Nested allele-specific PCR primers distinguish genetic groups of Uncinula necator.

Isolates of the obligately biotrophic fungus Uncinula necator cluster in three distinct genetic groups (groups I, II, and III). We designed PCR primers specific for these groups in order to monitor field populations of U. necator. We used the nucleotide sequences of the gene that encodes eburicol 14alpha-demethylase (CYP51) and of the ribosomal DNA internal transcribed spacer 1 (ITS1), ITS2, and 5. 8S regions. We identified four point mutations (three in CYP51 and one in ITS1) that distinguished groups I and II from group III based on a sample of 132 single-spore isolates originating from Europe, Tunisia, Israel, India, and Australia. We developed a nested allele-specific PCR assay in which the CYP51 point mutations were used to detect and distinguish groups I and II from group III in crude mildewed samples from vineyards. In a preliminary study performed with samples from French vineyards in which isolates belonging to genetic groups I and III were present, we found that a shift from a population composed primarily of group I isolates to a population composed primarily of group III isolates occurred during the grapevine growing season.

PCR cloning and detection of point mutations in the eburicol 14alpha-demethylase (CYP51) gene from Erysiphe graminis f. sp. hordei, a "recalcitrant" fungus.

Molecular studies of some micro-organisms are hampered by the difficulty of obtaining sufficient amounts of nucleic acids. A cloning strategy based on PCR has therefore been used to clone the eburicol 14alpha-demethylase (CYP51) gene of the obligate fungus Erysiphe graminis f. sp. hordei (Egh) using minute amounts of genomic DNA. The CYP51 gene encodes the enzymatic target of a major group of fungicides. Sequencing CYP51 from different Egh isolates revealed the occurrence of two alleles for this gene. An allele-specific PCR assay was developed to detect each CYP51 allele.

Rapid isolation of both double-stranded RNA and PCR-suitable DNA from the obligate biotrophic phytopathogenic fungus Uncinula necator using a commercially available reagent.

A method for rapid extraction of both double-stranded RNA (dsRNA) and DNA from an obligate biotrophic phytopathogenic fungus is described. Lyophilised fungal material is incubated in a commercial guanidium thiocyanate reagent. Proteins and cell debris are centrifuged by chloroform precipitation. After precipitation in isopropanol and washing in 75% ethanol, nucleic acids are resuspended in water (10 microl/mg fungal dry weight). DsRNA is directly visualised by agarose gel electrophoresis. DNA contained in 10-fold dilutions of the samples proved to be suitable for PCR-based experiments.

A mutation in the 14 alpha-demethylase gene of Uncinula necator that correlates with resistance to a sterol biosynthesis inhibitor.

We investigated the molecular basis of resistance of the obligate biotrophic grape powdery mildew fungus Uncinula necator to sterol demethylation-inhibiting fungicides (DMIs). The sensitivity of 91 single-spore field isolates of U. necator to triadimenol was assessed by using a leaf disc assay. Resistance factors (RF) ranged from 1.8 to 26.0. The gene encoding the target of DMIs (eburicol 14 alpha-demethylase) from five sensitive and seven resistant isolates was cloned and sequenced. A single mutation, leading to the substitution of a phenylalanine residue for a tyrosine residue at position 136, was found in all isolates exhibiting an RF higher than 5. No mutation was found in sensitive or weakly resistant (RF, < 5) isolates. An allele-specific PCR assay was developed to detect the mutation. Among the 91 isolates tested, only isolates with RF higher than 5 carried the mutation. Three of the 19 resistant isolates and all sensitive and weakly resistant isolates did not possess the mutation. The mutation at codon 136 is thus clearly associated with high levels of resistance to triadimenol.

Cloning and sequence analysis of the eburicol 14alpha-demethylase gene of the obligate biotrophic grape powdery mildew fungus.

In order to obtain molecular data concerning field resistance of Uncinula necator, the causal agent of grape powdery mildew, to sterol demethylation inhibitors, a major group of fungicides, the gene encoding the target of these compounds (eburicol 14alpha-demethylase) was cloned and sequenced from this obligately biotrophic phytopathogenic fungus. This single-copy gene encodes a 524 amino acid protein which displays high similarity to other known sterol 14alpha-demethylases (CYP51s). The coding sequence is interrupted by two short introns at positions identical to introns in Penicillium italicum CYP51, which is the only other known CYP51 gene in which introns have been identified. Intron excision was verified by cDNA sequencing.

RAPD Analysis Provides Insight into the Biology and Epidemiology of Uncinula necator.

ABSTRACT Ninety isolates of grape powdery mildew (Uncinula necator) from Europe (sixty-two) and India (twenty-eight) were collected. Ten of the sixty-two European isolates originated from mycelium overwintering in dormant buds ("flagshoots"). Mating types were determined, and genetic variation was assessed by random amplified polymorphic DNA (RAPD). Forty-one European isolates, including all "flagshoot" isolates, were mating type +, and twenty-one were mating type -. All Indian isolates were mating type -. Phenetic analysis based on 414 amplicons revealed three main groups. Most European isolates (53) clustered together. Nine flagshoot isolates clustered in a second distinct group. These isolates, which coexisted with other isolates in the field, may represent a genetically isolated biotype of U. necator. Indian isolates clustered into two groups. The first group (15 isolates) was a subgroup of the group containing European nonflagshoot isolates. The second group (12 isolates) was distinct from the other groups. These two groups of Indian isolates may represent genetically isolated populations with different climatic tolerances. A polymerase chain reaction primer pair, derived from a RAPD fragment specific to the Indian isolates, proved to be suitable for field studies.

Inhibition by the fungicide fenpropimorph of cholesterol biosynthesis in 3T3 fibroblasts.

Fenpropimorph (N-[3-(p-t-butylphenyl)-2-methylpropyl]-cis-2,6-dimethylmorpholine), a morpholine fungicide known to be an inhibitor of sterol biosynthesis in fungi and in higher plants, was demonstrated to be an efficient inhibitor of cholesterol biosynthesis in cultured Swiss 3T3 fibroblasts. Treatment of the mammalian cells with fenpropimorph resulted in a dose-dependent inhibition of [14C]acetate incorporation into the C27 sterols [IC50 (concentration causing half-maximal inhibition) = 0.5 microM], which was accompanied by an accumulation of polar sterols and a decrease in cellular hydroxymethylglutaryl-CoA reductase activity. Exposure of the cells to the drug affected cell growth. Analysis of the sterols in the growth-arrested and in the pulse-labelled cells indicate that fenpropimorph has, in the sterol-biosynthetic pathway, target enzymes in mammalian cells different from those in the other phyla. Whereas in plants and fungi fenpropimorph mainly affects sterol isomerases and reductases, in the fibroblasts its main target seems to be the demethylation of lanosterol.