Final Report Summary - BRIO (Banking Rhizosphere Micro-Organisms. <br/>European - Russian initiative to set up a network of rhizosphere microbiological resources centres)
Executive Summary:
CONTEXT
Soil microbes play key roles in ecosystems and exert multiple beneficial as well as detrimental functions impacting yield and quality of food.
Rhizosphere and endophytic microbes play a central role in promoting plant growth and health via acquisition and recycling of nutrients, modulation of plant hormonal balance, direct or indirect protection of the plant from detrimental organisms (e.g. pathogens), protection against abiotic stress (e.g. drought, heavy metals), and improvement of soil structure.
A holistic view of the rhizosphere microbiome is necessary to increase the knowledge about all micro-life interacting with the roots and with each other. Such understanding improves the effectiveness of solutions against stress induced by biological (pests and diseases) as well as non-biological (water shortage, pollutants, etc.) factors, offering opportunities for agro-environment sustainable management.
To support the study of the rhizosphere microbiome there is a need for optimised pool of microbial material, data, information and know how easily accessible, and technically as well as legally fit for use in biotechnology. BRIO aims at providing scientists with access to such a large array of rhizosphere micro-organisms, related data and expertise.
BRIO Objectives & Realization of PERN
The objectives of BRIO are:
I. to organize cooperation between specialised collections holding beneficial micro-organisms isolated from the rhizosphere,
II. to constitute a common wide-range pool of microbial diversity exploitable for research and industry: the Pan-European Rhizosphere resources Network (PERN).
Two types of activities were performed to achieve these objectives :
• Activities aiming at optimising the structure and organisation of the network, i.e. the framework including a common policy regarding regulatory and ownership issues and the IT system. The preliminary database structure developed during the first year has been extended to other data related to possible uses of the material. Outcomes of work on common protocols, common procedures and joint data management are already visible on the website www.pern-brio.eu . In addition, network structuring and accession and distribution policies development are going on. Specific activities have been organised in common during the third year, including a field survey in Russia and specific training focused on culture collections needs. Contacts have been made with other collections in the Russian Federation, in connection with the World Data Center for Microorganisms (WDCM) and the World Federation for Culture Collection. A MoU has been signed between WDCM and the Skryabin Institute of Biochemistry and Physiology of Microorganisms, host institute of the VKM collection. The maintenance of the BRIO website is ensured by the SPP-PS BCCM webmaster. The maintenance of the database is ensured by the VKM computer scientist. Contents is updated by the PERN members.
• Activities focused on the contents of the collection, i.e. the quality of the microbiological material and related data. All partners have biological material and related data, expertise, equipment and facilities to share and to organise in common. The work consists in organizing these assets to propose an optimum range of beneficial rhizosphere microbial resources organised around three main applications: biofertilisers, biopesticides, and bioremediation. The first assessment of the existing stocks made during the 1st year provided a set of 595 strains. Partners have designed a strategy to optimize every partner's holding and to build together a set of complementary stocks merged as one virtual collection. At the end of April 2014, the online catalogue posted on the project website www.pern-brio.eu listed more than 1100 strains. These BRIO collections constitute the Pan-European Rhizosphere resources Network (PERN) core, a significant modular repository of rhizosphere microbial resources publicly accessible for research.
Project Context and Objectives:
PROJECT CONTEXT
Soil microbes play key roles in ecosystems and exert multiple functions, from detrimental (as pathogens) to beneficial (e.g. plant growth promoters and pathogen antagonists), impacting yield and quality of food. They influence a large number of important ecosystem processes, including nutrient acquisition (Smith & Read 1997; Sprent 2001), nitrogen cycling (Tiedje 1988; Kowalchuk & Stephen 2001), carbon cycling (Hogberg et al. 2001) and soil formation (Rillig & Mummey 2006). Moreover, soil microbes represent the unseen majority in soil and comprise a large portion of the genetic diversity on Earth (Whitman et al. 1998).
Nowadays, increasing attention is devoted to rhizosphere and endophytic microbes which play a central role in promoting plant growth and health via:
- acquisition and recycling of nutrients important to plant growth,
- modulation of plant hormonal balance,
- direct or indirect protection of the plant from detrimental organisms (e.g. pathogens),
- protection against abiotic stress (e.g. drought, heavy metals), and
- improvement of soil structure.
It has been stated that “the ultimate agricultural goal in studies of the biology of the soil-root interface, must be the manipulation of micro-organisms in this zone to increase plant health and growth” (Rovira, 1979). Research should aim at improving our knowledge of the interactions between plants and microbes and of sustained management of these microbes to benefit the plant-food-consumer chain. This knowledge could help to reduce excessive use of agrochemicals alleviating hazardous effects of agricultural production on the environment.
Sound environmental and agricultural practices should favour balanced composition of micro-organisms population in the rhizosphere. A well balanced microbial population favours colonization of soil and nutrients uptake, limits pressure of pathogens (biotic stress), supports the development of commensal or symbiotic relationship between plant and micro-organisms, etc, with a good tempering effect regarding abiotic stress (good water retention capacity, available macro- and micronutrients).
A holistic view of the rhizosphere microbiome is necessary to increase the knowledge about all micro-life interacting with the roots and with each other. Such understanding improves the effectiveness of solutions against stress induced by biological (pests and diseases) as well as non-biological (water shortage, pollutants, etc.) factors, offering opportunities for agro-environment sustainable management.
To support the study of the rhizosphere microbiome there is a need for optimised pool of microbial material, data, information and know how easily accessible, and technically as well as legally fit for use in biotechnology. BRIO aims at providing scientists with access to such a large array of rhizosphere micro-organisms, related data and expertise.
PROJECT OBJECTIVES
The objectives of BRIO are:
1. To establish long term links and synergies between Russian and other European Biological Resources Centres preserving microbial material isolated from plants' rhizosphere. It is expected that BRIO contributes to facilitate collaborations, to increase synergy between teams studying rhizosphere via the Pan-European Rhizosphere resources Network (PERN). The original core of PERN is made of the BRIO participants.
2. To constitute a common pool of rhizosphere micro-biodiversity exploitable by research and industry. BRIO participants have built an initial pool of rhizosphere microbial resources, related data and human expertise available via the BRIO website www.pern-brio.eu The BRIO website is a HUB making these resources available for scientists. Facilitating the preservation and use of well documented, quality micro-organisms of the rhizosphere fosters the sustainable exploitation of renewable biological resources, a major objective of the European Knowledge Based Bio-Economy.
Collaboration between culture collections having material from West-European ecosystems and Russian Biological Resources Centres having micro-organisms from East-European biotopes forms a network of collections offering a wide range of micro-organisms coming from a broad spectrum of ecological zones spread over an extensive geographical area.
Project Results:
RESULTS & ACHIEVEMENTS grouped per objective and presented by work packages (WP)
The project's activities are grouped in 2 sets of 4 work packages
I. Work Packages 5 to 8, corresponding to Objective 1, included optimization of the databases structure and linkages, the design of a common policy regarding regulatory and ownership issues, as well as external communication and dissemination, in order to consolidate the network and optimize the coordination between the partners.
II. Work Packages 1 to 4, corresponding to Objective 2, focused on the contents of the collection, i.e. the quality of the microbiological material and related data in order to optimize the preservation and characterization of the microorganisms of the rhizosphere and the related data.
I. Objective I. Structure and organisation of the network, legal framework and database system.
WP5. NETWORK STRUCTURING AND LONG TERM SUSTAINABILITY
Coordination & networking
During the project 6 major meetings were held. All Partners have taken part in discussions at the BRIO project meetings where issues of network structuring and long term sustainability were discussed. All aspects of the future development of the network were discussed during three of these meetings.
Partner 8 (IBPPM) hosted the 1st Annual meeting , Partner 7 (IEGM) hosted the 2nd annual BRIO meeting, Partner 6 (VKM) hosted the 3rd annual meeting.
Coordinating partner 1 (SPP-PS) hosted the first and the final meeting in Brussels. Video-conferences using SPP PS bridging service facility for multipoint video conferencing were held in between.
Exchange of biological material and scientific visits
For instance, Partners 7 (IEGM) and 5 (UNITO) have exchanged their collection holdings. In particular, during Prof. Maria Kuyukina’s visit to UNITO on 22 October 2013 to give the seminar “Importance of Hydrocarbon-Oxidizing Rhodococcus for Petroleum Microbiology and Bioremediation” and Practicum on morphological identification of Rhodococcus actinobacteria, six actinobacterial strains were transferred to UNITO for educational purposes. Also, 26 fungal strains isolated from oil-contaminated soil and rhizosphere samples during the common field survey in Perm, as well as 2 fungal strains isolated from codeine-containing fermentation medium were transferred to UNITO during the final BRIO meeting in Brussels, for research and educational purposes. Material Transfer Agreements (MTAs) for Non-Commercial Purposes were signed between IEGM and UNITO.
Strain exchange between IBPPM collection and MUT collection and between IBPPM collection and LMG collection was carried out. In the first case the main objective of the exchange is a collaborative research concerning revealing of ligninolytic enzymes and their xenobiotic-degrading activities. In the second case the taxonomic belonging of Azospirillum sp. strains and rhizosphere strains newly isolated during the field surveys was clarified.
In the collaboration between IBPPM Collection and LMG collection Dr. Ekaterina Dubrovskaya visited UGent and received training on application of MALDI-TOF mass-spectrometry for bacterial identification and classification.
Cooperation with private companies, such as an agreement between Priroda-Perm Company and IEGM was set up to perform development works on oil sludge remediation and to get access to clean up sites of oil-contaminated soil. The parties jointly discussed the bioremediation scheme and transfer of an oil-degrading biopreparation developed by IEGM team considering the requirements of a particular contamination.
During the 1st annual BRIO meeting in Saratov, Priroda-Perm scientists presented the company's bioremediation activities, in which the IEGM team was involved as a consultant.
Collection of strains from common field survey
A common field survey was organised as a team building effort during which several multidisciplinary activities were done: the bio-prospecting survey sensu stricto, common laboratory activities for identification and characterization, testing administrative load and organising appropriate accession and distribution policy for the network, experiencing and applying the regulatory framework relevant for the collected microbiological material, its isolation from in situ, its transfer and its conservation.
A collection of strains from the common field survey in Perm was created that included 12 actinobacterial strains and 26 fungal strains isolated from oil-contaminated soil and rhizosphere. The actinobacterial strains are maintained in IEGM, while the fungal strains are maintained in MUT (UNITO) according to collection specializations. The information on these strains is included into collection’s databases and are available in the BRIO catalogue.
Results and outcomes of the common field survey in Perm were presented by Partner 7 (IEGM) at the 2nd BRIO meeting.
Set up of PERN
The participation of all BRIO Participants to the Pan-European Rhizosphere resources Network (PERN) is effective since the 3rd annual meeting organised in February 2014 in Moscow and Pushchino.
Various instruments and activities are in place or in development such as:
a) General accession and distribution policies,
b) Common online catalogue and website;
c) Development of a network of experts related to PERN,
d) Programme of collaboration with global networking activities such as the Global Catalogue of Microorganisms,
e) Plan for extension of the network in the Russian Federation as well as with other non-European partners.
WP6. UPGRADING DATABASE STRUCTURE
To optimize ICT management and database structure the project foresaw the creation of four main databases:
A. 1 database focused on the microbiological material and being the basis of the online catalogue, and
B. 3 other databases to store information related to experts in biofertilization, biopesticides and bioremediation.
At the end of the project these 4 database have been merged into a single database system with several sub-databases.
A. During period 1, Partner 6 (VKM) ICT expert, Dr Alexander Vasilenko made a comparative study of all fields of various standards in use to record data related to (micro) biological material, from the Minimum Data Set (MDS) for the "strain passport" up to the Extended Data Set (EDS).
The part of the database dedicated to the microbiological material is developed in conjunction with other international developments, namely the CODATA Task Group "Advancing Informatics for Microbiology", the WFCC (World Federation for Culture Collections) Global Catalogue of Micro-organisms (WFCC-GCM) and TRUST, the update of the MOSAICC code of conduct concerning the data necessary to implement the administrative and legal obligations set by the Nagoya Protocol. The outcomes of these works impact on the structure of databases developed for biological material including the BRIO database.
- The VKM ICT expert is involved in CODATA Task Group "Advancing Informatics for Microbiology". The main application field of this Task Group is in the analysis of the existing standards for database structure, ontology, and semantic of database fields for the purpose of microbial data integration. - VKM is also contributing to the creation of a European Biological Resource Centres Information System (in the framework of FP7 Infrastructure project Microbial Resource Research Infrastructure - MIRRI).
- Besides Partner 6 (VKM), Partner 1 (SPP PS) is contributing to WFCC GCM. The WFCC GCM is developed and managed by the World Data Centre for Microorganisms (WDCM), the WFCC ICT centre.
- Partner 6 (VKM) and Partner 1 (SPP PS) are involved in the update of the MOSAICC project (funded in 1999 by DG Research) via the initiative called TRUST - Transparent User-friendly System of Transfer.
These complementary efforts are essential to ensure full compatibility between projects funded by DG Research.
Having initiated the discussion with regard to the composition of the data fields and adjustment of catalogue data standard to OECD Minimal Data Set (MDS) and WDCM MDS, VKM has coordinated the following phases:
- First, once the standard and composition of data fields were agreed between participants, the BRIO database was created including the following steps:
- construction of the main table,
- construction of user requests,
- posting the primary database in Extranet.
- Secondly, all participants have loaded their data into BRIO database.
For instance, Partner 5 (UNITO) has selected 106 strains from the MUT collection to include in the BRIO database: 48 strains potentially useful for biofertilizers, 4 strains with biopesticides / biocontrol properties and 68 strains for bioremediation applications. This dataset contains the taxonomic information, isolation and identification data, storage and growth conditions, and strain properties with literature references when available.
Thirdly, with all data loaded, VKM performed the following activities:
- Transformation of fragments delivered by participants to BRIO database format and feedback to participants.
- Corrections / Additions from participants.
- Preparation of participants' database replica.
The first BRIO database containing data of 595 strains was posted by VKM on Extranet.
B. Concerning the three subdatabases storing information related to experts in biofertilization, biopesticides and bioremediation, it was agreed by the participants to merge these three databases into one subdatabase by developing database fields allowing segregation between various uses and thus segregation between experts. Taking into consideration that the structure must allow multiple entries - multiple outcomes linkage, not one to one. In other words, more than one expert can have the more than one field of interest. It was also decided to link these compiled databases with the list of strains.
A. & B. The project developments showed that one robust unique integrated database divided in sub-tables is the most appropriate construction because it allows advanced searches using and combining criteria such as strains characteristics, field of expertise of the "resources people" , kind of biotechnological use, taxonomic information, etc.
Merging the four databases as sub-tables of a unique structure makes them fully interoperable and simultaneously searchable. That enables for instance the users to find the experts who may answer their enquiries about specific kind of microorganisms.
Partner 6 VKM further detailed the fields regarding the use of bio-properties (biofertilisers, biopesticides and bioremediation). Outcomes of Work package 1 on stock survey were regularly used to update the database; strains were added to the strain sub-table and other kind of sub-tables are prepared to link the strains to complementary information.
Partner 7 (IEGM) in collaboration with Partner 6 (VKM) have developed a specific "strain property" field in the BRIO catalogue in a format which consists of two terms: a verb indicating the activity and a noun (or a word-combination) indicating the subject(s) of action (e.g. degrades polyaromatics or accumulates heavy metals). So, the user can search the particular strain of interest by both terms using the strain property search form at http://www.pern-brio.eu/catalogue
Partner 8 (IBPPM)'s list of appropriate strains for the BRIO database was upgraded. This includes 51 strains possessing high biodegrading abilities towards different pollutants, and plant-growth-promoting abilities towards different plants. This dataset contains taxonomic information, isolation and identification data, storage conditions, growth conditions, and strain properties with literature references when available.
Website
All partners have contributed to the project website structure and design as major external communication media. All partners have delivered content for the website as agreed. It was decided to make the website largely bilingual (E/RU) and the Russian partners have taken on the task of translating much of the content to ensure a wider dissemination and easier use by Russian researchers.
WP7. REGULATORY FRAMEWORK AND IPR MANAGEMENT
It aims at defining common policies regarding the deposit of strains into a collection of PERN and the distribution of microbiological material of the PERN collections. These policies are translated via respectively Material Accession Agreement and Material Transfer Agreement. These basic legal reference documents serve Russian and European collections to cope with regulations and administrative requirements concerning microbiological material. All Partners have taken part in discussions where regulatory issues and issues of IPR management were discussed.Partner 6 (VKM) collected information regarding laws and regulations (sanitary rules, transport regulations, patent issues, etc.) impacting on exchanges and uses of microbial material in Russia and compared the European and Russian legal framework related to access and transfer of microbiological material was done.
In short, most of regulations and legal documents of EU and Russian Federation are similar especially with regard to international Agreements (Convention on Biological Diversity, Biological and Toxin Weapons Convention, Plants quarantine, Budapest Treaty, etc.). A major constraint is the need for translation. That is done partly in the BRIO website which presents abstracts of the legal texts in English to make the document accessible to non-Russian users.
The most significant differences between European and Russian regulations were observed in the area of biosafety. In Europe all microorganisms are divided into risk classes 1 to 4. The risk class 1 includes non-pathogenic microorganisms (for humans, animal and plants) as well as opportunistic ones. According to European rules, all the microorganisms that are not included in risk classes 2-4 belongs to risk class 1.
In the contrary, in Russia only microorganisms known to induce diseases are qualified for their epidemiological risk. Group 1 encompasses the most hazardous, while the group 4 include the least hazardous microorganisms. The microorganisms that are not assigned to the pathogenicity groups 1-4 are assumed to be safe. Considering the items in lists of microorganisms belonging to analogous groups of risk/pathogenicity one can also see significant differences between them in European and Russian documents.
Based on the analysis of the documents for protection of intellectual property in area of biotechnology in EU and Russian Federation it may be concluded that the basic rules are quite similar. There are however, some differences in details. The Directive 98/44/EC Article 13 paragraphs 2 and 3 describe the rules for access to the deposited strains of microorganisms by third parties. In Russian Federation these rules are not yet provided. In practices, VKM collection requests official permission from depositors for this issue.
The laws for transfer of microorganisms are equal for Europe and Russia but there are differences in the departmental regulations.
Exchanges of strains have occurred during the BRIO project to test the administrative burden. For instance, partners 3 (UGent), 6 (VKM) and 8 (IBPPM) have tested the Russian administrative requirements by sending strains from Russia to Europe. A first shipment of two strains was requested by Partner 3 (UGent) from VKM collection on 6 Feb. 2014 and arrived on 31 March 2014 accompanied by 16 documents, nearly all in Russian; a second shipment of 11 strains was requested on 19 March 2014 and arrived on 10 April 2014 (accompanied by 35 documents for 11 strains).
Partner 7 (IEGM) has collaborated with Partner 5 (UNITO) to exchange strains from their culture collections. Two MTAs (of 22 October 2013 and 28 April 2014) produced using a model MTA designed by Partner 1 (SPP PS) for non-commercial purposes were signed between IEGM and UNITO, according to which 12 bacterial and 28 fungal strains were transferred from IEGM collection to MUT (UNITO) for educational and research purposes.
Partner 6 VKM has also continued activities related to the development of the common access and distribution policy in the PERN network. It has organized a WFCC seminar at Pushchino (June, 2013) under the coordination of Partner 1 SPP-PS about the Global Catalogue of Microorganisms, the latest development of the Nagoya Protocol and the TRUST system, TRansparent Users-friendly System of Transfer designed to implement the Nagoya Protocol in microbiology. The other seminar held also in June 2013 but in Moscow was timed to coincide with the signing of a Memorandum of Understanding (MoU) between the World Data Center for Microorganisms (WDCM) from the World Federation for Culture Collection (WFCC) and the Skryabin Institute of Biochemistry and Physiology of Microorganisms, Russian Academy of Sciences. The MoU is aiming at facilitating further cooperation directly between the VKM and WDCM, and structuring of the Russian network of collections. Relevant issues have been discussed. VKM's experts have participated in several local meetings at the RF Ministry of Education and Science in Moscow and in subsequent e-mail discussions focused on legal access to biodiversity (concerned with the latest development of the Nagoya Protocol), establishing biological resource centres in Russia and the Russian network of microbial collections, as well as activities related to improvement Russian regulations in the area of biosafety. In addition, VKM representative Prof. Lev Kalakoutskii provided presentation concerning with legal aspects of access to genetic resources and the required infrastructure at the round table conducted by the Committee on Science and High Technology of Russian State Duma (March, 2014).
