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. 2001 Jul;67(7):2966-72.
doi: 10.1128/AEM.67.7.2966-2972.2001.

Identification of deoxynivalenol- and nivalenol-producing chemotypes of Gibberella zeae by using PCR

T Lee  1 , D W Oh, H S Kim, J Lee, Y H Kim, S H Yun, Y W Lee
Affiliations

Identification of deoxynivalenol- and nivalenol-producing chemotypes of Gibberella zeae by using PCR

T Lee et al. Appl Environ Microbiol. 2001 Jul.

Abstract

Gibberella zeae, a major cause of cereal scab, may be divided into two chemotypes based on production of the trichothecenes deoxynivalenol (DON) and nivalenol (NIV). We cloned and sequenced the gene cluster for trichothecene biosynthesis from each chemotype. G. zeae H-11 is a DON producer isolated from corn, and G. zeae 88-1 is a NIV producer from barley. We sequenced a 23-kb gene cluster from H-11 and a 26-kb cluster from 88-1, along with the unlinked Tri101 genes. Each gene cluster contained 10 Tri gene homologues in the same order and transcriptional directions as those of Fusarium sporotrichioides. Between H-11 and 88-1 all of the Tri homologues except Tri7 were conserved, with identities ranging from 88 to 98% and 82 to 99% at the nucleotide and amino acid levels, respectively. The Tri7 sequences were only 80% identical at the nucleotide level. We aligned the Tri7 genes and found that the Tri7 open reading frame of H-11 carried several mutations and an insertion containing 10 copies of an 11-bp tandem repeat. The Tri7 gene from 88-1 carried neither the repeat nor the mutations. We assayed 100 G. zeae isolates of both chemotypes by PCR amplification with a primer pair derived from the Tri7 gene and could differentiate the chemotypes by polyacrylamide gel electrophoresis. The PCR-based method developed in this study should provide a simple and reliable diagnostic tool for differentiating the two chemotypes of G. zeae.

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Figures

FIG. 1
FIG. 1
PCR strategies for amplification of trichothecene biosynthesis gene clusters from G. zeae strains H-11 (A) and 88-1 (B). Block arrows indicate the locations of Tri ORFs and their transcription directions within the gene cluster. The connecting thick lines represent noncoding DNA regions. The inverted arrowheads indicate PCR primer locations and orientations. Numbers above the thin lines bounded by inverted arrowheads indicate the approximate sizes (in kilobases) of amplified PCR products. Numbers next to or below the inverted arrowheads indicate the primers used (see Table 1 for primer names and sequences), and the letters indicate restriction enzyme sites: H, HindIII; K, KpnI; M, MseI; Nc, NcoI; Ns, NsiI; P, PstI; S, SpeI; Xa, XbaI; Xm, XmnI. Note that Tri101 is not part of the cluster and is not shown here.
FIG. 2
FIG. 2
PCR amplification patterns of the inserted regions of Tri7 from DON- or NIV-producing isolates of G. zeae on a 5% polyacrylamide gel. Lanes 1 and 20, 100-bp DNA ladder; lane 2, 88-1 (NIV chemotype); lanes 3 to 7, Korean NIV chemotypes. Lanes 8 to 19 show DON chemotypes with various numbers of repeats (in parentheses): lane 8, Korean (2); lane 9, United States (3); lane 10, Korean (4); lane 11, United States (5); lane 12, Korean (6); lane 13, Korean (7); lane 14, United States (8); lane 15, United States (9); lane 16, Korean (10); lane 17, United States (11); lane 18, United States (13); lane 19, United States (16)
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