Quantitative resistance increases the durability of
Quantitative resistance increases the durability of qualitative resistance to Leptosphaeria maculans in Brassica napus
Hortense Brun 1,Anne-Marie Che
`vre 2,Bruce DL Fitt 3,Stephen Powers 3,Anne-Laure Besnard 1,Magali Ermel 1,Virginie Huteau 1,Bruno Marquer 1,Fre
′de ′rique Eber 2,Michel Renard 2and Didier Andrivon 11
INRA,Agrocampus Ouest,Univ.Rennes1,UMR1099BiO3P (Biology of Organisms and Populations applied to Plant Protection),F–35653Le Rheu,France;2INRA,Agrocampus Ouest,Univ.Rennes1,UMR118APBV (Ame
′lioration des Plantes et Biotechnologies Ve ′ge ′tales),F–35653Le Rheu,France;3
Rothamsted Research,Harpenden,Herts AL52JQ,UK
Author for correspondence:
Hortense Brun
Tel:+33223485185
Email:Hortense.Brun@rennes.inra.fr Received:10June 2009Accepted:20August 2009
New Phytologist (2009)
doi :10.1111/j.1469-8137.2009.03049.x
Key words:Brassica napus (oilseed rape),durable disease resistance,Leptosphaeria maculans (blackleg or phoma stem canker),plant–pathogen coevolution,R -gene mediated resistance,sustainable disease control.
Summary
?It has frequently been hypothesized that quantitative resistance increases the durability of qualitative (R -gene mediated)resistance but supporting experimental evidence is rare.To test this hypothesis,near-isogenic lines with ?without the R -gene Rlm6introduced into two Brassica napus cultivars differing in quantitative resistance to Leptosphaeria maculans were used in a 5-yr ?eld experiment.
?Recurrent selection of natural fungal populations was done annually on each of the four plant genotypes,using crop residues from each genotype to inoculate sep-arately the four series of ?eld trials for ?ve consecutive cropping seasons.Severity of phoma stem canker was measured on each genotype and frequencies of aviru-lence alleles in L.maculans populations were estimated.
?Recurrent selection of virulent isolates by Rlm6in a susceptible background rendered the resistance ineffective by the third cropping season .By contrast,the resistance was still effective after 5yr of selection by the genotype combining this gene with quantitative resistance.No signi?cant variation in the performance of quantitative resistance alone was noted over the course of the experiment.
?We conclude that quantitative resistance can increase the durability of Rlm6.We recommend combining quantitative resistance with R -gene mediated resis-tance to enhance disease control and crop production.
Introduction
Crop protection against pathogens that cause epidemic dis-eases is a major asset for global food security and sustainable crop https://www.wendangku.net/doc/9c15975262.html,ing resistant cultivars remains the best method to grow a crop with limited pesticide applications and low production costs.Two main types of resistance are generally described.Quantitative resistance (QR)is usually controlled by multiple genetic factors (quantitative trait loci or QTL)(Lindhout,2002;Stuthman et al.,2007).It leads to a reduction in symptom severity and ?or epidemic pro-gress over time,which can sometimes result in high levels of protection.Quantitative resistance is usually less effective when environmental or plant tissue conditions are favour-able to disease (Geiger &Heun,1989;Zadoks,1993).By
contrast,R -gene mediated resistance is often total and con-ferred by single dominant R gene.R -gene mediated resis-tance is under gene-for-gene recognition mechanisms (Flor,1955)that trigger hypersensitive response (HR).Both types of resistance coexist according to the pathosystem in a num-ber of crops,as well as in the corresponding wild genetic resources (Stuthman et al.,2007).
Many breeding programs and strategies have been devel-oped to improve cultivar resistance with the objective of resistance durability (Delourme et al.,2006;Rimmer,2006;Stuthman et al.,2007)because genetic resistance is most useful for growers if it is durable.Johnson (1981)de?ned durable resistance as ‘a resistance that remains effec-tive during its prolonged and widespread use in an environ-ment favourable to the disease’.This de?nition implies
that
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resistance durability can only be assessed retrospectively (i.e.after commercial use of resistant genotypes).Therefore,it does not allow predictive inferences,which are important to manage the construction and deployment of resistant culti-vars.Resistance type (quantitative resistance vs R -gene med-iated resistance)is often used as a surrogate –but not entirely adequate –predictor for durability.Indeed,R -gene mediated resistance is often isolate-speci?c and thus exerts a strong selection pressure on pathogen populations that adapt rapidly through selection and multiplication of viru-lent isolates.Pathogens with high evolutionary potential thus give the highest risk of sudden resistance breakdown (McDonald &Linde,2002),resulting in a succession of ‘boom and bust’cycles (Vanderplank,1968),which has been observed in various agricultural pathosystems (e.g.Sprague et al.,2006).Quantitative resistance is most often regarded as isolate-non speci?c and thus postulated to be more durable than R -gene mediated resistance (Lindhout,2002;Stuthman et al.,2007;Poland et al.,2008).How-ever,a number of examples demonstrate that R -gene medi-ated resistance can sometimes be long-lasting (Christ et al.,1987),but also that quantitative resistance can be eroded by increased aggressiveness (i.e.quantitative pathogenicity;see Vanderplank,1968)in pathogen populations faced for long periods with quantitative resistance (Andrivon et al.,2007;Stuthman et al.,2007).A recurrent concern for plant breeders and plant pathologists is thus to identify the best way to use resistance factors to construct cultivars with the highest resistance level and the best possible intrinsic dura-bility,and deployment strategies for such resistant cultivars in space and time to maximize durability.
The question of resistance durability can be approached as a problem of adaptive response in pathogen populations to selection exerted by resistant hosts (McDonald &Linde,2002).Strategies to maximize durability should therefore both limit the selection of the more pathogenic variants of the pathogen and reduce pathogen population sizes (Mundt et al.,2002).We postulate that one of the strategies likely to achieve this is to introduce major resistance gene (R genes)into cultivars with high levels of quantitative resis-tance,with a triple expected effect:to enhance the disease control provided by quantitative resistance by using R genes to control all avirulent fractions of the pathogen popula-tion;to limit selection for virulent isolates,as quantitative resistance slows down the rate of epidemic development and thus decreases the severity of the disease and the effec-tive population size;and to maintain a satisfactory level of protection when the R gene is ?nally overcome.
