Fumonisin Synthesis Research Articles

Functional analysis of cutinase transcription factors in Fusarium verticillioides

Fusarium verticillioides is an important pathogen of maize and causes serious yield losses and food safety issues worldwide. F. verticillioides produces highly toxic mycotoxin Fumonisin B1 (FB1) in infested commodities which makes these food and feeds unsafe for humans and animals. For pathogenic fungi to successfully penetrate its plant hosts, the pathogen secretes hydrolytic enzymes that can facilitate penetration into the plant cutin layer. However, there is limited information on how cutinases transcriptionally regulated to impact F. verticillioides pathogenicity. In this study, our aim is to functionally characterize cutinase transcription factors that regulate key cutinase activities that are directly associated with F. verticillioides pathogenicity and FB1 biosynthesis. Gene deletion of cutinase transcription factor FvCTF1α did not affect the growth and morphology of the fungal mycelia on CMII medium, whereas the conidiation, utilization of sodium acetate and sodium oleate, stress tolerance against cell wall interfering agent, and the cutinase and pectinase activities in the ΔFvctf1α mutant were negatively impacted. FvCtf1α regulates the expression of induced cutinase genes FvCUT1 and FvCUT4 by binding to their GC-rich promoters. In addition, FvCtf1α, containing a novel function in regulating FB1, interacts with the promoter of FvFUM1 and FvFUM6 to down-regulate the expression of FvFUM1 and FvFUM6, resulting in decreased production of FB1 in the ΔFvctf1α strain. ΔFvctf1α exhibited decreased pathogenicity in maize due to the down-regulation of pathogenicity-related genes as well as key downstream cutinase genes FvCUT3 and FvCUT4 in F. verticillioides. We also demonstrated that FvCtf1α regulated FvCUT3 and FvCUT4 differently; FvCUT4 via direct regulation while FvCUT3 via indirect regulation by interacting with FvFarB, a homologous protein of FvCtf1α. Moreover, RNA-seq analysis showed that FvCtf1α was associated with many pathways, such as fatty acid metabolism, carbon source utilization, cell wall integrity, oxidative stress, and fumonisin synthesis in F. verticillioides. Our study demonstrated that FvCtf1α was not only involved in the regulation of cutinases but also a broad spectrum of pathways that ultimately affect F. verticillioides virulence and mycotoxin biosynthesis.

Sphinganine-Analog Mycotoxins (SAMs): Chemical Structures, Bioactivities, and Genetic Controls.

Sphinganine-analog mycotoxins (SAMs) including fumonisins and A. alternata f. sp. Lycopersici (AAL) toxins are a group of related mycotoxins produced by plant pathogenic fungi in the Fusarium genus and in Alternaria alternata f. sp. Lycopersici, respectively. SAMs have shown diverse cytotoxicity and phytotoxicity, causing adverse impacts on plants, animals, and humans, and are a destructive force to crop production worldwide. This review summarizes the structural diversity of SAMs and encapsulates the relationships between their structures and biological activities. The toxicity of SAMs on plants and animals is mainly attributed to their inhibitory activity against the ceramide biosynthesis enzyme, influencing the sphingolipid metabolism and causing programmed cell death. We also reviewed the detoxification methods against SAMs and how plants develop resistance to SAMs. Genetic and evolutionary analyses revealed that the FUM (fumonisins biosynthetic) gene cluster was responsible for fumonisin biosynthesis in Fusarium spp. Sequence comparisons among species within the genus Fusarium suggested that mutations and multiple horizontal gene transfers involving the FUM gene cluster were responsible for the interspecific difference in fumonisin synthesis. We finish by describing methods for monitoring and quantifying SAMs in food and agricultural products.

The in vitro Production Potentialities of Secondary Toxic Metabolites by the Fungal Factory Fusarium verticillioides Is, Fortunately, Largely Underestimated in Fields: Pioneering Study on Fumonisins.

