FUM1 Gene Research Articles

Association between FUM expression and fumonisin contamination in maize from silking to harvest

Fusarium verticillioides is the main fumonisin-producing species of fungi within the Fusarium fujikuroi species complex (FFSC), leading to notorious economic losses. The current study evaluated the relative expression of FUM1, FUM3, FUM6, FUM7, FUM8, FUM13, FUM14, FUM15 and FUM19 in comparison to the frequency of F. verticillioides and fumonisin contamination in maize samples from silking to harvest. Positive correlation was not observed between FUM relative expression and the isolation of F. verticillioides and between FUM relative expression and FB1/FB2 contamination. However, the probability of amplifying FUM genes varies according to the kernel maturity stage, with the highest probability in milk stage. It was also observed a positive correlation between the mean levels of FB1 and the kernel maturity stage. The results may indicate that the relative expression of FUM could be a complementary tool to aid in the detection of fumonisin contamination in early stages of maize growth. This is the first report involving FUM expression during the maturation of maize grains, and its correlation with F. verticillioides contamination and fumonisin accumulation.

Mycotoxins: A Fungal Genomics Perspective.

The chemical and enzymatic diversity in the fungal kingdom is staggering. Large-scale fungal genome sequencing projects are generating a massive catalog of secondary metabolite biosynthetic genes and pathways. Fungal natural products are a boon and bane to man as valuable pharmaceuticals and harmful toxins. Understanding how these chemicals are synthesized will aid the development of new strategies to limit mycotoxin contamination of food and feeds as well as expand drug discovery programs. A survey of work focused on the fumonisin family of mycotoxins highlights technological advances and provides a blueprint for future studies of other fungal natural products. Expressed sequence tags led to the discovery of new fumonisin genes (FUM) and hinted at a role for alternatively spliced transcripts in regulation. Phylogenetic studies of FUM genes uncovered a complex evolutionary history of the FUM cluster, as well as fungi with the potential to synthesize fumonisin or fumonisin-like chemicals. The application of new technologies (e.g., CRISPR) could substantially impact future efforts to harness fungal resources.

Site-saturation engineering of proline 474 in pyruvate carboxylase from Rhizopus oryzae to elevate fumaric acid production in engineered Saccharomyces cerevisiae cells

Pyruvate carboxylase (PYC) plays an important role in the production of various carboxylic acids, because it links the high-capacity glycolytic pathway in Saccharomyces cerevisiae to the synthetic pathway of the desired product. In this study, site-saturation engineering of proline 474 in PYC from Rhizopus oryzae (RoPYC) was conducted to improve fumaric acid production in S. cerevisiae. We constructed a fumaric acid production strain with reduced ethanol yield by deleting the FUM1 gene in pdc1adh1 cells, and RoPYC was found to be more beneficial for fumaric acid production than ScPYC2. In particular, among the 19 RoPYC mutants, only overexpression of the P474N mutant in the pdc1adh1fum1 cells led to a significant increase in fumaric acid production with a yield of 314.5±3.8mgl−1 at 96h of fermentation, when compared with the wild-type RoPYC (252±17.0mgl−1). Further investigation showed that the P474N mutant possessed a higher (13.3%, p<0.05) specific activity. Supplementation of 56μgl−1 biotin into the fermentation broth produced the best yield of fumaric acid (448.4±2.1mgl−1) at 96 h of fermentation. To the best of our knowledge, this study is the first to report on the mutant PYC P474N, which achieved high fumaric acid production, thus acting as a model for accomplishing further improvement in carboxylic acid production.

Variation in Fumonisin and Ochratoxin Production Associated with Differences in Biosynthetic Gene Content in Aspergillus niger and A. welwitschiae Isolates from Multiple Crop and Geographic Origins.