The expected bene?ts of combining R -gene mediated resistance and quantitative resistance in a single cultivar have been investigated using mathematical models of patho-gen evolution (Kiyosawa,1982;Pietravalle et al.,2006)but rarely con?rmed experimentally.A recent paper,using suc-cessive arti?cial reinoculations of known viral isolates under
controlled conditions,showed delayed emergence of viru-lent variants on hosts combining R -gene mediated resistance and quantitative resistance relative to hosts with R -gene mediated resistance alone (Palloix et al.,2009).This paper describes work to investigate experimentally the ability of a combination of an R gene and quantitative resistance in a single cultivar to increase the durability of the R gene by delaying the selection of virulent isolates in natural fungal populations under ?eld conditions.We also compared the potential for evolution of pathogen populations to render ineffective the resistance conferred by an R gene or by quantitative resistance alone.The Brassica napus –Leptosp-haeria maculans pathosystem and the multiyear recurrent scheme described by Brun et al.(2000)were used to test the hypothesis.
Materials and Methods
The pathosystem Brassica napus –Leptosphaeria maculans
Leptosphaeria maculans is a heterothallic ascomycete causing phoma stem canker of oilseed rape,a disease of worldwide importance (Fitt et al.,2006).The fungus survives on infected crop residues for several years and produces both sexual and asexual fruiting bodies (pseudothecia and pycni-dia,respectively).Ascospores are discharged over several months (mainly in autumn and winter in Europe)from pseudothecia formed on residues,and can spread the patho-gen from ?eld to ?eld (West et al.,2001;Fitt et al.,2006).Conidia constitute the secondary inoculum,which contam-inates neighbouring plants by rain splash (Travadon et al.,2007).Infection by either ascospores or conidia causes leaf lesions,from which the fungus systemically reaches the stem base where it initiates crown canker (Hammond &Lewis,1987).The predominance of ascospores produced on resi-dues every year in the primary inoculum explains the high genetic variability observed in most populations of L.macu-lans (Hayden &Howlett,2005).
Two kinds of resistance are described in Brassica napus.There is R -gene mediated resistance,caused by a set of Rlm genes (Delourme et al.,2006;Huang et al.,2006a).Lep-tosphaeria maculans populations adapt rapidly to such a resistance,so that newly deployed resistant cultivars lose their effectiveness only 3–4yr after their release (Rouxel et al.,2003;Sprague et al.,2006).By contrast,quantitative resistance operates during the symptomless growth of the pathogen along leaf petioles and in stem tissues (Huang et al.,2009)and cannot be assessed before spring,when it decreases the severity of stem base cankers (Delourme et al.,2006).While both major gene-mediated and quantitative resistance against L.maculans may operate in a similar man-ner at the molecular level (Staal et al.,2008;Persson et al.,2009),in Brassica napus ,at the phenotype level,they
appear
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to operate in different tissues at different stages during the process of disease development(Huang et al.,2006a, 2009).
Near-isogenic lines of Brassica napus with?without Rlm6
A highly effective resistance introgressed from Brassica juncea into B.napus through interspeci?c crosses(Che`vre et al.,1997)segregates as a single gene called Rlm6and operates against many European isolates of L.maculans in both controlled and?eld conditions(Somda et al.,1996). Nevertheless,a multiyear?eld experiment has demonstrated that this resistance is not durable when introgressed into a highly susceptible background:recurrent selection of natu-ral L.maculans populations on this genotype,called‘MX’, leads to a rapid increase in the frequency of virulent isolates (Somda et al.,1999;Brun et al.,2000).The French National Institute for Agricultural Research(INRA)has thus decided not to release for commercial use the improved lines carrying Rlm6,but to keep them as research tools. Consequently,a large proportion of L.maculans popula-tions in France and the rest of Europe still contain avirulent AvrLm6isolates(Balesdent et al.,2006;Stachowiak et al., 2006),which allows work to study the selection exerted on L.maculans populations by Rlm6in different genetic back-grounds.There is evidence that Rlm6operates in B.napus against L.maculans soon after penetration of leaf stomata by the pathogen,preventing growth from leaf to stem tissues(Huang et al.,2006a).
The‘MX’line(spring type)was used as the progenitor to introduce Rlm6into winter-type oilseed rape cultivars Sam-oura?¨giving the Samoura?¨MX line(Che`vre et al.,1997),cv. Darmor,which carries several quantitative trait loci(QTLs) that give it good quantitative resistance to L.maculans(Pilet et al.,1998)and the susceptible cv.Eurol,to generate the DarmorMX and EurolMX lines,respectively.‘Eurol’carries the resistance genes Rlm2and Rlm3and‘Darmor’carries Rlm9;L.maculans populations in France are100%virulent against these three genes(Balesdent et al.,2006).‘Eur-olMX’was obtained by crossing‘Samoura?¨MX’with ‘Eurol’,followed by?ve backcrosses to‘Eurol’and nine sel?ng generations.Similarly,‘DarmorMX’was obtained by crossing‘Samoura?¨MX’with‘Darmor’,three backcrosses to‘Darmor’and two to four sel?ng generations.Both ‘DarmorMX’and‘EurolMX’were selected using molecular assisted selection on backcross and sel?ng generations (Che`vre et al.,1997);homozygous lines were con?rmed by cotyledon tests before seed multiplication.There is evidence that the QTL associated with quantitative resistance against L.maculans that are present in‘Darmor’and not‘Eurol’operate in B.napus stem tissues to slow down colonization and stem canker formation(Huang et al.,2009).Therefore, for the purposes of this paper,‘Eurol’and‘Darmor’will be regarded as providing a susceptible background and a quantitative resistant background,respectively,for the Rlm6-mediated resistance.
Design of the5-yr?eld experiment
Two pairs of near-isogenic lines(NILs),‘Eurol’?’EurolMX’and‘Darmor’?’DarmorMX’were included in all?eld trials. The susceptible cv.Eurol was useful as a common control, to compare severity of stem canker epidemics between trials. The durability experiment was established in Brittany(wes-tern France)during?ve consecutive cropping seasons (2002?2003to2006?2007).It began with an initial trial inoculated with a local pathogen population,which simu-lates the?rst year of cultivation of cultivars with a new resistance gene.It was followed by four separate4-yr?eld trials(PHO1,PHO2,PHO3and PHO4),each corre-sponding to recurrent selection of the L.maculans popula-tions by one of the four genotypes(Fig.1).Each series of trials simulates consecutive years of commercial cultivation of one oilseed rape genotype in adjacent?elds.Temperature and rainfall data were recorded daily at the INRA Le Rheu site,within10km of the trials.
