Amylopectin induces fumonisin B1 production by Fusarium verticillioides during colonization of maize kernels

Fusarium verticillioides, a fungal pathogen of maize, produces fumonisin mycotoxins that adversely affect human and animal health. Basic questions remain unanswered regarding the interactions between the host plant and the fungus that lead to the accumulation of fumonisins in maize kernels. In this study, we evaluated the role of kernel endosperm composition in regulating fumonisin B1 (FB1) biosynthesis. We found that kernels lacking starch due to physiological immaturity did not accumulate FB1. Quantitative polymerase chain reaction analysis indicated that kernel development also affected the expression of fungal genes involved in FB1 biosynthesis, starch metabolism, and nitrogen regulation. A mutant strain of F. verticillioides with a disrupted a-amylase gene was impaired in its ability to produce FB1 on starchy kernels, and both the wild-type and mutant strains produced significantly less FB1 on a high-amylose kernel mutant of maize. When grown on a defined medium with amylose as the sole carbon source, the wild-type strain produced only trace amounts of FB1, but it produced large amounts of FB1 when grown on amylopectin or dextrin, a product of amylopectin hydrolysis. We conclude that amylopectin induces FB1 production in F. verticillioides. This study provides new insight regarding the interaction between the fungus and maize kernel during pathogenesis and highlights important areas that need further study.     

Influence of kernel age on fumonisin B1 production in maize by Fusarium moniliforme.

Production of fumonisins by Fusarium moniliforme on naturally infected maize ears is an important food safety concern due to the toxic nature of this class of mycotoxins. Assessing the potential risk of fumonisin production in developing maize ears prior to harvest requires an understanding of the regulation of toxin biosynthesis during kernel maturation. We investigated the developmental-stage-dependent relationship between maize kernels and fumonisin B1 production by using kernels collected at the blister (R2), milk (R3), dough (R4), and dent (R5) stages following inoculation in culture at their respective field moisture contents with F. moniliforme. Highly significant differences (P 相似文献   

Characterization of Four Clustered and Coregulated Genes Associated with Fumonisin Biosynthesis in Fusarium verticillioides

Fumonisins are mycotoxins that cause several fatal animal diseases, including cancer in rats and mice. These toxins are produced by several Fusarium species, including the maize pathogen Fusarium verticillioides, and can accumulate in maize infected with the fungus. We have identified four F. verticillioides genes (FUM6, FUM7, FUM8, and FUM9) adjacent to FUM5, a previously identified polyketide synthase gene that is required for fumonisin biosynthesis. Gene disruption analysis revealed that FUM6 and FUM8 are required for fumonisin production and Northern blot analysis revealed that expression of all four recently identified genes is correlated with fumonisin production. Nucleotide sequence analysis indicated that the predicted FUM6 translation product is most similar to cytochrome P450 monooxygenase-P450 reductase fusion proteins and the predicted products of FUM7, FUM8, and FUM9 are most similar to type III alcohol dehydrogenases, class-II alpha-aminotransferases, and dioxygenases, respectively. Together, these data are consistent with FUM5 through FUM9 being part of a fumonisin biosynthetic gene cluster in F. verticillioides.     

Transformation-mediated complementation of a FUM gene cluster deletion in Fusarium verticillioides restores both fumonisin production and pathogenicity on maize seedlings

The filamentous ascomycete Fusarium verticillioides is a pathogen of maize and produces the fumonisin mycotoxins. However, a distinct population of F. verticillioides is pathogenic on banana and does not produce fumonisins. Fumonisin-producing strains from maize cause leaf lesions, developmental abnormalities, stunting, and sometimes death of maize seedlings, whereas fumonisin-nonproducing banana strains do not. A Southern analysis of banana strains did not detect genes in the fumonisin biosynthetic gene (FUM) cluster but did detect genes flanking the cluster. Nucleotide sequence analysis of the genomic region carrying the flanking genes revealed that the FUM cluster was absent in banana strains except for portions of FUM21 and FUM19, which are the terminal genes at each end of the cluster. Polymerase chain reaction analysis confirmed the absence of the cluster in all banana strains examined. Cotransformation of a banana strain with two overlapping cosmids, which together contain the entire FUM cluster, yielded fumonisin-producing transformants that were pathogenic on maize seedlings. Conversely, maize strains that possess the FUM cluster but do not produce fumonisins because of mutations in FUM1, a polyketide synthase gene, were not pathogenic on maize seedlings. Together, the data indicate that fumonisin production may have been lost by deletion of the FUM cluster in the banana population of F. verticillioides but that fumonisin production could be restored by molecular genetic complementation. The results also indicate that fumonisin production by F. verticillioides is required for development of foliar disease symptoms on maize seedlings.     

