Periodic Reporting for period 2 - PhageFire (An effective and environmentally friendly solution to control fire blight disease caused by Erwinia amylovora in pome fruit crops)
Période du rapport: 2021-08-01 au 2022-07-31
Fire Blight is a devastating disease caused by the bacterium Erwinia amylovora in rosaceous plants.
In some countries, E. amylovora is considered a quarantine organism and it is mandatory to uproot the orchards. In the affected areas, it can provoke yield losses of up to 95%, causing costs of ca. 4,3 - 8,1 M€.
E. amylovora responds poorly to the few available treatments which traditionally include rapid removal of infected branches or even cutting the whole tree off, combined with the preventing use of copper-bases pesticides and antibiotic sprays. However, first approach causes significant financial losses and the use of copper-based pesticides is limited in several EU countries. Additionally, antibiotics are forbidden in the EU because of the development of antibiotic-resistant bacteria and its limited efficiency.
Coming from the clear need to improve control strategies of this disease, Bacteriophages are natural viruses which exclusively prey on pathogenic bacteria. They are harmless for humans and the environment. Therefore, they are a promising treatment option to prevent infections by E. amylovora. So, PhageFire, being a phage-based pesticides, will suppose one step towards a real and effective solution against Fire Blight that will enhance significantly the quality, productivity and competitiveness of the fruit sector.
In some countries, E. amylovora is considered a quarantine organism and it is mandatory to uproot the orchards. In the affected areas, it can provoke yield losses of up to 95%, causing costs of ca. 4,3 - 8,1 M€.
E. amylovora responds poorly to the few available treatments which traditionally include rapid removal of infected branches or even cutting the whole tree off, combined with the preventing use of copper-bases pesticides and antibiotic sprays. However, first approach causes significant financial losses and the use of copper-based pesticides is limited in several EU countries. Additionally, antibiotics are forbidden in the EU because of the development of antibiotic-resistant bacteria and its limited efficiency.
Coming from the clear need to improve control strategies of this disease, Bacteriophages are natural viruses which exclusively prey on pathogenic bacteria. They are harmless for humans and the environment. Therefore, they are a promising treatment option to prevent infections by E. amylovora. So, PhageFire, being a phage-based pesticides, will suppose one step towards a real and effective solution against Fire Blight that will enhance significantly the quality, productivity and competitiveness of the fruit sector.
During this year, PhageFire consortium has achieved the planned goals. Partners have dedicated much time to set up a proper formulation for the phage cocktail to carry out field trials. The consortium has faced a contamination of samples, however, researchers have turned over the situation and converted this contamination in part of the solution. They have worked on extra tasks achieving new findings and knowledge about E. amylovora phages. Apart of identification, characterization, propagation and scale-up production of the phages, partners have developed fast identification methods that can be implemented as quality control during production of phages. Besides, studies about the host receptor specificity and the synergy between phages have been carried out. This work has led to a phage cocktail that has been tested on apples and pear during the season 2022. Field trials have been promising and have provided information to enhance the most efficient cocktail till next season.
Additionally, partners are studying the application for emergency use authorization among the different regulations for commercializing a new biopesticide in the EU member states. Following this regulation, the consortia have outlined a work plan. When formulate is ready PhageFire consortium will gather necessary data to complete dossier for the product. Meanwhile field trial permits in Spain and Hungary for season 2023 has been pursued and information to perform field trials in some more EU member states is being gathered.
Although WP1 was completed it was discovered that bacteriophage samples were contaminated. The consortium has taken measures to avoid this happens again by implementing a quality control system. Meanwhile the consortium has isolated phages again from the contaminated samples by purifying them. This process is very tough, however the consortium achieved the purification successfully obtaining pure phages. These phages were characterized and Zhaw identified different receptors in E. amylovora that are used by different phages. This selection is key for the design of a phage cocktail, because combinatorial effects (additive and synergistic) can be exploited for the control of the bacteria. Zhaw also determined the compatibility of different phages in phage cocktails. Based on these data, 3 different phages were selected for the phage cocktail. However, the work is still ongoing to improve PhageFire efficiency and efficacy.
Moreover, ZHAW defined the formulation to test in the field trials of season 2022, which includes the most promising phages, a UV protectant and a surfactant that enhance the performance of the phages. In addition, desiccation resistant phages were identified and added to the phage cocktail, if needed.
In WP3, the consortium has performed a detailed analysis of the (bio)pesticide regulatory framework in the EU to determine the strategy and next steps of the project. During this period, the permits required to perform the first round of field trials in 2022 season in pome fruit orchards in Spain and Hungary were successfully obtained. In addition, an updated assessment on the requirements to obtain the permits to perform the second round of field trials in other EU countries during 2023 was also performed.
