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Projects / Programmes source: ARIS

Developing simple, rapid and on-site methods for plant pathogens detection

Research activity

Code Science Field Subfield
4.06.00  Biotechnical sciences  Biotechnology   

Code Science Field
T490  Technological sciences  Biotechnology 

Code Science Field
4.01  Agricultural and Veterinary Sciences  Agriculture, Forestry and Fisheries 
Keywords
plant pathogens, biodiversity, on-site detection, NAIMA, quarantine organisms, high throughput sequencing, diagnostic systems
Evaluation (rules)
source: COBISS
Researchers (11)
no. Code Name and surname Research area Role Period No. of publicationsNo. of publications
1.  23611  PhD Tanja Dreo  Biotechnology  Researcher  2010 - 2013  499 
2.  12688  PhD Kristina Gruden  Biotechnology  Researcher  2010 - 2013  983 
3.  27827  PhD Jon Gutierrez Aguirre  Biotechnology  Head  2010 - 2013  363 
4.  08076  MSc Anton Kovač  Biochemistry and molecular biology  Researcher  2010 - 2013  18 
5.  07632  PhD Minka Kovač  Biochemistry and molecular biology  Researcher  2010 - 2013  111 
6.  34214  PhD Rok Lenarčič  Biotechnology  Researcher  2012 - 2013  28 
7.  23610  PhD Nataša Mehle  Biotechnology  Researcher  2010 - 2013  532 
8.  29178  PhD Dany Morisset  Biotechnology  Researcher  2010 - 2013  121 
9.  24505  PhD Manca Pirc  Biotechnology  Researcher  2010 - 2013  206 
10.  05229  PhD Maja Ravnikar  Biotechnology  Researcher  2010 - 2013  1,366 
11.  22582  PhD Nataša Toplak  Biotechnology  Researcher  2010 - 2013  168 
Organisations (2)
no. Code Research organisation City Registration number No. of publicationsNo. of publications
1.  0105  National Institute of Biology  Ljubljana  5055784  13,335 
2.  1775  OMEGA svetovanje, inženiring, razvoj in raziskovanje d.o.o. (Slovene)  Ljubljana  5808642  293 
Abstract
Currently, over 275 quarantine organisms are listed in the European Community. As an impact of climate change and expansion of trade networks, significant increase in establishing plant pathogens is expected in the next ten years. Because they evolve in response to control methods, plant pathogens find new reservoirs and host plants that must be identified for efficient plant disease control.   Plant pathogen detection is often done visually, followed by laboratory confirmation and subsequent monitoring. Before symptom development, detection tools are the only available solutions. Reliance on sometimes lengthy laboratory testing can cause significant delays in preventive actions. A relatively small number of detection tools is currently available for field use with many having serious shortcomings in terms of sensitivity, complexity or cost. Thus, there is a real need for rapid, simple and robust detection methods that can be used in the field to allow early detection of plant pathogens by inspection services and plant epidemiologists in discovering new host plants.   Both singleplex (detection of a single target) and multiplex (detection of multiple targets) methods based on RNA/DNA detection will be developed. Multiplex methods give the possibility to rapidly screen for/identify a larger panel of plant pathogens whilst a singleplex method can be used to confirm the identity of a specific pathogen. Recently we developed in our laboratory (Morisset et al., 2008, Nucl. Acids Res., 36 (18):e118), NAIMA, which is a detection method that has shown performance comparable to real-time PCR in detection of genetically modified organisms. Because of its isothermal nature omitting the use of expensive thermal cycling devices and demonstrated suitability for multiplexing, we will develop NAIMA for on-site simultaneous detection of several plant pathogens. Also, NAIMA will be used for fast singleplex real-time detection in combination with an adequate portable system. Finally, the next-generation sequencing system (SOLiD) will be tested as an alternative confirmation tool.   The project aims at “exporting” laboratory detection tools to the field in order to make robust and accurate results readily available also to field specialists and non-specialists. In this project, we will focus on the detection of plant pathogens threatening plants of great economic interest for the Republic of Slovenia and European Union such as the potato brown rot and potato ring rot bacteria or the potato spindle tuber viroid. The developed methods and techniques will be generic and therefore adaptable to a larger panel of plant, human and animal pathogens. Within other potential domains of application are human health and veterinarian services, human disease epidemiologists, food inspection (for e.g. food pathogens, GMOs, novel food/food additives).   Slovene researchers, specialists in plant virology, plant bacteriology, molecular biology, and microbiology diagnostics will participate in the project enabling efficient interdisciplinary approach. Our co-financer Omega d.o.o. is involved in state-of-art molecular diagnostics (SOLiD). For the project realization we have all the needed research equipment. In the field of plant pathology and plant molecular biology we will extend our collaboration with important European institutions, like the Food and Environment Research Agency (FERA) (Dr. Neil Boonham) in UK. In the field of biotechnology, our long standing experience of GMO detection, and our collaborators with high experience in diagnostic systems, such as FERA (LFD application) and Lumora Ltd. (portable real-time systems, on-site DNA/RNA extraction) will ensure precious information exchange for the good of the project.
Significance for science
