Susceptibility of Maize to Stalk Rot Caused by Fusarium graminearum Deoxynivalenol and Zearalenone Mutants.

Fusarium graminearum is a destructive pathogen of cereals that can cause stalk rot in maize. Stalk rot results in yield losses due to impaired grain filling, premature senescence, and lodging, which limits production and harvesting of ears. In addition, mycotoxins can make infected tissues unfit for silage. Our objectives were to evaluate the natural variation in stalk rot resistance among maize inbreds, to establish whether deoxynivalenol (DON)- and zearalenone (ZEA)-deficient strains are pathogenic on a panel of diverse inbreds, and to quantify the accumulation of DON in infected stalk tissue. Wild-type F. graminearum and mycotoxin mutants (DON and ZEA) were used to separately inoculate stalks of 9-week-old plants of 20 inbreds in the greenhouse. Plants were evaluated for lesion area at the inoculation point at 0, 2, 14, and 28 days postinoculation and tissues around lesions were sampled to determine the DON content. Regardless of their ability to produce DON or ZEA, all tested F. graminearum strains caused stalk rot; however, significant differences in disease levels were detected. Among the tested inbreds, Mp717 was resistant to all three F. graminearum strains while Mp317 and HP301 were only partially resistant. Accumulation of DON was significantly lower in infected stalks of the resistant and partially resistant inbreds than the susceptible inbreds. Analysis of the 20 inbreds using data from 17 simple-sequence repeats revealed population structure among the individuals; however, there was no association between genetic clustering and stalk rot resistance. These findings are an additional step toward breeding maize inbreds suitable for planting in fields infested with F. graminearum.

Hexanoate synthase, a specialized type I fatty acid synthase in aflatoxin B1 biosynthesis.

In fungi, fatty acids are biosynthesized by large multifunctional enzyme complexes, the fatty acid synthases (FASs), which catalyze chain assembly in an iterative manner. Many fungal secondary metabolites contain fatty acid moieties, and it is often unclear whether they are recruited from primary metabolism or are biosynthesized de novo by secondary metabolic FASs. The most convincing evidence of such a dedicated FAS comes from the biosyntheses of aflatoxin (AF) and sterigmatocystin (ST) in certain species of the filamentous fungus Aspergillus. Incorporation studies in AF and genetic analyses of ST and AF biosynthesis strongly suggest that their biosyntheses begin with the production of a C6 fatty acid by a specialized FAS. The genes encoding the alpha (hexA) and beta (hexB) subunits of this hexanoate synthase (HexS) from the AF pathway in Aspergillus parsiticus SU-1 were cloned and both their gDNAs and cDNAs were sequenced and their transcriptional ends analyzed. Translated amino acid sequences are predicted to result in proteins of 181.3 and 210.5 kDa, for HexA and HexB, respectively. Comparison of the HexA and HexB sequences with those of the ST FAS subunits and primary metabolic FASs indicated that the secondary metabolic enzymes are members of a well-defined subclass of the FAS family. Phylogenetic predictions and an analysis of GC-bias in AF and ST pathway genes compared with primary metabolic Aspergillus genes were used as a basis to propose a route for the evolution of the AF and ST clusters.

Novel procedure for identification of compounds inhibitory to transcription of genes involved in mycotoxin biosynthesis.

A novel assay is described for the identification and isolation of compounds that inhibit the transcription of genes involved in mycotoxin biosynthesis. The thin-layer chromatography-based assay was used to screen plant extracts for compounds that would inhibit the expression of the beta-glucuronidase reporter gene under the control of an aflatoxin biosynthesis gene promoter in Aspergillus parasiticus. The assay was used to track purification of an inhibitory compound, cp2, from extracts of black pepper (Piper nigrum). Cp2 did not inhibit mycelial growth or the expression of the beta-tubulin gene but did inhibit aflatoxin biosynthesis at the transcriptional level. Applications of cp2 to the control of mycotoxins are discussed.

Physical and transcriptional map of an aflatoxin gene cluster in Aspergillus parasiticus and functional disruption of a gene involved early in the aflatoxin pathway.

Two genes involved in aflatoxin B1 (AFB1) biosynthesis in Aspergillus parasiticus, nor-1 and ver-1, were localized to a 35-kb region on one A. parasiticus chromosome and to the genomic DNA fragment carried on a single cosmid, NorA. A physical and transcriptional map of the 35-kb genomic DNA insert in cosmid NorA was prepared to help determine whether other genes located in the nor-1-ver-1 region were involved in aflatoxin synthesis. Northern (RNA) analysis performed on RNA isolated from A. parasiticus SU1 grown in aflatoxin-inducing medium localized 14 RNA transcripts encoded by this region. Eight of these transcripts, previously unidentified, showed a pattern of accumulation similar to that of nor-1 and ver-1, suggesting possible involvement in AFB1 synthesis. To directly test this hypothesis, gene-1, encoding one of the eight transcripts, was disrupted in A. parasiticus CS10, which accumulates the aflatoxin precursor versicolorin A, by insertion of plasmid pAPNVES4. Thin-layer chromatography revealed that gene-1 disruptant clones no longer accumulated versicolorin A. Southern hybridization analysis of these clones indicated that gene-1 had been disrupted by insertion of the disruption vector. These data confirmed that gene-1 is directly involved in AFB1 synthesis. The predicted amino acid sequence of two regions of gene-1 showed a high degree of identity and similarity with the beta-ketoacyl-synthase and acyltransferase functional domains of polyketide synthases, consistent with a proposed role for gene-1 in polyketide backbone synthesis.

Structural and functional analysis of the nor-1 gene involved in the biosynthesis of aflatoxins by Aspergillus parasiticus.

The nor-1 gene was cloned previously by complementation of a mutation (nor-1) in Aspergillus parasiticus SU-1 which blocked aflatoxin B1 biosynthesis, resulting in the accumulation of norsolorinic acid (NA). In this study, the nucleotide sequences of the cDNA and genomic DNA clones encompassing the coding region of the nor-1 gene were determined. The transcription initiation and polyadenylation sites of nor-1 were located by primer extension and RNase protection analyses and by comparison of the nucleotide sequences of the nor-1 genomic and cDNA clones. A plasmid, pNA51-82, was created for one-step disruption of the nor-1 gene by inserting a functional copy of the nitrate reductase (niaD) gene from A. parasiticus into the coding region of the nor-1 gene. Transformation of A. parasiticus NR-3 (niaD Afl+) with pNA51-82 resulted in niaD+ transformants that accumulated NA and produced reduced levels of aflatoxin as determined by thin-layer chromatography and enzyme-linked immunosorbent assay analyses of extracts from mycelia and the growth medium. Southern analysis of genomic DNA isolated from the NA-accumulating transformants indicated that the wild-type nor-1 gene in the chromosome had been replaced by the nonfunctional allele carried on pNA51-82. This recombinational inactivation event provides direct evidence that the nor-1 gene is functionally involved in aflatoxin biosynthesis. Comparison of the predicted nor-1 amino acid sequence with sequences in the GenBank and EMBL databases suggested that the protein is a member of the family of short-chain alcohol dehydrogenases, consistent with its proposed function as a keto reductase.