The fungi Aspergillus niger and A. welwitschiae are morphologically indistinguishable species used for industrial fermentation and for food and beverage production. The fungi also occur widely on food crops. Concerns about their safety have arisen with the discovery that some isolates of both species produce fumonisin (FB) and ochratoxin A (OTA) mycotoxins. Here, we examined FB and OTA production as well as the presence of genes responsible for synthesis of the mycotoxins in a collection of 92 A. niger/A. welwitschiae isolates from multiple crop and geographic origins. The results indicate that (i) isolates of both species differed in ability to produce the mycotoxins; (ii) FB-nonproducing isolates of A. niger had an intact fumonisin biosynthetic gene (fum) cluster; (iii) FB-nonproducing isolates of A. welwitschiae exhibited multiple patterns of fum gene deletion; and (iv) OTA-nonproducing isolates of both species lacked the ochratoxin A biosynthetic gene (ota) cluster. Analysis of genome sequence data revealed a single pattern of ota gene deletion in the two species. Phylogenetic analysis suggest that the simplest explanation for this is that ota cluster deletion occurred in a common ancestor of A. niger and A. welwitschiae, and subsequently both the intact and deleted cluster were retained as alternate alleles during divergence of the ancestor into descendent species. Finally, comparison of results from this and previous studies indicate that a majority of A. niger isolates and a minority of A. welwitschiae isolates can produce FBs, whereas, a minority of isolates of both species produce OTA. The comparison also suggested that the relative abundance of each species and frequency of FB/OTA-producing isolates can vary with crop and/or geographic origin.

Influence of Butylated Hydroxyanisole on the Growth, Hyphal Morphology, and the Biosynthesis of Fumonisins in Fusarium proliferatum.

Fusarium proliferatum as a common fungus pathogen in foods can produce toxic fumonisins, which can cause animal diseases and increase risks of human cancers. On contrary, butylated hydroxyanisole (BHA) as a synthetic antioxidant offers a clue for preventing growth of fungal species and inhibiting production of mycotoxins. Unfortunately, information of the inhibitory mechanism of BHA on Fusarium species is still limited. In this study, influence of BHA treatment on growth and inhibition of fumonisin production in relation to the expression of the fumonisin biosynthesis-related genes of the F. proliferatum ZYF was investigated, which revealed that BHA had a negative influence on growth and fumonisin production of F. proliferatum. To further elucidate the mechanism of BHA on the growth of F. proliferatum, scanning electron microscopy (SEM) and transmission electron microscopy (TEM) were used to examine the F. proliferatum hyphae. The BHA treatment induced the loss of cytoplasm and cellular constituents, as well as distortion of mycelia, but it did not directly degrade the fumonisin. Furthermore, the BHA treatment markedly inhibited the expressions of FUM1 (a polyketide synthase encoding gene) and FUM8 (an aminotransferase encoding gene) genes, which resulted in the depression of metabolic pathway of F. proliferatum. The transcriptional analyses of the FUM1 and FUM8 genes confirmed a correlation between the fumonisin production and its gene expression. This study provided some insights into mechanisms of production of fumonisin and feasible prevention to reduce fumonisin contamination in favor of human and animal health.

Fusarium proliferatum strains change fumonisin biosynthesis and accumulation when exposed to host plant extracts

Fumonisin concentrations in mycelia and media were studied in liquid Fusarium proliferatum cultures supplemented with host plant extracts. Furthermore, the kinetics of fumonisin accumulation in media and mycelia collected before and after extract addition was analysed as well as the changes in the expression of the FUM1 gene. Fumonisin content in culture media increased in almost all F. proliferatum strains shortly after plant extracts were added. The asparagus extract induced the highest FB level increase and the garlic extract was the second most effective inducer. Fumonisin level decreased constantly until 14th day of culturing, though for some strains also at day 8th an elevated FB level was observed. Pineapple extract induced the highest increase of fum1 transcript levels as well as fumonisin synthesis in many strains, and the peas extract inhibited fungal growth and fumonisin biosynthesis. Moreover, fumonisins were accumulated in mycelia of studied strains and in the respective media.