The results of the project will have a relevant impact to the scientific fields of the plant pathogen diagnostics and epidemiology, and they will also add scientific advances to other diagnostics fields, where the application of on-site isothermal detection would be beneficial. In the molecular diagnostics as well as in other related fields, current trends and demands are focusing mainly on the reduction in size, simplicity of use, and high throughput. The results of the project are represented by new methods for detection of plant pathogens (PSTVd and Ralstonia solanacearum): 1) NAIMA method, enabling simultaneous multiplex amplification and detection of RNA and DNA targets; 2) LAMP method, enabling fast and simple pathogen detection; 3) detection of pathogens based on data gathered by high-throughput next generation sequencing. NAIMA and LAMP methods will equip epidemiologists with on-site, isothermal molecular methods that will assist them to study pathogens behaviour as well as, for example, bacterial communities in the field, detection and discovery of new pathogens’ reservoirs and host plants. Simultaneous multiplex amplification of RNA and DNA targets with NAIMA method represents an additional advantage, because only one reaction is needed for detection of both types of targets. The NAIMA method is the first method enabling isothermal amplification of RNA and DNA targets, thus it is expected to be highly cited. All LAMP and NAIMA methods are at the same time very simple and don’t require complex equipment, therefore they could be used on-site by staff with no special technical skill. Due to the simplicity and short time needed for analysis, these new methods are cost-effective, what will have an impact on the development of further methods. The shown usefulness of next generation sequencing approach makes it a new alternative tool for detection of nucleic acids that can become a landmark in the field of plant pathogen diagnostics as high-throughput sequencing is expected to become more and more affordable in the near future. In addition to the above fields of research, the outcome of the project will contribute to the development of other fields where there is a need for simultaneous multiplex DNA and RNA amplification and where simple, on-site molecular methods would represent a benefit. Such research fields include environmental virology and bacteriology, food safety diagnostics (GMO, food pathogens...), human and animal disease diagnostics, human/animal epidemiology studies and others, therefore, the project results will be applicable to a wide spectrum of research.
Significance for the country
The results of the project will have a direct impact for the economy and society of the Republic of Slovenia, as we have reached the aim of improving the early detection (and therefore eradication) of quarantine pests that constitute a real economic and ecological threat to the Slovenian agriculture as well as in the European Union and worldwide. The results of the project are newly available diagnostic methods that will be robust and fast but also focusing on simplicity and cost-effectiveness. With the help of these results the early front line detection and confirmation of plant pathogens will greatly assist in designing timely and efficient plant protection measures thus decreasing the socio-economical impacts of plant pathogen threat. The following stakeholder groups will benefit from the outcomes of the project: • inspection services and plant protection associations (by decreasing cost and time of analysis) • growers and breeders of plant material (better and faster detection avoiding the spread of pathogens) • exporters/importers (less delay for trade) • society (prevention of diseases that damage plants and the environment). Furthermore, the results of this project will benefit to the plant epidemiologists, providing new and cheaper tools for the discovery of new pathogens reservoirs and, for the study of plant-pathogens interactions, therefore helping the epidemiologists in protecting the Slovenian natural plant diversity. Thanks to its long-standing collaboration with different Slovenian stakeholders, the Department of Biotechnology and Systems Biology has already and will continue advertising the tools developed in the project and will promote their use to the different stakeholders of the plant protection and plant epidemiology fields in the Republic of Slovenia and abroad, as plant pathogens are a global threat to agriculture and plant trade, as well as plant diversity. Moreover, the inclusion of the company OMEGA d.o.o. as a partner in the project is an additional example of the importance of close collaboration and knowledge exchange between Slovenian biotech companies and research organizations, strategy, which at NIB is considered essential for an optimal national R&D development. The use of generated results can be extended to other applications in Slovenian network of research and development, thus inviting new users of the technology. This will therefore contribute to a further development of OMEGA d.o.o activities in the republic of Slovenia. Finally, the applications and benefits of the project results are not limited to the fields of plant protection and plant epidemiology, as fast and cost effective detection methods are needed in numerous fields of socio-economical importance such as veterinarian and human health diagnostics and epidemiology, food safety, bio-security. Combining the strength and skills of researchers from both GMO diagnostics and plant pathogens diagnostics units, with the highly valuable expertise from our international collaborators and the high technology provided by OMEGA d.o.o. facilitated an outcome of very high quality, ensuring the reputation of Slovenian research as one of the leaders in on-site diagnostics and applied biotechnology. In this way, Slovenian science and high technology companies will be promoted through the rest of the world via scientific publications and applications. Moreover, providing cost-effective and easy-to-use diagnostics tools may help their diffusion and use in developing countries where the current high technology tools are not affordable. In that sense, the project will also have a long-term positive effect for the promotion of Slovenian research.
Most important scientific results Annual report 2010, 2011, 2012, final report, complete report on dLib.si
Most important socioeconomically and culturally relevant results Annual report 2010, 2011, 2012, final report, complete report on dLib.si
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