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International audience; Aphanomyces euteiches Drechsler is an oomycete pathogen of leguminous crops that causes root rot, a severe disease of pea (Pisum sativum L.) worldwide. An improved understanding of the genetic structure of A. euteiches populations would increase knowledge of pathogen evolution and assist in the design of strategies to develop pea cultivars and germplasm with stable disease resistance. Twenty six primers pairs were used to amplify Sequence Related Amplified Polymorphisms (SRAP) among 49 A. euteiches isolates sampled from pea. A total of 190 polymorphic SRAP bands were generated, of which 82 were polymorphic between all the A. euteiches isolates. The percentage of polymorphic bands per primer pair ranged from 22 to 75%. According to the PIC value estimated for each marker, 60% of the SRAP markers were highly to reasonably informative (PIC > 0.25). Genetic structure of A. euteiches populations sampled in different American and French locations showed low to high genetic diversity within populations. The largest variation occurred within countries, with a total estimated genetic diversity of 0.477 and 0.172 for American and French populations, respectively. This was particularly evident from a principal component analysis (PCA) and a Minimum Spanning Networks (MSN) based on genetic profiles of isolates, which generated two different clusters, one corresponding to the French isolates and four American isolates (MV1, MV5, MV7, Ath3), and the other to American isolates. A. euteiches populations from cultivated pea in France appeared as a single unstructured population, whereas American isolates of A. euteiches diverged into three different populations.

Background Understanding the effects of resistance QTL on pathogen development cycle is an important issue for the creation of QTL combination strategies to durably increase disease resistance in plants. The oomycete pathogen Aphanomyces euteiches, causing root rot disease, is one of the major factors limiting the pea crop in the main producing countries. No commercial resistant varieties are currently available in Europe. Resistance alleles at seven main QTL were recently identified and introgressed into pea agronomic lines, resulting in the creation of Near Isogenic Lines (NILs) at the QTL. This study aimed to determine the effect of main A. euteiches resistance QTL in NILs on different steps of the pathogen life cycle. Results NILs carrying resistance alleles at main QTL in susceptible genetic backgrounds were evaluated in a destructive test under controlled conditions. The development of root rot disease severity and pathogen DNA levels in the roots was measured during ten days after inoculation. Significant effects of several resistance alleles at the two major QTL Ae-Ps7.6 and Ae-Ps4.5 were observed on symptom appearance and root colonization by A. euteiches. Some resistance alleles at three other minor-effect QTL (Ae-Ps2.2, Ae-Ps3.1 and Ae-Ps5.1) significantly decreased root colonization. The combination of resistance alleles at two or three QTL including the major QTL Ae-Ps7.6 (Ae-Ps5.1/Ae-Ps7.6 or Ae-Ps2.2/Ae-Ps3.1/Ae-Ps7.6) had an increased effect on delaying symptom appearance and/or slowing down root colonization by A. euteiches and on plant resistance levels, compared to the effects of individual or no resistance alleles. Conclusions This study demonstrated the effects of single or multiple resistance QTL on delaying symptom appearance and/or slowing down colonization by A. euteiches in pea roots, using original plant material and a precise pathogen quantification method. Our findings suggest that single resistance QTL can act on multiple or specific steps of the disease development cycle and that their actions could be pyramided to increase partial resistance in future pea varieties. Further studies are needed to investigate QTL effects on different steps of the pathogen life cycle, as well as the efficiency and durability of pyramiding strategies using QTL which appear to act on the same stage of the pathogen cycle. Electronic supplementary material The online version of this article (doi:10.1186/s12870-016-0822-4) contains supplementary material, which is available to authorized users.