Interplay between pathway-specific and global regulation of the fumonisin gene cluster in the rice pathogen Fusarium fujikuroi.

The rice pathogenic fungus Fusarium fujikuroi is known to produce a large variety of secondary metabolites. Besides the gibberellins, causing the bakanae effect in infected rice seedlings, the fungus produces several mycotoxins and pigments. Among the 47 putative secondary metabolite gene clusters identified in the genome of F. fujikuroi, the fumonisin gene cluster (FUM) shows very high homology to the FUM cluster of the main fumonisin producer Fusarium verticillioides, a pathogen of maize. Despite the high level of cluster gene conservation, total fumonisin FB1 and FB2 levels (FBx) produced by F. fujikuroi were only 1-10% compared to F. verticillioides under inducing conditions. Nitrogen repression was found to be relevant for wild-type strains of both species. However, addition of germinated maize kernels activated the FBx production only in F. verticillioides, reflecting the different host specificity of both wild-type strains. Over-expression of the pathway-specific transcription factor Fum21 in F. fujikuroi strongly activated the FUM cluster genes leading to 1000-fold elevated FBx levels. To gain further insights into the nitrogen metabolite repression of FBx biosynthesis, we studied the impact of the global nitrogen regulators AreA and AreB and demonstrated that both GATA-type transcription factors are essential for full activation of the FUM gene cluster. Loss of one of them obstructs the pathway-specific transcription factor Fum21 to fully activate expression of FUM cluster genes.

Polyphasic Approach Including MALDI-TOF MS/MS Analysis for Identification and Characterisation of Fusarium verticillioides in Brazilian Corn Kernels.

Fusarium verticillioides is considered one of the most important global sources of fumonisins contamination in food and feed. Corn is one of the main commodities produced in the Northeastern Region of Brazil. The present study investigated potential mycotoxigenic fungal strains belonging to the F. verticillioides species isolated from corn kernels in 3 different Regions of the Brazilian State of Pernambuco. A polyphasic approach including classical taxonomy, molecular biology, MALDI-TOF MS and MALDI-TOF MS/MS for the identification and characterisation of the F. verticillioides strains was used. Sixty F. verticillioides strains were isolated and successfully identified by classical morphology, proteomic profiles of MALDI-TOF MS, and by molecular biology using the species-specific primers VERT-1 and VERT-2. FUM1 gene was further detected for all the 60 F. verticillioides by using the primers VERTF-1 and VERTF-2 and through the amplification profiles of the ISSR regions using the primers (GTG)5 and (GACA)4. Results obtained from molecular analysis shown a low genetic variability among these isolates from the different geographical regions. All of the 60 F. verticillioides isolates assessed by MALDI-TOF MS/MS presented ion peaks with the molecular mass of the fumonisin B1 (721.83 g/mol) and B2 (705.83 g/mol).

Light inhibition of fumarase in Arabidopsis leaves is phytochrome A–dependent and mediated by calcium

Inhibition of fumarase activity in the light has been studied in Arabidopsis in relation to the involvement of phytochrome. Using knockout phytochrome mutants, we observed that the main regulator of FUM1 gene transcription, encoding the mitochondrial form of fumarase, is phytochrome A. The active form of phytochrome A suppressed FUM1 expression, while the expression of the FUM2 gene encoding the cytosolic form of fumarase was unaffected both in darkness and in light. The nuclear concentration of Ca2+ was modulated by red and far-red light. We suggest that the signal transduction mechanism operates via Ca2+ activation of expression of the gene encoding the transcription factor PIF3, which binds to promoters of phytochrome-regulated genes and inhibits FUM1 expression.

Molecular phylogeny, pathogenicity and toxigenicity of Fusarium oxysporum f. sp. lycopersici.

