Fumonisin Biosynthesis Research Articles

Pyrrocidine, a molecular off switch for fumonisin biosynthesis.

Sarocladium zeae is a fungal endophyte of maize and can be found co-inhabiting a single seed with Fusarium verticillioides, a major mycotoxigenic food safety threat. S. zeae produces pyrrocidines A and B that inhibit the growth of F. verticillioides and may limit its spread within the seed to locations lacking S. zeae. Although coinhabiting single seeds, the fungi are generally segregated in separate tissues. To understand F. verticillioides’ protective physiological response to pyrrocidines we sequenced the F. verticillioides transcriptome upon exposure to purified pyrrocidine A or B at sub-inhibitory concentrations. Through this work we identified a F. verticillioides locus FvABC3 (FVEG_11089) encoding a transporter critical for resistance to pyrrocidine. We also identified FvZBD1 (FVEG_00314), a gene directly adjacent to the fumonisin biosynthetic gene cluster that was induced several thousand-fold in response to pyrrocidines. FvZBD1 is postulated to act as a genetic repressor of fumonisin production since deletion of the gene resulted in orders of magnitude increase in fumonisin. Further, pyrrocidine acts, likely through FvZBD1, to shut off fumonisin biosynthesis. This suggests that S. zeae is able to hack the secondary metabolic program of a competitor fungus, perhaps as preemptive self-protection, in this case impacting a mycotoxin of central concern for food safety.

A loop-mediated isothermal amplification (LAMP) based assay for the rapid and sensitive group-specific detection of fumonisin producing Fusarium spp

Fumonisins are mycotoxins that contaminate maize and maize-based food products, and feed. They have been associated with nerve system disorders in horses, pulmonary edema in swine as well as neural tube defects and esophageal cancer in humans. The fum1 gene codes for a polyketide synthase involved in the biosynthesis of fumonisins. It is present in the genomes of all fumonisin producing Fusarium spp. Reliable detection of fum1 can provide an estimate of the toxicological potential of cultures and food sources. Therefore, a fum1 specific LAMP assay was developed and tested with purified DNA of 48 different species from the Fusarium fujikuroi species complex (FFSC). The fum1 gene was detected in 22 species among which F. fujikuroi, F. globosum, F. nygamai, F. proliferatum, F. subglutinans and F. verticillioides were the most prominent fumonisin producers. None out of 92 tested non-Fusarium species showed cross reactions with the new assay. The lowest limit of detection (LOD) was 5 pg of genomic DNA per reaction for F. fujikuroi, F. nygamai and F. verticillioides. Higher LODs were found for other LAMP positive species. Apart from pure genomic DNA, the LAMP assay detected fumonisin-producers when 103 conidia/reaction were used as template after mechanical lysis. LAMP-results were well correlated with FB1 production. This is the first report on fumonisin production in strains of F. annanatum, F. coicis, F. mundagurra, F. newnesense, F. pininemorale, F. sororula, F. tjataeba, F. udum and F. werrikimbe. Usefulness of the LAMP assay was demonstrated by analyzing fumonisin contaminated maize grains. The new LAMP assay is rapid, sensitive and reliable for the diagnosis of typical fumonisin producers and can be a versatile tool in HACCP concepts that target the reduction of fumonisins in the food and feed chain.

First report on the detection of fumonisin biosynthetic (FUM1) gene in Fusarium verticillioides and F. proliferatum associated with sugarcane diseases

Several Fusarium verticillioides and F. proliferatum strains produce fumonisin, a mycotoxin that negatively affects animals and humans. These two Fusarium species also cause Pokkah boeng and are associated with red rot-like disease, two important sugarcane diseases. In this study, we determined the presence of FUM1, a gene that is required for fumonisin biosynthesis, in 21 isolates of plant pathogenic Fusarium species associated with sugarcane Pokkah boeng and the red rot-like disease. Out of the 21 Fusarium isolates, four F. proliferatum and one F. verticillioides isolates conferred the FUM1 gene as detected by repeated polymerase chain reaction assay. The result indicates that pathogenicity and fumonisin biosynthesis are likely two separate events as other isolates, which did not confer the FUM1 gene, still induced Pokkah boeng and red rot. This study reports, for the first time, the occurrence of fumonisin-producing F. verticillioides and F. proliferatum that cause Pokkah boeng and red rot-like disease in sugarcane.

