WP coordinator: CSIC

Other partner involved: all

Objectives
Research coordination of the project. Financial management of ARIMNet research funding, which will be handled directly between national researchers and their national funding organisations in each participating country.

Methodology and study materials
T0.1: Facilitate close interaction between the Partners being the single point of contact between the ARIMNet Call Office and the Researchers
T0.2: Draw a Consortium Agreement in order to manage the delivery of the project activities, finances, Intellectual Property Rights (IPR) and to avoid disputes which might be detrimental to the completion of the project
T0.3: Redirect specific parts of the project if required by the progress of the Work packages
T0.4: Compile and submit reports/deliverables to the ARIMNet Call Office on behalf of the Research Consortium
T0.5: Three meetings (a kick-off, mid-term meeting and final ones) will be organised to share and capitalise experience to the benefit of the ARIMNet community and beyond
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Deliverables
D0.1: Consortium agreement
D0.2: Interim monitoring
D0.3: Final report to be delivered to the Call Office within two months following project conclusion
D0.4: Plan meetings calendar

WP coordinator: INRAT

Other partners involved: CSIC, CRRGC, INRA, INRAM, IAV, ARC, ITQB

Objectives:
– Inventory of existing data on constraints for chickpea, common bean, faba bean, lentil and pea production
– Inventory of existing data with regard to agronomic performance, resistance, adaptability and nutritional value
– Definition of desired phenotypes suitable for each area

Methodology and study materials
T1.1: INRAT will coordinate the inventory of existing data on regional priorities, agronomic performance of cultivars or landraces available and constraints for chickpea, common bean, faba bean, lentil and pea production. All partners will prospect and provide the information from their own area during the first 6 months of the project.
T1.2: Based on this inventory, a relevant collection of about 10 genotypes per crop and country will be composed to serve as a basis of the present project delivered at month 12 of the project. Based on the demand set per region a different ideotype (ideal genotype) will be defined between months 12 to 24.

Deliverables
D1.1: Definition of phenotypes and genotypes adapted to sustainable agriculture demands in different regions
D1.2: Information and protocols for field evaluation of agronomic traits and resistance to biotic and abiotic constrains
D1.3: Selection of the most representative lines/varieties that might serve as source gene-pool for genotype enhancement

WP coordinator: CSIC

Other partners involved: INRA, INRAT, CRRGC, INRAM, ARC, ITQB

Objectives
– Development of reliable screening methods for the most relevant biotic stresses: Ascochyta blights, broomrapes, rusts, fusarium wilts and chocolate spot both under field and growth chamber conditions.
– Identification of sources of resistance
– Characterisation of the resistance mechanisms at the cellular, physiological and molecular level
– Multilocation studies on stability of resistance

Methodology and study materials
T2.1: During year 1 different screening methods will be validated to establish a standard method that is quick, convenient and reliable for breeding.
T2.2: Adapted varieties as well as germplasm collections from each country will be tested, under both field and growth chamber conditions, to identify sources of resistance to major diseases (ascochyta blights, rusts, broomrapes, fusarium wilts and chocolate spot).
T2.3: Stability of the resistance (about 40 entries per crop) will be studied in multi-environment experiments, and in growth chamber against a range of isolates of contrasting virulence during years 2 and 3. Partner 1 (CSIC) will be responsible to coordinate the ring tests. Participating partners will be CSIC, CRRGC, INRAM, INRAT and ARC for chocolate spot (B. fabae); CSIC, CRRGC, INRA-IGEPP, INRAM and INRAT for ascochyta blights (Ascochyta rabiei, A. fabae, A. lentis and M. pinodes); CSIC, CRRGC, INRAM, INRAT and ARC for broomrapes (Orobanche crenata and O. foetida); CSIC, CRRGC, INRAM, INRAT and UKAFR for rusts (Uromyces appendiculatus, U. ciceris-arietini, U. viciae-fabae and U. pisi); and CSIC, CRRGC, INRA-IGEPP, INRAM, INRAT and ARC for fusariums (F. oxysporum ff.ssp.).
T2.4: Interesting accessions will be crossed with elite cultivars varieties by each partner to produce pre-breeding material that will be submitted to national breeding programmes that should be completed outside this project.