The present study aimed at the molecular characterization of pathogenic and non pathogenic F. oxysporum f. sp. lycopersici strains isolated from tomato. The causal agent isolated from symptomatic plants and soil samples was identified based on morphological and molecular analyses. Pathogenicity testing of 69 strains on five susceptible tomato varieties showed 45% of the strains were highly virulent and 30% were moderately virulent. Molecular analysis based on the fingerprints obtained through ISSR indicated the presence of wide genetic diversity among the strains. Phylogenetic analysis based on ITS sequences showed the presence of at least four evolutionary lineages of the pathogen. The clustering of F. oxysporum with non pathogenic isolates and with the members of other formae speciales indicated polyphyletic origin of F. oxysporum f. sp. lycopersici. Further analysis revealed intraspecies variability and nucleotide insertions or deletions in the ITS region among the strains in the study and the observed variations were found to be clade specific. The high genetic diversity in the pathogen population demands for development of effective resistance breeding programs in tomato. Among the pathogenic strains tested, toxigenic strains harbored the Fum1 gene clearly indicating that the strains infecting tomato crops have the potential to produce Fumonisin.

FUM Gene Expression Profile and Fumonisin Production by Fusarium verticillioides Inoculated in Bt and Non-Bt Maize.

This study aimed to determine the levels of fumonisins produced by Fusarium verticillioides and FUM gene expression on Bt (Bacillus thuringiensis) and non-Bt maize, post harvest, during different periods of incubation. Transgenic hybrids 30F35 YG, 2B710 Hx and their isogenic (30F35 and 2B710) were collected from the field and a subset of 30 samples selected for the experiments. Maize samples were sterilized by gamma radiation at a dose of 20 kGy. Samples were then inoculated with F. verticillioides and analyzed under controlled conditions of temperature and relative humidity for fumonisin B1 and B2 (FB1 and FB2) production and FUM1, FUM3, FUM6, FUM7, FUM8, FUM13, FUM14, FUM15, and FUM19 expression. 2B710 Hx and 30F35 YG kernel samples were virtually intact when compared to the non-Bt hybrids that came from the field. Statistical analysis showed that FB1 production was significantly lower in 30F35 YG and 2B710 Hx than in the 30F35 and 2B710 hybrids (P < 0.05). However, there was no statistical difference for FB2 production (P > 0.05). The kernel injuries observed in the non-Bt samples have possibly facilitated F. verticillioides penetration and promoted FB1 production under controlled conditions. FUM genes were expressed by F. verticillioides in all of the samples. However, there was indication of lower expression of a few FUM genes in the Bt hybrids; and a weak association between FB1 production and the relative expression of some of the FUM genes were observed in the 30F35 YG hybrid.

Resistance to Fusarium verticillioides and fumonisin accumulation in maize inbred lines involves an earlier and enhanced expression of lipoxygenase (LOX) genes

Fusarium verticillioides causes ear rot in maize and contaminates the kernels with the fumonisin mycotoxins. It is known that plant lipoxygenase (LOX)-derived oxylipins regulate defence against pathogens and that the host-pathogen lipid cross-talk influences the pathogenesis. The expression profiles of fifteen genes of the LOX pathway were studied in kernels of resistant and susceptible maize lines, grown in field condition, at 3, 7 and 14 days post inoculation (dpi) with F. verticillioides. Plant defence responses were correlated with the pathogen growth, the expression profiles of fungal FUM genes for fumonisin biosynthesis and fumonisin content in the kernels. The resistant genotype limited fungal growth and fumonisin accumulation between 7 and 14 dpi. Pathogen growth became exponential in the susceptible line after 7 dpi, in correspondence with massive transcription of FUM genes and fumonisins augmented exponentially at 14 dpi. LOX pathway genes resulted strongly induced after pathogen inoculation in the resistant line at 3 and 7 dpi, whilst in the susceptible line the induction was reduced or delayed at 14 dpi. In addition, all genes resulted overexpressed before infection in kernels of the resistant genotype already at 3 dpi. The results suggest that resistance in maize may depend on an earlier activation of LOX genes and genes for jasmonic acid biosynthesis.