In Vitro Fumonisin Biosynthesis and Genetic Structure of Fusarium verticillioides Strains from Five Mediterranean Countries.

Investigating the in vitro fumonisin biosynthesis and the genetic structure of Fusarium verticillioides populations can provide important insights into the relationships between strains originating from various world regions. In this study, 90 F. verticillioides strains isolated from maize in five Mediterranean countries (Italy, Spain, Tunisia, Egypt and Iran) were analyzed to investigate their ability to in vitro biosynthesize fumonisin B1, fumonisin B2 and fumonisin B3 and to characterize their genetic profile. In general, 80% of the analyzed strains were able to biosynthesize fumonisins (range 0.03–69.84 μg/g). Populations from Italy, Spain, Tunisia and Iran showed a similar percentage of fumonisin producing strains (>90%); conversely, the Egyptian population showed a lower level of producing strains (46%). Significant differences in fumonisin biosynthesis were detected among strains isolated in the same country and among strains isolated from different countries. A portion of the divergent FUM1 gene and of intergenic regions FUM6-FUM7 and FUM7-FUM8 were sequenced to evaluate strain diversity among populations. A high level of genetic uniformity inside the populations analyzed was detected. Apparently, neither geographical origin nor fumonisin production ability were correlated to the genetic diversity of the strain set. However, four strains from Egypt differed from the remaining strains.

The Impacts of Asparagus Extract Fractions on Growth and Fumonisins Biosynthesis in Fusarium Proliferatum.

Asparagus is a genus consisting of over two hundred species of perennial plants. Fusarium proliferatum is a major asparagus pathogen and it biosynthesizes a variety of mycotoxins, of which fumonisins B are prevalent. Our previous studies on F. proliferatum strains indicated that asparagus extract affects the expression of FUM1 gene, encoding polyketide synthase, a key enzyme of the FUM gene cluster governing the biosynthesis of fumonisins. An asparagus-derived F. proliferatum strain increased fumonisin B1 production after extract fractions’ addition, reaching the maximum 2 or 24 h after treatment. The cultures yielded between 40 and 520 mg of dry weight of mycelia after 14 days of cultivation. The differences in fungal biomass amounts between the whole extract and its fractions may result from synergistic effect of all bioactive compounds present in asparagus extract. Among extract fractions, the methanolic fraction had the highest effect on the dry weight of the mycelium reaching about a 13-fold increase compared to the control. Furthermore, we measured the relative expression of the FUM1 gene. Due to the possible antifungal activity of tested extract fractions, future research will be focused on the identification of the Asparagus officinalis L. compounds responsible for this activity.

Characterization of non-canonical G beta-like protein FvGbb2 and its relationship with heterotrimeric G proteins in Fusarium verticillioides.

Fusarium verticillioides is a fungal pathogen that is responsible for maize ear rot and stalk rot diseases worldwide. The fungus also produces carcinogenic mycotoxins, fumonisins on infested maize. Unfortunately, we still lack clear understanding of how the pathogen responds to host and environmental stimuli to trigger fumonisin biosynthesis. The heterotrimeric G protein complex, consisting of canonical Gα, Gβ and Gγ subunits, is involved in transducing signals from external stimuli to regulate downstream signal transduction pathways. Previously, we demonstrated that Gβ protein FvGbb1 directly impacts fumonisin regulation but not other physiological aspects in F. verticillioides. In this study, we identified and characterized a RACK1 (Receptor for Activated C Kinase 1) homolog FvGbb2 as a putative Gβ-like protein in F. verticillioides. The mutant exhibited severe defects not only in fumonisin biosynthesis but also vegetative growth and conidiation. FvGbb2 was positively associated with carbon source utilization and stress agents but negatively regulated general amino acid control. While FvGbb2 does not interact with canonical G protein subunits, it may associate with diverse proteins in the cytoplasm to regulate vegetative growth, virulence, fumonisin biosynthesis and stress response in F. verticillioides.

