Effects of Bunium persicum essential oil on the reduction of spore germination, growth, and expression of FUM1 and FUM14 genes in Fusarium verticillioides isolates.

BACKGROUND AND PURPOSE: Black Cumin of Kerman (Bunium persicum) is an Iranian plant that is commonly used as an antispasmodic, carminative, and antimicrobial substance. The present study aimed to assess different components of the essence of B. persicum and its effect on antifungal activity, spore germination inhibition, and expressions of FUM1 and FUM14 genes in Fusarium verticillioides strains. MATERIALS AND METHODS: The essence was extracted by hydrodistillation and analyzed through gas chromatography-mass spectroscopy. A broth microdilution method was used for the determination of the minimum inhibitory concentration (MIC). In addition, the expression of FUM1 and FUM14 genes of toxigenic F. verticillioides was assessed by using the real-time polymerase chain reaction (RT-PCR) technique. RESULTS: Based on the findings, most of the essence consisted of γ-terpinene (15.56%), propanal, and 2-methyl-3-phenyl (14.18%). The oil showed a good antifungal activity (mean MIC value: 2556.8 µg/ml) as well as the inhibition of spore germination and mycelial growth (P<0.05). The RT-PCR demonstrated that the expression levels of FUM1 and FUM14 of B. persicum-treated F. verticillioides were 0.43 and 0.53 folds lower than the control samples, respectively. CONCLUSION: These findings revealed that the essential oil of B. persicum has different components responsible for the inhibition of mycelial growth and spore germination of F. verticillioides as well as reduction of expressions of FUM1 and FUM14 genes involving fumonisin production.

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.

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.

Evaluating Fumonisin Gene Expression in Fusarium verticillioides.

Transcript levels of key genes in a biosynthetic pathway are often taken as a proxy for metabolite production. This is the case of FUM1, encoding the first dedicated enzyme in the metabolic pathway leading to the production of the mycotoxins Fumonisins by fungal species belonging to the genus Fusarium. FUM1 expression can be quantified by different methods; here, we detail a protocol based on quantitative reverse transcriptase polymerase chain reaction (RT-qPCR), by which relative or absolute transcript abundance can be estimated in Fusaria grown in vitro or in planta. As very seldom commercial kits for RNA extraction and cDNA synthesis are optimized for fungal samples, we developed a protocol tailored for these organisms, which stands alone but can be also easily integrated with specific reagents and kits commercially available.

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

OBJECTIVES: 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. MATERIALS AND METHODS: 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. RESULTS: 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. CONCLUSIONS: 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.

Host extract modulates metabolism and fumonisin biosynthesis by the plant-pathogenic fungus Fusarium proliferatum.

Fusarium proliferatum is a common pathogen able to infect a broad range of agriculturally important crops. Recently, some evidence for genetic variance among the species genotypes in relation to their plant origin has been reported. Mycotoxin contamination of plant tissues is the most important threat caused by F. proliferatum and fumonisins B (FBs) are the principal mycotoxins synthesized. The toxigenic potential of the pathogen genotypes is variable and also the reaction of different host plant species on the infection by pathogen is different. The objective of present study was to evaluate the impact of the extracts on the growth and fumonisin biosynthesis by 32 F. proliferatum strains originating from different host species (A-asparagus, M-maize, G-garlic, PS-pea and P-pineapple), and how it changes the secondary metabolism measured by fumonisin biosynthesis. The average strain dry weight was 65.2 mg for control conditions and it reached 180.7 mg, 100.5 mg, 76.6 mg, 126.2 mg and 51.1 mg when pineapple, asparagus, maize, garlic and pea extracts were added, respectively. In the second experiment the extracts were added after 5 days of culturing of the representative group of strains, displaying diverse reaction to the extract presence. Also, the influence of stationary vs. shaken culture was examined. Mean biomass amounts for shaken cultures of 15 chosen strains were as follows: 37.4 mg of dry weight for control culture (C), 219.6 mg (P), 113 mg (A), 93.6 mg (M), 62 mg (G) and 48 mg (PS), respectively. For stationary cultures, the means were as follows: C-57.4 mg, P-355.6 mg, A-291.6 mg, M-191.1 mg, G-171.1 mg and PS-58.9 mg. Few strains showed differential growth when stationary/shaken culture conditions were applied. Almost all strains synthesized moderate amounts of fumonisins in control conditions-less than 10 ng/µL, regardless of the origin and host species. Few strains were able to produce over 100 ng/µL of FBs when pineapple extract was added, twelve strains synthesized more than 10 ng/µL under asparagus extract induction and the pea extract was the most efficient inhibitor of fumonisin biosynthesis. The general impact of the extracts on the fungal biomass amounts was similar, regardless of the host plant origin of the fungal genotypes studied. The evaluation of FBs content has shown differential reaction of some strains, which may contribute to their aggressiveness and pathogenicity.