Inoculum production One oilseed rape stem base includ-ing tap root(c.30cm long)was the unit of inoculum and was described as a residue.Residues were uprooted at random from plots in June before harvest and40plants per genotype taken from three central rows of each plot (i.e.80plants per genotype per block)were scored for stem canker severity.Residues of each genotype were then stored separately outdoors on permeable canvas sheets during July and August to allow maturation of L.maculans pseudothecia and favour ascospore production.
Field plot design and inoculation All four genotypes were sown in early September,in all trials in a randomized block design with four blocks,with2m-wide paths between blocks.Each NIL was sown in two adjacent plots per block to ensure that there were enough stem base residues to inoc-ulate the trials in the following cropping season.Individual plots measured1.5·4m and included?ve rows of plants. In the initial trial(2002?2003),all plots were inoculated with two residues m)2of oilseed rape comprising,in equal proportions,residues of susceptible cultivars Samoura?¨, Shogun,Glacier and Lirabon highly infected in the previous cropping season by a natural local L.maculans population. The PHO1,PHO2,PHO3and PHO4series were done from harvest years2004to2007at sites at least1km away from each other to avoid cross-contamination.Each trial was inoculated2–3wk after sowing by scattering two resi-dues m)2collected from plots of the appropriate genotype in the corresponding series at the end of the previous season (Fig.
1):
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PHO1:‘Eurol’residues –evolution of a local pathogen population with little selection by host resistance;
PHO2:‘EurolMX’residues –evolution of the same popu-lation under selection by a major gene (Rlm6)introduced into a susceptible background;
PHO3:‘Darmor’residues –evolution of that population under selection by quantitative resistance;
PHO4:‘DarmorMX’residues,evolution of the population under joint selection by a major gene (Rlm6)and quantita-tive resistance.
To prevent an increase in isolates virulent against Rlm6in local L.maculans populations (and hence interference by virulent external inoculum),all remaining residues from plots sown to genotypes with Rlm6or from plots inoculated with ‘EurolMX’or ‘DarmorMX’residues were uprooted at the end of each cropping season and burned before the for-mation of pseudothecia.
Trials in the same series could be sown on a neighbouring part of the same piece of ?eld but residues from the previous trial were always ploughed in before sowing the following one.All trials were surrounded by crops of winter barley.Assessment of leaf lesions Disease incidence (%plants with at least one leaf lesion)and severity (number of leaf
lesions per plant scored)were assessed in each trial once in each cropping season,with the date of assessment (normally in November)depending on the development of leaf lesions.Leaf lesions were counted on both pairs of NILs,on all leaves of each individual plant assessed in a sample of 30plants per block per genotype.To obtain representative samples from each plot,three samples of 10consecutive plants were taken from sites evenly distributed across each plot.Whenever possible,three blocks were assessed (90plants in total)but sometimes two (2005:60plants in total)or all four blocks (2002:120plants in total)were assessed.Stem canker assessment Stem canker incidence (%plants with stem base canker)and severity (DI,disease index)were assessed 2–3wk before harvest (mid June).Eighty plants per genotype and per block were uprooted,and scored on a 1–6scale (Aubertot et al.,2004)for stem canker presence and severity,based on the extent of internal symptoms at the stem base of each plant.Stem canker incidence was calculated as the proportion of plants in classes 2–6(i.e.including those showing even minute symptoms),and disease severity was assessed as DI,computed as a sum of weighted proportions of plants in each class:DI ?R i eni ?ci T=N [ni ,number of plants in class
rape cultivars from local crops
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(i=1….6);ci,weighting coef?cient(0for class1,1for class2,3for class3,5for class4,7for class5and9for class 6);N,total number of plants scored].The temperature and rainfall data were?tted to the weather-based model of Evans et al.(2008)to predict the development of epidem-ics,including severity of canker at harvest,on cultivars with or without quantitative resistance to L.maculans in each cropping season.
Seedling pathogenicity tests to determine virulence frequencies in L.maculans populations
Isolates In autumn2002,50single-ascospore isolates were obtained from the four susceptible cultivars used as inoculum in the initial trial.In the following cropping seasons,25single-ascospore isolates per genotype were recovered every autumn(2003–2005)from‘Eurol’,‘Eur-olMX’,‘Darmor’or‘DarmorMX’residues used as inocu-lum.Single ascospores were obtained by placing a small piece of stem tissue bearing pseudothecia on the cover of a Petri dish,over water agar(20%)supplemented with streptomycin sulphate(0.1g l)1),for12–18h.Single as-cospores deposited onto the medium were cut out indi-vidually,under a binocular microscope,with a very sharp glass needle and transferred to malt agar(20%,20%) supplemented with streptomycin sulphate(0.1g l)1). Only one isolate per residue(plant)was assessed for viru-lence at seven Avr loci using cotyledon tests in controlled conditions.Sometimes it proved dif?cult to obtain resi-dues of‘EurolMX’or‘DarmorMX’with pseudothecia.In 2003,there were only14and10residues with pseudo-thecia for‘EurolMX’and‘DarmorMX’,respectively,from >50residues assessed per genotype.For‘DarmorMX’, there were nine and seven isolates collected in the autumn of2004and2005,respectively.Moreover,pseu-dothecia of other species,mainly Leptosphaeria biglobosa (Shoemaker&Brun,2001)and Fusarium spp.,and py-cnidia(visually similar to pseudothecia)were also present on these‘MX’lines,further reducing the number of iso-lates of L.maculans recovered from these genotypes.
