H2020 VIROPLANT Project: VIROME NGS ANALYSIS OF PESTS AND PATHOGENS FOR PLANT PROTECTION
- Type Project
- Status Filled
- Execution 2018 -2021
- Assigned Budget 3.331.580,00 €
- Scope Europeo
- Main source of financing H2020
- Project website Proyecto VIROPLANT
Next-generation sequencing (NGS) technology has proven instrumental in elucidating the complexity and role of the microbiome associated with plants and animals. The role of the virome as a subcomponent of the microbiome is beginning to be unraveled thanks to new ways of analyzing data and the recognition of its importance in shaping biological systems. The goal of VIROPLANT is to apply NGS technology and empirical biological experiments to develop novel, environmentally friendly virus-based control strategies for managing diseases transmitted by bacteria, fungi, and insect vectors (phytoplasmas and viruses), as well as insect pests.
To date, plant protection using viruses has relied on limited and effective model systems, such as baculoviruses for Lepidoptera and a mycovirus for chestnut blight control. Furthermore, viruses that infect bacteria (phages) are also cutting-edge tools with potential to address antibiotic-resistant bacterial strains in human and veterinary medicine. VIROPLANT will create a database of novel viruses (and biotechnological products derived from them) to expand the arsenal for controlling plant diseases caused by major biotic stresses. Different NGS and bioinformatics approaches will be applied to explore virome diversity, including latent infections that could be leveraged for novel biocontrol strategies.
Risk assessment protocols for virome-based control strategies will be implemented. VIROPLANT will address social and gender perceptions regarding the use of virome-based techniques as alternatives to traditional pesticides. A business plan will be included for representative categories of virome-based control strategies. Small and medium-sized enterprises and stakeholders will participate in the commercialization of the most promising products. The consortium will consist of experts in insect viromes, phage therapy, and mycoviruses from different agroecological systems across Europe.
VIROPLANT isolated and characterized 33 novel bacteriophages that are good candidates for a phage biocontrol approach for several important bacterial plant diseases, such as bacterial wilt of roses, bacterial canker of cherry and kiwifruit, and crazy root diseases of cucurbits. For some of them, such as bacterial canker of kiwifruit, translation to a specific in vivo application remains to be determined due to challenges in defining a correct delivery method. However, for pathogens of cherry and for the rose system, phage cocktails are ready to be contacted by interested companies for commercialization. The standardized methodology for environmental risk assessment for phage therapy tested in VIROPLANT could be further exploited by stakeholders involved in regulation (e.g., EFSA). Regarding the use of mycoviruses to limit biotrophic fungal/oomycete diseases, the main exploitable results obtained at VIROPLANT are:
- The description of viromes can be exploited by scientists to develop new theories of viral evolution. We have characterized more than 1,000 new viral segments corresponding to approximately 600 new mycovirus species. Their sequences are deposited in public repositories (GenBank) and described in peer-reviewed publications, and could be exploited for specific biocontrol applications. Several hypovirulent strains of B. cinerea with biocontrol potential were identified, and some hypovirulence-inducing mycoviral candidates were also characterized. An infectious clone of Botrytis F virus was successfully developed, which will be exploited for the construction of a viral expression vector and VIGS. A VIGS vector with the potential to produce dsRNA for use in RNAi strategies against fungi was developed. The vector, based on the CHV1 infectious clone, demonstrated its efficacy in endogenous silencing and can be exploited by other scientists for reverse genetics strategies. Regarding the search for new viruses for insect pest and vector control, the exploitable results of VIROPLANT are: A comprehensive approach for virus identification and study in agrosystems. Two new VIGS insect virus vectors.
- The development and reproduction of thrips populations with specific persistent viruses that can interfere with tospovirus transmission (synergy or antagonism).
- In the case of plant mitoviruses, virus-associated resilience to abiotic stress could become a strategy for improving plant yield, i.e., resistance breeding with mitochondrial viruses. VIROPLANT also conducted research on the economic and regulatory landscape for the commercialization of virus-based plant protection products (PPPs).
