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Introduction

Fusarium species are ubiquitous fungi extensively distributed in soil, plants and various organic substrates. This genus is an important plant pathogen, which causes different diseases and is responsible for important economic losses on crops. In humans, the Fusarium species causes a broad range of diseases, including superficial, locally invasive, or disseminated infections. Disseminated infections occur almost exclusively in severely immunocompromised patients and, currently, disseminated infections are the second most common mold that causes invasive fungal infections in immunosuppressed hosts, and is associated with high morbidity and mortality rates (1,2). Furthermore, the Fusarium species causes allergic diseases, such as sinusitis in immunocompetent individuals and mycotoxicosis following ingestion of food that is contaminated with toxin-producing Fusarium (3,4). This genus contains >70 species (5); a literature review of 259 cases of fusariosis between 2001 and 2005 demonstrated that 12 species were associated with infection. The F. solani species complex was the most common (50% of cases), followed by the F. oxysporum species complex (20% of cases) and F. verticillioides and F. moniliforme (10% of cases for each) (6).

Morphological identification of the Fusarium species is the primary, but most difficult, step in the detection procedure. However, for the species that cannot be reliably recognized by morphological characterization, additional analysis, such as DNA sequencing and species-specific polymerase chain reaction (PCR) assays, must be performed.

Translation elongation factor (TEF) 1-α consistently presents as a single-copy gene in the Fusarium genus. This gene demonstrates a high level of sequence polymorphism among the closely associated species of Fusarium, even compared with the intron-rich portions of protein-coding genes, such as β-tubulin, calmodulin and histone H3. Therefore, TEF has become the choice marker as a single-locus detection tool in Fusarium (7,8). The strategy that was developed in the present study consisted of novel PCR-restriction fragment length polymorphism (RFLP) analysis for detecting DNA polymorphisms in the TEF-1α gene and for discrimination of the Fusarium genus.

Materials and methods

Microorganisms

Fifty strains of Fusarium spp. (including environmental, clinical and reference isolates) were used in the present study. The following reference strains were used: F. solani complex PTCC 5284, F. solani complex PTCC 5285, F. oxysporum complex IBRC-M 30067, F. oxysporum complex PTCC 5115, F. verticillioides PFCC 53–131, F. verticillioides PFCC 15–89, F. proliferatum PFCC 48–125, F. proliferatum PFCC 12–86 and F. fujikuroi PTCC 5144. The environmental strains were obtained from soil, and two strains used in the present study were clinical, which included F. solani complex PTCC 5284 and B988.

DNA extraction

Thick spore suspension (1 ml) was inoculated in Ehrlenmeyer flasks containing yeast extract peptone dextrose medium and incubated on an incubator shaker at 200 rpm under agitation for 72 h at 25°C for mycelia growth. The harvested mycelia were washed with 0.5 M EDTA and sterile dH2O. The mycelia were ground into a fine powder using liquid nitrogen and a mortar and pestle.

Approximately 100 mg powdered mycelium was transferred into a 1.5-ml tube containing 400 µl lysis buffer (100 mM Tris-HCl, pH 8.0, 30 mM EDTA, pH 8.0, 5% SDS w/v). After microtubes were boiled at 100°C for 20 min, 3 M acetate potassium (150 µl) was added to each tube. The suspension was maintained at −20°C for 10 min and centrifuged at 14,000 × g in 4°C for 10 min. The supernatant was carefully transferred to a fresh 1.5-ml Eppendorf tube and 250 µl phenol:chloroform:isoamyl alcohol (25:24:1, v/v) was added. The microtube was vortexed briefly and centrifuged at 4°C at 14,000 × g for 10 min. After transferring the supernatant to a 1.5-ml microtube, 250 µl chloroform:isoamyl alcohol was added. The tubes were briefly vortexed and centrifuged at 4°C at 14,000 × g for 10 min. The supernatant was transferred to a fresh microtube, an equal volume of ice-cold 2-propanol was added, maintained at −20°C for 10 min and centrifuged at 14,000 × g for 10 min. The upper aqueous phase was discarded and the pellet was washed with 70% ethanol (300 µl). The ethanol was discarded and the DNA pellets were air dried and resuspended in 50 µl dH2O.