Deliverables
D2.1: Rapid and reliable screening methods suitable for selection and breeding
D2.2: Development of breeding material with improved resistance tested at a wide range of environments and pathogen populations
D2.3: Catalogue of sources of resistance

WP coordinator: INRA-LEG

Other partners involved: CSIC, IAV, INRAT, CRRGC, ARC, ITQB

Objectives:
– Development of reliable screening methods for resistance to drought and salt stresses in legumes
– Validation of drought and salt tolerance in present chickpea, faba bean, lentil and pea material
– Development of genotypes with improved drought tolerance

Methodology and study materials
T3.1: Experimental conditions for investigating pea response to drought and salinity will be established (INRA-LEG), in collaboration with INRAT (year 1). The High Troughput Plant Phenotyping Platform (PPHD) located in Dijon (France), allows the detailed analysis of up to 2000 plants grown under various climatic conditions, through dynamically, but non-destructively characterization by image analysis. RGB visible imaging methodologies will allow gathering details on the architecture of the shoot and root system, while Near Infra Red imaging will allow the measure of soil and plant water status.
T3.2: Screening methods for abiotic stress tolerance will be chosen (year 1). To this purpose, indirect methods (non-destructive and low-cost) such as gas-exchange measurements, chlorophyll fluorescence and canopy temperature depression (CTD) will be tested as indirect, rapid screening methods. They will be confronted to fine phenotyping methods obtained on the PPHD. Indeed, detailed information on plant water status in reaction to limited water availability or in reaction to salinity will be aquired automatically on the platform. In parallel, non-invasive methods will complete the phenotyping such as measurements of the photosynthetic activity, or N2 fixation, as well as destructive methods including membrane permeability and carbon isotope discrimination (Delta C13). The indirect method the most representative of the fine phenotyping method will be chosen for the next tasks.
T3.3: Experimental conditions, and the indirect method for screening for drought and salt tolerance will be adapted from T3.1 and T3.2, respectively, to screen chickpea (CRRGC, INRAT), common bean (ITQB), faba bean (CRRGC, IAV and INRAT), lentil (INRAT), pea (CSIC, CRRGC and INRA-LEG,) genotypes for tolerance to drought and salt stresses. Field trials will comprise one site across years 2 and 3, with two treatments, irrigated vs. drought, both under near field (rain-out shelter) and field condition (natural drought). In addition, plants in controlled-environment chambers will be subjected to either non-stress or drought / salt-stress conditions.
T3.4: Mechanisms underlying pea resistance to drought will be identified (years 2 and 3) using an ecophysiological framework which includes water-, nitrogen- and carbon- fluxes analyses within the plant, on several contrasted genotypes inoculated with Rhizobium strains shown to be drought-tolerant (INRA-LEG and INRAT). As a complementary approach, the understanding of physiological and molecular basis underlying salinity tolerance will also be studied, with a particular focus on ROS production, and oxidative damage (ARC).

Deliverables:
D3.1: Standardized experimental conditions for drought and salt tolerance
D3.2: Rapid methods for screening for drought and salinity tolerance
D3.3: Identification of sources of resistance to drought and salinity (on various species, and in combination with particular Rhizobium strains)

WP coordinator: INRA-IGEPP

Other partners involved: CSIC, INRAT, CRRGC, INRAM, IAV, ARC

Objectives
– To identify genes/QTL controlling resistances to biotic (ascochyta, broomrape, fusarium, rust) and abiotic (drought) stresses (below referred to as target stresses) in pea and faba bean relevant for the development of these crops in mediterranean areas.
– To assess the stability of these QTL across pathogen variation, environments, and resistance sources
– To identify genes/QTL controlling plant architectural and developmental traits likely to contribute to the control of biotic and abiotic stresses
– To suggest choice stress resistance and architectural genes/QTL combinations for breeding for mediterranean areas
– To identify and validate candidate genes for their involvement in stress resistance or tolerance