Partner 7 (IEGM) has promoted the establishment of biological resource centres (BRCs) in Russia. Prof. Irina Ivshina as a member of the expert group on developing BRCs in Russia has participated in the group meeting at the RF Ministry of Education and Science in Moscow on 28 May 2014, where results of annual inventory of current biological collections and further activities related to the formation of national BRCs were discussed.
Scientists face heavy administrative burden to exchange strains. Regardless of the user or the supplier, transfers from and to Russia are mainly centralized now by VKM.
IEGM has written the article "Current Situation and Challenges of Specialized Microbial Resource Centres in Russia" published in the journal Microbiology (2012. 81: 509−516). It considers the urgency of establishing national specialized centres based on target long-term funding and territorial location (e.g. the Far Eastern National Centre of Marine Bacteria, Siberian National Centre of Luminous Bacteria, Ural National Centre of Microbial Resources, etc.).
Furthermore, IEGM has highlighted the problems and opportunities relevant to collection-associated activities in Russia demonstrated by the example of the IEGM collection in the interview "Microbial Collections: Challenges and Opportunities" to the newspaper Science of Ural (the Ural Branch of the Russian Academy of Sciences, 2012. May, 13: 4.
IEGM has stressed the operation of the Russian specialized collections in accordance with the major goals under the Complex Program of Biotechnologies Development in RF “BIO-2020” is discussed in the article Infinite Role of Infinitely Small in the public science journal Science in Russia (2013. January-February 1(193): 9−15.
WP8 PROJECT MANAGEMENT AND DISSEMINATION
Project management, support to partners
The Participant Portal User's Guide is exhaustive but for some particular tasks it was more efficient to have a digest of the instructions dealing with one single action. Coordination has issued digests of instructions to help partners to fill in the financial statements online. During the time of the project changes occurred in the Research Participants Portal website layout and functionalities. Therefore, like during the first and second period, coordination has issued digests of instructions for the last reporting. A set of Microsoft Word® files with step by step instructions concerning the use of the Participant Portal system. The latest digests concern: Instructions for adding form C 3rd period.docx
and Adjustment to previous form C.docx
Internal Communication
Several videoconferences have been held by using the Gotomeeting™ software of Citrix® to solve specific issues and prepare the meetings.
Communication between the partners was excellent, making use of several tools:
- Email communication for written exchanges on particular topics,
- Videoconferencing for consultation on bottlenecks, focusing on sticking points
- Project Intranet providing for E-storage and sharing of files and documents
- Meetings
- The BRIO website
Website
The domain name: www.brionet.eu was the only one used during the project. Since the PERN network is set, there are now two domain names for PERN: www.brionet.eu and www.pern-brio.eu.
The BRIO website was designed and managed in house by SPP PS ICT expert. It is developed on open source software. SPP PS has written the source code to publish a searchable catalogue online, next to setting up the website itself.
The Alpha version of the website was made accessible to the participants for testing since spring 2013. The Beta version of the website was presented at month 30, six months before schedule. The website GO LIVE was made at the final BRIO meeting on 25 April 2014, on schedule.
The contents of the website is available in two languages (English and Russian) and comprises information on BRIO, its partners and funders, protocols for scientific experiments, a list of scientific experts and their fields of expertise, legal documentation, news, contact information, related links...
The website hosts the searchable BRIO Catalogue. More than 1.100 well documented microbial strain records are available online. The catalogue data can be searched via a specialised web database application developed for BRIO by VKM and SPP-PS. The catalogue can also be browsed. Therefore 'taxonomic' and 'strain properties' catalogue browsers were implemented.
The BRIO website is now converted in PERN and its content will be continuously maintained after the end of the current BRIO project.
Meetings
As foreseen during period 1 and 2, the original meeting planning has been reshuffled. Six major meetings have been organised.
Meeting 1 - BRIO Kick-off meeting - 6 and 7 June 2011, Brussels, Belgium
It included sessions corresponding to the
Content task force (CTF) Meeting on extensive stock review preparation and
Framework task force (FTF) Meeting. It aimed at organising the consortium, in complement to talks related to specific work packages. Special sessions were devoted to:
- designing a dissemination and exploitation action plan,
- setting up contingency plan for the various stumbling block identified at administrative and technical levels,
- setting up the Extranet workspace as soon as possible,
Meeting 2 - BRIO first Annual meeting - 8 to 11 October 2012, Saratov, Russian Federation
It was organised by participant IBPPM (Institute of Biochemistry and Physiology of Plants and Microorganisms). The meeting was originally planned at month 12 but postponed.
This meeting included:
• a closed workshop for the project participants only; during this day, in complement to the Work Package work summaries, the following theme were discussed :
– the extensive stock review progress originally planned at month 12, but postponed to month 18. It is the Content task force (CTF) meeting
– the plan for enhancement of individual collections
– the field survey to be organised in August/September 2013
• a closed workshop with participation of representatives of Russian private companies as scheduled as: Biofertilisers expert group meeting, Biopesticides experts group meeting, Bioremediation experts group,
Meeting 3 - BRIO intermediary ad hoc meeting - 17-21 June 2013, Moscow and Pushchino, Moscow Region, Russian Federation
This mission included three main activities:
- The signing of a Memorandum of Understanding (MoU) between the World Data Center for Microorganisms (WDCM) from the World Federation for Culture Collection (WFCC) and the Skryabin Institute of Biochemistry and Physiology of Microorganisms, Russian Academy of Sciences, Host institution of Participant 6 (VKM). The purpose of the MoU is to facilitate further cooperation directly between the VKM All Russian Culture Collection and WDCM as well as indirectly with the PERN network. It is a step towards structuring of the Russian network of collections. It is also important because it facilitates the linkages between the BRIO database and the Global Catalogue of Microorganisms.
- A work session dedicated more specifically to Regulatory framework. Comparison between Russian laws and EU laws as well as understanding the technical differences between the Russian and EU system of biosafety/biosecurity system is important.
- A WFCC Seminar at Pushchino, about the Global Catalogue of Microorganisms, the latest development of the Nagoya Protocol and the TRUST system, TRansparent Users-friendly System of Transfer designed to implement the Nagoya Protocol in microbiology. These activities are relevant for the development of the common access and distribution policy in the PERN network.
Participants to the meeting were from the coordination SPP-PS and participant VKM.
Meeting 4 - BRIO 2nd annual meeting - 9 to 13 September 2013, Perm, Russian Federation
Representatives of all participants participated to the meeting . The programme included 4 parts
A. Common field survey to collect strains in situ
This field trip and sampling for microbial strains was organised on 9 September 2013, with a visit to the Kokuy oilfield, with company “Priroda-Perm”, a visit to the waste treatment plant and of a natural park. Samples were taken from these three sites.
B. The follow up of the BRIO work package progress
The programme of the 2 days closed meeting encompassed all work packages. The sketch of the website structure was proposed by SPP PS and approved. It was decided to wait for the beta version to make more precise adjustments. Exchange of protocols for rhizosphere microorganism isolation, identification and preservation was done and a uniform protocol form to include into the website was prepared.
C. The public seminar
The public seminar was held on the 3rd day 12 September at the Perm State National Research University (PSNRU). The rector of PSNRU introduced the seminar. The BRIO coordinator made a lecture on the Relationship between the microbial culture collections and their users, as a means to improve quality and reproducibility in scientific testing. These academic talks were followed by a discussion chaired by the head of partner 7 IEGM. After a visit to the PSNRU laboratories and botanical garden, participant 7, IEGM, organised a round-table discussion concerning
- BRIO and the development of the network in Russia.
- Russian rhizosphere network connected to the WFCC Global Catalogue initiative.
D. Training course in mycology
The training course in mycology was held on September 13, at the IEGM collection laboratory and chaired by Prof. Cristina Varese, partner 5 (UNITO) and Dr Fritz Oehl, partner 4 (ART).
Meeting 5 - BRIO 3rd Annual meeting - 10 to 14 February 2014, Moscow, Russian Federation
Representatives of all participants participated to the meeting. The agenda of the meeting included 2 parts:
A. Reviewing of the work packages progresses during closed meeting and the endorsement by the participants of the several deliverables, these steps constituted the milestones:
- Regulatory framework and IPR management. Information on Russian and European laws impacting on access to and transfer of microbiological resources. Discussion and endorsement of the Common accession and distribution policy proposal
- Network structuring and long term sustainability.Future developments on long term sustainability were discussed.
- Meeting on survey outcomes. The field trip organised in September 2013 during the previous annual meeting was considered fully successful because the collected samples yielded several strains of relevant microorganisms. The administrative workflow to get these strains exported from Russia and imported in another European country was tested. All strains have not been characterized. It has been agreed to work on it after the end of the EC subsidized project phase.
- Exchange of protocols for rhizosphere microorganism isolation/identification/ preservation. Discussion on a unique format of the protocols for the website
- Parallel programming of the activities.
- Set of common fields between database of rhizosphere microbes and databases of relevant parameters (geographical distribution of diseases, soil information, target plants for microbial biofertilizers, etc.)
- Database subset of microbiological resources (for use as biofertilizers, biopesticides and bioremediation agents).
- Extensive stock review: Presentation of Catalogue.
- Round-table discussion: Proposals for the future development of the project.
B. Scientific conference
- Conference at the G.K. Skryabin Institute of Biochemistry and Physiology of Microorganisms, Russian Academy of Sciences, Pushchino, Moscow Region. Culture Collections at the crossroad of biodiversity and biotechnology activities
- Visit of VKM and other laboratories of the G.K. Skryabin Institute of Biochemistry and Physiology of Microorganisms, Pushchino, Moscow Region
- Visit to Radio Astronomy Observatory, Russian Academy of Sciences, Pushchino
Meeting 6 - Final meeting - 24 to 28 April 2014, Brussels, Belgium
Programme
Visit of BCCM/ MUCL collection and Laboratory of Microbiology.
Project outcomes overview, per Work packages
The updated catalogue was put online in March 2014 during the meeting. The BRIO website that hosts the online catalogue The central communication instrument is. Firstly access was limited to the BRIO participants, then after testing, it was made publicly accessible. BRIO website check and design of maintenance, updating and optimization programme.
The meetings were highly productive and constructive. They were absolutely necessary to get the final version of deliverables approved.
Visa for travel to Russia were not always easy to get. One should ask for a multiple entries visa open for the length of the project.
Thanks to the possibility of SPP-PS to hold the meetings in its own facilities, costs of the meetings in Brussels were limited.
Some partners have benefited from their joint participation to other international conferences to improvise work sessions. This helped to move faster, especially on the structure of the database and on the subsets of strains for biofertilization, biopesticides and bioremediation.
All participants consider that having meetings in Russia is very important for the Russian participants:
- It helps the teams based in relatively remote city of Russia to feel fully part of the project;
- It raises the profile of the Russian teams;
- It allows a better understanding by the European Union's participant of the working environment of their Russian counterparts
- Through the organization of public information sessions such as the one organized in Saratov, it better disseminate the project outcomes as well as other information related to the Framework programme of the European Commission, among others, the MIRRI ESFRI initiative.
- Finally, it has permitted the participation of representatives of local Russian SME to the meeting. This participation improved dramatically the understanding of the situation of these companies in this difficult economic time, focusing on the necessity to shorten the delay between upstream research outputs and downstream developments supporting innovative commercial activities.
- It has also highlighted unexpected impact of minor scientific progresses on the operation of SME. For example, change in taxonomical name of specific micro-organisms can result in costly additional administrative burden such as the obligation to renew authorization for commercialization of products because a new taxonomical name induces a new formulation of biofertilizers or biopesticides.
II. Objective 2. Building up the holdings of the microbial collections, with special care fro quality of microbiological material as well as accuracy of related data.
Activities were grouped in work packages 1 to 4:
In Work Package 1 an extensive stock review was performed to have an inventory of microbiological material in the collections of every participants and to report it according to an assessment grid ultimately structured as a Minimum Data Set (MDS). The list of strains provided by the participants has been consolidated into one database and form the core of the PERN database.
Work packages 2, 3 and 4 were executed in parallel.
Their objectives were similar:
a) To structure the holdings of rhizosphere micro-organisms and to list them in the BRIO catalogue
b) To organize the scientific expertise by establishing a group of experts concerning rhizosphere micro-organisms used as biofertilisers, biopesticides or in bioremediation.
Their focuses were complementary:
Work package 2 focuses on biofertilizers,
Work package 3 looks for biopesticides
Work package 4 aims at bioremediation
These efforts lead to a better offer of biopesticides, biofertilizers and bioremediation agents of the rhizosphere and the related data to the public or private scientific users.
WP1. EXTENSIVE STOCK REVIEW
Inventory of the partner’s holdings
The first phase focused on making an inventory of microbiological material already available in the collections of every participant and to report it according to an assessment grid. The assessment grids for holdings is a list of fields necessary but sufficient to describe unambiguously a strain, to differentiate it from any other similar strain and to give a first hint regarding its possible use in biotechnology (Bio-remediation/Bio-fertilizers/Bio-pesticides). The assessment grid corresponds to the Minimum Data Set (MDS).
To make the census, the participants have screened their holdings by means of the grid and for one or more of the following criteria: strains isolated from rhizosphere and with proven effectiveness in at least one of the three applications (Bio-remediation/Bio-fertilizers/Bio-pesticides).
The lists of strains selected by every participant during the stock review have been consolidated into one provisional database that forms the core of the PERN database. The first set listed 595 strains. This first list was presented at the first annual meeting in October 2012 in Saratov, Russia.
The inventory of microbiological material went on and was consolidated into an upgraded version of the database under coordination of partner 6 (VKM). At month 35, when made publicly accessible at www.pern-brio.eu for the first time, the catalogue comprised 770 strains with biofertilizer potential, 81 strains with biopesticide potential, and 291 strains with bioremediation potential.
Partner 2 UCL/MBLA is active in the field of microbiology applied to food processes and environments. It hosts the ‘Mycothèque of UCL’ (MUCL), member of the Belgian Co-ordinated Collections of Micro-organisms (BCCM). MUCL is the 4th largest world collection of fungi, yeasts and arbuscular mycorrhizal fungi (AMF) of agro-food and environmental interest. It also hosts the Glomeromycota in vitro Collection (GINCO), the largest world collection of AMF strictly cultured in vitro. GINCO holds hundreds of AMF and about 213 have been incorporated in the BRIO catalogue.
Partner 3 UGent hosts the BCCM/LMG bacteria collection which holds many reference strains of rhizobia and these may all be included in the BRIO catalogue. The BCCM/LMG collection is a general bacterial collection of over 30.000 cultures, belonging to more than 4000 species in more than 800 genera. With the aim of enriching the BRIO catalogue, these strains need to be verified for biofertiliser, biocontrol or bioremediation applications. Given the large numbers of strains, to document the three different applications, the work was addressed in two phases.
The first phase was at genus level: for each genus literature was searched (using Thomson Reuters Web of Science) for reports of its use as biofertiliser, in biocontrol or bioremediation applications. This way, 868 bacterial genera were verified and 198 of them were found to have one or several applications: 64 genera are used in plant growth promotion, 87 in biocontrol and 158 in bioremediation. After elimination of non-soil genera, 148 genera remained.
In the second phase species and strain level was addressed. The 148 genera documented specifically for biofertilization, biocontrol or bioremediation applications that were withheld from the first phase are represented by 8.878 strains. These need to be verified for the same three applications by literature searches. This is done first at species level and next at strain level. Positive strains are marked for inclusion in the BRIO catalogue.
The inventory of these rhizobia has been verified and a first dataset including 122 strains belonging to Ensifer and Rhizobium was sent to Participant 6 (VKM) for inclusion. Further strains of the other rhizobial genera were included latter in the project.
Partner 4, ART entered the Swiss Collection of Arbuscular mycorrhizal fungi (SAF) into the WFCC database (WDCM, World Data Centre for Microorganisms, Culture Collections World Wide) under the registered number 1032: http://www.wfcc.info/ccinfo/collection/by_id/1032. In April 2013, 202 AM fungal isolates were registered in SAF. For 100 of these isolates a 'passport' was created and has been made available for the purposes of BRIO data base on 4 November 2012. The SAF database itself went online with these first 100 isolates at the end of 2013.
Partner 5, UNITO hosts the MUT collection which holds more than 5000 strains of filamentous fungi. During the second year of the project, MUT continued to cryopreserve and/or freeze-dry the largest possible number of fungal strains kept at the MUT in active growth condition. At present 1520 strains are lyophilized and 1280 cryopreserved. Moreover, in the last year of the project 435 strains has been inserted in MUT.
A list of 106 strains has been selected for their properties as biofertiliser, biopesticides or for bioremediation purposes; all their data have been shared with the other participants to set-up a BRIO database. All these strains have been checked for their vitality and subjected to extensive systematic reviews (i.e. Cladosporium) carrying out a polyphasic re-identification by means of morpho-physiological and molecular approaches.
During the project period, the MUT collection has been amended with fungal cultures isolated from different polluted areas and from agricultural soils in Italy. Several of these strains have been identified for biofertilization, biocontrol or bioremediation applications and hence were added to the BRIO database.
Partner 6 (VKM) hosts the All-Russian collection of microorganisms (VKM) which encompasses bacteria, archaea and fungi, including yeasts (about 20000 strains integrally). Among these, more then 7000 strains are presented in VKM catalogue available at www.vkm.ru and the remaining ones represent the 'non-catalogued stock' (the strains which are waiting for their precise identification). About 100 strains selected from VKM collection and included in PERN database contains bacteria belonging to 15 genera and yeasts of 8 genera. The strains were documented or predicted to have biotechnological potential and can be used as biofertilizers, biopesticides, bioremediation or sources for development of genetically modified plant growth promoters. The strains were isolated from rhizosphere (soil), root nodules of actinorhizal plants, aerial part of plants (plant tissue and phyloplane), phyloplane, plant galls induced by nematodes on different part of plant, etc.
The long term preservation of all these strains have been checked. The strains were either verified for their identification or re-identified.
The collection of partner 7 (IEGM) has been enhanced by microbial cultures isolated from soil and rhizosphere samples taken during the common field survey on 9 September 2013 in Kungur region, Perm Krai, Russia. In total, 12 strains of hydrocarbon-oxidizing actinobacteria and 26 strains of hydrocarbon-oxidizing filamentous fungi were isolated from oily waste, oil-contaminated soil and plant rhizosphere using mineral media supplemented with hydrocarbons as the sole carbon source. Taxonomic positions of plants were determined from which rhizosphere microorganisms were isolated. The information on these new strains is included into the collection database.
The IBPPM collection (partner 8) currently maintains over than 300 strains of bacteria. A part of IBPPM-collection is not presented in the on-line catalogue. During the project the IBPPM collection has been enlarged with newly isolated rhizosphere strains having agriculture and/or ecological significance. Thirteen strains were isolated in the course of field survey in Saratov region, 5 strains were isolated in the course of field survey in Perm, and 4 strains were isolated from root zone of Cucurbita pepo and Cucumis sativus. The taxonomic position of these isolates is recognized. They were assigned to Bacillus pumilus (3 strains) and Bacillus licheniformis (1 strain). “Strain passports” for these bacteria were generated and formalized according to the provisional standards worked out in the framework of the BRIO project. All strains will be verified for their ability to be used as biofertilizers, or bioremediation agents.
Sharing and improving of protocols
All partners have provided a number of protocols relevant for isolation, preservation and study of rhizosphere microorganisms. These methods are now publicly available at http://www.pern-brio.eu/protocols/ . The protocols were divided under three sections which are:
- Sampling and isolation
- Preservation and maintenance
- Characterization and identification
Partner 2 UCL has provided two innovative protocols for the long-term preservation at ultra-low temperature of AMF and ECM. These organisms remain particularly complex to keep in culture collections and thus the development of novel procedures for the preservation at temperatures arresting metabolism is requested. This was achieved and published in a major review paper (Mycorrhiza 2014).
