Alexandre Fediaevsky
email: alexandre.fediaevsky@agriculture.gouv.fr

Didier Calavas

Patrick Gasqui

Katayoun Moazami-Goudarzi

Pascal Laurent

Jean- Noël Arsac

Christian Ducrot

Carole Moreno

Quantitative estimation of genetic risk for atypical scrapie in French sheep and potential consequences of the current breeding programme for resistance to scrapie on the risk of atypical scrapie

d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

Background

Bovine spongiform encephalopathy (BSE) is a zoonotic transmissible spongiform encephalopathy (TSE), which may have spread to sheep and goat populations. This situation has prompted European countries to organise control and surveillance of TSEs in small ruminants in which BSE cannot be clinically distinguished from scrapie.

In sheep, genetic susceptibility to classical scrapie (CS) is supported by polymorphic variations at codons 136, 154 and 171 of the PRNP gene, which encodes the normal cellular protein prion (PrP C ) [START_REF] Dawson | Guidance on the use of PrP genotyping as an aid to the control of clinical Scrapie[END_REF]. The main alleles defined by these three codons can be classified by increasing risk as follows ARR < AHQ < ARQ ≈ ARH < VRQ [START_REF] Palhière | Allele frequencies of the PrP gene in 29 French sheep breeds. Possible use in selection for resistance to scrapie[END_REF]. This knowledge has made it possible to implement eradication programmes throughout Europe based on positive selection of the most resistant allele (ARR) and negative selection of the most susceptible one (VRQ) [START_REF] Palhière | Allele frequencies of the PrP gene in 29 French sheep breeds. Possible use in selection for resistance to scrapie[END_REF][START_REF]Commission Regulation (EC) No 999/2001 of the European Parliament and of the Council of 22 May 2001 laying down rules for the prevention, control and eradication[END_REF].

Recently, an atypical form of scrapie (AS) has been detected and has challenged such programmes since animals genetically resistant to CS are affected [START_REF] Efsa | Opinion of the scientific panel on biological hazards on classification of atypical transmissible spongiform encephalopathy (TSE) cases in small ruminants[END_REF][START_REF] Benestad | Cases of scrapie with unusual features in Norway and designation of a new type Nor98[END_REF][START_REF] Efsa | Opinion of the Scientific Panel on Biological Hazards of the European Food Safety Authority on the Breeding programme for TSE resistance in sheep[END_REF][START_REF] Baylis | Transmissible spongiform encephalopathies: scrapie control under new strain[END_REF]. However, although it is generally accepted that CS is an infectious and contagious disease [START_REF] Detwiler | The epidemiology of scrapie[END_REF], the contagiousness of AS is questioned. Indeed, the marker specific for AS disease is not detected outside the central nervous system [START_REF] Benestad | Cases of scrapie with unusual features in Norway and designation of a new type Nor98[END_REF][START_REF] Hopp | A case-control study of scrapie Nor98 in Norwegian sheep flocks[END_REF][START_REF] Green | Demographic risk factors for classical and atypical scrapie in Great Britain[END_REF][START_REF] Fediaevsky | A descriptive study of the prevalence of atypical and classical scrapie in sheep in 20 European countries[END_REF][START_REF] Luhken | Epidemiological and genetical differences between classical and atypical scrapie cases[END_REF][START_REF] Fediaevsky | A case-control study on the origin of atypical scrapie in sheep, France[END_REF][START_REF] Fediaevsky | Discrepant epidemiological patterns between classical and atypical scrapie in sheep flocks under French TSE control measures[END_REF], even in cases where AS has been experimentally transmitted to transgenic mice [START_REF] Dur | A newly identified type of scrapie agent can naturally infect sheep with resistant PrP genotypes[END_REF] and sheep [START_REF] Simmons | Experimental transmission of atypical scrapie to sheep[END_REF].

Furthermore, most AS cases have been observed in animals with genotypes showing low susceptibility to CS. Several studies have shown that AS susceptibility is highly associated with PrP codons 141 (L/F) and 154 (R/ H) [START_REF] Moum | Polymorphisms at codons 141 and 154 in the ovine prion protein gene are associated with scrapie Nor98 cases[END_REF][START_REF] Benestad | Atypical/Nor98 scrapie: properties of the agent, genetics, and epidemiology[END_REF]. It is important to note that AS cases have been detected in ALRR carriers but rarely in VLRQ carriers.

A descriptive study has reported that the estimation of AS prevalence in Europe is similar, whatever the country or tested population and amounts to about 6 cases per 10,000 tests. Conversely, CS prevalence appears to be heterogeneous both between countries and, within countries, between fallen stock and healthy slaughter [START_REF] Fediaevsky | A descriptive study of the prevalence of atypical and classical scrapie in sheep in 20 European countries[END_REF]. This could hide a relatively high prevalence of AS in the most susceptible genotypes. In addition, estimation of the prevalence of AS in ALRR-ALRR animals is strategically important to assess the impact of current breeding programmes on the AS prevalence in the general population. In France, breeders of flocks participating in breeding programmes for CS resistance are encouraged to join in a voluntary scrapie certification scheme, for which it is also compulsory to send animals to breed selection centres (Internal circular from French Ministry of Agriculture, DGAL/SDSPA/N2006-8093). This certification scheme implies testing all fallen stock over 18 months, which increases the risk of detecting AS cases in these flocks compared to general flocks.

We have carried out a case control study by using data from the TSEs surveillance programme to estimate the risk of AS associated with the PrP genotypes in the French sheep population and to compare it with the risk of CS. In addition, we have used our dataset to estimate the prevalence of AS per genotype and the risk for a fully CS resistant flock (100% ALRR-ALRR) to be detected positive for AS under certain assumptions.

Methods

Selection of cases

Cases were recruited among the animals tested for TSEs in France from 1 st January 2002 to 31 st December 2007. Each year since 2002, a number of adult (over 18 months old) sheep from unknown TSE status flocks was randomly selected for TSE detection at abattoirs (healthy slaughter) or at rendering plants (fallen stock). Details concerning the French active surveillance programme are described in Cazeau [START_REF] Cazeau | Prévalence des formes classique et atypique de tremblante chez les petits ruminants en France entre 2002 et 2006[END_REF]. Between 2002 and 2007, 859,157 samples were analysed within this programme, among which 532,500 were tested with the recommended tests for AS detection [START_REF]European Food Safety Autority: Scientific Report of the European Food Safety Authority on the Evaluation of Rapid post mortem TSE Tests intended for Small Ruminants[END_REF][START_REF]European Food Safety Autority: Scientific Report of the European Food Safety Authority on the Evaluation of Rapid post mortem TSE Tests intended for Small Ruminants (2)[END_REF].

All positive samples were confirmed and typed by the National reference laboratory for animal TSEs of AFSSA Lyon using EU approved reference methods [START_REF] Arsac | Similar biochemical signatures and prion protein genotypes in atypical scrapie and Nor98 cases, France and Norway[END_REF]. For all positive cases the presence of BSE was dismissed by discriminatory tests; the cases detected with the tests recommended for AS were typed in order to classify them as AS or CS; other positive cases were assumed to be CS. During that period, 404 cases of AS and 395 cases of CS were confirmed. Among these cases, DNA was available or could be extracted from the central nervous system samples kept at AFSSA Lyon for respectively 248 AS and 245 CS cases.

