FvSTUA is a Key Regulator of Sporulation, Toxin Synthesis, and Virulence in Fusarium verticillioides.

Fusarium verticillioides is one of the most important pathogens of maize, causing rot and producing fumonisin mycotoxins during infection. Ingestion of fumonisin-contaminated corn causes underperformance and even fatal toxicity in livestock and is associated with neural tube birth defects, growth stunting in children, and some cancers. StuA, an APSES-class transcription factor, is a major developmental transcriptional regulator in fungi. It has been shown to regulate crucial developmental processes, such as sporulation, virulence, and mycotoxin synthesis among others. In this study, the role of FvSTUA in F. verticillioides was examined by characterizing ∆FvstuA deletion mutants functionally and transcriptomally. The deletion mutants exhibited reduced vegetative growth, stunted aerial hyphae, and significant reductions in microconidiation. Macroconidiation and hydrophobicity of the deletion strains were reduced as well. Additionally, fumonisin production and virulence of the deletion mutants were greatly reduced. Transcriptomic analysis revealed downregulation of expression of several genes in the fumonisin and fusarin C biosynthetic clusters and differential expression of genes involved in conidiation and virulence. Nuclear localization of FvSTUA supported its likely function as a transcription factor. Together, our results indicate that FvSTUA plays a global role in transcriptional regulation in F. verticillioides influencing morphogenesis, toxin production, and virulence.

Subclinical Doses of Combined Fumonisins and Deoxynivalenol Predispose Clostridium perfringens-Inoculated Broilers to Necrotic Enteritis.

Fumonisins (FB) and deoxynivalenol (DON) are mycotoxins which may predispose broiler chickens to necrotic enteritis (NE). The objective of this study was to identify the effects of subclinical doses of combined FB and DON on NE. A total of 480 day-old male broiler chicks were divided into four treatment groups; 1) control group (basal diet + Clostridium perfringens); 2) necrotic enteritis group (basal diet + Eimeria maxima + C. perfringens); 3) FB + DON group (basal diet + 3 mg/kg FB + 4 mg/kg DON + C. perfringens); and 4) FB + DON + NE group (basal diet + 3 mg/kg FB + 4 mg/kg DON + E. maxima + C. perfringens). Birds in NE and FB + DON + NE groups received 2.5 × 103 E. maxima on day 14. All birds were inoculated with C. perfringens on days 19, 20, and 21. On day 35, birds in the NE, FB + DON, and FB + DON + NE groups had 242, 84, and 339 g lower BWG and a 19-, 2-, and 22-point increase in FCR respectively, than in the control group. Subclinical doses of FB + DON increased (p < 0.05) the NE lesion scores compared to the control group on day 21. On day 21, birds in the NE, FB + DON, and FB + DON + NE groups had increased (p < 0.05) serum FITC-D, lower (p < 0.05) jejunal tight junction protein mRNA, and increased (p < 0.05) cecal tonsil IL-1 mRNA compared to control group. On day 21, birds in the NE group had decreased (p < 0.05) villi height to crypt depth ratio compared to the control group and the presence of FB + DON in NE-induced birds further decreased the villi height to crypt depth ratio. Birds in the NE, FB + DON, and FB + DON + NE groups had increased (p < 0.05) C. perfringens, lower (p < 0.05) Lactobacillus loads in the cecal content, and a lower (p < 0.05) CD8+: CD4+ cell ratio in the cecal tonsils compared to the control group. It can be concluded that subclinical doses of combined FB and DON predispose C. perfringens-inoculated birds to NE, and the presence of FB + DON in NE-induced birds exacerbated the severity of NE.

FDB2 encodes a member of the arylamine N-acetyltransferase family and is necessary for biotransformation of benzoxazolinones by Fusarium verticillioides.

AIMS: To clone and characterize genes from the mycotoxigenic fungus, Fusarium verticillioides, which are associated with its ability to biotransform allelopathic benzoxazolinones produced by maize, wheat, and rye. METHODS AND RESULTS: Suppression subtractive hybridization identified F. verticillioides genes up-regulated in response to 2-benzoxazolinone (BOA), including a cluster of genes along chromosome 3. One of these genes, putatively encoding an arylamine N-acetyltransferase (NAT), was highly represented in the subtracted library and was of particular interest since previous analyses identified the FDB2 locus as possibly encoding transferase activity. The gene was subcloned and complemented a natural fdb2 mutant. Conversely, disruption of the gene eliminated the ability of F. verticillioides to metabolize BOA. Other genes in the cluster also were assessed using a complementation assay. Metabolic profiles of fdb2 mutants suggest that minor acylation activity occurred independently of the NAT activity encoded by FDB2. CONCLUSIONS: The previously defined FDB2 locus was functionally associated with the gene encoding putative NAT activity, and the FDB2 gene was essential for biotransformation of BOA. The flanking gene FDB3 encodes a putative Zn(II)2Cys6 transcription factor and contributes to efficient BOA biotransformation but was not essential. SIGNIFICANCE AND IMPACT OF THE STUDY: Biotransformation of benzoxazolinones by F. verticillioides may enhance its ecological fitness in maize field environments and our results provide greater understanding of the genes that modulate the biotransformation process. Additionally, this is the first homologue of the NAT gene family to be characterized in a filamentous fungus.

Toxicity of endophyte-infected tall fescue alkaloids and grass metabolites on Pratylenchus scribneri.