PCR amplification

The primer sets, Fu3f (5′-GGTATCGACAAGCGAACCAT-3′) and Fu3r (5′-TAGTAGCGGGGAGTCTCGAA-3′) was used to amplify an ~420-bp DNA fragment of the TEF-1α gene (9). PCR reactions were performed with a volume of 50 µl, comprised of 5 µl 10X reaction buffer, 2.2 mM MgCl2, 200 µM each dNTP, 2.5 units of Taq DNA polymerase (CinnaGen, Tehran, Iran), 30 ng template DNA and 50 pmol of each primer.

An initial denaturation step for 5 min at 94°C was followed by 30 cycles of denaturation at 94°C for 1 min, annealing at 58°C for 1 min and extension at 68°C for 2 min. The amplified PCR product (5 µl) was electrophoresed on 1% agarose gel in TAE buffer at 100 V for 1 h and stained with ethidium bromide. The PCR amplification of TEF-1α gene resulted in an ~420-bp fragment.

RFLP analysis

Digestion with one restriction enzyme was not sufficient to discriminate the 420-bp DNA fragment of the TEF-1α gene in the Fusarium species. Therefore, double digestion with two restriction enzymes, XhoI and SduI (Thermo Fisher Scientific, Inc., Waltham, MA, USA) was used for discrimination. The restriction digestion reaction was performed in a total volume of 20 µl containing 5 units of each enzyme, 2 µl Buffer O (Thermo Fisher Scientific, Inc.), 5 µl PCR product, and Ultrapure water (CinnaGen, Karaj, Iran) to reach a volume of 20 µl. Digested PCR products were electrophoresed at 50 V for 3 h on 2% agarose gel in TAE buffer and stained with ethidium bromide.

Results

PCR amplification of the TEF-1α gene

The PCR amplification of TEF-1α gene with Fu3f and Fu3r primers produced a unique band of ~420 bp for all tested Fusarium isolates (Fig. 1). The TEF-1α gene fragment was sequenced for certain isolates, including the reference strains. The BLAST search in NCBI (https://blast.ncbi.nlm.nih.gov/Blast.cgi) demonstrated the TEF-1α gene fragment from five clinically important Fusarium reference strains, including F. solani species complex, F. oxysporum species complex, F. verticillioides, F. proliferatum and F. fujikuroi exhibited 99% homology with the associated sequences deposited in the GenBank database.

Figure 1. Agarose gel electrophoresis of transcription elongation factor-1α gene products (420 bp) of the Fusarium species. Lane M, 100-bp ladder; lane 1, F. oxysporum complex IBRC-M 30067; lane 2, F. verticillioides PFCC 15–89; lane 3, F. proliferatum PFCC 48–125; lane 4, F. fujikuroi PTCC 5144; lane 5, F. solani complex PTCC 5284. TEF, transcription elongation factor.

Restriction patterns for the Fusarium strains

Double digestion of the fragment with restriction enzymes, XhoI and SduI clearly discriminated the F. solani species complex, F.oxysporum species complex, F. verticillioides, F. proliferatum and F. fujikuroi from each other (Table I and Fig. 2).

Figure 2. Agarose gel electrophoresis of transcription elongation factor-1α gene products (420 bp) of the Fusarium species following double digestion with XhoI and SduI. Lane M, 100-bp ladder; lane 1, F. oxysporum complex IBRC-M 30067; lane 2, F. verticillioides PFCC 15–89; lane 3, F. proliferatum PFCC 48–125; lane 4, F. fujikuroi PTCC 5144; lane 5, F. solani complex PTCC 5284.

Table I. Restriction fragment size (bp) of the Fusarium species TEF-1α gene, double digested with two restriction enzymes, XhoI and SduI.

Table I.