In2004?2005,the composition of the overall inoculum received by PHO1and PHO2trials was assessed.This was the inoculum from residues of either‘Eurol’or‘EurolMX’and from external airborne ascospores.Thus,‘Drakkar’(without any known major resistance genes)was sown in autumn2004in PHO1and in PHO2trials.Ten leaves of ‘Drakkar’with leaf lesions were sampled on10separated plants per block from three blocks each of PHO1and PHO2.One pycnidial isolate per leaf lesion and per plant (i.e.30isolates per trial)was transferred to malt agar and included in the seedling pathogenicity tests.In2004?2005 natural inoculum(i.e.from external airborne ascospores) was also investigated in a trial sown with‘Drakkar’without using any crop residue inoculum.This trial was established at least4km away from any of the four trial series.Isolates were obtained and assessed according to the same procedure. Seedling pathogenicity tests The differential host set com-prised seven genotypes:‘MT29’(Rlm1,9),‘Eurol’(Rlm2, 3),line‘22.1.1’(Rlm3),‘Falcon’(Rlm4),line‘150.2.1’(Rlm5),‘EurolMX’(Rlm2,3,6),‘Darmor’(Rlm9),to iden-tify virulence?avirulence alleles at Avr loci(AvrLm1to AvrLm6and AvrLm9)in each isolate.The protocol described in Che`vre et al.(2008)was then used for pro-duction of inoculum and assessment of avirulence pro?les. Frequencies of the avirulence alleles and of different races (combinations of AvrLm genes)were calculated. Statistical analysis
For each series and trial,for data inspection and presenta-tion,the mean number of leaf lesions(severity)obtained per plant,the mean percentage(incidence)of plants with leaf lesions,the mean stem canker severity(DI),and the mean percentage(incidence)of plants with stem canker were calculated across the blocks sampled for each geno-type.The data per cropping season and per trial were sub-mitted to ANOVA including the effect of the genotypes and taking account of the blocks.Following ANOVA,the standard error of the difference(SED)between means was output and selected means for the genotypes were compared using the least signi?cant difference(LSD)between means at the 5%level of signi?cance.
A position and parallelism regression analysis was used to examine the trends in three variables(incidence and severity of leaf lesions,and stem DI)over cropping season.For each variable,this assessed the statistical signi?cance of an overall linear trend and whether this trend was the same(i.e.paral-lel)but differently positioned(shifted)for the background (‘Eurol’or‘Darmor’)or the MX status(without?with Rlm6)or for both these factors(which would result in four parallel lines).Finally,the analysis was used to assess the signi?cance of the trend being different for each or both of the factors,the latter situation resulting in four separate lines.The best(most parsimonious)model is found using F-tests to assess the additional variance accounted for by changing from a single line to parallel lines and then,if nec-essary,from parallel lines to separate lines.
The relationship between(DI)and the number of leaf lesions preceding this for‘Eurol’and‘EurolMX’and taking data from the initial trial(2002?2003)and PHO1and PHO2(2003?2004to2006?2007)was examined using a non-linear least squares regression,including assessment(F-tests)of whether separate curves were statistically signi?cant for the two cultivars.The GENSTAT(11th edition;Lawes Agricultural Trust(Rothamsted Research)VSN Interna-tional Ltd.,Hemel Hempstead,UK)statistical system was used for all statistical
analyses.
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Results
Effects of quantitative and qualitative resistance on seasonal changes in composition of L.maculans populations
The avirulence alleles present in the natural L.maculans populations near Rennes,detected on ‘Drakkar’in autumn 2004,were similar to those in the population in autumn 2002,at the start of the experiment,except for Avrlm4(Table 1).The alleles AvrLm2,AvrLm3and AvrLm9were never observed in L.maculans isolates.There was little dif-ference between populations from different sources of deb-ris in the frequencies of AvrLm4and AvrLm5,except for natural inoculum sampled from ‘Drakkar’(Table 1).Isolates carried three or four AvrLm alleles out of the seven AvrLm alleles that could be detected with this differential host set.The greatest number of races per population was 11,in the initial inoculum (Table 2);the most frequent race was Av5–6.
There was no effect of quantitative resistance on the com-position of L.maculans populations,with similar frequen-cies of avirulence ?virulence alleles in L.maculans populations selected on ‘Eurol’or ‘Darmor’in PHO1or PHO3series.These frequencies ?uctuated a little seasonally but were similar to those observed in the initial inoculum and in natural inoculum sampled with ‘Drakkar’in 2004.By contrast,and as expected,the qualitative Rlm6resistance greatly affected the composition of L.maculans populations.Although few pseudothecia were observed on ‘EurolMX’residues in autumn 2003in PHO2,single ascospore isolates obtained from these residues were all virulent (a vrLm6)to Rlm6and the residues with pseudothecia increased thereaf-ter.Similarly,few single ascospore isolates (7–10)could be recovered from ‘DarmorMX’residues in PHO4.Frequen-cies of avirulence ?virulence alleles were otherwise similar in populations collected from ‘EurolMX’residues (PHO2)and from ‘DarmorMX’residues (PHO4)(Table 1).
Surprisingly,the frequency of the avirulence allele AvrLm1had greatly increased at the same time as the fre-quency of the virulence allele avrLm6increased in autumn 2003in PHO2and PHO4compared with PHO1and PHO3.For example,its frequency increased from 31%in the initial population in autumn 2002to 92%on ‘Eur-olMX’residues,and remained high thereafter.The number of L.maculans races detected ranged from two to eight (Table 2).The most frequent races were Av5–6in PHO1and PHO3and Av1–5in PHO2and PHO4.This re?ects the selection against AvrLm6and for AvrLm1by the pres-ence of Rlm6.Based on the expected proportion of recom-bination given a linkage distance between AvrLm1and AvrLm6of 6cM (Fudal et al.,2007),v 2tests on the observed frequencies of isolates having neither or both AvrLm1and AvrLm6compared with frequencies of isolates
having one or other allele suggested a highly signi?cant dif-ference between observed and expected frequencies in all cases (P <0.001,Table 2),with more isolates having one or the other allele than expected.
Less than 1%of leaf lesions on ‘Eurol’were caused by isolates virulent against Rlm6in PHO1and PHO3,what-ever the cropping season,the genotype used as inoculum and the severity of stem canker (Table 3).By contrast,almost all leaf lesions on ‘Eurol’were caused by virulent isolates in autumn 2003in PHO2and PHO4with residues carrying Rlm6,indicating that few isolates came from exter-nal inoculum.During the period 2003–2006,the mean number of leaf lesions per plant on ‘EurolMX’caused by virulent isolates remained stable in PHO4compared with PHO2.It is surprising that the proportion of virulent isolates in the L.maculans population required to render Rlm6resistance ineffective at harvest 2005was similar in autumn 2004,whether it was assessed by counting numbers of leaf lesions on ‘EurolMX’?’Eurol’(34.7%)or by obtaining 23isolates from leaf lesions on ‘Drakkar’sown in PHO2and analysing them in cotyledon pathogenicity tests (39.1%)(data not shown).