Partner 3 UGent has provided a protocol for the isolation of rhizobia from legume root nodules.
Partner 4 ART has provided two protocols for the isolation of AM fungal spores and for the extraction and staining of AM fungal root colonization.
Partner 5 UNITO has prepared 2 protocols for the isolation and detection of filamentous fungi potentially involved in soli bioremediation.
Partner 6 VKM has provided a protocol for isolation of of actinorhizal plant root nodule bacteria.
Partner 7 IEGM has provided a protocol of identification of ecologically significant Rhodococcus species based on free fatty acid composition. IEGM has prepared 2 protocols about the isolation of PHA-degrading and surfactant degrading microorganisms.
Partner 8 IBPPM has prepared protocols for detection and isolation of plant-growth-promoting rhizobacteria and azospirilla. Protocols using selective media for preservation and characterization of PAH-degrading and surfactant degrading microorganisms have been set up.
Partner 1 (SPP-PS) has published the protocols on the website. All these methods are now publicly available on the BRIO website. English and Russian versions are available.
Pilot Field survey
The field survey was held at the occasion of the second project meeting in Perm, Russia, 8-13 Sept. 2013. Partner 7 (IEGM) organized a common field survey on 9 September 2013 in Kungur region, Perm Krai, Russia. This field survey was aimed to visit the Priroda-Perm Company waste treatment & bioremediation site at Kokuy and to collect samples of oil-contaminated soil/waste, oil-resistant plant rhizosphere, endemic plant rhizosphere and nodule-carrying plants. Totally, 11 samples in triplicate were collected with the appropriate sampling information recorded (e.g. geographical origin, including GPS coordinates, isolation substrate, taxonomically identified plants). The samples collected were preserved at the IEGM collection in the air-dried state and intended to be transferred to all partners for isolation of specific groups of agro-environmentally relevant microorganisms (according to WP2, WP3, and WP4) to increase partners’ collection holdings. Additionally, small portions of the freshly taken samples were cryo-preserved at -84oC for future metagenomic studies. Because of the non-harmonized European-Russian postal regulations on environmental samples (in particular soil samples), timely sample transfer to European partners was not possible. Therefore, the samples were transferred within Russia to partners 6 (VKM) and 8 (IBPPM); and partner 6 (VKM) was requested to provide documents necessary for the sample release from Russia to abroad.
The partner 8 (IBPPM) collection has been enlarged with newly isolated rhizosphere strains having agriculture and/or ecological significance. Thirteen strains were isolated in the course of field survey in Saratov region, 5 strains were isolated in the course of field survey in Perm, and 4 strains were isolated from seedlings roots of Cucurbita pepo and Cucumis sativus grown under lab conditions.
Online catalogue of strains
The catalogue is available at http://www.pern-brio.eu/catalogue/ . It is effectively modulated into subsets pertinent to the main biotechnological fields of application: biofertilizers and bioremediation. It is searchable for several phenotypic and technical features.
After the lifespan of the EC subsidized part of the project, an objective quantitative indicator of the health of the network will be the number of strains incorporated regularly in the public catalogue.
WP2. BIOFERTILISERS - WP3 BIOPESTICIDES - WP4 BIOREMEDIATION
A. Organising the collections
In work package 1 all partners' holdings have been screened for strains with one or more potential use. In work packages 2, 3 and 4 the best strains are selected and added to the BRIO catalogue.
B. Identifying the experts
For each of these fields of application experts have been asked to join a virtual reference group. These experts, including Russian biotechnology companies are listed in the online expert list at http://www.pern-brio.eu/experts/
C. Enhancement of specific collections.
WP 2 BIOFERTILISERS
Selection of strains
Partner 2 (UCL) selected strains originating from temperate to tropical and Andean ecosystems (via several EU-funded projects such as VALORAM and COMMINANDES). A number of strains have been evaluated for improved nutrition and growth of agricultural important crops.
The collection of Partner 3 (UGent) holds many rhizobial strains in this category. Most of these are type strains or strains from other continents. None of them originates from Belgium. Therefore as part of this task Partner 3 (UGent) has enhanced its holdings by depositing in the public collection a number of rhizobial isolates from Belgium isolated over the past five years. A representative selection of 52 strains was chosen for inclusion in the BCCM/LMG collection and the BRIO catalogue. These strains were revived from frozen state, the purity and the identity was checked. Finally 43 viable strains with confirmed identification were passed on to BCCM/LMG for inclusion in the collection. Viability of the lyophilized cultures was performed as final control. Strains will be made available in the BRIO catalogue.
Partner 4 (ART), has another remarkable, growing collection of 214 strains of AMF. Strains originate from arable land and grassland in Middle Europe and recently also from new habitats or niches (e.g. deeper soil layers, or AMF strains with a very long life cycle). The ART AMF collection complements the collection at MUCL in that the isolates originate from Middle Europe and its strong focus on the functional diversity of AMF. Several AMF isolates have been found as promising biofertilisers.
Saprotrophic fungi: Partner 5 (UNITO), has a large collection (about 1000 strains) of fungi isolated from the rhizosphere, the rhizoplane and inside the roots of many herbaceous and woody plants. These will be further characterized for important features to be exploited as biofertilisers.
Partners UGent, VKM and IEGM focused on bacteria, the following groups of biofertilizers are represented in the partners’ collections:
Partner 6 (VKM) maintains about 30 Frankia strains (along with many other actinobacteria isolated from root nodules of actinorhyzal plants of different geographical origin).
Partner 3 (UGent) has expertise in isolation and identification and holds a rich representation of rhizobial reference strains in the BCCM/LMG bacteria collection that is hosted by UGent.
Partner 8 (IBPPM), maintains the largest Russian collection of Azospirillum, including isolates that degrade various classes of hydrocarbons.
The BRIO catalogue holds 775 strains of biofertilizer strains, composed of subsets from the different partners:
Partner 2 UCL: 213 MUCL strains
Partner 3 UGent: 325 LMG strains
Partner 4 ART: 116 ART strains
Partner 5 UNITO: 41 MUT strains
Partner 6 VKM: 41 VKM strains
Partner 8 IBPPM 39 strains
Further experiments were performed and novel strains that were received from other scientists were added to the collection of biofertilizers with: 3 novel Rhizobium strains from Bangladesh, 3 novel Bosea and Tardiphaga strains from Russia (St. Peterburg), 3 novel Azospirillum strains from India and 2 novel Azospirillum strains from Russia (Saratov).
These organisms have been checked for purity, genus identification and determination of closest relatives to establish species identity or novel species status. They will be described and deposited in the BCCM/LMG bacteria collection and be made available in the BRIO database once their characterization is completed.
Partner 7 (IEGM) has provided plant rhizosphere and nodule-carrying plant samples to partners 6 (VKM) and 8 (IBPPM) for isolation of microorganisms with potential biofertilizer properties.
IBPPM collection has been enhanced by seven type strains of Azospirillum genus (A. halopreference, A. doberanire, A. canadense, A. melinis, A. oryzae, A. palatum, and A. zeae). These strains have been received from LMG collection within the framework of strain exchange. In its turn LMG collection has been received seven rhizosphere strains including newly isolated during the field survey in Saratov region.
Taxonomic characterisation of strains of particular interest.
Partner 3 (UGent) performed a taxonomic identification of 52 Belgian rhizobial strains that had been selected for inclusion in the collection and the BRIO catalogue, with the aim of enhancing the collection with strains of European (Belgian) origin. This identification consisted of partial sequencing of the 16S rRNA gene for comparison with earlier data so as to assess the authenticity and identity of the strain.
Partner 3 also characterized and identified several other sets of strains with the objective to further enrich its collection or at the request of other partners.
1. Three novel Rhizobium strains were received from Dr. Harun-o-Rashid, Soil Microbiology Laboratory, Bangladesh Institute of Nuclear Agriculture (BINA), Mymensingh, Bangladesh.
2. Three novel Bosea and Tardiphaga strains were received from Dr. Vera Safronova, All Russian Research Institute for Agricultural Microbiology, St. Peterburg, Russia.
3. Three novel Azospirillum strains were received from Dr. Mahesh S. Dharne NCIM Resource Centre, CSIR-National Chemical Laboratory, Maharashtra, India.
4. During the final month of the project UGent received two novel Azospirillum strains from Dr. Anna Muratova, IBPPM, Saratov, Russia (Partner 8). These strains had been sent via the VKM collection in Pushchino and had gone through the lengthy administrative process to receive all the required permits and approvals. The characterization of these strains will continue in collaboration with Dr. Muratova.
Among the newly isolated microorganisms from the field survey, 13 strains manifested biofertilizer properties. All of them were capable of growing on nitrogen-free medium, and may be considered as potential nitrogen-fixers. Nine strains were capable of solubilisation of Ca3(PO4)2 demonstrating the appearance of clearing zones around the colonies grown on selective agar medium, and may be considered as Pi-solubilizers. In addition, taxonomic and functional characterizations have been supplemented for some strains with distinct biofertilizer properties
Among the plant-associated bacteria maintained in the IBPPM (Partner 8) collection, direct evidence for ability to promote plant growth has been obtained for 19 strains of the genera Alcaligenes, Azospirillum, Comamonas, Enterobacter, Moraxella, and Pseudomonas. The plant species tested were as follows: in the case of Azospirillum inoculation, Triticum aestivum (cv. Saratovskaya 29), Lolium perenne, and Sorghum bicolor; in all other cases, Sorghum saсcharatum, Sorghum technicum L., and Helianthus annus L. However, more than 40 strains can be considered to be potential biofertilisers, because they have one or several plant-growth-promoting traits.
Capacity building.
Partner 2 (UCL) has prepared a protocol for the long-term preservation at ultra-low temperature of AMF grown both in vivo and in vitro. In the same paper, a protocol for the long-term preservation at also -130 °C of ectomycorrhizal fungi (ECM) was proposed. This protocol was assayed on 100 strains and worked for more than 95% of these. These protocols for AMF and ECM are used in MUCL.
Partner 3 (UGent) has prepared a protocol for the isolation of rhizobia from legume root nodules. This protocol is now available from the project website at http://www.pern-brio.eu/protocols/
Partner 4 (ART) investigated how mycorrhizal fungi and different mycorrhizal strains influence nutrient cycling. In particular it was tested whether mycorrhizal fungi can I. enhance plant nutrient uptake and contribute to the reduction of nutrient leaching losses (e.g. induced by rain). This later aspect is important because mycorrhizal fungi could help to enhance the nutrient use efficiency in (agricultural) ecosystems, a fact that will become increasingly important as phosphorus fertilisers are expected to become depleted and nitrogen fertilisers will become more expensive. First results indicate that mycorrhizal fungi can enhance nutrient use efficiency and thus contribute to the sustainability of farming systems. However, results are soil type dependent. Furthermore, ART has isolated about 100 strains of bacteria and fungi from the rhizosphere of clover and from the soil. Currently, experiments are initiated to investigate the ecological function of these isolates (activities as biofertiliser or biopesticides and effects on plant growth). Isolates with activities as biofertilisers or biopesticides will be included in the BRIO database.
Partner 6 (VKM) prepared a protocol for the isolation of Frankia from root nodules of actinorhizal plants.
One of the bioremediation experts invited by Partner 7 (IEGM) and listed in the online expert list at http://www.brionet.eu/experts/ Prof. Irina A. Arkhipchenko from the All-Russia Research Institute for Agricultural Microbiology, has also biofertilizer specialization.
The taxonomic identification of two rhizosphere strains newly isolated during the field survey in Saratov region was performed by Partner 3 (UGent) in BCCM/LMG. Within the framework of the collaboration with BCCM/LMG collection Dr. E.V. Dubrovskaya has got training on application of MALDI-TOF mass spectrometry for bacterial identification and classification. Dr. Dubrovskaya has visited UGent from 6 till 13 April 2014. It is expected that cooperation between IBPPM (partner 8) and the BCCM/LMG collection will be continued.
Standard protocols for plant-growth-promoting rhizobacteria and for azospirilla detection and isolation using selective media, preservation and characterization have been set up in view of the common field survey. These protocols are available on the website.
Biofertilisers can be selected through the search options developed for the online catalogue accessible at the BRIO webportal http://www.pern-brio.eu/catalogue Updates are made over time, including, when available, links to metagenomic data.
WP3 BIOPESTICIDES AND DISEASES PREVENTION
Selection of strains
Partner 2 (UCL) confirms that the AMF strains documented for biofertilization also present potential as biopesticide. A limited number of strains documented in the database have been specifically and extensively documented and tested for their bioprotection effects against Rhizoctonia solani and Phytophthora infestans, two major pathogens in potato and against nematodes (Radopholus similis, Meloidogyne spp. and pratylenchus goodeyi) and phytopathogenous fungi (Cylindrocladium spathiphylli) in banana.
The laboratory of mycology is leading a national programme on the utilization of filamentous fungi, yeasts and arbuscular mycorrhizal fungi on the control of major plant pathogens. This project is first focused on potato and Phytophthora infestans. 150 fungi were tested for their biocontrol properties against fungi plant pathogens. 92 presented strong inhibition towards P. infestans. Interestingly 8 strains were particularly efficient (Chaetomium globosum, Emericella nidulans, Gliocladium catenulatum, Hypomyces rosellus, Lecanicillium dimorphum, Penicillium aurantiogriseum, Talaromyces flavus,Trichoderma Virens). 4 strains inhibit P. infestans with the fungal extract (Clavispora reshetovae, Phytophthora parasitica, Saccharomyces cerevisiae, Ustilago maydis). These strains will be included in the BRIO catalogue.
Partner 3 (UGent) has documented its strain holdings for strains used in applications in the areas of biopesticides or disease prevention. So far there are 34 strains with documented biocontrol properties that are available at the LMG collection and that have now been included in the BRIO catalogue. Of these 34 strains, two strains have anti-nematode properties, while 30 strains have antifungal properties, and two strains have antibacterial properties.
In February 2014 the SAF collection of Partner 4 (ART) went online at Agroscope homepage (http://www.agroscope.admin.ch/grandes-cultures-systemes-pastoraux/05911/07581/index.html) with 132 available isolates comprising the 21 AM fungal species. These 132 isolates entered the BRIO catalogue. One formerly undescribed AM fungal species, Glomus compressum, was published online on 3.6.2014 and could be added to the SAF collection (SAF203).
Forty-eight of the SAF isolates had been tested with six replicates per treatment for their potential to suppress three agriculturally important weeds (Echinochloa grus-galli, Solanum nigrum and Papaver rhoeas). Up to three isolates per AM fungal species were screened including 11 AMF genera and about 20 AM fungal species. 15 isolates, generated at Agroscope have been sequenced on the ribosomal gene by Oehl and co-workers in the frame of a Swiss project in April 2014. The data will be continuously added to the BRIO catalogue.
Two additional isolates from another fungal collection at Agroscope were added to the BRIO catalogue that are registered in Switzerland as biopesticides, INH Myc 8 (Beauveria brongniartii) and INH Myc 36 (Metarhizium anisopliae). These isolates are on the Swiss markets under the product names Beauveria-Schweizer and Metarhizium Schweizer (see BRIO catalogue). However, no correlation can so far be made between the biogeography of the pests (the harmful insects Melolontha melolontha and Agriotes spp.) and the two biopesticides controlling these.
A multidisciplinary group of experts at Agroscope works on rhizosphere micro-organisms that protect against soilborn diseases or which suppress aggressive agricultural weeds (i.e. act as biopesticides). Besides AM fungi, these experts use entomopathogenic fungi to suppress harmful soil insects (e.g. Melolontha melolontha and Agriotes spp.), or bacteria to supress harmful pathogenic soil fungi like Rhizoctonia solani and Alternaria alternata. A first list of experts was provided for BRIO on 18 October 2012.
Partner 5 (UNITO) selected 4 fungal strains with documented biocontrol properties that are available at the MUT collection and that are now included in the BRIO catalogue. One strains has anti-nematode properties, three strains have antifungal properties.
During the second year of activity, partner 5 (UNITO) hosted Dr Fritz Oehl from ART for a seminar and discussed with him bioremediation issues. UNITO team proposed Prof. Davide Spadaro of the Department of Agricultural, Forest and Food Science of the University of Turin to be on the list of experts for biopesticides. He has expertise mainly on mycotoxins and postharvest disease.
Partner 6 (VKM) selected more then 50 strains with documented or potential biocontrol properties that have been included in the BRIO catalogue. These encompass Streptomyces strains with high insecticide activities against spider mites, mosquito larvae and flies. The effective biocontrol activity against peach aphid and greenhouse whitefly was shown for Streptomyces sp. VKM Ac-2626 (water extract of mycelium). Some actinobacteria have antifungal activity against phytopathogenic fungi (Fusarium moniliforme, Fusarium oxysporum, Drechslera sorokiniana, Puccinia albescens). Antifungal activities was also shown for some yeast strains (Cryptococcus spp.) that produce 'micocins'. Several bacterial strains presumably assigned to a new non-pathogenic subspecies of Clavibacter michiganensis and two strains from plant pathogenic species/subspecies (Rathaibacter tritici and Clavibacter mihiganensis subsp. insidiosus) showed antagonism towards many plant pathogenic actinobacteria (genera Clavibacter, Curtobacterium, Rathayibacter) due to production of antibacterial agents ('bacteriocins'). Some above actinobacterial strains from rhizosphere and plant tissues (e.g. representatives of Clavibacter, Curtobacterium, Microbacterium) may also be biofertilizers.
Partner 7 (IEGM) has reviewed its collection holdings for actinobacterial strains with potential applications in the areas of biopesticides or disease prevention. Rhodococcus erythropolis strains with documented N-acyl homoserine lactone degrading activity will be included in the catalogue.
Partner 8 (IBPPM) has 4 strains with documented biocontrol properties and they are included in the BRIO catalogue.
WP4. BIOREMEDIATION
Selection of strains - Taxonomic and functional characterization of rhizospheric microorganisms from polluted environments
291 strains of microorganisms belonging to different genera of bacteria, yeasts, and filamentous fungi have been included in the catalogue.
Partner 5 (UNITO) carried out the taxonomical identification for 51 fungal strains and all of them have been characterized for the capability to degrade and detoxify recalcitrant organic compounds. Six fungi have been selected as potential candidates for bioremediation applications and will be further studied to understand the mechanisms involved in the processes.
Partner 7 (IEGM) has studied taxonomic and functional characteristics of isolated strains of actinobacteria, such as oxygenase activity, resistance to heavy metal salts, biodegrading activities towards individual hydrocarbons, crude oil and oil products, and other pollutants (nitrogen-containing heterocyclic compounds, phenol derivatives, etc.). Young researcher Dr. Anastasiia Krivoruchko visited Partner 3 (UGent) for MALDI-TOF characterisation of actinobacteria in November 2013. These results were used to confirm the taxonomic status of actinobacteria isolated from contaminated soils. Relevant information on strain properties useful for bioremediation was included into the BRIO catalogue.
Bioremediation properties were found among 9 newly isolated strains. Degradative activity toward crude oil (21 to 51% for 7 days) has been shown for 5 of them. One strain was able to utilize phenanthrene.
In addition, taxonomic and functional characterizations have been supplemented for some strains with distinct pollutant-degrading properties. In particular, the taxonomic position of Ensifer meliloti P221 that is PAH-degrader has been confirmed. This is the first time that the dioxygenation of phenanthrene at the C9 and C10 atoms, proven by identification of characteristic metabolites has been reported for a bacterium of the Ensifer genus.
In the course of screening studies four new strains manifesting tolerance to Cd2+ have been isolated from rhizosphere of heavy metals accumulating plants Solanum nigrum and Bidens tripartite. These strains are tolerance to 0.2-1.0 mM Cd2+.
Standard protocols for isolation of PAH-degrading rhizospheric microorganisms using selective media, for preservation and characterization have been set up.
Evaluation of the ecologically significant factors limiting the bioremediation processes
Partner 5 (UNITO) has evaluated several ecologically significant factors limiting the wastewater bioremediation processes by selected fungi with special reference to textile industry and landfill leachate ones. These trials allowed to understand that among the different parameters, the most important ones in limiting the fungal activity are: pH higher than 7 and low nutrient availability. Moreover, several experiments clearly demonstrated that the selection of appropriate carriers can highly increase the degradation and detoxification activity.