Isolation and characterization of Fusarium verticillioides from maize in eastern China

In 2012, we isolated mycotoxigenic fungi from maize kernels sourced in Jiangsu and Anhui provinces, China. Fusarium spp. accounted for most isolates detected, followed by Aspergillus and Penicillium spp. Of the Fusarium species, Fusarium verticillioides was the most prevalent, as revealed by direct PCR of DNA from kernel samples, followed by F. proliferatum and F. graminearum. Most F. verticillioides strains contained the FUM1 gene, the product of which plays a key role in fumonisin biosynthesis. In terms of mating type idiomorphs, about 80 % of all strains carried the MAT-1 allele, and 20 % carried MAT-2. The genetic structures of 192 representative F. verticillioides isolates were determined via an analysis of eight simple sequence repeat markers. This analysis revealed high-level gene diversity but low linkage disequilibrium in three populations grouped by the geographical areas in which they were collected. The three populations exhibited low-level genetic differentiation and high gene flow, indicating that geographical differences minimally influenced population differentiation; all three populations were potentially components of a larger, randomly mating population.

Efficacy of Cuminum cyminum essential oil on FUM1 gene expression of fumonisin-producing Fusarium verticillioides strains.

The purpose of this study was to evaluate the effect of Cuminum cyminum (C. cyminum) essential oil on the growth and FUM1 gene expression of fumonisin-producing Fusarium verticillioides (F. verticillioides) strains isolated from maize. All fungal strains were cultured on potato dextrose agar (PDA) slopes at 30°C for 7 days. The antifungal activity was evaluated by broth microdilution assay. One set of primers was F. verticillioides species specific, which selectively amplified the intergenic space region of rDNA. The other set of primers was specific to FUM1 gene region of fumonisin-producing F. verticillioides. FUM1 transcript levels were quantified using a reverse transcription-polymerase chain reaction (RT-PCR) protocol. Minimum inhibitory concentration (MIC) values of C. cyminum oil against F. verticillioides strains varied from 0.195 to 0.781 µl.ml(-1) (mean MIC value: 0.461 µl.ml(-1)) indicating 54.5% of the fungal strains inhibited at 0.390 µl.ml(-1). PCR analysis of FUM1 gene expression revealed that DNA fragment of 183 bp was amplified in all the isolates of F. verticillioides before treatment with C. cyminum essential oil. Based on RT-PCR analyses, reduction in the expression of fumonisin biosynthetic genes was significant only for FUM1 gene (p<0.05), while no effect was observed on ITS gene. This study showed that all F. verticillioides isolates were susceptible to C. cyminum essential oil, indicating a significant reduction in the growth of fungal isolates. In addition, this oil completely inhibited the expression of FUM1 gene in concentrations dose-dependently.

Pathogenicity and potential toxigenicity of seed-borne Fusarium species on soybean and pea.

The effect of 15 Fusarium species on seed germination and early plant development was examined in a laboratory study, together with the potential toxigenicity of selected isolates and the possibility of T-2 toxin production on soybean and pea grain. In germination tests, 33 out of 47 isolates of Fusarium chlamydosporum, F. pseudograminearum, F. sporotrichioides, F. crookwellense, F. verticillioides, F. equiseti, F. semitectum, F. solani, F. poae, F. sambucinum and F. compactum significantly reduced the number of normal soybean seedlings, while only six out of 48 isolates of F. sporotrichioides, F. semitectum and F. chlamydosporum significantly reduced the number of normal pea seedlings. When inoculated on plants grown in Hoagland's media, nearly all Fusarium isolates caused necrosis of soybean and pea root, but neither of them significantly reduced shoot and root dry mass of pea plants, or shoot dry mass of soybean plants. Amplification of tri5 and FUM1 genes, required for trichothecene and fumonisin biosynthesis, was carried out by PCR in 45 Fusarium isolates from soybean and 40 isolates from pea. Positive tri5 PCR reaction was recorded in 19 isolates of F. sporotrichioides, F. crookwellense, F. pseudograminearum, F. poae, F. sambucinum, F. culmorum, F. equiseti and F. chlamydosporum. Positive FUM1 PCR reaction was recorded in 35 out of 38 F. verticillioides and F. proliferatum isolates. The content of T-2 toxin produced by eight F. sporotrichioides isolates inoculated on autoclaved soybean, pea and barley grain ranged from 69.4 to 2595.5 μg/kg. No significant differences were determined between T-2 toxin production on soybean and barley grain, nor on pea and barley grain.

