Fungal dye-decolorizing peroxidase diversity: roles in either intra- or extracellular processes.

Fungal dye-decolorizing peroxidases (DyPs) have found applications in the treatment of dye-contaminated industrial wastes or to improve biomass digestibility. Their roles in fungal biology are uncertain, although it has been repeatedly suggested that they could participate in lignin degradation and/or modification. Using a comprehensive set of 162 fully sequenced fungal species, we defined seven distinct fungal DyP clades on basis of a sequence similarity network. Sequences from one of these clades clearly diverged from all others, having on average the lower isoelectric points and hydropathy indices, the highest number of N-glycosylation sites, and N-terminal sequence peptides for secretion. Putative proteins from this clade are absent from brown-rot and ectomycorrhizal species that have lost the capability of degrading lignin enzymatically. They are almost exclusively present in white-rot and other saprotrophic Basidiomycota that digest lignin enzymatically, thus lending support for a specific role of DyPs from this clade in biochemical lignin modification. Additional nearly full-length fungal DyP genes were isolated from the environment by sequence capture by hybridization; they all belonged to the clade of the presumably secreted DyPs and to another related clade. We suggest focusing our attention on the presumably intracellular DyPs from the other clades, which have not been characterized thus far and could represent enzyme proteins with novel catalytic properties. KEY POINTS: • A fungal DyP phylogeny delineates seven main sequence clades. • Putative extracellular DyPs form a single clade of Basidiomycota sequences. • Extracellular DyPs are associated to white-rot fungi.

Datamining and functional environmental genomics reassess the phylogenetics and functional diversity of fungal monosaccharide transporters.

Sugar transporters are essential components of carbon metabolism and have been extensively studied to control sugar uptake by yeasts and filamentous fungi used in fermentation processes. Based on published information on characterized fungal sugar porters, we show that this protein family encompasses phylogenetically distinct clades. While several clades encompass transporters that seemingly specialized on specific "sugar-related" molecules (e.g., myo-inositol, charged sugar analogs), others include mostly either mono- or di/oligosaccharide low-specificity transporters. To address the issue of substrate specificity of sugar transporters, that protein primary sequences do not fully reveal, we screened "multi-species" soil eukaryotic cDNA libraries for mannose transporters, a sugar that had never been used to select transporters. We obtained 19 environmental transporters, mostly from Basidiomycota and Ascomycota. Among them, one belonged to the unusual "Fucose H+ Symporter" family, which is only known in Fungi for a rhamnose transporter in Aspergillus niger. Functional analysis of the 19 transporters by expression in yeast and for two of them in Xenopus laevis oocytes for electrophysiological measurements indicated that most of them showed a preference for D-mannose over other tested D-C6 (glucose, fructose, galactose) or D-C5 (xylose) sugars. For the several glucose and fructose-negative transporters, growth of the corresponding recombinant yeast strains was prevented on mannose in the presence of one of these sugars that may act by competition for the binding site. Our results highlight the potential of environmental genomics to figure out the functional diversity of key fungal protein families and that can be explored in a context of biotechnology. KEY POINTS: • Most fungal sugar transporters accept several sugars as substrates. • Transporters, belonging to 2 protein families, were isolated from soil cDNA libraries. • Environmental transporters featured novel substrate specificities.

Multi-metal tolerance of DHHC palmitoyl transferase-like protein isolated from metal contaminated soil.

The microbiota inhabiting in metal polluted environment develops strong defense mechanisms to combat pollution and sustain life. Investigating the functional genes of the eukaryotic microbiota inhabiting in these environments by using metatranscriptomics approach was the focus of this study. Size fractionated eukaryotic cDNA libraries (library A, < 0.5 kb, library B, 0.5-1.0 kb, and library C, > 1.0 kb) were constructed from RNA isolated from the metal contaminated soil. The library C was screened for Cadmium (Cd) tolerant genes by using Cd sensitive yeast mutant ycf1Δ by functional complementation assay, which yielded various clones capable of growing in Cd amended media. One of the Cd tolerant clones, PLCg39 was selected because of its ability to grow at high concentrations of Cd. Sequence analysis of PLCg39 showed homology with DHHC palmitoyl transferases, which are responsible for addition of palmitoyl groups to proteins and usually possess metal coordination domains. The cDNA PLCg39 was able to confer tolerance to Cd-sensitive (ycf1Δ), Copper-sensitive (cup1Δ) and Zn-sensitive (zrc1Δ) yeast mutants when grown at different concentrations of Cd (40-100 µM), Cu (150-1000 µM) and Zn (10-13 mM), respectively. The DHHC mutant akr1Δ transformed with PLCg39 rescued from the metal sensitivity indicating the role of DHHC palmitoyl transferase in metal tolerance. This study demonstrated that PLCg39 acts as a potential metal tolerant gene which could be used in bioremediation, biosensing and other biotechnological fields.