Genotyping of CS and AS cases

During that period, the PrP genotype of all cases was routinely determined, when suitable samples were available, at codons 136, 154 and 171 with a technique that could not differentiate ARH and ARQ alleles. Further analyses were conducted to specify alleles at position 141 and 171 by the Taq man method [START_REF] Livak | Towards fully automated genome-wide polymorphism screening[END_REF][START_REF] Kwok | High-throughput genotyping assay approaches[END_REF]. It is generally agreed that this method provides accurate, reproducible and reliable results. For example, in human large-scale association studies, the error rate is estimated to be less than 0.3% [START_REF] Olivier | High-throughput genotyping of single nucleotide polymorphisms using new biplex invader technology[END_REF] or even 0.05% [START_REF] Ranade | Highthroughput genotyping with single nucleotide polymorphisms[END_REF]. These analyses were conducted by the National reference laboratory for PRNP genotyping (Labogena, Jouy-en-Josas, France) which is certified for this test by the French standard COFRAC (NF EN ISO/CEI 17025). PrP polymorphisms at codons 136, 141, 154 and 171 of the 248 AS cases were analysed after direct sequencing of the coding region of the PRP gene encompassing codons 92-282. These analyses were conducted by INRA (Jouy-en-Josas, France). For more details concerning primers and sequencing see [START_REF] Moreno | Which PrP haplotypes in a French sheep population are the most susceptible to atypical scrapie?[END_REF].

Selection of controls

Controls were recruited among animals that were tested for the presence of TSEs with a negative result. Controls for AS were animals tested with one of the tests recommended for the detection of AS and controls for CS were animals tested with any rapid test.

Genotyping of controls

As a legal requirement [START_REF]Commission Regulation (EC) No 999/2001 of the European Parliament and of the Council of 22 May 2001 laying down rules for the prevention, control and eradication[END_REF], during the TSE active surveillance programme 2002-2007, a subset of 3,347 animals was randomly selected for PrP genotyping. We used this sample as genotype controls. The genotyping of this control dataset was conducted by Labogena at codons 136, 154 and 171 with the limits specified before (missing information for codons 141 and 171). No significant difference of PrP frequencies at codons 136, 154 and 171 was observed between the present control dataset and estimates from an external data set of the general French ovine population [START_REF] Moreno | Which PrP haplotypes in a French sheep population are the most susceptible to atypical scrapie?[END_REF]. In addition, distributions in both datasets were in Hardy-Weinberg equilibrium. For these two reasons, we inferred F141 and H171 information from the external data set to our control data set. In order to take into account a possible uncertainty on the estimation of F141 and H171 in the external dataset, we considered that the estimated values for F141 and H171 were random variables with beta-multinomial distributions with means and variances equalling those estimated from the external dataset. Finally, we took into account that uncertainty in the inference process by bootstrapping 1,000 times the control dataset.

Demographic data

In order to adjust for potential confounding variables, we collected information on the stream of surveillance (healthy slaughter/fallen stock) and the dentition of the animals (2-4 definitive incisors (DI)/5-7 DI/8 DI) which is a poor proxy for age [START_REF] Cocquyt | Variability in the eruption of the permanent incisor teeth in sheep[END_REF] but was the only information available. This led to exclude some animals for which information was missing (two cases of CS and nine controls).

Multivariate analysis

For each type of scrapie, we fitted multivariate logistic regression models to assess the risk associated to genotypes with ALRQ-ALRQ as the baseline category, because this genotype is assumed to be the ancestral genotype. We adjusted the models on the surveillance stream with healthy slaughter as the baseline category, and on dentition, with the class corresponding to the youngest animals as the baseline category. The model outputs were odds ratio (OR) which can be interpreted as an approximation of the relative risk in the context of rare diseases. We could not estimate OR associated with the genotypes in which no case was detected. The value of the OR and their 95% confidence intervals for the 1,000 datasets were computed using the Rubin method [START_REF] Roderick | Statistical Analysis with Missing Data[END_REF].

Estimation of prevalence

We estimated the prevalence of AS for each genotype. Because the proportion of animals genotyped was different for cases and controls, we defined it as the national prevalence (404/532,500 for AS) times the proportion of the genotype among cases, divided by the frequency of the genotype in the general population. Using a Bayesian approach, we considered that each of these proportions followed a beta distribution with mean and variance estimated by the observations. We derived the median and the 95% credibility interval of prevalences based on 2,000 iterations obtained after convergence.

Estimation of the probability of case detection at flock level

We have considered the risk for a fully ALRR-ALRR flock to be detected positive for AS. We assumed that in any flock the individual risk of AS depended only on the genotype, the age and the surveillance stream. Considering that the animals tested from a given flock had the same distribution of age and surveillance stream as the general population, the average individual risk for an ALRR-ALRR animal could be estimated by the prevalence previously estimated for this genotype. Since the occurrence of AS in a flock was defined as an independent event, the number of cases in a flock, X, followed a binomial distribution. The probability for at least one animal being AS positive among n animals tested was:

Using the previous methods to estimate the prevalence per genotype, we derived the median of the probability and its 95% credibility interval for n varying from 1 to 1,000, based on 2,000 iterations obtained after convergence.

All the statistical analyses were done with R for Windows [START_REF]R: A language and environment for statistical computing[END_REF] and Winbugs [START_REF] Lunn | WinBUGS -A Bayesian modelling framework: Concepts, structure, and extensibility[END_REF].

Results

After exclusion of missing information on genotypes and dentition, 248 AS cases, 245 CS cases and 3,317 controls were used to estimate OR and prevalence (Table 1). Cases of CS and/or AS were detected in all genotypes.

There were no AS cases among the ALRR-VLRQ, ALRQ-ALRH, ALRQ-VLRQ, and VLRQ-VLRQ genotypes. The ALHQ-ALHQ, AFRQ-ALHQ and AFRQ-AFRQ genotypes were associated with the highest risks of AS compared to ALRQ-ALRQ. Within CS cases, the VLRQ-VLRQ animals presented the highest risk compared to ALRQ-ALRQ. ALRR carriers and to a lesser extent ALHQ carriers were the most resistant to CS.

The AS prevalence estimates for the most susceptible genotypes (AFRQ/ALHQ, ALHQ/ALHQ and AFRQ/ AFRQ) were respectively 11, 25 and 31 times higher than the prevalence for the general population (Table 1). The AS prevalence estimate for ALRR-ALRR animals was not significantly different from that in the general population.

The animals tested at fallen stock were significantly more at risk for CS but not for AS. Adjustment on the dentition of the animals improved the fit of the model for AS (log-likelihood ratio test of models with and without dentition: p-value <0.003), the oldest animals were more at risk but OR was not significantly different from 1 at 5%. However, this could be due to a lack of power since comparison with the intermediate category showed that the OR associated with the oldest category was significantly different from 1. For CS there was no observed effect of age. The probability for a 100% ALRR-ALRR flock to be detected as positive for AS was not negligible when 100 animals were tested (Figure 1). For instance, a flock with a constant size of 400 ALRR-ALRR adult ewes and an adult mortality rate of 5% would have 200 animals tested over a 10 year-period of time. Under these conditions, the cumulative probability over 10 years to detect at least one AS case out of 200 animals tested for TSEs would be 10%.