Restriction fragment size (bp) of the Fusarium species TEF-1α gene, double digested with two restriction enzymes, XhoI and SduI.

Fusarium species	TEF-1α fragment prior to digestion (bp)	XhoI and SduI (bp)
F. oxysporum species complex	420	45,62,103,170
F. verticillioides	420	6,30,56,47,55,186
F. proliferatum	420	25,168,187
F. fujikuroi	420	27,62,99,192
F. solani species complex	420	308,110

[i] TEF, transcription elongation factor.

The restriction patterns of one clinical and six environmental Fusarium strains following double digestion using XhoI and SduI are presented in Fig. 3. The digestion of the 420-bp fragment from these strains demonstrated different patterns. Strains E4, E16 and E25 were sequenced. A BLAST search showed that strains E4 and E16 exhibited 100% homology with the F. equiseti and F. solani species complex, respectively and strain E25 exhibited 99% homology with F. incarnatum. Therefore, the restriction pattern strain E16 (Fig. 3) was similar to the F. solani complex PTCC 5284 (Fig. 2).

Figure 3. Agarose gel electrophoresis of transcription elongation factor-1α gene products (420 bp) of the Fusarium species (lane 1, clinical isolate; lanes 2–7, environmental isolates) following double digestion with XhoI and SduI. Lane M, 100-bp ladder; lane1, B988; lane 2, E4; lane 3, E16; lane 4, E17; lane 5, E18; lane 6, E20; lane 7, E25.

Discussion

Identification of filamentous fungi at the species level using classical techniques, such as morphological methods, is difficult and time-consuming. Novel rapid techniques are required in order to verify the Fusarium genus on time, particularly for clinical administration of patients. Rapid molecular approaches, such as PCR, DNA hybridization and DNA microarray have been developed and they may replace the classical methods. The major advantages of molecular approaches are their specificity and that they are completely discriminative even for closely associated species (8,9).

The majority of molecular techniques are PCR-based, where the primers are typically directed to conserved regions of the ribosomal DNA gene, particularly towards the internal transcribed spacer (ITS) regions. With regard to Fusarium spp., analysis of ITS sequencing is considered unreliable for detection of strains, as they contain two paralogous, discrepant ITS sequence forms, which may cause confusion (10,11). The TEF-1α gene has shown optimal results for the identification of Fusarium spp. (12–14).

Guevara-Suarez et al (15) used a TEF-1α gene fragment and performed a multi-locus sequence analysis of the ITS region with the RNA-dependent polymerase subunit II (Rpb2) genes, and recognized the phylogenetic species and circulating haplotypes for Fusarium isolates from onychomycosis. The pathogenic isolates to the pecan tree were identified, based on the TEF-1α gene, as belonging to the F. chlamydosporum species complex, F. graminearum species complex, F. proliferatum, and F. oxysporum (16). A TEF-1α-RFLP technique was described for the identification of the three clades of F. oxysporum (17). The particularly effective TEF-1α gene of the Fusarium spp. encouraged the present development of a PCR-RFLP technique as an advanced, simple and reliable method for determination and discrimination of the clinically important Fusarium species.

In the current study, molecular identification was performed using the TEF-1α gene and RFLP, and it was possible to discriminate between all five clinically important Fusarium species. However, further analyses are required for discrimination between other Fusarium species.

The Primer set, TEF-Fu3 resulted in an ~420-bp product for five of the Fusarium species, including F. solani species complex, F. oxysporum species complex, F. verticillioides, F. proliferatum and F. fujikuroi. RFLP, using double digestion with two restriction enzymes, XhoI and SduI differentiated between the species. This method may facilitate detection, verify the Fusarium genus, and be applied for disease control. This PCR-RLFP method is rapid, economical and efficient for detection and discrimination of the Fusarium genus.

Acknowledgements

The present study was supported by the Health Research Institute, Infectious and Tropical Diseases Research Center, Ahvaz Jundishapur University of Medical Sciences (Ahvaz, Iran) (grant no. 92118).

References