IGS-RFLP analysis and development of molecular markers for identification of Fusarium poae, Fusarium langsethiae, Fusarium sporotrichioides and Fusarium kyushuense

The intergenic spacer (IGS) regions of the rDNA of several Fusarium spp. strains obtained from the collaborative researchers (Int. J. Food Microbiol. (2003)) were amplified by polymerase chain reaction (PCR), and an IGS-RFLP analysis was performed. Restriction digestion with AluI, MspI and PstI allowed differentiation between the related Fusarium poae and Fusarium kyushuense species. Fusarium langsethiae was also separated from Fusarium sporotrichioides (including var. minus) on the basis of the banding patterns after MspI digestion, while specific XhoI, AluI and MspI restriction patterns were found in the IGS amplicons of F. sporotrichioides var. minus. According to the phylogenetic analysis of IGS-RFLP patterns, F. langsethiae (except for one strain), F. sporotrichioides, F. poae and F. kyushuense strains formed four well-supported clades with high-bootstrap values. Based on the sequence differences in the IGS region, species-specific primers were designed for the F. langsethiae/F. sporotrichioides group and for F. poae. The specificity and sensitivity of the primers were tested on various Fusarium species and isolates, and on several other important fungal genera associated with cereals. The F. poae-specific primers, designed in this study, showed the same specificity as primers Fp82f/Fp82r developed previously. The two phylogenetic subgroups of F. langsethiae, found by IGS sequencing analysis, were separated on the basis of size differences of the amplification products with primers CNL12/PulvIGSr specific for the F. langsethiae/F. sporotrichioides group. RFLP analysis of the amplified IGS region is a useful molecular assay for characterisation and a phylogenetic study of several related Fusarium species-F. langsethiae, F. sporotrichioides, F. sporotrichioides var. minus, F. poae and F. kyushuense. The primers designed in this study were highly specific and allowed identification of F. poae and the F. langsethiae/F. sporotrichioides group.     

An integrated taxonomic study of Fusarium langsethiae, Fusarium poae and Fusarium sporotrichioides based on the use of composite datasets

An integrated systematic study was carried out to clarify the taxonomical position and relationship of Fusarium langsethiae to other taxa within the Fusarium section Sporotrichiella. Strains of this species were compared with strains of the closely related species Fusarium poae and Fusarium sporotrichioides using a composite dataset. This set consisted of DNA sequences derived from the ribosomal internal transcribed spacer (ITS) regions, partial sequences of the ribosomal intergenic spacer (IGS) region, the beta-tubulin and translation elongation factor-1 alpha (EF-1alpha) genes, AFLP fingerprints, chromatographic data on secondary metabolites and morphological data and growth characteristics. From these combined data, a consensus matrix was calculated by taking the mean of all pairwise distances between single isolates over all separate datasets. The consensus matrix was used as the basis for the construction of a UPGMA dendrogram and a multidimensional scaling, both of which revealed a clear separation of the three taxa. Partial IGS, EF-1alpha and beta-tubulin sequence-as well as chromatography-and AFLP-derived similarities turned out to be comparably consistent, while ITS sequence- and morphology-derived similarity matrices were rather divergent.     

Phylogenetic analyses of the Fusarium poae, Fusarium sporotrichioides and Fusarium langsethiae species complex based on partial sequences of the translation elongation factor-1 alpha gene

Phylogenetic relationships between four Fusarium species were studied using parts of the nuclear translation elongation factor-1 alpha (EF-1alpha) gene as a phylogenetic marker. Sequences from 12 isolates of Fusarium poae, 10 isolates of Fusarium sporotrichioides and 12 isolates of Fusarium langsethiae yielded 4, 5 and 5 haplotypes, respectively. In addition, we included one isolate of Fusarium kyushuense. The aligned sequences were subjected to neighbor-joining (NJ), maximum parsimony and maximum likelihood (ML) analyses. The results from the different analyses were highly concordant. The EF-1alpha-based phylogenies support the classification of F. langsethiae as a separate taxon in the section Sporotrichiella of Fusarium, as the closest sister taxon to F. sporotrichioides, while F. kyushuense is the sister taxon to F. poae. This corresponds well with the ability of F. langsethiae and F. sporotrichioides to produce T-2 and HT-2 toxins. In contrast, morphological characters indicate a closer relationship between F. langsethiae and F. poae on the one hand, and between F. sporotrichioides and F. kyushuense on the other hand.     