During this season the phages selected in conjunction with the adjuvants were joined in a cocktail tested in three different field trials in Spain and Hungary in apple and pear orchards. Results were promising despite the weather conditions this year being special, with drought and heat waves. Next season (2023) field trials will be performed again even in some other regions to validate the results.
On WP5, the consortium has optimized the fermentation procedures and defined the best growth conditions for E. amylovora and phage production by testing different media composition, temperatures, and incubation times, for each of the selected phages. This task included the definition of protocols and quality control mechanisms required to produce high volumes of purified phage suspension for commercialization in large volumes. For this first round of field trials during 2022, three different phages concentrations were tested to decide the ideal dose of the product.
Communication and dissemination are important part of the project, and main objective during this period has been to increase awareness of the project. To get the target, the consortium has used the social media, the project devoted webpage and the beneficiaries´ own webpages and has participated in Micrope conference. With the coordination of the Consortium members PhageFire is more known nowadays.
Additionally, partners are studying the application for emergency use authorization among the different regulations for commercializing a new biopesticide in the EU member states. Following this regulation, the consortia have outlined a work plan. When formulate is ready PhageFire consortium will gather necessary data to complete dossier for the product. Meanwhile field trial permits in Spain and Hungary for season 2023 has been pursued and information to perform field trials in some more EU member states is being gathered.
Although WP1 was completed it was discovered that bacteriophage samples were contaminated. The consortium has taken measures to avoid this happens again by implementing a quality control system. Meanwhile the consortium has isolated phages again from the contaminated samples by purifying them. This process is very tough, however the consortium achieved the purification successfully obtaining pure phages. These phages were characterized and Zhaw identified different receptors in E. amylovora that are used by different phages. This selection is key for the design of a phage cocktail, because combinatorial effects (additive and synergistic) can be exploited for the control of the bacteria. Zhaw also determined the compatibility of different phages in phage cocktails. Based on these data, 3 different phages were selected for the phage cocktail. However, the work is still ongoing to improve PhageFire efficiency and efficacy.
Moreover, ZHAW defined the formulation to test in the field trials of season 2022, which includes the most promising phages, a UV protectant and a surfactant that enhance the performance of the phages. In addition, desiccation resistant phages were identified and added to the phage cocktail, if needed.
In WP3, the consortium has performed a detailed analysis of the (bio)pesticide regulatory framework in the EU to determine the strategy and next steps of the project. During this period, the permits required to perform the first round of field trials in 2022 season in pome fruit orchards in Spain and Hungary were successfully obtained. In addition, an updated assessment on the requirements to obtain the permits to perform the second round of field trials in other EU countries during 2023 was also performed.
During this season the phages selected in conjunction with the adjuvants were joined in a cocktail tested in three different field trials in Spain and Hungary in apple and pear orchards. Results were promising despite the weather conditions this year being special, with drought and heat waves. Next season (2023) field trials will be performed again even in some other regions to validate the results.
On WP5, the consortium has optimized the fermentation procedures and defined the best growth conditions for E. amylovora and phage production by testing different media composition, temperatures, and incubation times, for each of the selected phages. This task included the definition of protocols and quality control mechanisms required to produce high volumes of purified phage suspension for commercialization in large volumes. For this first round of field trials during 2022, three different phages concentrations were tested to decide the ideal dose of the product.
Communication and dissemination are important part of the project, and main objective during this period has been to increase awareness of the project. To get the target, the consortium has used the social media, the project devoted webpage and the beneficiaries´ own webpages and has participated in Micrope conference. With the coordination of the Consortium members PhageFire is more known nowadays.
PhageFire project is having an impact in the partners allowing them to increase their network since the project applies a “multi-actor approach” joining growers and scientific, practical and commercial experts. PhageFire project is giving an effective solution against FB to growers that will be natural, environmentally friendly, safe and cost-effective based in a state of the art principle, phage therapy. This kind of production is closer to the customers’ demand nowadays since it reduces health risks associated to chemicals toxicity. Finally, PhageFire project is driving the advancement of phage knowledge. Results got from the experiments of this project are useful for the scientific society. This project is contributing to settle the basis of a lot of know-how in the behaviour of phages, their characteristics and their management. New literature will be published available for researchers that will help to advance in this new strategy of control of pathogen microorganisms. The development and market arrival of PhageFire solution will be a key precedent helping drawing private investment to continue research.
PhageFire project will also have a positive social impact through job creation since during project execution, and later to respond to increasing product demands in the near future, the Consortium estimates that about 22 new job positions will be created
PhageFire project will also have a positive social impact through job creation since during project execution, and later to respond to increasing product demands in the near future, the Consortium estimates that about 22 new job positions will be created