Fum3p, a 2-ketoglutarate-dependent dioxygenase required for C-5 hydroxylation of fumonisins in Fusarium verticillioides

Fumonisins are polyketide-derived mycotoxins produced by several agriculturally important Fusarium species. The B series fumonisins, FB(1), FB(2), FB(3), and FB(4), are fumonisins produced by wild-type Fusarium verticillioides strains, differing in the number and location of hydroxyl groups attached to the carbon backbone. We characterized the protein encoded by FUM3, a gene in the fumonisin biosynthetic gene cluster. The 33-kDa FUM3 protein (Fum3p) was heterologously expressed and purified from Saccharomyces cerevisiae. Yeast cells expressing the Fum3p converted FB(3) to FB(1), indicating that Fum3p catalyzes the C-5 hydroxylation of fumonisins. This result was verified by assaying the activity of Fum3p purified from yeast cells. The C-5 hydroxylase activity of purified Fum3p required 2-ketoglutarate, Fe(2+), ascorbic acid, and catalase, all of which are required for 2-ketoglutarate-dependent dioxygenases. The protein also contains two His motifs that are highly conserved in this family of dioxygenases. Thus, Fum3p is a 2-ketoglutarate-dependent dioxygenase required for the addition of the C-5 hydroxyl group of fumonisins.     

FUM9 Is Required for C-5 Hydroxylation of Fumonisins and Complements the Meitotically Defined Fum3 Locus in Gibberella moniliformis

Deletion of the Gibberella moniliformis FUM9 gene resulted in mutants that produce only fumonisins that lack a C-5 hydroxyl group. This phenotype is identical to that of previously described mutants with defective alleles at the meiotically defined Fum3 locus. Transformation with a wild-type FUM9 gene into a Fum3-defective mutant restored wild-type fumonisin production. These results indicate that the FUM9 protein catalyzes the C-5 hydroxylation of fumonisins and that FUM9 and the Fum3 locus are the same gene.     

The TRI11 Gene of Fusarium sporotrichioides Encodes a Cytochrome P-450 Monooxygenase Required for C-15 Hydroxylation in Trichothecene Biosynthesis

Several genes in the trichothecene biosynthetic pathway of Fusarium sporotrichioides have been shown to reside in a gene cluster. Sequence analysis of a cloned DNA fragment located 3.8 kb downstream from TRI5 has led to the identification of the TRI11 gene. The nucleotide sequence of TRI11 predicts a polypeptide of 492 residues (M_r = 55,579) with significant similarity to members of the cytochrome P-450 superfamily. TRI11 is most similar to several fungal cytochromes P-450 (23 to 27% identity) but is sufficiently distinct to define a new cytochrome P-450 gene family, designated CYP65A1. Disruption of TRI11 results in an altered trichothecene production phenotype characterized by the accumulation of isotrichodermin, a trichothecene pathway intermediate. The evidence suggests that TRI11 encodes a C-15 hydroxylase involved in trichothecene biosynthesis.     

FUM9 is required for C-5 hydroxylation of fumonisins and complements the meitotically defined Fum3 locus in Gibberella moniliformis

Deletion of the Gibberella moniliformis FUM9 gene resulted in mutants that produce only fumonisins that lack a C-5 hydroxyl group. This phenotype is identical to that of previously described mutants with defective alleles at the meiotically defined Fum3 locus. Transformation with a wild-type FUM9 gene into a Fum3-defective mutant restored wild-type fumonisin production. These results indicate that the FUM9 protein catalyzes the C-5 hydroxylation of fumonisins and that FUM9 and the Fum3 locus are the same gene.     

Fdb1 and Fdb2, Fusarium verticillioides loci necessary for detoxification of preformed antimicrobials from corn

Fusarium verticillioides is a fungus of significant economic importance because of its deleterious effects on plant and animal health and on the quality of their products. Corn (Zea mays) is the primary host for F. verticillioides, and we have investigated the impact of the plant's antimicrobial compounds (DIMBOA, DIBOA, MBOA, and BOA) on fungal virulence and systemic colonization. F. verticillioides is able to metabolize these antimicrobials, and genetic analyses indicated two loci, Fdb1 and Fdb2, were involved in detoxification. Mutation at either locus caused sensitivity and no detoxification. In vitro physiological complementation assays resulted in detoxification of BOA and suggested that an unknown intermediate compound was produced. Production of the intermediate compound involved Fdbl, and a lesion in fdb2 preventing complete metabolism of BOA resulted in transformation of the intermediate into an unidentified metabolite. Based on genetic and physiological data, a branched detoxification pathway is proposed. Use of genetically characterized detoxifying and nondetoxifying strains indicated that detoxification of the corn antimicrobials was not a major virulence factor, since detoxification was not necessary for development of severe seedling blight or for infection and endophytic colonization of seedlings. Production of the antimicrobials does not appear to be a highly effective resistance mechanism against F. verticillioides.     

