser et al. (1) and the BLAST tool (48). Aligned sequences showed identical results. All se- quences are deposited at NCBI database (GenBank IDs: KJ170153- KJ170227).

The efficacy of the paper method for subsequent DNA extraction and sequencing was lower compared to the other methods due to the variability of results ob- tained with the paper. As paper may induce different colourations in the strains, it probably activates second- ary metabolite pathways that may produce molecules that inhibit the PCR. PCR amplification had an effect on the sequence quality afterwards. For this reason the use of the paper is not ideal for our purposes.

In order to study the application of the method to other fungi, the method was also tested on 69 strains of Zymoseptoria tritici (data not shown) confirming the ef- fectiveness and speed of this method.

Conclusion

In conclusion, we demonstrated that coupling the growth of mycelium on Miracloth with microwave pro- cessing is a valuable, cheap and reliable method for large screenings of Fusarium isolates in order to deter- mine the toxigenic potential of the strains. We verified that direct sequencing of the products and a multiplex with 4 primers could be performed with 100 % effi- ciency on 75 Fusarium strains. We could not reproduce recent results reported by Ben Amar et al. (41) of direct PCR with our Master Mix. It seems that the method de- veloped by those authors relies on the operator's ability to collect similar amounts of mycelium in all assays. For this reason, the use of a surface with defined area for collecting the mycelium guaranteed better reproducibi- lity for large screenings. Given the easy handling, rapid preparation of samples and the high reproducibility of the results for the identification and characterisation of strains, the method proposed here will be further used in currently running chemotyping studies aiming at the characterisation of Fusarium chemotypes over Europe.

Acknowledgements

We would like to thank Sylvain Legay and Laurent Solinhac for running the sequencer and Servane Contal and Boris Untereiner for the technical assistance. This study was realized in the framework of the ANTREPP project.

References

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