The main challenge for the  REMIRUCULA research  team is the increase of knowledge on the mechanisms underlying downy mildew resistance in the interaction D. tenuifoliaHyaloperonospora sp., in order to propose effective solutions to control this devastating disease. Another challenging aspect is the search for new resistant sources with the identification of resistant and partially resistant genotypes to downy mildew which delay the disease progression and are frequently resistant sources more durable. 

The strategy of this project is a multidisciplinary and complementary approach that involves different research groups working in close commitment with a commercial Portuguese grower of baby leaf. 

Responsibility: INIAV (Braga and Oeiras)

Specific objective of Task 1:

1.1. To establish a collection of wild rocket (Diplotaxis tenuifolia) seeds and Hyaloperonospora sp. isolates

  • A collection of about 180 wild rocket accessions was organized. The sources are: i) germplasm banks (Germany, Australia, Austria, Hungary and Israel), ii) seed companies (CN SEEDS - United Kingdom, ENZA ZADEN – Spain, and ISI SEMENTI S.P.A. - Italy), iii) wild rocket producers (VITACRESS - Portugal and DUNCAN FAMILY FARMS - USA), iv) local trade, and v) field harvests in continental Portugal.
  • Four seeds' prospecting and collecting missions were carried out in the northern and central regions of continental Portugal. The missions took place from May to July 2019 and covered 3 sub-regions: i) Litoral Norte Minhoto and Costa Nova (RUC – 1-2019); ii) Trás-os-Montes and Beira Alta (RUC – 2-2019); and iii) Beira Baixa and Alto Alentejo (RUC – 3-2019). Twenty-seven accessions of wild rocket of the genera Diplotaxis and Eruca were obtained, with the respective harvest locations identified by GPS coordinates. After the accessions’ processing (drying, selection and cleaning), the seeds were included in the wild rocket collection. Later, the accessions were sown in a protected environment for multiplication of seeds and species identification based on morphological characters.
  • All the collection accessions have a registration number and are associated with a database that combines all the available information about the samples.
  • About 140 accessions in the collection are Diplotaxis tenuifolia (wild rocket), while the remaining ones are Diplotaxis muralis and Eruca sp. (cultivated rocket). The accessions from continental Portugal field harvests are being identified.
  • A collection of eight isolates of Hyaloperonospora sp. was organized. The isolates were harvested from infected field Diplotaxis tenuifolia plants in the Alentejo region, at different dates. After laboratory processing (inoculation, isolation and multiplication) the isolates were kept at -18 °C for future characterization.
  • The harvesting of new isolates of Hyaloperonospora sp. to be continued. These isolates will join the downy mildew collection.

Responsibility: INIAV (Oeiras)

Specific objectives of Task 2:

2.1. To develop a robust protocol for downy mildew rocket phenotyping, in controlled conditions

  • The protocol for testing downy mildew resistance describes the methodology for assessing the resistance of young wild rocket plants and the optimal conditions for establishing the infection of this plant-pathogen interaction. The protocol establishes: i) plant material (sowing method and plant age), ii) growth conditions (day / night temperature, relative humidity, photoperiod, type and light intensity), and iii) evaluation method (inoculation methodology, inoculum concentration, duration of the different stages and evaluation scale).

2.2. To screen the wild rocket collection for downy mildew resistance

  • The evaluation of downy mildew resistance of 179 wild rocket accessions was carried out in 3-4 replications. Depending on the germination power and the number of available seeds, 40 to 60 plants per accessions were evaluated, by registering the infection observation in the cotyledons and the first two leaves. The accessions were grouped according to their resistance level to the infection, allowing an overview of the susceptibility of the wild rocket collection to downy mildew infection. Some preliminary results were presented as a poster communication in the “International Meeting of the Portuguese Society of Genetics”, held on 23-24 January 2020 at the University of Algarve.
  • Throughout the tests, resistant and susceptible plants were selected for seed multiplication to be used in the following activities.