Diversity in metabolite production by Fusarium langsethiae, Fusarium poae, and Fusarium sporotrichioides

The production of mycotoxins and other metabolites by 109 strains of Fusarium langsethiae, Fusarium poae, Fusarium sporotrichioides, and F. kyushuense was investigated independently in four laboratories by liquid or gas chromatography analyses of cultural extracts with UV diode array, electron capture, or mass spectrometric detection systems. From the compiled results, it was found that F. langsethiae consistently produced the trichothecenes diacetoxyscirpenol (DAS), T-2 toxin (T-2), HT-2 toxin (HT-2), and neosolaniol (NEO) and, to a lesser extent, some additional trichothecene derivatives. F. langsethiae also produced culmorins, chrysogine (CHRYS), aurofusarin (AUF), and enniatin (EN). F. sporotrichioides showed a metabolite profile similar to that of F. langsethiae, while F. poae had a different profile as 41 of 49 strains produced nivalenol (NIV) and other 8-keto trichothecenes, in addition to DAS and derivatives of this metabolite. Only a trace amount of NIV was detected from one strain of F. kyushuense. In summary, all the three core taxa of this joint study were found to produce trichothecenes. Fusarin C (F-C) was not detected from F. langsethiae, but it was produced by F. poae and F. sporotrichioides. Aurofusarin was only detected from a few strains of F. langsethiae, while nearly all strains of F. poae and F. sporotrichioides produced this compound. In contrast, chrysogine was not detected from F. poae, but was produced by the other two taxa. Production of enniatins was scattered among the three main taxa of this study, whereas beauvericin (BEA) was produced by many strains of F. poae and F. sporotrichioides. Only one odd strain of F. langsethiae (IBT 9959) produced beauvericin. However, the status of this strain is uncertain. By a polyphasic approach using species-specific metabolite profiles, the fruity odour of F. poae, and morphological observations, it was concluded that F. langsethiae, F. poae, and F. sporotrichioides should be regarded as three significant taxa at a species level.     

The use of tri5 gene sequences for PCR detection and taxonomy of trichothecene-producing species in the Fusarium section Sporotrichiella

Purified DNA from isolates of Fusarium poae, Fusarium sporotrichioides, Fusarium kyushuense and Fusarium langsethiae was used as a template to amplify a 658-bp fragment from the trichodiene synthase (tri5) gene of these fungi with the gene-specific PCR primer pair Tox5-1/Tox5-2. Fragments obtained were isolated and sequenced. DNA sequence alignments revealed high similarity between the sequences derived from F. sporotrichioides and F. langsethiae (98.7%) and less similarity between the latter species and F. poae (90.9%). Phylogenetic analysis of the aligned sequences using the tri5 sequence of Fusarium pseudograminearum as an outgroup revealed clear separation between one group consisting of F. poae and F. kyushuense and another consisting of F. sporotrichioides and F. langsethiae. The two latter species could not be distinguished phylogenetically on the basis of their tri5 sequences. Taxon-specific reverse primers were designed from the aligned sequences and combined with the tri5 gene-specific forward primer Tox5-1. The new reverse primers enabled specific amplification of a fragment of approximately 400 bp from DNA isolated from F. sporotrichioides, F. poae, F. langsethiae and F. kyushuense, respectively. All primers were tested for cross-reactivity with DNA from 26 fungal species potentially capable of producing trichothecenes. Only the primer designed for F. langsethiae cross-reacted with F. sporotrichioides. PCR assays were applied in analysis of artificially and naturally infected samples of barley and oats. On artificially infected barley, species were selectively detected by the corresponding primers. In naturally infected oats, F. langsethiae was identified by the combination of two PCR assays designed for detection of F. sporotrichioides and F. langsethiae, respectively.     