A CRISPR-Cas9 System for Genome Editing of Fusarium proliferatum

Fusarium proliferatum causes diverse diseases of many economically important plants. The fungus produces several mycotoxins of which the fumonisins are the most toxic. Currently, deletion of key genes for mycotoxin biosynthesis is a laborious and time-consuming procedure. We developed a novel CRISPR/Cas9-based genome-editing tool for the direct delivery of preassembled Cas9 ribonucleoproteins into protoplasts of F. proliferatum. Our CRISPR–Cas9 system couples a site-specific double-strand DNA break mediated by two Cas9 ribonucleoproteins with microhomology recombination requiring only 50-bp regions flanking the target gene. This system reduces the risk of off-target mutations and minimizes the risk of altering any gene adjacent to the target region. We used this tool to delete a polyketide synthase gene (FUM1) required for fumonisin biosynthesis. The mutants generated are no longer able to produce fumonisins, confirming the key role of FUM1 in fumonisin biosynthesis. Our CRISPR-Cas9 system is an important new tool for genetic studies of Fusarium.

Potential use of machine learning methods in assessment of Fusarium culmorum and Fusariumproliferatum growth and mycotoxin production in treatments with antifungal agents

Fusarium-controlling fungicides are necessary to limit crop loss. Little is known about the effect of antifungal formulations at sub-lethal doses, and their interaction with abiotic factors, on Fusarium culmorum and F. proliferatum development and on zearalenone and fumonisin biosynthesis, respectively. In the present study different treatments based on sulfur, trifloxystrobin and demethylation inhibitor fungicides (cyproconazole, tebuconazole and prothioconazole) under different environmental conditions, in Maize Extract Medium, are assayed in vitro. Several machine learning methods (neural networks, random forest and extreme gradient boosted trees) have been applied for the first time for modeling growth of F. culmorum and F. proliferatum and zearalenone and fumonisin production, respectively. The most effective treatment was prothioconazole, 250 g/L + tebuconazole, 150 g/L. Effective doses of this formulation for reduction or total growth inhibition ranged as follows ED50 0.49–1.70, ED90 2.57–6.02 and ED100 4.0–8.0 µg/mL, depending on the species, water activity and temperature. Overall, the growth rate and mycotoxin levels in cultures decreased when doses increased. Some treatments in combination with certain aw and temperature values significantly induced toxin production. The extreme gradient boosted tree was the model able to predict growth rate and mycotoxin production with minimum error and maximum R2 value.

Effects of Disruption of Five FUM Genes on Fumonisin Biosynthesis and Pathogenicity in Fusarium proliferatum.

The mycotoxin fumonisin is known to be harmful to humans and animals, and thus it is desirable to reduce fumonisin content in crop products. We explored the functions of several genes that function in fumonisin biosynthesis (FUM1, FUM6, FUM8, FUM19, and FUM21) in Fusarium proliferatum and found that deletion of FUM1, FUM6, FUM8, or FUM21 results in a severe reduction in fumonisin biosynthesis, while loss of FUM19 does not. In addition, fumonisin-deficient strains display significantly decreased pathogenicity. Co-cultivation of the ΔFUM1, ΔFUM6, ΔFUM8, and ΔFUM19 mutants restores fumonisin synthesis. However, co-cultivation was unable to restore fumonisin synthesis in the ΔFUM21 strain. The relative expression levels of three key FUM genes (FUM1, FUM6, and FUM8) differed significantly in each mutant strain; notably, the expression levels of these three genes were significantly down-regulated in the ΔFUM21 strain. Taken together, our results demonstrate that FUM1, FUM6, FUM8, and FUM21 are essential for fumonisin synthesis, and FUM19 is non-essential. Partial mutants lost the ability to synthesize fumonisin, the co-culture of the mutants was able to restore fumonisin biosynthesis. While the pathogenicity of F. proliferatum is affected by many factors, inhibition of the synthesis of the mycotoxin fumonisin will weaken the pathogenicity of rice spikelet rot disease (RSRD).

Genetic regulation of aflatoxin, ochratoxin A, trichothecene, and fumonisin biosynthesis: A review.

Mycotoxins are a significant food safety concern. Aflatoxins, trichothecenes, fumonisins, and ochratoxin A are considered the most important mycotoxins due to their frequent occurrence in food products and their well-known toxicity. The regulation of mycotoxin biosynthesis occurs mainly at transcriptional level, and specific regulators have been described in every biosynthetic cluster. Secondary metabolite production, including mycotoxin synthesis, is also regulated by general regulator pathways affected by light, osmotic stress and oxidative stress, among others. This review is focused on this genetic regulation of mycotoxin biosynthesis by specific genes and global regulators.

Effects of Different Carbon Sources on Fumonisin Production and FUM Gene Expression by Fusarium proliferatum.