Combined effects of benomyl and environmental factors on growth and expression of the fumonisin biosynthetic genes FUM1 and FUM19 by Fusarium verticillioides.

Fusarium verticillioides is predominantly responsible of fumonisin contamination of maize and other cereals in Mediterranean climatic regions. This study examined the interaction of the fungicide benomyl, at ED50 and ED90 concentrations (effective doses of benomyl to reduce growth by 50% and 90%, respectively), with a range of temperatures (20-35 °C) and water potentials (-0.7, -2.8 and -7.0 MPa) compatible with current and foreseen climate change scenarios for these regions on growth and fumonisin biosynthesis in in vitro assays. The expression of fumonisin biosynthetic genes (FUM1 and FUM19) was quantified by real time RT-PCR. FUM1 encodes a polyketide synthase and FUM19 an ABC-type transporter, located both in the fumonisin biosynthetic cluster. The ED50 and ED90 concentrations obtained at 25 °C were 0.93 mg/L and 3.30 mg/L, respectively. Benomyl affected growth and fumonisin gene expression differently but it generally reduced fungal growth and fumonisin biosynthesis and both were significantly affected by temperature and water potential. This indicated that efficacy of benomyl might be compromised at certain conditions, although at similar or lower levels than other fungicides tested. Both fumonisin biosynthetic genes had similar expression patterns in all treatments and their correlation was positive and significant. The results suggested that Mediterranean climatic scenarios might suffer an additional negative impact of climate change by reducing the efficacy of antifungals used to control pathogens and toxigenic fungi.

Transcriptional changes in developing maize kernels in response to fumonisin-producing and nonproducing strains of Fusarium verticillioides.

Fusarium verticillioides infects maize producing ear rot, yield loss and the accumulation of fumonisins. In the present study, a transcriptomic approach was employed to investigate the molecular aspects of the interaction of susceptible/resistant maize genotypes with fumonisin-producing/nonproducing strains of F. verticillioides over a time course of 4 days after inoculation. The fumonisin-nonproducing strain led transcription in susceptible maize kernels, starting from 48h post inoculation, with a peak of differentially expressed genes at 72h after inoculation. Pathogen attack altered the mRNA levels of approximately 1.0% of the total number of maize genes assayed, with 15% encoding proteins having potential functions in signal transduction mechanisms, and 9% in the category of transcription factors. These findings indicate that signalling and regulation pathways were prominent in the earlier phases of kernel colonization, inducing the following expression of defense genes. In the resistant maize genotype, the fum1 mutant of F. verticillioides, impaired in this polyketide synthase gene (PKS), provoked a delayed and weakened activation of defense and oxidative stress-related genes, compared to the wild-type strain. The inability to infect resistant kernels may be related to the lack of PKS activity and its association with the lipoxygenase pathway. Plant and fungal 9-lipoxygenases had greater expression after fum1 mutant inoculation, suggesting that PKS plays an indirect effect on pathogen colonization by interfering with the lipid mediated cross-talk between host and pathogen.

Potential effects of environmental conditions on the efficiency of the antifungal tebuconazole controlling Fusarium verticillioides and Fusarium proliferatum growth rate and fumonisin biosynthesis.

Fusarium verticillioides and Fusarium proliferatum are important phytopathogens which contaminate cereals in the Mediterranean climatic region with fumonisins. In this study we examined the interaction between the fungicide efficacy of tebuconazole and water potential (Ψw) (-0.7-7.0MPa)×temperature (20-35°C) on growth and FUM1 gene expression by real time RT-PCR (an indicator of fumonisin biosynthesis) in strains of both Fusarium species. Concentrations of tebuconazole required to reduce growth by 50 and 90% (ED50 and ED90 values) were determined. Growth of strains of both species was largely reduced by tebuconazole, with similar efficacy profiles in the interacting water potential×temperature conditions. In contrast, FUM1 expression was not generally reduced by tebuconazole. Moreover, sub-lethal doses in combination with mild water stress and temperatures less than 35°C significantly induced FUM1 expression with slight differences in both species. These results suggest that the efficacy of antifungal compounds to reduce mycotoxin risk would be more effective if consideration is given to both growth rate and toxin biosynthesis in relation to interacting environmental conditions. This is the first study linking fungicide efficacy of tebuconazole with environmental factor effects on control of growth and FUM1 gene expression of F. verticillioides and F. proliferatum.