ABSTRACT Neotyphodium coenophialum, an endophytic fungus associated with tall fescue grass, enhances host fitness and imparts pest resistance. This symbiotum is implicated in the reduction of stresses, including plant-parasitic nematodes. To substantiate this implication, toxicological effects of root extracts, polyphenolic fraction, ergot, and loline alkaloids from endophyte-infected tall fescue were investigated using Pratylenchus scribneri, a nematode pest of tall fescue. In vitro bioassays and greenhouse studies were used as tests for effects of root fractions and compounds on motility and mortality of this lesion nematode. Greenhouse studies revealed that endophyte-infected tall fescue grasses are essentially nonhosts to P. scribneri, with root populations averaging 3 to 17 nematodes/pot, compared with 4,866 and 8,450 nematodes/pot for noninfected grasses. The in vitro assay indicated that root extracts from infected tall fescues were nematistatic. Polyphenols identified in extracts included chlorogenic acid, 3,5-dicaffeoylquinic acids, caffeic acid, and two unidentified compounds, but these were not correlated with endophyte status, qualitatively or quantitatively. Tests of several ergot alkaloids revealed that ergovaline and alpha-ergocryptine were nematicidal at 5 and 50 microg/ml, respectively, while ergocornine and ergonovine were nematistatic at most concentrations. Loline (N-formylloline), the pyrrolizidine alkaloid tested, was nematicidal (50 to 200 microg/ml). The ecological benefits of the metabolites tested here should assist in defining their role in deterring this nematode species while offering some probable mechanisms of action against plant-parasitic nematodes in general.

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.

The conserved global regulator VeA is necessary for symptom production and mycotoxin synthesis in maize seedlings by Fusarium verticillioides.

The veA or velvet gene is necessary for biosynthesis of mycotoxins and other secondary metabolites in Aspergillus species. In addition, veA has also been demonstrated to be necessary for normal seed colonization in Aspergillus flavus and Aspergillus parasiticus. The present study shows that veA homologues are broadly distributed in fungi, particularly in Ascomycetes. The Fusarium verticillioides veA orthologue, FvVE1, is also required for the synthesis of several secondary metabolites, including fumonisin and fusarins. This study also shows that maize plants grown from seeds inoculated with FvVE1 deletion mutants did not show disease symptoms, while plants grown from seeds inoculated with the F. verticillioides wildtype and complementation strains clearly showed disease symptoms under the same experimental conditions. In this latter case, the presence of lesions coincided with accumulation of fumonisins in the plant tissues, and only these plant tissues had elevated levels of sphingoid bases and their 1-phosphate derivatives, indicating inhibition of ceramide synthase and disruption of sphingolipid metabolism. The results strongly suggest that FvVE1 is necessary for pathogenicity by F. verticillioides against maize seedlings. The conservation of veA homologues among ascomycetes suggests that veA could play a pivotal role in regulating secondary metabolism and associated pathogenicity in other fungi.

Detoxification of corn antimicrobial compounds as the basis for isolating Fusarium verticillioides and some other Fusarium species from corn.

The preformed antimicrobial compounds produced by maize, 2,4-dihydroxy-7-methoxy-2H-1,4-benzoxazin-3-one and its desmethoxy derivative 2,4-dihydroxy-2H-1,4-benzoxazin-3-one, are highly reactive benzoxazinoids that quickly degrade to the antimicrobials 6-methoxy-2-benzoxazolinone (MBOA) and 2-benzoxazolinone (BOA), respectively. Fusarium verticillioides (= F. moniliforme) is highly tolerant to MBOA and BOA and can actively transform these compounds to nontoxic metabolites. Eleven of 29 Fusarium species had some level of tolerance to MBOA and BOA; the most tolerant, in decreasing order, were F. verticillioides, F. subglutinans, F. cerealis (= F. crookwellense), and F. graminearum. The difference in tolerance among species was due to their ability to detoxify the antimicrobials. The limited number of species having tolerance suggested the potential utility of these compounds as biologically active agents for inclusion within a semiselective isolation medium. By replacing the pentachloronitrobenzene in Nash-Snyder medium with 1.0 mg of BOA per ml, we developed a medium that resulted in superior frequencies of isolation of F. verticillioides from corn while effectively suppressing competing fungi. Since the BOA medium provided consistent, quantitative results with reduced in vitro and taxonomic efforts, it should prove useful for surveys of F. verticillioides infection in field samples.

Identification of intermediate and branch metabolites resulting from biotransformation of 2-benzoxazolinone by Fusarium verticillioides.

Detoxification of the maize (Zea mays) antimicrobial compound 2-benzoxazolinone by the fungal endophyte Fusarium verticillioides involves two genetic loci, FDB1 and FDB2, and results in the formation of N-(2-hydroxyphenyl)malonamic acid. Intermediate and branch metabolites were previously suggested to be part of the biotransformation pathway. Evidence is presented here in support of 2-aminophenol as the intermediate metabolite and 2-acetamidophenol as the branch metabolite, which was previously designated as BOA-X. Overall, 2-benzoxazolinone metabolism involves hydrolysis (FDB1) to produce 2-aminophenol, which is then modified (FDB2) by addition of a malonyl group to produce N-(2-hydroxyphenyl)malonamic acid. If the modification is prevented due to genetic mutation (fbd2), then 2-acetamidophenol may accumulate as a result of addition of an acetyl group to 2-aminophenol. This study resolves the overall chemistry of the 2-benzoxazolinone detoxification pathway, and we hypothesize that biotransformation of the related antimicrobial 6-methoxy-2-benzoxazolinone to produce N-(2-hydroxy-4-methoxyphenyl)malonamic acid also occurs via the same enzymatic modifications. Detoxification of these antimicrobials by F. verticillioides apparently is not a major virulence factor but may enhance the ecological fitness of the fungus during colonization of maize stubble and field debris.