Development of leaf lesions and stem canker in the initial trial (2002?2003)
In the initial trial,there were large numbers of leaf lesions in autumn on ‘Eurol’and ‘Darmor’,with 96%and 98%of plants with at least one leaf lesion and averages of 7.6and 8.9leaf lesions per plant,respectively.By contrast,‘EurolMX’and ‘DarmorMX’had only 8%and 4%of plants with leaf lesions and averages of 0.1and 0.04leaf lesions per plant,respectively.These differences in leaf lesions between lines with Rlm6resistance against L.maculans and cultivars without it were re?ected in differences in the incidence (data not presented)and severity of stem base cankers in June 2003.Stem cankers were severe on ‘Eurol’(DI =7.64)but very slight on ‘EurolMX’and ‘DarmorMX’(DI =0.70and 0.39,respectively).The severity of canker on ‘Darmor’was intermediate (DI =3.67).
Effects of quantitative resistance on seasonal changes in leaf lesions and stem canker (‘Darmor’vs ‘Eurol’)There was a clear relationship between the severity of stem canker shortly before harvest and the severity of leaf lesions in the previous autumn over the period 2002?2003to 2006?2007in ‘Eurol’and ‘EurolMX’,with an increasing number of leaf lesions,up to four per plant,associated with increasing severity of stem canker (Fig 2).Above this threshold of four leaf lesions per plant in November,an increase in severity of phoma leaf lesions did not result in increased severity of stem canker;the relation was best described by an asymptotic exponential curve,with
an
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T a b l e 1P e r c e n t a g e o f t h e a v i r u l e n c e a l l e l e a t e a c h o f t h e f o u r A v r l o c i i n L e p t o s p h a e r i a m a c u l a n s p o p u l a t i o n s s a m p l e d i n a u t u m n ,e i t h e r b y o b t a i n i n g s i n g l e -a s c o s p o r e i s o l a t e s f r o m b a s a l s t e m o i l s e e d r a p e r e s i d u e s o f i n i t i a l i n o c u l u m (2002?2003c r o p p i n g s e a s o n )a n d o f g e n o t y p e s u s e d a s i n o c u l u m i n P H O 1t o P H O 4,e a c h c o m p r i s i n g a s e r i e s o f t r i a l s i n t h e f o l l o w i n g c r o p p i n g s e a s o n s (2003?2004,2004?2005,2005?2006)o r b y o b t a i n i n g s i n g l e p y c n i d i a l i s o l a t e s f r o m l e a f l e s i o n s o n c v .‘D r a k k a r ’(2004?2005).
F r e q u e n c y (%)o f a v i r u l e n c e a l l e l e i n p o p u l a t i o n 1
A v r L m g e n e
I n i t i a l i n o c u l u m 2
N a t u r a l i n o c u l u m 3
(D r a k k a r )‘E u r o l ’r e s i d u e s (P H O 1)
‘E u r o l M X ’r e s i d u e s (P H O 2)‘D a r m o r ’r e s i d u e s (P H O 3)‘D a r m o r M X ’r e s i d u e s (P H O 4)
2002?20032004?20052003?20042004?20052005?20062003?20042004?20052005?20062003?20042004?20052005?20062003?20042004?20052005?2006
A v r L m 1319212428924
9692212412708957A v r L m 4180111216234421882000A v r L m 5844610068100929276100848480100100A v r L m 68491100100800009292761000T o t a l n o .o f i s o l a t e s t e s t e d
4922
2825251324252425251097
1
A l l e l e f r e q u e n c i e s i n i s o l a t e s w e r e a s s e s s e d i n c o n t r o l l e d c o n d i t i o n s o n c o t y l e d o n s o f a d i f f e r e n t i a l h o s t s e t a b l e t o d e t e c t s e v e n a v i r u l e n c e a l l e l e s (A v r L m 1,A v r L m 2,A v r L m 3,A v r L m 4,A v -r L m 5,A v r L m 6,A v r L m 9).T h e f r e q u e n c i e s o f a v i r u l e n t a l l e l e s A v r L m 2,A v r L m 3a n d A v r L m 9w e r e a l l 0%,s o t h e s e d a t a a r e n o t p r e s e n t e d .2I n i t i a l i n o c u l u m c o m p r i s e d t h e r e s i d u e s o f s u s c e p t i b l e o i l s e e d r a p e c u l t i v a r s S a m o u r a ?¨,G l a c i e r ,S h o g u n a n d L i r a b o n s a m p l e d l o c a l l y a f t e r h a r v e s t i n 2002.3C u l t i v a r D r a k k a r w i t h n o k n o w n R -g e n e s w a s s o w n a t l e a s t 4k m a w a y f r o m t h e c l o s e s t o f t h e t r i a l s P H O 1t o P H O 4,i n a s e p a r a t e ?e l d t r i a l w i t h n o o i l s e e d r a p e r e s i d u e s p r e s e n t ,t o a s s e s s t h e f r e q u e n c i e s o f d i f f e r e n t a l l e l e s i n n a t u r a l i n o c u l u m n e a r R e n n e s ,F r a n c e .4T h e p o p u l a t i o n s w i t h t h e g r e a t e s t f r e q u e n c i e s o f A v r L m 1o r A v r L m 6a l l e l e s a r e s h o w n i n b o l d t y p e
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T a b l e 2P e r c e n t a g e o f e a c h r a c e i n L e p t o s p h a e r i a m a c u l a n s p o p u l a t i o n s s a m p l e d i n a u t u m n ,e i t h e r b y o b t a i n i n g s i n g l e -a s c o s p o r e i s o l a t e s f r o m b a s a l s t e m o i l s e e d r a p e r e s i d u e s o f i n i t i a l i n o c u l u m (2002?2003c r o p p i n g s e a s o n )a n d o f g e n o t y p e s u s e d a s i n o c u l u m i n P H O 1t o P H O 4,e a c h c o m p r i s i n g a s e r i e s o f t r i a l s i n t h e f o l l o w i n g c r o p p i n g s e a s o n s (2003?2004,2004?2005,2005?2006)o r b y o b t a i n i n g s i n g l e p y c n i d i a l i s o l a t e s f r o m l e a f l e s i o n s o n c v .D r a k k a r (2004?2005)
F r e q u e n c y (%)o f r a c e i n p o p u l a t i o n 1
L .m a c u l a n s r a c e
I n i t i a l i n o c u l u m 2
N a t u r a l i n o c u l u m 3
(D r a k k a r )
‘E u r o l ’r e s i d u e s (P H O 1)‘E u r o l M X ’r e s i d u e s (P H O 2)‘D a r m o r ’r e s i d u e s (P H O 3)‘D a r m o r M X ’r e s i d u e s (P H O 4)
2002?20032004?20052003?20042004?20052005?20062003?20042004?20052005?20062003?20042004?20052005?20062003?20042004?20052005?2006
A v -4
00000000004000A v 1-0500078240401500A v 4-00004000004000A v 5-40000748844151143A v 6-10410240000044000A v 1–400000000004000A v 1–5-850012576
8364008318957A v 1–6-00040000080000A v 2–5-20000000000000A v 4–5-20000000400000A v 4–6-20040000000000A v 5–6-4846684448000566472000A v 1–4–5-000002144000800A v 1–5–6-802116160001680000A v 4–5–6-80114160001240800A v 1–4–5–6-60040000440000W e a k l y a g g r e s s i v e 5