Background Genome-wide association (GWA) mapping has recently emerged as a valuable approach for refining the genetic basis of polygenic resistance to plant diseases, which are increasingly used in integrated strategies for durable crop protection. Aphanomyces euteiches is a soil-borne pathogen of pea and other legumes worldwide, which causes yield-damaging root rot. Linkage mapping studies reported quantitative trait loci (QTL) controlling resistance to A. euteiches in pea. However the confidence intervals (CIs) of these QTL remained large and were often linked to undesirable alleles, which limited their application in breeding. The aim of this study was to use a GWA approach to validate and refine CIs of the previously reported Aphanomyces resistance QTL, as well as identify new resistance loci. Methods A pea-Aphanomyces collection of 175 pea lines, enriched in germplasm derived from previously studied resistant sources, was evaluated for resistance to A. euteiches in field infested nurseries in nine environments and with two strains in climatic chambers. The collection was genotyped using 13,204 SNPs from the recently developed GenoPea Infinium® BeadChip. Results GWA analysis detected a total of 52 QTL of small size-intervals associated with resistance to A. euteiches, using the recently developed Multi-Locus Mixed Model. The analysis validated six of the seven previously reported main Aphanomyces resistance QTL and detected novel resistance loci. It also provided marker haplotypes at 14 consistent QTL regions associated with increased resistance and highlighted accumulation of favourable haplotypes in the most resistant lines. Previous linkages between resistance alleles and undesired late-flowering alleles for dry pea breeding were mostly confirmed, but the linkage between loci controlling resistance and coloured flowers was broken due to the high resolution of the analysis. A high proportion of the putative candidate genes underlying resistance loci encoded stress-related proteins and others suggested that the QTL are involved in diverse functions. Conclusion This study provides valuable markers, marker haplotypes and germplasm lines to increase levels of partial resistance to A. euteiches in pea breeding. Electronic supplementary material The online version of this article (doi:10.1186/s12864-016-2429-4) contains supplementary material, which is available to authorized users.

The consequence of 10 successive monocultural cycles involving different legume species/cultivars on the inoculum potential (IP) of soils naturally infested by Aphanomyces euteiches was investigated under greenhouse conditions. The results showed that the IP of a soil naturally infested by A.euteiches can be significantly modified not only by the non-host or host status of crop species but also by the level of resistance of the cultivar. Susceptible species/cultivars (pea, lentil and susceptible cultivars of vetch and faba bean) are very favourable to pathogen multiplication, and continuous cultivation of each of these increased the IP values of a soil with a moderate initial IP (from 1 center dot 9 to 3 center dot 5 after 10 cycles). Conversely, non-host species and resistant cultivars of vetch or faba bean contributed to reducing the IP values of soils irrespective of the initial IP (from 1 center dot 9 to 0 center dot 5 and from 4 to 2, respectively, after 10 cycles). Aphanomyces root rot severity values on the resistant legume species/cultivars were not affected by the successive cultural cycles. This study, which showed that the IP of A.euteiches in soil can be reduced by planting appropriate legume species and cultivars in greenhouse conditions, will be useful for defining better crop successions for legumes.

International audience; The main objective of the study was to describe the horizontal and vertical distribution of Aphanomyces root rot in a naturally infested pea field. Measurements of inoculum potential clearly indicated a horizontal distribution of inoculum among several foci in the field, these foci differing in size and disease intensity. A highly significant relationship was observed between disease severity on plants during the cropping season and soil inoculum potential. In terms of the vertical distribution of inoculum in the soil, detection was maximal at a depth of 10 to 40cm, but inoculum was detected down to a depth of 60cm. Generally, inoculum potential was lowest for the layers at depths of 0 to 10 and 50 to 60cm. Inoculum distribution and the value of the methodologies used are discussed in terms of possible use for epidemiology and disease forecasting.

International audience; Marker-assisted backcrossing was used to generate pea NILs carrying individual or combined resistance alleles at main Aphanomyces resistance QTL. The effects of several QTL were successfully validated depending on genetic backgrounds. Quantitative trait loci (QTL) validation is an important and often overlooked step before subsequent research in QTL cloning or marker-assisted breeding for disease resistance in plants. Validation of QTL controlling partial resistance to Aphanomyces root rot, one of the most damaging diseases of pea worldwide, is of major interest for the future development of resistant varieties. The aim of this study was to validate, in different genetic backgrounds, the effects of various resistance alleles at seven main resistance QTL recently identified. Five backcross-assisted selection programs were developed. In each, resistance alleles at one to three of the seven main Aphanomyces resistance QTL were transferred into three genetic backgrounds, including two agronomically important spring (Eden) and winter (Isard) pea cultivars. The subsequent near-isogenic lines (NILs) were evaluated for resistance to two reference strains of the main A. euteiches pathotypes under controlled conditions. The NILs carrying resistance alleles at the major-effect QTL Ae-Ps4.5 and Ae-Ps7.6, either individually or in combination with resistance alleles at other QTL, showed significantly reduced disease severity compared to NILs without resistance alleles. Resistance alleles at some minor-effect QTL, especially Ae-Ps2.2 and Ae-Ps5.1, were also validated for their individual or combined effects on resistance. QTL × genetic background interactions were observed, mainly for QTL Ae-Ps7.6, the effect of which increased in the winter cultivar Isard. The pea NILs are a novel and valuable resource for further understanding the mechanisms underlying QTL and their integration in breeding programs.