Specific detection of Fusarium langsethiae and related species by DGGE and ARMS-PCR of a beta-tubulin (tub1) gene fragment

Fusarium langsethiae was recently described as a new toxigenic Fusarium species, which morphologically resembles Fusarium poae, but exhibits a mycotoxin pattern related to Fusarium sporotrichioides. To develop tools for early and specific detection of F. langsethiae and distinguishing it from related species of section Sporotrichiella and Discolor (F. poae, F. sporotrichioides, Fusarium kyushuense, Fusarium robustum, Fusarium sambucinum and Fusarium tumidum) sequence variations in their beta-tubulin-encoding (tub1) gene were employed to design two PCR-based methods, denaturing gradient gel electrophoresis (DGGE) and amplification refractory mutation system (ARMS)-PCR. DGGE reliably separated all these strains, even from mixtures and in the presence of DNA from their natural hosts Zea mais, Triticum aestivum and Avena sativa. In addition, a tetraprimer ARMS-PCR, which employs two primer pairs to amplify, respectively, two different fragments of tub1 in a single PCR reaction resulted in rapid differentiation between F. langsethiae, F. sporotrichioides and F. poae according to the number of amplicons (four, two and one, respectively). These two methods will thus be worthwhile tools in the specific detection of F. langsethiae in infected crops.     

Molecular studies to identify the Fusarium species responsible for HT-2 and T-2 mycotoxins in UK oats

High levels of Fusarium mycotoxins HT-2 and T-2 have been detected in UK oats since surveys started in 2002. Fusarium langsethiae and the closely related species F. sporotrichioides have previously been associated with the contamination of cereals with type A trichothecenes HT-2 and T-2 in Nordic countries. Preliminary microbiological analysis of UK oat samples with high concentrations of HT-2 and T-2 detected and isolated F. langsethiae and F. poae but not the other type A trichothecene producing species F. sporotrichioides, F. sibiricum and F. armeniacum. Two hundred and forty oat flour samples with a known mycotoxin profile were selected from a previous four year study (2002-2005) to cover the full concentration range from below the limit of quantification (<20 μg/kg) to 9,990 μg/kg HT-2+T-2 combined. All samples were analysed for the DNA of F. langsethiae, F. poae and F. sporotrichioides based on previously published PCR assays. F. langsethiae was detectable in nearly all samples; F. poae was detected in 90% of samples whereas F. sporotrichioides was not detected in any sample. A real-time PCR assay was developed to quantify F. langsethiae DNA in plant material. The assay could quantify as low as 10(-4)ngF. langsethiae DNA/μl. Based on this assay and a previously published assay for F. poae, both species were quantified in the oat flour samples with known HT-2+T-2 content. Results showed a good regression (P<0.001, r(2)=0.60) between F. langsethiae DNA and HT-2+T-2 concentration. F. poae DNA concentration was not correlated to HT2+T2 concentration (P=0.448) but was weakly correlated to nivalenol concentration (P<0.001, r(2)=0.09). Multiple regression with F. langsethiae and F. poae DNA as explanatory variates identified that both F. langsethiae and F. poae DNA were highly significant (P<0.001) but F. poae DNA only accounted for an additional 4% of the variance and the estimate was negative, indicating that higher concentrations of F. poae DNA were correlated with slightly lower concentrations of HT2+T2 detected. A stronger regression (P<0.001, r(2)=0.77) between F. langsethiae DNA and HT-2+T-2 was obtained after extraction and quantification of DNA and mycotoxins from individual oat grains. The results from this study provide strong evidence that F. langsethiae is the primary, if not sole, fungus responsible for high HT-2 and T-2 in UK oats.     

Characterization of Fusarium spp. isolates by PCR-RFLP analysis of the intergenic spacer region of the rRNA gene (rDNA)