UNITO has characterized and identified several strains of fungi and bacteria with the objective to further enrich its collection or at the request of other partners. During the stay of Dr. Maria Kuyukina in December 2013, several strains of Rhodococcus belonging to the IEGM collection have been characterized for the capability to produce oxidative enzymes such as laccases and peroxidases. 29 fungal strains isolated from polluted soil and able to degrade organopollutants have been identified at species level. Moreover, 4 fungal strains isolated from Russian polluted soil and preserved at the IBPPM, Saratov where identified at species level by the Russian team that took advantages of different protocols provided by MUT.
Partner 7 (IEGM) contributed to
- The database subset on bioremediation. New characteristics for the IEGM hydrocarbon-oxidizing rhizosphere strains obtained by the MALDI-TOF MS technique were included into the database for the strains relevant to bioremediation. These actinobacteria possess unique enzymatic systems for biodegradation of mono- and polyaromatic hydrocarbons. These data can be used for selecting the appropriate cultures for hydrocarbon-contaminated soil bioremediation. Based on the data obtained, a subset of the IEGM database relevant to bioremediation was created to be included into the BRIO database. This bioremediation database subset contains the information on the IEGM strain properties useful for bioremediation, such as biodegradation profiles, biosurfactant production, heavy metal accumulation and resistance to harsh environmental conditions.
- Identification of bioremediation experts. Leading expert in petroleum-contaminated soil remediation Prof. Arkhipchenko I.A. (All-Russia Research Institute for Agricultural Microbiology, St. Petersburg, Russia), agreed to be involved. The list of experts also includes Khudokormov A.A. PhD, head of Biotechnology Center , Kuban State University (Krasnodar, Russia), Mr. Cherepanov A.V. and Ms Ageeva A.R leading specialists at Priroda-Perm Company (Perm, Russia). Over a decade they have been involved in decontamination, treatment and disposal of petroleum wastes during oil extraction, preparation, transportation, and refining. They are also involved in petroleum-contaminated soil reclamation, elimination of slurry ponds followed by their reclamation. The above experts are experienced in practical tasks, economic necessity of solutions, thus they are essential as consultants on issues such as product quality management, registration and certification of biopreparations.
The collection of Participant 8 - IBPPM includes 34 taxonomically different strains of plant-associated bacteria able to degrade various technogenic pollutants. Degradative activity toward crude oil (20 to 65% for 14 days) has been shown for 16 strains of the genera Azospirillum, Brevundimonas, and Pseudomonas. Degradative activity toward diesel oil (20 to 45% for 5 days) has been found for 8 strains of the genera Arthrobacter, Bacillus, Mycobacterium, Pseudomonas, and Rhodococcus. Degradative activity toward polycyclic aromatic hydrocarbons (anthracene, fluorene, naphthalene, and phenanthrene) has been observed for 10 strains assigned to the genera Acidovorax, Arthrobacter, Mycobacterium, Pimelobacter, Pseudomonas, and Sinorhizobium.
During the whole period of the project Partner 8 together with Scientific Production Association “Ecosphere” Ltd. conducted field surveys of oil-contaminated sites in the Saratov region. As a result of monitoring the evaluation of the ecologically significant factors limiting the bioremediation process have been done. It was found that the most significant factor limiting the bioremediation process in the Saratov region is humidity, because this region is in the arid zone. Soils are mainly chernozems and chestnut soils and are characterized by a large buffer capacity and a high number of microorganisms.
Field observations and isolation of strains degrading petroleum hydrocarbons from soils of Saratov region allow to conclude that these soils have a high potential for self-healing: the number of hydrocarbon-oxidizing microorganisms reaches 105 (in bulk soil) and 106 (in rhizosphere soil ) cells per gram. Phytoremediation or biostimulation are often preferable bioremediation approaches, excluding introduction of active biopreparations, and aimed at stimulating indigenous pollutant degrading populations. Monitoring of plant species most frequently encountered in oil-polluted areas identifies potential phytoremediation plants such as Medicago sativa, Medicago falcate, Lolium perenne, Calamagrostis epigeios, Sorghum bicolor. Analysis of prevalence of rhizospheric hydrocarbon-degrading microorganisms allowed us to identify the plant species that support the highest population of these microorganisms in their root zone: Calamagrostis epigeios, Medicago sativa, Medicago falcate, Phragmites australis, Atriplex tatarica.
Capacity building.
Partner 3 (UGent) has hosted twice Dr. Anastasiia Krivoruchko,from partner 7 (IEGM) for training in MALDI-TOF characterisation of bacteria (13/11 to 5/12/2012 and November 2013). This work contributed to the characterization and identification of rhodococci from contaminated soils.
Partner 5 (UNITO) hosted Dr. Maria Kuyukina from the IEGM team in December 8-10, 2012. During this stay, a discussion was carried out on the compatibility of the methods for isolating hydrocarbon-oxidizing bacteria and fungi. According to this discussion, protocols of hydrocarbon-oxidizing bacteria detection and isolation using selective media are currently worked on at the IEGM to be combined with the MUT protocols of hydrocarbon-oxidizing fungi isolation and detection. A combined protocol is available at the BRIO website.
Development of rhizosphere-enhanced bioremediation associations for multifunctional remediation
Partner 2 (UCL) has a number of AMF listed in the catalogue which offer special interest for bioremediation of pollutants such as hydrocarbons.
Partner 2 is also leading a national initiative on the isolation, identification and application of arbuscular mycorrhizal fungi and other filamentous fungi from the Yasuni national park in Ecuador. This park is a hotspot of diversity but is also exploited for hydrocarbons. In this project, partner 2 (UCL) is sampling polluted soils for fungi. Several species has been isolated and are in the process of identification. In a further step, these strains will be tested for bioremediation and included in the BRIO catalogue.
During the final month of the project the UNITO team (partner 5) received one fungal strain from the IEGM team, Perm, Russia. These strains had been isolated as “contaminants” from a trial devoted to identify bacteria able to degrade pharmaceutical organopollutants. The Russian isolate is a fungal consortium of two species endowed of bioremediation capabilities: Trametes versicolor and Aureobasidium pullulans. The two strains are currently under evaluation at IEGM to assess their potential in bioremediation and their possible cooperation with bacterial strains.
Partner 7 (IEGM) has developed actinobacterial associations based on IEGM collection strains suitable for oil-contaminated soil and water bioremediation. One association was tested in laboratory soil microcosms and provided an effective reduction in oil contamination under cold conditions. The association developed was recommended for multifunctional remediation (restoring all natural functions of soil) of oil-contaminated soils in cold climate regions (Urals, Russia) and is currently field tested by the Priroda-Perm company under the joint demonstration project on field-scale bioremediation. This demonstration project is aimed to evaluate the efficacy of bioremediation using a selected bacterial association, and to demonstrate its advantages to SMEs and large industrial companies dealing with the oil-contaminated soil clean-up. Another actinobacterial association was developed for oil-contaminated wastewater purification and is currently under laboratory bioreactor testing.
Partner 7 (IEGM) has also studied actinobacterial associations made of the IEGM strains able to degrade pharmaceutical pollutants, in particular codeine-containing waste, to be used in the wastewater treatment process. During laboratory trials, an air-born fungus culture was isolated from the fermentation medium, which was able to efficiently degrade codeine. This culture was sent to Partner 5 (UNITO) for taxonomic characterization and deposition in the UNITO-MUT collection. Partners 5 and 7 intend to carry out further collaboration research using selected actinobacterial and fungal cultures to develop associations useful for pharmaceutical wastewater purification and contaminated site bioremediation.
Some strains from Partner 8 (IBPPM) collection were studied on their capacity to improved phytoremediation effectiveness. Rhizosphere-enhanced plant-microbial associations for phytoremediation of contaminated soil were developed for PAH-contaminated soil, for petroleum-contaminated soil, for glyphosate-contaminated soil and for soil contaminated with Pb, Cd, Ni and As.
IBPPM collection has been enhanced by five fungal strains with ligninolytic enzymes xenobiotic-degrading activities. These strains have been isolated in Saratov region during the field survey. These strains were identified in collaboration with Dr. Cristina Varese.
Database subset of IBPPM collection used for bioremediation includes 34 oil-degrading strains, 12 PAH-degrading strains, 8 synthetic surfactant-degrading strains, 10 heavy metals resistant strains and 10 phosphonates-resistant or degrading strains.
Metagenomic data.
The database allows storage and management of metagenomic data. Yet no metagenomic data from soils are currently available to the partners, or have been analysed for biofertilizer presence or activity.
Oil-contaminated soil and rhizosphere samples taken during the common field survey in Perm were cryopreserved by Partner 7 (IEGM) for future metagenomic studies. Also, total bacterial DNA isolated from 2 fresh soil and 2 rhizosphere samples was cryopreserved and tested by direct PCR.
For each strain isolated from polluted sites and potentially useful for bioremediation, the information on contaminated substrate and geographic origin is provided.
Such as for biofertilizers, biopesticides, these strains for bioremediationcan be searched in the BRIO by using either the taxon browser or the strain properties browser.
Potential Impact:
A. IMPACT
1. Direct and future impact of the outcomes related to BRIO Objective 1: To organize cooperation between specialised collections.
BRIO builds long term links and synergies between major Russian and EU Biological Resources Centres. BRIO not only contributes to increase the synergy and to foster collaborative efforts within the group of BRIO, but also acts as an initial core around which complementary efforts crystallize and develop into further collaborative efforts. It opens new perspectives of collaboration between Russian and EU teams who will access the resources of and collaborate with the Pan-European Rhizosphere resources Network (PERN). PERN is conceived as a resources hub, according the OECD recommendations (3).
CONNECTIONS AND COLLABORATIONS
Establishing PERN enables further cooperation and exchanges at global level.
- Via the coordinator (SPP-PS) and Russian partner 6 (VKM) connections have been established with the World Data Centre for Microorganisms (WDCM) in the field of bioinformatics.
- All partners are registered in the World Federation for Culture Collections (WFCC). All partners also join the Global Catalogue of Microorganisms for connecting catalogue into one global portal.
- Via the coordination (SPP-PS), partners have incorporated the developments of the TRUST (1) initiative related to the implementation of the Nagoya Protocol in the acquisition and distribution policy (Accession & Transfer). It included the organization of an intermediary ad hoc meeting of June 2013 in Moscow were approved by the participants.
- Connection with the European Infrastructure MIRRI - Microbial Resource Research Infrastructure is ensured via the participation of BRIO partners VKM, UGent and SPP-PS to the MIRRI preparatory phase.
2. Direct and future impact of the outcomes related to BRIO Objective 2: To constitute a common pool of micro-biodiversity exploitable for research and industry: the Pan-European Rhizosphere resources Network (PERN).
WINDOW TO THE RHIZOSPHERE RESEARCH & INNOVATION COMMUNITY
BRIO participants are associated to create a pool of common rhizosphere microbial resources, related data and human expertise. They manage a one-stop shop HUB making these resources available for scientists. Putting together these material, data and expertise facilitates multidisciplinary research.
The focus is laid on biofertilisers, biopesticides and bioremediation to help scientists to cope with challenges in agriculture, forestry and environment under changing climate conditions. Facilitating the access to and the use of well documented, quality micro-organisms of the rhizosphere fosters the exploitation of renewable biological resources, a major objective of the European Knowledge Based Bio-Economy (2).
The project impacts on cumulative cutting-edge research via:
1. Support to collaborative scientific research:
- Development of long-term links between Russian and other partners beyond the project life;
- Development of collaborative synergies with industries to help develop eco-efficient products using rhizosphere micro-organisms and communities in promoting plant growth, suppressing soil borne disease and priming plant biotic defences.
2. Conservation of the natural resources:
- Underpinning cumulative research by ensuring long term preservation of the microbial components of the rhizosphere in reputed West European and Russian collections;
- Use the expertise of PERN partners for management of fungal and bacterial germplasm collections;
- Providing soil microbial micro-flora for R&D;
- Linking the consortium with industry for transfer of technology.
3. Facilitated communication and exchanges
The website is the portal to the resources gathered and organised during the project
(a) PERN ensures conservation of micro-organisms associated in the plant-microbes-soil systems, facilitating further use by scientists from academies and enterprises (including SME) in EU and Russia.
=>The website host a searchable catalogue
(b) PERN contributes to research on better understanding of rhizosphere diversity and its effect on soil fertility and suppression of pathogens. That will generate research outcomes useful for the development of eco-efficient technologies and management practices, reducing the adverse effects of human activities on the environment.
=> The website posts article relevant to the rhizosphere study.
(c) PERN shares knowledge and strengthen capacities in the area of upstream and downstream research in soil microbiology. It organises and generates resources and knowledge applicable globally. It promote exploitation of the beneficial properties of rhizosphere micro-organisms For instance, oil-polluted areas are a problem encountered in all continents, BRIO partners and thus PERN members have experience in different part of the globe, in different eco-systems, under different climatic and edaphic conditions. Combining expertise and comparing solutions applied for particular situation brings innovative approach. PERN members are scanning the possibilities for funding and partnership to further extent the impact of the newly developed tools.
=> The website proposes lists of experts in the three core field of application: bio-remediation, bio-fertilization, and bio-control.
In summary the project outcomes have been made accessible via the website. Website update and maintenance, including catalogue database optimization is ensured by Russian partner VKM and coordinator SPP-Ps with the voluntary contribution of the PERN members.
B. CONTRIBUTION TO THE EU POLICY AND OBJECTIVES IN R& I.
PERN participates in the exploration and subsequent exploitation of renewable biological resources (i.e. micro-organisms, plants and animals), contributing to the European knowledge based bio-economy (2).
Since this project is by essence cross-border and focused on microbial diversity resources it has to abide to the provisions of the Convention of Biological Diversity (CBD) and the Nagoya Protocol, contributing to the ex situ conservation of Biological Diversity and the fair and equitable sharing of benefits:
BRIO contributes to the commitments of the EU by conforming to the Convention of Biological Diversity (CBD) more particularly articles 9, 16, and 19.
Like already explained in the previous section of the report, the acquisition and distribution policy designed to be common to the PERN members design a non-discriminatory framework for the access to genetic resources, and for the fair and equitable sharing of the benefits arising out of their utilization. PERN hosts EU and Russian world-classes international culture collections which follow these rules and more particularly the recommendations of the EU funded code of conduct MOSAICC (5) and the guidelines of the TRUST(1) system.
Following the Nagoya Protocol, PERN members will implement the EU Regulation 511/2014 concerning fair and equitable sharing of benefits which can be monetary and/or preferably non-monetary and directed in such way as to promote conservation and sustainable use of microbial resources of the rhizosphere.
C. DISSEMINATION AND EXPLOITATION OF PROJECT RESULTS
Website and online catalogue.
Since a major objective of BRIO is to provide for publicly available rhizosphere resources, the project website is the central instrument to provide access to the projects results, mainly via the online catalogue of strains, and also through complementary sources of information and links.
The online catalogue is the basis instrument for exploitation of the project results since it will allow users to access well-documented technically and legally fit-for-use rhizosphere microbial resources and related information.
As part of the website, the project Intranet serves as internal communication only accessible to partners and the European Commission.
Contacts with private companies have been made, more specifically concerning existing applications in the fields of biofertilisers, biopesticides and bioremediation. Contacts are maintained via the PERN members who usually collaborate with them.
Contacts with other Russian resources centres are ongoing. The Russian members of PERN are actively promoting the necessary structuration of the Russian network of microbial collections. Depending on available funding and opportunities PERN partners will invite other research teams to joint activities as in a capacity building effort.
Other activities include participation to workshop, training sessions. It is worth noting that although not scheduled in the project life, training sessions have been already organised by taking advantages of the travel of the PERN members to BRIO meeting or to other conference.
_______________________________________________________
(1) TRUST stand for Transparent User-friendly System of Transfer see http://bccm.belspo.be/projects/trust
(2) Bio-economy is defined as that part of economic activities “which captures the latent value in biological processes and renewable bio-resources to produce improved health and sustainable growth and development”.
(3) See recommendations in document: OECD Best Practice Guidelines for Biological resource Centre, 2007, OECD, Paris. http://www.oecd.org/dataoecd/6/27/38778261.pdf
(4) Regulation (EU) No 511/2014 of the European Parliament and of the Council of 16 April 2014 on compliance measures for users from the Nagoya Protocol on Access to Genetic Resources and the Fair and Equitable Sharing of Benefits Arising from their Utilization in the Union
(5) MOSAICC stands for “Micro-organisms Sustainable use and Access regulation International Code of Conduct”. It is a voluntary Code of Conduct to support the implementation of the Convention on Biological Diversity (CBD, Rio de Janeiro, 5 June 1992) at the microbial level, in accordance with other relevant rules of international and national laws. See http://www.cbd.int/abs/instruments/ or http://bccm.belspo.be/projects/mosaicc.
List of Websites:
PROJECT WEBSITE: www.pern-brio.eu and www.brionet.eu
PARTNERS CONTACT DETAILS
Partner 1 - Federal Public Planning Service - Science Policy (SPP-PS), Belgium
Avenue Louise, 231 Louizalaan
B-1050 Brussels, Belgium
Contact: Philippe Desmeth
Tel: +32 2 238 3713
Fax: +32 2 230 5912
E-mail: philippe.desmeth@belspo.be
Partner 2 - Mycothèque de l’Université catholique de Louvain (UCL), Belgium
Mycothèque de l'Université catholique de Louvain, UCL
Croix du Sud 2, box L7.05.06
B-1348 Louvain-la-Neuve, Belgium
Contact: Prof. Dr. Stéphane Declerck
Tel.: +32 10 473742
Fax: +32 10 451501
E mail: bccm-mucl@uclouvain.be
Partner 3 - Laboratorium of Microbiology (LM-UGent), Belgium
Laboratorium voor Microbiologie, Vakgroep Biochemie en Microbiologie
Fac. Wetenschappen, Universiteit Gent
K.L. Ledeganckstraat 35
B-9000 Gent, Belgium
Contact: Prof. Dr. Anne Willems
Tel.: +32 9 2645103
Fax: +32 9 2645092
E-mail: Anne.Willems@UGent.be
Partner 4 - Agroscope Reckenholz-Tänikon, Research Station (ART), Switzerland
Ecological Farming Systems group
Swiss Federal Research Institute Agroscope
Reckenholzstrasse 191
CH 8046 Zurich, Switzerland
Contacts: Prof. Dr. Marcel van der Heijden, Dr. Fritz Oehl
Tel.: +41 44 377 7278
Fax: +41 44 377 7201
Email: marcel.vanderheijden@art.admin.ch
E-mail: fritz.oehl@art.admin.ch
Partner 5 - Dipartimento di Biologia Vegetale- Università degli Studi di Torino (UNITO), Italy
Dipartimento di Scienze della Vita e Biologia dei Sistemi
Università degli Studi di Torino
viale Mattioli, 25
10125 Torino, Italia
Contact: Prof. Dr. Giovanna Cristina Varese
Tel: +39 011 6705984
Fax: +39 011 6705962
Email: cristina.varese@unito.it
Partner 6 - Skryabin Institute of Biochemistry and Physiology of microorganisms - Russian Academy of Sciences, Russian Federation
All Russian Collection of Micro-organisms (VKM)
Prospekt Nauki, 5
142290, Pushchino, Moscow region, Russian Federation
Contact: Dr. Lyudmila Evtushenko
Tel.: +7 4967 730924
E-mail: evtushenko@ibpm.pushchino.ru
Partner 7 - Institute of Ecology and Genetics of Micro-organisms (IEGM) - Ural Branch of the Russian Academy of Sciences, Russian Federation
Laboratory of Alkanotrophic Microorganisms
Regional Specialized Collection of Alkanotrophic Micro-organisms
13, Golev Street,
614081 Perm, Russian Federation
Contact: Prof. Dr. Irena Borisovna Ivshina
Tel. +7 342 244 6714
Fax: +7 342 244 6711
E-mail: ivshina@iegm.ru
Partner 8 - Institute of Biochemistry and Physiology of Plants and Micro-organisms - Russian Academy of Sciences (IBPPM RAS), Russian Federation
Collection of Bacteria
Prospekt Entuziastov, 13
410049 Saratov City, Russian Federation
Contact: Prof. Dr. Olga Turkovskaya
Tel.: +7 8452 970786
Fax: +7 8452 970383
Email: ovtur@ibppm.sgu.ru
CONTEXT
Soil microbes play key roles in ecosystems and exert multiple beneficial as well as detrimental functions impacting yield and quality of food.