Variation in the fumonisin biosynthetic gene cluster in fumonisin-producing and nonproducing black aspergilli

The ability to produce fumonisin mycotoxins varies among members of the black aspergilli. Previously, analyses of selected genes in the fumonisin biosynthetic gene (fum) cluster in black aspergilli from California grapes indicated that fumonisin-nonproducing isolates of Aspergillus welwitschiae lack six fum genes, but nonproducing isolates of Aspergillus niger do not. In the current study, analyses of black aspergilli from grapes from the Mediterranean Basin indicate that the genomic context of the fum cluster is the same in isolates of A. niger and A. welwitschiae regardless of fumonisin-production ability and that full-length clusters occur in producing isolates of both species and nonproducing isolates of A. niger. In contrast, the cluster has undergone an eight-gene deletion in fumonisin-nonproducing isolates of A. welwitschiae. Phylogenetic analyses suggest each species consists of a mixed population of fumonisin-producing and nonproducing individuals, and that existence of both production phenotypes may provide a selective advantage to these species. Differences in gene content of fum cluster homologues and phylogenetic relationships of fum genes suggest that the mutation(s) responsible for the nonproduction phenotype differs, and therefore arose independently, in the two species. Partial fum cluster homologues were also identified in genome sequences of four other black Aspergillus species. Gene content of these partial clusters and phylogenetic relationships of fum sequences indicate that non-random partial deletion of the cluster has occurred multiple times among the species. This in turn suggests that an intact cluster and fumonisin production were once more widespread among black aspergilli.

Pathogenicity and genetic diversity of Fusarium oxysporum isolates from corms of Crocus sativus

Crocus sativus is a triploid sterile plant and autumnal flowering geophyte with corms. As a subterranean organ, the corm is susceptible to diseases caused by fungi, bacteria, nematodes and viruses. Fusarium corm rot incited by F. oxysporum is the most destructive disease in saffron, having caused severe yield losses in saffron producer's countries. Infected plants die off early, resulting in reduction of corm yield, quality and flower and stigma production. Little is known about the formae speciales attacking saffron and less about the relationship among the fungus and the possible hosts. In this study we investigate the formae speciales which colonize saffron and their relationship with some members of iridaceae such as C. vernus (ornamental crocus), gladiolus and narcissus using pathogenicity tests; we determine whether different pathogenic isolates of F. oxysporum can be distinguished by ISSR, and we analyze the genetic relationships and variability among some isolates of these pathogens. We found two formae speciales iridiacearum and croci which attack different iridaceous crops and Crocus sp., respectively and we suggested the creation of a new formae speciales saffrani which shows only pathogenicity on saffron corms. From ISSR analysis, the unweighted paired group method with arithmetic averages cluster analysis (UPGMA) was used to discriminate the F. oxysporum isolates, we were able to differentiate among f. sp. from saffron corms and other formae speciales of F. oxysporum but not among formae speciales iridiacearum, croci and saffrani. Furthermore, we searched for fum1, tri5, tri7 and tri13 genes using PCR assays, however, all the isolates from saffron corms were negative.