In the present study, 44 Fusarium spp. isolates (5 Fusarium culmorum, 7 Fusarium graminearum, 1 Fusarium cerealis, 1 Fusarium poae, 26 Fusarium oxysporum, and 4 Gibberella fujikuroi species complex) were characterized morphologically, physiologically and genetically. All except one (Dutch Collection: CBS 620.72) were isolated from different hosts grown in various Spanish localizations. Morphological characterization was made according to macroscopic and microscopic aspects. Physiological characterization was based on their ability to produce zearalenone (ZEA) and type B trichothecenes (deoxynivalenol, nivalenol and 3-acetyldeoxynivalenol). ZEA was determined by liquid chromatography and trichothecenes by gas chromatography. Confirmation was carried out by liquid chromatography-ion trap-mass spectrometry (ZEA) or gas chromatography-mass spectrometry (trichothecenes). Molecular characterization of isolates was performed using an optimized, simple and low-cost method for isolation of DNA from filamentous fungi and polymerase chain reaction-restriction fragment length polymorphisms (PCR-RFLP) of the intergenic spacer region (IGS) of the rRNA gene (rDNA). The results indicate that F. graminearum, F. culmorum and F. cerealis isolates were high ZEA and type B trichothecene producers, the F. poae isolate produced very low level of nivalenol while F. oxysporum and the G. fujikuroi complex isolates did not show this ability. Restriction patterns of the IGS region did not show any relationship with the host, geographic origin of the isolate and mycotoxin-producing capacity. However, the haplotypes obtained with six restriction enzymes (CfoI, AluI, HapII, XhoI, EcoRI and PstI) permitted to discern the six assayed Fusarium species. Therefore, this is a rapid and suitable methodology that allows closely related strains to group and to estimate the genetic relationships between the groups.     

Fusarium langsethiae sp. nov. on cereals in Europe

A new species of Fusarium, Fusarium langsethiae, is described, illustrated and discussed. This species is isolated from kernels of oats, wheat and barley in several European countries. Morphologically, the species resembles Fusarium poae. It is differentiated from F. poae by slower growth, less aerial mycelium and absence of odour; its napiform or globose conidia are borne in the aerial mycelium on the agar surface on often bent phialides which exhibit sometimes more than one opening, whereas those of F. poae are produced on straight monophialides mostly in the aerial mycelium. No sporodochial conidia are formed by F. langsethiae even under near-UV light (nUV). Based on morphological characters, the species is placed in the section Sporotrichiella.     

Species-specific PCR-based assays for the detection of Fusarium species and a comparison with the whole seed agar plate method and trichothecene analysis

Species-specific PCR was used for the identification of nine Fusarium species in pure mycelial culture. A PCR-based method was compared with the whole seed agar plate method and trichothecene analysis for three toxin-producing Fusarium species using 85 grain samples of wheat, barley, oat, corn and rye. A simple SDS-based DNA extraction system followed by potassium acetate precipitation resulted in consistent PCR amplification of DNA fragments from cultures and grain samples. The species-specific PCR assays correctly identified pure cultures of Fusarium avenaceum ssp. avenaceum (9 isolates), Fusarium acuminatum ssp. acuminatum (12 isolates), Fusarium crookwellense (7 isolates), Fusarium culmorum (12 isolates), Fusarium equiseti (11 isolates), Fusarium graminearum (77 isolates), Fusarium poae (10 isolates), Fusarium pseudograminearum (23 isolates), and Fusarium sporotrichioides (10 isolates). Multiplex PCR was developed for the simultaneous detection of F. culmorum, F. graminearum and F. sporotrichioides, the three most important trichothecene producing species in Canada. In grain samples, results of PCR assays for these same three species related well with whole seed agar plate method results and determination of Fusarium trichothecenes. The PCR assay described in this study can be used for routine detection and identification of Fusarium spp. in Canada.     

烟草镰刀菌根腐病的病原鉴定

为了探明烟草镰刀菌根腐病的病原菌分类地位,2015-2017年从福建省三明市主要烟区采集的烟草根腐病病样中分离获得31个镰孢菌属菌株（F1~F31）。对所分离的菌株进行形态学甄别、系统进化分析、种特异分子鉴定及致病性测定,结果表明分离菌株显微形态特征与尖孢镰刀菌（Fusarium oxysporum）和共享镰刀菌（F. commune）相似;采用贝叶斯法（Bayesian Analysis）对4株分离菌F-04、F-18、F-19和F-28的rDNA基因内间隔区（IGS）基因作系统发育分析,结果显示这4个菌株同F. commune聚为一类并与F. oxysporum明确区分;特异性引物扩增鉴定结果表明分离菌株均为F. commune。通过室内与田间接种试验验证了分离菌株F. commune的致病性,明确其为烟草根腐病的主要病原物,这是我国首次报道共享镰刀菌F. commune引起烟草根腐病。      