Expression profile analysis of wild-type and fcc1 mutant strains of Fusarium verticillioides during fumonisin biosynthesis

Fusarium verticillioides produces a group of mycotoxins known as fumonisins that are associated with a variety of mycotoxicoses in humans and animals. In this study, DNA microarrays were constructed with expressed sequence tags (ESTs) from F. verticillioides. To identify genes with patterns of expression similar to the fumonisin biosynthetic (FUM) genes, the microarray was probed with labeled cDNAs originating from a wild-type strain and a fcc1 mutant grown on maize and in a defined medium adjusted to either pH 3 or pH 8. The comparative analyses revealed differential expression of genes corresponding to 116 ESTs when the fungal strains were grown on maize. Under different pH conditions, 166 ESTs were differentially expressed, and 19 ESTs were identified that displayed expression patterns similar to the FUM ESTs. These results provide candidate genes with potential roles in fumonisin biosynthesis.     

The FvMK1 mitogen-activated protein kinase gene regulates conidiation, pathogenesis, and fumonisin production in Fusarium verticillioides

Fusarium verticillioides is one of the most important fungal pathogens to cause destructive diseases of maize worldwide. Fumonisins produced by the fungus are harmful to human and animal health. To date, our understanding of the molecular mechanisms associated with pathogenicity and fumonisin biosynthesis in F. verticillioides is limited. Because MAP kinase pathways have been implicated in regulating diverse processes important for plant infection in phytopathogenic fungi, in this study we identified and functionally characterized the FvMK1 gene in F. verticillioides. FvMK1 is orthologous to FMK1 in F. oxysporum and GPMK1 in F. graminearum. The Fvmk1 deletion mutant was reduced in vegetative growth and production of microconidia. However, it was normal in sexual reproduction and increased in the production of macroconidia. In infection assays with developing corn kernels, the Fvmk1 mutant was non-pathogenic and failed to colonize through wounding sites. It also failed to cause stalk rot symptoms beyond the inoculation sites on corn stalks, indicating that FvMK1 is essential for plant infection. Furthermore, the Fvmk1 mutant was significantly reduced in fumonisin production and expression levels of FUM1 and FUM8, two genes involved in fumonisin biosynthesis. The defects of the Fvmk1 mutant were fully complemented by re-introducing the wild type FvMK1 allele. These results demonstrate that FvMK1 plays critical roles in the regulation of vegetative growth, asexual reproduction, fumonisin biosynthesis, and pathogenicity.     

Production of Fumonisins by Fusarium Verticillioides Strains on Solid and in a Defined Liquid Medium — Effects of L-Methionine and Inoculum

In order to evaluate the toxicological and carcinogenic effects of fumonisins, large amounts of fumonisins need to be purified, which requires optimal conditions for production in culture. Five strains of F. verticillioides were compared for their ability to produce fumonisins in solid and liquid media with and without the addition of methionine, a fumonisin precursor. Inoculations were made either with lyophilized cultures or a concentrated inoculum. Growth in liquid medium, measured by biomass, was directly correlated to total fumonisin production when a lyophilized inoculum was used. Fumonisin production was stimulated by the addition of 0.2% L-methionine to solid media for most strains. Levels ranged from 1500-3900 mg/kg in rice, and 2900-12500 mg/kg in maize cultures inoculated with lyophilized cultures; 200-4800 mg/kg in rice, and 1500-4200 mg/kg in maize cultures inoculated with concentrated inoculum. Strains that produced relatively high levels of fumonisins in solid media did not necessarily do so in liquid medium and vice versa. Total fumonisin levels in liquid medium ranged from 40-590 mg/l inoculated with lyophilized cultures and < 1-110 mg/l inoculated with concentrated inoculum, without adding the precursor. F. verticillioides strains therefore varied in their ability to produce fumonisins, and conditions for production need to be optimized individually for each strain.     