P X P X p p p n n ( ) ( ) .( ) ( ) ≥ = - = = - - = -- 1 1 0 1 1 1 1 0 (1)

Discussion

This study was aimed at estimating the genetic risk for AS in French sheep and potential consequences of the current breeding programme for the ARR allele on the risk of atypical scrapie. The analysis was conducted on a sheep population submitted to TSE active surveillance and we have considered the ancestral genotype ALRQ-ALRQ as the baseline reference. Our results were congruent with those from other studies either regarding ranking AS susceptibility [START_REF] Luhken | Epidemiological and genetical differences between classical and atypical scrapie cases[END_REF][START_REF] Moum | Polymorphisms at codons 141 and 154 in the ovine prion protein gene are associated with scrapie Nor98 cases[END_REF][START_REF] Moreno | Which PrP haplotypes in a French sheep population are the most susceptible to atypical scrapie?[END_REF][START_REF] Saunders | PrP genotypes of atypical scrapie cases in Great Britain[END_REF] or AS genotype specific prevalence [START_REF] Mcintyre | No temporal trends in the prevalence of atypical scrapie in British sheep, 2002-2006[END_REF]. We detected a significant risk of AS for sheep carrying the ALRR-ALRR genotype.

The risk and prevalence of AS for AFRQ-AFRQ, AFRQ-ALHQ and ALHQ-ALHQ genotypes were the highest. Besides, AFRQ-AFRQ and ALHQ-ALHQ homozygotes had a much higher risk than AFRQ-ALRQ and ALHQ-ALRQ heterozygotes which is not indicative of a dominant effect.

The effect of the VLRQ allele towards AS was not obvious. No or very few cases of AS have been detected among VLRQ carriers, depending on the other allele, but VLRQ carriers are also relatively rare. So the apparent protective effect cannot be properly confirmed and such an effect would need to be assessed through a larger study, at the European scale for example.

Our estimations were based on a control dataset and a case dataset both originating from the population submitted to TSE active surveillance which is representative of adult animals collected at rendering plants or slaughtered for human consumption. Both datasets were randomly selected and we did not identify any selection bias linked with the exclusion of missing data within case and control datasets.

A strong assumption to establish such an estimation is that population and risk are homogenously distributed. In France, this assumption appears to be verified for AS but not for CS for which an important spatial heterogeneity is observed [START_REF] Fediaevsky | Discrepant epidemiological patterns between classical and atypical scrapie in sheep flocks under French TSE control measures[END_REF][START_REF] Fediaevsky | Does atypical scrapie pop ut at random? 12th Symposium on Veterinary Epidemiology and Economics[END_REF]. This could be due to differential exposure to CS infectious agent and/or different strains of CS having different behaviours in terms of genetic susceptibility [START_REF] Dawson | Guidance on the use of PrP genotyping as an aid to the control of clinical Scrapie[END_REF][START_REF] Hunter | Scrapie: uncertainties, biology and molecular approaches[END_REF]. For this reason, we have decided not to present genotype specific prevalence for CS. Matching cases and controls could have been an option. However that was not possible in our design, which used the subjects retrospectively selected in compulsory TSE active surveillance.

Our knowledge of the general French sheep population genotype structure was based on an extensive survey for the codons 136, 154 and 171 but it was based on an unbiased but much smaller sample for the codon 141. We took into account that difference of accuracy and the resulting uncertainty by bootstrapping the control dataset, which eventually increased the robustness of our results.

The maintenance of the polymorphism of the PRNP gene raises question. This could be explained by an absence of selection due to the late onset of clinical signs in the life of farm animals. Alternatively, it could be maintained by a balancing selection process [START_REF] Slate | Molecular evolution of the sheep prion protein gene[END_REF][START_REF] Woolhouse | A centuries-long epidemic of scrapie in British sheep?[END_REF]. The weak associations between PrP alleles and the production performances or health traits found so far [START_REF] Sweeney | The evidence of associations between prion protein genotype and production, reproduction, and health traits in sheep[END_REF] would hardly contribute to this selection. However, recent results have shown the selective advantages of susceptible PrP alleles on survival traits or on physical breed characteristics [START_REF] Sawalha | Scrapie-resistant sheep show certain coat colour characteristics[END_REF][START_REF] Sawalha | Lambs with scrapie susceptible genotypes have higher postnatal survival[END_REF]. Another explanation may be found in heterozygous advantage and/or frequency dependency. The frequency dependent selection is supported by the differences of genetic susceptibility associated to PrP alleles towards the numerous strains of scrapie that prevail in the different subpopulations. This diversity includes AS and the many strains of CS [START_REF] Bruce | TSE strain variation[END_REF]. There is also a possible advantage of heterozygous compared to homozygous individuals, specific to AS and suggested by the lower risk for AFRQ-ALHQ animals compared to ALHQ or AFRQ homozygous animals found in our study and in Great Britain [START_REF] Efsa | Opinion of the scientific panel on biological hazards of the European Food Safety Authority on the breeding programme for TSE resistance in sheep (and Annexe I, II and III)[END_REF].

The susceptibility to AS conferred by the ALRR allele questions the long term consequences of the breeding programme for CS resistance on AS prevalence in the sheep population and particularly in the flocks deeply engaged in this programme. The AS prevalence estimated for the ALRR-ALRR subpopulation (0.60‰, CI95%: 0.45-0.78) was not significantly different from the current average national prevalence (0.76‰, CI95%: 0.69-0.83). Therefore in standing conditions and assuming AS occurrence is not dependent on infectious exposure, increasing frequency of the ALRR allele in the general population should not result in an increase of the global AS prevalence in the sheep population.

Despite the low prevalence of AS in ALRR-ALRR animals, the probability of detecting a positive case is not negligible when the number of animals tested increases as shown by Figure 1. Farms fully engaged in ALRR-ALRR selection tend to test more animals due to their participation in the scrapie certification scheme, thus the probability to detect AS in their flock should be consid- ered. The consequence is that they face a risk of being placed under inadequate control measures.

The assumption of a common risk of AS in farms is supported by the homogenous distribution of the disease and similarities of prevalence per genotype in France and in Europe [START_REF] Fediaevsky | A descriptive study of the prevalence of atypical and classical scrapie in sheep in 20 European countries[END_REF][START_REF] Cazeau | Prévalence des formes classique et atypique de tremblante chez les petits ruminants en France entre 2002 et 2006[END_REF][START_REF] Mcintyre | No temporal trends in the prevalence of atypical scrapie in British sheep, 2002-2006[END_REF]. This is partly challenged by the existence of some potential risk factors, such as mineral feeding [START_REF] Hopp | A case-control study of scrapie Nor98 in Norwegian sheep flocks[END_REF][START_REF] Fediaevsky | A case-control study on the origin of atypical scrapie in sheep, France[END_REF]. However, in a recent case-control study, the farm level risk factors were much less important than the genetic factors [START_REF] Fediaevsky | A case-control study on the origin of atypical scrapie in sheep, France[END_REF]. Actually a number of studies argue for the hypothesis that AS could develop in the absence of exposure to an infectious agent [START_REF] Benestad | Cases of scrapie with unusual features in Norway and designation of a new type Nor98[END_REF][START_REF] Hopp | A case-control study of scrapie Nor98 in Norwegian sheep flocks[END_REF][START_REF] Green | Demographic risk factors for classical and atypical scrapie in Great Britain[END_REF][START_REF] Fediaevsky | A descriptive study of the prevalence of atypical and classical scrapie in sheep in 20 European countries[END_REF][START_REF] Luhken | Epidemiological and genetical differences between classical and atypical scrapie cases[END_REF][START_REF] Fediaevsky | A case-control study on the origin of atypical scrapie in sheep, France[END_REF][START_REF] Simmons | Atypical scrapie in sheep from a UK research flock which is free from classical scrapie[END_REF], even if a doubt persists given the limited knowledge on the physiopathology of AS. Genotype appears as a very strong component for risk of AS and possibly age too. To have a better understanding of their respective impact on the development of the disease it would be useful to study the age-genotype interactions. This would especially help to assess age-specific penetrance of the different genotypes and to have an insight on the survival of animals with very susceptible genotypes. Unfortunately to carry out such assessments, more detailed data on age of cases and control populations and larger populations are required. Careful monitoring of AS positive flocks as planned by new European approaches on the control of AS could help address this issue in the future [START_REF]Commission Regulation (EC) No 999/2001 of the European Parliament and of the Council of 22 May 2001 laying down rules for the prevention, control and eradication[END_REF].