Fusarium sibiricum sp. nov, a novel type A trichothecene-producing Fusarium from northern Asia closely related to F. sporotrichioides and F. langsethiae

Production of type A trichothecenes has been reported in the closely related species Fusarium langsethiae and F. sporotrichioides. Here, we characterized a collection of Fusarium isolates from Siberia and the Russian Far East (hereafter Asian isolates) that produce high levels of the type A trichothecene T-2 toxin and are similar in morphology to the type A trichothecene-producing F. langsethiae, and to F. poae which often produces the type B trichothecene nivalenol. The Asian isolates possess unique macroscopic and microscopic characters and have a unique TG repeat in the nuclear ribosomal intergenic spacer (IGS rDNA) region. In Asian isolates, the TRI1-TRI16 locus, which determines type A versus type B trichothecene production in other species, is more similar in organization and sequence to the TRI1-TRI16 locus in F. sporotrichioides and F. langsethiae than to that in F. poae. Phylogenetic analysis of the TRI1 and TRI16 gene coding regions indicates that the genes in the Asian isolates are more closely related to those of F. sporotrichioides than F. langsethiae. Phylogenetic analysis of the beta-tubulin, translation elongation factor, RNA polymerase II and phosphate permease gene sequences resolved the Asian isolates into a well-supported sister lineage to F. sporotrichioides, with F. langsethiae forming a sister lineage to F. sporotrichioides and the Asian isolates. The Asian isolates are conspecific with Norwegian isolate IBT 9959 based on morphological and molecular analyses. In addition, the European F. langsethiae isolates from Finland and Russia were resolved into two distinct subgroups based on analyses of translation elongation factor and IGS rDNA sequences. Nucleotide polymorphisms within the IGS rDNA were used to design PCR primers that successfully differentiated the Asian isolates from F. sporotrichioides and F. langsethiae. Based on these data, we formally propose that the Asian isolates together with Norwegian isolate IBT 9959 comprise a novel phylogenetic species, F. sibiricum, while the two subgroups of F. langsethiae only represent intraspecific groups.     

Production of trichothecene mycotoxins by Australian Fusarium species.

Australian isolates of Fusarium species were grown on potato dextrose agar. Trichothecenes produced by these species were extracted by ethyl acetate followed by methanol and a silica gel column was used to clean-up the extract. The extracted samples were derivatized by acetylation with trifluoroacetic anhydride and the derivatives analysed by gas chromatography/mass spectrometry (GC/MS). Multiple ion detection was used to trace ions characteristic of the trichothecenes expected to be present. Quantitation of those found was based on a known mass of pentabromophenol that was added as an internal standard. Eight species of Fusarium (nineteen strains) were surveyed, of which three species, F. acuminatum, F. equiseti and F. sporotrichioides, produced the trichothecenes scirpentriol, diacetoxyscirpenol, neosolaniol, HT-2 toxin, T-2 toxin, T-2 tetraol and deoxynivalenol. Wheat samples were inoculated with four different species of Fusarium, F. acuminatum, F. equiseti, F. graminearum and F. sporotrichioides, and in these samples diacetoxyscirpenol, neosolaniol, HT-2 toxin and T-2 toxin were found.     

Effect of fungal strain and cereal substrate on in vitro mycotoxin production by Fusarium poae and Fusarium avenaceum.

Single conidia strains of Fusarium poae and Fusarium avenaceum were investigated for their ability to synthesise mycotoxins in vitro. In a first experiment, rice was inoculated with three strains of F. poae and three strains of F. avenaceum. In a second experiment, the same strains were cultivated on four different cereal substrates. For both experiments, the colonised substrates were analysed using liquid chromatography coupled with tandem mass spectrometric detection (LC-MS/MS) for the content of Fusarium mycotoxins. On rice, a strong effect of strains on mycotoxin content was found. With the analyses of different cereal types, substantial substrate effects were observed. For F. poae, these effects were highly dependent on the strain. The results of this study are of benefit for risk assessment concerning naturally infected grain samples since substrate effects could also be triggered by different cereal species or varieties, and thus could lead to variable mycotoxin contamination in cereal products intended for human consumption.     