The ectomycorrhizal basidiomycete Hebeloma cylindrosporum undergoes early waves of transcriptional reprogramming prior to symbiotic structures differentiation.

To clarify the early molecular interaction between ectomycorrhizal partners, we performed a RNA-Seq study of transcriptome reprogramming of the basidiomycete Hebeloma cylindrosporum before symbiotic structure differentiation with Pinus pinaster. Mycorrhiza transcriptome was studied for comparison. By reference to asymbiotic mycelium, 47 and 46 genes were specifically upregulated over fivefold (p ≤ 0.05) upon rhizosphere colonization and root adhesion respectively. Other 45 were upregulated throughout the symbiotic interaction, from rhizosphere colonization to differentiated mycorrhizas, whereas 274 were specifically upregulated in mycorrhizas. Although exoproteome represents 5.6% of H. cylindrosporum proteome, 38.5% of the genes upregulated upon pre-infectious root colonization encoded extracellular proteins. The proportion decreased to 23.5% in mycorrhizas. At all studied time points, mycorrhiza-induced small secreted proteins (MiSSPs), representing potential effectors, were over-represented among upregulated genes. This was also the case for carbohydrate-active enzymes (CAZymes). Several CAZymes were upregulated at all studied stages of the interaction. Consistent with a role in fungal morphogenesis and symbiotic interface differentiation, CAZymes over-expressed before and upon root attachment targeted fungal and both fungal and plant polysaccharides respectively. Different hydrophobins were upregulated upon early root adhesion, in mycorrhizas or throughout interaction. The functional classification of genes upregulated only in mycorrhizas pointed to intense metabolic activity and nutritional exchanges.

RNA extraction from decaying wood for (meta)transcriptomic analyses.

Wood decomposition is a key step of the terrestrial carbon cycle and is of economic importance. It is essentially a microbiological process performed by fungi and to an unknown extent by bacteria. To gain access to the genes expressed by the diverse microbial communities participating in wood decay, we developed an RNA extraction protocol from this recalcitrant material rich in polysaccharides and phenolic compounds. This protocol was implemented on 22 wood samples representing as many tree species from 11 plant families in the Angiosperms and Gymnosperms. RNA was successfully extracted from all samples and converted into cDNAs from which were amplified both fungal and bacterial protein coding genes, including genes encoding hydrolytic enzymes participating in lignocellulose hydrolysis. This protocol applicable to a wide range of decomposing wood types represents a first step towards a metatranscriptomic analysis of wood degradation under natural conditions.

Metal induction of a Pisolithus albus metallothionein and its potential involvement in heavy metal tolerance during mycorrhizal symbiosis.

Metallothioneins (MTs) are small, cysteine-rich peptides involved in intracellular sequestration of heavy metals in eukaryotes. We examined the role in metal homeostasis and detoxification of an MT from the ectomycorrhizal fungus Pisolithus albus (PaMT1). PaMT1 encodes a 35 amino acid-long polypeptide, with 7 cysteine residues; most of them part of a C-x-C motif found in other known basidiomycete MTs. The expression levels of PaMT1 increased as a function of increased external Cu and Cd concentrations and were higher with Cu than with Cd. Heterologous complementation assays in metal-sensitive yeast mutants indicated that PaMT1 encodes a polypeptide capable of conferring higher tolerance to both Cu and Cd. Eucalyptus tereticornis plantlets colonized with P. albus grown in the presence of Cu and Cd showed better growth compared with those with non-mycorrhizal plants. Higher PaMT1 expression levels were recorded in mycorrhizal plants grown in the presence of Cu and Cd compared with those in control mycorrhizal plants not exposed to heavy metals. These data provide the first evidence to our knowledge that fungal MTs could protect ectomycorrhizal fungi from heavy metal stress and in turn help the plants to establish in metal-contaminated sites.