Rhizosphere and endophytic microbes play a central role in promoting plant growth and health via acquisition and recycling of nutrients, modulation of plant hormonal balance, direct or indirect protection of the plant from detrimental organisms (e.g. pathogens), protection against abiotic stress (e.g. drought, heavy metals), and improvement of soil structure.
A holistic view of the rhizosphere microbiome is necessary to increase the knowledge about all micro-life interacting with the roots and with each other. Such understanding improves the effectiveness of solutions against stress induced by biological (pests and diseases) as well as non-biological (water shortage, pollutants, etc.) factors, offering opportunities for agro-environment sustainable management.
To support the study of the rhizosphere microbiome there is a need for optimised pool of microbial material, data, information and know how easily accessible, and technically as well as legally fit for use in biotechnology. BRIO aims at providing scientists with access to such a large array of rhizosphere micro-organisms, related data and expertise.
BRIO Objectives & Realization of PERN
The objectives of BRIO are:
I. to organize cooperation between specialised collections holding beneficial micro-organisms isolated from the rhizosphere,
II. to constitute a common wide-range pool of microbial diversity exploitable for research and industry: the Pan-European Rhizosphere resources Network (PERN).
Two types of activities were performed to achieve these objectives :
• Activities aiming at optimising the structure and organisation of the network, i.e. the framework including a common policy regarding regulatory and ownership issues and the IT system. The preliminary database structure developed during the first year has been extended to other data related to possible uses of the material. Outcomes of work on common protocols, common procedures and joint data management are already visible on the website www.pern-brio.eu . In addition, network structuring and accession and distribution policies development are going on. Specific activities have been organised in common during the third year, including a field survey in Russia and specific training focused on culture collections needs. Contacts have been made with other collections in the Russian Federation, in connection with the World Data Center for Microorganisms (WDCM) and the World Federation for Culture Collection. A MoU has been signed between WDCM and the Skryabin Institute of Biochemistry and Physiology of Microorganisms, host institute of the VKM collection. The maintenance of the BRIO website is ensured by the SPP-PS BCCM webmaster. The maintenance of the database is ensured by the VKM computer scientist. Contents is updated by the PERN members.
• Activities focused on the contents of the collection, i.e. the quality of the microbiological material and related data. All partners have biological material and related data, expertise, equipment and facilities to share and to organise in common. The work consists in organizing these assets to propose an optimum range of beneficial rhizosphere microbial resources organised around three main applications: biofertilisers, biopesticides, and bioremediation. The first assessment of the existing stocks made during the 1st year provided a set of 595 strains. Partners have designed a strategy to optimize every partner's holding and to build together a set of complementary stocks merged as one virtual collection. At the end of April 2014, the online catalogue posted on the project website www.pern-brio.eu listed more than 1100 strains. These BRIO collections constitute the Pan-European Rhizosphere resources Network (PERN) core, a significant modular repository of rhizosphere microbial resources publicly accessible for research.
Project Context and Objectives:
PROJECT CONTEXT
Soil microbes play key roles in ecosystems and exert multiple functions, from detrimental (as pathogens) to beneficial (e.g. plant growth promoters and pathogen antagonists), impacting yield and quality of food. They influence a large number of important ecosystem processes, including nutrient acquisition (Smith & Read 1997; Sprent 2001), nitrogen cycling (Tiedje 1988; Kowalchuk & Stephen 2001), carbon cycling (Hogberg et al. 2001) and soil formation (Rillig & Mummey 2006). Moreover, soil microbes represent the unseen majority in soil and comprise a large portion of the genetic diversity on Earth (Whitman et al. 1998).
Nowadays, increasing attention is devoted to rhizosphere and endophytic microbes which play a central role in promoting plant growth and health via:
- acquisition and recycling of nutrients important to plant growth,
- modulation of plant hormonal balance,
- direct or indirect protection of the plant from detrimental organisms (e.g. pathogens),
- protection against abiotic stress (e.g. drought, heavy metals), and
- improvement of soil structure.
It has been stated that “the ultimate agricultural goal in studies of the biology of the soil-root interface, must be the manipulation of micro-organisms in this zone to increase plant health and growth” (Rovira, 1979). Research should aim at improving our knowledge of the interactions between plants and microbes and of sustained management of these microbes to benefit the plant-food-consumer chain. This knowledge could help to reduce excessive use of agrochemicals alleviating hazardous effects of agricultural production on the environment.
Sound environmental and agricultural practices should favour balanced composition of micro-organisms population in the rhizosphere. A well balanced microbial population favours colonization of soil and nutrients uptake, limits pressure of pathogens (biotic stress), supports the development of commensal or symbiotic relationship between plant and micro-organisms, etc, with a good tempering effect regarding abiotic stress (good water retention capacity, available macro- and micronutrients).
A holistic view of the rhizosphere microbiome is necessary to increase the knowledge about all micro-life interacting with the roots and with each other. Such understanding improves the effectiveness of solutions against stress induced by biological (pests and diseases) as well as non-biological (water shortage, pollutants, etc.) factors, offering opportunities for agro-environment sustainable management.
To support the study of the rhizosphere microbiome there is a need for optimised pool of microbial material, data, information and know how easily accessible, and technically as well as legally fit for use in biotechnology. BRIO aims at providing scientists with access to such a large array of rhizosphere micro-organisms, related data and expertise.
PROJECT OBJECTIVES
The objectives of BRIO are:
1. To establish long term links and synergies between Russian and other European Biological Resources Centres preserving microbial material isolated from plants' rhizosphere. It is expected that BRIO contributes to facilitate collaborations, to increase synergy between teams studying rhizosphere via the Pan-European Rhizosphere resources Network (PERN). The original core of PERN is made of the BRIO participants.
2. To constitute a common pool of rhizosphere micro-biodiversity exploitable by research and industry. BRIO participants have built an initial pool of rhizosphere microbial resources, related data and human expertise available via the BRIO website www.pern-brio.eu The BRIO website is a HUB making these resources available for scientists. Facilitating the preservation and use of well documented, quality micro-organisms of the rhizosphere fosters the sustainable exploitation of renewable biological resources, a major objective of the European Knowledge Based Bio-Economy.
Collaboration between culture collections having material from West-European ecosystems and Russian Biological Resources Centres having micro-organisms from East-European biotopes forms a network of collections offering a wide range of micro-organisms coming from a broad spectrum of ecological zones spread over an extensive geographical area.
Project Results:
RESULTS & ACHIEVEMENTS grouped per objective and presented by work packages (WP)
The project's activities are grouped in 2 sets of 4 work packages
I. Work Packages 5 to 8, corresponding to Objective 1, included optimization of the databases structure and linkages, the design of a common policy regarding regulatory and ownership issues, as well as external communication and dissemination, in order to consolidate the network and optimize the coordination between the partners.
II. Work Packages 1 to 4, corresponding to Objective 2, focused on the contents of the collection, i.e. the quality of the microbiological material and related data in order to optimize the preservation and characterization of the microorganisms of the rhizosphere and the related data.
I. Objective I. Structure and organisation of the network, legal framework and database system.
WP5. NETWORK STRUCTURING AND LONG TERM SUSTAINABILITY
Coordination & networking
During the project 6 major meetings were held. All Partners have taken part in discussions at the BRIO project meetings where issues of network structuring and long term sustainability were discussed. All aspects of the future development of the network were discussed during three of these meetings.
Partner 8 (IBPPM) hosted the 1st Annual meeting , Partner 7 (IEGM) hosted the 2nd annual BRIO meeting, Partner 6 (VKM) hosted the 3rd annual meeting.
Coordinating partner 1 (SPP-PS) hosted the first and the final meeting in Brussels. Video-conferences using SPP PS bridging service facility for multipoint video conferencing were held in between.
Exchange of biological material and scientific visits
For instance, Partners 7 (IEGM) and 5 (UNITO) have exchanged their collection holdings. In particular, during Prof. Maria Kuyukina’s visit to UNITO on 22 October 2013 to give the seminar “Importance of Hydrocarbon-Oxidizing Rhodococcus for Petroleum Microbiology and Bioremediation” and Practicum on morphological identification of Rhodococcus actinobacteria, six actinobacterial strains were transferred to UNITO for educational purposes. Also, 26 fungal strains isolated from oil-contaminated soil and rhizosphere samples during the common field survey in Perm, as well as 2 fungal strains isolated from codeine-containing fermentation medium were transferred to UNITO during the final BRIO meeting in Brussels, for research and educational purposes. Material Transfer Agreements (MTAs) for Non-Commercial Purposes were signed between IEGM and UNITO.
Strain exchange between IBPPM collection and MUT collection and between IBPPM collection and LMG collection was carried out. In the first case the main objective of the exchange is a collaborative research concerning revealing of ligninolytic enzymes and their xenobiotic-degrading activities. In the second case the taxonomic belonging of Azospirillum sp. strains and rhizosphere strains newly isolated during the field surveys was clarified.
In the collaboration between IBPPM Collection and LMG collection Dr. Ekaterina Dubrovskaya visited UGent and received training on application of MALDI-TOF mass-spectrometry for bacterial identification and classification.
Cooperation with private companies, such as an agreement between Priroda-Perm Company and IEGM was set up to perform development works on oil sludge remediation and to get access to clean up sites of oil-contaminated soil. The parties jointly discussed the bioremediation scheme and transfer of an oil-degrading biopreparation developed by IEGM team considering the requirements of a particular contamination.
During the 1st annual BRIO meeting in Saratov, Priroda-Perm scientists presented the company's bioremediation activities, in which the IEGM team was involved as a consultant.
Collection of strains from common field survey
A common field survey was organised as a team building effort during which several multidisciplinary activities were done: the bio-prospecting survey sensu stricto, common laboratory activities for identification and characterization, testing administrative load and organising appropriate accession and distribution policy for the network, experiencing and applying the regulatory framework relevant for the collected microbiological material, its isolation from in situ, its transfer and its conservation.
A collection of strains from the common field survey in Perm was created that included 12 actinobacterial strains and 26 fungal strains isolated from oil-contaminated soil and rhizosphere. The actinobacterial strains are maintained in IEGM, while the fungal strains are maintained in MUT (UNITO) according to collection specializations. The information on these strains is included into collection’s databases and are available in the BRIO catalogue.
Results and outcomes of the common field survey in Perm were presented by Partner 7 (IEGM) at the 2nd BRIO meeting.
Set up of PERN
The participation of all BRIO Participants to the Pan-European Rhizosphere resources Network (PERN) is effective since the 3rd annual meeting organised in February 2014 in Moscow and Pushchino.
Various instruments and activities are in place or in development such as:
a) General accession and distribution policies,
b) Common online catalogue and website;
c) Development of a network of experts related to PERN,
d) Programme of collaboration with global networking activities such as the Global Catalogue of Microorganisms,
e) Plan for extension of the network in the Russian Federation as well as with other non-European partners.
WP6. UPGRADING DATABASE STRUCTURE
To optimize ICT management and database structure the project foresaw the creation of four main databases:
A. 1 database focused on the microbiological material and being the basis of the online catalogue, and
B. 3 other databases to store information related to experts in biofertilization, biopesticides and bioremediation.
At the end of the project these 4 database have been merged into a single database system with several sub-databases.
A. During period 1, Partner 6 (VKM) ICT expert, Dr Alexander Vasilenko made a comparative study of all fields of various standards in use to record data related to (micro) biological material, from the Minimum Data Set (MDS) for the "strain passport" up to the Extended Data Set (EDS).
The part of the database dedicated to the microbiological material is developed in conjunction with other international developments, namely the CODATA Task Group "Advancing Informatics for Microbiology", the WFCC (World Federation for Culture Collections) Global Catalogue of Micro-organisms (WFCC-GCM) and TRUST, the update of the MOSAICC code of conduct concerning the data necessary to implement the administrative and legal obligations set by the Nagoya Protocol. The outcomes of these works impact on the structure of databases developed for biological material including the BRIO database.
- The VKM ICT expert is involved in CODATA Task Group "Advancing Informatics for Microbiology". The main application field of this Task Group is in the analysis of the existing standards for database structure, ontology, and semantic of database fields for the purpose of microbial data integration. - VKM is also contributing to the creation of a European Biological Resource Centres Information System (in the framework of FP7 Infrastructure project Microbial Resource Research Infrastructure - MIRRI).
- Besides Partner 6 (VKM), Partner 1 (SPP PS) is contributing to WFCC GCM. The WFCC GCM is developed and managed by the World Data Centre for Microorganisms (WDCM), the WFCC ICT centre.
- Partner 6 (VKM) and Partner 1 (SPP PS) are involved in the update of the MOSAICC project (funded in 1999 by DG Research) via the initiative called TRUST - Transparent User-friendly System of Transfer.
These complementary efforts are essential to ensure full compatibility between projects funded by DG Research.
Having initiated the discussion with regard to the composition of the data fields and adjustment of catalogue data standard to OECD Minimal Data Set (MDS) and WDCM MDS, VKM has coordinated the following phases:
- First, once the standard and composition of data fields were agreed between participants, the BRIO database was created including the following steps:
- construction of the main table,
- construction of user requests,
- posting the primary database in Extranet.
- Secondly, all participants have loaded their data into BRIO database.
For instance, Partner 5 (UNITO) has selected 106 strains from the MUT collection to include in the BRIO database: 48 strains potentially useful for biofertilizers, 4 strains with biopesticides / biocontrol properties and 68 strains for bioremediation applications. This dataset contains the taxonomic information, isolation and identification data, storage and growth conditions, and strain properties with literature references when available.
Thirdly, with all data loaded, VKM performed the following activities:
- Transformation of fragments delivered by participants to BRIO database format and feedback to participants.
- Corrections / Additions from participants.
- Preparation of participants' database replica.
The first BRIO database containing data of 595 strains was posted by VKM on Extranet.
B. Concerning the three subdatabases storing information related to experts in biofertilization, biopesticides and bioremediation, it was agreed by the participants to merge these three databases into one subdatabase by developing database fields allowing segregation between various uses and thus segregation between experts. Taking into consideration that the structure must allow multiple entries - multiple outcomes linkage, not one to one. In other words, more than one expert can have the more than one field of interest. It was also decided to link these compiled databases with the list of strains.
A. & B. The project developments showed that one robust unique integrated database divided in sub-tables is the most appropriate construction because it allows advanced searches using and combining criteria such as strains characteristics, field of expertise of the "resources people" , kind of biotechnological use, taxonomic information, etc.
Merging the four databases as sub-tables of a unique structure makes them fully interoperable and simultaneously searchable. That enables for instance the users to find the experts who may answer their enquiries about specific kind of microorganisms.
Partner 6 VKM further detailed the fields regarding the use of bio-properties (biofertilisers, biopesticides and bioremediation). Outcomes of Work package 1 on stock survey were regularly used to update the database; strains were added to the strain sub-table and other kind of sub-tables are prepared to link the strains to complementary information.
Partner 7 (IEGM) in collaboration with Partner 6 (VKM) have developed a specific "strain property" field in the BRIO catalogue in a format which consists of two terms: a verb indicating the activity and a noun (or a word-combination) indicating the subject(s) of action (e.g. degrades polyaromatics or accumulates heavy metals). So, the user can search the particular strain of interest by both terms using the strain property search form at http://www.pern-brio.eu/catalogue
Partner 8 (IBPPM)'s list of appropriate strains for the BRIO database was upgraded. This includes 51 strains possessing high biodegrading abilities towards different pollutants, and plant-growth-promoting abilities towards different plants. This dataset contains taxonomic information, isolation and identification data, storage conditions, growth conditions, and strain properties with literature references when available.
Website
All partners have contributed to the project website structure and design as major external communication media. All partners have delivered content for the website as agreed. It was decided to make the website largely bilingual (E/RU) and the Russian partners have taken on the task of translating much of the content to ensure a wider dissemination and easier use by Russian researchers.
WP7. REGULATORY FRAMEWORK AND IPR MANAGEMENT
It aims at defining common policies regarding the deposit of strains into a collection of PERN and the distribution of microbiological material of the PERN collections. These policies are translated via respectively Material Accession Agreement and Material Transfer Agreement. These basic legal reference documents serve Russian and European collections to cope with regulations and administrative requirements concerning microbiological material. All Partners have taken part in discussions where regulatory issues and issues of IPR management were discussed.Partner 6 (VKM) collected information regarding laws and regulations (sanitary rules, transport regulations, patent issues, etc.) impacting on exchanges and uses of microbial material in Russia and compared the European and Russian legal framework related to access and transfer of microbiological material was done.
In short, most of regulations and legal documents of EU and Russian Federation are similar especially with regard to international Agreements (Convention on Biological Diversity, Biological and Toxin Weapons Convention, Plants quarantine, Budapest Treaty, etc.). A major constraint is the need for translation. That is done partly in the BRIO website which presents abstracts of the legal texts in English to make the document accessible to non-Russian users.
The most significant differences between European and Russian regulations were observed in the area of biosafety. In Europe all microorganisms are divided into risk classes 1 to 4. The risk class 1 includes non-pathogenic microorganisms (for humans, animal and plants) as well as opportunistic ones. According to European rules, all the microorganisms that are not included in risk classes 2-4 belongs to risk class 1.
In the contrary, in Russia only microorganisms known to induce diseases are qualified for their epidemiological risk. Group 1 encompasses the most hazardous, while the group 4 include the least hazardous microorganisms. The microorganisms that are not assigned to the pathogenicity groups 1-4 are assumed to be safe. Considering the items in lists of microorganisms belonging to analogous groups of risk/pathogenicity one can also see significant differences between them in European and Russian documents.
Based on the analysis of the documents for protection of intellectual property in area of biotechnology in EU and Russian Federation it may be concluded that the basic rules are quite similar. There are however, some differences in details. The Directive 98/44/EC Article 13 paragraphs 2 and 3 describe the rules for access to the deposited strains of microorganisms by third parties. In Russian Federation these rules are not yet provided. In practices, VKM collection requests official permission from depositors for this issue.
The laws for transfer of microorganisms are equal for Europe and Russia but there are differences in the departmental regulations.
Exchanges of strains have occurred during the BRIO project to test the administrative burden. For instance, partners 3 (UGent), 6 (VKM) and 8 (IBPPM) have tested the Russian administrative requirements by sending strains from Russia to Europe. A first shipment of two strains was requested by Partner 3 (UGent) from VKM collection on 6 Feb. 2014 and arrived on 31 March 2014 accompanied by 16 documents, nearly all in Russian; a second shipment of 11 strains was requested on 19 March 2014 and arrived on 10 April 2014 (accompanied by 35 documents for 11 strains).
Partner 7 (IEGM) has collaborated with Partner 5 (UNITO) to exchange strains from their culture collections. Two MTAs (of 22 October 2013 and 28 April 2014) produced using a model MTA designed by Partner 1 (SPP PS) for non-commercial purposes were signed between IEGM and UNITO, according to which 12 bacterial and 28 fungal strains were transferred from IEGM collection to MUT (UNITO) for educational and research purposes.
Partner 6 VKM has also continued activities related to the development of the common access and distribution policy in the PERN network. It has organized a WFCC seminar at Pushchino (June, 2013) under the coordination of Partner 1 SPP-PS about the Global Catalogue of Microorganisms, the latest development of the Nagoya Protocol and the TRUST system, TRansparent Users-friendly System of Transfer designed to implement the Nagoya Protocol in microbiology. The other seminar held also in June 2013 but in Moscow was timed to coincide with the signing of a Memorandum of Understanding (MoU) between the World Data Center for Microorganisms (WDCM) from the World Federation for Culture Collection (WFCC) and the Skryabin Institute of Biochemistry and Physiology of Microorganisms, Russian Academy of Sciences. The MoU is aiming at facilitating further cooperation directly between the VKM and WDCM, and structuring of the Russian network of collections. Relevant issues have been discussed. VKM's experts have participated in several local meetings at the RF Ministry of Education and Science in Moscow and in subsequent e-mail discussions focused on legal access to biodiversity (concerned with the latest development of the Nagoya Protocol), establishing biological resource centres in Russia and the Russian network of microbial collections, as well as activities related to improvement Russian regulations in the area of biosafety. In addition, VKM representative Prof. Lev Kalakoutskii provided presentation concerning with legal aspects of access to genetic resources and the required infrastructure at the round table conducted by the Committee on Science and High Technology of Russian State Duma (March, 2014).
