Identification of Fusarium species has always been difficult due to confusing phenotypic classification systems. We have developed a fluorescent-based polymerase chain reaction assay that allows for rapid and reliable identification of five toxigenic and pathogenic Fusarium species. The species includes Fusarium avenaceum, F. culmorum, F. equiseti, F. oxysporum and F. sambucinum. The method is based on the PCR amplification of species-specific DNA fragments using fluorescent oligonucleotide primers, which were designed based on sequence divergence within the internal transcribed spacer region of nuclear ribosomal DNA. Besides providing an accurate, reliable, and quick diagnosis of these Fusaria, another advantage with this method is that it reduces the potential for exposure to carcinogenic chemicals as it substitutes the use of fluorescent dyes in place of ethidium bromide. Apart from its multidisciplinary importance and usefulness, it also obviates the need for gel electrophoresis.
Mycotoxigenic Fusarium species are ubiquitous fungi, infecting a wide range of crop plants resulting in significant quantitative and qualitative losses to global agriculture . Fusaria are known to produce deleterious mycotoxins resulting in toxin contamination of crop products . The consumption of mycotoxin-contaminated food and feed products pose an acute risk to human and animal health, as these mycotoxins are carcinogenic and can potentially impair the immune system . Recent outbreaks of diseases caused by these fungi pose a great problem for the agricultural industry and are potentially threatening to the global food supply [4,5]. Therefore, there is need for rapid identification and speedy implementation of the control measures of diseases caused by these fungi.
The accurate identification of Fusarium species has always been problematic even for expert mycologists. This is because of the contradictory classification systems proposed by various researchers, primarily based on cultural and morphological characters [6–10] that could be highly variable depending on the media and cultural conditions. In addition, degeneration of the cultures and production of mutants may further add to the problems in fungal identification and diagnosis.
In recent years, the increasing use of molecular methods in fungal diagnostics has emerged as a possible answer to the problems associated with the existing phenotypic identification systems [11,12]. One of the most robust and informative techniques used in fungal diagnosis is nucleotide sequencing , where DNA sequence variations have been used to design species-specific primers and/or probes. This investigation was, therefore, initiated to develop molecular markers for the identification of five toxigenic and pathogenic Fusarium species that would have the sensitivity of the polymerase chain reaction (PCR), would be easy in use, would be reproducible, and would provide species-specific resolution.
Materials and methods
Fungal isolates, DNA extraction and sequencing of ITS region
Ninety-two isolates of five toxigenic Fusarium species were tested (Table 1). Fungal tissue for DNA extraction was harvested from the isolates grown on filter paper placed over potato-dextrose agar plates and ground in liquid nitrogen. The total genomic DNA was extracted using a CTAB method , re-suspended in 1×Tris–EDTA and stored at −20°C. The internal transcribed spacer (ITS) region of nuclear ribosomal DNA (nrDNA) was amplified using PCR primers ITS4 and ITS5 . Amplification reactions were carried out in a 20 µl volume containing 1×PCR buffer (100 mM Tris–Cl, pH 8.0, 500 mM KCl, and 0.8% Nonidet P40, Helena Biosciences, UK), 1 U Taq polymerase (Bioline, UK), 0.2 mM each dNTP, 2 mM MgCl2, 0.35 µM each primer and 20 ng of genomic DNA. PCR was performed in a thermocycler (GeneAmp PCR System 9700, Applied Biosystems, Foster City, CA, USA) with the following conditions: initial denaturation for 1 min at 95°C was followed by 25 cycles of 94°C for 1 min, 52°C for 30 s, 72°C for 1 min with a final extension of 7 min at 72°C. PCR products were sequenced in both directions following the standard procedure  using an ABI Prism 377 automated DNA sequencer and an ABI Prism Big Dye Terminator Cycle Sequencing Ready Reaction Kit (Applied Biosystems, Foster City, CA, USA). The ITS sequences were deposited to the GenBank database under accession numbers AY147281 to AY147372, respectively.