Comparative genomics, proteomics and transcriptomics give new insight into the exoproteome of the basidiomycete Hebeloma cylindrosporum and its involvement in ectomycorrhizal symbiosis.

Extracellular proteins play crucial roles in the interaction between mycorrhizal fungi and their environment. Computational prediction and experimental detection allowed identification of 869 proteins constituting the exoproteome of Hebeloma cylindrosporum. Small secreted proteins (SSPs) and carbohydrate-active enzymes (CAZymes) were the two major classes of extracellular proteins. Twenty-eight per cent of the SSPs were secreted by free-living mycelia and five of the 10 most abundant extracellular proteins were SSPs. By contrast, 63-75% of enzymes involved in nutrient acquisition were secreted. A total of 150 extracellular protein-coding genes were differentially expressed between mycorrhizas and free-living mycelia. SSPs were the most affected. External environmental conditions also affected expression of 199 exoproteome genes in mycorrhizas. SSPs displayed different patterns of regulation in response to presence of a host plant or other environmental signals. Several of the genes most overexpressed in the presence of organic matter encoded oxidoreductases. Hebeloma cylindrosporum has not fully lost its ancestral saprotrophic capacities but rather adapted them not to harm its hosts and to use soil organic nitrogen. The complex and divergent patterns of regulation of SSPs in response to a symbiotic partner and/or organic matter suggest various roles in the biology of mycorrhizal fungi.

A fungal conserved gene from the basidiomycete Hebeloma cylindrosporum is essential for efficient ectomycorrhiza formation.

We used Agrobacterium-mediated insertional mutagenesis to identify genes in the ectomycorrhizal fungus Hebeloma cylindrosporum that are essential for efficient mycorrhiza formation. One of the mutants presented a dramatically reduced ability to form ectomycorrhizas when grown in the presence of Pinus pinaster. It failed to form mycorrhizas in the presence of glucose at 0.5 g liter(-1), a condition favorable for mycorrhiza formation by the wild-type strain. However, it formed few mycorrhizas when glucose was replaced by fructose or when glucose concentration was increased to 1 g liter(-1). Scanning electron microscopy examination of these mycorrhizas revealed that this mutant was unable to differentiate true fungal sheath and Hartig net. Molecular analyses showed that the single-copy disrupting T-DNA was integrated 6,884 bp downstream from the start codon, of an open reading frame potentially encoding a 3,096-amino-acid-long protein. This gene, which we named HcMycE1, has orthologs in numerous fungi as well as different other eukaryotic microorganisms. RNAi inactivation of HcMycE1 in the wild-type strain also led to a mycorrhizal defect, demonstrating that the nonmycorrhizal phenotype of the mutant was due to mutagenic T-DNA integration in HcMycE1. In the wild-type strain colonizing P. pinaster roots, HcMycE1 was transiently upregulated before symbiotic structure differentiation. Together with the inability of the mutant to differentiate these structures, this suggests that HcMycE1 plays a crucial role upstream of the fungal sheath and Hartig net differentiation. This study provides the first characterization of a fungal mutant altered in mycorrhizal ability.

Construction of sized eukaryotic cDNA libraries using low input of total environmental metatranscriptomic RNA.

BACKGROUND: Construction of high quality cDNA libraries from the usually low amounts of eukaryotic mRNA extracted from environmental samples is essential in functional metatranscriptomics for the selection of functional, full-length genes encoding proteins of interest. Many of the inserts in libraries constructed by standard methods are represented by truncated cDNAs due to premature stoppage of reverse transcriptase activity and preferential cloning of short cDNAs. RESULTS: We report here a simple and cost effective technique for preparation of sized eukaryotic cDNA libraries from as low as three microgram of total soil RNA dominated by ribosomal and bacterial RNA. cDNAs synthesized by a template switching approach were size-fractionated by two dimensional agarose gel electrophoresis prior to PCR amplification and cloning. Effective size selection was demonstrated by PCR amplification of conserved gene families specific of each size class. Libraries of more than one million independent inserts whose sizes ranged between one and four kb were thus produced. Up to 80% of the insert sequences were homologous to eukaryotic gene sequences present in public databases. CONCLUSIONS: A simple and cost effective technique has been developed to construct sized eukaryotic cDNA libraries from environmental samples. This technique will facilitate expression cloning of environmental eukaryotic genes and contribute to a better understanding of basic biological and/or ecological processes carried out by eukaryotic microbial communities.

Paleomicrobiology: Tracking the past microbial life from single species to entire microbial communities.