Fusarium proliferatum can infect many crops and then produce fumonisins that are very harmful to humans and animals. Previous study indicates that carbon sources play important roles in regulating the fumonisin biosynthesis. Unfortunately, there is limited information on the effects of carbon starvation in comparison with the carbon sources present in the host of fumonisin production in F. proliferatum. Our results indicated that F. proliferatum cultivated in the Czapek’s broth (CB) medium in the absence of sucrose could greatly induce production of fumonisin, while an additional supplementation of sucrose to the culture medium significantly reduced the fumonisin production. Furthermore, cellulose and hemicellulose, and polysaccharide extracted from banana peel, which replaced sucrose as the carbon source, can reduce the production of fumonisin by F. proliferatum. Further work showed that these genes related to the synthesis of fumonisin, such as FUM1 and FUM8, were significantly up-regulated in the culture medium in the absence of sucrose. Consistent with fumonisin production, the expressions of FUM gene cluster and ZFR1 gene decreased after the addition of sucrose. Moreover, these genes were also significantly down-regulated in the presence of cellulose, hemicellulose or polysaccharide extracted from peel. Altogether, our results suggested that fumonisin production was regulated in F. proliferatum in response to different carbon source conditions, and this regulation might be mainly via the transcriptional level. Future work on these expressions of the fumonisin biosynthesis-related genes is needed to further clarify the response under different carbon conditions during the infection of F. proliferatum on banana fruit hosts. The findings in this study will provide a new clue regarding the biological effect of the fumonisin production in response to environmental stress.

A Natural Variation of Fumonisin Gene Cluster Associated with Fumonisin Production Difference in Fusarium fujikuroi

Fusarium fujikuroi, a member of the Fusarium fujikuroi species complex, stands out as a rice bakanae disease pathogen with a high production of gibberellic acid. Not all, but some F. fujikuroi strains are known to produce a carcinogenic mycotoxin fumonisin. Fumonisin biosynthesis is dependent on the FUM cluster composed of 16 FUM genes. The FUM cluster was detected not only from a fumonisin producing strain, but also from a fumonisin nonproducing strain that does not produce a detectable level of fumonisin. Genetic mapping indicated the causative mutation(s) of fumonisin nonproduction is present in the FUM cluster of the fumonisin nonproducing strain. Comparative analyses of FUM genes between the fumonisin producing and the nonproducing strains and gene complementation indicated that causative mutation of fumonisin nonproduction is not a single occurrence and the mutations are distributed in FUM21 and FUM7. Our research revealed a natural variation in the FUM cluster involving fumonisin production difference in F. fujikuroi.

Aspergillus species from Brazilian dry beans and their toxigenic potential

Aspergilli are common contaminants of food and feed and a major source of mycotoxins. In this study, 87 Aspergillus strains were isolated from beans from 14 different cities in Brazil and identified to the species level based on partial calmodulin and β-tubulin sequence data. All green spored isolates belonged to section Flavi and were identified as A. flavus (n = 39) or A. pseudocaelatus (n = 1). All black spored isolates belonged to section Nigri and were identified as A. niger (n = 24) or A. luchuensis (n = 10), while the yellow spored strains were identified as A. westerdijkiae (n = 7), A. ostianus (n = 3), A. ochraceus (n = 1) or A. wentii (n = 2). The toxigenic potential of these Aspergillus strains from beans was studied by the prospection of genes in three of the major mycotoxin clusters: aflatoxin (seven genes checked), ochratoxin A (four genes) and fumonisin (ten genes and two intergenic regions). Genes involved in the biosynthesis of aflatoxin were only detected in A. flavus isolates: 17/39 A. flavus isolates proved to contain all the aflatoxin genes tested, the others missed one or more genes. The full complement of fumonisin biosynthesis genes was identified in all A. niger isolates. Finally, no genes for ochratoxin A were detected in any of the isolates. Our work suggests that aflatoxin production by some A. flavus strains and fumonisin production by A. niger isolates form the largest mycotoxin risks in Brazilian beans.