Evolving dual targeting of a prokaryotic protein in yeast.

Dual targeting is an important and abundant phenomenon. Indeed, we estimate that more than a third of the yeast mitochondrial proteome is dual localized. The enzyme fumarase is a highly conserved protein in all organisms with respect to its sequence, structure, and enzymatic activity. In eukaryotes, it is dual localized to the cytosol and mitochondria. In Saccharomyces cerevisiae, the dual localization of fumarase is achieved by the reverse translocation mechanism; all fumarase molecules harbor a mitochondrial targeting sequence (MTS), are targeted to mitochondria, begin their translocation, and are processed by mitochondrial processing peptidase in the matrix. A subset of these processed fumarase molecules in transit is then fully imported into the matrix, whereas the majority moves back into the cytosol by reverse translocation. The proposed driving force for fumarase distribution is protein folding during import. Here, we asked how reverse translocation could have evolved on a prokaryotic protein that had already acquired expression from the nuclear genome and a targeting sequence. To address this question, we used, as a model, the Escherichia coli FumC Class II fumarase, which is homologous to eukaryotic fumarases (∼58% identity and ∼74% similarity to the yeast Fum1). Starting with an exclusively mitochondrial targeted FumC (attached to a strong MTS), we show that two randomly acquired mutations within the prokaryotic FumC sequence are sufficient to cause substantial dual targeting by reverse translocation. In fact, the unmutated MTS-FumC also has some ability to be dual targeted but only at low temperatures. Our results suggest that in this case, evolution of dual targeting by reverse translocation is based on naturally occurring and fortuitously conserved features of fumarase folding.

Phylogenetic analysis, fumonisin production and pathogenicity of Fusarium fujikuroi strains isolated from rice in the Philippines.

BACKGROUND: Fusarium fujikuroi Nirenberg is a maize and rice pathogen causing important agricultural losses and produces fumonisins - mycotoxins which pose health risk to humans and farm animals. However, little information is available about the phylogenetics of this species and its ability to produce fumonisins in rice. We studied 32 strains isolated from rice in the Philippines and performed a phylogenetic analysis using the partial sequence of Elongation Factor 1 alpha (EF-1α) including isolates belonging to closely related species. Fumonisin B1 (FB1 ) production was analyzed in 7-day-old cultures grown in fumonisin-inducing medium by an enzyme-linked immunosorbent assay-based method and by real-time reverse transcriptase-polymerase chain reaction using primers for FUM1 gene, a key gene in fumonisin biosynthesis. RESULTS: Nucleotide diversities per site (π) were 0.00024 ± 0.00022 (standard deviation) for the 32 F. fujikuroi strains from the Philippines and 0.00189 ± 0.00143 for all 34 F. fujikuroi strains, respectively. F. fujikuroi isolates grouped into one cluster separated from the rest of isolates belonging to the closely related F. proliferatum and showed very low variability, irrespective of their geographic origin. The cluster containing strains of F. proliferatum showed higher intraspecific variability than F. fujikuroi. Thirteen of the 32 strains analyzed were FB1 producers (40.62%), with production ranging from 0.386 to 223.83 ppm. All isolates analyzed showed FUM1 gene expression above 1 and higher than the CT value of the non-template control sample. Both seedling stunting and elongation were induced by the isolates in comparison with the control. CONCLUSION: F. fujikuroi are distinct from F. proliferatum isolates based on phytogenetic analysis and are potential fumonisin producers because all are positive for FUM1 gene expression. No relationship between fumonisin production and pathogenicity could be observed.

Natural Occurrence of Toxigenic Fusarium proliferatum on Paddy (Oryza sativa L.) in Karnataka, India.

Contamination of paddy seeds (rice with husk) by Fusarium species can cause spoilage and subsequent production of mycotoxins, especially fumonisins that affect human and animal health. A mycological study was conducted to evaluate the natural occurrence of fumonisin B1 produced by Fusarium proliferatum on paddy grown in different geographic regions of Karnataka (India). A total of 65 isolates of F. proliferatum from paddy samples were analysed by polymerase chain reaction (PCR). One set of primers, Fp3-F and Fp4-R was employed to identify the species F. proliferatum, and another set of primers, FUM1 was employed to determine the fumonisin producing ability of the isolates. All 65 isolates of F. proliferatum scored positive with both set of primers, producing amplified products of the expected sizes. Furthermore, thin layer chromatography (TLC) analysis detected fumonisin B1 (FB1) in all of the PCR positive isolates of F. proliferatum.