By deciphering information encoded in degraded ancient DNA extracted from up to million-years-old samples, molecular paleomicrobiology enables to objectively retrace the temporal evolution of microbial species and communities. Assembly of full-length genomes of ancient pathogen lineages allows not only to follow historical epidemics in space and time but also to identify the acquisition of genetic features that represent landmarks in the evolution of the host-microbe interaction. Analysis of microbial community DNA extracted from essentially human paleo-artefacts (paleofeces, dental calculi) evaluates the relative contribution of diet, lifestyle and geography on the taxonomic and functional diversity of these guilds in which have been identified species that may have gone extinct in today's human microbiome. As for non-host-associated environmental samples, such as stratified sediment cores, analysis of their DNA illustrates how and at which pace microbial communities are affected by local or widespread environmental disturbance. Description of pre-disturbance microbial diversity patterns can aid in evaluating the relevance and effectiveness of remediation policies. We finally discuss how recent achievements in paleomicrobiology could contribute to microbial biotechnology in the fields of medical microbiology and food science to trace the domestication of microorganisms used in food processing or to illustrate the historic evolution of food processing microbial consortia.

Identification of DNA Viruses in Ancient DNA from Herbarium Samples.

Herbaria encompass millions of plant specimens, mostly collected in the nineteenth and twentieth centuries that can represent a key resource for investigating the history and evolution of phytopathogens. In the last years, the application of high-throughput sequencing technologies for the analysis of ancient nucleic acids has revolutionized the study of ancient pathogens including viruses, allowing the reconstruction of historical genomic viral sequences, improving phylogenetic based molecular dating, and providing essential insight into plant virus ecology. In this chapter, we describe a protocol to reconstruct ancient plant and soil viral sequences starting from highly fragmented ancient DNA extracted from herbarium plants and their associated rhizospheric soil. Following Illumina high-throughput sequencing, sequence data are de novo assembled, and DNA viral sequences are selected, according to their similarity with known viruses.

Soil metatranscriptomics for mining eukaryotic heavy metal resistance genes.

Heavy metals are pollutants which affect all organisms. Since a small number of eukaryotes have been investigated with respect to metal resistance, we hypothesize that many genes that control this phenomenon remain to be identified. This was tested by screening soil eukaryotic metatranscriptomes which encompass RNA from organisms belonging to the main eukaryotic phyla. Soil-extracted polyadenylated mRNAs were converted into cDNAs and 35 of them were selected for their ability to rescue the metal (Cd or Zn) sensitive phenotype of yeast mutants. Few of the genes belonged to families known to confer metal resistance when overexpressed in yeast. Several of them were homologous to genes that had not been studied in the context of metal resistance. For instance, the BOLA ones, which conferred cross metal (Zn, Co, Cd, Mn) resistance may act by interfering with Fe homeostasis. Other genes, such as those encoding 110- to 130-amino-acid-long, cysteine-rich polypeptides, had no homologues in databases. This study confirms that functional metatranscriptomics represents a powerful approach to address basic biological processes in eukaryotes. The selected genes can be used to probe new pathways involved in metal homeostasis and to manipulate the resistance level of selected organisms.

Herbaria preserve plant microbiota responses to environmental changes.

Interaction between plants and their microbiota is a central theme to understand adaptation of plants to their environment. Considering herbaria as repositories of holobionts that preserved traces of ancient plant-associated microbial communities, we propose to explore these historical collections to evaluate the impact of long-lasting global changes on plant-microbiota interactions.

Optimized assay and storage conditions for enzyme activity profiling of ectomycorrhizae.

The aim of a joint effort by different research teams was to provide an improved procedure for enzyme activity profiling of field-sampled ectomycorrhizae, including recommendations on the best conditions and maximum duration for storage of ectomycorrhizal samples. A more simplified and efficient protocol compared to formerly published procedures was achieved by using manufactured 96-filter plates in combination with a vacuum manifold and by optimizing incubation times. Major improvements were achieved by performing the series of eight enzyme assays with a single series of root samples instead of two series, reducing the time needed for sample preparation, minimizing error-prone steps such as pipetting and morphotyping, and facilitating subsequent DNA analyses due to the reduced sequencing effort. The best preservation of samples proved to be storage in soil at 4-6 °C in the form of undisturbed soil cores containing roots. Enzyme activities were maintained for up to 4 weeks under these conditions. Short-term storage of washed roots and ectomycorrhizal tips overnight in water did not cause substantial changes in enzyme activity profiles. No optimal means for longer-term storage by freezing at -20 °C or storage in 100% ethanol were recommended.