200007004002300N u m b e r o f r a c e s 114375544787622T o t a l n o .i s o l a t e s t e s t e d 50222825251424252525251397S i g n i ?c a n c e 7
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1
F r e q u e n c i e s o f r a c e s i n p o p u l a t i o n s w e r e a s s e s s e d b y t e s t i n g i s o l a t e s i n c o n t r o l l e d c o n d i t i o n s o n c o t y l e d o n s o f a d i f f e r e n t i a l h o s t s e t a b l e t o d e t e c t s e v e n a v i r u l e n c e a l l e l e s (A v r L m 1,A v r L m 2,A v r L m 3,A v r L m 4,A v r L m 5,A v r L m 6,A v r L m 9).2I n i t i a l i n o c u l u m c o m p r i s e d t h e r e s i d u e s o f s u s c e p t i b l e o i l s e e d r a p e c u l t i v a r s S a m o u r a ?¨,
G l a c i e r ,S h o g u n a n d L i r a b o n s a m p l e d l o c a l l y a f t e r h a r v e s t i n 2002.3C u l t i v a r D r a k k a r w i t h n o k n o w n R -g e n e s w a s s o w n a t l e a s t a t 4k m a w a y f r o m t h e c l o s e s t o f t h e t r i a l s P
H O 1t o P H O 4,i n a s e p a r a t e ?e l d t r i a l w i t h n o o i l s e e d r a p e r e s i d u e s p r e s e n t ,t o a s s e s s t h e f r e q u e n c i e s o f d i f f e r e n t a l l e l e s i n n a t u r a l i n o c u l u m n e a r R e n n e s .4A v i s t h e d e n o m i n a t i o n o f L .m a c u l a n s r a c e s a c c o r d i n g t o t h e n o m e n c l a t u r e o f B a l e s d e n t e t a l .,2006.5W e a k l y a g g r e s s i v e i s o l a t e s g a v e t o o f e w s y m p t o m s t o b e a b l e t o d i f f e r e n t i a t e b e t w e e n v i r u l e n t a n d a v i r u l e n t i s o l a t e s w h a t e v e r t h e r e s i s t a n t h o s t a s s e s s e d .6T h e m o s t f r e q u e n t r a c e i n e a c h t r i a l i s i n b o l d t y p e .7P -v a l u e f o r v 2t e s t c o m p a r i n g o b s e r v e d f r e q u e n c i e s o f o c c u r r e n c e o f A v 1a n d A v 6a l l e l e s i n i s o l a t e p o p u l a t i o n s t o f r e q u e n c i e s e x p e c t e d f o r t w o g e n e s s e p a r a t e d b y a l i n k a g e d i s t a n c e o f 6c M (F u d a l e t a l .,2007)
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asymptote of 4.95for DI as leaf lesions increased.By con-trast,for ‘Darmor’there was no relationship between sever-ity of leaf lesions and stem base canker severity (data not presented).
By contrast with autumn 2002,the overall conditions in 2003were less favourable for the development of leaf lesions and although the incidence of leaf lesions was similar (Fig.3a),there were fewer lesions per plant on ‘Eurol’and ‘Darmor’(Fig.4a)in PHO1than in 2002.The position and parallelism regression analyses suggested that there was little effect of quantitative resistance on the incidence (Fig.3)or severity (Fig.4)of leaf lesions in autumn.The data for ‘Eurol’and ‘Darmor’were ?tted by common lines within MX status (i.e.without ?with Rlm6)for all four series over the period 2003–2006for incidence (Fig.3),and for three of the four series of trials for severity (Fig.4).There was a trend towards a greater number of leaf lesions on ‘Darmor’than ‘Eurol’but the difference was signi?cant (P =0.005,F -test)only in PHO3(Fig.4c).However,the regressions showed a seasonal increase with time in the inci-dence and severity of leaf lesions on ‘Eurol’and ‘Darmor’in all four series over the period 2003–2006.For example,in PHO1the incidence of leaf lesions on ‘Eurol’increased from 71%(autumn 2004)to 100%(autumn 2005),and the average number of leaf lesions per plant increased from 2.2in autumn 2004to 15.5in autumn 2005.By contrast,the regression analyses showed signi?cant (P <0.05,F -tests)effects of quantitative resistance on sever-ity of stem canker,with more severe canker on ‘Eurol’than on ‘Darmor’in all four series (Fig.5).However,there was no difference between ‘Eurol’and ‘Darmor’in incidence of stem base canker except in the 2004?2005cropping season (data not presented).The 2003?2004cropping season was less favourable for the development of stem canker than the 2002?2003season,with the severity of stem canker on ‘Eurol’in (PHO1)half that in the previous season (Fig.5a).These differences between the two cropping seasons were con?rmed by the predictions for severity of stem cankers made from the weather data using the model of Evans et al.(2008)(data not presented).The regression analysis shows a seasonal increase with time in the severity of stem canker on ‘Eurol’and ‘Darmor’over the period 2003–2006in (PHO1,Fig.5a),(PHO2,Fig.5b)and (PHO3,Fig.5c).By contrast,there was no increase with time in DI in the series with ‘DarmorMX’inoculum,with some canker on ‘Eurol’and little on ‘Darmor’(PHO4,Fig.5d).‘Darmor’resistance was more effective against stem canker develop-ment when inoculum concentration was low,as in the crop-ping seasons 2003?2004and 2004?2005,with DI =3.63and DI =3.29for ‘Eurol’compared with DI =1.51and DI =1.30for ‘Darmor’in PHO1.In addition,‘Darmor’resistance was effective against virulent populations selected
Table 3Seasonal changes in the percentage of leaf lesions caused by virulent (avrLm6)isolates of L.maculans assessed in autumn as the number of leaf lesions per oilseed rape plant on ‘EurolMX’divided by the number on ‘Eurol’in each trial
Trial Genotype used as inoculum Autumn No.of leaf lesions per plant on EurolMX No.of leaf lesions per plant on ‘Eurol’Leaf lesions EurolMX ?[no.of lesions Eurol]·1001Initial Susceptible 20020.127.58 1.55PHO1
Eurol
20030.01 4.460.2220040.00 2.210.0020050.0015.520.002006–––2
PHO2EurolMX
20030.460.41113.112004 1.52 4.3834.702005 2.34 4.9547.172006 5.629.0062.48PHO3Darmor
20030.01 3.940.2520040.01 4.060.2520050.02 4.290.352006–––PHO4DarmorMX
2003 1.28 1.3693.8920040.290.4465.4120050.15 2.047.372006
0.35
8.96
3.94
Seasonal changes in the percentage of leaf lesions caused by virulent (avrLm6)isolates of Leptosphaeria maculans .1
The mean numbers of leaf lesions per plant assessed once in autumn on at least 60plants of ‘EurolMX’(leaf lesions due to only virulent isolates avrLm6)and of ‘Eurol’(leaf lesions caused by avirulent AvrLm6plus avrLm6isolates)in each trial were used to estimate the percentage (%)of leaf lesions caused by virulent isolates.2
Not
assessed.