Tri16 Is Required for Esterification of Position C-8 during Trichothecene Mycotoxin Production by Fusarium sporotrichioides

We previously characterized Tri1, a gene required for hydroxylation of the C-8 position during trichothecene mycotoxin biosynthesis in Fusarium sporotrichioides NRRL 3299. Sequence analysis of the region surrounding Tri1 revealed a gene, named Tri16, which could encode an acyltransferase. Unlike the wild-type parent strain NRRL 3299, which accumulates primarily T-2 toxin along with low levels of diacetoxyscirpenol (DAS) and neosolaniol (NEO) and trace amounts of 8-propionyl-neosolaniol (P-NEO) and 8-isobutyryl-neosolaniol (B-NEO), mutants containing a disruption of Tri16 were blocked in the production of the three C-8 esterified compounds T-2 toxin, P-NEO, and B-NEO and accumulated the C-8-hydroxylated compound NEO along with secondary levels of DAS. These data indicate that Tri16 encodes an acyltransferase that catalyzes the formation of ester side groups at C-8 during trichothecene biosynthesis. We also report the presence of a Tri16 ortholog in Gibberella pulicaris R-6380 that is likely linked to a presumably inactive ortholog for Tri1.     

GAC1, a gene encoding a putative GTPase-activating protein, regulates bikaverin biosynthesis in Fusarium verticillioides

Fusarium verticillioides (teleomorph Gibberella moniliformis) is an ascomycete known to produce a variety of secondary metabolites, including fumonisins, fusaric acid and bikaverin. These metabolites are synthesized when the fungus is under stress, notably nutrient limitations. To date we have limited understanding of the complex regulatory process associated with fungal secondary metabolism. In this study we generated a collection of F. verticillioides mutants by using REMI (restriction enzyme mediated integration) mutagenesis and in the process identified a strain, R647, that carries a mutation in a gene designated GAC1. Mutation in the GACI locus, which encodes a putative GTPase activating protein, resulted in the increased production of bikaverin, suggesting that GAC1 is negatively associated with bikaverin biosynthesis. Complementation of R647 with the wildtype GAC1 gene restored the bikaverin production level to that of the wild-type progenitor, demonstrating that gac1 mutation was directly responsible for the overproduction of bikaverin. We also demonstrated that AREA, encoding global nitrogen regulator, and PKS4, encoding polyketide synthase, are downstream genes that respectively are regulated positively and negatively by GAC1. Our results suggest that GAC1 plays an important role in signal transduction regulating bikaverin production in F. verticillioides.     

Production of Beauvericin, Moniliformin, Fusaproliferin, and Fumonisins B1, B2, and B3 by Fifteen Ex-Type Strains of Fusarium Species

Fifteen Fusarium species were analyzed by high-performance liquid chromatography for the production of six mycotoxins in corn grits cultures. Production of mycotoxins ranged from 66 to 2,500 μg/kg for fumonisin B₁, 0.6 to 1,500 μg/g for moniliformin, 2.2 to 720 μg/g for beauvericin, and 12 to 130 μg/g for fusaproliferin. Fumonisin B₂ (360 μg/kg) was produced by two species, fumonisin B₃ was not detected in any of the 15 species examined, and Fusarium bulbicola produced none of the six mycotoxins that we analyzed.     

Control of Fusarium verticillioides on corn with a combination of Bacillus subtilis TM3 formulation and botanical pesticides

The aim of this study was to evaluate the effectiveness of the combination of Bacillus subtilis TM3 formulation with botanical pesticides in suppressing Fusarium verticilloides infection in corn. The research was carried out at the Plant Pathology Laboratory and the Experimental Farm of Indonesian Cereals Research Institute (ICERI) from February to November 2019. The research consisted of two stages, namely an in vitro test of antagonists of botanical pesticides against F. verticilloides using 5 types of plant extracts namely betel leaf extract, turmeric, galangal, cosmos, and clove leaf. The second stage was to test the effectiveness of the combination of the formulation of B. subtilis TM3 with the best 3 types of plant extracts in vitro testing in suppressing F. verticilloides infection in plants. The results of the in vitro study showed that the plant extracts of betel leaf, clove leaf and galangal had the best inhibitory ability on the mycelia growth of F. verticilloides. Meanwhile, the field test found that the application of the B. subtilis TM3 formulation, either alone or in combination with plant extracts, was able to suppress F. verticilloides infection. The combination of B. subtilis TM3 formulation with betel leaf extract showed the best inhibition of 20% against stem rot disease and 13.33% against corn cob rot. This treatment did not affect production quantitatively, but was able to suppress the decline in seed quality due to F. verticilloides infection. Seeds grown by the Plastic Rolled Paper Test (PRPT) method were not only infected with F. verticilloides, but also infected with other seed-borne pathogens, such as Aspergillus niger and A. falvus. The presence of these two pathogens did not inhibit the growth of F. verticilloides in kernels.     