Protection from metal toxicity by Hsp40-like protein isolated from contaminated soil using functional metagenomic approach.

Pollution in the environment due to accumulation of potentially toxic metals results in deterioration of soil and water quality, thus impacting health of all living organisms including microbes. In the present investigation, a functional metagenomics approach was adopted to mine functional genes involved in metal tolerance from potentially toxic metal contaminated site. Eukaryotic cDNA library (1.0-4.0 kb) was screened for the genes providing tolerance to cadmium (Cd) toxicity through a functional complementation assay using Cd-sensitive Saccharomyces cerevisiae mutant ycf1Δ. Out of the 98 clones able to recover growth on Cd-supplemented selective medium, one clone designated as PLCc43 showed more tolerance to Cd along with some other clones. Sequence analysis revealed that cDNA PLCc43 encodes a 284 amino acid protein harbouring four characteristic zinc finger motif repeats (CXXCXGXG) and showing partial homology with heat shock protein (Hsp40) of Acanthamoeba castellanii. qPCR analysis revealed the induction of PLCc43 in the presence of Cd, which was further supported by accumulation of Cd in ycf1Δ/PLCc43 mutant. Cu-sensitive (cup1Δ), Zn-sensitive (zrc1Δ) and Co-sensitive (cot1Δ) yeast mutant strains were rescued from sensitivity when transformed with cDNA PLCc43 indicating its ability to confer tolerance to various potentially toxic metals. Oxidative stress tolerance potential of PLCc43 was also confirmed in the presence of H2O2. Present study results suggest that PLCc43 originating from a functional eukaryotic gene of soil community play an important role in detoxification of potentially toxic metals and may be used as biomarker in various contaminated sites.

Exploring the Diversity of Fungal DyPs in Mangrove Soils to Produce and Characterize Novel Biocatalysts.

The functional diversity of the New Caledonian mangrove sediments was examined, observing the distribution of fungal dye-decolorizing peroxidases (DyPs), together with the complete biochemical characterization of the main DyP. Using a functional metabarcoding approach, the diversity of expressed genes encoding fungal DyPs was investigated in surface and deeper sediments, collected beneath either Avicennia marina or Rhizophora stylosa trees, during either the wet or the dry seasons. The highest DyP diversity was observed in surface sediments beneath the R. stylosa area during the wet season, and one particular operational functional unit (OFU1) was detected as the most abundant DyP isoform. This OFU was found in all sediment samples, representing 51-100% of the total DyP-encoding sequences in 70% of the samples. The complete cDNA sequence corresponding to this abundant DyP (OFU 1) was retrieved by gene capture, cloned, and heterologously expressed in Pichia pastoris. The recombinant enzyme, called DyP1, was purified and characterized, leading to the description of its physical-chemical properties, its ability to oxidize diverse phenolic substrates, and its potential to decolorize textile dyes; DyP1 was more active at low pH, though moderately stable over a wide pH range. The enzyme was very stable at temperatures up to 50 °C, retaining 60% activity after 180 min incubation. Its ability to decolorize industrial dyes was also tested on Reactive Blue 19, Acid Black, Disperse Blue 79, and Reactive Black 5. The effect of hydrogen peroxide and sea salt on DyP1 activity was studied and compared to what is reported for previously characterized enzymes from terrestrial and marine-derived fungi.

Molecular evidence for widespread occurrence of Foraminifera in soils.

Environmental SSU rDNA-based surveys are contributing to the dramatic revision of eukaryotic high-level diversity and phylogeny as the number of sequence data increases. This ongoing revolution gives the opportunity to test for the presence of some eukaryotic taxa in environments where they have not been found using classical microscopic observations. Here, we test whether the foraminifera, a group of single-celled eukaryotes, considered generally as typical for the marine ecosystems are present in soil. We performed foraminiferal-specific nested PCR on 20 soil DNA samples collected in contrasted environments. Unexpectedly, we found that the majority of the samples contain foraminiferal SSU rDNA sequences. In total, we obtained 49 sequences from 17 localities. Phylogenetic analysis clusters them in four groups branching among the radiation of early foraminiferal lineages. Three of these groups also include sequences originated from previous freshwater surveys, suggesting that there were up to four independent colonization events of terrestrial and/or freshwater ecosystems by ancestral foraminifera. As shown by our data, foraminifera are a widespread and diverse component of soil microbial communities. Yet, identification of terrestrial foraminiferal species and understanding of their ecological role represent an exciting challenge for future research.