2.3. To characterize at the histological and cellular level the contrasting downy mildew genotypes

  • The protocol for histological analysis of resistant/susceptible wild rockets plants infected with Hyaloperonospora sp. is being optimized. The available results showed that the pathogen infect the plant by entering the upper leaf page and developing the intercellular mycelium in the leaves. The resistant wild rocket genotype quickly develops a brownish necrosis zone that prevents further development of the pathogen. In the case of susceptible genotypes, despite the formation of some necroses, the pathogen finishes their life cycle with the sporulation of Hyaloperonospora sp. on the bottom leaf page.
  • The assessment of the wild rocket collection (task 2.2) will allow the identification of resistant wild rocket accessions for the analysis of lipids, and help to verify the effects of the pathogen in the lipid component.

Responsibility: INIAV (Oeiras)

Specific objectives of Task 3:

3.1. Testing a set of a rocket accessions under field conditions in a Portuguese baby leaf producer.

  • After evaluating the downy mildew resistance of the rocket collection in controlled environment (activity 2.2), forty wild rocket genotypes were selected to be tested in the adult phase under organic farming production.
  • The different accessions were sown in the Experimental Greenhouses of INIAV (Oeiras) in multi-cell trays, where they were grown for 22 days. On October 15, 2021, they were planted in a polytunnel at the Vitacress Portugal, SA company in Odemira (Alentejo, south Portugal).
  • The plants were individually weekly monitored in the field and the onset of flowering and the appearance of signs of downy mildew on the leaves were recorded.
  • After the last evaluation of plants, on 3.12.2021, the leaves were harvest and after 16 days of regrowth, the accessions were evaluated for agronomic characteristics (vigour, plant habit, leaf serration).
  • The results obtained will be presented in the publication “Downy mildew evaluation in wild rocket genotypes [Diplotaxis tenuifolia (L.) DC.] under field and controlled condition” to be presented at the International Horticulture Congress (IHC-2022), held in Angers (France) from 14 to 20 August 2022.

Responsibility: UAlg

Specific objectives of Task 4:

4.1. Molecular characterization of the wild rocket (D. tenuifolia) accessions

  • The genomic DNA of 100 accessions (~10 plants per access) was extracted and prepared for molecular markers analysis. Forty of these accessions were already characterized by molecular markers.
  • To address the lack of genomic information on D. tenuifolia and the consequent lack of specific tools (e.g. specific, not random, DNA markers) for genomic analyses, a program of NGS was implemented which resulted in:
  1. Genome Assembly (UALg DiploT.01) ( https://www.ncbi.nlm.nih.gov/assembly/GCA_014822095.1/ )
  2. A SRX8354034 was uploaded to ncbi containing all reads of this project: 105.7M reads, 15.3 Gbp
    https://www.ncbi.nlm.nih.gov/sra/?term=SRX8354034 )
  3. Five hundred microsatellite sequences useful for the establishment of an identical number of microsatellite (SSR) markers were identified and uploaded to the public databases (www.ncbi.nlm.nih.gov/search/all/?term=diplotaxis+tenuifolia+microsatellite)
  4. Multiple RAPD primers were assessed among some samples. The selected primers are being used for a basic genetic analysis of 100 accessions.
  5. Based on the produced novel genomic information, primers were designed for 20 microsatellite markers, among which 8 SSR markers were validated as producing simple and clear PCR products. These markers are presently being used for more accurate characterization of the 100 accessions of the germplasm collection and identification of markers linked to identified resistance loci.

4.2. Molecular characterization of the collection of Hyaloperonospora sp. isolates

  • The different populations of Hyaloperonospora sp. preserved at -18 °C are being multiplied in a susceptible Diplotaxis tenuifolia host to be further analysed by microsatellites and genome sequencing.

4.3. Taxonomic characterization of Hyaloperonospora sp. isolates from wild rocket

  • The molecular characterization of the Hyaloperonospora sp. will allow pathogen taxonomy and assignment of the species-specific epithet.