Conclusion

Finally, AS occurrence appears to be heavily dependent on the PrP genotype, possibly modulated by some environmental risk factors which are to be further explored.

Even if the open reading frame of the PRNP gene has a strong influence on the occurrence of AS it might not be the unique genetic factor of susceptibility. Other regions of the PRNP or other genes could be involved either independently or in synergy and this hypothesis is also worth to be investigated.

  Quantitative estimation of genetic risk for atypical scrapie in French sheep and potential consequences of the current breeding programme for resistance to scrapie on the risk of atypical scrapie. Alexandre Fediaevsky, Didier Calavas, Patrick Gasqui, Katayoun Moazami-Goudarzi, Pascal Laurent, Jean-Noël Arsac, Christian Ducrot, Carole Moreno To cite this version: Alexandre Fediaevsky, Didier Calavas, Patrick Gasqui, Katayoun Moazami-Goudarzi, Pascal Laurent, et al.. Quantitative estimation of genetic risk for atypical scrapie in French sheep and potential consequences of the current breeding programme for resistance to scrapie on the risk of atypical scrapie.. Genetics Selection Evolution, 2010, 42, pp.14. 10.1186/1297-9686-42-14. hal-00509571

Figure 1

 1 Figure 1 Probability for a 100% ALRR-ALRR flock to have at least one AS case detected according to the number of animals tested. Plain line: median of probability, dashed lines: 95% credibility interval of probabilty

Table 1 : Risk of AS and CS according to genotypes

 1

	AS	CS

INRA, Centre de Clermont-Theix, 63122 Saint Genès Champanelle, France Full list of author information is available at the end of the article

Acknowledgements

We thank the French Ministry of Agriculture (DGAl) for its financial and technical support. This work was financially supported by French funding (Groupement d'Intérêt Scientifique PRION and Agence nationale de la recherche).

Competing interests

The authors declare that they have no competing interests.

Authors' contributions

AF, PG, CD, DC and CM conceived the study, participated in its design and coordination. AF and CM performed the strategy of the selection of animals and carried out the statistical analysis. JNA performed the scrapie status confirmation and the strain determination. PL and KMG performed the polymorphism genotyping work by sequencing. AF, PG, CD, DC, CM, JNA and KMG helped to draft the manuscript. All authors read and approved the final manuscript.

Author Details


S Éverine Fontaine

Florent Remuson

Laurence Fraissinet-Tachet

Annie Micoud

Roland Marmeisse

Delphine Melayah
email: delphine.melayah@univ-lyon1.fr

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

Keywords: Venturia inaequalis, apple scab, QoI, fungicide resistance, allele-specific PCR, molecular diagnostics

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.

INTRODUCTION

Apple scab, caused by the fungus Venturia inaequalis (Cooke) Winter, is characterised by brown to olive-coloured spots on apple tree leaves and fruits. This major disease in apple tree orchards is mainly controlled through fungicide treatments. Quinone outside inhibiting (QoI) fungicides are one of the latest families of chemicals that play an important role in plant protection against many phytopathogenic fungi including V. inaequalis. These molecules specifically inhibit cell respiration by binding at the ubiquinol oxidation centre (Qo site) of the mitochondrial cytochrome bc1 complex. 1,[START_REF] Bartlett | The strobilurin fungicides[END_REF] Resistance to QoI fungicides has been detected in several fungal pathogens such as Blumeria graminis Speer [START_REF] Sierotzki | Point mutation in cytochrome b gene conferring resistance to strobilurin fungicides in Erysiphe graminis f. sp. tritici field isolates[END_REF] and Mycosphaerella fijiensis Morelet [START_REF] Sierotzki | Mode of resistance to respiration inhibitors at the cytochrome bc1 complex of Mycosphaerella fijiensis field isolates[END_REF] or Oomycetes such as Pseudoperonospora cubensis (Burk. & Curt.) Rostosev. 5 -7 QoI resistance in V. inaequalis was first reported in 1997 in European field and experimental trials. [START_REF] Färber | Sensitivity of Venturia inaequalis to trifloxystrobin[END_REF] In France, the commercial QoI fungicides used to control V. inaequalis correspond to strobilurins. Although strobilurins have been successfully used on apple trees to control scab since 1998, the first cases of QoI resistance that occurred in French commercial apple tree orchards were reported in 2002 (unpublished results from the Service R égional Rh ône-Alpes de la Protection des Vég étaux, France). Several mechanisms leading to QoI resistance have been reported in V. inaequalis. One known mode of resistance is the metabolisation of kresoxim-methyl, a commercial strobilurin molecule, by esterases and cytochrome P450 monooxygenases. [START_REF] Jabs | New strobilurin resistance mechanism in apple scab (Venturia inaequalis)[END_REF] Under laboratory conditions, induction of the alternative respiration pathway has also been observed during conidia germination. [START_REF] Olaya | Differential responses of germinating Venturia inaequalis conidia to kresoxim-methyl[END_REF] This latter phenomenon could possibly occur in apple tree orchards and contribute to resistance in the field. [START_REF] Färber | Sensitivity of Venturia inaequalis to trifloxystrobin[END_REF] Finally, the commonly found target-site mutation referred to as the G143A mutation, which accounts for a high level of resistance to QoI in many plant pathogens, has also been identified in QoI-resistant V. inaequalis isolates. This point mutation occurs in the mitochondrial cytochrome b (Cyt b) gene and results in a change of glycine to alanine at position 143 of the protein. [START_REF] Zheng | Characterization of laboratory mutants of Venturia inaequalis resistant to the strobilurin-related fungicide kresoxim-methyl[END_REF] However, Steinfeld et al. [START_REF] Steinfeld | Sensitivity of mitochondrial respiration to different inhibitors in Venturia inaequalis[END_REF] obtained an isolate of V. inaequalis that presented a high level of resistance to QoI without the G143A mutation from an experimental orchard in Switzerland. As the alternative oxidase pathway was not found to be overexpressed in this isolate, additional mutations in genes encoding components of the cytochrome bc1 complex were suspected.

Monitoring of QoI resistance from field-collected infected material (leaves, apples) is of practical importance to evaluate the efficiency of these molecules and to reorient fungicide treatments in the corresponding orchards. Currently, detection of V. inaequalis QoI-resistant isolates relies on in vitro test systems based on spore germination on fungicide-amended media. As viability of V. inaequalis conidia recovered from commercial orchards treated with pesticides is usually low, alternative monitoring methods are needed. PCR-based detection is a valuable tool when the fungicideresistant phenotype is clearly associated with a known point mutation. This is the case for the single nucleotide polymorphism that characterises the G143A mutation in the Cyt b gene of several fungal QoI-resistant pathogens including V. inaequalis.

The objectives of this study were to develop a rapid molecular test to detect the G143A mutation in field-collected apple scab lesions and to use this method to monitor this point mutation in apple tree orchards in France. Two methods based on the amplification of a portion of the Cyt b gene were developed. The first one requires the digestion of PCR products with a restriction enzyme that specifically cleaves the amplified fragment at the site of the mutation. The second one is an allele-specific PCR detection using an oligonucleotide primer that specifically binds to the mutant sequence.