Partner 7 (IEGM) has promoted the establishment of biological resource centres (BRCs) in Russia. Prof. Irina Ivshina as a member of the expert group on developing BRCs in Russia has participated in the group meeting at the RF Ministry of Education and Science in Moscow on 28 May 2014, where results of annual inventory of current biological collections and further activities related to the formation of national BRCs were discussed.
Scientists face heavy administrative burden to exchange strains. Regardless of the user or the supplier, transfers from and to Russia are mainly centralized now by VKM.
IEGM has written the article "Current Situation and Challenges of Specialized Microbial Resource Centres in Russia" published in the journal Microbiology (2012. 81: 509−516). It considers the urgency of establishing national specialized centres based on target long-term funding and territorial location (e.g. the Far Eastern National Centre of Marine Bacteria, Siberian National Centre of Luminous Bacteria, Ural National Centre of Microbial Resources, etc.).
Furthermore, IEGM has highlighted the problems and opportunities relevant to collection-associated activities in Russia demonstrated by the example of the IEGM collection in the interview "Microbial Collections: Challenges and Opportunities" to the newspaper Science of Ural (the Ural Branch of the Russian Academy of Sciences, 2012. May, 13: 4.
IEGM has stressed the operation of the Russian specialized collections in accordance with the major goals under the Complex Program of Biotechnologies Development in RF “BIO-2020” is discussed in the article Infinite Role of Infinitely Small in the public science journal Science in Russia (2013. January-February 1(193): 9−15.
WP8 PROJECT MANAGEMENT AND DISSEMINATION
Project management, support to partners
The Participant Portal User's Guide is exhaustive but for some particular tasks it was more efficient to have a digest of the instructions dealing with one single action. Coordination has issued digests of instructions to help partners to fill in the financial statements online. During the time of the project changes occurred in the Research Participants Portal website layout and functionalities. Therefore, like during the first and second period, coordination has issued digests of instructions for the last reporting. A set of Microsoft Word® files with step by step instructions concerning the use of the Participant Portal system. The latest digests concern: Instructions for adding form C 3rd period.docx
and Adjustment to previous form C.docx
Internal Communication
Several videoconferences have been held by using the Gotomeeting™ software of Citrix® to solve specific issues and prepare the meetings.
Communication between the partners was excellent, making use of several tools:
- Email communication for written exchanges on particular topics,
- Videoconferencing for consultation on bottlenecks, focusing on sticking points
- Project Intranet providing for E-storage and sharing of files and documents
- Meetings
- The BRIO website
Website
The domain name: www.brionet.eu was the only one used during the project. Since the PERN network is set, there are now two domain names for PERN: www.brionet.eu and www.pern-brio.eu.
The BRIO website was designed and managed in house by SPP PS ICT expert. It is developed on open source software. SPP PS has written the source code to publish a searchable catalogue online, next to setting up the website itself.
The Alpha version of the website was made accessible to the participants for testing since spring 2013. The Beta version of the website was presented at month 30, six months before schedule. The website GO LIVE was made at the final BRIO meeting on 25 April 2014, on schedule.
The contents of the website is available in two languages (English and Russian) and comprises information on BRIO, its partners and funders, protocols for scientific experiments, a list of scientific experts and their fields of expertise, legal documentation, news, contact information, related links...
The website hosts the searchable BRIO Catalogue. More than 1.100 well documented microbial strain records are available online. The catalogue data can be searched via a specialised web database application developed for BRIO by VKM and SPP-PS. The catalogue can also be browsed. Therefore 'taxonomic' and 'strain properties' catalogue browsers were implemented.
The BRIO website is now converted in PERN and its content will be continuously maintained after the end of the current BRIO project.
Meetings
As foreseen during period 1 and 2, the original meeting planning has been reshuffled. Six major meetings have been organised.
Meeting 1 - BRIO Kick-off meeting - 6 and 7 June 2011, Brussels, Belgium
It included sessions corresponding to the
Content task force (CTF) Meeting on extensive stock review preparation and
Framework task force (FTF) Meeting. It aimed at organising the consortium, in complement to talks related to specific work packages. Special sessions were devoted to:
- designing a dissemination and exploitation action plan,
- setting up contingency plan for the various stumbling block identified at administrative and technical levels,
- setting up the Extranet workspace as soon as possible,
Meeting 2 - BRIO first Annual meeting - 8 to 11 October 2012, Saratov, Russian Federation
It was organised by participant IBPPM (Institute of Biochemistry and Physiology of Plants and Microorganisms). The meeting was originally planned at month 12 but postponed.
This meeting included:
• a closed workshop for the project participants only; during this day, in complement to the Work Package work summaries, the following theme were discussed :
– the extensive stock review progress originally planned at month 12, but postponed to month 18. It is the Content task force (CTF) meeting
– the plan for enhancement of individual collections
– the field survey to be organised in August/September 2013
• a closed workshop with participation of representatives of Russian private companies as scheduled as: Biofertilisers expert group meeting, Biopesticides experts group meeting, Bioremediation experts group,
Meeting 3 - BRIO intermediary ad hoc meeting - 17-21 June 2013, Moscow and Pushchino, Moscow Region, Russian Federation
This mission included three main activities:
- The signing of a Memorandum of Understanding (MoU) between the World Data Center for Microorganisms (WDCM) from the World Federation for Culture Collection (WFCC) and the Skryabin Institute of Biochemistry and Physiology of Microorganisms, Russian Academy of Sciences, Host institution of Participant 6 (VKM). The purpose of the MoU is to facilitate further cooperation directly between the VKM All Russian Culture Collection and WDCM as well as indirectly with the PERN network. It is a step towards structuring of the Russian network of collections. It is also important because it facilitates the linkages between the BRIO database and the Global Catalogue of Microorganisms.
- A work session dedicated more specifically to Regulatory framework. Comparison between Russian laws and EU laws as well as understanding the technical differences between the Russian and EU system of biosafety/biosecurity system is important.
- A WFCC Seminar at Pushchino, about the Global Catalogue of Microorganisms, the latest development of the Nagoya Protocol and the TRUST system, TRansparent Users-friendly System of Transfer designed to implement the Nagoya Protocol in microbiology. These activities are relevant for the development of the common access and distribution policy in the PERN network.
Participants to the meeting were from the coordination SPP-PS and participant VKM.
Meeting 4 - BRIO 2nd annual meeting - 9 to 13 September 2013, Perm, Russian Federation
Representatives of all participants participated to the meeting . The programme included 4 parts
A. Common field survey to collect strains in situ
This field trip and sampling for microbial strains was organised on 9 September 2013, with a visit to the Kokuy oilfield, with company “Priroda-Perm”, a visit to the waste treatment plant and of a natural park. Samples were taken from these three sites.
B. The follow up of the BRIO work package progress
The programme of the 2 days closed meeting encompassed all work packages. The sketch of the website structure was proposed by SPP PS and approved. It was decided to wait for the beta version to make more precise adjustments. Exchange of protocols for rhizosphere microorganism isolation, identification and preservation was done and a uniform protocol form to include into the website was prepared.
C. The public seminar
The public seminar was held on the 3rd day 12 September at the Perm State National Research University (PSNRU). The rector of PSNRU introduced the seminar. The BRIO coordinator made a lecture on the Relationship between the microbial culture collections and their users, as a means to improve quality and reproducibility in scientific testing. These academic talks were followed by a discussion chaired by the head of partner 7 IEGM. After a visit to the PSNRU laboratories and botanical garden, participant 7, IEGM, organised a round-table discussion concerning
- BRIO and the development of the network in Russia.
- Russian rhizosphere network connected to the WFCC Global Catalogue initiative.
D. Training course in mycology
The training course in mycology was held on September 13, at the IEGM collection laboratory and chaired by Prof. Cristina Varese, partner 5 (UNITO) and Dr Fritz Oehl, partner 4 (ART).
Meeting 5 - BRIO 3rd Annual meeting - 10 to 14 February 2014, Moscow, Russian Federation
Representatives of all participants participated to the meeting. The agenda of the meeting included 2 parts:
A. Reviewing of the work packages progresses during closed meeting and the endorsement by the participants of the several deliverables, these steps constituted the milestones:
- Regulatory framework and IPR management. Information on Russian and European laws impacting on access to and transfer of microbiological resources. Discussion and endorsement of the Common accession and distribution policy proposal
- Network structuring and long term sustainability.Future developments on long term sustainability were discussed.
- Meeting on survey outcomes. The field trip organised in September 2013 during the previous annual meeting was considered fully successful because the collected samples yielded several strains of relevant microorganisms. The administrative workflow to get these strains exported from Russia and imported in another European country was tested. All strains have not been characterized. It has been agreed to work on it after the end of the EC subsidized project phase.
- Exchange of protocols for rhizosphere microorganism isolation/identification/ preservation. Discussion on a unique format of the protocols for the website
- Parallel programming of the activities.
- Set of common fields between database of rhizosphere microbes and databases of relevant parameters (geographical distribution of diseases, soil information, target plants for microbial biofertilizers, etc.)
- Database subset of microbiological resources (for use as biofertilizers, biopesticides and bioremediation agents).
- Extensive stock review: Presentation of Catalogue.
- Round-table discussion: Proposals for the future development of the project.
B. Scientific conference
- Conference at the G.K. Skryabin Institute of Biochemistry and Physiology of Microorganisms, Russian Academy of Sciences, Pushchino, Moscow Region. Culture Collections at the crossroad of biodiversity and biotechnology activities
- Visit of VKM and other laboratories of the G.K. Skryabin Institute of Biochemistry and Physiology of Microorganisms, Pushchino, Moscow Region
- Visit to Radio Astronomy Observatory, Russian Academy of Sciences, Pushchino
Meeting 6 - Final meeting - 24 to 28 April 2014, Brussels, Belgium
Programme
Visit of BCCM/ MUCL collection and Laboratory of Microbiology.
Project outcomes overview, per Work packages
The updated catalogue was put online in March 2014 during the meeting. The BRIO website that hosts the online catalogue The central communication instrument is. Firstly access was limited to the BRIO participants, then after testing, it was made publicly accessible. BRIO website check and design of maintenance, updating and optimization programme.
The meetings were highly productive and constructive. They were absolutely necessary to get the final version of deliverables approved.
Visa for travel to Russia were not always easy to get. One should ask for a multiple entries visa open for the length of the project.
Thanks to the possibility of SPP-PS to hold the meetings in its own facilities, costs of the meetings in Brussels were limited.
Some partners have benefited from their joint participation to other international conferences to improvise work sessions. This helped to move faster, especially on the structure of the database and on the subsets of strains for biofertilization, biopesticides and bioremediation.
All participants consider that having meetings in Russia is very important for the Russian participants:
- It helps the teams based in relatively remote city of Russia to feel fully part of the project;
- It raises the profile of the Russian teams;
- It allows a better understanding by the European Union's participant of the working environment of their Russian counterparts
- Through the organization of public information sessions such as the one organized in Saratov, it better disseminate the project outcomes as well as other information related to the Framework programme of the European Commission, among others, the MIRRI ESFRI initiative.
- Finally, it has permitted the participation of representatives of local Russian SME to the meeting. This participation improved dramatically the understanding of the situation of these companies in this difficult economic time, focusing on the necessity to shorten the delay between upstream research outputs and downstream developments supporting innovative commercial activities.
- It has also highlighted unexpected impact of minor scientific progresses on the operation of SME. For example, change in taxonomical name of specific micro-organisms can result in costly additional administrative burden such as the obligation to renew authorization for commercialization of products because a new taxonomical name induces a new formulation of biofertilizers or biopesticides.
II. Objective 2. Building up the holdings of the microbial collections, with special care fro quality of microbiological material as well as accuracy of related data.
Activities were grouped in work packages 1 to 4:
In Work Package 1 an extensive stock review was performed to have an inventory of microbiological material in the collections of every participants and to report it according to an assessment grid ultimately structured as a Minimum Data Set (MDS). The list of strains provided by the participants has been consolidated into one database and form the core of the PERN database.
Work packages 2, 3 and 4 were executed in parallel.
Their objectives were similar:
a) To structure the holdings of rhizosphere micro-organisms and to list them in the BRIO catalogue
b) To organize the scientific expertise by establishing a group of experts concerning rhizosphere micro-organisms used as biofertilisers, biopesticides or in bioremediation.
Their focuses were complementary:
Work package 2 focuses on biofertilizers,
Work package 3 looks for biopesticides
Work package 4 aims at bioremediation
These efforts lead to a better offer of biopesticides, biofertilizers and bioremediation agents of the rhizosphere and the related data to the public or private scientific users.
WP1. EXTENSIVE STOCK REVIEW
Inventory of the partner’s holdings
The first phase focused on making an inventory of microbiological material already available in the collections of every participant and to report it according to an assessment grid. The assessment grids for holdings is a list of fields necessary but sufficient to describe unambiguously a strain, to differentiate it from any other similar strain and to give a first hint regarding its possible use in biotechnology (Bio-remediation/Bio-fertilizers/Bio-pesticides). The assessment grid corresponds to the Minimum Data Set (MDS).
To make the census, the participants have screened their holdings by means of the grid and for one or more of the following criteria: strains isolated from rhizosphere and with proven effectiveness in at least one of the three applications (Bio-remediation/Bio-fertilizers/Bio-pesticides).
The lists of strains selected by every participant during the stock review have been consolidated into one provisional database that forms the core of the PERN database. The first set listed 595 strains. This first list was presented at the first annual meeting in October 2012 in Saratov, Russia.
The inventory of microbiological material went on and was consolidated into an upgraded version of the database under coordination of partner 6 (VKM). At month 35, when made publicly accessible at www.pern-brio.eu for the first time, the catalogue comprised 770 strains with biofertilizer potential, 81 strains with biopesticide potential, and 291 strains with bioremediation potential.
Partner 2 UCL/MBLA is active in the field of microbiology applied to food processes and environments. It hosts the ‘Mycothèque of UCL’ (MUCL), member of the Belgian Co-ordinated Collections of Micro-organisms (BCCM). MUCL is the 4th largest world collection of fungi, yeasts and arbuscular mycorrhizal fungi (AMF) of agro-food and environmental interest. It also hosts the Glomeromycota in vitro Collection (GINCO), the largest world collection of AMF strictly cultured in vitro. GINCO holds hundreds of AMF and about 213 have been incorporated in the BRIO catalogue.
Partner 3 UGent hosts the BCCM/LMG bacteria collection which holds many reference strains of rhizobia and these may all be included in the BRIO catalogue. The BCCM/LMG collection is a general bacterial collection of over 30.000 cultures, belonging to more than 4000 species in more than 800 genera. With the aim of enriching the BRIO catalogue, these strains need to be verified for biofertiliser, biocontrol or bioremediation applications. Given the large numbers of strains, to document the three different applications, the work was addressed in two phases.
The first phase was at genus level: for each genus literature was searched (using Thomson Reuters Web of Science) for reports of its use as biofertiliser, in biocontrol or bioremediation applications. This way, 868 bacterial genera were verified and 198 of them were found to have one or several applications: 64 genera are used in plant growth promotion, 87 in biocontrol and 158 in bioremediation. After elimination of non-soil genera, 148 genera remained.
In the second phase species and strain level was addressed. The 148 genera documented specifically for biofertilization, biocontrol or bioremediation applications that were withheld from the first phase are represented by 8.878 strains. These need to be verified for the same three applications by literature searches. This is done first at species level and next at strain level. Positive strains are marked for inclusion in the BRIO catalogue.
The inventory of these rhizobia has been verified and a first dataset including 122 strains belonging to Ensifer and Rhizobium was sent to Participant 6 (VKM) for inclusion. Further strains of the other rhizobial genera were included latter in the project.
Partner 4, ART entered the Swiss Collection of Arbuscular mycorrhizal fungi (SAF) into the WFCC database (WDCM, World Data Centre for Microorganisms, Culture Collections World Wide) under the registered number 1032: http://www.wfcc.info/ccinfo/collection/by_id/1032. In April 2013, 202 AM fungal isolates were registered in SAF. For 100 of these isolates a 'passport' was created and has been made available for the purposes of BRIO data base on 4 November 2012. The SAF database itself went online with these first 100 isolates at the end of 2013.
Partner 5, UNITO hosts the MUT collection which holds more than 5000 strains of filamentous fungi. During the second year of the project, MUT continued to cryopreserve and/or freeze-dry the largest possible number of fungal strains kept at the MUT in active growth condition. At present 1520 strains are lyophilized and 1280 cryopreserved. Moreover, in the last year of the project 435 strains has been inserted in MUT.
A list of 106 strains has been selected for their properties as biofertiliser, biopesticides or for bioremediation purposes; all their data have been shared with the other participants to set-up a BRIO database. All these strains have been checked for their vitality and subjected to extensive systematic reviews (i.e. Cladosporium) carrying out a polyphasic re-identification by means of morpho-physiological and molecular approaches.
During the project period, the MUT collection has been amended with fungal cultures isolated from different polluted areas and from agricultural soils in Italy. Several of these strains have been identified for biofertilization, biocontrol or bioremediation applications and hence were added to the BRIO database.
Partner 6 (VKM) hosts the All-Russian collection of microorganisms (VKM) which encompasses bacteria, archaea and fungi, including yeasts (about 20000 strains integrally). Among these, more then 7000 strains are presented in VKM catalogue available at www.vkm.ru and the remaining ones represent the 'non-catalogued stock' (the strains which are waiting for their precise identification). About 100 strains selected from VKM collection and included in PERN database contains bacteria belonging to 15 genera and yeasts of 8 genera. The strains were documented or predicted to have biotechnological potential and can be used as biofertilizers, biopesticides, bioremediation or sources for development of genetically modified plant growth promoters. The strains were isolated from rhizosphere (soil), root nodules of actinorhizal plants, aerial part of plants (plant tissue and phyloplane), phyloplane, plant galls induced by nematodes on different part of plant, etc.
The long term preservation of all these strains have been checked. The strains were either verified for their identification or re-identified.
The collection of partner 7 (IEGM) has been enhanced by microbial cultures isolated from soil and rhizosphere samples taken during the common field survey on 9 September 2013 in Kungur region, Perm Krai, Russia. In total, 12 strains of hydrocarbon-oxidizing actinobacteria and 26 strains of hydrocarbon-oxidizing filamentous fungi were isolated from oily waste, oil-contaminated soil and plant rhizosphere using mineral media supplemented with hydrocarbons as the sole carbon source. Taxonomic positions of plants were determined from which rhizosphere microorganisms were isolated. The information on these new strains is included into the collection database.
The IBPPM collection (partner 8) currently maintains over than 300 strains of bacteria. A part of IBPPM-collection is not presented in the on-line catalogue. During the project the IBPPM collection has been enlarged with newly isolated rhizosphere strains having agriculture and/or ecological significance. Thirteen strains were isolated in the course of field survey in Saratov region, 5 strains were isolated in the course of field survey in Perm, and 4 strains were isolated from root zone of Cucurbita pepo and Cucumis sativus. The taxonomic position of these isolates is recognized. They were assigned to Bacillus pumilus (3 strains) and Bacillus licheniformis (1 strain). “Strain passports” for these bacteria were generated and formalized according to the provisional standards worked out in the framework of the BRIO project. All strains will be verified for their ability to be used as biofertilizers, or bioremediation agents.
Sharing and improving of protocols
All partners have provided a number of protocols relevant for isolation, preservation and study of rhizosphere microorganisms. These methods are now publicly available at http://www.pern-brio.eu/protocols/ . The protocols were divided under three sections which are:
- Sampling and isolation
- Preservation and maintenance
- Characterization and identification
Partner 2 UCL has provided two innovative protocols for the long-term preservation at ultra-low temperature of AMF and ECM. These organisms remain particularly complex to keep in culture collections and thus the development of novel procedures for the preservation at temperatures arresting metabolism is requested. This was achieved and published in a major review paper (Mycorrhiza 2014).
Partner 3 UGent has provided a protocol for the isolation of rhizobia from legume root nodules.