Common root rot (Aphanomyces euteiches Drechs.) has become a very destructive disease in the French pea crops since 1993. For an accurate investigation of the virulence variability among French A. euteiches populations and between French and foreign populations, a new set of differential pea genotypes was developed. Thirty-three American and European pea lines, displaying different levels of resistance, were screened in a growth chamber against two French isolates. Symptoms (disease severity from 0 to 5, evaluating symptom surface on roots and epicotyl) and percentage of top fresh weight (inoculated/uninoculated top fresh weight ratio) were measured. From this screening 12 relatively resistant lines, from various genetic backgrounds, were identified along with a highly susceptible control. This set of 13 genotypes was inoculated under controlled conditions with 14 isolates from France, Sweden, USA, Canada and New Zealand, to investigate genotype–isolate interactions. Root symptoms were rated (disease severity), and a susceptibility/resistance threshold was established at disease severity = 1. Significant quantitative interactions were observed, and five 'resistance patterns' were identified, leading to a set of six pea genotypes: Baccara (susceptible), Capella, MN313, 902131, 552 and PI180693. Fields trials of this set in 1999 and 2000 gave the same resistance rankings than in growth chamber conditions. This set will allow more accurate assessments of the variability in virulence/aggressiveness of A. euteiches isolates from France and foreign countries, and further investigations of the epidemiological and genetic basis of pea–A. euteiches interactions.

International audience; Aphanomyces euteiches is a widespread oomycete pathogen causing root rot in a wide range of leguminous crops. Losses can reach up to 100% for pea culture and there is currently no registered pesticide for its control. Crop management remains the most efficient tool to control root rot, and avoidance of infested soil seems to be the optimal solution. A test was developed to identify fields suitable for pea crops, consisting of the determination of the inoculum potential of soil using baiting plants. A new rapid, specific, and sensitive molecular method is described allowing the quantification of less than 10 oospores per gram of soil. This challenge is achieved by a real-time polymerase chain reaction procedure targeting internal transcribed spacer 1 from the ribosomal DNA operons. A preliminary study based on typical soils from northwestern France demonstrated that the A. euteiches oospore density in soil is related to the inoculum potential. Furthermore, this method has proved sensitive enough to accurately study the influence of biotic factors that may govern the actual emergence of root rot.

The role of infected seed in the epidemiology of Ascochyta blight of pea, caused by Mycosphaerella pinodes, was studied both under growth chamber and field conditions, using healthy seeds, naturally infected seeds and artificially infected seeds. Results suggest that infected seeds caused serious losses, as a result of poor germination and high transmission of the disease, to parts of the plants under soil level. Foot rot symptoms often caused the death of young seedlings. Losses were increased by low temperatures during the early stage of crop development. M. pinodes progressed from seeds to aerial parts of the plants, but no Ascochyta blight symptoms occurred, the disease remaining near to the basal parts of the plants as a foot rot symptom. This suggests that seeds cannot be regarded as a source of contamination in the epidemiology of the disease.