MATERIALS AND METHODS

Fungal isolates

Two strains of V. inaequalis were isolated from infected apple tree leaves and maintained in pure culture on potato dextrose agar medium (Table 1). The QoI-sensitive single-spore isolate SFR2, collected in July 2002 originated from an orchard that had never been treated with fungicides. Single-spore isolate 160, collected in June 2003 from a commercial orchard in south France, displayed a high resistance factor (RF = 283) towards kresoxim-methyl (see Section 2.4). In addition to V. inaequalis isolates, strains of Botrytis cinerea Pers., Rhizopus sp. and Penicillium sp. were used to test for the specificity of the PCR primers (Table 1).

A total of 317 field samples were collected from 2004 to 2007, and 291 were analysed (see supporting information in Table S1). They originated from commercial and experimental French orchards where strobilurin treatments were suspected to be ineffective to control the pathogen. Infected apple leaves collected from orchards that had never been treated with any fungicides were used as control QoI-sensitive field samples.

For each orchard, V. inaequalis spores were collected from 30-50 lesion-bearing leaves or fruits with a brush. Spore suspensions were adjusted in distilled water to 1 × 10 5 conidia mL -1 . For each orchard, 30 lesions on leaves (ca 0.6 × 0.6 cm) or fruits (ca 100 mg) were collected (1 lesion per leaf or fruit), pooled and stored at -20 • C until DNA extraction. In addition, individual leaf lesions were frozen separately. The latter samples were used as starting material to evaluate the detection thresholds of both PCR methods.

DNA extraction

Frozen mycelia as well as mixed and unique lesion samples were ground in liquid nitrogen using a mortar and pestle. Fungal DNA or DNA from a plant-fungus mixture was extracted and purified using Nucleospin Plant DNA extraction kit (Macherey Nagel) according to the manufacturer's recommendations. DNA was resuspended in 100 µL of elution buffer and quantified using a NanoDrop  ND-1000 spectrophotometer.

PCR-based detection 2.3.1 Cleaved amplified polymorphic sequence (CAPS) assay

The PCR primers PS1 and PR1 (Table 2) were designed using the published sequence of the V. inaequalis Cyt b gene (GenBank accession number AF004559). [START_REF] Zheng | Characterization of the mitochondrial cytochrome b gene from Venturia inaequalis[END_REF] They hybridise in exons 3 and 4 of the gene, respectively, and amplify a 488 bp fragment containing the region of interest, i.e. codon 143 site of the G143A mutation. This point mutation changes the sequence 5 -GGTGC-3 into 5 -GCTGC-3 , which is specifically recognised by the restriction endonuclease Fnu4HI. PCR amplifications were performed in a 25 µL volume containing 1 µL of DNA (1-10 ng), 0.5 µM of each primer, 200 µM of each dNTP, 0.5 mM magnesium chloride and 2 U of Taq-DNA polymerase in the appropriate buffer (Invitrogen). An initial denaturation step at 94 • C for 3 min was followed by 35 cycles that each included a denaturation at 94 • C for 30 s, a primer annealing step at 55 • C for 45 s and a polymerisation step at 72 • C for 50 s. Amplifications were terminated by a 5 min final extension at 72 • C. PCR products from the sensitive (SFR2) and resistant (160) isolates were directly sequenced using the BigDye3.1 Terminator sequencing kit (Applied Biosystems) and analysed on an ABI3730XL capillary sequencer (Applied Biosystems) at Genoscreen Services (Lille, France). A quantity of 10 µL of the PCR products was digested overnight by 1.5 U of Fnu4HI (New England Biolabs) at 37 • C. Restriction fragments were separated on 2.5% agarose gels in 0.5 × TBE and stained with ethidium bromide. Gels were visualised using a UV transilluminator and photographed using the Gel Doc 2000 apparatus (BioRad).

Allele-specific PCR (AS-PCR)

Allele-specific PCR primers that would preferentially bind to the mutant sequence were designed. The backbone sequence of these primers corresponds to primer G143AMM0 for which the 3 G nucleotide is complementary to the C residue specific to the mutant allele (Table 2). Enhanced allelic discrimination was tested by introducing an additional mismatch in the 3 subterminal region of the primer, thus creating primers G143AMM1, G143AMM2, G143AMM3 and G143AMM4 (Table 2). These allele-specific primers were used in combination with primer PS1 and tested to amplify a 433 bp DNA fragment. All AS-PCR reactions included, as an amplification control, a second set of primers, namely PS-exon7 and PR-exon7 (Table 2). This primer pair allows, from any V. inaequalis Cyt b alleles, the amplification of a 238 bp sequence located some 5 kb downstream of the G143 mutation. PCR amplifications were performed in a 25 µL volume containing 0.2 µM of allele-specific primers and 0.1 µM of control primers (PS-exon7 and PR-exon7). Other AS-PCR components included 1 µL of DNA (1-10 ng), 400 µM of each dNTP, 0.5 mM of magnesium chloride and 2 U of Taq-DNA polymerase in the appropriate buffer (Invitrogen). An initial denaturation at 94 • C for 3 min was followed by 30 cycles that each included denaturation at 94 • C for 30 s, a primer annealing step at 60 • C for 45 s, and extension at 72 • C for 50 s. Amplifications were followed by a final extension of 3 min at 72 • C. PCR products were separated on 1% agarose gels in 0.5×TBE and stained with ethidium bromide.

Detection thresholds of PCR assays

Total DNA extracted from leaves colonised by a QoI-sensitive fungal isolate was mixed at different ratios with DNA extracted from leaves contaminated by a G143A positive isolate. CAPS assay and AS-PCR were carried out as described above using 3 ng of mixed DNA samples as template.

Spore germination assay

The QoI fungicide used in bioassays was kresoxim-methyl (BASF). It was of technical-grade quality (97.8% purity). Stock solutions of kresoxim-methyl were prepared in ethanol, and varying volumes of these were added to water agar to achieve final concentrations that ranged from 1 × 10 -3 to 3 × 10 2 mg AI L -1 of medium. Randomly selected spore suspensions were also spread on kresoxim-methylcontaining medium amended with salicylhydroxamic acid (SHAM) at a final concentration of 30 mg L -1 .

A freshly prepared conidial suspension was spread on each of the different media at a density of about 230 conidia per 19.6 cm 2 Petri dish. Plates were incubated in the dark, at 20-22 • C, for 48 h. A total of 100 conidia were then scored as either germinated or not. For each fungicide concentration, the germination rate was calculated as the percentage germination on the fungicidesupplemented medium divided by the mean of percentage germination on the control, fungicide-free, medium. The 50% effective concentration (EC 50 ) was calculated using the regression curves obtained by graphically plotting the germination rates against the log-transformed value of the fungicide concentrations. The resistance factor (RF) corresponded to the EC 50 value of the studied orchard divided by the EC 50 value obtained from control orchards that had never been treated with strobilurins. For each analysis, the minimal inhibitory concentration (MIC) was determined.

RESULTS

PCR-based detection of the G143A mutation in Venturia inaequalis

Cleaved Amplified polymorphic sequence (CAPS) assay

The preliminary set of this experimental work consisted of controlling the primer specificity towards V. inaequalis. Primer pair PS1-PR1 amplified a 488 bp fragment from DNA samples extracted from pure cultures of both QoI-sensitive (isolate SFR2) and resistant (isolate 160) V. inaequalis mycelia, as well as from DNA extracted from V. inaequalis-infected apple leaves and fruits collected in orchards. No amplification was obtained using DNA from non-infected apple tree leaves or other fungi such as Botrytis cinerea, Penicillium sp. and Rhizopus sp. (data not shown).