The potentially useful outcomes of this analysis are: The results of the techno-economic system analysis can guide companies wishing to invest in virus-based PPPs and inform funding agencies about the need and urgency of supporting research in this field. Gaps in regulation and research for introducing phage-based PPPs to the EU market have been identified, which should be addressed by regulatory agencies. A preliminary three-tiered protocol for the environmental impact of phage-based PPPs is proposed. VIROPLANT also considered ways to monitor, and possibly improve, the social acceptance of virus-based PPPs. The results are of utmost importance for policymakers and those involved in technological innovation in the field of biotechnology. The path is paved toward conditions for improving the acceptability process.
The dissemination activities implemented in the project are: 24 peer-reviewed open access scientific publications (which obtained a total of 246 citations), 4 articles published in local journals for farmers, 25 participations in national and international scientific conferences (9 posters and 16 oral presentations), 13 seminars for academics and 16 meetings with stakeholders.
The overall objective of VIROPLANT is to study and propose integrated virus-based biocontrol strategies. Growing conflicts between farmers and society at large over the use of pesticides in highly urbanized areas dedicated to grape and fruit cultivation are a cause for great concern. Safer alternatives are needed. VIROPLANT has the following specific objectives:
- To find biocontrol tools for bacterial plant diseases that replace copper, the only chemical method routinely available and permitted in Europe against bacteria.
- Find alternatives to the use of chemical fungicides to control plant diseases caused by fungi.
- Reduce the use of insecticides to control pests that cause direct and indirect damage to plants.
- Explore the potential of recent technology (RNA interference = RNAi) to limit the use of fungicides and pesticides in agriculture.
- To evaluate the potential resilience to biotic stress caused by the presence of covert/cryptic viruses in model crops (tomato) and in a specific experimental system.
- Evaluate the environmental risk of phage therapy against bacterial plant pathogens.
- Identification and measurement of generational and gender-specific factors for the social acceptance of natural and biotechnologically engineered virome-based tools.
- Identification of new business and market opportunities at regional and European level for virus-based biological control agents (VCAs).
In summary, VIROPLANT was able to identify and molecularly characterize a large number of novel viruses that infect bacteria, fungi, and insects. Some of them were also biologically characterized, and their potential for biocontrol was evaluated. Some encouraging results were obtained specifically for bacteriophages that will be used to control bacterial plant diseases. Virus-induced gene silencing (VIGS) approaches did not result in viable biocontrol tools, for both technical and regulatory reasons.
A potential market and communication strategy were identified to overcome reluctance to adopt virus-based biocontrol agents. Regulatory burden, coupled with low social acceptability, remains a significant obstacle to developing further steps.
Next-generation sequencing (NGS) technology has proven instrumental in elucidating the complexity and role of the microbiome associated with plants and animals. The role of the "virome" as a subcomponent of the microbiome is beginning to be revealed due to new ways of analyzing data and the recognition of its importance in shaping biological systems.
The goal of VIROPLANT is to apply NGS technology and empirical biological experiments to develop new, environmentally friendly virus-based control strategies for managing diseases transmitted by bacterial, fungal, and insect vectors (phytoplasmas and viruses), as well as insect pests. To date, plant protection using viruses has relied on limited successful model systems, such as baculovirus for Lepidoptera and a mycovirus for chestnut blight control.
Furthermore, viruses that infect bacteria (phages) are also cutting-edge tools with the potential to combat antibiotic-resistant bacterial strains in human and veterinary medicine. VIROPLANT will create a database of novel viruses (and virus-derived biotechnological products) to enhance the arsenal for controlling plant diseases caused by the most important biotic stresses. Different NGS and bioinformatics approaches will be applied to explore virome diversity, including latent infections that can potentially be exploited for new biocontrol strategies. Risk assessment protocols for virome-based model control strategies will be implemented.
VIROPLANT will address social and gender-specific perceptions of the use of virome-based techniques as alternatives to traditional pesticides. A business plan will be included for representative categories of virome-based control strategies. Small and medium-sized enterprises and stakeholders will be involved in the commercialization of the most promising products. The consortium will include experts in insect viromes, phage therapy, and mycoviruses from different agroecological systems across Europe.