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on ‘EurolMX’residues in PHO2,as its DI was less than those of the other lines.Although canker severity was always less on ‘Darmor’than on ‘Eurol’,the production of ascosp-ores on residues of both cultivars caused similar seasonal patterns of disease in their respective series of trials PHO1(Fig.5a)and PHO3(Fig.5c).All lines assessed developed similar DI in the two series of trials.Moreover,DI on ‘Dar-mor’in PHO3increased similarly with time to that in PHO1.
Effects of qualitative resistance on seasonal changes in leaf lesions and stem canker (‘EurolMX’vs ‘Eurol’;‘DarmorMX’vs ‘Darmor’)
The regression analyses showed that both ‘EurolMX’and ‘DarmorMX’always developed fewer leaf lesions and less severe stem canker than their recurrent cultivars ‘Eurol’and ‘Darmor’in PHO1(Figs 3a,4a,5a)and PHO3(Figs 3c,4c,5c)series.This was also the case for the initial 2002?2003trial (P <0.05,LSD test).Moreover,there was no difference in severity of leaf lesions or stem canker between the genotypes with Rlm6‘EurolMX’and ‘Dar-morMX’in PHO1and PHO3series.However,a few leaf lesions were observed on these Rlm6lines in the 2002?2003
trial and subsequently in the PHO1and PHO3series,sug-gesting that isolates virulent against Rlm6existed at a low frequency in the local population.When ‘EurolMX’or ‘DarmorMX’residues were used as inoculum in the PHO2and PHO4series,respectively,there was a low incidence (Fig.3b,d)and severity (Fig.4b,d)of leaf lesions on plants assessed in autumn 2003,compared with those inoculated with ‘Eurol’residues (PHO1,Figs 3a,4a).However,the DI had increased greatly on ‘EurolMX’by the third year (2004?2005)of the PHO2series,showing that the Rlm6resistance in this line was no longer effective.The incidence of plants with leaf lesions on ‘Eurol’,‘Darmor’and,to a les-ser extent,on ‘EurolMX’and ‘DarmorMX in autumn from 2004onwards was much greater than in autumn 2003(Fig.3b).As a consequence,there was little difference in DI the following summer between ‘EurolMX’and ‘Eurol’in this series from the 2004?2005cropping season onwards (Fig.5b).Although the epidemic was not severe in PHO2in 2004?2005because of the use of ‘EurolMX’residues as inoculum,the severity of epidemics increased in subsequent seasons.
Effects of combining quantitative and qualitative resis-tance on seasonal changes in leaf lesions and stem canker (‘DarmorMX’vs ‘EurolMX’)
The combination of quantitative and qualitative resistance in ‘DarmorMX’was effective in control of leaf lesions and stem canker.For example,in PHO1in the autumn of 2005,leaf lesions were abundant on all plants of ‘Darmor’whereas only 1.7%of ‘DarmorMX’plants had leaf lesions (Fig.4a).Similarly,in that year,the incidence and severity of stem canker were considerably less on ‘DarmorMX’(27%of plants with symptoms,DI =0.27)than on ‘Eurol’(inci-dence 86%,DI =5.87)(P <0.05,LSD test,for both incidence and severity).‘DarmorMX’resistance remained effective throughout the PHO4series with few leaf lesions and little stem canker developing at least until the 5th year of the experiment (Figs 3d,4d,5d).It was dif?cult to ?nd pseudothecia of L.maculans on ‘DarmorMX’residues over the 4yr of its use as inoculum,and most of the pseudothecia found on this line were of L.biglobosa (data not shown).
