Jenny A. Butters

The sterol composition of powdery mildews

Abstract The sterol composition of conidia of powdery mildews of apple ( Podosphaera leucotricha ), barley ( Erysiphe graminis f.sp. hordei ), and cucurbits ( Sphaerotheca fuliginea ) was very similar, and simple compared with that of other plant pathogenic fungi. Ergosta-5,24(24 1 )-dien-3β-ol was the principal sterol in all three species, accompanied by much smaller amounts of 5α-ergosta-7,24(24 1 )-dien-3β-ol in apple and barley mildews, and cholesterol in the cucurbit mildew. All the ergostane derivatives found share the structural feature of a 24(24 1 )-double bond, suggesting that in powdery mildews sterol side-chain biosynthesis does not proceed beyond the initial introduction of C-24 1 .

Screening for tolerance to bavistin, a benzimidazole fungicide containing methyl benzimidazol-2-yl carbamate (MBC), in Beauveria bassiana: Sequence analysis of the β-tubulin gene to identify mutations conferring tolerance

The entomopathogenic Beauveria bassiana is a potential biopesticide. Fungicide-tolerant isolates of this fungus would have an added advantage of being compatible with the conventional chemical methods of pest control. Therefore, 30 isolates of the fungus were screened for tolerance to bavistin a commonly used benzimidazole fungicide containing methyl benzimidazol-2-yl carbamate (MBC). Germination and growth bioassays in the presence of 0.05% bavistin were conducted for screening. Three tolerant isolates were identified, showing tolerance up to 2% bavistin. Mutation in the beta-tubulin gene is known to confer tolerance to MBC; the nine known mutation sites in the gene involved were sequenced in the tolerant strains. The beta-tubulin gene from codons 1-405 was amplified using two pairs of degenerate primers, designed for the conserved region of the beta-tubulin gene and sequenced. From the sequence suitable primers were designed for the regions flanking the nine known sites involved in mutations conferring MBC tolerance. The amplified products with these primers from the MBC-tolerant isolates were sequenced and in two (ARSEF 1315 and ARSEF 1316) a mutation was detected in the 198 codon resulting in replacement of glutamic acid with lysine. In the third tolerant isolate (ITCC 913) no mutation could be detected in any of the nine known sites conferring tolerance to MBC. To complete the sequencing of the beta-tubulin gene, the remaining part (from codon 405 onwards) of the gene was isolated by a three-prime gene walk. The gene sequence showed a close homology to fungal beta-tubulin genes with four introns.

Resistance to benzimidazole can be caused by changes in β-tubulin isoforms†

The summary reports work that indicates that resistance in Rhynchosporium secalis to benzimidazole fungicides could result from substitution of the normal wild-type benA β-tubulin gene by other β-tubulin isoforms. © 1999 Society of Chemical Industry

Isozyme uniformity in Erysiphe graminis f.sp. hordei

Isozyme analysis was used to search for variation in barley powdery mildew ( Erysiphe graminis f.sp hordei ). Sixteen different enzymes were examined in 2·0% Triton extracts of conidia, using several different electrophoretic techniques coupled with standard enzyme-specific staining methods. Two enzymes were monomorphic; all other enzymes had at least two isozymes. No significant isozyme polymorphism was found in seventy-five barley mildew isolates collected from geographically well-separated regions of the world including Europe, Japan, the Middle East, N. America, N. Africa and New Zealand. Detailed analysis of mildew populations from two U.K. sites about 100 km apart, and where considerable variation existed in both fungicide sensitivity and virulence, also revealed no variation for isozyme markers. Isozyme variation was observed between different formae speciales of E. graminis and also between other powdery mildew genera. Although isozyme variation might provide useful taxonomic criteria for distinguishing mildew species infecting one host, so far the technique has provided no useful markers for genetic work.

Mechanism of Resistance to Fungicides

Fungicide resistance has been reported from well over 300 fungi, although the vast majority of these cases involve laboratory-resistant mutants, rather than fungi isolated from field crops. So far, only in some 25 plant pathogens has resistance become a serious practical problem. Whilst studies of laboratory mutants, and clinically resistant human yeast pathogens, have provided information on the molecular mechanisms of resistance, similar results from resistant strains collected from field populations is extremely limited. Cross resistance studies suggest that much of this resistance is target based, reflecting changes in one or just a few genes. Only in the case of benzimidazole resistance, and the β-tubulin target, is sufficient information available to understand aspects of the molecular basis of resistance. Even so, knowledge of how the amino acid changes clustered at codons 198 and 200 of β-tubulin affect benzimidazole binding and alter pathogenicity, must await more detailed structural analysis of the β-tubulin protein, and its association with α-tubulin to form tubulin filaments. To date, no fungicide target protein has been overexpressed and crystallized to provide a three-dimensional structure of fungicide binding sites although models are available which describe sites for azole fungicides in sterol 14α-demethylase. Nevertheless, sufficient information is becoming available at a molecular level to exploit rapid diagnostic methods to improve monitoring programmes. Progress is likely to be slow, however, since the molecular systems needed to analyse gene function in plant pathogens are at present very poorly developed, or even non-existent.

The mechanism of resistance to sterol 14α demethylation inhibitors in a mutant (Erg 40) of Ustilago maydis.

Resistance to DMI fungicides is a problem in both agriculture and medicine. Several mechanisms of resistance exist, but, as yet, few have been characterised in field resistant strains of plant pathogens. One approach to evaluating the role of mutations in the sterol 14α demethylase (14DM) target site requires cloning this gene and confirming its identity by complementation in an appropriate mutant. The azole-resistant mutant, Erg 40, of Ustilago maydis which is totally blocked at the 14α demethylation step in sterol biosynthesis seems to be suitable for such expression studies. Transformation of Erg 40 with a plasmid containing the yeast 14α demethylase (CYP51A1) gene removed the block in sterol biosynthesis and generated azole-sensitive transformants. Detailed analysis of these transformants failed to detect the presence of the yeast gene and suggested, instead, that changes in sterol biosynthesis resulted simply from the transformation protocol and not from the incorporation of extracellular DNA. Subsequent sequence analysis has revealed a mutation in the 14α demethylase gene of Erg 40. The results suggest that azole resistance in Erg 40 is not simply controlled by this mutation but involves some additional regulatory function, and consequently Erg 40 is not suitable for complementation studies with CYP51A1 genes. © 2000 Society of Chemical Industry

Isolation and characterization of α-tubulin genes from Septoria tritici and Rhynchosporium secalis, and comparative analysis of fungal α-tubulin sequences

The alpha-tubulin genes from Septoria tritici and Rhynchosporium secalis have been cloned and sequenced. The predicted amino acid sequence and intron structure showed strong homology with other known filamentous fungal alpha-tubulins. Comparison of sixteen fungal alpha-tubulin sequences based on amino acid sequence homology and intron structure identified five groups of proteins. Group 1 consists of filamentous fungi, including S. tritici and R. secalis, the dimorphic fungus Histoplasma capsulatum, and Pneumocystis carinii. Group 2 includes two divergent isoforms from Neurospora crassa and Aspergillus nidulans. Group 3 includes the yeast Saccharomyces cerevisiae and the dimorphic fungus Candida albicans. Group 4 contains the single yeast Schizosaccharomyces pombe. Group 5 includes the only Basidiomycete, Schizophyllum commune. This analysis supports the classification of P carinii as a primitive Ascomycete. The presence of an additional glycine residue between the second and third amino acid found only in Group 2 proteins may indicate a functionally distinct fungal isotype. Implications in terms of structure-function relationships for alpha-tubulin molecules are discussed.