Publication Inra prise en compte dans l'analyse bibliométrique des publications scientifiques mondiales sur les Fruits, les Légumes et la Pomme de terre. Période 2000-2012. http://prodinra.inra.fr/record/256699; International audience; Eight germplasm lines of green pea (Pisum sativum L.)-RIL 846-07 (Reg. No. GP-99, PI 660729), RIL 847-08 (Reg. No. GP-100, PI 660730), RIL 847-22 (Reg. No. GP-101, PI 660731), RIL 847-28 (Reg. No. GP-102, PI 660732), RIL 847-45 (Reg. No. GP-103, PI 660733), RIL 847-50 (Reg. No. GP-104, PI 660734), RIL 847-53 (Reg. No. GP-105, PI 660735), and RIL 847-68 (Reg. No. GP-106, PI 660736)-were developed and released jointly by the USDA-ARS, the French National Institute for Agricultural Research (INRA), and North Dakota State University. They were selected from an F-8-derived recombinant inbred line (RIL) population developed from a cross between 'Dark Skin Perfection' and 90-2131. These lines all have high levels of partial resistance to root rot caused by Aphanomyces euteiches Drechs. f. sp. pisi when screened in 12 environments of 10 infested field nurseries across 4 yr and four locations in the USA and France and with two reference isolates in controlled conditions. On the basis of a disease index derived from the 12 environments (location-years), these lines had improved partial resistance compared with either parent. RIL 847-028, RIL 847-050, RIL 847-053, and RIL 847-068 performed better in U.S. field nurseries than in the French nurseries. Conversely, RIL 846-007, RIL 847-008, RIL 847-022, and RIL 847-045 performed better in the French field nurseries than in the U.S. nurseries. The eight lines also have acceptable agronomic characteristics: they have white flowers, straight blunt green pods, and green cotyledons; are clear seeded and semidwarf; and flower at the 14th-16th node in 57-61 d. The release of these germplasms will assist the breeding and development of either smooth-or wrinkle-seeded cultivars for the fresh or freezer pea and dry pea markets, respectively, with improved partial resistance to Aphanomyces root rot.

Publication Inra prise en compte dans l'analyse bibliométrique des publications scientifiques mondiales sur les Fruits, les Légumes et la Pomme de terre. Période 2000-2012. http://prodinra.inra.fr/record/256699; International audience; Partial resistances, often controlled by quantitative trait loci (QTL), are considered to be more durable than monogenic resistances. Therefore, a precursor to developing efficient breeding programs for polygenic resistance to pathogens should be a greater understanding of genetic diversity and stability of resistance QTL in plants. In this study, we deciphered the diversity and stability of resistance QTL to Aphanomyces euteiches in pea towards pathogen variability, environments and scoring criteria, from two new sources of partial resistance (PI 180693 and 552), effective in French and USA infested fields. Two mapping populations of 178 recombinant inbred lines each, derived from crosses between 552 or PI 180693 (partially resistant) and Baccara (susceptible), were used to identify QTL for Aphanomyces root rot resistance in controlled and in multiple French and USA field conditions using several resistance criteria. We identified a total of 135 additive-effect QTL corresponding to 23 genomic regions and 13 significant epistatic interactions associated with partial resistance to A. euteiches in pea. Among the 23 additive-effect genomic regions identified, five were consistently detected, and showed highly stable effects towards A. euteiches strains, environments, resistance criteria, condition tests and RIL populations studied. These results confirm the complexity of inheritance of partial resistance to A. euteiches in pea and provide good bases for the choice of consistent QTL to use in marker-assisted selection schemes to increase current levels of resistance to A. euteiches in pea breeding programs