Discussion
This work provides experimental evidence,for an arable crop grown in successive seasons,that the combination of qualitative effective major gene and quantitative polygenic resistance to a pathogen both improves control of the dis-ease and increases durability of the qualitative resistance.When combined with quantitative resistance in ‘Darmor-MX’,the qualitative Rlm6resistance provided effective control of phoma stem canker until at least the 5th year,2yr longer than when it was deployed in a susceptible
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background ‘EurolMX’.This conclusion,based on direct experimental evidence for a ?eld crop attacked by a fungal disease,is consistent with both theoretical predictions (Pietravalle et al.,2006)and glasshouse work with a virus disease (Palloix et al.,2009).Moreover,an effect of quanti-tative resistance has been suggested by Brun et al.(2000)to explain the superior durability of the R gene from B.nigra compared with that from B.juncea,introgressed into B.napus lines either with or without quantitative resistance,respectively.The bene?t of this combination of R -gene mediated resistance and quantitative resistance may have involved a decrease in airborne inoculum concentration because the quantitative resistance limited the number of pseudothecia formed,and a decrease in number of leaf lesions because operation of Rlm6eliminated leaf lesions caused by the avirulent fraction of the pathogen population.This interpretation is supported by the small numbers of pseudothecia on residues and of leaf lesions observed on ‘DarmorMX’throughout the years in PHO4whereas no such restriction occurred on ‘EurolMX’in PHO2.The results of this experiment,in which only c.40%of the L.maculans population was virulent in the third season when severe epidemics occurred on ‘EurolMX’in PHO2,suggests that population size of virulent isolates is more important in determining the effectiveness of qualitative resistance than the frequency of virulent isolates.The con-siderable decrease (by comparison with 2002?2003)in severity of stem canker on all host genotypes (including the susceptible ‘Eurol’)in trials inoculated in 2003and 2004with inoculum from the ‘MX’lines provides good evidence that R genes act by reducing pathogen population sizes.The rate of loss in effectiveness of resistance in ‘EurolMX’in PHO2was comparable to that of other major genes in susceptible backgrounds in commercial agricultural crops.Resistance derived from Brassica rapa var.sylvestris intro-duced into ‘Surpass’was ineffective and associated with devastating epidemics within 2yr after its cultivation over large areas in Australia (Sprague et al.,2006).Resistance conferred by Rlm1introduced into several commercial cul-tivars in France was also rendered ineffective after 2–3yr
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(Rouxel et al.,2003).This indicates that the results obtained in small ?eld experiments can adequately re?ect the mechanisms acting over large geographical scales during the commercial use of resistant cultivars.
There was no evidence that quantitative background resistance affected the surprising increase in the frequency of isolates avirulent (AvrLm1)against Rlm1associated with the increase in frequency of isolates that were virulent (av-rLm6)against Rlm6;similar phenomena were observed in both PHO2and PHO4.One explanation may be that selection for virulence at the AvrLm6locus was indirectly linked to selection against virulence at the AvrLm1locus (if selection for virulence avrlm6occurred in Avrlm1iso-lates,which represented 30%of the initial population).Another explanation might be that there is a greater ?tness cost of virulence at both loci (avrLm1?avrLm6isolates)than of virulence at only the AvrLm6locus (AvrLm1?av-rLm6isolates).It is likely that selection at the two loci may be linked,since they are located within 6cM of each other on the L.maculans genome (Fudal et al.,2007).Furthermore,there is evidence of a ?tness cost of virulence at the AvrLm1locus (Huang et al.,in press),as at the AvrLm4locus (Huang et al.,2006b).Like evolution for virulence at the AvrLm1locus,evolution for virulence at the AvrLm6locus often involves deletion of the gene (Fu-dal et al.,2007,2009).It is important to understand which mechanism is responsible for the link between Av-rLm1and avrLm6,because either mechanism can be exploited in the management of resistance against L.macu-lans but they require different breeding and cultivar deployment strategies.Negatively linked indirect selection offers the possibility of ‘recycling’ineffective resistance genes in successive cycles of selection but requires that dif-ferent R genes are kept separate in different cultivars for them to be effective (Andrivon &de Vallavieille-Pope,1993).By contrast,?tness costs that favour monovirulent isolates against multivirulent isolates would suggest that pyramiding of several R genes in a single cultivar should be an effective strategy to control the disease (Stukenbrock &McDonald,2008).
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These results suggest that the quantitative resistance in ‘Darmor’did not exert selection for speci?c virulence in the pathogen population,as the frequency of avirulence ?viru-lence alleles in isolates from ‘Darmor’residues did not change over the years.It was similar to that of the initial L.maculans population and to that recovered from the suscep-tible cultivar ‘Eurol’.This may be explained by the fact that,while qualitative R -gene resistance operates in the leaf tis-sues (Huang et al.,2006a),the quantitative resistance against L.maculans operates later in disease development,in leaf petiole and stem tissues (Fitt et al.,2006;Huang et al.,2009).Whereas quantitative resistance is generally consid-ered to be durable (Poland et al.,2008),there is experimen-tal data to show that some pathogens can adapt to gradually erode such polygenic resistance and render it ineffective (Andrivon et al.,2007).However,in our ?eld experiment,the ‘Darmor’resistance was still effective after 5yr of recur-rent selection on L.maculans populations.Nevertheless,it is important to widen the genetic base of quantitative resistance against L.maculans in oilseed rape,as the quanti-tative resistance in European oilseed rape is mainly based on ‘Jet Neuf’resistance.‘Jet Neuf’was cultivated as the only cultivar in France and the rest of Europe from 1977–1983,without erosion of its resistance (Delourme et al.,2006).This lasting performance of resistance ?ts well with John-son’s (1981)de?nition of durable resistance.Nevertheless,‘Jet Neuf’also carries Rlm4and there are some avirulent Av-rLm4isolates currently found in European populations of L.maculans (Stachowiak et al.,2006).Therefore,the dura-bility of ‘Jet Neuf’resistance may have been derived from a combination of an ef?cient qualitative major gene with quantitative resistance.
Breeders may be reluctant to put in extra effort required to combine polygenic quantitative resistance with an effective major gene resistance in new cultivars,as there is no immediate bene?t in breeding cultivars with only an effective major resistance gene.However,our results clearly show that this strategy bene?ts yields in the long-term by
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extending the durability of resistance,so that new resistance genes can be effectively deployed for longer periods.Such durable resistance to crop diseases provided by combining quantitative and qualitative resistance can help to avoid the devastating ‘boom and bust’cycles (Stukenbrock &McDonald,2008)and hence make an essential contribu-tion to global food security.
Acknowledgements
We acknowledge the European Union (QKL5-CT-2002-01813,SECURE project)and the French National Insti-tute for Agricultural Research (INRA)for funding the investigations under ?eld and controlled conditions at Le Rheu from 2002to 2006and the 2006?2007trials,respec-tively.We thank the UK Biotechnology and Biological Research Council and Department for Environment,food and Rural Affairs for supporting the work at Rothamsted and N.Evans for managing the SECURE project.We are grateful to H.Picault,C.Domin and the Experimental Unit INRA UE787,for performing the agronomic work needed for the ?eld experiment.We thank L.Bousset for providing leaves of ‘Drakkar’in autumn 2004allowing characterization of natural L.maculans populations and M.H.Balesdent for providing winter oilseed rape line 22.1.1(Rlm3)and Brassica juncea line150.2.1(Rlm5)to be included in the differential host set.
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