Partner 4 ART has provided two protocols for the isolation of AM fungal spores and for the extraction and staining of AM fungal root colonization.
Partner 5 UNITO has prepared 2 protocols for the isolation and detection of filamentous fungi potentially involved in soli bioremediation.
Partner 6 VKM has provided a protocol for isolation of of actinorhizal plant root nodule bacteria.
Partner 7 IEGM has provided a protocol of identification of ecologically significant Rhodococcus species based on free fatty acid composition. IEGM has prepared 2 protocols about the isolation of PHA-degrading and surfactant degrading microorganisms.
Partner 8 IBPPM has prepared protocols for detection and isolation of plant-growth-promoting rhizobacteria and azospirilla. Protocols using selective media for preservation and characterization of PAH-degrading and surfactant degrading microorganisms have been set up.
Partner 1 (SPP-PS) has published the protocols on the website. All these methods are now publicly available on the BRIO website. English and Russian versions are available.
Pilot Field survey
The field survey was held at the occasion of the second project meeting in Perm, Russia, 8-13 Sept. 2013. Partner 7 (IEGM) organized a common field survey on 9 September 2013 in Kungur region, Perm Krai, Russia. This field survey was aimed to visit the Priroda-Perm Company waste treatment & bioremediation site at Kokuy and to collect samples of oil-contaminated soil/waste, oil-resistant plant rhizosphere, endemic plant rhizosphere and nodule-carrying plants. Totally, 11 samples in triplicate were collected with the appropriate sampling information recorded (e.g. geographical origin, including GPS coordinates, isolation substrate, taxonomically identified plants). The samples collected were preserved at the IEGM collection in the air-dried state and intended to be transferred to all partners for isolation of specific groups of agro-environmentally relevant microorganisms (according to WP2, WP3, and WP4) to increase partners’ collection holdings. Additionally, small portions of the freshly taken samples were cryo-preserved at -84oC for future metagenomic studies. Because of the non-harmonized European-Russian postal regulations on environmental samples (in particular soil samples), timely sample transfer to European partners was not possible. Therefore, the samples were transferred within Russia to partners 6 (VKM) and 8 (IBPPM); and partner 6 (VKM) was requested to provide documents necessary for the sample release from Russia to abroad.
The partner 8 (IBPPM) collection has been enlarged with newly isolated rhizosphere strains having agriculture and/or ecological significance. Thirteen strains were isolated in the course of field survey in Saratov region, 5 strains were isolated in the course of field survey in Perm, and 4 strains were isolated from seedlings roots of Cucurbita pepo and Cucumis sativus grown under lab conditions.
Online catalogue of strains
The catalogue is available at http://www.pern-brio.eu/catalogue/ . It is effectively modulated into subsets pertinent to the main biotechnological fields of application: biofertilizers and bioremediation. It is searchable for several phenotypic and technical features.
After the lifespan of the EC subsidized part of the project, an objective quantitative indicator of the health of the network will be the number of strains incorporated regularly in the public catalogue.
WP2. BIOFERTILISERS - WP3 BIOPESTICIDES - WP4 BIOREMEDIATION
A. Organising the collections
In work package 1 all partners' holdings have been screened for strains with one or more potential use. In work packages 2, 3 and 4 the best strains are selected and added to the BRIO catalogue.
B. Identifying the experts
For each of these fields of application experts have been asked to join a virtual reference group. These experts, including Russian biotechnology companies are listed in the online expert list at http://www.pern-brio.eu/experts/
C. Enhancement of specific collections.
WP 2 BIOFERTILISERS
Selection of strains
Partner 2 (UCL) selected strains originating from temperate to tropical and Andean ecosystems (via several EU-funded projects such as VALORAM and COMMINANDES). A number of strains have been evaluated for improved nutrition and growth of agricultural important crops.
The collection of Partner 3 (UGent) holds many rhizobial strains in this category. Most of these are type strains or strains from other continents. None of them originates from Belgium. Therefore as part of this task Partner 3 (UGent) has enhanced its holdings by depositing in the public collection a number of rhizobial isolates from Belgium isolated over the past five years. A representative selection of 52 strains was chosen for inclusion in the BCCM/LMG collection and the BRIO catalogue. These strains were revived from frozen state, the purity and the identity was checked. Finally 43 viable strains with confirmed identification were passed on to BCCM/LMG for inclusion in the collection. Viability of the lyophilized cultures was performed as final control. Strains will be made available in the BRIO catalogue.
Partner 4 (ART), has another remarkable, growing collection of 214 strains of AMF. Strains originate from arable land and grassland in Middle Europe and recently also from new habitats or niches (e.g. deeper soil layers, or AMF strains with a very long life cycle). The ART AMF collection complements the collection at MUCL in that the isolates originate from Middle Europe and its strong focus on the functional diversity of AMF. Several AMF isolates have been found as promising biofertilisers.
Saprotrophic fungi: Partner 5 (UNITO), has a large collection (about 1000 strains) of fungi isolated from the rhizosphere, the rhizoplane and inside the roots of many herbaceous and woody plants. These will be further characterized for important features to be exploited as biofertilisers.
Partners UGent, VKM and IEGM focused on bacteria, the following groups of biofertilizers are represented in the partners’ collections:
Partner 6 (VKM) maintains about 30 Frankia strains (along with many other actinobacteria isolated from root nodules of actinorhyzal plants of different geographical origin).
Partner 3 (UGent) has expertise in isolation and identification and holds a rich representation of rhizobial reference strains in the BCCM/LMG bacteria collection that is hosted by UGent.
Partner 8 (IBPPM), maintains the largest Russian collection of Azospirillum, including isolates that degrade various classes of hydrocarbons.
The BRIO catalogue holds 775 strains of biofertilizer strains, composed of subsets from the different partners:
Partner 2 UCL: 213 MUCL strains
Partner 3 UGent: 325 LMG strains
Partner 4 ART: 116 ART strains
Partner 5 UNITO: 41 MUT strains
Partner 6 VKM: 41 VKM strains
Partner 8 IBPPM 39 strains
Further experiments were performed and novel strains that were received from other scientists were added to the collection of biofertilizers with: 3 novel Rhizobium strains from Bangladesh, 3 novel Bosea and Tardiphaga strains from Russia (St. Peterburg), 3 novel Azospirillum strains from India and 2 novel Azospirillum strains from Russia (Saratov).
These organisms have been checked for purity, genus identification and determination of closest relatives to establish species identity or novel species status. They will be described and deposited in the BCCM/LMG bacteria collection and be made available in the BRIO database once their characterization is completed.
Partner 7 (IEGM) has provided plant rhizosphere and nodule-carrying plant samples to partners 6 (VKM) and 8 (IBPPM) for isolation of microorganisms with potential biofertilizer properties.
IBPPM collection has been enhanced by seven type strains of Azospirillum genus (A. halopreference, A. doberanire, A. canadense, A. melinis, A. oryzae, A. palatum, and A. zeae). These strains have been received from LMG collection within the framework of strain exchange. In its turn LMG collection has been received seven rhizosphere strains including newly isolated during the field survey in Saratov region.
Taxonomic characterisation of strains of particular interest.
Partner 3 (UGent) performed a taxonomic identification of 52 Belgian rhizobial strains that had been selected for inclusion in the collection and the BRIO catalogue, with the aim of enhancing the collection with strains of European (Belgian) origin. This identification consisted of partial sequencing of the 16S rRNA gene for comparison with earlier data so as to assess the authenticity and identity of the strain.
Partner 3 also characterized and identified several other sets of strains with the objective to further enrich its collection or at the request of other partners.
1. Three novel Rhizobium strains were received from Dr. Harun-o-Rashid, Soil Microbiology Laboratory, Bangladesh Institute of Nuclear Agriculture (BINA), Mymensingh, Bangladesh.
2. Three novel Bosea and Tardiphaga strains were received from Dr. Vera Safronova, All Russian Research Institute for Agricultural Microbiology, St. Peterburg, Russia.
3. Three novel Azospirillum strains were received from Dr. Mahesh S. Dharne NCIM Resource Centre, CSIR-National Chemical Laboratory, Maharashtra, India.
4. During the final month of the project UGent received two novel Azospirillum strains from Dr. Anna Muratova, IBPPM, Saratov, Russia (Partner 8). These strains had been sent via the VKM collection in Pushchino and had gone through the lengthy administrative process to receive all the required permits and approvals. The characterization of these strains will continue in collaboration with Dr. Muratova.
Among the newly isolated microorganisms from the field survey, 13 strains manifested biofertilizer properties. All of them were capable of growing on nitrogen-free medium, and may be considered as potential nitrogen-fixers. Nine strains were capable of solubilisation of Ca3(PO4)2 demonstrating the appearance of clearing zones around the colonies grown on selective agar medium, and may be considered as Pi-solubilizers. In addition, taxonomic and functional characterizations have been supplemented for some strains with distinct biofertilizer properties
Among the plant-associated bacteria maintained in the IBPPM (Partner 8) collection, direct evidence for ability to promote plant growth has been obtained for 19 strains of the genera Alcaligenes, Azospirillum, Comamonas, Enterobacter, Moraxella, and Pseudomonas. The plant species tested were as follows: in the case of Azospirillum inoculation, Triticum aestivum (cv. Saratovskaya 29), Lolium perenne, and Sorghum bicolor; in all other cases, Sorghum saсcharatum, Sorghum technicum L., and Helianthus annus L. However, more than 40 strains can be considered to be potential biofertilisers, because they have one or several plant-growth-promoting traits.
Capacity building.
Partner 2 (UCL) has prepared a protocol for the long-term preservation at ultra-low temperature of AMF grown both in vivo and in vitro. In the same paper, a protocol for the long-term preservation at also -130 °C of ectomycorrhizal fungi (ECM) was proposed. This protocol was assayed on 100 strains and worked for more than 95% of these. These protocols for AMF and ECM are used in MUCL.
Partner 3 (UGent) has prepared a protocol for the isolation of rhizobia from legume root nodules. This protocol is now available from the project website at http://www.pern-brio.eu/protocols/
Partner 4 (ART) investigated how mycorrhizal fungi and different mycorrhizal strains influence nutrient cycling. In particular it was tested whether mycorrhizal fungi can I. enhance plant nutrient uptake and contribute to the reduction of nutrient leaching losses (e.g. induced by rain). This later aspect is important because mycorrhizal fungi could help to enhance the nutrient use efficiency in (agricultural) ecosystems, a fact that will become increasingly important as phosphorus fertilisers are expected to become depleted and nitrogen fertilisers will become more expensive. First results indicate that mycorrhizal fungi can enhance nutrient use efficiency and thus contribute to the sustainability of farming systems. However, results are soil type dependent. Furthermore, ART has isolated about 100 strains of bacteria and fungi from the rhizosphere of clover and from the soil. Currently, experiments are initiated to investigate the ecological function of these isolates (activities as biofertiliser or biopesticides and effects on plant growth). Isolates with activities as biofertilisers or biopesticides will be included in the BRIO database.
Partner 6 (VKM) prepared a protocol for the isolation of Frankia from root nodules of actinorhizal plants.
One of the bioremediation experts invited by Partner 7 (IEGM) and listed in the online expert list at http://www.brionet.eu/experts/ Prof. Irina A. Arkhipchenko from the All-Russia Research Institute for Agricultural Microbiology, has also biofertilizer specialization.
The taxonomic identification of two rhizosphere strains newly isolated during the field survey in Saratov region was performed by Partner 3 (UGent) in BCCM/LMG. Within the framework of the collaboration with BCCM/LMG collection Dr. E.V. Dubrovskaya has got training on application of MALDI-TOF mass spectrometry for bacterial identification and classification. Dr. Dubrovskaya has visited UGent from 6 till 13 April 2014. It is expected that cooperation between IBPPM (partner 8) and the BCCM/LMG collection will be continued.
Standard protocols for plant-growth-promoting rhizobacteria and for azospirilla detection and isolation using selective media, preservation and characterization have been set up in view of the common field survey. These protocols are available on the website.
Biofertilisers can be selected through the search options developed for the online catalogue accessible at the BRIO webportal http://www.pern-brio.eu/catalogue Updates are made over time, including, when available, links to metagenomic data.
WP3 BIOPESTICIDES AND DISEASES PREVENTION
Selection of strains
Partner 2 (UCL) confirms that the AMF strains documented for biofertilization also present potential as biopesticide. A limited number of strains documented in the database have been specifically and extensively documented and tested for their bioprotection effects against Rhizoctonia solani and Phytophthora infestans, two major pathogens in potato and against nematodes (Radopholus similis, Meloidogyne spp. and pratylenchus goodeyi) and phytopathogenous fungi (Cylindrocladium spathiphylli) in banana.
The laboratory of mycology is leading a national programme on the utilization of filamentous fungi, yeasts and arbuscular mycorrhizal fungi on the control of major plant pathogens. This project is first focused on potato and Phytophthora infestans. 150 fungi were tested for their biocontrol properties against fungi plant pathogens. 92 presented strong inhibition towards P. infestans. Interestingly 8 strains were particularly efficient (Chaetomium globosum, Emericella nidulans, Gliocladium catenulatum, Hypomyces rosellus, Lecanicillium dimorphum, Penicillium aurantiogriseum, Talaromyces flavus,Trichoderma Virens). 4 strains inhibit P. infestans with the fungal extract (Clavispora reshetovae, Phytophthora parasitica, Saccharomyces cerevisiae, Ustilago maydis). These strains will be included in the BRIO catalogue.
Partner 3 (UGent) has documented its strain holdings for strains used in applications in the areas of biopesticides or disease prevention. So far there are 34 strains with documented biocontrol properties that are available at the LMG collection and that have now been included in the BRIO catalogue. Of these 34 strains, two strains have anti-nematode properties, while 30 strains have antifungal properties, and two strains have antibacterial properties.
In February 2014 the SAF collection of Partner 4 (ART) went online at Agroscope homepage (http://www.agroscope.admin.ch/grandes-cultures-systemes-pastoraux/05911/07581/index.html) with 132 available isolates comprising the 21 AM fungal species. These 132 isolates entered the BRIO catalogue. One formerly undescribed AM fungal species, Glomus compressum, was published online on 3.6.2014 and could be added to the SAF collection (SAF203).
Forty-eight of the SAF isolates had been tested with six replicates per treatment for their potential to suppress three agriculturally important weeds (Echinochloa grus-galli, Solanum nigrum and Papaver rhoeas). Up to three isolates per AM fungal species were screened including 11 AMF genera and about 20 AM fungal species. 15 isolates, generated at Agroscope have been sequenced on the ribosomal gene by Oehl and co-workers in the frame of a Swiss project in April 2014. The data will be continuously added to the BRIO catalogue.
Two additional isolates from another fungal collection at Agroscope were added to the BRIO catalogue that are registered in Switzerland as biopesticides, INH Myc 8 (Beauveria brongniartii) and INH Myc 36 (Metarhizium anisopliae). These isolates are on the Swiss markets under the product names Beauveria-Schweizer and Metarhizium Schweizer (see BRIO catalogue). However, no correlation can so far be made between the biogeography of the pests (the harmful insects Melolontha melolontha and Agriotes spp.) and the two biopesticides controlling these.
A multidisciplinary group of experts at Agroscope works on rhizosphere micro-organisms that protect against soilborn diseases or which suppress aggressive agricultural weeds (i.e. act as biopesticides). Besides AM fungi, these experts use entomopathogenic fungi to suppress harmful soil insects (e.g. Melolontha melolontha and Agriotes spp.), or bacteria to supress harmful pathogenic soil fungi like Rhizoctonia solani and Alternaria alternata. A first list of experts was provided for BRIO on 18 October 2012.
Partner 5 (UNITO) selected 4 fungal strains with documented biocontrol properties that are available at the MUT collection and that are now included in the BRIO catalogue. One strains has anti-nematode properties, three strains have antifungal properties.
During the second year of activity, partner 5 (UNITO) hosted Dr Fritz Oehl from ART for a seminar and discussed with him bioremediation issues. UNITO team proposed Prof. Davide Spadaro of the Department of Agricultural, Forest and Food Science of the University of Turin to be on the list of experts for biopesticides. He has expertise mainly on mycotoxins and postharvest disease.
Partner 6 (VKM) selected more then 50 strains with documented or potential biocontrol properties that have been included in the BRIO catalogue. These encompass Streptomyces strains with high insecticide activities against spider mites, mosquito larvae and flies. The effective biocontrol activity against peach aphid and greenhouse whitefly was shown for Streptomyces sp. VKM Ac-2626 (water extract of mycelium). Some actinobacteria have antifungal activity against phytopathogenic fungi (Fusarium moniliforme, Fusarium oxysporum, Drechslera sorokiniana, Puccinia albescens). Antifungal activities was also shown for some yeast strains (Cryptococcus spp.) that produce 'micocins'. Several bacterial strains presumably assigned to a new non-pathogenic subspecies of Clavibacter michiganensis and two strains from plant pathogenic species/subspecies (Rathaibacter tritici and Clavibacter mihiganensis subsp. insidiosus) showed antagonism towards many plant pathogenic actinobacteria (genera Clavibacter, Curtobacterium, Rathayibacter) due to production of antibacterial agents ('bacteriocins'). Some above actinobacterial strains from rhizosphere and plant tissues (e.g. representatives of Clavibacter, Curtobacterium, Microbacterium) may also be biofertilizers.
Partner 7 (IEGM) has reviewed its collection holdings for actinobacterial strains with potential applications in the areas of biopesticides or disease prevention. Rhodococcus erythropolis strains with documented N-acyl homoserine lactone degrading activity will be included in the catalogue.
Partner 8 (IBPPM) has 4 strains with documented biocontrol properties and they are included in the BRIO catalogue.
WP4. BIOREMEDIATION
Selection of strains - Taxonomic and functional characterization of rhizospheric microorganisms from polluted environments
291 strains of microorganisms belonging to different genera of bacteria, yeasts, and filamentous fungi have been included in the catalogue.
Partner 5 (UNITO) carried out the taxonomical identification for 51 fungal strains and all of them have been characterized for the capability to degrade and detoxify recalcitrant organic compounds. Six fungi have been selected as potential candidates for bioremediation applications and will be further studied to understand the mechanisms involved in the processes.
Partner 7 (IEGM) has studied taxonomic and functional characteristics of isolated strains of actinobacteria, such as oxygenase activity, resistance to heavy metal salts, biodegrading activities towards individual hydrocarbons, crude oil and oil products, and other pollutants (nitrogen-containing heterocyclic compounds, phenol derivatives, etc.). Young researcher Dr. Anastasiia Krivoruchko visited Partner 3 (UGent) for MALDI-TOF characterisation of actinobacteria in November 2013. These results were used to confirm the taxonomic status of actinobacteria isolated from contaminated soils. Relevant information on strain properties useful for bioremediation was included into the BRIO catalogue.
Bioremediation properties were found among 9 newly isolated strains. Degradative activity toward crude oil (21 to 51% for 7 days) has been shown for 5 of them. One strain was able to utilize phenanthrene.
In addition, taxonomic and functional characterizations have been supplemented for some strains with distinct pollutant-degrading properties. In particular, the taxonomic position of Ensifer meliloti P221 that is PAH-degrader has been confirmed. This is the first time that the dioxygenation of phenanthrene at the C9 and C10 atoms, proven by identification of characteristic metabolites has been reported for a bacterium of the Ensifer genus.
In the course of screening studies four new strains manifesting tolerance to Cd2+ have been isolated from rhizosphere of heavy metals accumulating plants Solanum nigrum and Bidens tripartite. These strains are tolerance to 0.2-1.0 mM Cd2+.
Standard protocols for isolation of PAH-degrading rhizospheric microorganisms using selective media, for preservation and characterization have been set up.
Evaluation of the ecologically significant factors limiting the bioremediation processes
Partner 5 (UNITO) has evaluated several ecologically significant factors limiting the wastewater bioremediation processes by selected fungi with special reference to textile industry and landfill leachate ones. These trials allowed to understand that among the different parameters, the most important ones in limiting the fungal activity are: pH higher than 7 and low nutrient availability. Moreover, several experiments clearly demonstrated that the selection of appropriate carriers can highly increase the degradation and detoxification activity.