Effect of fungal strain and cereal substrate on in vitro mycotoxin production by Fusarium poae and Fusarium avenaceum

Single conidia strains of Fusarium poae and Fusarium avenaceum were investigated for their ability to synthesise mycotoxins in vitro. In a first experiment, rice was inoculated with three strains of F. poae and three strains of F. avenaceum. In a second experiment, the same strains were cultivated on four different cereal substrates. For both experiments, the colonised substrates were analysed using liquid chromatography coupled with tandem mass spectrometric detection (LC-MS/MS) for the content of Fusarium mycotoxins. On rice, a strong effect of strains on mycotoxin content was found. With the analyses of different cereal types, substantial substrate effects were observed. For F. poae, these effects were highly dependent on the strain. The results of this study are of benefit for risk assessment concerning naturally infected grain samples since substrate effects could also be triggered by different cereal species or varieties, and thus could lead to variable mycotoxin contamination in cereal products intended for human consumption.     

A PCR-Based Assay for the Detection and Differentiation of Potential Fumonisin-Producing Fusarium verticillioides Isolated from Indian Maize Kernels

One hundred and three Fusarium isolates from maize samples collected from different districts of Karnataka state, India, were analyzed with genus-specific, species-specific, and potential fumonisin specific oligonucleotide primers. One set of genus-specific primers ITS F and ITS R based on a highly conserved ITS region of the genus Fusarium were used to differentiate Fusarium species from closely related genera. All the Fusarium species tested scored positive with the ITS pair of primers. Detection and identification of Fusarium verticillioides species was done by using a newly designed reverse primer VERT-R (5′- CGA CTC ACG GCC AGG AAA CC ?3′) based on an intergenic spacer sequence (IGS) combined with an already designed forward primer VERTF-1 (5′-GCG GGA ATT CAA AAG TGG CC -3′) published previously. Out of 103 Fusarium species tested, 83 isolates of F. verticillioides scored positive for VERTF-1/ VERT-R species-specific pair of primers. Further to discriminate potential fumonisin-producing and nonproducing strains of F. verticillioides, the VERTF-1/VERTF-2 set of primers [VERTF-1 (5′-GCG GGA ATT CAA AAG TGG CC -3′) and VERTF-2 (5′-GAG GGC GCG AAA CGG ATC GG -3′)] were used. 64 isolates of F. verticillioides scored positive for VERTF-1/ VERTF-2 pair of primers. In total, three primers, one forward primer VERTF-1 and two reverse primers VERT-R and VERTF-2, were used for the confirmation of F. verticillioides up to the species level and the second pair of primers were used to confirm the potential for fumonisin production. The developed PCR assay should provide a powerful tool for the detection and differentiation of potential fumonisin-producing F. verticillioides strains in a population.     

Cryptic subspecies and beauvericin production by Fusarium subglutinans from Europe

Fusarium subglutinans is a maize ear rot pathogen and producer of beauvericin and other mycotoxins. This species has recently been split into two major phylogenetic within-species groups based on RFLP DNA sequence polymorphisms identified in the histone H3 and beta-tubulin sequences. A Pan European collection of the fungus originating mostly from maize was subjected to phylogenetic analysis by RFLP grouping and to chemical analysis for beauvericin production. Of the 62 isolates belonging to Group 1, 48 (77%) produced from 10 to 532 microg/g of beauvericin, whereas none of the 39 Group 2 isolates synthesized detectable amounts of the mycotoxin. The association between RFLP group and beauvericin production is consistent with the existence of two reproductively isolated subgroups within F. subglutinans and indicates that the toxicological risk of isolates of F. subglutinans depends on the group with which they are affiliated.     