This study presents fungi infrequently viewed as fungal factories for secondary metabolite production resources such as mycotoxins in Ascomycota. Additionally, we demonstrated that biochemical warfare of Fusarium verticillioides factory against animal cells is not only due to mycotoxins such as fumonisins, but acute cytotoxic firing is based on different excreted secondary metabolite series, potentially leading to animal and human diseases. In this study, fumonisins, which can be followed by in situ localization, quantification, or expression of the key gene implicated in their synthesis, are used to understand secondary metabolite production by this fungus. It is known that F. verticillioides produces mycotoxins such as fumonisins on cereals, but until now, there is no evidence demonstrating a method to totally block fumonisin production on feed and food. In this paper, we explained, what was never clearly established before, that fumonisin production depends on two bottlenecks. The fumonisin synthesis and secretion in fungal articles of the mycelium are medium-independent and follow the fungal cell cycle developmental program (ontogenesis). Conversely, the fumonisin excretion into the medium depends on its composition, which also impacts fumonisin biosynthesis level. Using a high-pressure freezing method, we showed that, in non-permissive fumonisin excretion (NPFE) medium, FB1 is sequestered inside extra-vesicles and in the first third of the cell wall next to the plasmalemma, leading to the hypothesis that the fungus develops mechanisms to protect its cytosolic homeostasis against this cytotoxic. In permissive fumonisin excretion (PFE) medium, leading to very high quantities of excreted fumonisins, FB1 localized inside extra-vesicles, crosses the entire cell wall thickness, and then releases into the medium. Our results demonstrated a delayed and lower expression of Fvpks gene in mycelium developed on NPFE medium as compared to PFE medium. Conversely, higher amounts of fumonisins were accumulated in NPFE-grown mycelium than in PFE-grown mycelium. Thus, our results demonstrated for the first time that we have to take into account that the synthesis and secretion inside the article of secondary metabolites depend on the occurrence of cryptic biochemical specialized articles, differentiated in the mycelium. However, those are not morphologically different from other colonial hyphae.

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).

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.

Grain colonization by fumonisin-producing Fusarium spp. and fumonisin synthesis in South African commercial maize in relation to prevailing weather conditions

Fumonisins are mycotoxins produced primarily by Fusarium verticillioides and F. proliferatum on maize. These mycotoxins are secondary, carcinogenic metabolites with a worldwide distribution. This study was performed to evaluate the relationship between weather variables, the colonization of grain by F. verticillioides and resultant fumonisin contamination. Grain colonization by Fusarium spp. was determined using quantitative real-time PCR (qPCR) and contamination with fumonisins using HPLC analysis. Results indicated high natural infection by fumonisin-producing Fusarium spp. and fumonisin concentrations in warmer production areas such as Northern Cape, North West and Free State Provinces. Site-specific weather data, including temperature, radiation, humidity, rainfall and evapo-transpiration were provided by the ARC-Institute for Soil Water and Climate's meteorology office. Stepwise multiple regression analysis selected the variables mean maximum temperature and mean minimum humidity (days 1–14 post-silking) as having significant relationships with colonization of maize kernels by fumonisin-producing Fusarium spp.. Further relationships were calculated using the non-linear, 3-dimensional Lorentzian equation (Sigmaplot 10.0). The optimum temperature for colonization of maize by Fusarium spp. were 28.97, 32.14 and 30.40 °C and the optimum minimum humidities were calculated at 27.29, 31.86 and 29.74% respectively over the three recorded seasons. The application of correlation analysis and stepdown multiple regression analysis on sequential means of weather data and the inclusion of observed fungal biomass as a variable, suggested two phases in the development of fumonisins in maize kernels i.e. colonization of maize tissues during the early post-silking stage, followed by fumonisin production during the dough stage of grain fill. Based on this statistical model it would appear that fumonisin production by a specific biomass of fumonisin producing Fusarium spp. on grain was influenced largely by temperature and less so by rainfall. Optimum fumonisin concentrations were obtained with fungal biomasses of 39.94 pg during 2007, 47.65 pg during 2008 and 90.69 pg during 2009. Optimum temperatures for fumonisin production were 30.33, 31.12 and 29.80 °C for 2007–2009, respectively.Although these statistical models were not consistent over seasons with regard to actual prediction values, they were consistent regarding time of fungal infection and fumonisin production. It is evident that additional driving variables need to be identified.

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.

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.

The effects of cultivar and prophylactic fungicide spray for leaf diseases on colonisation of maize ears by fumonisin producing Fusarium spp. and fumonisin synthesis in South Africa