Methodology and study materials
T4.1: Development of specific standard markers for mapping in pea: SSR markers will be developped from pea EST sequences available in public databases and that will be made available in the course of the project from current international initiatives (pea cDNA and gDNA public sequencing projects currently carried on in USA, Canada, France). Both these eSSR, previous public and published SSR markers from the Agrogene consortium, and available SNP markers will be used for mapping.
T4.2: In addition, a new set of 384 SNP markers designed within resistance genes identified (following transcriptomic or protein profiling previously published studies on target stresses), or to identify in the project course studies in T4.8, and known genes controlling plant architecture and development will be established and used for mapping. This will involve a listing of candidate genes for resistance, sequencing of these genes on a set of 16 pea parental accessions and SNP choice and design.
T4.3: Development and/or complementation of molecular maps will be conducted with SSR and SNP markers on 4 existing pea RIL populations (3 provided by INRA and 1 by CSIC) segregating for quantitative resistance to M.pinodes or for resistance to Orobanche and/or rust. Mapping will be performed using mainly markers likely to be used as bridge markers between maps (SSR and SNP markers obtained in T4.1 and T4.2) to allow comparative mapping between populations. Linkage studies will also be carried on F2 and RIL faba bean populations segregating for resistance to Orobanche.
T4.4: Phenotyping RIL and F2 pea and faba bean populations (diverse genetic backgrounds) for resistance to target stresses and plant architectural traits using methodologies developped in WP2 (biotic stresses, plant architecture) and WP3 (abiotic stresses) both in the field under multienvironment field trials and under growth chamber controlled conditions. Parental lines of RIL and F2 populations will be scored for all the biotic and abiotic stresses included in the project to discern whether these RIL populations also segregates for resistance/tolerance to additional stresses.
T4.5: Analysis of QTLs stability in different environments, towards pathogen variability, as well as in diverse genetic backgrounds (comparative analysis between populations using SSR and SNP bridge markers). Analysis of colocations between QTL controlling resistance to different biotic and abiotic constraints, and genes/QTL controlling plant architecture and development.
T4.6: Assessement of genetic progress for resistance to target stresses in large sets of accessions (192 independent pea cultivars, 300 faba bean local populations) adapted to cropping in various countries covering the mediterranean basin through (i) phenotyping for resistance and plant architecture using methodologies developped in WP2 (biotic stresses, architecture) and WP3 (abiotic stresses) both in the field and under controlled conditions; (ii) classification using anonymous SSR markers; (iii) a posteriori control of presence/absence of favourable alleles at QTL controlling resistance to stresses and architectural traits.
T4.7: Marker Assisted Breeding. Once the genes and QTLs have been identified for each stress and assessed for stability, the next step will be to select a set of markers linked to these QTLs that could be used for the simultaneous selection for different stresses. First crosses will be done for pyramiding of multiple resistances into elite cultivars and selection will be aided by these set of markers.
T4.8: Further transcriptomic or proteomic studies (CDNA-AFLP, SuperSAGE, microarrays, protein profiling) will be set up in order to identify genes or proteins differentially expressed between a resistant and a susceptible accessions to target stresses. The combination of these techniques will allow the establishment of a global and detailed picture of all genes expressed or differentially regulated during this particular interactions giving relevant information about the mechanisms of resistance acting at the molecular level. In addition, it will allow the full characterisation of candidate genes selected from the global view of the interaction, and assumed to be implicated in resistance.
T4.9: Validation of candidate genes from published, existing and planned (T4.8) transcriptomic and proteomic studies for (i) potential involvement in individual and crossed stresses, through the validation of gene expression in resistant / susceptible accessions following different stress applications by qRT-PCR (ii) development and mapping of genic molecular markers (cf T4.1) and analysis of colocalisations with QTL.

Deliverables:
D4.1: List of polymorphic eSSR and SNP markers for genetic mapping and pea map comparisons
D4.2: A set of SNP markers in pea designed in genes involved in stress resistance and plant architecture and development, likely to allow the rapid mapping or diversity study of resistance genes.
D4.3: High density pea linkage maps from populations segregating for resistance biotic and abiotic factors
D4.4: Identification, and determination of the genetic effects of the QTLs controlling polygenic traits
D4.5: Information on QTL stability across diverse field locations, pathogen variability and plant genetic backgronds
D4.6: Development of standard markers for pyramiding and rapid screening
D4.7: List of candidate genes likely to be involved in resistances to stresses.