Responsibility: ITQB NOVA

Specific objectives of Task 5:

5.1. Evaluation of metabolome of resistant and partially resistant wild rocket (D. tenuifolia) to downy mildew

  • After evaluating the resistance of the rocket collection to downy mildew carried out under controlled environment (activity 2.2), 8 resistant and susceptible wild rocket genotypes were selected for characterization of the metabolomics profiles.
  • A total of 80 plants (10 plants per accession) were grown in small pots (9x9x9-cm) for 28 days in a controlled environment.
  • The plants were separated into two groups and subjected to different treatments (inoculated vs. control). In the inoculated group, the plants were infected by spraying with a conidia suspension of Hyaloperonospora spp. Seven days after inoculation, the leaves of each plant were collected separately for analysis by gas chromatography and spectrophotometry (GC-TOF-MS).
  • At the final of this study, the metabolomics profiles of the different accessions are expected to be obtained and to assess how these metabolites are affected by the presence of the pathogen.

Responsibility: INIAV (Oeiras)

Specific objectives of Task 6:

6.1. To promote and develop an open website to show the project scope, main research findings and outreach activities

  • The project team, in close collaboration with INIAV's IT department, developed an online platform ( https://projects.iniav.pt/REMIRucula/ ) allocated on the Institute's website. This website shows the most relevant information on the development of the REMIRUCULA project.

6.2. To coordinate the organization of a workshop and a technical-scientific seminar

  • A kick-off meeting was carried out at INIAV (Oeiras) at the beginning of the project, which brought together all the members of the REMIRUCULA project. In this meeting, the different activities to be carried out within the project scope and the coordination of these activities were enunciated and discussed.
  • Due to current restrictions resulting from COVID-19 pandemic, the interaction among the project team members has been made virtually.

6.3. To coordinate the transfer of knowledge to the scientific community, a curator database and a possible patent

  • The results obtained have been presented in several scientific meetings, as posters, and published in peer-reviewed journals. They have also been presented at events to the general public, such as “Researchers Night”, the GREEN-IT platform (activity 6.4).

6.4. To implement the outreach activities aiming different stakeholders – farmers, industry, policy-makers – and the general public

  • On September 27, 2019, the team of REMIRUCULA project at INIAV (Oeiras) participated in the European Researchers Night (NEI 2019) which took place at the National Museum of Natural History and Science (MUHNAC-UL), Príncipe Real, Lisbon, Portugal. The activity consisted on the presentation of a poster and roll-up focusing “The importance of rocket in the Mediterranean diet" to familiarize the consumers, and the general public, on the crop production and health benefits.
  • On January 23-24, 2020, the poster communication entitled "Establishment of a wild rocket (Diplotaxis tenuifolia) germplasm collection and its preliminary assessment for downy mildew resistance and genetic diversity" was presented to the "International Meeting of the Portuguese Society of Genetics (IMPSG)" at the University of Algarve in Faro (Portugal).
  • An article was published in the Vida Rural magazine (Coelho P.S., Pereira A.L., Carranca C., Scotti P., Lopes V., Boto C., Reis J., Leitão J. 2021. Míldio na rúcula selvagem – o porquê de investigar esta doença. Vida Rural 1867:44-50. https://www.researchgate.net/publication/352769754).
  • An article was published in the Voz do Campo magazine (Paula S. Coelho, Ana L. Pereira, Corina Carranca, Paula Scotti, Carlos Gaspar, Violeta Lopes, José Leitão. 2021. O míldio é um problema grave na produção de rúcula selvagem (Diplotaxis tenuifolia L.). Voz do Campo 249: 40-42. https://www.researchgate.net/publication/352466011).
  • On 1-2 July 2021, three posters were presented at the “II International Meeting of the Portuguese Society of Genetics (IMPSG)” which took place online at the University of Trás-os-Montes e Alto Douro in Vila Real (Portugal).
  • On september 24, 2021, the REMIRucula team participated in the European Researchers' Night (NEI Oeiras 2021) which took place at Marina de Oeiras, in Oeiras. The activity consisted of the presentation of the crop (flower, siliques and seeds), a panel on types of baby leaves, a roll-up on “The Importance of Wild Rocket in the Mediterranean Diet”, and dissemination of preliminary results under the project, namely the identification of accessions that are more resistant to downy mildew disease. This activity intended to familiarize consumers about the benefits of wild rocket for human health and alert to the main disease caused by the oomycete Hyaloperonospora sp..