Tasks

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 with 182 accessions was organized. The origins 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) collecting missions in Portugal mainland.
• Four seeds' prospecting and collecting missions were carried out in the Northern and Central regions of Portugal (mainland). 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 were obtained with the respective collecting 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 identification based on morphological characters and seeds multiplication.
• All the seeds in the collection have a registration number and are associated with a database that combines all the available information about the accessions.

• 132 accessions in the collection are Diplotaxis tenuifolia (wild rocket) while the remaining ones are from other species (Diplotaxis spp., Eruca spp., Brassica spp.).

• A collection with 28 isolates of Hyaloperonospora sp. was organized. The isolates were collected from infected Diplotaxis tenuifolia field plants in the Alentejo region, at different dates. After laboratory processing (inoculation, isolation and multiplication) the isolates were kept at -18 °C for characterization.
• The prospection of new isolates of Hyaloperonospora sp. is in progress. 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) plant 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 downy mildew resistance of 182 accessions was evaluated in 3 replications. Depending on the germination power and the number of available seeds, 40 to 60 plants per accessions were evaluated, by registering the infection in the cotyledons and the first two leaves. The accessions were grouped according to their level of resistance, allowing an overview of the susceptibility of the collection to downy mildew infection.
• 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 wild rocket genotypes contrasting in their response to downy mildew

• The protocol for histological analysis of resistant/susceptible wild rockets plants infected with downy mildew (Hyaloperonospora) was defined. The results showed that the pathogen infected the plant by entering the upper leaf page and developing intercellular mycelium. The resistant wild rocket genotype quickly developed a brownish necrosis zone that prevented further development of the pathogen. In the case of susceptible genotypes, despite the formation of some necroses, the pathogen finishes its life cycle with the sporulation on the bottom leaf page.
• The assessment of the wild rocket collection (task 2.2) allowed the selection of accessions for the analysis of lipids, and 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 (task 2.2), 40 wild rocket genotypes were selected to be tested in the adult phase under organic farming production.
• The different accessions were sown in multi-cell trays, and cultivated in the Experimental Greenhouses of INIAV (Oeiras, Portugal) 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 monitored weekly in the field, individually, and the onset of flowering and the appearance of signs of downy mildew on the leaves were registered.
• After the final evaluation of plants, on 3.12.2021, the leaves were harvested and after 16 days of regrowth, the accessions were evaluated for agronomic characteristics (vigour, plant habit, leaf serration).

Responsibility: UAlg

Specific objectives of Task 4:

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

• Genomic DNA of 100 accessions (~10 plants per access) was extracted and prepared for molecular markers analysis.
• To address the lack of genomic information on tenuifolia and the consequent lack of specific tools for genomic analyses (e.g. specific, not random, DNA markers), 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 were 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 were 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 preserved at -18 °C were multiplied in a susceptible Diplotaxis tenuifolia host and analyzed by microsatellites and genome sequencing.

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

• The molecular characterization of the Hyaloperonospora allowed the taxonomic classification of the pathogen to assign 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 under controlled environment (task 2.2), eight 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 Seven days later, the leaves of each plant were collected separately for analysis by gas chromatography and spectrophotometry (GC-TOF-MS).
• The metabolomics profiles of the different accessions were obtained and the effects of pathogen infection were evaluated.

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 in the former 6 months of the project (https://projects.iniav.pt/REMIRucula/) allocated on the Institute's website and regularly updated. 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.
• A technical-scientific seminar was organized at the end of the project (4 December 2022) held at University of Algarve, Faro, Portugal.

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

• The results obtained in the project were presented in several scientific meetings, as posters and published in peer-reviewed journals. They have also been disseminated at events to the general public.

