Genetic variation and population structure in black-grass (Alopecurus myosuroides Huds.), a successful, herbicide-resistant, annual grass weed of winter cereal fields.

Black-grass (Alopecurus myosuroides) is an allogamous grass weed common in cereal fields of northern Europe, which developed resistance to a widely used family of herbicides, the ACCase-inhibiting herbicides. Resistance is caused by mutations at the ACCase gene and other, metabolism-based, mechanisms. We investigated the genetic structure of 36 populations of black-grass collected in one region of France (Côte d'Or), using 116 amplified fragment length polymorphism (AFLP) loci and sequence data at the ACCase gene. The samples were characterized for their level of herbicide resistance and genotyped for seven known ACCase mutations conferring resistance. All samples contained herbicide-resistant plants, and 19 contained ACCase mutations. The genetic diversity at AFLP loci was high (H(T) = 0.246), while differentiation among samples was low (F(ST) = 0.023) and no isolation by distance was detected. Genetic diversity within samples did not vary with the frequency of herbicide resistance. A Bayesian algorithm was used to infer population structure. The two genetic clusters inferred were not associated with any geographical structure or with herbicide resistance. A high haplotype diversity (H(d) = 0.873) and low differentiation (G(ST) = 0.056) were observed at ACCase. However, haplotype diversity within samples decreased with the frequency of ACCase-based resistance. We suggest that the genetic structure of black-grass is affected by its recent expansion as a weed. Our data demonstrate that the strong selection imposed by herbicides did not modify the genome-wide genetic structure of an allogamous weed that probably has large effective population sizes. Our study gives keys to a better understanding of the evolution of successful, noxious weeds in modern agriculture.

SNP markers for black-grass ( Alopecurus myosuroides Huds.) genotypes resistant to acetyl CoA-carboxylase inhibiting herbicides.

Chloroplastic acetyl CoA-carboxylase (ACCase) is the target of widely used, specific graminicide herbicides: cyclohexanediones (CHDs) and aryloxyphenoxypropionates (APPs). Resistance to these compounds is a worldwide, increasing problem. Population genetic studies aimed at understanding the dynamics of this situation and the diffusion of resistance genes within and between weed populations are challenging because biological assays are not adequate for this purpose, and because different mechanisms of resistance confer a similar resistance phenotype. Molecular markers for specifically detecting resistance genes are therefore urgently needed to conduct such studies. For this purpose, we cloned and sequenced the whole gene encoding chloroplastic ACCase in Alopecurus myosuroides Huds. (Black-grass). We identified two point mutations at nucleotide 5,341 that both cause an isoleucine-leucine substitution at position 1,781. Three bi-directional allele-specific PCR assays were developed, each detecting two distinct ACCase alleles with a single PCR reaction. The sensitivity of 1,190 seedlings of A. myosuroides to one CHD and one APP was determined. Genotyping revealed that, although resistant plants were only selected by APPs, the (1,781)Leu ACCase allele is a widespread, dominant gene of resistance to both APPs and CHDs. No other ACCase allele associated with resistance could be identified in this work. Useful applications of allele-specific PCR markers are population genetic studies as well as routine molecular diagnosis of herbicide resistance.

PCR assays that identify the grapevine dieback fungus Eutypa lata.

Eutypa lata is the causal fungal agent of Eutypa dieback, a serious grapevine necrotic disease. The erratic and delayed (1 to 2 months) appearance of characteristic conidia on culture media and the presence of numerous microorganisms in decaying wood make it difficult either to identify or to detect E. lata in grapevine wood samples. We designed six pairs of PCR primers for diagnosis of E. lata. Three primer pairs were derived from ribosomal DNA internal transcribed spacer sequences, and three pairs were derived from randomly amplified polymorphic DNA fragments. The six primer pairs could be used to amplify DNAs extracted from all of the E. lata isolates tested. They did not amplify DNAs from fungi and bacteria representing more than 50 different species of microorganisms associated with grapevine. We developed a simple protocol, leading to a rapid release of DNA, that enabled us to identify E. lata from pure or mixed cultures as well as from grapevine wood samples. Identification of E. lata in wood was achieved within a few hours, instead of the several weeks required for classical cultures on agar medium. We believe that the procedure described here can be adapted to detect other microorganisms involved in woody plant diseases.

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.