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New biological control agents against European fruit tree canker disease (Neonectria ditissima) in apple: from microbiome analysis towards product development

Periodic Reporting for period 1 - BioNeedit (New biological control agents against European fruit tree canker disease (Neonectria ditissima) in apple: from microbiome analysis towards product development)

Reporting period: 2019-04-01 to 2021-03-31

Apple is the most important fresh fruit cultivation in Europe with more than 11.5 M tonnes produced per year, equivalent to the 32% of the total fresh fruit production. Due to the intensification of its production, apple orchards have been increasingly affected by pests and diseases. European fruit tree canker, caused by the fungal pathogen Neonectria ditissima, is after apple scab the most devastating disease in apple production in Europe. To date, chemical control is the essential component in the European fruit tree canker management. Due to the commitment of the EU agri-food sector in reducing the use of chemicals pesticides, new biological control strategies have to be implemented to achieve a sustainable management of this disease. The subsequent reduction in use of pesticides will reduce the exposure of workers in agriculture and the environment to toxic chemical agents, and will increase the confidence of consumers in eating healthy and high-quality food. With this purpose, BioNeedit contributes in the development of a new biocontrol product for the management of European fruit tree canker. This project ensures that the selected microorganisms not only have antagonistic properties, but also fulfil the basic criteria regarding commercial production, safety and registration of plant protection products, as well as the ecological needs for the applications in the field.
A collection of 520 fungal isolates consisting of yeasts and hyphal fungi were obtained from 105 samples of five different countries and 30 different apple varieties. The obtained candidates were tested for their spore production at laboratory scale, their ecological characteristics (cold tolerance, drought tolerance, and UV resistance) and safety issues (no growth/germination at human body temperature). A total of 252 candidates tested were able to fulfil the combined selection criteria of spore production, ecological characteristics and safety issues. Candidates were identified based on DNA sequencing. Most of the candidates belonged to few species: Alternaria spp., Aureobasidium pullulans, Cladosporium spp., Fusarium spp., Vishniacozyma spp., Didymella macrostoma and Penicillium spp. The remaining candidates belonged to 37 different taxonomical groups, in many cases represented by only one or very few isolates. The potential risks to humans, animals, plants, the environment (including ground water) and non-target organisms were preliminary assessed using information from public databases. A total of 26 species were classified as ‘Go’ and continued in this study, because no risks were identified in the preliminary risk assessment. A patent search was done for fungal groups assessed with a ‘Go’ decision based on risks assessments, to judge options for the protection of intellectual property rights for isolates of the given species and to identify possible already protected use. A total of 158 candidates belonging to 13 taxonomic groups were selected to continue in the screening process. A bioassay protocol was designed to test the large collection of candidates. ‘Elstar’ apple branches were used. Fresh cuts were made on the branches which were inoculated with a N. ditissima macroconidia suspension and 24 hrs later with a spore or cell suspension of the corresponding candidate. After four weeks of incubation at 17°C, 16 hrs light per day and HR > 90%, branches were assessed for their canker symptom severity and for the DNA concentration of N. ditissima using a qPCR assay developed at Wageningen University and Research (WUR). A total of 20 candidates representing 14 species reduced the presence of canker symptom and the concentration of N. ditissima DNA on the branches. A final bioassay with these 20 candidates was performed. After the results obtained in the two rounds of bioassays, four candidates belonging to one species showed extraordinary results in reducing canker symptom severity and N. ditissima DNA concentration in both bioassays and were selected as potential antagonists of N. ditissima. These four candidates were sent to the company e-nema for assessing their up-scale biomass production in bioreactors. The results of the experiment to assess the spore production in liquid medium showed that two of the four candidates produced more than 108 spores ml-1 in five days. These two candidates are the most promising antagonists against N. ditissima: they have fulfilled the selection criteria stablished in the screening program developed in BioNeedit.

Despite the difficulties of the last year of the project due to the corona crisis, different activities have been carried out to exploit and disseminated of the obtained results. One scientific manuscript will be submitted in a high impact Q1 journal by June 2021. BioNeedit was mentioned as a practical case in the book chapter Elena G., Köhl J. Screening Strategies for Selection of New Microbial Antagonists of Plant Pathogens (2020). In: De Cal A., Melgarejo P., Magan N. (Eds.) How Research Can Stimulate the Development of Commercial Biological Control Against Plant Diseases. Springer, Switzerland, pp 165-181. One oral presentation and one poster were submitted in two international conferences on biological control. Two presentations were given in meetings addressed to Dutch and International fruit growers. One workshop was organised for Dutch stakeholders and advisors on apple production. An article was published in the Dutch journal for growers Fruitteelt. One workshop for PhD students was organised about how to apply for a MSCA fellowship. A presentation on biological control was given to high-school students. The participation of women in Science has been promoted thought different activities in the frame of IgR, the internal commission of Research and Gender at RAICEX (Network of Spanish Researchers Abroad). Different publications have been made on different social media, including a video on YouTube (https://www.youtube.com/watch?v=lM6tMateiUw).
The new findings of BioNeedit contribute to the development of a new biological control product, which will better complete the biocontrol options in the integrated pest management of European fruit tree canker. Two fungal isolates were found as potential antagonists against N. ditissima following a systematic stepwise antagonist screening program. The candidates showed promising antagonistic behaviour in the bioassays in planta and they fulfilled major criteria with regards to spore production and safety as well as ecological needs. The obtained results now allow to perform field experiments to evaluate the potential of these antagonists to control N. ditissima and to reduce canker symptom development. Successful results in the field experiments will allow the registration of a new biological control product against European fruit tree canker within few years. This will enhance the biocontrol options in apple production as a strong alternative to fungicide use that are being restricted or even no longer permitted in the near future.
The fellow, Georgina Elena Jiménez, performing bioassays in planta using 'Elstar' apple branches