WP coordinator: INRA-IGEPP

Other partners involved: CSIC, IAV, INRAT, CRRGC, INRAM

Objectives
– To develop molecular tools in order to study genetic variability in pathogens/parasite populations using different PCR-based markers,
– To identify the primary inoculum sources implicated in the development of epidemic,
– To study the epidemic process involved in the spatiotemporal development of legume pathogens,
– To identify architectural features of plant that will help to control disease development,
– To evaluate the impact of cultural practices and control method combinations for the management of the disease

Methodology and study materials
T5.1: Development of specific standard markers for pathogen population genetic studies: SSR markers or others markers (SNP,…) will be developped from sequences available in public databases and that will be made available in the course of the project from current international initiatives. Both these eSSR, or other markers will be used to study population genetic variations at different spatial and temporal scales (evolution of population structure during the growing season, comparison of the genetic structure in different locations of a same country, comparison of populations from different countries).
T5.2: Development of a landscape epidemiology study to identify the origin and the impact of different primary inoculums on the epidemic development of Ascochyta spp. Disease and the genetic structure of their populations. This task will particularly focus on the impact of wild legume species as potential sources of inoculums during the growing season. Based on a standard sampling procedure and the use of molecular markers, a 2 years field experiments will be developed for all the partners. This sampling will help us to define wild legume species that will act as a reservoir plant for pathogens. Controlled conditions experiments will be performed to characterize host specificity of these different strains.
T5.3: Dissection of the underlying mechanisms implicated in new control methods for disease reduction. Specific experiments will be developed in controlled conditions to determine which epidemic component is modified by the new control methods. Studies of plant (rain simulator) or roots architectures (rhizotron systems) effects on dispersal process of disease will be considered. Vertical progression of splash dispersal diseases in relation to the interference induced by non host canopy or particular on inoculum dispersal will also be investigated.
T5.4: Complementation of resistance with other methods for disease management of legume crops. Field experiments will be performed at different spatial and temporal scales to evaluate the impact of cultural practices and control method combinations towards disease. At a field scale, resistance associated with architectural plant features (aerial or root part) will be associated to evaluate their impact on spatiotemporal epidemic development. Measurement will concern the disease, microclimate, and architectural features. Selected resistant cultivars and various sowing dates would also be associated in field experiments conducted at various locations and countries to study the epidemiological effect of delayed sowing combined with available levels of resistance.
T5.5: Standard differential sets for race identification will be refined to allow systematic studies on the virulence spectrum of pathogen populations (rust, broomrape and ascochyta) in Mediterranean basin. Knowledge of the genetic structure of populations of plant pathogens is needed to aid in decision making strategies regarding implementation of effective control strategies and to ensure a durable efficiency of resistant cultivars. Knowledge on the population structure and pathogenic variation between strains collected in the different countries will help to constitute this standard differential set for future screening programs.

Deliverables
D5.1: Clarification of the genetic variation and adaptive potential of ascochyta species
D5.2: Identification of new control levers to better control disease development.
D5.3: Determine control method combinations that will decrease disease development.
D5.4: Identification of architectural features that will limit the spatiotemporal dispersal of the disease.
D5.5: Development of standard differential sets for race identification.

WP coordinator: UNINA

Other partners involved: CSIC, ENSA

Objectives
– Development of protocols to extract and purify plant and fungal metabolites
– Determination of the structure of bioactive plant and fungi metabolites using advanced spectroscopic and optical methods
– Optimization of chemical reactions to prepare derivative for structures-activity relationships studies