Fumonisins are mycotoxins produced primarily by Fusarium verticillioides and Fusarium proliferatum on maize. These are secondary, carcinogenic metabolites and have been reported on maize worldwide. Field trials were conducted during 2010/2011 and 2011/2012 in six diverse maize production areas of South Africa to study the efficacy of an existing prophylactic fungicide regime for the control of foliar diseases, on the infection of grains of seven cultivars by fumonisin producing Fusarium spp. and fumonisin synthesis. Seven cultivars were selected to include both yellow and white, Bt and non-Bt and regionally adapted varieties. Azoxystrobin + difenoconazole (strobilurin, 200 g/L + triazole, 125 g/L) was applied 40–45 days after planting followed by flusilazole + carbendazim (silicone triazole, 125 g/L + benzimidazole, 250 g/L) with petroleum as adjuvant, 28–30 days later. Fumonisins in harvested kernels were analysed using High Performance Liquid Chromatography (HPLC) and fumonisin producing Fusarium spp. were quantified by means of quantitative Real Time PCR (qPCR). Mean natural colonisation of maize kernels by fumonisin producing Fusarium spp. was highest at Makhathini (33,696 pg/0.5 g of milled maize kernels) and the lowest at Potchefstroom (179 pg/0.5 g of milled maize kernels). Cultivars differed in susceptibility to fungal colonisation and fumonisin synthesis with PAN6P-110, DKC80-10 and CRN3505 proving most susceptible and LS8521B and DKC78-15B most resistant. Mean fumonisin contamination was highest at Makhathini (23.62 ppm) and lowest at Buffelsvlei (1.50 ppm). Analysis of variance showed no significant differences in colonisation of grain by fumonisin producing Fusarium spp. between sprayed and control treatments. Sprayed treatments had significantly higher fumonisin levels compared with unsprayed treatments. A highly significant cultivar × environment interaction was recorded for fungal colonisation. Highly significant environment×fungicide-treatment×cultivar interactions were recorded for fumonisin production. The strong interaction between cultivar and environment may be due to cultivar adaptation/behaviour under different environmental conditions. The use of a prophylactic fungicide spray regime for control of leaf diseases in maize did not reduce Fusarium ear rot in maize, however, significantly elevated fumonisin levels were recorded.

LDS1-produced oxylipins are negative regulators of growth, conidiation and fumonisin synthesis in the fungal maize pathogen Fusarium verticillioides

Oxylipins are fatty acid-derived signaling compounds produced by all eukaryotes so far investigated; in mycotoxigenic fungi, they modulate toxin production and interactions with the host plants. Among the many enzymes responsible for oxylipin generation, Linoleate Diol Synthase 1 (LDS1) produces mainly 8-hydroperoxyoctadecenoic acid and subsequently different di-hydroxyoctadecenoic acids. In this study, we inactivated a copy of the putative LDS1 ortholog (acc. N. FVEG_09294.3) of Fusarium verticillioides, with the aim to investigate its influence on the oxylipin profile of the fungus, on its development, secondary metabolism and virulence. LC-MS/MS oxylipin profiling carried out on the selected mutant strain revealed significant quali-quantitative differences for several oxylipins when compared to the WT strain. The Fvlds1-deleted mutant grew better, produced more conidia, synthesized more fumonisins and infected maize cobs faster than the WT strain. We hypothesize that oxylipins may act as regulators of gene expression in the toxigenic plant pathogen F. verticillioides, in turn causing notable changes in its phenotype. These changes could relate to the ability of oxylipins to re-shape the transcriptional profile of F. verticillioides by inducing chromatin modifications and exerting a direct control on the transcription of secondary metabolism in fungi.

Fusarium verticillioides and maize interaction in vitro: relationship between oxylipin cross-talk and fumonisin synthesis

Fusarium verticillioides is one of the most important fungal pathogens causing ear and stalk rot in maize. Even if frequently asymptomatic, it can produce a harmful series of compounds named fumonisins. Plant and fungal oxylipins play a crucial role in determining the outcome of the interaction between the pathogen and its host. Moreover, oxylipins are factors able to modulate the secondary metabolism in fungi. To uncover the existence of the relationship between oxylipin production and fumonisin synthesis in F. verticillioides, we analysed some molecular and physiological parameters, such as the expression of genes whose products are related to oxylipin synthesis (i.e. lipoxygenase, diol synthases and fatty acid oxidase), the oxylipin profile of both cracked maize and the pathogen by using a lipidomic approach (i.e. combining LC-TOF and LC-MS/MS approaches with a robust statistical analysis) and the synthesis of fumonisin B1. The results suggested a close relationship between the modification of the pathogen oxylipin profile with the fumonisin synthesis. Notably, a modification of the oxylipin profile of the pathogen during its growth on cracked maize can be demonstrated. The switch in oxylipin synthesis could indicate that the ‘presence’ of maize determinants (e.g. plant cell wall fragments and/or lipids) was able to promote the modification of the pathogen lifestyle, also by adapting the secondary metabolism, notably fumonisin synthesis.