Species composition of and fumonisin production by the Fusarium fujikuroi species complex isolated from Korean cereals

To assess the risk of fumonisin contamination in Korean cereals, we isolated colonies of the Fusarium fujikuroi species complex (FFSC) from barley, maize, rice and soybean samples from 2011 to 2015. A total of 878 FFSC strains were isolated mostly from maize and rice, and species identity of the isolates were determined using the DNA sequence of the translation elongation factor 1-α (TEF-1α) and RNA polymerase II (RPB2) genes. Fusaria recovered from Korean cereals included F. fujikuroi (317 isolates and a frequency of 36%), F. proliferatum (212 isolates and 24.1%), F. verticillioides (170 isolates and 19.4%), F. concentricum (86 strains and 9.8%), F. andiyazi (56 isolates and 6.4%), F. subglutinans (28 isolates and 3.2%), F. thapsinum (5 isolates and 0.6%), and F. circinatum (2 isolates and 0.2%). The rice samples were dominated by F. fujikuroi (47.4%), F. proliferatum (27.3%), and F. concentricum (15.1%), whereas maize samples were dominated by F. verticillioides (33.9%), F. fujikuroi (25.3%), and F. proliferatum (21.1%). A phylogenetic analysis of 70 representative isolates demonstrated that each species was resolved as genealogically exclusive in the ML tree. Fumonisin production potential was evaluated using a PCR assay for the fumonisin biosynthesis gene, FUM1 in all of the isolates. Most of the isolates tested (94%) were positive for FUM1. All of the isolates assigned to F. fujikuroi, F. proliferatum, F. verticillioides and F. thapsinum were positive for FUM1 irrespective of their host origin. Seventy-seven representative isolates positive for FUM1 were examined for fumonisin production in rice medium. The majority of F. proliferatum (26/27, 96.3%), F. verticillioides (16/17, 94.1%) and F. fujikuroi (19/25, 76.0%) produced both FB1 and FB2. Notably, 16 of 19 fumonisin-producing F. fujikuroi produced >1000μg/g of fumonisins (FB1+FB2) in rice medium, which is higher than that in previous reports. These results suggest that F. fujikuroi can produce high levels of fumonisins similar to F. verticillioides and F. proliferatum.

Influence of water activity and temperature on growth and fumonisin production by Fusarium proliferatum strains on irradiated wheat grains

Wheat is the most important cereal consumed by the Argentine population. In previous studies performed in durum and common wheat grains in this country it has been observed fumonisin contamination as well as high incidence of Fusarium proliferatum. Fumonisins are toxic fungal metabolites, and consumption of fumonisin-contaminated maize has been epidemiologically associated with oesophageal cancer and neural tube defects in some human populations. Using irradiated wheat-grains, the effects of abiotic factors, temperature (15, 25, and 30°C) and water activity (aW; 0.995, 0.98, 0.96, 0.94, 0.92, and 0.88), on mycelial growth and fumonisin biosynthesis were compared for three F. proliferatum strains isolated from wheat grains in Argentina. Although all isolates showed similar profiles of growth, the fumonisin production profiles were slightly different. Maximum growth rates were obtained at the highest aW (0.995) and 25°C, with growth decreasing as the aW of the medium was reduced. Maximum amounts of total fumonisins (FB1, FB2 and FB3) were produced at 0.995 aW and 15°C for 2 strains, and at 25°C and 0.995 aW for the third one. Fumonisins concentrations varied considerably depending on the aW and temperature interactions assayed. Studied strains showed different fumonisin production profiles. Two-dimensional profiles of aW by temperature interactions were developed from these data to identify areas where conditions indicate a significant risk of fumonisins accumulation on wheat. As a result, temperature and aW conditions that resulted in fumonisins production are those found during wheat grain development (especially milk and dough stages) in the field. This is the first study made using irradiated wheat grains and provides useful baseline data on conditions representing a low or a high risk for fumonisins contamination of wheat grains which is of concern because this cereal is destined mainly for human consumption.

Abstract B15: Fumonisins and oesophageal cancer: A proteomic and transcriptomic analyses of molecular mechanisms associated with fumonisin production by Fusarium verticillioides MRC 826 in maize