En Europe, les protéagineux peuvent contribuer à faire face aux besoins en protéines végétales pour l'alimentation animale. Dans ce contexte, la stabilisation et l'amélioration du rendement du pois protéagineux sont des objectifs primordiaux des généticiens et des sélectionneurs. Ces objectifs pourraient notamment être atteints au travers du développement de variétés d'hiver qui ont un cycle de développement plus long que les pois de printemps, actuellement les plus cultivés. Cependant, le développement de la culture se heurte à plusieurs facteurs limitants, dont des stress biotiques (pourriture racinaire liée à Aphanomyces euteiches, ascochytose liée à Mycosphaerella pinodes) et abiotiques (gels hivernaux). L’amélioration pour la tolérance au gel et pour les résistances à l'ascochytose et à Aphanomyces s’appuie sur la connaissance de la variabilité des sources de résistance, l’analyse des déterminants génétiques ou QTL contrôlant ces résistances, et l’usage des marqueurs moléculaires pour combiner ces facteurs génétiques afin de développer des résistances efficaces et durables. La connaissance de la diversité de ces facteurs, de leur stabilité vis à vis des environnements et vis à vis de la variabilité des stress, et des mécanismes sous-jacents permet d’orienter le choix des QTL à combiner en sélection. Les lignées résistantes créées pourront être évaluées pour leur adaptation à différents environnements dont les principaux facteurs limitants sont décrits au sein de réseaux expérimentaux, et pour l’effet de leur culture sur l’adaptation éventuelle des populations pathogènes., In Europe, pulses can contribute to face needs in protein supply for animal feed. Field pea yield stabilization and improvement are essential objectives for geneticists and breeders, which could be reached through the development of autumn-sown varieties, with a longer development cycle than spring peas. However, pea crop development faces several limiting factors, among which biotic (root rot due to Aphanomyces euteiches, ascochyta blight due to Mycosphaerella pinodes) and abiotic (winter frost) stresses. The improvement for the tolerance to frost and for partial resistances to ascochyta blight and to Aphanomyces relies on the knowledge of the variability of sources of resistance, analysis of genetic factors or QTL controlling resistance or tolerance, and use of molecular markers to combine these genetic factors in order to develop efficient and durable resistances. The knowledge of the diversity of these factors, of their stability towards environments and stress variability, and of underlying mechanisms allows to target the choice of the QTL to combine together within breeding programs. The created partially resistant lines will be screened for their adaptation to various environments within field trial networks whose main limiting factors are described, and for the effect of their cropping on the possible adaptation of pathogen populations.

International audience; A higher understanding of genetic and genomic bases of partial resistance in plants and their diversity regarding pathogen variability is required for a more durable management of resistance genetic factors in sustainable cropping systems. In this study, we investigated the diversity of genetic factors involved in partial resistance to Aphanomyces euteiches, a very damaging pathogen on pea and alfalfa, in Medicago truncatula. A mapping population of 178 recombinant inbred lines, from the cross F83005.5 (susceptible) and DZA045.5 (resistant), was used to identify quantitative trait loci for resistance to four A. euteiches reference strains belonging to the four main pathotypes currently known on pea and alfalfa. A major broad-spectrum genomic region, previously named AER1, was localized to a reduced 440 kb interval on chromosome 3 and was involved in complete or partial resistance, depending on the A. euteiches strain. We also identified 21 additive and/or epistatic genomic regions specific to one or two strains, several of them being anchored to the M. truncatula physical map. These results show that, in M. truncatula, a complex network of genetic loci controls partial resistance to different pea and alfalfa pathotypes of A. euteiches, suggesting a diversity of molecular mechanisms underlying partial resistance.

A real-time PCR assay using 136F/211R primers and 161T TaqMan® probe for the detection and quantification of Aphanomyces euteiches in soil is presented. The specificity of primers was tested on 105 different A. euteiches isolates, mainly from France. A calibration curve was established with a plasmid pHS1 resulting from the target region cloned into the pCR4 Topo vector (Invitrogen). The target copy number was evaluated and was constant whatever the isolate. A DNA-based method was able to discriminate between different artificial infestation levels in soil with small SDs thus validating the relevance of the extraction and amplification method in soil samples. Furthermore, a good correlation was observed between inoculum quantity in soil estimated by qPCR and root rot severity in plant evaluated by bioassays. These steps are essential when considering the feasibility of using a DNA-based method as a fast and accurate way to evaluate inoculum quantity in soil.

International audience; Ditylenchus dipsaci, the stem nematode of alfalfa (Medicago sativa), Mycosphaerella pinodes, cause of Ascochyta blight in pea (Pisum sativum) and Aphanomyces euteiches, cause of pea root rot, result in major yield losses in French alfalfa and pea crops. These diseases are difficult to control and the partial resistances currently available are not effective enough. Medicago truncatula, the barrel medic, is the legume model for genetic studies, which should lead to the identification and characterization of new resistance genes for pathogens. We evaluated a collection of 34 accessions of M. truncatula and nine accessions from three other species (two from M. italica, six from M. littoralis and one from M. polymorpha) for resistance to these three major diseases. We developed screening tests, including standard host references, for each pathogen. Most of the accessions tested were resistant to D. dipsaci, with only three accessions classified as susceptible. A very high level of resistance to M. pinodes was observed among the accessions, none of which was susceptible to this pathogen. Conversely, a high level of variation, from resistant to susceptible accessions, was identified in response to infection by A. euteiches

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