Methodology and study materials

T6.1: Search and exploitation of allelopathy for broomrape control: root exudates will be obtained from hydroponically grown seedlings of nonhosts (oat and rye with known allelopathic effect) and hosts (faba bean, pea, lentil and chickpea) will be extracted. Bioguided-fractionation of crude organic extract will be performed with column and thin layer chromatography in order to isolate and identify the active metabolites responsible for inhibition of weed germination and growth. Structural characterisation will be carried out using advanced spectroscopic methods (IR, UV, but essentially 1D and 2D 1H- and 13C-NMR and EI- and ESI-MS), optical (optical rotation and CD) methods. When needed, the structure of new metabolites will be confirmed through the preparation of their key derivatives
T6.2: Structure-activity relationship study: some suitable derivatives of fungal metabolites as phyllostin and scytolide produced by Phyllosticta cirsii which showed a stimulatory activity on Orobanche in a preliminary investigation, are currently being in preparation in the laboratory of UNINA by modification of their funtionalities. Inhibitory and stimulatory activity of available derivatives will be tested in a range of Orobanche spp.
T6.3. Search and exploitation of allelopathy for fungal disease control: isolation and chemical characterization of metabolites from different legumes, resistant to different pathogens, with putative antifungal activity (phytoalexins). Assessment of the compound activity in the development of fungal pathogens. Extracts of plants showing significant inhibitory effects in various aerial fungi will be extracted with the most suitable organic solvent. The crude extract will be fractionated using bioguide-combined chromatographic methods as in details reported above. The pure bioctive metabolites will be used to test their inhibitory effect on different fungal pathogens, both, in vivo by spraying susceptible plants and in vitro in petri dishes. Chemical structure of the inhibitors will be determined by spectroscopic (IR, UV, but essentially 1D e 2D 1H- and 13C-NMR and EI- and ESI-MS), optical (optical rotation and CD) and chemical methods.
T6.4: Exploitation of phytotoxins isolated from pea and lentil pathogens as tool for crop protection: toxic liquid and/or solid fungal culture filtrates will be obtained from inoculated tissue. The lypophilic bioactive metabolites will be purified by extraction with most suitable organic solvent, followed by classic chromatographic fractionation (using both normal and reverse phases supports); the hydrophilic ones will be purified by fractionated precipitation combined with suitable chromatographic methods. The structure determination of phytotoxins will be performed using spectroscopic (IR, UV, but essentially 1D e 2D 1H- and 13C-NMR and EI- and ESI-MS), and optical (optical rotation and CD) methods. In some cases their structure will be confirmed using chemical methods based on the preparation of key derivatives through their functional group transformation. The purification processes of phytotoxins will be checked with opportune biological assays on host and non-host plant organs. The same assays will be used for the activity characterization of purified phytotoxins. For these last the micotoxic and antimicrobic activity will be also assayed.
T6.5: Search and exploitation of allelopathy for control of broomrape and dodder and plant pathogens: a previous screening made by ENSA partner identified several Mediterranean plants (Inula viscosa in particular) that produced low molecular weight lipophlic metabolites with herbicidal activity against two broomrapes species (P. ramosa, O. crenata), dodder (C. campestris) and several plant pathogens (fungal and bacterial diseases). This will be studied further by identifiying and characterizing the phytotoxic metabolites following the procedures described in T6.4.
Tasks T6.1, T6.2, T6.3 and T6.4 will be performed by UNINA in collaboration with CSIC and T6.5 in collaboration with ENSA. These partners will supply dried plants and root plant exudates, liquid and/or solid lyophilized fungal culture filtrates and will carry out in vitro bioassays and field experiments.

Deliverables
D6.1: Protocols for the extraction and purification of bioactive metabolites (phytotoxins, herbicides, phytoalexins)
D6.2: Structure determination of new bioactive compounds with original carbon skeleton
D6.3: Characterization of phytotoxic, antifungal, mycotoxic and allelopatic activities of the pure metabolites isolated
D6.4: Preparation of key derivatives for structure confirmation and structure-activity relationships studies

WP coordinator: IAV

Partners involved: all

Objectives
WP7 will ensure that the scientific innovations developed in the project are transferred effectively to endusers. A particular focus will be to ensure international coordination of research with other ongoing research programs and with previous and on-going Mediterranean and EU research. All project partners will contribute to the dissemination activities.

– Organisation of Workshops at the end of the project/together with the final meeting for farmer associations to explain the recommendations resulting from the project
– Diffusion of enhanced chickpea, common bean, faba bean, lentil and pea germplams to interested parties
– Creating awareness at the end market of the initiative, its goals and results through popular magazines and a project website

Methodology and study materials
T7.1: Dissemination of information to endusers by a workshop, demonstration plots and extension workers visiting farms where food legumes are grown.
T7.2: The scientific aspects of the project will be published in international journals of such as Crop Protection, Plant Pathology, Euphytica, Plant Breeding, Molecular Breeding, Phytochemistry, or Pest Management Science
T7.3: Build and maintain MEDLEG project open website for dissemination activities. It will be used as the repository of all information concerning the project and actively used to monitor project goals and targets for all work packages. It will be a source of information for all stakeholders with publications that are focused on meeting their specific needs and interests.

Deliverables
D7.1: Methodology and biotechnological protocols transference to facilitate the selection of interesting genotypes
D7.2: Scientific papers, divulgation articles in farmer journals and contributions to conferences
D7.3: Project web site