In vitro control of growth and fumonisin production by Fusarium verticillioides and F. proliferatum using antioxidants under different water availability and temperature regimes

AIMS: To examine the effect of butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), trihydroxybutyrophenone and propylparaben (PP) (at concentrations of 1-20 mmol l(-1)) on growth of and fumonisin production by Argentinian strains of Fusarium verticillioides and F. proliferatum. METHODS AND RESULTS: Studies on lag phases prior to growth, relative growth rates and fumonisin concentrations were carried out in vitro in relation to water activity (0.995-0.93 a(w)) and temperature (18 and 25 degrees C) on a maize meal agar. Overall, PP was the antioxidant which was most effective at inhibiting strains of both species. The lag phase prior to growth and growth rates were significantly decreased by PP and BHA at 10 and 20 mmol l(-1), regardless of the temperature or aw level tested. Total fumonisin production was higher at 0.98 a(w) and decreased by about 45-50% at 0.995 and 0.95 a(w). Overall, BHT only inhibited fumonisin production at 0.95 aw at 10 and 20 mmol l(-1), while BHA was effective at most a(w) levels tested at 10 and 20 mmol l(-1). Propylparaben completely inhibited fumonisin production by both F. verticillioides and F. proliferatum at > 1 mmol l(-1), regardless of the temperature or a(w) level. Small interstrain differences in the levels of inhibition by the antioxidants were observed for three F. verticillioides and four F. proliferatum strains at 0.995, 0.98 and 0.95 a(w). Propylparaben and BHA completely inhibited the growth of both species at the concentrations evaluated, regardless of the a(w) level. CONCLUSIONS: Two antioxidants show promise for the control of growth of and fumonisin production by these species over a wide range of environmental conditions. SIGNIFICANCE AND IMPACT OF THE STUDY: Potential exists for using such food-grade preservatives for prevention of mycotoxigenic fungi and their toxins entering the food chain.     

In vitro influence of bacterial mixtures on Fusarium verticillioides growth and fumonisin B1 production: effect of seeds treatment on maize root colonization

AIMS: Enterobacter cloacae, Microbacterium oleovorans, Pseudomonas solanacearum and Bacillus subtilis were investigated in order to evaluate: (i) the inoculum size of two bacterial mixtures on Fusarium verticillioides growth and fumonisin B1 production in vitro at different water activities and (ii) the efficacy of a seed treatment with the best bacterial mixture on F. verticillioides root colonization in greenhouse studies. METHODS AND RESULTS: The influence of bacterial mixtures (1 = E. cloacae and M. oleovorans and 2 = P. solanacearum and B. subtilis) to antagonize 13 F. verticillioides strains at different inoculum concentrations (10(8), 10(9) and 10(10) cells ml(-1)) and water activities (0.937, 0.955 and 0.982 aW) were examined. Antibiosis, growth rate and fumonisin B1 production were determined. Bacterial mixture 1 proved to exert the most effective control. Seed treatment with mixture 1 at 10(8) cells ml(-1) had the best inhibitory effect on F. verticillioides root colonization. CONCLUSIONS: These results suggest that the combination E. cloacae and M. oleovorans has the potential for the biological control of F. verticillioides as a maize seed inoculant. SIGNIFICANCE AND IMPACT OF THE STUDY: The application of this knowledge contributes to prevent the vertical transmission of F. verticillioides.     