Turning viruses into biocontrol agents to combat bacteria, fungi, and insects that attack crops could reduce our reliance on chemicals and pave the way for next-generation pesticides. Viruses are viewed by most people as an absolute danger and threat. After all, they cause diseases and, as we've seen with COVID-19, pandemics. However, the reality is that the vast majority of viral species are completely harmless; in fact, some can be beneficial and play essential roles in many ecosystems. One virus, for example, works against chestnut canker, a fungal disease that attacks chestnut trees.
This virus, which is harmless to the tree, acts as a kind of natural biocontrol, rendering the fungus less able to cause disease. “I am a plant virologist by training, and I was interested in seeing if there were possibilities for using viruses in agriculture,” explains VIROPLANT project coordinator Massimo Turina of the National Research Council in Italy. “The use of chemicals in agriculture is unsustainable, especially in more urbanized areas, and antibiotics against bacteria cannot be used due to antimicrobial resistance. The EU also wants to phase out the use of copper. It is therefore important that we find new approaches to protecting crops.” Ecological niche analysis The aim of the VIROPLANT project was therefore to examine the potential of new virus-based biocontrol agents that target bacteria, fungi, and insect pests. Turina notes that there are currently only five virus-based biocontrol products on the EU market for controlling insects. "Much work has already been done on the ecological role of viruses in the marine environment, aided by new high-throughput sequencing techniques," says Turina. "We wanted to extend this approach to other ecological niches."
To do this, Turina and her team identified lesions on leaves where the plants were being attacked. Bacterial diseases that attack kiwis, beans, tomatoes, and cucumbers were some of the project's case studies. Insect pests that also transmit viruses and phytoplasmas to important crops such as grapevines, peppers, and onions were also included. The team also collected downy and powdery mildew. These samples were taken back to the lab and analyzed using genetic sequencing technology. This allowed the team to characterize the genetic makeup of a specific ecological niche and search for viruses that could be developed into biocontrol agents. Promising candidates were subsequently used in trials. A New Era in Biocontrol “We were able to describe the genomes of thousands of new viruses in detail by analyzing bacteria, fungi, and insects,” Turina adds. “We found numerous candidates that could be used against specific diseases and that could one day be transformed into products.” The findings could also influence human medicine. There is growing interest in these bacteriophages (viruses that attack bacteria) as a viable alternative to antibiotics. "Going forward, it will be crucial to address the social acceptance of virus-based biocontrol agents," he notes.
We also need to analyze their commercial potential and work toward a more streamlined regulatory framework.” Turina points out that biocontrol agents face the same regulatory burdens as chemical pesticides. He believes this is something that needs to be examined. Finally, Turina and his team were able to identify previously undiscovered virus types. “We found what could be missing links in viral evolution,” he says. “This is very interesting from a fundamental science perspective. With further research, this could help scientists better understand the origin and evolution of viruses.”
VIROPLANT took the first steps to bring to the European market the first phage cocktail for a crop disease; studied the possibility of using circular ssDNA mycoviruses directly as fungicides against Botrytis cinerea; developed the first mycovirus-based vector for successful VIGS in fungi; expanded the arsenal of virus-based tools available for insect pest biocontrol beyond the limited host range of baculoviruses; analyzed the specific aspects behind the acceptance of gender/generational bias and provided targeted communication strategies to overcome the reluctance to adopt virus-based products. IMPACTS: VIROPLANT had a major impact in reshaping the evolutionary history of mycoviruses; VIROPLANT identified a novel insect virus-based strategy to interfere with tospovirus transmission; potentially reduced the dependence on chemical crop protection products; introduced new products with higher specificity and improved environmental performance.
IMPACTS ON SOCIETY: improved social awareness, i.e., improved general acceptance of virus-based gene therapy. - improved perception of viruses and virus-based products in the learning process in school curricula.
- CONSIGLIO NAZIONALE DELLE RICERCHE (CNR)
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