Metabarcoding on both environmental DNA and RNA highlights differences between fungal communities sampled in different habitats.

In recent years, metabarcoding has become a key tool to describe microbial communities from natural and artificial environments. Thanks to its high throughput nature, metabarcoding efficiently explores microbial biodiversity under different conditions. It can be performed on environmental (e)DNA to describe so-called total microbial community, or from environmental (e)RNA to describe active microbial community. As opposed to total microbial communities, active ones exclude dead or dormant organisms. For what concerns Fungi, which are mostly filamentous microorganisms, the relationship between DNA-based (total) and RNA-based (active) communities is unclear. In the present study, we evaluated the consequences of performing metabarcoding on both soil and wood-extracted eDNA and eRNA to delineate molecular operational taxonomic units (MOTUs) and differentiate fungal communities according to the environment they originate from. DNA and RNA-based communities differed not only in their taxonomic composition, but also in the relative abundances of several functional guilds. From a taxonomic perspective, we showed that several higher taxa are globally more represented in either "active" or "total" microbial communities. We also observed that delineation of MOTUs based on their co-occurrence among DNA and RNA sequences highlighted differences between the studied habitats that were overlooked when all MOTUs were considered, including those identified exclusively by eDNA sequences. We conclude that metabarcoding on eRNA provides original functional information on the specific roles of several taxonomic or functional groups that would not have been revealed using eDNA alone.

The dominant Hc.Sdh (R) carboxin-resistance gene of the ectomycorrhizal fungus Hebeloma cylindrosporum as a selectable marker for transformation.

In an attempt to get a marker gene suitable for genetical transformation of the ectomycorrhizal fungus Hebeloma cylindrosporum, the gene Hc.Sdh (R) that confers carboxin-resistance was isolated from a UV mutant of this fungus. It encodes a mutant allele of the Fe-S subunit of the succinate dehydrogenase gene that carries a single amino acid substitution known to confer carboxin-resistance. This gene was successfully used as the selective marker to transform, via Agrobacterium tumefaciens, monokaryotic and dikaryotic strains of H. cylindrosporum. We also successfully transformed hygromycin-resistant insertional mutants. Transformation yielded mitotically stable carboxin-resistant mycelia. This procedure produced transformants, the growth of which was not affected by 2 microg l(-1) carboxin, whereas wild-type strains were unable to grow in the presence of 0.1 microg l(-1) of this fungicide. This makes the carboxin-resistance cassette much more discriminating than the hygromycin-resistance one. PCR amplification and Southern blot hybridisation indicated that more than 90% of the tested carboxin-resistant mycelia contained the Hc.Sdh (R) cassette, usually as a single copy. The AGL-1 strain of A. tumefaciens was a much less efficient donor than LBA 1126; the former yielded ca. 0-30% transformation frequency, depending on fungal strain and resistance cassette used, whereas the latter yielded ca. 60-95%.

Monitoring of Venturia inaequalis harbouring the QoI resistance G143A mutation in French orchards as revealed by PCR assays.

BACKGROUND: Genetic resistance to QoI fungicides may account for recent failures to control Venturia inaequalis (Cooke) Winter in French orchards. Two PCR-based assays were developed to detect the G143A point mutation in the fungal mitochondrial cytochrome b gene. The mutation is known to confer a high level of resistance to QoI fungicides. Occurrence of the G143A mutation in French field isolates collected from 2004 to 2007 was monitored. RESULTS: The QoI-resistant cytochrome b allele was specifically detected either following the cleavage of the amplified marker by a restriction endonuclease (CAPS assay) or its amplification using an allele-specific PCR primer. Using either method, the G143A mutation was found in 42% of the 291 field samples originating from French orchards in which apple scab proved difficult to be controlled. Monitoring of the G143A mutation in orchards located in 15 French administrative regions indicated that the mutation was detected at least once in nine of the regions, and its presence ranged from 33% to 64% of the orchards analysed in 2004 and in 2007 respectively. CONCLUSION: The PCR-based methods developed in this study efficiently reveal the presence of the G143A mutation in French V. inaequalis field populations.