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

• On 27 September 2019, the team of REMIRucula project at INIAV 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 of a poster presentation and roll-up focusing on “The importance of rocket in the Mediterranean diet" with the scope to familiarize the consumers, and the general public, on the health benefits of rocket consumption.
• On 23-24 January 2020, a poster communication was presented to the "International Meeting of the Portuguese Society of Genetics (IMPSG)" at the University of Algarve, Faro, Portugal. (https://researchgate.net/publication/339973853).
• 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). Voz do Campo 249: 40-42. https://www.researchgate.net/publication/352466011).
• Publication of a book chapter in “Advances in Plant Breeding Strategies: Vegetable Crops” edited by Springer (https://doi.org/10.1007/978-3-030-66969-0).
• On 1-2 July 2021, three posters were presented virtually to the “II International Meeting of the Portuguese Society of Genetics (IMPSG)” at the University of Trás-os-Montes e Alto Douro, Vila Real, Portugal.
• On 24 September 2021, the REMIRucula team participated in the “European Researchers' Night (NEI Oeiras 2021)” which took place at the Marina de Oeiras, Portugal. The activity consisted on the presentation of the wild rocket plant, a poster on type of baby leaves, a roll-up on “The Importance of Wild Rocket in the Mediterranean Diet”, and dissemination of preliminary results obtained under the project, namely the identification of accessions that are more resistant to downy mildew disease. This activity intended to familiarize consumers with health benefits of consuming wild rocket and alerting to the main disease caused in the plant by the oomycete Hyaloperonospora
• On 27-28 July 2022, two posters were presented to the “III International Meeting of the Portuguese Society of Genetics (IMPSG)” at the Universidade de Évora, Portugal.
• A scientific article was published in the Agronomy Journal (Coelho P.S., Reis J.M., Pereira A.L., Vairinhos A., Lopes V., Leitão J.M. 2022. Downy mildew resistance and genetic variability in a wild rocket germplasm collection. Agronomy Journal 114(6): 3083–3095. https://doi.org/10.1002/agj2.21190).
• On 17 August 2022, a poster was presented with a short oral presentation to the “XXXI International Horticultural Congress: IHC2022” to "International Symposium on Sustainable Control of Pests and Diseases", Angers, France, 14-20th of August (Coelho P.S., Pereira A.L., Reis J., Carranca C., Lopes V.R., Leitão J.M. 2022. Downy mildew evaluation in wild rocket genotypes [Diplotaxis tenuifolia (L.) DC.] under field and controlled conditions., https://www.researchgate.net/publication/363021869)
• On 6 September 2022, a poster was presented to the “XII Encontro Internacional de Fitossociologia”, in Escola Superior Agrária de Castelo Branco, Portugal (Gaspar C., Lopes V., Figueiredo P., Coelho P., Leitão J. 2022. Colheita e conservação de parentes selvagens de espécies cultivadas (CWR) de brássica. Book of abstracts, pp. 110-111. ISBN 978-989-333734. https://www.researchgate.net/publication/363885649).
• On 8 September 2022, a poster was presented to the “2^nd Nordic Metabolomics Conference 2022”, Copenhagen, Denmark (Rodrigues A.M., Pereira A.L., Coelho P.S., António C. 2022. Primary metabolite profiling of wild rocket Diplotaxis tenuifolia accessions with differential responses to downy mildew disease. Book of abstracts, pp. 37-38. https://www.researchgate.net/publication/363885650).
• A scientific article was published in the Acta of the “XXXI International Horticultural Congress: IHC2022” (Coelho P.S., Pereira A.L., Reis J., Carranca C., Lopes V.R., Leitão J.M. 2022. Downy mildew evaluation in wild rocket genotypes [Diplotaxis tenuifolia (L.) DC.] under field and controlled conditions. Angers, France, 14-20th of August. Acta Horticulturae (Accepted for publication on 11.06.2022).
• On 14 October 2022, REMIRucula project team participated in the “Festival Internacional de Ciência - FICA”, Oeiras, Portugal.
• On 4 November 2022, the “Seminar for the Presentation of the Final Results of the REMIRucula Project” was held at the University of Algarve, in Faro, Portugal, with the presentation of 6 oral communications by project members and 2 by invitation.
• A scientific article was published in Plants (Reis J.M., Pereira R.J., Coelho P.S., Leitão J.M. 2022. Assessment of wild rocket (Diplotaxis tenuifolia (L.) DC.) germplasm accessions by NGS identified SSR and SNP markers. Plants 11(24): 3482. https://doi.org/10.3390/plants11243482).