Growth inhibition of a Fusarium verticillioides GUS strain in corn kernels of aflatoxin-resistant genotypes

Two corn genotypes, GT-MAS:gk and MI82, resistant to Aspergillus flavus infection/aflatoxin contamination, were tested for their ability to limit growth of Fusarium verticillioides. An F. verticillioides strain was transformed with a beta-glucuronidase (GUS) reporter gene (uidA) construct to facilitate fungal growth quantification and then inoculated onto endosperm-wounded and non-wounded kernels of the above-corn lines. To serve as a control, an A. flavus strain containing the same reporter gene construct was inoculated onto non-wounded kernels of GT-MAS:gk. Results showed that, as in a previous study, non-wounded GT-MAS:gk kernels supported less growth (six- to ten-fold) of A. flavus than did kernels of a susceptible control. Also, non-wounded kernels of GT-MAS:gk and M182 supported less growth (two- to four-fold) of F. verticillioides than did susceptible kernels. Wounding, however, increased F. verticillioides infection of MI82, but not that of GT-MAS:gk. This is in contrast to a previous study of A. flavus, where wounding increased infection of GT-MAS:gk rather than M182 kernels. Further study is needed to explain genotypic variation in the kernel response to A. flavus and F. verticillioides kernel infections. Also, the potential for aflatoxin-resistant corn lines to likewise inhibit growth of F. verticillioides needs to be confirmed in the field.     

Synthesis of C-22, C-23-3H-labeled 3 alpha,7 alpha,12 alpha-trihydroxy-5 beta-cholestane

This report describes an efficient synthesis of C-22, C-23-(3)H-labeled 3alpha,7alpha,12alpha-trihydroxy-5beta-cholestane. - Somanathan, R., and S. Krisans. Synthesis of C-22, C-23-(3)H-labeled 3alpha,7alpha,12alpha-trihydroxy-5beta-cholestane.     

Survival of Phytophthora cinnamomi and Fusarium verticillioides in commercial potting substrates for ornamental plants

Live plants, particularly when accompanied by soil or potting substrates, are considered the main pathway for international spread of plant pathogens. Modern, rapid shipping technologies for international plant trade increase the probability of plant pathogen survival during transport and the subsequent chances of disease outbreaks in new locations. The survival of two model pathogens, an Oomycete, Phytophthora cinnamomi, and a filamentous fungus, Fusarium verticillioides, was studied in two different commercial potting substrates (peat and peat‐free) under glasshouse conditions in the absence of a plant host. Survival rates were analysed at 2, 7, 12 and 17 months after substrate inoculation. Fusarium verticillioides had the longest survival rate, and was still present at 17 months. In contrast, P. cinnamomi survived up to 7 months but was not recovered after 12 or 17 months. There was no significant difference in the number of colony‐forming units (CFUs) of either pathogen in the two substrates, except at 2 months, when higher numbers were recovered from peat substrates.     

Role of AREA, a regulator of nitrogen metabolism, during colonization of maize kernels and fumonisin biosynthesis in Fusarium verticillioides

Fumonisin B1 (FB(1)) biosynthesis is repressed in cultures containing ammonium as the nitrogen source and when grown on blister kernels, the earliest stages of kernel development. In this study AREA, a regulator of nitrogen metabolism, was disrupted in Fusarium verticilliodes. The mutant (DeltaareA) grew poorly on mature maize kernels, but grew similar to wild type (WT) with the addition of ammonium phosphate. FB(1) was not produced by DeltaareA under any condition or by the WT with added ammonium phosphate. Constitutive expression of AREA (strain AREA-CE) rescued the growth and FB(1) defects in DeltaareA. Growth of WT, DeltaareA, and AREA-CE on blister-stage kernels was similar. After 7 days of growth, none of the strains produced FB(1) and the pH of the kernel tissues was 8.0. Addition of amylopectin to the blister kernels resulted in a pH near 6.6 and FB(1) production by WT and AREA-CE. The results support the hypothesis that FB(1) biosynthesis is regulated by AREA. Also the failure to produce FB(1) in blister kernels is due to high pH conditions generated because of an unfavorable carbon/nitrogen environment.     

Tri1 Encodes the Cytochrome P450 Monooxygenase for C-8 Hydroxylation during Trichothecene Biosynthesis in Fusarium sporotrichioides and Resides Upstream of Another New Tri Gene

Many Fusarium species produce one or more agriculturally important trichothecene mycotoxins, and the relative level of toxicity of these compounds is determined by the pattern of oxygenations and acetylations or esterifications on the core trichothecene structure. Previous studies with UV-induced Fusarium sporotrichioides NRRL 3299 trichothecene mutants defined the Tri1 gene and demonstrated that it was required for addition of the oxygen at the C-8 position during trichothecene biosynthesis. We have cloned and characterized the Tri1 gene from NRRL 3299 and found that it encodes a cytochrome P450 monooxygenase. The disruption of Tri1 blocks production of C-8-oxygenated trichothecenes and leads to the accumulation of 4,15-diacetoxyscirpenol, the same phenotype observed in the tri1 UV-induced mutants MB1716 and MB1370. The Tri1 disruptants and the tri1 UV-induced mutants do not complement one another when coinoculated, and the Tri1 gene sequence restores T-2 toxin production in both MB1716 and MB1370. The DNA sequence flanking Tri1 contains another new Tri gene. Thus, Tri1 encodes a C-8 hydroxylase and is located either in a new distal portion of the trichothecene gene cluster or in a second separate trichothecene gene cluster.