Incidence of Fusarium spp. and Levels of Fumonisin B1 in Maize in Western Kenya

Maize kernel samples were collected in 1996 from smallholder farm storages in the districts of Bomet, Bungoma, Kakamega, Kericho, Kisii, Nandi, Siaya, Trans Nzoia, and Vihiga in the tropical highlands of western Kenya. Two-thirds of the samples were good-quality maize, and one-third were poor-quality maize with a high incidence of visibly diseased kernels. One hundred fifty-three maize samples were assessed for Fusarium infection by culturing kernels on a selective medium. The isolates obtained were identified to the species level based on morphology and on formation of the sexual stage in Gibberella fujikuroi mating population tests. Fusarium moniliforme (G. fujikuroi mating population A) was isolated most frequently, but F. subglutinans (G. fujikuroi mating population E), F. graminearum, F. oxysporum, F. solani, and other Fusarium species were also isolated. The high incidence of kernel infection with the fumonisin-producing species F. moniliforme indicated a potential for fumonisin contamination of Kenyan maize. However, analysis of 197 maize kernel samples by high-performance liquid chromatography found little fumonisin B₁ in most of the samples. Forty-seven percent of the samples contained fumonisin B₁ at levels above the detection limit (100 ng/g), but only 5% were above 1,000 ng/g, a proposed level of concern for human consumption. The four most-contaminated samples, with fumonisin B₁ levels ranging from 3,600 to 11,600 ng/g, were from poor-quality maize collected in the Kisii district. Many samples with a high incidence of visibly diseased kernels contained little or no fumonisin B₁, despite the presence of F. moniliforme. This result may be attributable to the inability of F. moniliforme isolates present in Kenyan maize to produce fumonisins, to the presence of other ear rot fungi, and/or to environmental conditions unfavorable for fumonisin production.     

In planta reduction of maize seedling stalk lesions by the bacterial endophyte Bacillus mojavensis

Maize (Zea mays L.) is susceptible to infection by Fusarium verticillioides through autoinfection and alloinfection, resulting in diseases and contamination of maize kernels with the fumonisin mycotoxins. Attempts at controlling this fungus are currently being done with biocontrol agents such as bacteria, and this includes bacterial endophytes, such as Bacillus mojavensis . In addition to producing fumonisins, which are phytotoxic and mycotoxic, F. verticillioides also produces fusaric acid, which acts both as a phytotoxin and as an antibiotic. The question now is Can B. mojavensis reduce lesion development in maize during the alloinfection process, simulated by internode injection of the fungus? Mutant strains of B. mojavensis that tolerate fusaric acid were used in a growth room study to determine the development of stalk lesions, indicative of maize seedling blight, by co-inoculations with a wild-type strain of F. verticillioides and with non-fusaric acid producing mutants of F. verticillioides. Lesions were measured on 14-day-old maize stalks consisting of treatment groups inoculated with and without mutants and wild-type strains of bacteria and fungi. The results indicate that the fusaric-acid-tolerant B. mojavensis mutant reduced stalk lesions, suggesting an in planta role for this substance as an antibiotic. Further, lesion development occurred in maize infected with F. verticillioides mutants that do not produce fusaric acid, indicating a role for other phytotoxins, such as the fumonisins. Thus, additional pathological components should be examined before strains of B. mojavensis can be identified as being effective as a biocontrol agent, particularly for the control of seedling disease of maize.     

A fumonisin biosynthetic gene cluster in Fusarium oxysporum strain O-1890 and the genetic basis for B versus C fumonisin production

Most species of Fusarium that produce fumonisin mycotoxins produce predominantly B fumonisins (FBs). However, Fusarium oxysporum strain O-1890 produces predominantly C fumonisins (FCs). In this study, the nucleotide sequence of the fumonisin biosynthetic gene (FUM) cluster in strain O-1890 was determined. The order and orientation of FUM genes were the same as in the previously described clusters in Fusarium verticillioides and Fusarium proliferatum. Coding regions of F. oxysporum and F. verticillioides FUM genes were 88-92% identical, but regions flanking the clusters did not share significant identity. The FUM cluster gene FUM8 encodes an alpha-oxoamine synthase, and fum8 mutants of F. verticillioides do not produce fumonisins. Complementation of a fum8 mutant with the F. verticillioidesFUM8 restored FB production. Complementation with F. oxysporumFUM8 also restored production, but the fumonisins produced were predominantly FCs. These data indicate that different orthologues of FUM8 determine whether Fusarium produces predominantly FBs or FCs.