Sequences of the PCR products from the sensitive and resistant isolates were identical to the published sequence of V. inaequalis Cyt b gene, except for a guanine to cytosine transversion in the sequence from the isolate 160 responsible for the typical G143A mutation conferring QoI resistance (Fig. 1). This mutation created an Fnu4HI (5 -GCTGC-3 ) restriction site in the mutant sequence. Upon digestion with this endonuclease, two DNA fragments of 412 and 76 bp were obtained with the PCR fragment from the resistant isolate 160, whereas, as expected, the PCR product from the sensitive isolate SFR2 was not cleaved (Fig. 2), allowing CAPS analysis on composite samples. As a control for population analysis, DNA amplified from lesions collected in several control orchards that had never been treated with strobilurins could not be digested by Fnu4HI. On the other hand, DNA amplified from numerous samples collected in orchards in which strobilurin treatments failed to control apple scab were frequently partially digested by Fnu4HI (Fig. 2). Partial digestions probably resulted from the coamplification of sensitive and resistant Cyt b alleles, as the field- collected samples are composite samples made up of 30 different apple scab lesions.

The sensitivity of the CAPS method was tested by establishing different ratios of DNA extracted from leaves infected with a QoIresistant (R) isolate with DNA extracted from leaves infected with a QoI-sensitive (S) isolate. The QoI-resistant allele could not be detected after Fnu4HI digestion for R : S ratios equal to or below 1 : 9 (w : w) (Fig. 3).

Allele-specific PCR (AS-PCR)

In order to improve the sensitivity of the PCR detection method, the authors developed an AS-PCR method. For this purpose, primer PS1, which should hybridise to any V. inaequalis Cyt b allele, was combined with a second primer designed to bind specifically to the sequence of the mutant allele conferring QoI resistance. The first AS primer designed, G143AMM0 (Table 2), differed from the wild-type sequence by having a G at its 3 end, which corresponds to the G143A mutation. Using the primer pair PS1-G143AMM0, both wild-type and mutant alleles were equally amplified at either annealing temperature tested (data not shown). Therefore, four additional AS primers that differed from G143AMM0 by a second mismatch in position -1, -2 or -3 relative to the 3 end of the primer were designed (Table 2). In combination with primer PS1, these four primers amplified a 433 bp fragment from both sensitive and resistant isolates at an annealing temperature of 55 • C. Allelic discrimination, i.e. specific amplification of the QoI-resistant allele, was achieved using any of the four primer pairs at the annealing temperature of 60 • C (Fig. 4). Primer G143AMM1 was then chosen as the AS primer for all subsequent experiments. To make sure that the absence of amplification of a 433 bp fragment did not result from PCR inhibition, all AS-PCR reactions included a second primer pair (PS-exon7 and PR-exon7), which allowed the amplification of a 238 bp fragment from all Cyt b alleles (Fig. 4).

The sensitivity of the AS-PCR protocol was evaluated by amplifying Cyt b mutant allele DNA molecules when mixed at different ratios with wild-type allele molecules. The mutant allele was amplified at the lowest ratio tested, 1% of DNA extracted from leaves infected with a QoI-resistant isolate to 99% of DNA extracted from leaves infected with a QoI-sensitive isolate (Fig. 5), thus showing a higher sensitivity of the AS-PCR detection method compared with CAPS analysis.

Spore germination assay versus molecular detection methods

Among the 317 samples collected from orchards from 2004 to 2007, only 284 samples were analysed for QoI resistance by both the bioassay and the PCR approaches. The 33 remaining samples were only tested by PCR approaches. Here, 74% of spore germination assays could not be interpreted owing to an excessively low quantity of spores collected from the leaves or to a very low germination rate on QoI-free medium, although an unusually low threshold value of 10% was used. In contrast, only in 8% of these samples did PCR amplification of the Cytb fragment fail. This could be ascribed to degradation of the DNA extracted from excessively old lesion tissues or leaf samples not well preserved between their collection and delivery in the laboratory.

The QoI-sensitive isolate SFR2 showed 50% mycelia growth inhibition when using 0.06 mg L -1 of kresoxim-methyl (Table 1). Comparatively, QoI-resistant isolate 160 exhibited an EC 50 for mycelium of 8.5 mg L -1 of QoI fungicide. The present data are consistent with the baseline sensitivity (0.09 mg L -1 ) and the mean EC 50 value (6.09 mg L -1 ) found in V. inaequalis populations exposed to kresoxim-methyl in Quebec orchards. [START_REF] Jobin | Incidence of myclobutanil-and kresoximmethyl-insensitive isolates of Venturia inaequalis in Quebec orchards[END_REF] Using kresoxim-methyl, the EC 50 for spores collected in French orchards that had never been treated with strobilurin varied between 0.001 and 0.006 mg L -1 (Table 3). These EC 50 values are in accordance with literature data found for sensitive isolates (0.0006-0.007 mg L -1 ). [START_REF] Färber | Sensitivity of Venturia inaequalis to trifloxystrobin[END_REF][START_REF] Steinfeld | Sensitivity of mitochondrial respiration to different inhibitors in Venturia inaequalis[END_REF] As illustrated in Fig. 6, spores collected in 46 of the 73 analysed orchards had EC 50 values that were at least 10 times higher (up to more than 10 4 times) than the values recorded for sensitive fungal isolates. For the resistant groups of orchards that were considered from in vitro germination tests as being infected by QoI-resistant isolates, molecular analyses made it possible to detect the presence of the G143A mutation in 35 (76%) of them. For the remaining 11 resistant orchards, the G143A mutation could not be detected by a PCR approach, in spite of the fact that their EC 50 and MIC values were respectively 10-10 000 and 5-10 000 times higher than values recorded for sensitive fungal isolates (mean EC 50 = 0.004 mg L -1 ; highest MIC = 0.03 mg L -1 ).

For the second group of the 27 remaining orchards, for which the EC 50 values of the V. inaequalis spores ranged from 0.002 to 0.04 mg L -1 of kresoxim-methyl, the G143A mutation could be detected only in four of them.

Occurrence of the G143A mutation in French orchards from 2004 to 2007

From 2004 to 2007, infected leaves were sampled in 281 different commercial orchards in which fungicide treatments failed to limit the spread of apple scab, and 255 were successfully analysed by either PCR-based method. A total of 29 orchards were analysed over 2 years (22 orchards) or 3 years (7 orchards). In total, this represented 291 sample analyses. In 2004 and 2005 the samples were exclusively analysed using the CAPS method. In 2006, 67 samples were analysed by the CAPS method and 20 using AS-PCR (Table 4). Among the 67 initially analysed by CAPS, 21 were found to be positive and 46 to be negative for the mutation. Among these latter, 33 of them were reanalysed by AS-PCR, and five of them (15%) turned out to be positive for the mutation using this latter method. In 2007, all samples were analysed by AS-PCR. a Twenty unique samples + 33 that were found to be negative in the CAPS assay. b Among which 5 were found to be negative by the CAPS method. a Localities are all located in the French administrative region Rh ône-Alpes. b Percentage germination refers to the value obtained on fungicide-free medium. c EC 50 refers to the fungicide concentration that leads to a 50% inhibition of the germination rate. d MIC is the minimal concentration of fungicide that entirely inhibits spore germination. The G143A mutation was detected in 15 orchards (32%) in 2004, 43 (34%) in 2005, 42 (48%) in 2006 and 21 (64%) in 2007. Four out of the 238 samples analysed by the CAPS method revealed that the amplified 488 bp fragment was entirely digested by Fnu4HI, thus suggesting that, in these four situations, the G143A mutation predominated among the different V. inaequalis isolates present in each of these four orchards. For the 29 orchards analysed over 2 or 3 years, the mutation was detected in the first year of monitoring for 12 of them. Except for one orchard, the mutation continued to be detected over this time period, in spite of an alleged cessation of strobilurin treatments in these orchards.