Morphological, chemical and molecular differentiation of Fusarium equiseti isolated from Norwegian cereals

The morphological variation, secondary metabolite profiles and restriction fragment length polymorphisms (RFLPs) of PCR amplified intergenic spacer (IGS) ribosomal DNA (rDNA) were studied in 27 isolates of Fusarium equiseti, 25 isolated from Norwegian cereals and 2 from soil obtained from the IBT culture collection (BioCentrum, Technical University of Denmark). All 27 isolates were tested for production of fusarochromanone (FUSCHR), zearalenone (ZEA) and the trichothecenes: 15-monoacetoxy-scirpentriol (MAS), diacetoxy-scirpenol (DAS), T-2 and HT-2 toxins, T2-triol, neosolaniol (NEO), deoxynivalenol (DON), nivalenol (NIV) and 4-acetylnivalenol (Fus-X). The trichothecenes were analysed by GC-MS in a selected ion monitoring mode, while FUSCHR was determined by ion pair HPLC with fluorometric detection and production of ZEA by TLC. For amplification of IGS rDNA primers CNL12 and CNS1 were applied. IGS rDNA was digested with the four restriction enzymes: AvaII, CfoI, EcoRI and Sau3A. In addition, we sequenced the IGS rDNA region of three of the Norwegian isolates. There were two morphological types among the Norwegian strains of F. equiseti, type I with short apical cells (dominating) and type II with long apical cells, with four haplotypes identified based on the RFLP data. Variation in secondary metabolite profiles within and between the morphological groups was observed and the levels of produced toxins were: FUSCHR 3000-42,500 and 25-30 ng/g, NIV 20-2500 and 120-700 ng/g, FUS-X 20-15,000 and 0 ng/g, DAS 30-7500 and 0-600 ng/g, and MAS 10-600 and 0-500 ng/g, for strains with short and long apical cells, respectively. NEO was detected in 16/27 strains tested (all morphotype I). All but four strains of type I (these four lacked a restriction site for EcoRI) had identical RFLP profiles. The isolates of type II had two haplotypes. The IGS sequence similarity data indicated differences between these morphotypes corresponding to two separate lineages apparently at the species level.     

Identification of fungi using DNA sequences: an approach to identify Fusarium species isolated from domestic unpolished rice

Molecular approaches are being developed to provide for the rapid and objective identification of fungi. We attempted the identification of Fusarium species by a genetic analysis to validate practically the utility of a molecular approach for fungal identification and to reveal its limitations, and sequenced three regions, the 5' end of the 28S rRNA gene (D2 region) and the internal transcribed spacer 1 and 2 (ITS1 and ITS2) regions, in the rRNA genes. The DNA sequences of 38 Fusarium strains isolated from domestic unpolished rice were compared for similarity with entries in the GenBank. Based on this comparison, it was estimated that all these three regions, as a minimum, must be compared with the database to identify Fusaria at the species level. According to the combinations of sequences in the three regions, the 38 isolates were classified into 13 groups. Out of the 13 groups, 6 groups (20 isolates in total) could be identified as definite species based only on the sequence data. For the other 6 groups (17 isolates in total), candidate species were limited on the basis of the sequence similarity, and then the isolates were identified at the species level with the aid of morphology. Only one isolate could not be identified. These results verified that DNA sequence comparison with the GenBank database is useful for the identification of Fusarium species.     

Characterization of Fusarium verticillioides strains by PCR‐RFLP analysis of the intergenic spacer region of the rDNA

Thirty‐three Fusarium verticillioides strains from diverse origins and hosts have been analysed for fumonisin production and characterized in order (i) to detect the variability present in this species and (ii) to discriminate among isolates. The method used was a combination of polymerase chain reaction (PCR) and restriction fragment length polymorphism (RFLP) generated by restriction endonucleases applied to the IGS region (intergenic spacer of rDNA). All the F. verticillioides strains associated with crops produced fumonisins B₁ and B₂ except those isolated from banana. Analysis of the IGS region by PCR‐RFLP proved to be useful to detect variability within F. verticillioides and allowed discrimination of two related groups of isolates belonging to distinct lineages differing in fumonisin production and host preferences: the fumonisin‐producing group associated with cereals and the fumonisin non‐producing group associated with banana. The method used facilitates early detection and characterization of F. verticillioides strains required to control both types of pathogens and to evaluate plant exposure to the toxin, quality of the raw material to be processed and the potential fumonisin contamination in order to prevent fumonisins entering the food chain. Copyright © 2005 Society of Chemical Industry