Abstract Fusarium verticillioides is a plant pathogenic fungus that causes ear rot disease of maize and produces the carcinogenic mycotoxins, the fumonisins that are hazardous to both human and animal health. Fumonisin B1 (FB1) is the most prevalent and generally occurs at the highest levels in contaminated maize intended for human consumption. The association between fumonisin contaminated maize and maize products and human disease has raised food safety and public health concerns worldwide. Fumonisins are associated with oesophageal and liver cancer in humans and has been classified as Group 2B (potentially carcinogenic to humans) by the International Agency of the Research on Cancer. With a worldwide research focus centered on the reduction of mycotoxin exposure to humans and animals, the current study investigated the molecular mechanisms associated with fumonisin biosynthesis of the fungus in maize in order to identify potential targets for prevention. Several subcultures, showing genetic homogeneity, have been shown to produce varying amounts of the fumonisins in maize cultures. These subculture strains are therefore unique and present ideal opportunities to investigate the molecular regulatory basis of fumonisin production. Differences in gene regulation and protein expression in one high and one low fumonisin producing strain were investigated in maize cultures and in plants utilizing a susceptible maize cultivar. Quantitative real-time PCR as well as proteomic analyses were performed on maize cultures after 7, 14, 21, and 28 days, to monitor differences in the expression of the FUM genes (FUM1, FUM6, FUM8 and FUM21) as well as the total proteomic profiles. FUM1, FUM6 and FUM8 respectively showed a 5.2-, 3.1-, and 4.5-fold increase in gene expression levels in the high fumonisin producing strain, which corroborate with the higher fumonisin concentration. Expression of FUM21, one of the regulatory genes, did not differ significantly (P<0.05) between the two strains. Preliminary proteomic analyses detected a total of 729 proteins whereas 117 are unique to the high toxin producing strain and 100 unique to the low producing strain which could provide important information on differences regarding the regulation of fumonisin production. For the in planta study, the maize cobs were infected at the blister stage and harvested 10 weeks after inoculation, where after fumonisin and real-time PCR analyses were conducted Preliminary results indicated a higher virulence as well as an increase in production by the high fumonisin producing strain. This study will provide important information on the elucidation of the molecular factors involved in the regulation of fumonisin biosynthesis in maize which could identify specific targets to control fumonisin production. Citation Format: M Lilly, JF Alberts, JP Rheeder, WCA Gelderblom. Fumonisins and oesophageal cancer: A proteomic and transcriptomic analyses of molecular mechanisms associated with fumonisin production by Fusarium verticillioides MRC 826 in maize [abstract]. In: Proceedings of the AACR International Conference: New Frontiers in Cancer Research; 2017 Jan 18-22; Cape Town, South Africa. Philadelphia (PA): AACR; Cancer Res 2017;77(22 Suppl):Abstract nr B15.

Analysis of the global regulator Lae1 uncovers a connection between Lae1 and the histone acetyltransferase HAT1 in Fusarium fujikuroi.

The fungus Fusarium fujikuroi causes "bakanae" disease of rice due to its ability to produce gibberellins (GAs), a family of plant hormones. Recent genome sequencing revealed the genetic capacity for the biosynthesis of 46 additional secondary metabolites besides the industrially produced GAs. Among them are the pigments bikaverin and fusarubins, as well as mycotoxins, such as fumonisins, fusarin C, beauvericin, and fusaric acid. However, half of the potential secondary metabolite gene clusters are silent. In recent years, it has been shown that the fungal specific velvet complex is involved in global regulation of secondary metabolism in several filamentous fungi. We have previously shown that deletion of the three components of the F. fujikuroi velvet complex, vel1, vel2, and lae1, almost totally abolished biosynthesis of GAs, fumonisins and fusarin C. Here, we present a deeper insight into the genome-wide regulatory impact of Lae1 on secondary metabolism. Over-expression of lae1 resulted in de-repression of GA biosynthetic genes under otherwise repressing high nitrogen conditions demonstrating that the nitrogen repression is overcome. In addition, over-expression of one of five tested histone acetyltransferase genes, HAT1, was capable of returning GA gene expression and GA production to the GA-deficient Δlae1 mutant. Deletion and over-expression of HAT1 in the wild type resulted in downregulation and upregulation of GA gene expression, respectively, indicating that HAT1 together with Lae1 plays an essential role in the regulation of GA biosynthesis.

Carbon Sources Influence Fumonisin Production in Fusarium proliferatum.