The present analyses clearly indicated the repeated occurrence of the G143A mutation in multiple apple-producing regions in France all through the sampling years (Fig. 7). Indeed, in 2004 the mutation was recorded in three (33%) regions out of the nine from which samples were analysed: Midi-Pyrén ées, Rh ône-Alpes and Provence-Alpes-C ôte d'Azur. In 2005, five (42%) regions out of 12 contained the mutation, six (54%) out of 11 in 2006 and all seven in 2007. In the two administrative regions where the G143A mutation had prevailed since 2004 (the Midi-Pyr én ées and Rh ône-Alpes regions), a global increase in the percentages of orchards with the G143A mutation was noticed, ranging from 73% in 2004 to 86% in 2006 in Midi-Pyr én ées and from 67% in 2004 to 84% in 2006 in Rh ône-Alpes (Fig. 7).

DISCUSSION

As one of the major pathogens of apple trees, it is necessary to monitor the occurrence, origin and distribution of fungicide resistance in V. inaequalis in order to optimise the use of pesticides in apple tree orchards. Single-target-site QoI fungicides have become popular to control apple scab. Recent reports of QoI resistance in the field have suggested that mutations conferring this phenotype could have been selected several times independently and/or were disseminating among pathogen populations. 15 -17 As shown in this study, spore germination assays on fungicidesupplemented media, although effective to estimate resistance levels, fail to give interpretable results in many cases owing to very low spore viability in field-collected samples. Indeed, field samples have often been exposed to additional fungicides other than QoI molecules, which, in addition to delays between collection time in the field and processing in the laboratory, may affect spore viability. Nevertheless, spore germination assays demonstrate that, in about 25% of the samples that were processed, the failure properly to control apple scab could not be attributed to QoI resistance, as both the EC 50 and the RF values for these samples were similar to the values recorded for samples collected in control orchards contaminated by QoI-sensitive V. inaequalis isolates.

Detection of the G143A mutation by the CAPS method and/or AS-PCR has already been reported for several plant pathogens such as Magnaporthe grisea (Hebert) Barr, [START_REF] Avila-Adame | Characterization of spontaneous mutants of Magnaporthe grisea expressing stable resistance to the Qo-inhibiting fungicide azoxystrobin[END_REF] Erysiphe graminis DC [START_REF] Fraaije | Following the dynamics of strobilurin resistance in Blumeria graminis f. sp. tritici using quantitative allele-specific real-time PCR measurements with the fluorescent dye SYBR Green I[END_REF] and Alternaria sp., [START_REF] Ma | An allele-specific PCR assay for detecting azoxystrobin-resistant Alternaria isolates from pistachio in California[END_REF] but not for V. inaequalis. The AS-PCR developed here proved to be at least 10 times more sensitive than the CAPS method based on the difference in detection level in the test samples containing different ratios of the mutated allele. This was confirmed in the samples from the orchards: the G143A mutation was detected in mixed lesion samples that were first interpreted as free of the mutation by CAPS. Nevertheless, this latter method may provide additional information compared with the AS-PCR, i.e. the presence of both the wild-type and the mutant Cyt b allelebearing isolates occurring in a unique leaf sample. The digestion profiles indicate that in most cases (98%) the samples contain both the wild-type and the mutant Cyt b alleles. This situation may result from the coexistence in the samples of leaf lesions colonised by either mutant or wild-type sensitive strains. Alternatively, leaf lesions may have been colonised by heteroplasmic fungal strains containing in their mycelia a mixed population of mitochondria with or without the G143A mutation. Both hypotheses could be tested by analysing separately different lesions from the corresponding orchards as well as single spore isolates, as it cannot be ruled out that a single lesion could result from a coinfection by a QoI-sensitive and a resistant isolate. Heteroplasmy has been demonstrated for fungicide-resistant isolates originating from commercial orchards. [START_REF] Dux | Sensitivity of Venturia inaequalis populations to anilinopyrimidine, DMI and QoI fungicides[END_REF] As anticipated, the present results clearly show a strong association between the occurrence of the G143A mutation and the detection of QoI resistance by in vitro spore germination assays. Furthermore, this mutation was also observed in many other samples that could not be studied by the in vitro assay. Altogether, these results demonstrate that the main cause of the failure to control apple scab using QoI fungicides arises from the spread of fungal strains harbouring this mutation. In a few cases, discrepancies between the spore germination assays and the molecular detection of the G143A mutation were observed. A high level of resistance in the absence of the G143A mutation could indicate that an alternative resistance mechanism is operating. Other resistance mechanisms have indeed been reported in V. inaequalis and/or in other fungi. Change from phenylalanine to leucine at position 129 of the Cyt b polypeptide (mutation F129L) has been shown to occur in field populations of Pyricularia grisea Sacc. [START_REF] Grasso | Cytochrome b gene structure and consequences for resistance to Qo inhibitor fungicides in plant pathogens[END_REF][START_REF] Kim | Field resistance to strobilurin (QoI) fungicides in Pyricularia grisea caused by mutations in the mitochondrial cytochrome b gene[END_REF] The present authors failed to detect this point mutation by sequencing the corresponding segment of the V. inaequalis gene amplified from one sample showing a moderate level of resistance without harbouring the G143A mutation (data not shown). The alternative oxidase pathway may also operate, thus leading to resistance, as previously demonstrated in V. inaequalis, [START_REF] Olaya | Differential responses of germinating Venturia inaequalis conidia to kresoxim-methyl[END_REF][START_REF] Jobin | Incidence of myclobutanil-and kresoximmethyl-insensitive isolates of Venturia inaequalis in Quebec orchards[END_REF] although, in the present case, inclusion of SHAM in the spore germination medium did not modify strobilurin sensitivity (data not shown). Further experiments would be needed to identify the other mechanisms of QoI resistance in V. inaequalis.

In contrast, PCR-based detection of the G143A mutation in samples that were shown to be QoI-sensitive in spore germination assays is more difficult to interpret. It could be hypothesised that these samples were a mixture of lesions colonised by predominantly sensitive isolates versus QoI-resistant ones, and that only the former contributed, owing to germination bias, to the spore germination assay, whereas DNA from both lesions contributed to the molecular assay.

In addition to being more reliable than the spore germination assays, molecular monitoring is less time consuming. It was performed on 291 samples collected in the main apple-producing regions of France in orchards in which apple scab could not be controlled by the use of strobilurin. These orchards are, however, not representative of French orchards in terms of apple scab control. Multiple detection of QoI resistance in the French appleproducing regions raises the epidemiological question about its origin, i.e. frequent occurrence of independent G143A mutational events versus the long-distance dissemination of a single or of very few G143A alleles.

Figure 1 .

 1 Figure 1. Partial alignment of the cytochrome b sequences from the QoIsensitive (SFR2) and resistant (160) Venturia inaequalis isolates to the GeneBank AF004559 sequence. Note the G to C transversion at nucleotide position 5437 of the Cyt b gene, leading to the amino acid change glycine to alanine responsible for QoI resistance.

Figure 2 .

 2 Figure 2. Fnu4HI digestion of the 488 bp PCR fragment of the cytochrome b gene generated by primer pair PS1-PR1 using DNA extracted from QoI-resistant Venturia inaequalis isolate 160 (lane 2), QoI-sensitive isolate SFR2 (lane 3) and apple scab lesions collected in an orchard where resistant isolates predominated (r; lane 4) or were absent (s; lane 5). MW, 1 kb DNA ladder (Fermentas).