Molecular detection and genetic characterisation of fumonisin producingfusariumisolates from rice cultivars

Fusarium species mainly produce fumonisins group of mycotoxins which are classified as Group 2B human carcinogen by International Agency for Research on Cancer (IARC). In poor storage conditions, Fusarium species producing fumonisins can infect rice or paddy (Oryza sativa L.) which is the highest produced and consumed staple food in India. A rapid molecular method using primer Fum5F and Fum6R detected 85% fumonisin producers among 28 Fusarium isolates from Indian rice cultivars. Genetic variability of the isolates was studied by PCR based RAPD assay using 13 random primers. A total of 169 polymorphic bands were obtained by 13 markers with an average polymorphism information content (PIC) of 0.665 and overall polymorphism of 88%. Primer 3B showed a polymorphism of 96% with PIC value of 0.66 and it amplified 26 scorable fragments hence may be useful for the analysis of genetic variation among Fusarium isolates. Four strains (F47, F90, F92 and F96) in which fum gene wasn't amplified by Fum5F and Fum6R and supposed to be non producer of fumonisin have been consistently placed in one separate group by RAPD primers. Genetic variation of toxic Fusarium in rice from India is less studied. RAPD proved to be a suitable tool for depicting Polymorphism among the isolates. The high genetic variability among the Fusarium isolates used in the current study is a matter of concern considering the importance of Rice in India.

Fumonisin and T-2 toxin production of Fusarium spp. isolated from complete feed and individual agricultural commodities used in shrimp farming

Fusarium spp. are plant pathogens producing fumonisins and trichothecenes that both affect human and animal health. In the present study, 40 fungal strains were isolated and species identified from 35 shrimp feed samples and from 61 agricultural raw materials. F. verticillioides was the predominant species (85%) mostly found in corn and soybean meal, while no Fusarium contamination was detected in shrimp feed. Levels of 10% of F. oxysporum were isolated from peanut and 5% of F. equiseti contamination in corn and peanut. To determine the ability of toxin production, enzyme-linked immunosorbent assay, polymerase chain reaction, and ultra-pressure liquid chromatography-tandem mass spectrometry were performed. All but four of the fumonisin-producing strains contained the FUM1 gene. No Fusarium synthesized T-2 toxin nor contained the Tri5 gene. This survey brings more data on mycotoxin contamination in the food chain of animal feed production, and leads to the awareness of the use of contaminated raw materials in shrimp farming.

Molecular identification and characterization ofFusariumspp. associated with sorghum seeds

Fusarium spp. are not only pathogenic to plants but are also known as toxin producers that negatively affect animal and human health. The identification of Fusarium spp. remains one of the most critical issues in fungal taxonomy. In this study, different strains of Fusarium spp. were isolated from sorghum seed samples and identified at the molecular level by tef-1α gene amplification. A multiplex polymerase chain reaction (mPCR) assay was developed to differentiate toxigenic and non-toxigenic Fusarium spp. by designing a primer for the Fum21 gene along with the Fum1 and Fum8 genes. A competitive direct enzyme-linked immunosorbent assay (CD-ELISA) was employed to assess the fumonisin-producing ability of Fusarium spp. Phylogenetic analyses were performed using partial sequences of tef-1α and inter-simple sequence repeat (ISSR) markers of different Fusarium spp. All 27 isolates of Fusarium spp. were positive for the tef-1α gene and revealed the presence of F. verticillioides, F. thapsina and F. cf. incarnatum-equiseti complex. The standardized mPCR assay distinguished toxigenic and non-toxigenic F. verticillioides. Further, mPCR fumonisin-positive F. verticillioides isolates were also positive by CD-ELISA. The tef-1α gene sequence was found to be useful in revealing intraspecific polymorphism to some extent. ISSR markers revealed a high level of polymorphism among different isolates of Fusarium spp., and the dendrogram of ISSR analyses grouped the 27 isolates into two major clusters. The present method provided rapid and reliable detection of fumonisin-producing Fusarium spp. The mPCR assay could be an alternative strategy to current conventional mycotoxin analytical techniques and a reliable tool for high-throughput monitoring of major mycotoxin-producing fungi during the processing steps of food and feed commodities.