Fusarium proliferatum is a worldwide fungal pathogen that produces fumonisins which are harmful to animal and human health. However, environmental factors affecting fumonisin biosynthesis in F. proliferatum are not well understood. Based on our preliminary results, in this study, we investigated the effect of sucrose or mannose as the sole carbon source on fumonisin B (FB) production by F. proliferatum and studied their underlying mechanisms via proteome and gene expression analysis. Our results showed that mannose, used as the sole carbon source, significantly blocked fumonisin B1 and B2 production by F. proliferatum as compared with the use of sucrose. Fifty-seven differentially expressed proteins were successfully identified. The downregulated proteins in the mannose-cultured strain were mainly involved in carbon metabolism, response to stress, and methionine metabolism, as compared with the sucrose-cultured strain. Moreover, quantitative real-time PCR analysis indicated that expression of several key genes involved in FB biosynthetic pathway and in transcription regulation were significantly downregulated in the mannose-cultured F. proliferatum, whereas expression of histone deacetylation-related genes were significantly upregulated. These results suggested that the blockage of FB biosynthesis by mannose was associated with the decreases in conversion of acetyl-CoA to polyketide, methionine biosynthesis, and NADPH regeneration. More importantly, milder oxidative stress, downregulated expression of genes involved in biosynthetic pathway and transcription regulation, and upregulated expression of genes with histone deacetylation possibly were responsible for the blockage of FB biosynthesis in F. proliferatum.

Genetic structure of Fusarium verticillioides populations and occurrence of fumonisins in maize grown in Southern Brazil

The fumonisins are a group of mycotoxins produced primarily by Fusarium spp. There are several different forms of fumonisins, among them fumonisins B1 and B2 are the most common and economically important forms in maize. The aim of this study was to investigate the presence of fumonisins and Fusarium spp. in kernels of four maize genotypes grown in two Southern Brazilian locations. Fumonisins B1 and B2 were detected in all samples, with levels ranging from 0.4 to 9.1 μg × g−1. Of the 3840 maize kernels examined, 77.0% were infected with Fusarium spp., and F. verticillioides was the most prevalent species (98.1%). In addition, we found that approximately 95% of the isolates of F. verticillioides harbor essential genes for fumonisin biosynthesis (FUM1 and FUM8). Next, we investigated the genetic structure of F. verticillioides populations based on molecular data generated by the AFLP technique, which revealed a high genetic variability. Statistical analyses have shown that a significant part of the genetic differentiation was associated with the maize growing location.

Proteomics analysis of Fusarium proliferatum under various initial pH during fumonisin production

Fusarium proliferatum as a fungal pathogen can produce fumonisin which causes a great threat to animal and human health. Proteomic approach was a useful tool for investigation into mycotoxin biosynthesis in fungal pathogens. In this study, we analyzed the fumonisin content and mycelium proteins of Fusarium proliferatum cultivated under the initial pH5 and 10. Fumonisin production after 10days was significantly induced in culture condition at pH10 than pH5. Ninety nine significantly differently accumulated protein spots under the two pH conditions were detected using two dimensional polyacrylamide gel electrophoresis and 89 of these proteins were successfully identified by MALDI-TOF/TOF and LC-ESI-MS/MS analysis. Among these 89 proteins, 45 were up-regulated at pH10 while 44 were up-accumulated at pH5. At pH10, these proteins were found to involve in the modification of fumonisin backbone including up-regulated polyketide synthase, cytochrome P450, S-adenosylmethionine synthase and O-methyltransferase, which might contribute to the induction of fumonisin production. At pH5, these up-regulated proteins such as l-amino-acid oxidase, isocitrate dehydrogenase and citrate lyase might inhibit the condensation of fumonisin backbone, resulting in reduced production of fumonisins. These results may help us to understand the molecular mechanism of the fumonisin synthesis in F. proliferatum. Biological significanceTo extend our understanding of the mechanism of the fumonisin biosynthesis of F. proliferatum, we reported the fumonisin production in relation to the differential proteins of F. proliferatum mycelium under two pH culture conditions. Among these 89 identified spots, 45 were up-accumulated at pH10 while 44 were up-accumulated at pH5. Our results revealed that increased fumonisin production at pH10 might be related to the induction of fumonisin biosynthesis caused by up-regulation of polyketide synthase, cytochrome P450, S-adenosylmethionine synthase and O-methyltransferase. Meanwhile, the up-regulation of l-amino-acid oxidase, isocitrate dehydrogenase and citrate lyase at pH5 might be related to the inhibition of the condensation of fumonisin backbone, resulting in reduced production of fumonisin. These results may help us to understand better the molecular mechanism of the fumonisin synthesis in F. proliferatum and then broaden the current knowledge of the mechanism of the fumonisin biosynthesis.