Figure 3 .

 3 Figure 3. Sensitivity of the CAPS method. DNA extracted from individual lesions caused by QoI-resistant (R) and QoI-sensitive (S) isolates were mixed at different ratios (from 1 : 99 to 40 : 60, w : w) before amplification of a Cyt b 488 bp DNA fragment which was digested with Fnu4HI. The 100% sensitive and 100% resistant corresponded to DNA extracted from mycelium of the QoI-sensitive isolate SFR2 and the QoI-resistant isolate 160, respectively. MW, 1 kb DNA ladder (Fermentas).

Figure 4 .

 4 Figure 4. Allele-specific PCR amplification of the G143A Cyt b allele.DNA extracted from Venturia inaequalis QoI-resistant (a) and sensitive (b) isolates were used as templates for the PCR amplifications which used primer PS1 in combination with one of the five allele-specific primers G143AMM0 to G143AMM4 that specifically bind to the mutant cytochrome b sequence. As a positive amplification control, primers PS-exon7 and PR-exon7, which amplify a 238 bp fragment, were included in all PCR reactions. Primer G143AMM0, which differs from the wild-type sequence by one nucleotide at its 3 end, was not specific at an annealing temperature of 60 • C. MW, 1 kb DNA ladder (Fermentas).

Figure 5 .

 5 Figure 5. Sensitivity of the allele-specific PCR method. DNA extracted from QoI-resistant (R) and QoI-sensitive (S) isolates were mixed at different ratios (from 1 : 99 to 80 : 20, w : w) before coamplification at 60 • C by primer pair PS1-G143AMM1 of a G143A-specific 433 bp fragment and by primer pair PS-exon7 and PR-exon7 of a control 238 bp fragment. The 100% sensitive and 100% resistant corresponded to DNA extracted from mycelium of the QoI-sensitive isolate SFR2 and the QoI-resistant isolate 160, respectively. MW, 1 kb DNA ladder (Fermentas).

Figure 6 .

 6 Figure 6. Distribution of the studied apple tree orchards according to the sensitivity of their Venturia inaequalis populations to kresoxim-methyl, as determined by spore germination assays. An arbitrary threshold value of 0.04 mg L -1 of fungicide (tenfold EC 50 and twofold MIC of sensitive isolates) is usually used to separate the 'sensitive' from the 'resistant' fungal populations. Grey bars represent the number of orchards in which the G143A mutation was detected and white bars the number of orchards in which it could not be detected using either PCR-based method.

Figure 7 .

 7 Figure 7. Distribution of the G143A mutation detected by PCR-based methods in French Venturia inaequalis populations sampled from 2004 to 2007. The number of populations where the point mutation was detected over the number of sampled orchards is indicated. The French administrative regions are as follows: Al, Alsace; Aq, Aquitaine; BN, Basse Normandie; Bo, Bourgogne; Br, Bretagne; Ce, Centre; HN, Haute Normandie; Li, Limousin; Lo, Lorraine; LR, Languedoc-Roussillon; MP, Midi-Pyrénées; PACA, Provence-Alpes-C ôte d'Azur; PC, Poitou-Charentes; PL, Pays de Loire; RA, Rh ône-Alpes.

Table 1 .

 1 Geographic origin and sampling year of the fungal isolates used in this study

	Isolates	Sampling year	French administrative region (locality)	a (mg L -1 ) EC 50
	Venturia inaequalis
	SFR2	2002	Rh ône-Alpes (Saint	0.06
			Marcel d'Urfé)
	160	2003	Midi-Pyrénées	8.5
			(Montauban)
	Botrytis cinerea	2003	Rh ône-Alpes (Saint	nd b
			Genis Laval)
	Rhizopus sp.	2003	Rh ône-Alpes (Saint	nd b
			Didier sous Riverie)
	Penicillium sp.	2003	Rh ône-Alpes (Saint	nd b
			Didier sous Riverie)
	a EC 50 refers to the concentration of kresoxim-methyl that leads to a
	50% inhibition of the mycelia growth rate.
	b nd = not determined.

Table 2 .

 2 Primers used in this study. For the allele-specific primers G143AMM0 to G143AMM4, the introduced mismatch bases relative to the Venturia inaequalis Cyt b wild-type sequence are underlined

	Name	Orientation	Sequence (5 → 3 )	Nucleotide position a
	PS1	Forward	GTTACAGCCTTCCTGGGTTAT	5025-5045
	PR1	Reverse	AGGCCTCCCCACAGAAATTCG	5513-5495
	G143AMM0	Reverse	GGTTTGTGATGACAGTTGCAG	5457-5437
	G143AMM1	Reverse	GGTTTGTGATGACAGTTGCTG	5457-5437
	G143AMM2	Reverse	GGTTTGTGATGACAGTTACAG	5457-5437
	G143AMM3	Reverse	GGTTTGTGATGACAGTTGTAG	5457-5437
	G143AMM4	Reverse	GGTTTGTGATGACAGTTGGAG	5457-5437
	PS-exon7	Forward	GGTGTTATAGCTATGTTTGCAGCTA	10 394-10 418
	PR-exon7	Reverse	CAAGAGGTACAATTCACAAAGTGAG	10 631-10 605

a Position as referred to the GeneBank accession AF004559.

Table 4 .

 4 Prevalence of the Venturia inaequalis Cyt b G143A mutation in orchards sampled from 2004 to 2007, as revealed by either the CAPS method or allele-specific PCR (AS-PCR)

			CAPS		AS-PCR
	Sampling	No. of	G143A mutation	No. of	G143A mutation
	year	orchards	present	orchards	present
	2004	46	15	-	-
	2005	125	43	-	-
	2006	67	21	53 a	21 b
	2007	-	-	33	21
	2004-2007	238	79	86	42

Table 3 .

 3 Sensitivity to kresoxim-methyl of Venturia inaequalis spores collected in control orchards that had never been treated with strobilurins

	Orchard	Sampling year	Geographic origin a	Germination b (%)	EC 50	c (mg L -1 )	M I C d (mg L -1 )
	FR B	2005	Saint Marcel d'Urf é	10		0.006	0.03
	FR B	2007	Saint Marcel d'Urf é	20		0.005	0.03
	FR M	2005	Saint Marcel d'Urf é	12		0.002	0.01
	FR M	2006	Saint Marcel d'Urf é	13		0.003	0.01
	FR M	2007	Saint Marcel d'Urf é	14		0.006	0.03
	FR V	2005	Saint Marcel d'Urf é	10		0.001	0.01
	FR V	2007	Saint Marcel d'Urf é	15		0.005	0.03
	VB	2004	Givors	28		0.004	0.03
	CB	2004	Saint Jean le Vieux	20		0.005	0.01
	Mean (± SD)			15.7 (±5.5)	0.004 (±0.002)	0.02 (±0.01)

PCR-based detection of Qol resistance in Venturia inaequalis www.soci.org

www.interscience.wiley.com/journal/ps

c 2008 Society of Chemical Industry Pest Manag Sci 2009; 65: 74-81

Sauter H, Steglich W and Anke T, Strobilurins: evolution of a new class of active substances. Angew Chem Internat Ed 38:1328-1349 (1999).

SUPPORTING INFORMATION

Supporting information may be found in the online version of this article.

ACKNOWLEDGEMENT

The authors are grateful to Pierre Leroux and Anne Sophie Walker from INRA of Versailles for their helpful advice during this work. They also thank all the technicians and growers for collecting and sending samples of leaves.