|Fusarium species||Number of isolates||Primer name||Primer sequence||Fluorescent dye|
|Fusarium species||Number of isolates||Primer name||Primer sequence||Fluorescent dye|
Design and evaluation of species-specific PCR primers
DNA sequences were visually edited after initial alignment by MEGALIGN in the Lasergene programme (DNAStar Inc., Madison, WI, USA). Species-specific primers were designed based on the DNA sequence variability found within the ITS region (Table 1). The specificity of the primers for Fusarium species was also validated through BLAST searching the primer sequences against the NCBI sequence database (http://www.ncbi.nlm.nih.gov/BLAST). All the upstream primers were conjugated at their proximal end with different fluorescent dyes (Table 1). The fluorophores were chosen so as to have minimal spectral overlap and can be excited with ultraviolet wavelengths. Each PCR primer pair was tested with DNA from at least three different isolates of each species. The PCR reactions were prepared as described above. The amplification regime was 1 min at 94°C followed by 25 cycles of 94°C for 1 min, 58°C for 30 s, and 72°C for 1 min with a final extension of 7 min at 72°C. Fragments were resolved on a 2% metaphor agarose gel without ethidium bromide staining. The technique was further modified to preclude the need for gel electrophoresis. The PCR products were purified using Qiagen PCR Purification Kit (Qiagen Inc., UK) to remove excess, unused and unconjugated dye, and unused primers. Subsequently, the amplified colour was visualised under ultraviolet light in Eppendorf tubes, which were photographed with a hand-held Polaroid camera using Polaroid Colour Film Type 689 (Polaroid Inc, UK).
Results and discussion
Identification of Fusarium species based on cultural and morphological features is often complicated and confusing, even for expert taxonomists, for example the quorn protein fungus Fusarium venenatum (strain ATCC20334) was identified as F. sulphureum, F. crookewellense, F. venenatum and F. graminearum by different Fusarium taxonomists . Fungal identification is still achieved through traditional phenotypic typing that requires an expert taxonomist, takes a long time, and has often led to mis-identification of species due to paucity and plasticity of the characters used, loss of cultural viability and degeneration of the cultures .
This study was initiated to develop and evaluate the efficacy and usefulness of PCR primers designed from the sequences of the nrDNA ITS region for differentiation of five toxigenic and pathogenic Fusarium species. The PCR primers developed in this study exhibited species-specific resolution (Fig. 1). The five Fusarium species could be differentiated from each other on the basis of a single PCR amplification with high confidence and precision. Species-specific fluorescent PCR primers amplified an expected size DNA fragment only from the isolates of Fusarium species for which the primer was originally designed (Fig. 1). The resulting colour was clearly visible after exciting the electrophoresed metaphor agarose gel with ultraviolet wavelength (Fig. 2a). The primer pairs designed for F. culmorum amplified a fragment of 245 bp, whereas, fragments of 315 bp, 340 bp, 389 bp, and 314 bp were amplified from the isolates of F. sambucinum, F. oxysporum, F. equiseti, and F. avenaceum, respectively (Fig. 1).
The molecular markers developed in this study have potential to overcome the problems associated with existing methods and can even be used by non-expert biologists with a high degree of certainty. Thus, we have achieved our objective of developing genomic markers that have the sensitivity of PCR and detect DNA sequence polymorphisms corresponding to the Fusarium species examined.
The purified PCR amplicon was clearly detectable in Eppendorf tubes during excitation with ultraviolet light (Fig. 2b). Besides providing an accurate, reliable and quick diagnosis of these toxigenic and pathogenic Fusaria, another advantage with this method is that it obviates the need for gel electrophoresis as well as reduces the potential for exposure to carcinogenic chemicals as it substitutes the use of ethidium bromide with bound fluorescent dyes.
These primers can be used in the assessment of diseases and mycotoxin contamination of crop products using quantitative PCR . It is also possible to use these markers in detection and assay of Fusarium species from the environmental samples needed to verify the causes of allergies and other associated diseases. Finally, the benefits of such a simple and precise molecular marker would be substantial to researchers from wide areas of science including mycotoxicologists, taxonomists, and plant pathologists.
A research scholarship from Felix Foundation, UK, to P.K.M. is gratefully acknowledged. We thank L.W. Burgess (Australia), R.M. Clear (Canada), W. Blok (The Netherlands), J. Chelkowski (Poland), L. Carris (USA), B.M. Cooke (Ireland), A.G. Schilling (Germany), U. Thrane (Denmark), J. Saunders (UK), L. Chernin (Israel) and J.P. Verma (India) for providing fungal cultures. Technical assistance received from Moy Robson, George Gibbings, Alan Simmonds, Alice Davies and Shirley French is duly acknowledged.