UNITO has characterized and identified several strains of fungi and bacteria with the objective to further enrich its collection or at the request of other partners. During the stay of Dr. Maria Kuyukina in December 2013, several strains of Rhodococcus belonging to the IEGM collection have been characterized for the capability to produce oxidative enzymes such as laccases and peroxidases. 29 fungal strains isolated from polluted soil and able to degrade organopollutants have been identified at species level. Moreover, 4 fungal strains isolated from Russian polluted soil and preserved at the IBPPM, Saratov where identified at species level by the Russian team that took advantages of different protocols provided by MUT.
Partner 7 (IEGM) contributed to
- The database subset on bioremediation. New characteristics for the IEGM hydrocarbon-oxidizing rhizosphere strains obtained by the MALDI-TOF MS technique were included into the database for the strains relevant to bioremediation. These actinobacteria possess unique enzymatic systems for biodegradation of mono- and polyaromatic hydrocarbons. These data can be used for selecting the appropriate cultures for hydrocarbon-contaminated soil bioremediation. Based on the data obtained, a subset of the IEGM database relevant to bioremediation was created to be included into the BRIO database. This bioremediation database subset contains the information on the IEGM strain properties useful for bioremediation, such as biodegradation profiles, biosurfactant production, heavy metal accumulation and resistance to harsh environmental conditions.
- Identification of bioremediation experts. Leading expert in petroleum-contaminated soil remediation Prof. Arkhipchenko I.A. (All-Russia Research Institute for Agricultural Microbiology, St. Petersburg, Russia), agreed to be involved. The list of experts also includes Khudokormov A.A. PhD, head of Biotechnology Center , Kuban State University (Krasnodar, Russia), Mr. Cherepanov A.V. and Ms Ageeva A.R leading specialists at Priroda-Perm Company (Perm, Russia). Over a decade they have been involved in decontamination, treatment and disposal of petroleum wastes during oil extraction, preparation, transportation, and refining. They are also involved in petroleum-contaminated soil reclamation, elimination of slurry ponds followed by their reclamation. The above experts are experienced in practical tasks, economic necessity of solutions, thus they are essential as consultants on issues such as product quality management, registration and certification of biopreparations.
The collection of Participant 8 - IBPPM includes 34 taxonomically different strains of plant-associated bacteria able to degrade various technogenic pollutants. Degradative activity toward crude oil (20 to 65% for 14 days) has been shown for 16 strains of the genera Azospirillum, Brevundimonas, and Pseudomonas. Degradative activity toward diesel oil (20 to 45% for 5 days) has been found for 8 strains of the genera Arthrobacter, Bacillus, Mycobacterium, Pseudomonas, and Rhodococcus. Degradative activity toward polycyclic aromatic hydrocarbons (anthracene, fluorene, naphthalene, and phenanthrene) has been observed for 10 strains assigned to the genera Acidovorax, Arthrobacter, Mycobacterium, Pimelobacter, Pseudomonas, and Sinorhizobium.
During the whole period of the project Partner 8 together with Scientific Production Association “Ecosphere” Ltd. conducted field surveys of oil-contaminated sites in the Saratov region. As a result of monitoring the evaluation of the ecologically significant factors limiting the bioremediation process have been done. It was found that the most significant factor limiting the bioremediation process in the Saratov region is humidity, because this region is in the arid zone. Soils are mainly chernozems and chestnut soils and are characterized by a large buffer capacity and a high number of microorganisms.
Field observations and isolation of strains degrading petroleum hydrocarbons from soils of Saratov region allow to conclude that these soils have a high potential for self-healing: the number of hydrocarbon-oxidizing microorganisms reaches 105 (in bulk soil) and 106 (in rhizosphere soil ) cells per gram. Phytoremediation or biostimulation are often preferable bioremediation approaches, excluding introduction of active biopreparations, and aimed at stimulating indigenous pollutant degrading populations. Monitoring of plant species most frequently encountered in oil-polluted areas identifies potential phytoremediation plants such as Medicago sativa, Medicago falcate, Lolium perenne, Calamagrostis epigeios, Sorghum bicolor. Analysis of prevalence of rhizospheric hydrocarbon-degrading microorganisms allowed us to identify the plant species that support the highest population of these microorganisms in their root zone: Calamagrostis epigeios, Medicago sativa, Medicago falcate, Phragmites australis, Atriplex tatarica.
Capacity building.
Partner 3 (UGent) has hosted twice Dr. Anastasiia Krivoruchko,from partner 7 (IEGM) for training in MALDI-TOF characterisation of bacteria (13/11 to 5/12/2012 and November 2013). This work contributed to the characterization and identification of rhodococci from contaminated soils.
Partner 5 (UNITO) hosted Dr. Maria Kuyukina from the IEGM team in December 8-10, 2012. During this stay, a discussion was carried out on the compatibility of the methods for isolating hydrocarbon-oxidizing bacteria and fungi. According to this discussion, protocols of hydrocarbon-oxidizing bacteria detection and isolation using selective media are currently worked on at the IEGM to be combined with the MUT protocols of hydrocarbon-oxidizing fungi isolation and detection. A combined protocol is available at the BRIO website.
Development of rhizosphere-enhanced bioremediation associations for multifunctional remediation
Partner 2 (UCL) has a number of AMF listed in the catalogue which offer special interest for bioremediation of pollutants such as hydrocarbons.
Partner 2 is also leading a national initiative on the isolation, identification and application of arbuscular mycorrhizal fungi and other filamentous fungi from the Yasuni national park in Ecuador. This park is a hotspot of diversity but is also exploited for hydrocarbons. In this project, partner 2 (UCL) is sampling polluted soils for fungi. Several species has been isolated and are in the process of identification. In a further step, these strains will be tested for bioremediation and included in the BRIO catalogue.
During the final month of the project the UNITO team (partner 5) received one fungal strain from the IEGM team, Perm, Russia. These strains had been isolated as “contaminants” from a trial devoted to identify bacteria able to degrade pharmaceutical organopollutants. The Russian isolate is a fungal consortium of two species endowed of bioremediation capabilities: Trametes versicolor and Aureobasidium pullulans. The two strains are currently under evaluation at IEGM to assess their potential in bioremediation and their possible cooperation with bacterial strains.
Partner 7 (IEGM) has developed actinobacterial associations based on IEGM collection strains suitable for oil-contaminated soil and water bioremediation. One association was tested in laboratory soil microcosms and provided an effective reduction in oil contamination under cold conditions. The association developed was recommended for multifunctional remediation (restoring all natural functions of soil) of oil-contaminated soils in cold climate regions (Urals, Russia) and is currently field tested by the Priroda-Perm company under the joint demonstration project on field-scale bioremediation. This demonstration project is aimed to evaluate the efficacy of bioremediation using a selected bacterial association, and to demonstrate its advantages to SMEs and large industrial companies dealing with the oil-contaminated soil clean-up. Another actinobacterial association was developed for oil-contaminated wastewater purification and is currently under laboratory bioreactor testing.
Partner 7 (IEGM) has also studied actinobacterial associations made of the IEGM strains able to degrade pharmaceutical pollutants, in particular codeine-containing waste, to be used in the wastewater treatment process. During laboratory trials, an air-born fungus culture was isolated from the fermentation medium, which was able to efficiently degrade codeine. This culture was sent to Partner 5 (UNITO) for taxonomic characterization and deposition in the UNITO-MUT collection. Partners 5 and 7 intend to carry out further collaboration research using selected actinobacterial and fungal cultures to develop associations useful for pharmaceutical wastewater purification and contaminated site bioremediation.
Some strains from Partner 8 (IBPPM) collection were studied on their capacity to improved phytoremediation effectiveness. Rhizosphere-enhanced plant-microbial associations for phytoremediation of contaminated soil were developed for PAH-contaminated soil, for petroleum-contaminated soil, for glyphosate-contaminated soil and for soil contaminated with Pb, Cd, Ni and As.
IBPPM collection has been enhanced by five fungal strains with ligninolytic enzymes xenobiotic-degrading activities. These strains have been isolated in Saratov region during the field survey. These strains were identified in collaboration with Dr. Cristina Varese.
Database subset of IBPPM collection used for bioremediation includes 34 oil-degrading strains, 12 PAH-degrading strains, 8 synthetic surfactant-degrading strains, 10 heavy metals resistant strains and 10 phosphonates-resistant or degrading strains.
Metagenomic data.
The database allows storage and management of metagenomic data. Yet no metagenomic data from soils are currently available to the partners, or have been analysed for biofertilizer presence or activity.
Oil-contaminated soil and rhizosphere samples taken during the common field survey in Perm were cryopreserved by Partner 7 (IEGM) for future metagenomic studies. Also, total bacterial DNA isolated from 2 fresh soil and 2 rhizosphere samples was cryopreserved and tested by direct PCR.
For each strain isolated from polluted sites and potentially useful for bioremediation, the information on contaminated substrate and geographic origin is provided.
Such as for biofertilizers, biopesticides, these strains for bioremediationcan be searched in the BRIO by using either the taxon browser or the strain properties browser.
Potential Impact:
A. IMPACT
1. Direct and future impact of the outcomes related to BRIO Objective 1: To organize cooperation between specialised collections.
BRIO builds long term links and synergies between major Russian and EU Biological Resources Centres. BRIO not only contributes to increase the synergy and to foster collaborative efforts within the group of BRIO, but also acts as an initial core around which complementary efforts crystallize and develop into further collaborative efforts. It opens new perspectives of collaboration between Russian and EU teams who will access the resources of and collaborate with the Pan-European Rhizosphere resources Network (PERN). PERN is conceived as a resources hub, according the OECD recommendations (3).
CONNECTIONS AND COLLABORATIONS
Establishing PERN enables further cooperation and exchanges at global level.
- Via the coordinator (SPP-PS) and Russian partner 6 (VKM) connections have been established with the World Data Centre for Microorganisms (WDCM) in the field of bioinformatics.
- All partners are registered in the World Federation for Culture Collections (WFCC). All partners also join the Global Catalogue of Microorganisms for connecting catalogue into one global portal.
- Via the coordination (SPP-PS), partners have incorporated the developments of the TRUST (1) initiative related to the implementation of the Nagoya Protocol in the acquisition and distribution policy (Accession & Transfer). It included the organization of an intermediary ad hoc meeting of June 2013 in Moscow were approved by the participants.
- Connection with the European Infrastructure MIRRI - Microbial Resource Research Infrastructure is ensured via the participation of BRIO partners VKM, UGent and SPP-PS to the MIRRI preparatory phase.
2. Direct and future impact of the outcomes related to BRIO Objective 2: To constitute a common pool of micro-biodiversity exploitable for research and industry: the Pan-European Rhizosphere resources Network (PERN).
WINDOW TO THE RHIZOSPHERE RESEARCH & INNOVATION COMMUNITY
BRIO participants are associated to create a pool of common rhizosphere microbial resources, related data and human expertise. They manage a one-stop shop HUB making these resources available for scientists. Putting together these material, data and expertise facilitates multidisciplinary research.
The focus is laid on biofertilisers, biopesticides and bioremediation to help scientists to cope with challenges in agriculture, forestry and environment under changing climate conditions. Facilitating the access to and the use of well documented, quality micro-organisms of the rhizosphere fosters the exploitation of renewable biological resources, a major objective of the European Knowledge Based Bio-Economy (2).
The project impacts on cumulative cutting-edge research via:
1. Support to collaborative scientific research:
- Development of long-term links between Russian and other partners beyond the project life;
- Development of collaborative synergies with industries to help develop eco-efficient products using rhizosphere micro-organisms and communities in promoting plant growth, suppressing soil borne disease and priming plant biotic defences.
2. Conservation of the natural resources:
- Underpinning cumulative research by ensuring long term preservation of the microbial components of the rhizosphere in reputed West European and Russian collections;
- Use the expertise of PERN partners for management of fungal and bacterial germplasm collections;
- Providing soil microbial micro-flora for R&D;
- Linking the consortium with industry for transfer of technology.
3. Facilitated communication and exchanges
The website is the portal to the resources gathered and organised during the project
(a) PERN ensures conservation of micro-organisms associated in the plant-microbes-soil systems, facilitating further use by scientists from academies and enterprises (including SME) in EU and Russia.
=>The website host a searchable catalogue
(b) PERN contributes to research on better understanding of rhizosphere diversity and its effect on soil fertility and suppression of pathogens. That will generate research outcomes useful for the development of eco-efficient technologies and management practices, reducing the adverse effects of human activities on the environment.
=> The website posts article relevant to the rhizosphere study.
(c) PERN shares knowledge and strengthen capacities in the area of upstream and downstream research in soil microbiology. It organises and generates resources and knowledge applicable globally. It promote exploitation of the beneficial properties of rhizosphere micro-organisms For instance, oil-polluted areas are a problem encountered in all continents, BRIO partners and thus PERN members have experience in different part of the globe, in different eco-systems, under different climatic and edaphic conditions. Combining expertise and comparing solutions applied for particular situation brings innovative approach. PERN members are scanning the possibilities for funding and partnership to further extent the impact of the newly developed tools.
=> The website proposes lists of experts in the three core field of application: bio-remediation, bio-fertilization, and bio-control.
In summary the project outcomes have been made accessible via the website. Website update and maintenance, including catalogue database optimization is ensured by Russian partner VKM and coordinator SPP-Ps with the voluntary contribution of the PERN members.
B. CONTRIBUTION TO THE EU POLICY AND OBJECTIVES IN R& I.
PERN participates in the exploration and subsequent exploitation of renewable biological resources (i.e. micro-organisms, plants and animals), contributing to the European knowledge based bio-economy (2).
Since this project is by essence cross-border and focused on microbial diversity resources it has to abide to the provisions of the Convention of Biological Diversity (CBD) and the Nagoya Protocol, contributing to the ex situ conservation of Biological Diversity and the fair and equitable sharing of benefits:
BRIO contributes to the commitments of the EU by conforming to the Convention of Biological Diversity (CBD) more particularly articles 9, 16, and 19.
Like already explained in the previous section of the report, the acquisition and distribution policy designed to be common to the PERN members design a non-discriminatory framework for the access to genetic resources, and for the fair and equitable sharing of the benefits arising out of their utilization. PERN hosts EU and Russian world-classes international culture collections which follow these rules and more particularly the recommendations of the EU funded code of conduct MOSAICC (5) and the guidelines of the TRUST(1) system.
Following the Nagoya Protocol, PERN members will implement the EU Regulation 511/2014 concerning fair and equitable sharing of benefits which can be monetary and/or preferably non-monetary and directed in such way as to promote conservation and sustainable use of microbial resources of the rhizosphere.
C. DISSEMINATION AND EXPLOITATION OF PROJECT RESULTS
Website and online catalogue.
Since a major objective of BRIO is to provide for publicly available rhizosphere resources, the project website is the central instrument to provide access to the projects results, mainly via the online catalogue of strains, and also through complementary sources of information and links.
The online catalogue is the basis instrument for exploitation of the project results since it will allow users to access well-documented technically and legally fit-for-use rhizosphere microbial resources and related information.
As part of the website, the project Intranet serves as internal communication only accessible to partners and the European Commission.
Contacts with private companies have been made, more specifically concerning existing applications in the fields of biofertilisers, biopesticides and bioremediation. Contacts are maintained via the PERN members who usually collaborate with them.
Contacts with other Russian resources centres are ongoing. The Russian members of PERN are actively promoting the necessary structuration of the Russian network of microbial collections. Depending on available funding and opportunities PERN partners will invite other research teams to joint activities as in a capacity building effort.
Other activities include participation to workshop, training sessions. It is worth noting that although not scheduled in the project life, training sessions have been already organised by taking advantages of the travel of the PERN members to BRIO meeting or to other conference.
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(1) TRUST stand for Transparent User-friendly System of Transfer see http://bccm.belspo.be/projects/trust
(2) Bio-economy is defined as that part of economic activities “which captures the latent value in biological processes and renewable bio-resources to produce improved health and sustainable growth and development”.
(3) See recommendations in document: OECD Best Practice Guidelines for Biological resource Centre, 2007, OECD, Paris. http://www.oecd.org/dataoecd/6/27/38778261.pdf
(4) Regulation (EU) No 511/2014 of the European Parliament and of the Council of 16 April 2014 on compliance measures for users from the Nagoya Protocol on Access to Genetic Resources and the Fair and Equitable Sharing of Benefits Arising from their Utilization in the Union
(5) MOSAICC stands for “Micro-organisms Sustainable use and Access regulation International Code of Conduct”. It is a voluntary Code of Conduct to support the implementation of the Convention on Biological Diversity (CBD, Rio de Janeiro, 5 June 1992) at the microbial level, in accordance with other relevant rules of international and national laws. See http://www.cbd.int/abs/instruments/ or http://bccm.belspo.be/projects/mosaicc.
List of Websites:
PROJECT WEBSITE: www.pern-brio.eu and www.brionet.eu
PARTNERS CONTACT DETAILS
Partner 1 - Federal Public Planning Service - Science Policy (SPP-PS), Belgium
Avenue Louise, 231 Louizalaan
B-1050 Brussels, Belgium
Contact: Philippe Desmeth
Tel: +32 2 238 3713
Fax: +32 2 230 5912
E-mail: philippe.desmeth@belspo.be
Partner 2 - Mycothèque de l’Université catholique de Louvain (UCL), Belgium
Mycothèque de l'Université catholique de Louvain, UCL
Croix du Sud 2, box L7.05.06
B-1348 Louvain-la-Neuve, Belgium
Contact: Prof. Dr. Stéphane Declerck
Tel.: +32 10 473742
Fax: +32 10 451501
E mail: bccm-mucl@uclouvain.be
Partner 3 - Laboratorium of Microbiology (LM-UGent), Belgium
Laboratorium voor Microbiologie, Vakgroep Biochemie en Microbiologie
Fac. Wetenschappen, Universiteit Gent
K.L. Ledeganckstraat 35
B-9000 Gent, Belgium
Contact: Prof. Dr. Anne Willems
Tel.: +32 9 2645103
Fax: +32 9 2645092
E-mail: Anne.Willems@UGent.be
Partner 4 - Agroscope Reckenholz-Tänikon, Research Station (ART), Switzerland
Ecological Farming Systems group
Swiss Federal Research Institute Agroscope
Reckenholzstrasse 191
CH 8046 Zurich, Switzerland
Contacts: Prof. Dr. Marcel van der Heijden, Dr. Fritz Oehl
Tel.: +41 44 377 7278
Fax: +41 44 377 7201
Email: marcel.vanderheijden@art.admin.ch
E-mail: fritz.oehl@art.admin.ch
Partner 5 - Dipartimento di Biologia Vegetale- Università degli Studi di Torino (UNITO), Italy
Dipartimento di Scienze della Vita e Biologia dei Sistemi
Università degli Studi di Torino
viale Mattioli, 25
10125 Torino, Italia
Contact: Prof. Dr. Giovanna Cristina Varese
Tel: +39 011 6705984
Fax: +39 011 6705962
Email: cristina.varese@unito.it
Partner 6 - Skryabin Institute of Biochemistry and Physiology of microorganisms - Russian Academy of Sciences, Russian Federation
All Russian Collection of Micro-organisms (VKM)
Prospekt Nauki, 5
142290, Pushchino, Moscow region, Russian Federation
Contact: Dr. Lyudmila Evtushenko
Tel.: +7 4967 730924
E-mail: evtushenko@ibpm.pushchino.ru
Partner 7 - Institute of Ecology and Genetics of Micro-organisms (IEGM) - Ural Branch of the Russian Academy of Sciences, Russian Federation
Laboratory of Alkanotrophic Microorganisms
Regional Specialized Collection of Alkanotrophic Micro-organisms
13, Golev Street,
614081 Perm, Russian Federation
Contact: Prof. Dr. Irena Borisovna Ivshina
Tel. +7 342 244 6714
Fax: +7 342 244 6711
E-mail: ivshina@iegm.ru
Partner 8 - Institute of Biochemistry and Physiology of Plants and Micro-organisms - Russian Academy of Sciences (IBPPM RAS), Russian Federation
Collection of Bacteria
Prospekt Entuziastov, 13
410049 Saratov City, Russian Federation
Contact: Prof. Dr. Olga Turkovskaya
Tel.: +7 8452 970786
Fax: +7 8452 970383
Email: ovtur@ibppm.sgu.ru