<i>Fusarium verticillioides</i> and maize interaction <i>in vitro</i>: relationship between oxylipin cross-talk and fumonisin synthesis

Fusarium verticillioides is one of the most important fungal pathogens causing ear and stalk rot in maize. Even if frequently asymptomatic, it can produce a harmful series of compounds named fumonisins. Plant and fungal oxylipins play a crucial role in determining the outcome of the interaction between the pathogen and its host. Moreover, oxylipins are factors able to modulate the secondary metabolism in fungi. To uncover the existence of the relationship between oxylipin production and fumonisin synthesis in F. verticillioides, we analysed some molecular and physiological parameters, such as the expression of genes whose products are related to oxylipin synthesis (i.e. lipoxygenase, diol synthases and fatty acid oxidase), the oxylipin profile of both cracked maize and the pathogen by using a lipidomic approach (i.e. combining LC-TOF and LC-MS/MS approaches with a robust statistical analysis) and the synthesis of fumonisin B1. The results suggested a close relationship between the modification of the pathogen oxylipin profile with the fumonisin synthesis. Notably, a modification of the oxylipin profile of the pathogen during its growth on cracked maize can be demonstrated. The switch in oxylipin synthesis could indicate that the ‘presence’ of maize determinants (e.g. plant cell wall fragments and/or lipids) was able to promote the modification of the pathogen lifestyle, also by adapting the secondary metabolism, notably fumonisin synthesis.

Effects of temperature and water activity on FUM2 and FUM21 gene expression and fumonisin B production in Fusarium verticillioides

The fungus Fusarium verticillioides is a pathogen of maize and can produce the mycotoxins fumonisins. The present work examined the effect of abiotic factors on the expression of two fumonisin biosynthetic genes (FUM2 and FUM21) and fumonisin production in the fungus. Specifically, the effects of water activity (0.901-0.990), temperature (20-30 °C) and incubation time (7–21 days) were analysed in cultures of F. verticillioides strains ITEM 10027 and ITEM 1744. Fumonisin B was measured through liquid chromatography tandem mass spectrometry and gene expression was quantified with relative real-time PCR, using the Livak method. Fumonisin production increased with incubation time up to 21 days and the transcription of both genes was highest at 14 days; however intraspecific variability was observed. FUM2 and FUM21 expression was positively correlated with fumonisin synthesis at different water activity and temperature regimes. A higher transcription of FUM2 in comparison with FUM21 was observed for all the conditions considered. Incubation time played a significant role in fumonisin production and gene expression, with the highest contamination and gene expression after 21 and 14 days of incubation, respectively. Temperature significantly affected only FUM21 expression, with the optimum at 25 °C; while water activity did not have a significant effect.

ChemInform Abstract: Fumonisin, a Template for Methodology Development and Drug Discovery

AbstractReview: 60 refs.

Synthesis of Fumonisin B1

Fumonisin B1 is a mycotoxin isolated from the Fusarium verticilliodes. It is fatal to horses and pigs (”moldy corn poisoning") and it is associated with oesophagal cancer and birth defects in humans. Hydrolysis of the two tricarballylic esters in vivo followed by N-palmitoylation gives a compound which is more cytotoxic to HT29 ­human colon cancer cells than fumonisin B1.

A dynamic risk assessment model (FUMAgrain) of fumonisin synthesis by Fusarium verticillioides in maize grain in Italy

Fumonisin contamination of maize grain starts in the field. Forecasting Fusarium infection and fumonisin synthesis could allow operators in the field to control contamination during the growing season and to make the best agronomic decisions for high quality yields while respecting the limits imposed by the European Union. A research project to develop a decision support system for the control of field-phase fumonisin contamination began in Italy in 2003. This paper presents a preliminary version of the engine of the decision support tool: FUMAgrain, a dynamic risk assessment model developed with data from the north of Italy. The structure of FUMAgrain is based on the pathosystem formed by maize, F. verticillioides and Ostrinia nubilalis (European Corn Borer). The elements of the pathosystem are simulated by three sub-models: (i) maize development, (ii) F. verticillioides infection and fumonisin synthesis, (iii) European Corn Borer wounding activity on maize grain. Inputs to the model are (i) planting date, (ii) hourly meteorological data including temperature, relative humidity, wind speed and rain intensity, (iii) information on the phenological development of the hybrid planted (flowering and dry-down), and (iv) information about the chemical treatment against European Corn Borer. FUMAgrain gives an initial risk alert at the end of flowering based on the meteorological conditions during this phase. A second alert follows maturation when an assessment is made from (i) maize grain moisture, (ii) European Corn Borer damage to the ear, and (iii) fumonisin synthesis risk. Following calibration and validation with data FUMAgrain demonstrated its good capability to simulate fumonisin synthesis in maize grain in Italy (calibration: R 2 = 0.70; validation: R 2 = 0.71) and its usefulness for determining the optimal harvest date while respecting grain safety levels required by the international market and limiting moisture content, hence drying costs.

Structure, Synthesis, and Biosynthesis of Fumonisin B 1 and Related Compounds

Structure, Synthesis, and Biosynthesis of Fumonisin B 1 and Related Compounds

Enantioselective Total Synthesis of Fumonisin B2

Enantioselective Total Synthesis of Fumonisin B₂